HSD17B7 counters bone loss in estrogen deficiency via estrogen receptor stabilization and mediates the effect of raloxifene

HSD17B7 counters bone loss in estrogen deficiency via estrogen receptor stabilization and mediates the effect of raloxifene | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article HSD17B7 counters bone loss in estrogen deficiency via estrogen receptor stabilization and mediates the effect of raloxifene Young Jae Moon, Junyue Zhang, Yiping Song, Jeong-Hyun Koo, Si Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6158228/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Estrogen receptor (ER) α is a key regulator of osteoclasts in osteoporosis induced by estrogen deficiency. Although ERα is regulated through interactions with various coactivators, the precise mechanisms remain unknown. We used LC-MS/MS to screen proteins that bind to ERα and identified a physical interaction between HSD17B7 and ERα, specifically that ERα binds to the 119–172 domain of HSD17B7. This interaction blocked ubiquitin-proteasomal degradation of ERα and increased ERE activity. Estrogen-deficient mice lacking HSD17B7 in their preosteoclasts showed more severe bone loss than control mice. This was attributed to increased mitochondrial biogenesis through the activation of PLD1-mTOR signaling. Additionally, in preosteoclasts derived from patients with severe osteoporosis, HSD17B7 and ERα expressions were significantly reduced, compared with control subjects. Finally, raloxifene, which boosts ERα, did not inhibit bone loss without HSD17B7, confirming the modulation of ERα through HSD17B7. Therefore, HSD17B7 regulation is a novel therapeutic approach for alleviating estrogen-deficient osteoporosis. Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis HSD17B7 Estrogen Receptor alpha Osteoporosis Mitochondria Raloxifene Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Osteoporosis is a prevalent bone disease marked by decreased bone mineral density and the deterioration of bone microarchitecture, which significantly increases the risk of fractures, especially among postmenopausal and elderly people 1 . This condition arises from the uncoupling of bone formation and maintenance, driven by osteoblasts and osteocytes, and bone resorption, driven by osteoclasts, during bone remodeling. In postmenopausal women, the level of the sex hormone estrogen decreases rapidly, accelerating bone resorption by activating osteoclasts 2 . Aging also causes gradual bone loss due to changes in intrinsic factors of the bone (e.g., energy metabolism, oxidative stress, DNA damage, and osteocyte senescence) and external factors (e.g., the ovary and immune system) 3 – 5 . Aging and declines in sex hormones thus interact to produce an overall bone phenotype in the elderly that leads to osteoporosis. The estrogen–estrogen receptor (ER) complex plays a critical role in bone remodeling through several mechanisms. It not only suppresses osteoclast numbers by regulating the apoptosis and cellular metabolism of osteoclast precursors 6 , 7 , but it also inhibits osteoclastogenesis by regulating the receptor activator of NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG) 8 – 10 . Thus, estrogen supplementation is theoretically a treatment option for osteoporosis; however, the use of estrogen alone can increase the risk of endometrial, ovarian, and breast cancers 2 , 11 . Therefore, selective estrogen receptor modulators (SERMs) such as tamoxifen, raloxifene, and bazedoxifene are used in clinical practice as a form of estrogen replacement therapy 12 . These modulators, bound with co-activators, have estrogen-like activity in bone and anticancer effects 13 . Unliganded ERα also plays a major positive role in bone formation through the mechanical stimulation of osteoblasts 14 – 16 , and it has been reported to enhance estrogen response element (ERE) activity in the bone marrow of ovariectomized (OVX) mice 17 . However, the precise mechanisms of action that regulate the ERα complex bound to a SERM and the unliganded ERα complex are still not fully understood. For this work, we used LC-MS/MS to screen for proteins that bind unliganded ERα in preosteoclasts from OVX mice and found that 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7) binds unliganded ERα. HSD17B7 catalyzes the conversion of low-activity keto-steroid sex hormones into their high-activity hydroxylated forms, regulating estrogen and cholesterol synthesis 18 , 19 . More recently, it has been reported that HSD17B7 regulates cellular energy metabolism 20 . In this study, we confirm a physical interaction between HSD17B7 and ERα, specifically ERα binding at the 119–172 domain of HSD17B7. This interaction prevents the ubiquitin-proteasomal degradation of ERα and increases ERE activity. To investigate the osteoclastic role of HSD17B7 in osteoporosis, we generated osteoclast-specific HSD17B7 knockout mice (cKO) and performed OVX. The cKO OVX group showed more severe trabecular bone loss than control OVX mice. A mechanistic investigation through a transcriptomic analysis of preosteoclasts from cKO and control mice revealed significant upregulation of genes involved in oxidative phosphorylation and mitochondrial biogenesis, which are associated with the activation of the PLD1-mTOR pathway, in cKO preosteoclasts. At the cellular level, oxidative consumption was increased in cKO preosteoclasts, compared with control preosteoclasts, and that effect was reversed by the application of a PLD1 inhibitor, indicating that HSD17B7 regulates preosteoclast energy metabolism via the PLD1 signaling pathway. Additionally, protein expression levels of HSD17B7 and ERα were significantly reduced, compared with control patients, in preosteoclasts and femoral heads derived from osteoporosis patients, and those levels correlated positively with bone mineral density, confirming the importance of HSD17B7 in humans. Lastly, to verify that HSD17B7 is a potential SERM co-activator, we assessed raloxifene's therapeutic effect in cKO and control mice following OVX. Raloxifene inhibited OVX-induced bone loss in control mice, blocking PLD1 pathway activation and mitochondrial oxidation in preosteoclasts. However, those effects were not observed in HSD17B7-deficient cells and animal models. This study suggests that HSD17B7 enhances and upregulates ERα expression in preosteoclasts, thereby preventing bone loss by inhibiting osteoclast-mediated bone resorption. Therefore, HSD17B7 regulation is a novel therapeutic approach for alleviating osteoporosis. Materials and methods Animal experiments WT C57BL/6J mice were obtained from Damul Science (Daejeon, Korea). HSD17B7 +/- mice were obtained from Gempharmatech (Nanjing, China), HSD17B7 fl/fl mice were obtained from Cyagen (Santa Clara, CA, USA), and LysM-Cre mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Osteoclast-specific HSD17B7 knockout mice (HSD17B7 O c KO) mice were generated by mating HSD17B7 fl/fl mice with LysM-Cre mice. HSD17B7 fl/fl littermates served as controls. Animals were maintained on a 12-h light/dark cycle at 23±1°C with free access to food and water. The genotypes of the mice were determined by polymerase chain reaction (PCR) using tail tissues. Eight-week-old female mice were subjected to bilateral OVX or sham surgery, and the mice were sacrificed after 6 weeks. The bone parameters of cancellous and cortical bone from the distal femur were analyzed using a SKYSCAN 1076 Micro‐CT unit (SkyScan, Kontich, Belgium) installed in the Center for University-wide Research Facilities (CURF) at Jeonbuk National University. The study protocol was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (Permit No: JBUH-IACUC-2020-20). In vitro o steoclast differentiation Femoral CD11b+ bone marrow cells (BMCs) from 8-week-old mice were used for osteoclastogenesis. Briefly, mouse femoral bone marrow cells were extracted, and the cell suspensions were passed through a 70-µm mesh filter screen. Red blood cells were removed using ACK lysing buffer. The following day, the supernatant cells were collected and combined with CD11b (Invitrogen, Carlsbad, CA, USA), and anti-biotin microbeads (Miltenyi Biotec, San Diego, CA, USA) were used for positive sorting. The isolated cells were then seeded onto 6-well plates at a density of 1x10 6 and cultured in 30 ng/ml M-CSF (PeproTech, London, England) for 2 days. To induce osteoclast formation, we supplemented the growth medium with M-CSF (10 ng/ml) and RANKL (30 ng/ml, PeproTech). After 3 days of osteoclast differentiation, tartrate-resistant acid phosphatase (TRAP) multinucleated cells were detected using a TRAP staining kit (Cosmo Bio Co., Ltd., Tokyo, Japan). The procedures for human osteoclast extraction and osteoclastogenesis were similar to those in a previous study 21 . Briefly, human CD11b+ BMCs were extracted, and 25 ng/ml M-CSF and 50 ng/ml RANKL were added for osteoclast differentiation over 7 days. LC-MS/MS and peak alignment Immunoprecipitated proteins differentially expressed in CD11b+ BMCs from Sham and OVX mice were found by staining SDS gels with colloidal Coomassie blue (Abcam, Cambridge, UK). After the proteins were excised from the gel bands, they were subjected to gel trypsin digestion. The tryptic peptides were analyzed for related protein identification using a high-resolution mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific, Waltham, MA, USA) as described previously 22 . Patients We evaluated patients who underwent hip arthroplasty at Jeonbuk National University Hospital (Jeonju, South Korea) between August 2022 and August 2023. The inclusion and exclusion criteria were described previously 21 . Sixteen patients met the criteria. All cases were reviewed according to the World Health Organization Diagnostic Criteria for Osteoporosis 23 and the American Association of Clinical Endocrinologists staging system 24 . This study was performed with the approval of the institutional review board at Jeonbuk National University Hospital, and the requirement for informed consent was waived (IRB number, JBUH 2023-12-023). Histology Mouse femurs were fixed in 4% paraformaldehyde, decalcified, and soaked in 10% EDTA for 1 month until the bone softened evenly to complete the decalcification. The femurs were then embedded in paraffin, and cut into 5 μm transverse sections at different levels. The sections were stained with hematoxylin and eosin (H&E) and TRAP after rehydration. For immunofluorescence staining, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After being blocked with 5% BSA (GenDEPOT, Barker, TX, USA) to prevent non-specific staining, the slides were incubated with primary anti-HSD17B7 (14854-1-AP, ProteinTech Group, Chicago, IL, USA) and anti-cathepsin K (sc-48353, Santa Cruz Biochemicals, Dallas, TX, USA) overnight at 4°C. Alexa Fluor 488-conjugated anti-rabbit IgG (1:100 Invitrogen) and Alexa Fluor 594-conjugated anti-mouse IgG (1:100 Invitrogen) secondary antibodies were added to the sections to visualize the staining. DAPI (1:200 Invitrogen) was used for nuclear staining. Immunofluorescence images were taken with a Zeiss LSM 880 on an Airyscan confocal microscope (Carl Zeiss, Göttingen, Germany). The osteogenic ability of each group was shown by observing the formation rate of new mineralized bone. Ten days before sacrifice, mice were given an intraperitoneal injection of Calcein (30 mg/kg Sigma-Aldrich, St. Louis, MO, USA), and 3 days before sacrifice, they received a dose of Alizarin red (30 mg/kg Sigma-Aldrich) to form dual fluorescent labels. Sample preparation followed a previously described method 25 . Briefly, femurs were collected, fixed, and subsequently immersed in 5% aqueous potassium hydroxide (KOH) for 96 h. After being embedded in paraffin, each mineralized femur was cut into 5-μm slices with a microtome and observed with an APX100 microscope (Olympus, Waltham, MA, USA). ImageJ (Version 1.53a, National Institutes of Health, Bethesda, MD, USA) software was used to evaluate the bone formation rate of each bone surface (BFR/BS). Five samples in each group were tested. Enzyme-linked immunosorbent assay (ELISA) Blood was collected from Sham and OVX mice, and the serum was separated. Serum RANKL and OPG were measured using RANKL ELISA assay kits (Abcam) and OPG ELISA assay kits (R&D Systems, MN, USA), respectively, according to each manufacturer's protocols. Flow cytometry We first extracted femoral bone marrow cells without red blood cells and then incubated the resulting single-cell suspension with 1:200 anti-CD16/32 (FcγRIII/II Invitrogen) for 15 minutes to block the non-specific binding of Fc receptors. Antibody mixtures, CD11b (Invitrogen), HSD17B7 (ProteinTech Group), and ERα (R&D Systems, Minneapolis, MN, USA) typically diluted at 1:100, were added and incubated in the dark at 4°C for 30 minutes. Subsequently, appropriate secondary antibodies were added and incubated at room temperature for 30 minutes. Data were acquired with a FACS Aria III (BD, Franklin Lakes, NJ, USA). The analysis was performed using FlowJo software (Version 10.0.7, Tree Star, San Carlos, CA). Cell culture, transient transfection, and promoter luciferase assay Human embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM). To examine the relationships of HSD17B7, HEK293T cells were transfected with 1 μg of plasmid DNA containing Flag, MYC-HSD17B7 (#RC209534, OriGene Technologies, Rockville, MD), sh-HSD17B7 (sc-88433-SH, Santa Cruz), or HA-ERα (Applied Biological Materials Inc. Richmond, Canada) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). To evaluate promoter activity, 1 μg each of FasL and ERE promoter luciferase (Promega, Madison, WI, USA) were used. Briefly, HEK293T cells were transfected with plasmids encoding the Flag, MYC-HSD17B7, shHSD17B7, ERE-luc, FasL-luc, or Renilla luciferase reporter (pRL-TK-luc). After 24 more h, the cells were harvested in a reporter lysis buffer. Luciferase activity was determined in whole cell lysates using a luciferase assay kit (Promega, Madison, WI, USA). Subcellular fractionation, co-immunoprecipitation, and Western blotting We extracted proteins from tissues or cells using a protein extraction kit (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol. Nuclear, membrane, and cytoplasmic extracts were isolated using a subcellular protein fractionation kit (#78840, Thermo Fisher Scientific). Homogenates (20 μg for Western blotting) were separated by SDS-PAGE and transferred to nitrocellulose membranes. For co-immunoprecipitation, 400 μg of homogenates were immunoprecipitated with the indicated antibody at 4°C overnight. The immunocomplexes were pulled down using protein A/G agarose beads (#20421 Thermo Fisher Scientific) and separated by Western blotting. After blocking the samples with 5% skim milk, I incubated them with primary antibodies overnight at 4 °C. This was followed by a 1-hour incubation with an HRP-conjugated secondary antibody at room temperature. Then, the membranes were visualized with an enhanced chemiluminescence detection kit (Millipore, Billerica, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA). The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ERα (R&D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology). Molecular docking analysis We obtained the protein structures of ERα and HSD17B7 from the UniProt database and then processed them using PyMOL (Version 2.1, Schrödinger, Inc., New York, NY) and uploaded them to the HDOCK SERVER website (http://hdock.phys.hust.edu.cn/) for protein–protein docking. We selected the combination with the highest docking score and confidence score for simulating the docking site and used PyMOL for the visual analysis. For docking with RAL, I obtained the SDF format file of its main active ingredient from the PubChem database. We then collected the protein structures of ERα and HSD17B7 from the PDB database. I used PyMOL to optimize the targets by removing water molecules and small molecule ligands and then used AutoDock Tools (National Institutes of Health, Bethesda, MD, USA) to add hydrogen atoms and charge treatments, saving the files in pdbqt format. We then performed molecular docking using PyRx (version 0.9.7) software's internal vina, calculated the binding energy, and output the result files. We used PyMOL for result visualization. Osteogenic function analysis As previously described, primary osteoblasts were extracted from mouse femurs and cultured in osteogenic induction medium (MUXMX-90021 Cyagen) for 7 or 14 days 26 . The cells were fixed with 4% paraformaldehyde for 10 minutes. For alkaline phosphatase (ALP) staining, the 7-day cells were treated with a BCIP/NBT kit (K4151 ApexBio, Houston, USA). The stained images were captured with a digital camera and quantified by measuring the OD value at 405 nm with an alkaline phosphatase assay kit (ab83369 Abcam). For Alizarin red S (ARS) staining, cells cultured for 14 days were treated with 40 mM ARS (Sigma-Aldrich), pH 4.0, and the staining results were photographed. Subsequently, 10% chlorinated pivaloyl chloride (Sigma-Aldrich) was added, and quantification was performed by measuring the OD value at 560 nm. Mutagenesis of HSD17B7 Based on the docking prediction results for HSD17B7 and ERα, corresponding deletion mutants from the HSD17B7-MYC plasmid were generated (Gene Synthesis, Seoul, Korea). Specifically, HSD17B7 d5-45 , HSD17B7 d119-172 , HSD17B7 d192-200 , and HSD17B7 d274-302 mutants were generated by deleting the corresponding sequences starting from the N-terminal sequence of HSD17B7. Quantitative real-time PCR with reverse-transcription analysis Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. Total RNA samples (1 μg) were reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed in 384-well plates using an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). RNA from individual samples was analyzed separately. The primer sequences used in the PCR are listed in Table S1. Measurement of the oxygen consumption rate (OCR) The OCR was measured in an XF24 extracellular analyzer (Agilent Technologies, Santa Clara, CA, USA). CD11b + BMCs treated with M-CSF and RANKL for 3 days (2 × 10 5 cells per well) were seeded into 24-well plates. The next day, the medium was changed to analysis medium containing 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. The cells were incubated in a CO 2 -free incubator at 37℃ for 1 h and then sequentially exposed to oligomycin (1 μM), FCCP (1 μM), and rotenone (0.5 μM) from a Seahorse XF cell Mito stress kit (Agilent Technologies). Data were processed using Wave software (Agilent Technologies). TUNEL assay and assessment of intracellular reactive oxygen species (ROS) Following the manufacturer's instructions, a DeadEnd colorimetric TUNEL system (G7360, Promega Madison, WI, USA) was used to assess apoptosis in CD11b + osteoclasts. The nuclei were stained with hematoxylin. To measure intracellular ROS levels, CD11b + osteoclasts were incubated with 10 mM H2DCFDA (D399, Life Technologies) for 30 minutes. The nuclei were stained with Hoechst (HY-15559, MedChemExpress, Monmouth Junction, NJ, USA). Observations and 5–6 microscopic images were obtained using an APX100 microscope. ImageJ (Version 1.53a) software was then used to evaluate the ratio of positive cells to total cells in each field of view. Evaluation of mitochondrial membrane potential CD11b + osteoclasts were incubated with JC-10 (786-1549 G-Biosciences, St. Louis, USA) dissolved in DMEM for 30 min. We used the APX100 microscope to observe JC-10 polymers (Ex/Em=540/590 nm) and JC-10 monomers (EX/Em= 490/525 nm). ImageJ (Version 1.53a) software was used to evaluate the ratio of JC-10 polymer cells to JC-10 monomers cells in each field of view. Transmission electron microscopy (TEM) and image analysis To compare the mitochondrial morphology of WT mice and HSD17B7 Oc KO mice, CD11b+ BMCs treated with M-CSF and RANKL for 3 days were collected and observed using TEM. As previously described 27 , the samples were fixed, embedded, sectioned, and then observed and imaged using a Hitachi Bio-TEM (H-7650, Hitachi, Tokyo, Japan). Based on previous research 28 , ImageJ (Version 1.53a) and DigitalMicrograph software (Version 3.6, Gatan Microscopy Suite, USA) were used to calculate the number of mitochondria. RNA sequencing data analysis Mouse femur bone marrow cells from WT mice ( n =4) and HSD17B7 Oc KO mice ( n =4) were extracted and differentiated into preosteoclasts (1x10 6 cells) for RNA sequencing. Total RNA was isolated from WT and cKO preosteoclasts using an RNeasy mini kit (QIAGEN Valencia, CA, USA) with DNase treatment to remove genomic DNA contamination. Strand-specific RNA libraries were prepared using a KAPA mRNA HyperPrep kit (KAPA Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. The prepared libraries were sequenced on an Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA) to generate paired-end reads. The raw sequencing reads were processed to remove adapters and low-quality bases using standard quality control procedures. The processed reads were aligned to the mouse reference genome using StringTie to estimate gene abundances in the read counts. A differential gene expression analysis was performed using the DESeq2 package within the R (Version 4.3.2) statistical environment. Genes with adjusted p -values less than 0.05 were considered to be significantly differentially expressed. Gene enrichment, functional annotation, and pathway analyses for significant gene lists were performed using the g: Profiler pathway (https://biit.cs.ut.ee/gprofiler/). Statistical analysis The data are expressed as the mean ± standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance followed by Fisher’s post hoc analysis. The significance of differences between groups was determined using Student’s unpaired t -test. Linear regressions were performed with GraphPad Prism, version 9.00 (San Diego, CA, USA). A p -value less than 0.05 was considered significant. Results Preosteoclast ERα regulation was associated with HSD17B7 from old and OVX mice Based on the increase in ERE activity in bone marrow cells following OVX in young mice 17 , we used immunoprecipitation with an ERα antibody to verify which proteins interacted with unliganded ERα in myeloid cells six weeks after OVX. Next, the proteins bound to ERα were placed on a gel, and the peptides and proteins were identified and estimated by mass spectrometry and the Mascot program (Fig. 1a). The data suggest that HSD17B7 can specifically bind to unliganded ERα in the OVX state (Fig. 1b), a notion supported by our subsequent predicted docking module analysis (Fig. 1c). To explore that possibility, we performed co-immunoprecipitation assays after inserting the ERα and HSD17B7 plasmids. Those results confirmed a physical interaction between HSD17B7 and ERα (Fig. 1d). Because ERα is closely related to both aging and estrogen deficiency 21 , we checked the expression of ERα and HSD17B7 in CD11b + BMCs. CD11b + BMCs from young OVX mice exhibited a significant increase in ERα expression, compared with young Sham mice. Interestingly, the expression pattern of HSD17B7 was consistent with that of ERα (Fig. 1e). To reconfirm the expression patterns of ERα and HSD17B7 in CD11b + BMCs in OVX model, we extracted BMCs from the mice and performed flow cytometry. Those results show that the percentage change in the co-expression of ERα and HSD17B7 in CD11b + BMCs was consistent with the change in protein level (Fig. 1f, Fig. S1). These results suggest that HSD17B7 might act as a co-activator that binds to ERα and regulates its stability in CD11b + BMCs in mice. HSD17B7 interacts with ERα and stabilizes its protein expression We next investigated the molecular mechanisms by which HSD17B7 regulates ERα. Plasmid DNA expressing ERα and control vector or HSD17B7 was transfected into HEK293T cells, which were then stimulated with estrogen. In the control group, the protein level of ERα gradually decreased, but in cells overexpressing HSD17B7 under each test condition, the decrease rate was slower (Fig. 2a). Furthermore, in HEK293T cells with HSD17B7 knockdown, treatment with cycloheximide led to the rapid degradation of ERα, compared with the empty vector control (Fig. 2b), indicating that HSD17B7 increases the stability of ERα in both ligand-dependent and -independent degradation pathways. To further explore the role of HSD17B7 in stabilizing ERα, we evaluated ERα proteasomal degradation. When cells were treated with MG132, a potent proteasome inhibitor, the effect of the HSD17B7-mediated ERα protein expression change was abolished (Fig. 2c), suggesting that HSD17B7-mediated ERα expression depends on proteasomal protein degradation. Immunoprecipitation was then performed on total protein lysates using ERα antibodies, followed by immunoblotting with ubiquitin antibodies. Those results show that the overexpression of HSD17B7 reduced the ubiquitination of ERα, and HSD17B7 knockdown increased ERα ubiquitination (Fig. 2e). Additional results from luciferase reporter gene assays using FasL-Luc, which is an ERα target gene 29 , and ERE-Luc confirmed that HSD17B7 overexpression significantly increased ERα transcriptional activity, whereas shHSD17B7 overexpression did not (Fig. 2d). Next, we constructed HSD17b7 deletion mutants based on the results of our docking module analysis (Fig. S2) and the known functional sites of HSD17B7 20 to determine where ERα binds to HSD17B7 (Fig. 2f). Immunoprecipitation experiments revealed that, compared with the other HSD17B7 deletion mutants, HSD17B7 d119-172 had a weaker binding affinity with ERα (Fig. 2g), and the ubiquitination inhibition effect of HSD17B7 on ERα was not observed when the 119-172 region of HSD17b7 was deleted (Fig. 2i). Additionally, luciferase reporter gene assays using FasL-Luc and ERE-Luc confirmed that deleting amino acids 119–172 of HSD17B7 abolished ERα transcriptional activity (Fig. 2h). These results indicate that ERα binds to the 119-172 region of HSD17B7, thereby inhibiting the ubiquitin-proteasomal degradation of ERα and increasing its target transcriptional activity. HSD17B7 deficiency exacerbates bone defects mediated by osteoclasts rather than osteoblasts in OVX-induced bone loss To investigate the physiological role of HSD17B7 in bone, we generated mice deficient in HSD17B7 in their whole bodies. Because HSD17B7 homozygotes die at embryonic age 10.5 days 30 , we used HSD17B7 heterozygotes (HSD17B7 +/- ) and then performed bilateral OVX on both WT and HSD17B7 +/- mice. H&E staining and micro-CT analyses of the distal femur showed that the HSD17B7 +/- mice and their WT littermates had phenotypically normal bones at birth and similar bone mass at 3 months of age (Fig. 3a, b). However, after OVX surgery, the amount of trabecular bone loss in the HSD17B7 +/- mice was significantly greater than that in the WT mice. Consistently, the trabecular bone volume fraction and trabecular number were significantly lower in the HSD17B7 +/- mice than in the WT mice (Fig. 3a, b). To further investigate whether the bone loss was due to an increase in osteoclast numbers, we performed TRAP staining on trabecular bone at the distal femur and found that HSD17B7 deficiency led to a significant increase in the number of osteoclasts, compared with WT mice (Fig. 3a, c). Consistently, in vitro osteoclastogenesis induction showed the same trend (Fig. 3f). Meanwhile, a decrease in uterine weight and increase in the RANKL/OPG ratio confirmed the success of the OVX surgery. Still, we found no difference between the genotypes that underwent OVX, indicating no difference in estrogen levels or cytokines for osteoclastogenesis (Fig. 3d, e, Fig. S3a). To investigate the effect of HSD17B7 on osteogenesis capacity, Calcein-Alizarin red dual labeling was performed on both genotypes that underwent OVX. The bone-forming rate results confirmed that HSD17B7 +/- mice and their WT littermates had similar osteogenic capacity (Fig. 3g). Primary femoral osteoblasts from HSD17B7 +/- mice were cultured under osteogenic conditions and did not show differences in osteoblast differentiation or mineralization (Fig. 3h). Compared with WT mice, primary osteoblasts from HSD17B7 +/- mice exhibited significantly reduced HSD17B7 expression. However, the expression levels of other osteoblastic markers ( Col1a1, Bglap, Runx2, Spp1, and Osx ) were unaffected by the HSD17B7 deficiency in HSD17B7 +/- mice (Fig. S3b). Micro-CT analyses of cortical bone at the mid-shaft femur showed that tissue mineral density was significantly lower following OVX treatment (Fig. S3c, S3d). However, OVX-induced cortical bone loss showed almost no difference between the HSD17B7 +/- and WT mice (Fig. S3c, S3d), confirming that the loss of HSD17B7 in bone selectively affects trabecular bone during OVX-induced bone loss. OVX-induced cancellous bone loss is aggravated by HSD17B7 deficiency in preosteoclasts To further investigate the role of HSD17B7 in preosteoclasts, we crossed HSD17B7 fl/fl mice with LysM-Cre mice to generate myeloid-specific HSD17B7 knockout (cKO) mice (Fig. S4a). A Western blot analysis confirmed the specific knockout of HSD17B7 in the bone marrow (Fig. S4b). The cKO mice had a normal phenotype at birth and showed bone mass similar to their HSD17B7 fl/fl littermates at 3 months of age (Fig. 4a). We performed OVX on 3-month-old cKO and control mice and sacrificed them six weeks post-surgery. As expected, OVX resulted in greater bone loss in cKO mice than in control mice, and the mice treated with a sham operation showed no difference in bone mass (Fig. 4a). Consistently, microstructural evaluation of the trabecular bone reflected that the lack of myeloid cell–specific HSD17B7 exacerbated OVX-induced trabecular bone loss (Fig. 4b). In the OVX state, the number of osteoclasts was significantly higher in cKO mice than in control mice (Fig. 4a, c). Additionally, the protective mechanism that increased the expression of ERα and HSD17B7 in bone marrow cells after OVX in control mice was not observed in the cKO OVX mice (Fig. 4d). As in the HSD17B7 +/- mice, the genotypes did not show differences in estrogen levels or cytokines for osteoclastogenesis after OVX (Fig. 4e, f). To further investigate the localization of HSD17B7 in the bone marrow, we performed immunofluorescence staining on the trabecular bone of the distal femur. We found that HSD17B7 was co-expressed with the osteoclast marker cathepsin K, supporting the importance of HSD17B7 in osteoclasts (Fig. 4g). In vitro studies corroborated these results. In CD11b + BMCs cultured with M-CSF and RANKL, HSD17B7 and ERα were co-expressed (Fig. 4h), suggesting that HSD17B7 plays a crucial role in trabecular bone resorption by bone marrow osteoclasts. However, this inhibitory effect of HSD17B7 on the number of osteoclasts did not affect the osteoclast differentiation signaling pathway mediated by M-CSF and RANKL (Fig. S4c). HSD17B7 ablation increases preosteoclast mitochondrial content and oxidative phosphorylation capacity To elucidate the mechanism by which HSD17B7 regulates preosteoclasts, we treated CD11b + BMCs from cKO mice with RANKL for two days and then performed an RNA sequencing analysis. Considering that the mitochondrial OxPhos of preosteoclasts plays an essential role in the number and function of osteoclasts via the estrogen–ER complex 7, 14 , we focused on the expression of mitochondria-related genes. The heatmap in Fig. 5a displays the increased expression of mitochondrial complex genes in cKO mice, compared with control mice. A qPCR analysis confirmed alterations in mitochondrial biogenesis and OxPhos gene expression (Fig. 5b). These findings correlate well with the increased mitochondrial DNA content and promoted expression of OxPhos complexes found in preosteoclasts from cKO mice (Fig. 5c). To ascertain whether the increased mitochondrial content in cKO preosteoclasts was associated with elevated respiratory function, we conducted Seahorse XF mitochondrial stress tests on differentiated preosteoclasts from CD11b + BMCs with both genotypes. We observed a significant increase in maximum OCR in cKO cells, indicating that HSD17B7 regulates energy metabolism in preosteoclasts by modulating OxPhos (Fig. 5d). The results of measuring mitochondrial membrane potential, an indicator of mitochondrial activity, also showed higher levels in the cKO group than the control group (Fig. 5e). Consistently, an electron microscopy analysis of preosteoclasts revealed a higher number of healthier mitochondria in cKO mice than in control mice (Fig. 5f). These results were corroborated by in vitro studies. TUNEL-positive cells and ROS-positive cells were significantly reduced in preosteoclasts from cKO mice, compared with control mice, indicating that the apoptotic process within preosteoclasts of cKO mice was inhibited, and oxidative stress levels were decreased (Fig. 5g). HSD17B7 affects osteoclast function through the PLD1-mTOR signaling pathway To elucidate the molecular mechanisms by which HSD17B7 regulates osteoclast numbers and energy metabolism, we further analyzed the RNA sequencing data. The volcano plot in Fig. 6a shows genes that were upregulated (in red) or downregulated (in blue) in cKO mice, compared with control mice. The KEGG pathway analysis (https://www.genome.jp/kegg/pathway.html) performed on transcriptomes that increased in preosteoclasts from cKO mice revealed significant alterations in the PLD1 signaling pathway (Fig. 6b). Because PLD1 and mTOR have been reported to have important roles in osteoclast differentiation and energy metabolism 33–35 , we investigated the PLD1-mTOR pathway. We found that proteins related to the PLD1 pathway and PLD activity were consistently and significantly increased in cKO mice, compared with the control group (Fig. 6c, d). To confirm that PLD1 signaling affects osteoclast function and cellular metabolism, we treated cKO preosteoclasts with the PLD1 inhibitor VU0359595 to inhibit the PLD1 pathway. Preosteoclasts deficient in HSD17B7 have increased OxPhos in the PLD1-mTOR signal pathway, resulting in osteoclast activation, and a PLD1 inhibitor was confirmed to block that effect (Fig. 6e–h). These results indicate that HSD17B7 inhibits osteoclast metabolism and activity by regulating the PLD1-mTOR pathway. HSD17B7-ERα expression is attenuated in patients with severe osteoporosis To determine the clinical relevance of our findings so far, we cultured CD11b + BMCs from human subjects with M-CSF and RANKL for 7 days and then analyzed their expression of HSD17B7 and ERα. Consistent with the results from mouse studies, the expression of HSD17B7 and ERα in human preosteoclasts decreased with increasing age (Fig. 7a). There was a positive correlation between the expression of HSD17B7 and ERα, as well as between HSD17B7 and bone mineral density (BMD) and ERα and BMD (Fig. 7a), supporting the animal data that HSD17B7 increases both ERα protein levels and BMD. On the other hand, both HSD17B7 and ERα correlated negatively with serum type I collagen C-terminal telopeptide (CTX), a marker of osteoclast activity (Fig. 7a). To further examine the expression of HSD17B7 in osteoclasts from patients with osteoporosis, we performed H&E and TRAP staining on femoral heads, revealing that trabeculae were sparser and the number of mature osteoclasts was significantly higher in osteoporosis patients than in the control group. Consistent with the results of our in vivo and ex vivo experiments, HSD17B7 expression was high in osteoclasts from the control group and significantly lower in osteoclasts from patients with severe osteoporosis (Fig. 7b). Finally, we measured the expression of HSD17B7 and ERα in preosteoclasts from patients with severe osteoporosis and control subjects (Fig. 7c). Compared with the control subjects, the expression of HSD17B7 and ERα in preosteoclasts from osteoporosis patients, who had lower BMD, higher CTX, and older age than the control group, was significantly lower (Fig. 7d). These findings suggest that decreased expression of HSD17B7 and ERα in preosteoclasts is highly associated with osteoporosis in humans. A lack of HSD17B7 abolishes the osteoclast-inhibitory function of raloxifene It has been reported that SERMs bind to ER, inhibit osteoclast activity 12 , and block breast cancer cell proliferation because they inhibit PLD1 36,37 . Interestingly, our present study shows that HSD17B7 stabilizes ERα to activate ERα target genes and inhibits the PLD1-mTOR pathway to block osteoclast activity. In other words, we observed a significant overlap in the roles of HSD17B7 and SERMs. Thus, we hypothesized that HSD17B7 would be related to the effect of SERMs. We treated cKO mice and control mice with a SERM after OVX and observed the bone phenotype. For this experiment, we used AutoDock Vina to simulate molecular docking between HSD17B7 and raloxifene, with a binding energy of -8.5 kcal/mol indicating a significant potential for actual binding (Fig. S5). To determine the effect of raloxifene on cKO mice, we subcutaneously injected 8-week-old female cKO mice with raloxifene five days a week for five weeks during the OVX protocol 38,39 . An H&E staining and analysis of the distal femur showed that, following OVX treatment, raloxifene treatment increased trabecular bone in control mice but had minimal effects on cKO mice (Fig. 8a). Consistent with those changes, far fewer osteoclasts were formed in the presence of raloxifene in control mice, but raloxifene treatment did not lead to significant changes in the number of osteoclasts in cKO mice either in vivo or in vitro (Fig. 8a–c). These results suggest that raloxifene increases bone mass in estrogen-deficient mice, similar to the effects of estrogen on bone, but it does not reverse bone loss in HSD17B7-deficient mice. Previous results have demonstrated that 17β-estradiol inhibits mitochondrial function and promotes apoptosis in preosteoclasts[7]. Raloxifene also regulates mitochondrial-mediated apoptosis and inhibits mitochondrial and peroxisomal β-oxidation 40,41 . Therefore, we performed Seahorse XF mitochondrial stress tests on preosteoclasts to investigate whether raloxifene interacts with HSD17B7 in ways that affect mitochondrial function in preosteoclasts. We observed that raloxifene significantly reduced OCR in control preosteoclasts, but it did not affect cKO preosteoclasts (Fig. 8d). Similarly, raloxifene decreased mitochondrial membrane potential in control preosteoclasts but not in cKO preosteoclasts (Fig. 8e), suggesting that HSD17B7 might be an essential mediator of the mitochondrial function–regulating effects of raloxifene in preosteoclasts. Next, we evaluated the expression of the PLD1 pathway in preosteoclasts after raloxifene treatment. Interestingly, RAL-stimulated control preosteoclasts showed significantly reduced expression of PLD1-related protein and PLD activity, along with increased expression of HSD17B7 and ERα, compared with untreated control cells. However, that effect was absent in cKO preosteoclasts (Fig. 8f, g). Through co-immunoprecipitation studies after raloxifene treatment of control and cKO preosteoclasts, we observed an increase in the physical interaction between ERα and HSD17B7 in control mice but not cKO mice (Fig. 8h). These findings suggest that raloxifene exerts its effects via HSD17B7 in preosteoclasts and suppresses bone loss in estrogen-deficient osteoporosis by regulating the expression of HSD17B7-ERα. Discussion In this study, we identified a previously unrecognized function of HSD17B7 as an ERα-binding protein that serves as a coactivator to influence trabecular bone mass in estrogen deficiency. Specifically, deleting HSD17B7 in myeloid cells enhanced the number of osteoclasts and decreased trabecular bone mass post-OVX, compared with control OVX, mirroring observations in myeloid ERα KO mice 42 . During increased bone resorption, HSD17B7 expression levels paralleled those of ERα in preosteoclasts. However, HSD17B7 ablation accelerated bone resorption, as evidenced by elevated mitochondrial content and oxidative capacity in preosteoclasts. A transcriptome analysis combined with physiological data pinpointed PLD1 as the target through which HSD17B7 modulates mitochondrial OxPhos, aligning with previous research that highlighted the crucial role of the PLD1 and mTOR signaling pathway in metabolism 33 – 35 , 43 . An analysis of human subjects revealed that preosteoclasts from severely osteoporotic patients exhibited lower expression levels of HSD17B7 and ERα than those from controls, suggesting that HSD17B7 acts as a positive regulator in human osteoporosis. Lastly, we demonstrated that the selective ER modulator raloxifene had no therapeutic effect on estrogen deficiency–induced osteoporosis in cKO mice. Therefore, raloxifene's inhibitory effect on osteoclast activation is mediated through HSD17B7, which emphasizes the importance of the HSD17B7–ERα regulatory mechanism in bone homeostasis. In this study, we used LC-MS/MS to find that HSD17B7 binds to ERα and reconfirmed that finding by in vitro co-immunoprecipitation. This binding occurred at 119–172 on HSD17B7, which includes the substrate binding site and the docking prediction site, rather than the steroid and NADP binding sites of HSD17B7 20 . The binding of the two proteins enhanced the stability of ERα by inhibiting its ubiquitin-proteasomal degradation. Evidence from diverse expression patterns supports this mechanism of ERα regulation by HSD17B7. First, when young mice are rendered estrogen-deficient, a complementary increase in ERα is observed in myeloid cells 17 , 21 , at which time the expression of HSD17B7 is significantly increased. Second, in aged rodents, ERα expression is markedly reduced by non-ligand-dependent ubiquitination 44 , and HSD17B7 is also reduced in the same manner. Third, ERα expression is consistently reduced in HSD17B7 KO preosteoclasts, compared with control cells. Lastly, the expression of HSD17B7 and ERα in patient-derived preosteoclasts showed a significant positive correlation. Because ERα has various post-translational modification sites that regulate its ubiquitin-proteasomal degradation 45 – 47 , binding to HSD17B7 is thought to affect stability by inducing changes in those lysine and serine residues. On the other hand, it is also possible that ERα regulates HSD17B7. ERα bound to the ligand was recruited to the promoter of HSD17B7 and stimulated its expression 19 . Furthermore, we found that the expression of HSD17B7 increased when control preosteoclasts were treated with raloxifene. Thus, the regulatory mechanism of HSD17B7 and ERα is thought to be a feed-forward loop. Through a transcriptome analysis, we found that the phospholipase D signaling pathway was a significant mechanism in the increase in the number of osteoclasts in HSD17B7-deficient preosteoclasts. PLD1 is an upstream factor of the mitogenic activation of mTOR signaling and regulates cell growth through the PLD1-mTOR-S6K1 axis 35 . Consistent with these results, the osteoclastic activity of HSD17B7 knockout preosteoclasts accompanied the activation of the PLD1-mTOR-S6K1 axis, and that osteoclastic activity was suppressed by a PLD1 inhibitor. PLD1 is involved in synoviocyte activation and the expression of various cytokines in rheumatoid arthritis 48 , and PLD1 inhibitors have been reported to alleviate the symptoms of collagen-induced arthritis and inhibit osteoclast activity 33 , supporting our findings in this study. Meanwhile, PLD1-mTOR is a central regulator of not only cell growth but also cellular metabolism. PLD1 or mTOR inhibition suppresses oxygen consumption and mitochondrial capacity and increases ROS levels, leading to cell death 49 – 52 . HSD17B7 KO preosteoclasts showed increased OCR, enhanced mitochondrial biogenesis, and decreased ROS, which reduced the number of apoptotic cells. All of these metabolic-related preosteoclast phenotypes were reversed by a PLD1 inhibitor, suggesting that metabolic events associated with HSD17B7 are regulated by PLD1-mTOR signaling. Although we could not provide direct evidence that the phenotypes of HSD17B7 KO preosteoclasts were entirely related to ERα, we can speculate that the effects of HSD17B7 are highly associated with ERα based on our result that raloxifene, which binds to ERα and acts on preosteoclasts, did not improve the bone phenotype in HSD17B7 KO mice and on reports that increased metabolic activity in ERα KO preosteoclasts 7 and the overexpression of PLD1 were observed in ER-negative breast cancer cells 53 , 54 . Raloxifene, a SERM, exploits the positive effects of ERα on bone to alleviate osteoporosis caused by estrogen deficiency. It has been used extensively clinically and has shown various pharmacological properties, including anti-osteoporotic, antiviral, immunomodulatory, and anticancer activities 12 , 40 , 55 , 56 . We introduced raloxifene to investigate the role of HSD17B7 in estrogen-deficiency osteoporosis for three reasons. First, in cKO and control OVX models, the RANKL-to-OPG ratio remained the same between the OVX genotypes, and estrogen was reduced by the same amount. However, the degree of bone resorption was different, suggesting that ERα, which is highly related to HSD17B7 in preosteoclasts, might have estrogen-independent bone protective effects. Second, although the exact mechanism has not yet been fully elucidated, raloxifene is a PLD1 inhibitor 37 . Because HSD17B7 regulated osteoclast activity through the PLD1 signaling pathway, we expected raloxifene to be highly related to HSD17B7. Third, the in silico structure prediction and molecular docking analysis predicted that HSD17B7 and raloxifene would have a high binding affinity. We have demonstrated that raloxifene did not affect bone phenotypes in OVX mice lacking HSD17B7 in their preosteoclasts, indicating that the osteoprotective effect of raloxifene occurs through HSD17B7. The expression of HSD17B7 in preosteoclasts decreased with increasing age, which could explain the clinical decline seen in the effect of raloxifene on BMD with increasing age after menopause 57 , 58 . Thus, boosting the protein level of HSD17B7 could be one treatment method for improving the effect of raloxifene on osteoporosis. In summary, this study has demonstrated that HSD17B7 upregulates and increases ERα expression in preosteoclasts, inhibiting their metabolic activity and controlling bone resorption. Therefore, HSD17B7 regulation could be a novel therapeutic approach to alleviating osteoporosis in postmenopausal patients. Declarations Acknowledgements This work was supported by a grant from the National Research Foundation (NRF-2022R1C1C1006721, 2022R1A2C2005734), a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare (HR22C1832), by Fund of Biomedical Research Institute, Jeonbuk National University Hospital. Competing Interests The authors declare no competing interests. Author Contributions J.Z., Y.S., J.K., and S.C. performed experiments and analyzed the data. Y.J.M. designed experiment. S.Y. provided the clinical samples. J.Z and Y.J.M wrote the manuscript. K.Y.J., and J.R.K. supervised and conceived the project. All authors critically revised and approved the manuscript. Data abailability All data supporting the findings of this study are available within the article and its supplementary files. 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J Am Med Dir Assoc 13, 189.e1-189.e7 (2012). Additional Declarations There is no conflict of interest Supplementary Files Rawuncroppedgel.pdf Raw uncropped gel schematicdiagram.tif Schematic diagram Supplementalinformation.docx Supplemental information Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6158228","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":432956638,"identity":"87755490-196b-4307-b5b1-f026ef73d7fd","order_by":0,"name":"Young Jae Moon","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYFCCAxCKn+EMVICHWC2SDcRrgQKDAzClhLQYHDyd+ODjDht744Nnjz3mYbCTZ+A5+4CA4Wc3G848k5a47cC5dGMehmTDBt52A0Jatknzth1OMDtwxkyah4E5gYGfjZAXIFrsjRvAWuqJ18K4gQGs5XACA28bfi2SYL+0pSXOOHAuTXKOwXHDNp5j+LXw3Ti78cHHNht7/hlnj0m8qaiW5+dJw69F4cYBKEsCxACGFQGfMDDI9zdAWfwNeJSNglEwCkbBiAYAai5HGuh9nkQAAAAASUVORK5CYII=","orcid":"","institution":"Chonbuk National University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Young","middleName":"Jae","lastName":"Moon","suffix":""},{"id":432956639,"identity":"acb423dc-c00a-45b0-a434-117b8eb6006c","order_by":1,"name":"Junyue Zhang","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Junyue","middleName":"","lastName":"Zhang","suffix":""},{"id":432956640,"identity":"659bee09-cee8-452f-a656-f64b716d4c07","order_by":2,"name":"Yiping Song","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yiping","middleName":"","lastName":"Song","suffix":""},{"id":432956641,"identity":"59a29617-0718-4d6a-89ce-1ccbd9bc254b","order_by":3,"name":"Jeong-Hyun Koo","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Jeong-Hyun","middleName":"","lastName":"Koo","suffix":""},{"id":432956642,"identity":"810bbcc5-a175-4f08-b646-d0474271af81","order_by":4,"name":"Si Chen","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Si","middleName":"","lastName":"Chen","suffix":""},{"id":432956643,"identity":"ac95c46a-6437-47fd-b618-388e6c2fcdb6","order_by":5,"name":"Kyu Yun Jang","email":"","orcid":"https://orcid.org/0000-0002-5276-4446","institution":"Jeonbuk National University","correspondingAuthor":false,"prefix":"","firstName":"Kyu","middleName":"Yun","lastName":"Jang","suffix":""},{"id":432956644,"identity":"359ccf90-d517-480d-aed8-ae2a57026ae7","order_by":6,"name":"Sun-Jung Yoon","email":"","orcid":"","institution":"Jeonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Sun-Jung","middleName":"","lastName":"Yoon","suffix":""},{"id":432956645,"identity":"b5df1e59-a1ea-4239-842f-7e4ff39433ec","order_by":7,"name":"Jung Ryul Kim","email":"","orcid":"","institution":"Chonbuk National University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Jung","middleName":"Ryul","lastName":"Kim","suffix":""}],"badges":[],"createdAt":"2025-03-05 03:05:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6158228/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6158228/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79262870,"identity":"97e69360-2dc8-41b5-a29c-fdd6f0c5dd2e","added_by":"auto","created_at":"2025-03-26 09:48:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1003078,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHSD17B as a binding partner with ERα in preosteoclasts. a\u003c/strong\u003e Primary mouse CD11b+ bone marrow cell (BMC) proteins were pulled down by biotin-labeled ERα followed by proteomics. \u003cstrong\u003eb \u003c/strong\u003eMS/MS spectra of HSD17B7 peptides bound with ERα are shown.\u003cstrong\u003e c \u003c/strong\u003eThe predicted docking modules of HSD17B7 (blue) and ERα (red) were analyzed by AutoDock and exhibited with PyMol. The docking score was 307 and confidence score 0.9585, indicating a strong potential for real binding. \u003cstrong\u003ed \u003c/strong\u003eAfter transfecting HEK293T cells with HA-ERα or MYC-HSD17B7, a co-immunoprecipitation assay was performed to determine the physical interaction between HSD17B7 and ERα.\u003cstrong\u003e e \u003c/strong\u003eWestern blot analysis of the protein levels of HSD17B7 and ERα in BMCs from Sham/OVX from young mice; n = 3. \u003cstrong\u003ef \u003c/strong\u003eProportions of HSD17B7 and ERα in CD11b+ BMCs from Sham/OVX from young mice. Values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003cstrong\u003e \u003c/strong\u003eFig. 1a was created with Biorender.com.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/4c6d5cd5a085bb237a6268e0.png"},{"id":79262871,"identity":"8ea5b9b7-ee2c-428b-9c13-de28e05053ca","added_by":"auto","created_at":"2025-03-26 09:48:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1094474,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBlocking the ubiquitin-proteasomal degradation of ERα by HSD17B7. a\u003c/strong\u003e HEK293T cells transfected with ERα were treated with estrogen (E2, 10 nM) for the indicated times, and the relative protein levels of ERα were compared (n = 3). \u003cstrong\u003eb\u003c/strong\u003e HEK293T cells were transfected with empty vector or shRNA for HSD17B7. 24 h after transfection, the cells were treated with 20 μg/ml cycloheximide (CHX) or 20 μM MG132 for 0.5 to 2 h. The cell lysates were blotted with GAPDH antibody. \u003cstrong\u003ec\u003c/strong\u003e HEK293T cells were transfected with ERα and HSD17B7-OE or shHSD17B7 and then treated with MG132 (20 μM) for 2 h. The cell lysates were immunoblotted with ERα and HSD17B7antibodies. \u003cstrong\u003ed\u003c/strong\u003e HEK293T cells were co-transfected with HSD17B7-OE or shHSD17B7, and the FasL and ERE luciferase activity in the cell lysates was measured (n = 4). \u003cstrong\u003ee\u003c/strong\u003e HEK293T cells were co-transfected with Ub, ERα, and HSD17B7-OE or shHSD17B7. After 24 h, the cells were treated with E2 (10 nM) for 40 min in the presence of MG132 (2 μM), and total cell lysates were immunoprecipitated with anti-ERα antibodies and immunoblotted with anti-ubiquitin antibodies. \u003cstrong\u003ef–i \u003c/strong\u003eBased on the docking prediction results for HSD17B7 and ERα, potential docking sites were deleted from the HSD17B7-MYC plasmid. \u003cstrong\u003ef\u003c/strong\u003e Schemes for deleting N-terminal sequences of HSD17B7. \u003cstrong\u003eg\u003c/strong\u003e HEK293T cells were co-transfected with HA-ERα and WT or deletion-mutant HSD17B7. Co-immunoprecipitation was performed using anti-HA or anti-MYC antibodies, and immunoblots were visualized for HSD17B7 and ERα. \u003cstrong\u003eh\u003c/strong\u003e HEK293T cells were transfected with WT or deletion-mutant HSD17B7, and FasL and ERE luciferase activities were measured (n = 5). \u003cstrong\u003ei\u003c/strong\u003e HEK293T cells were co-transfected with Ub, ERα, and WT or deletion-mutant HSD17B7 for 24 h. After 24 h, the cells were treated with E2 (10 nM) for 40 min in the presence of MG132 (2 μM), and total cell lysates were immunoprecipitated using anti-ERα antibody and immunoblotted using anti-Ub antibody.\u003cstrong\u003e \u003c/strong\u003eValues presented are the mean ± SEM. *p \u0026lt; 0.05 versus EV.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/9bbffa40e6ec97e9c061190d.png"},{"id":79264781,"identity":"9fd1e9d7-6722-4ecb-866b-6980cc6c9a24","added_by":"auto","created_at":"2025-03-26 09:56:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1951786,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeficiency of HSD17B7 induces estrogen-deficiency osteoporosis by increasing the number of osteoclasts without affecting osteoblast function. a \u003c/strong\u003eRepresentative micro-CT reconstruction of cancellous bone, H\u0026amp;E staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e250\u003cstrong\u003e \u003c/strong\u003eµm) and TRAP staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e40 µm) images of distal femoral metaphyses (n = 4–5 animals/group). \u003cstrong\u003eb \u003c/strong\u003eCancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were determined by a micro-CT analysis. \u003cstrong\u003ec\u003c/strong\u003eOsteoclast numbers were analyzed in cancellous bone from the distal femur (n = 7). \u003cstrong\u003ed\u003c/strong\u003e The RANKL/OPG ratio in serum was determined (n = 3–4). \u003cstrong\u003ee\u003c/strong\u003eSix weeks after surgery, uterus weight was determined (n = 6). \u003cstrong\u003ef\u003c/strong\u003eRepresentative TRAP staining images after osteoclastogenesis induction. Scale bars \u003cstrong\u003e=\u003c/strong\u003e 40 µm. TRAP-positive multinucleate cells with three or more nuclei were counted as osteoclasts, and they were scored per field (n = 5). \u003cstrong\u003eg \u003c/strong\u003eCalcein-Alizarin red label staining image and quantitative analysis of BFR (scale bars \u003cstrong\u003e= \u003c/strong\u003e100\u003cstrong\u003e \u003c/strong\u003eµm).\u003cstrong\u003e h, i \u003c/strong\u003eRepresentative images of ARS and ALP staining and quantitative analysis (n=4–5). The values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus WT Sham, #p \u0026lt; 0.05 and ##p \u0026lt; 0.01, and ###p \u0026lt; 0.001 versus WT OVX.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/8dd01b2f8200d8615f4cd9fb.png"},{"id":79262874,"identity":"65e1a6b6-08ea-4b7b-b755-2c205e55476a","added_by":"auto","created_at":"2025-03-26 09:48:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2591139,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAggravation of OVX-induced cancellous bone loss caused by HSD17B7 deficiency in preosteoclasts. a \u003c/strong\u003eRepresentative micro-CT reconstruction of cancellous bone, H\u0026amp;E staining (scale bars \u003cstrong\u003e=\u003c/strong\u003e 250\u003cstrong\u003e \u003c/strong\u003eµm) and TRAP staining (scale bars \u003cstrong\u003e=\u003c/strong\u003e 40\u003cstrong\u003e \u003c/strong\u003eµm) images of distal femoral metaphyses (n = 4–5 animals/group). \u003cstrong\u003eb \u003c/strong\u003eCancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were determined by a micro-CT analysis. \u003cstrong\u003ec\u003c/strong\u003eOsteoclast numbers were analyzed in cancellous bone from the distal femur (n = 7). \u003cstrong\u003ed \u003c/strong\u003eWestern blot analysis of the protein levels of HSD17B7 and ERα in Sham/OVX mouse BMCs (n = 3). \u003cstrong\u003ee\u003c/strong\u003e The RANKL/OPG ratio in serum was determined (n = 5). \u003cstrong\u003ef\u003c/strong\u003e Six weeks after surgery, uterus weight was determined (n = 4). \u003cstrong\u003eg\u003c/strong\u003eRepresentative images of immunofluorescence staining of HSD17B7 (green) and cathepsin K (red) in distal femoral metaphyses. \u003cstrong\u003eh\u003c/strong\u003e Representative images of immunofluorescence staining of HSD17B7 (green) and ERα (red) in CD11b+ osteoclast cells (scale bars \u003cstrong\u003e= \u003c/strong\u003e20\u003cstrong\u003e \u003c/strong\u003eµm). The values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus Control Sham, #p \u0026lt; 0.05 and ###p \u0026lt; 0.001 versus Control OVX.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/57347315c15b01f143136a03.png"},{"id":79265544,"identity":"18e06a83-0361-4c28-8389-da481dd37d79","added_by":"auto","created_at":"2025-03-26 10:04:18","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1863863,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced mitochondrial function in HSD17B7-deficient preosteoclasts. a \u003c/strong\u003eHeatmap of the RNA-seq analysis of mitochondrial complex genes in CD11b+ Control/cKO BMCs treated with M-CSF (10 ng/ml) and RANKL (30 ng/ml).\u003cstrong\u003e b \u003c/strong\u003eqPCR for marker genes in CD11b+ Control /cKO BMCs treated with M-CSF (10 ng/ml) and RANKL (30 ng/ml) (n = 4). \u003cstrong\u003ec \u003c/strong\u003eRepresentative Western blot results for the OxPhos complex and quantitative qPCR analysis of mitochondrial DNA (mtDNA), using nuclear DNA (nDNA) as the standard (n = 5). \u003cstrong\u003ed \u003c/strong\u003eOCR curves in CD11b+ Control/cKO BMCs treated with M-CSF (10 ng/ml), RANKL (30 ng/ml), oligomycin, FCCP, and rotenone/antimycin A (n = 5). \u003cstrong\u003ee\u003c/strong\u003e CD11b+ BMCs were stained using JC-10 and observed via confocal microscopy. Representative images show merged polymeric (red) and monomeric (green) JC-10 signals. The ratio of polymeric to monomeric JC-10 was calculated. \u003cstrong\u003ef\u003c/strong\u003eRepresentative transmission electron micrographs of M-CSF (10 ng/ml) and RANKL (30 ng/ml)–treated CD11b+ Control/cKO BMCs and quantitative analysis showing the numbers of mitochondria with normal and abnormal morphology. \u003cstrong\u003eg\u003c/strong\u003eFragmentation of cellular DNA was detected by a TUNEL assay. Intracellular reactive oxygen species (ROS) were detected by H2DCFDA (green) (scale bars \u003cstrong\u003e= \u003c/strong\u003e40\u003cstrong\u003e \u003c/strong\u003eµm). The values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, and ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/3b14a0d649a3e0df2b206272.png"},{"id":79264782,"identity":"1fb884ea-7d23-44ae-9fd3-930179a1e697","added_by":"auto","created_at":"2025-03-26 09:56:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1237773,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEnhanced oxidative capacity of HSD17B7 cKO mice via the PLD1-mTOR signaling pathway. a\u003c/strong\u003e Volcano plot showing the log2 fold-difference in M-CSF (10 ng/ml) and RANKL (30 ng/ml)–treated CD11b+ Control/cKO BMCs, assessed using a StringTie analysis of RNA-seq data. Red and blue dots represent upregulated and downregulated DEGs, with \u0026gt; 2-fold change and p \u0026lt; 0.05, respectively. Each sample was analyzed in triplicate. \u003cstrong\u003eb\u003c/strong\u003e Bubble plots show significant changes in the KEGG analysis. \u003cstrong\u003ec \u003c/strong\u003eWestern blot analysis of the PLD1-mTOR signaling pathway. \u003cstrong\u003ed\u003c/strong\u003e PDL1 activity from preosteoclasts. \u003cstrong\u003ee\u003c/strong\u003e CD11b+ Control/cKO BMCs were treated with VU0359595 (1 μM) for 24 h, and the expression patterns of the PLD1-mTOR signaling pathway and OxPhos complex were determined via Western blotting. \u003cstrong\u003ef\u003c/strong\u003eqPCR for marker genes after treatment with VU0359595. \u003cstrong\u003eg\u003c/strong\u003e Representative TRAP staining images after osteoclastogenesis induction. Scale bars \u003cstrong\u003e=\u003c/strong\u003e 200 µm. TRAP-positive multinucleate cells with three or more nuclei were counted as osteoclasts, and they were scored per field (n = 5). \u003cstrong\u003eh \u003c/strong\u003eOCR curves in VU0359595-treated CD11b+ Control/cKO BMCs also treated with oligomycin, FCCP, and rotenone/antimycin A (n = 3). The values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus Control, #p \u0026lt; 0.05, ##p\u0026lt;0.01, ###p \u0026lt; 0.001 versus cKO.\u003c/p\u003e","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/a2262f6acfad4b8bcbe0147d.png"},{"id":79262884,"identity":"8c76fa58-9749-4f84-963a-50ed8ac66d2f","added_by":"auto","created_at":"2025-03-26 09:48:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":911277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAttenuation of HSD17B7-ERα expression in severely osteoporotic patients.\u003c/strong\u003e Human CD11b+ BMCs were treated with M-CSF (25 ng/ml) and RANKL (50 ng/ml) for 7 days. \u003cstrong\u003ea\u003c/strong\u003e Scatterplots of HSD17B7 expression and various bone parameters. The coefficient of determination was used to compare the association between HSD17B7 expression and the bone parameters, with red and blue representing negative and positive correlation, respectively (n = 16). \u003cstrong\u003eb\u003c/strong\u003e Representative H\u0026amp;E staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e250\u003cstrong\u003e \u003c/strong\u003eµm), TRAP staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e40\u003cstrong\u003e \u003c/strong\u003eµm) and immunofluorescence staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e40\u003cstrong\u003e \u003c/strong\u003eµm) images of osteoclasts in control subjects and osteoporotic patients. \u003cstrong\u003ec\u003c/strong\u003e Human CD11b+ BMCs were treated with M-CSF and RANKL for 7 days, and the expression of HSD17B7 and ERα proteins was analyzed using Western blotting. \u003cstrong\u003ed\u003c/strong\u003e Clinical parameters of control subject and osteoporotic patients. The values presented are the mean ± SEM. *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001 versus control. Control: T-score \u0026gt;-1, Osteoporosis: T-score \u0026lt;-2.5, Severe osteoporosis: T-score \u0026lt;-2.5 with bone fracture.\u003c/p\u003e","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/1ac75fb0fd411daf58bf4c73.png"},{"id":79262887,"identity":"577c899f-2864-40e0-85fe-7f7817e4d1a0","added_by":"auto","created_at":"2025-03-26 09:48:18","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2077180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe anti-bone-resorption effect of raloxifene in estrogen-deficient osteoporosis is mediated by HSD17B7. a \u003c/strong\u003eControl and cKO mice were treated with raloxifene (RAL) (0.5mg/kg) via subcutaneous injection 5 days/week for five weeks during the OVX program. Representative micro-CT reconstruction of cancellous bone, H\u0026amp;E staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e250\u003cstrong\u003e \u003c/strong\u003eµm), and TRAP staining (scale bars \u003cstrong\u003e= \u003c/strong\u003e40\u003cstrong\u003e \u003c/strong\u003eµm) images of distal femoral metaphyses (n = 4–5 animals/group). \u003cstrong\u003eb \u003c/strong\u003eCancellous bone volume (BV/TV, %), trabecular thickness (Tb.Th), trabecular number (Tb.N), trabecular separation (Tb.Sp), and uterus weight were determined by a micro-CT analysis. \u003cstrong\u003ec\u003c/strong\u003eRepresentative TRAP staining images after osteoclastogenesis induction. Scale bars \u003cstrong\u003e=\u003c/strong\u003e 100 µm (n = 5).\u003cstrong\u003ed \u003c/strong\u003eOCR curves from CD11b+ Control/cKO BMCs treated with RAL (3 μM), M-CSF (10 ng/ml), RANKL (30 ng/ml), oligomycin, FCCP, and rotenone/antimycin A (n = 3–4). \u003cstrong\u003ee\u003c/strong\u003e Calculation of the ratio of polymeric to monomeric JC-10. \u003cstrong\u003ef \u003c/strong\u003eCD11b+ Control/cKO BMCs were treated with RAL (3 μM) for 24 h, and the expression patterns of the PLD1-mTOR signaling pathway were determined via Western blotting. \u003cstrong\u003eg \u003c/strong\u003ePDL1 activity from preosteoclasts. \u003cstrong\u003eh \u003c/strong\u003eCo-IP and immunoblot analyses of CD11b+ Control/cKO BMCs treated with RAL. *p \u0026lt; 0.05, **p \u0026lt; 0.01 versus Control Sham or Control DMSO, #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001 versus Control OVX, cKO OVX, or cKO DMSO.\u003c/p\u003e","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/b6f3e567271c0a3ac739350d.png"},{"id":83102796,"identity":"ae9e4c9e-7fbf-4543-a652-d6fc40b5f77f","added_by":"auto","created_at":"2025-05-20 05:29:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":14024355,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/591a4b24-1158-4e0a-8097-78fbccd8f8ed.pdf"},{"id":79265543,"identity":"ae6aa5fd-c16b-4dec-8192-09433451aa01","added_by":"auto","created_at":"2025-03-26 10:04:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2316529,"visible":true,"origin":"","legend":"Raw uncropped gel","description":"","filename":"Rawuncroppedgel.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/65179fd816cdd3981bf8f240.pdf"},{"id":79262881,"identity":"de8dfbdf-c7ed-49b6-80ae-c332986ce4be","added_by":"auto","created_at":"2025-03-26 09:48:18","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1624700,"visible":true,"origin":"","legend":"Schematic diagram","description":"","filename":"schematicdiagram.tif","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/859f84891d2762700d8ff3a8.tif"},{"id":79262882,"identity":"f9478a45-f9a7-4093-841b-d28d610debea","added_by":"auto","created_at":"2025-03-26 09:48:18","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4713943,"visible":true,"origin":"","legend":"Supplemental information","description":"","filename":"Supplementalinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6158228/v1/e6baa154a7e4e1a533f7c9a1.docx"}],"financialInterests":"There is no conflict of interest","formattedTitle":"HSD17B7 counters bone loss in estrogen deficiency via estrogen receptor stabilization and mediates the effect of raloxifene","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoporosis is a prevalent bone disease marked by decreased bone mineral density and the deterioration of bone microarchitecture, which significantly increases the risk of fractures, especially among postmenopausal and elderly people\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. This condition arises from the uncoupling of bone formation and maintenance, driven by osteoblasts and osteocytes, and bone resorption, driven by osteoclasts, during bone remodeling. In postmenopausal women, the level of the sex hormone estrogen decreases rapidly, accelerating bone resorption by activating osteoclasts\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Aging also causes gradual bone loss due to changes in intrinsic factors of the bone (e.g., energy metabolism, oxidative stress, DNA damage, and osteocyte senescence) and external factors (e.g., the ovary and immune system)\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Aging and declines in sex hormones thus interact to produce an overall bone phenotype in the elderly that leads to osteoporosis.\u003c/p\u003e \u003cp\u003eThe estrogen\u0026ndash;estrogen receptor (ER) complex plays a critical role in bone remodeling through several mechanisms. It not only suppresses osteoclast numbers by regulating the apoptosis and cellular metabolism of osteoclast precursors\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, but it also inhibits osteoclastogenesis by regulating the receptor activator of NF-κB ligand (RANKL), macrophage colony-stimulating factor (M-CSF), and osteoprotegerin (OPG)\u003csup\u003e\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Thus, estrogen supplementation is theoretically a treatment option for osteoporosis; however, the use of estrogen alone can increase the risk of endometrial, ovarian, and breast cancers\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Therefore, selective estrogen receptor modulators (SERMs) such as tamoxifen, raloxifene, and bazedoxifene are used in clinical practice as a form of estrogen replacement therapy\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. These modulators, bound with co-activators, have estrogen-like activity in bone and anticancer effects\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Unliganded ERα also plays a major positive role in bone formation through the mechanical stimulation of osteoblasts\u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e, and it has been reported to enhance estrogen response element (ERE) activity in the bone marrow of ovariectomized (OVX) mice\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. However, the precise mechanisms of action that regulate the ERα complex bound to a SERM and the unliganded ERα complex are still not fully understood.\u003c/p\u003e \u003cp\u003eFor this work, we used LC-MS/MS to screen for proteins that bind unliganded ERα in preosteoclasts from OVX mice and found that 17β-hydroxysteroid dehydrogenase type 7 (HSD17B7) binds unliganded ERα. HSD17B7 catalyzes the conversion of low-activity keto-steroid sex hormones into their high-activity hydroxylated forms, regulating estrogen and cholesterol synthesis\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. More recently, it has been reported that HSD17B7 regulates cellular energy metabolism\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. In this study, we confirm a physical interaction between HSD17B7 and ERα, specifically ERα binding at the 119\u0026ndash;172 domain of HSD17B7. This interaction prevents the ubiquitin-proteasomal degradation of ERα and increases ERE activity. To investigate the osteoclastic role of HSD17B7 in osteoporosis, we generated osteoclast-specific HSD17B7 knockout mice (cKO) and performed OVX. The cKO OVX group showed more severe trabecular bone loss than control OVX mice. A mechanistic investigation through a transcriptomic analysis of preosteoclasts from cKO and control mice revealed significant upregulation of genes involved in oxidative phosphorylation and mitochondrial biogenesis, which are associated with the activation of the PLD1-mTOR pathway, in cKO preosteoclasts. At the cellular level, oxidative consumption was increased in cKO preosteoclasts, compared with control preosteoclasts, and that effect was reversed by the application of a PLD1 inhibitor, indicating that HSD17B7 regulates preosteoclast energy metabolism via the PLD1 signaling pathway. Additionally, protein expression levels of HSD17B7 and ERα were significantly reduced, compared with control patients, in preosteoclasts and femoral heads derived from osteoporosis patients, and those levels correlated positively with bone mineral density, confirming the importance of HSD17B7 in humans. Lastly, to verify that HSD17B7 is a potential SERM co-activator, we assessed raloxifene's therapeutic effect in cKO and control mice following OVX. Raloxifene inhibited OVX-induced bone loss in control mice, blocking PLD1 pathway activation and mitochondrial oxidation in preosteoclasts. However, those effects were not observed in HSD17B7-deficient cells and animal models. This study suggests that HSD17B7 enhances and upregulates ERα expression in preosteoclasts, thereby preventing bone loss by inhibiting osteoclast-mediated bone resorption. Therefore, HSD17B7 regulation is a novel therapeutic approach for alleviating osteoporosis.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cstrong\u003eAnimal experiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWT C57BL/6J mice were obtained from Damul Science (Daejeon, Korea). HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice were obtained from Gempharmatech (Nanjing, China), HSD17B7\u003csup\u003efl/fl\u003c/sup\u003e mice were obtained from Cyagen (Santa Clara, CA, USA), and LysM-Cre mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA). Osteoclast-specific HSD17B7 knockout mice (HSD17B7\u003csup\u003eO\u003c/sup\u003e\u003csup\u003ec\u003c/sup\u003e KO) mice were generated by mating HSD17B7\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003emice with LysM-Cre mice. HSD17B7\u003csup\u003efl/fl\u003c/sup\u003e littermates served as controls. Animals were maintained on a 12-h light/dark cycle at 23\u0026plusmn;1\u0026deg;C with free access to food and water. The genotypes of the mice were determined by polymerase chain reaction (PCR) using tail tissues. Eight-week-old female mice were subjected to bilateral OVX or sham surgery, and the mice were sacrificed after 6 weeks. The bone parameters of cancellous and cortical bone from the distal femur were analyzed using a SKYSCAN 1076 Micro‐CT unit (SkyScan, Kontich, Belgium) installed in the Center for University-wide Research Facilities (CURF) at Jeonbuk National University. The study protocol was approved by the Institutional Animal Care and Use Committee of Jeonbuk National University (Permit No: JBUH-IACUC-2020-20).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIn vitro\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eo\u003c/strong\u003e\u003cstrong\u003esteoclast differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemoral CD11b+ bone marrow cells (BMCs) from 8-week-old mice were used for osteoclastogenesis. Briefly, mouse femoral bone marrow cells were extracted, and the cell suspensions were passed through a 70-\u0026micro;m mesh filter screen. Red blood cells were removed using ACK lysing buffer. The following day, the supernatant cells were collected and combined with CD11b (Invitrogen,\u0026nbsp;Carlsbad, CA, USA), and anti-biotin microbeads (Miltenyi Biotec,\u0026nbsp;San Diego, CA, USA) were used for positive sorting. The isolated cells were then seeded onto 6-well plates at a density of 1x10\u003csup\u003e6\u003c/sup\u003e and cultured in 30 ng/ml M-CSF (PeproTech, London, England) for 2 days. To induce osteoclast formation, we supplemented the growth medium with M-CSF (10 ng/ml) and RANKL (30 ng/ml, PeproTech). After 3 days of osteoclast differentiation, tartrate-resistant acid phosphatase (TRAP) multinucleated cells were detected using a TRAP staining kit (Cosmo Bio Co., Ltd., Tokyo, Japan). The procedures for human osteoclast extraction and osteoclastogenesis were similar to those in a previous study\u003csup\u003e21\u003c/sup\u003e. Briefly, human CD11b+ BMCs were extracted, and 25 ng/ml M-CSF and 50 ng/ml RANKL were added for osteoclast differentiation over 7 days.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLC-MS/MS and peak alignment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunoprecipitated proteins differentially expressed in CD11b+ BMCs from Sham and OVX mice were found by staining SDS gels with colloidal Coomassie blue (Abcam,\u0026nbsp;Cambridge, UK). After the proteins were excised from the gel bands, they were subjected to gel trypsin digestion. The tryptic peptides were analyzed for related protein identification using a high-resolution mass spectrometer (LTQ-Orbitrap Velos, Thermo Fisher Scientific, Waltham, MA, USA) as described previously\u003csup\u003e22\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe evaluated patients who underwent hip arthroplasty at Jeonbuk National University Hospital (Jeonju, South Korea) between August 2022 and August 2023. The inclusion and exclusion criteria were described previously\u003csup\u003e21\u003c/sup\u003e. Sixteen patients met the criteria. All cases were reviewed according to the World Health Organization Diagnostic Criteria for Osteoporosis\u003csup\u003e23\u003c/sup\u003e and the American Association of Clinical Endocrinologists staging system\u003csup\u003e24\u003c/sup\u003e. This study was performed with the approval of the institutional review board at Jeonbuk National University Hospital, and the requirement for informed consent was waived (IRB number, JBUH 2023-12-023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse femurs were fixed in 4% paraformaldehyde, decalcified, and soaked in 10% EDTA for 1 month until the bone softened evenly to complete the decalcification. The femurs were then embedded in paraffin, and cut into 5 \u0026mu;m transverse sections at different levels. The sections were stained with hematoxylin and eosin (H\u0026amp;E) and TRAP after rehydration.\u003c/p\u003e\n\u003cp\u003eFor immunofluorescence staining, the sections were deparaffinized, rehydrated, and subjected to antigen retrieval. After being blocked with 5% BSA (GenDEPOT, Barker, TX, USA) to prevent non-specific staining, the slides were incubated with primary anti-HSD17B7 (14854-1-AP, ProteinTech Group, Chicago, IL, USA) and anti-cathepsin K (sc-48353, Santa Cruz Biochemicals, Dallas, TX, USA) overnight at 4\u0026deg;C. Alexa Fluor 488-conjugated anti-rabbit IgG (1:100 Invitrogen) and Alexa Fluor 594-conjugated anti-mouse IgG (1:100 Invitrogen) secondary antibodies were added to the sections to visualize the staining. DAPI (1:200 Invitrogen) was used for nuclear staining. Immunofluorescence images were taken with a Zeiss LSM 880 on an Airyscan confocal microscope (Carl Zeiss, G\u0026ouml;ttingen, Germany).\u003c/p\u003e\n\u003cp\u003eThe osteogenic ability of each group was shown by observing the formation rate of new mineralized bone. Ten days before sacrifice, mice were given an intraperitoneal injection of Calcein (30 mg/kg\u0026nbsp;Sigma-Aldrich, St. Louis, MO, USA), and 3 days before sacrifice, they received a dose of Alizarin red (30 mg/kg\u0026nbsp;Sigma-Aldrich) to form dual fluorescent labels. Sample preparation followed a previously described method\u003csup\u003e25\u003c/sup\u003e. Briefly, femurs were collected, fixed, and subsequently immersed in 5% aqueous potassium hydroxide (KOH) for 96 h. After being embedded in paraffin, each mineralized femur was cut into 5-\u0026mu;m slices with a microtome and observed with an APX100 microscope (Olympus, Waltham, MA, USA). ImageJ (Version 1.53a, National Institutes of Health, Bethesda, MD, USA) software was used to evaluate the bone formation rate of each bone surface (BFR/BS). Five samples in each group were tested.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood was collected from Sham and OVX mice, and the serum was separated. Serum RANKL and OPG were measured using RANKL ELISA assay kits (Abcam) and OPG ELISA assay kits (R\u0026amp;D Systems, MN, USA), respectively, according to each manufacturer\u0026apos;s protocols.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFlow cytometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe first extracted femoral bone marrow cells without red blood cells and then incubated the resulting single-cell suspension with 1:200 anti-CD16/32 (Fc\u0026gamma;RIII/II Invitrogen) for 15 minutes to block the non-specific binding of Fc receptors. Antibody mixtures, CD11b (Invitrogen), HSD17B7 (ProteinTech Group), and ER\u0026alpha; (R\u0026amp;D Systems, Minneapolis, MN, USA) typically diluted at 1:100, were added and incubated in the dark at 4\u0026deg;C for 30 minutes. Subsequently, appropriate secondary antibodies were added and incubated at room temperature for 30 minutes. Data were acquired with a FACS Aria III (BD, Franklin Lakes, NJ, USA). The analysis was performed using FlowJo software (Version 10.0.7, Tree Star, San Carlos, CA).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell culture, transient transfection, and promoter luciferase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuman embryonic kidney 293T (HEK293T) cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in Dulbecco\u0026rsquo;s modified Eagle medium (DMEM). To examine the relationships of HSD17B7, HEK293T cells were transfected with 1 \u0026mu;g of plasmid DNA containing Flag, MYC-HSD17B7 (#RC209534, OriGene Technologies, Rockville, MD), sh-HSD17B7 (sc-88433-SH, Santa Cruz), or HA-ER\u0026alpha; (Applied Biological Materials Inc. Richmond, Canada) using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). To evaluate promoter activity, 1 \u0026mu;g each of FasL and ERE promoter luciferase (Promega, Madison, WI, USA) were used. Briefly, HEK293T cells were transfected with plasmids encoding the Flag, MYC-HSD17B7, shHSD17B7, ERE-luc, FasL-luc, or Renilla luciferase reporter (pRL-TK-luc). After 24 more h, the cells were harvested in a reporter lysis buffer. Luciferase activity was determined in whole cell lysates using a luciferase assay kit (Promega, Madison, WI, USA). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSubcellular fractionation, co-immunoprecipitation, and Western blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe extracted proteins from tissues or cells using a protein extraction kit (#78510 or 78505, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer\u0026apos;s protocol. Nuclear, membrane, and cytoplasmic extracts were isolated using a subcellular protein fractionation kit (#78840, Thermo Fisher Scientific). Homogenates (20 \u0026mu;g for Western blotting) were separated by SDS-PAGE and transferred to nitrocellulose membranes. For co-immunoprecipitation, 400 \u0026mu;g of homogenates were immunoprecipitated with the indicated antibody at 4\u0026deg;C overnight. The immunocomplexes were pulled down using protein A/G agarose beads (#20421 Thermo Fisher Scientific) and separated by Western blotting. After blocking the samples with 5% skim milk, I incubated them with primary antibodies overnight at 4\u0026thinsp;\u0026deg;C. This was followed by a 1-hour incubation with an HRP-conjugated secondary antibody at room temperature. Then, the membranes were visualized with an enhanced chemiluminescence detection kit (Millipore, Billerica, USA). Immunoreactive bands were detected with an LAS-4000 imager (GE Healthcare Life Science, Pittsburgh, PA, USA). The primary antibodies used for Western blotting were HSD17B7, RANKL (1:1000 Santa Cruz Biotechnology), ER\u0026alpha; (R\u0026amp;D Systems), HA, MYC, PLD1, phosphorylated PLD1, mTOR, phosphorylated mTOR, S6K, phosphorylated S6K, S6, phosphorylated S6, ubiquitin (1:1000 Cell Signaling Technology, Danvers, MA, USA), Col1a1, OPN, OPG, total OxPhos, NFATC1 (1:2000 Abcam), and GAPDH (1:2000 Cell Signaling Technology).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMolecular docking analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe obtained the protein structures of ER\u0026alpha; and HSD17B7 from the UniProt database and then processed them using PyMOL\u0026nbsp;(Version 2.1, Schr\u0026ouml;dinger, Inc., New York, NY) and uploaded them to the HDOCK SERVER website (http://hdock.phys.hust.edu.cn/) for protein\u0026ndash;protein docking. We selected the combination with the highest docking score and confidence score for simulating the docking site and used PyMOL for the visual analysis.\u003c/p\u003e\n\u003cp\u003eFor docking with RAL, I obtained the SDF format file of its main active ingredient from the PubChem database. We then collected the protein structures of ER\u0026alpha; and HSD17B7 from the PDB database. I used PyMOL to optimize the targets by removing water molecules and small molecule ligands and then used AutoDock Tools (National Institutes of Health, Bethesda, MD, USA) to add hydrogen atoms and charge treatments, saving the files in pdbqt format. We then performed molecular docking using PyRx (version 0.9.7) software\u0026apos;s internal vina, calculated the binding energy, and output the result files. We used PyMOL for result visualization.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOsteogenic function analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs previously described, primary osteoblasts were extracted from mouse femurs and cultured in osteogenic induction medium (MUXMX-90021 Cyagen) for 7 or 14 days\u003csup\u003e26\u003c/sup\u003e. The cells were fixed with 4% paraformaldehyde for 10 minutes. For alkaline phosphatase (ALP) staining, the 7-day cells were treated with a BCIP/NBT kit (K4151 ApexBio, Houston, USA). The stained images were captured with a digital camera and quantified by measuring the OD value at 405 nm with an alkaline phosphatase assay kit (ab83369 Abcam). For Alizarin red S (ARS) staining, cells cultured for 14 days were treated with 40 mM ARS (Sigma-Aldrich), pH 4.0, and the staining results were photographed. Subsequently, 10% chlorinated pivaloyl chloride (Sigma-Aldrich) was added, and quantification was performed by measuring the OD value at 560 nm.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMutagenesis of HSD17B7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the docking prediction results for HSD17B7 and ER\u0026alpha;, corresponding deletion mutants from the HSD17B7-MYC plasmid were generated (Gene Synthesis, Seoul, Korea). \u0026nbsp;Specifically, HSD17B7\u003csup\u003ed5-45\u003c/sup\u003e, HSD17B7\u003csup\u003ed119-172\u003c/sup\u003e, HSD17B7\u003csup\u003ed192-200\u003c/sup\u003e, and HSD17B7\u003csup\u003ed274-302\u003c/sup\u003e mutants were generated by deleting the corresponding sequences starting from the N-terminal sequence of HSD17B7.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR with reverse-transcription analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies, Carlsbad, CA) according to the manufacturer\u0026rsquo;s instructions. Total RNA samples (1 \u0026mu;g) were reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Applied Biosystems, Foster City, CA, USA). Quantitative PCR was performed in 384-well plates using an ABI Prism 7900HT sequence detection system (Applied Biosystems, Foster City, CA, USA). RNA from individual samples was analyzed separately. The primer sequences used in the PCR are listed in Table S1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of the oxygen consumption rate (OCR)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe OCR was measured in an XF24 extracellular analyzer (Agilent Technologies, Santa Clara, CA, USA). CD11b\u003csup\u003e+\u003c/sup\u003e BMCs treated with M-CSF and RANKL for 3 days (2 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells per well) were seeded into 24-well plates. The next day, the medium was changed to analysis medium containing 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine. The cells were incubated in a CO\u003csub\u003e2\u003c/sub\u003e-free incubator at 37℃ for 1 h and then sequentially exposed to oligomycin (1 \u0026mu;M), FCCP (1 \u0026mu;M), and rotenone (0.5 \u0026mu;M) from a Seahorse XF cell Mito stress kit (Agilent Technologies). Data were processed using Wave software (Agilent Technologies).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTUNEL assay and assessment of intracellular reactive oxygen species (ROS)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the manufacturer\u0026apos;s instructions, a DeadEnd colorimetric TUNEL system (G7360, Promega\u0026nbsp;Madison, WI, USA) was used to assess apoptosis in CD11b\u003csup\u003e+\u003c/sup\u003e osteoclasts. The nuclei were stained with hematoxylin. To measure intracellular ROS levels, CD11b\u003csup\u003e+\u003c/sup\u003e osteoclasts were incubated with 10 mM H2DCFDA (D399, Life Technologies) for 30 minutes. The nuclei were stained with Hoechst (HY-15559, MedChemExpress, Monmouth Junction, NJ, USA). Observations and 5\u0026ndash;6 microscopic images were obtained using an APX100 microscope. ImageJ (Version 1.53a) software was then used to evaluate the ratio of positive cells to total cells in each field of view.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEvaluation of mitochondrial membrane potential\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCD11b\u003csup\u003e+\u003c/sup\u003e osteoclasts were incubated with JC-10 (786-1549 G-Biosciences, St. Louis, USA) dissolved in DMEM for 30 min. We used the APX100 microscope to observe JC-10 polymers (Ex/Em=540/590 nm) and JC-10 monomers (EX/Em= 490/525 nm). ImageJ (Version 1.53a) software was used to evaluate the ratio of JC-10 polymer cells to JC-10 monomers cells in each field of view.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM) and image analysis \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo compare the mitochondrial morphology of WT mice and HSD17B7\u003csup\u003eOc\u003c/sup\u003e KO mice, CD11b+ BMCs treated with M-CSF and RANKL for 3 days were collected and observed using TEM. As previously described\u003csup\u003e27\u003c/sup\u003e, the samples were fixed, embedded, sectioned, and then observed and imaged using a Hitachi Bio-TEM (H-7650, Hitachi, Tokyo, Japan). Based\u0026nbsp;on previous research\u003csup\u003e28\u003c/sup\u003e, ImageJ (Version 1.53a) and DigitalMicrograph software (Version 3.6, Gatan Microscopy Suite, USA) were used to calculate the number of mitochondria.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRNA sequencing data analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMouse femur bone marrow cells from WT mice (\u003cem\u003en\u003c/em\u003e=4) and HSD17B7\u003csup\u003eOc\u003c/sup\u003e KO mice (\u003cem\u003en\u003c/em\u003e=4) were extracted and differentiated into preosteoclasts (1x10\u003csup\u003e6\u003c/sup\u003e cells) for RNA sequencing. Total RNA was isolated from WT and cKO preosteoclasts using an RNeasy mini kit (QIAGEN Valencia, CA, USA) with DNase treatment to remove genomic DNA contamination. Strand-specific RNA libraries were prepared using a KAPA mRNA HyperPrep kit (KAPA Biosystems, Foster City, CA, USA) according to the manufacturer\u0026apos;s protocol. The prepared libraries were sequenced on an Illumina HiSeq 4000 instrument (Illumina, San Diego, CA, USA) to generate paired-end reads. The raw sequencing reads were processed to remove adapters and low-quality bases using standard quality control procedures. The processed reads were aligned to the mouse reference genome using StringTie to estimate gene abundances in the read counts. A differential gene expression analysis was performed using the DESeq2 package within the R (Version 4.3.2) statistical environment. Genes with adjusted \u003cem\u003ep\u003c/em\u003e-values less than 0.05 were considered to be significantly differentially expressed. Gene enrichment, functional annotation, and pathway analyses for significant gene lists were performed using the g: Profiler pathway (https://biit.cs.ut.ee/gprofiler/). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are expressed as the mean \u0026plusmn; standard error of the mean (SEM). Statistical comparisons were performed using one-way analysis of variance followed by Fisher\u0026rsquo;s post hoc analysis. The significance of differences between groups was determined using Student\u0026rsquo;s unpaired \u003cem\u003et\u003c/em\u003e-test. Linear regressions were performed with GraphPad Prism, version 9.00 (San Diego, CA, USA). A \u003cem\u003ep\u003c/em\u003e-value less than 0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003ePreosteoclast ER\u0026alpha; regulation was associated with HSD17B7 from old and OVX mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBased on the increase in ERE activity in bone marrow cells following OVX in young mice\u003csup\u003e17\u003c/sup\u003e, we used immunoprecipitation with an ER\u0026alpha; antibody to verify which proteins interacted with unliganded ER\u0026alpha; in myeloid cells six weeks after OVX. Next, the proteins bound to ER\u0026alpha; were placed on a gel, and the peptides and proteins were identified and estimated by mass spectrometry and the Mascot program (Fig. 1a). The data suggest that HSD17B7 can specifically bind to unliganded ER\u0026alpha; in the OVX state (Fig. 1b), a notion supported by our subsequent predicted docking module analysis (Fig. 1c). To explore that possibility, we performed co-immunoprecipitation assays after inserting the ER\u0026alpha; and HSD17B7 plasmids. Those results confirmed a physical interaction between HSD17B7 and ER\u0026alpha; (Fig. 1d). Because ER\u0026alpha; is closely related to both aging and estrogen deficiency\u003csup\u003e21\u003c/sup\u003e, we checked the expression of ER\u0026alpha; and HSD17B7 in CD11b\u003csup\u003e+\u003c/sup\u003e BMCs. CD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eBMCs from young OVX mice exhibited a significant increase in ER\u0026alpha; expression, compared with young Sham mice. Interestingly, the expression pattern of HSD17B7 was consistent with that of ER\u0026alpha; (Fig. 1e). To reconfirm the expression patterns of ER\u0026alpha; and HSD17B7 in CD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eBMCs in OVX model, we extracted BMCs from the mice and performed flow cytometry. Those results show that the percentage change in the co-expression of ER\u0026alpha; and HSD17B7 in CD11b\u003csup\u003e+\u003c/sup\u003e BMCs was consistent with the change in protein level (Fig. 1f, Fig. S1). These results suggest that HSD17B7 might act as a co-activator that binds to ER\u0026alpha; and regulates its stability in CD11b\u003csup\u003e+\u0026nbsp;\u003c/sup\u003eBMCs in mice.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSD17B7 interacts with ER\u0026alpha; and stabilizes its protein expression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next investigated the molecular mechanisms by which HSD17B7 regulates ER\u0026alpha;. Plasmid DNA expressing ER\u0026alpha; and control vector or HSD17B7 was transfected into HEK293T cells, which were then stimulated with estrogen. In the control group, the protein level of ER\u0026alpha; gradually decreased, but in cells overexpressing HSD17B7 under each test condition, the decrease rate was slower (Fig. 2a). Furthermore, in HEK293T cells with HSD17B7 knockdown, treatment with cycloheximide led to the rapid degradation of ER\u0026alpha;, compared with the empty vector control (Fig. 2b), indicating that HSD17B7 increases the stability of ER\u0026alpha; in both ligand-dependent and -independent degradation pathways. To further explore the role of HSD17B7 in stabilizing ER\u0026alpha;, we evaluated ER\u0026alpha; proteasomal degradation. When cells were treated with MG132, a potent proteasome inhibitor, the effect of the HSD17B7-mediated ER\u0026alpha; protein expression change was abolished (Fig. 2c), suggesting that HSD17B7-mediated ER\u0026alpha; expression depends on proteasomal protein degradation. Immunoprecipitation was then performed on total protein lysates using ER\u0026alpha; antibodies, followed by immunoblotting with ubiquitin antibodies. Those results show that the overexpression of HSD17B7 reduced the ubiquitination of ER\u0026alpha;, and HSD17B7 knockdown increased ER\u0026alpha; ubiquitination (Fig. 2e). Additional results from luciferase reporter gene assays using FasL-Luc, which is an ER\u0026alpha; target gene\u003csup\u003e29\u003c/sup\u003e, and ERE-Luc confirmed that HSD17B7 overexpression significantly increased ER\u0026alpha; transcriptional activity, whereas shHSD17B7 overexpression did not (Fig. 2d). Next, we constructed HSD17b7 deletion mutants based on the results of our docking module analysis (Fig. S2) and the known functional sites of HSD17B7\u003csup\u003e20\u003c/sup\u003e to determine where ER\u0026alpha; binds to HSD17B7 (Fig. 2f). Immunoprecipitation experiments revealed that, compared with the other HSD17B7 deletion mutants, HSD17B7 d119-172 had a weaker binding affinity with ER\u0026alpha; (Fig. 2g), and the ubiquitination inhibition effect of HSD17B7 on ER\u0026alpha; was not observed when the 119-172 region of HSD17b7 was deleted (Fig. 2i). Additionally, luciferase reporter gene assays using FasL-Luc and ERE-Luc confirmed that deleting amino acids 119\u0026ndash;172 of HSD17B7 abolished ER\u0026alpha; transcriptional activity (Fig. 2h). These results indicate that ER\u0026alpha; binds to the 119-172 region of HSD17B7, thereby inhibiting the ubiquitin-proteasomal degradation of ER\u0026alpha; and increasing its target transcriptional activity.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSD17B7 deficiency exacerbates bone defects mediated by osteoclasts rather than osteoblasts in OVX-induced bone loss\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the physiological role of HSD17B7 in bone, we generated mice deficient in HSD17B7 in their whole bodies. Because HSD17B7 homozygotes die at embryonic age 10.5 days\u003csup\u003e30\u003c/sup\u003e, we used HSD17B7 heterozygotes (HSD17B7\u003csup\u003e+/-\u003c/sup\u003e) and then performed bilateral OVX on both WT and HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice. H\u0026amp;E staining and micro-CT analyses of the distal femur showed that the HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice and their WT littermates had phenotypically normal bones at birth and similar bone mass at 3 months of age (Fig. 3a, b). However, after OVX surgery, the amount of trabecular bone loss in the HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice was significantly greater than that in the WT mice. Consistently, the trabecular bone volume fraction and trabecular number were significantly lower in the HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice than in the WT mice (Fig. 3a, b). To further investigate whether the bone loss was due to an increase in osteoclast numbers, we performed TRAP staining on trabecular bone at the distal femur and found that HSD17B7 deficiency led to a significant increase in the number of osteoclasts, compared with WT mice (Fig. 3a, c). Consistently, \u003cem\u003ein vitro\u003c/em\u003e osteoclastogenesis induction showed the same trend (Fig. 3f). Meanwhile, a decrease in uterine weight and increase in the RANKL/OPG ratio confirmed the success of the OVX surgery. Still, we found no difference between the genotypes that underwent OVX, indicating no difference in estrogen levels or cytokines for osteoclastogenesis (Fig. 3d, e, Fig. S3a).\u003c/p\u003e\n\u003cp\u003eTo investigate the effect of HSD17B7 on osteogenesis capacity, Calcein-Alizarin red dual labeling was performed on both genotypes that underwent OVX. The bone-forming rate results confirmed that HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice and their WT littermates had similar osteogenic capacity (Fig. 3g). Primary femoral osteoblasts from HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice were cultured under osteogenic conditions and did not show differences in osteoblast differentiation or mineralization (Fig. 3h). Compared with WT mice, primary osteoblasts from HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice exhibited significantly reduced HSD17B7 expression. However, the expression levels of other osteoblastic markers (\u003cem\u003eCol1a1, Bglap, Runx2, Spp1,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;Osx\u003c/em\u003e) were unaffected by the HSD17B7 deficiency in HSD17B7\u003csup\u003e+/-\u003c/sup\u003e mice (Fig. S3b). Micro-CT analyses of cortical bone at the mid-shaft femur showed that tissue mineral density was significantly lower following OVX treatment (Fig. S3c, S3d). However, OVX-induced cortical bone loss showed almost no difference between the HSD17B7\u003csup\u003e+/-\u003c/sup\u003e and WT mice (Fig. S3c, S3d), confirming that the loss of HSD17B7 in bone selectively affects trabecular bone during OVX-induced bone loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOVX-induced cancellous bone loss is aggravated by HSD17B7 deficiency in preosteoclasts\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo further investigate the role of HSD17B7 in preosteoclasts, we crossed \u003cem\u003eHSD17B7\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice with \u003cem\u003eLysM-Cre\u003c/em\u003e mice to generate myeloid-specific HSD17B7 knockout (cKO) mice (Fig. S4a). A Western blot analysis confirmed the specific knockout of HSD17B7 in the bone marrow (Fig. S4b). The cKO mice had a normal phenotype at birth and showed bone mass similar to their \u003cem\u003eHSD17B7\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e littermates at 3 months of age (Fig. 4a). We performed OVX on 3-month-old cKO and control mice and sacrificed them six weeks post-surgery. As expected, OVX resulted in greater bone loss in cKO mice than in control mice, and the mice treated with a sham operation showed no difference in bone mass (Fig. 4a). Consistently, microstructural evaluation of the trabecular bone reflected that the lack of myeloid cell\u0026ndash;specific HSD17B7 exacerbated OVX-induced trabecular bone loss (Fig. 4b). In the OVX state, the number of osteoclasts was significantly higher in cKO mice than in control mice (Fig. 4a, c). Additionally, the protective mechanism that increased the expression of ER\u0026alpha; and HSD17B7 in bone marrow cells after OVX in control mice was not observed in the cKO OVX mice (Fig. 4d). As in the HSD17B7\u003csup\u003e+/-\u003c/sup\u003emice, the genotypes did not show differences in estrogen levels or cytokines for osteoclastogenesis after OVX (Fig. 4e, f). To further investigate the localization of HSD17B7 in the bone marrow, we performed immunofluorescence staining on the trabecular bone of the distal femur. We found that HSD17B7 was co-expressed with the osteoclast marker cathepsin K, supporting the importance of HSD17B7 in osteoclasts (Fig. 4g). \u003cem\u003eIn vitro\u003c/em\u003e studies corroborated these results. In CD11b\u003csup\u003e+\u003c/sup\u003e BMCs cultured with M-CSF and RANKL, HSD17B7 and ER\u0026alpha; were co-expressed (Fig. 4h), suggesting that HSD17B7 plays a crucial role in trabecular bone resorption by bone marrow osteoclasts. However, this inhibitory effect of HSD17B7 on the number of osteoclasts did not affect the osteoclast differentiation signaling pathway mediated by M-CSF and RANKL (Fig. S4c).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSD17B7 ablation increases preosteoclast mitochondrial content and oxidative phosphorylation capacity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the mechanism by which HSD17B7 regulates preosteoclasts, we treated CD11b\u003csup\u003e+\u003c/sup\u003e BMCs from cKO mice with RANKL for two days and then performed an RNA sequencing analysis. Considering that the mitochondrial OxPhos of preosteoclasts plays an essential role in the number and function of osteoclasts via the estrogen\u0026ndash;ER complex\u003csup\u003e7, 14\u003c/sup\u003e, we focused on the expression of mitochondria-related genes. The heatmap in Fig. 5a displays the increased expression of mitochondrial complex genes in cKO\u0026nbsp;mice, compared with control mice. A qPCR analysis confirmed alterations in mitochondrial biogenesis and OxPhos gene expression (Fig. 5b). These findings correlate well with the increased mitochondrial DNA content and promoted expression of OxPhos complexes found in preosteoclasts from\u0026nbsp;cKO\u0026nbsp;mice (Fig. 5c). To ascertain whether the increased mitochondrial content in\u0026nbsp;cKO\u0026nbsp;preosteoclasts was associated with elevated respiratory function, we conducted Seahorse XF mitochondrial stress tests on differentiated preosteoclasts from CD11b\u003csup\u003e+\u003c/sup\u003e BMCs with both genotypes. We observed a significant increase in maximum OCR in cKO cells, indicating that HSD17B7 regulates energy metabolism in preosteoclasts by modulating OxPhos (Fig. 5d). The results of measuring mitochondrial membrane potential, an indicator of mitochondrial activity, also showed higher levels in the cKO group than the control group (Fig. 5e). Consistently, an electron microscopy analysis of preosteoclasts revealed a higher number of healthier mitochondria in cKO mice than in control mice (Fig. 5f). These results were corroborated by \u003cem\u003ein vitro\u003c/em\u003e studies. TUNEL-positive cells and ROS-positive cells were significantly reduced in preosteoclasts from cKO mice, compared with control mice, indicating that the apoptotic process within preosteoclasts of cKO mice was inhibited, and oxidative stress levels were decreased (Fig. 5g).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSD17B7 affects osteoclast function through the PLD1-mTOR signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo elucidate the molecular mechanisms by which HSD17B7 regulates osteoclast numbers and energy metabolism, we further analyzed the RNA sequencing data. The volcano plot in Fig. 6a shows genes that were upregulated (in red) or downregulated (in blue) in\u0026nbsp;cKO\u0026nbsp;mice, compared with control mice. The KEGG pathway analysis (https://www.genome.jp/kegg/pathway.html) performed on transcriptomes that increased in preosteoclasts from\u0026nbsp;cKO\u0026nbsp;mice revealed significant alterations in the PLD1 signaling pathway (Fig. 6b). Because PLD1 and mTOR have been reported to have important roles in osteoclast differentiation and energy metabolism\u003csup\u003e33\u0026ndash;35\u003c/sup\u003e, we investigated the PLD1-mTOR pathway. We found that proteins related to the PLD1 pathway and PLD activity were consistently and significantly increased in cKO mice, compared with the control group (Fig. 6c, d). To confirm that PLD1 signaling affects osteoclast function and cellular metabolism, we treated cKO preosteoclasts with the PLD1 inhibitor VU0359595 to inhibit the PLD1 pathway. Preosteoclasts deficient in HSD17B7 have increased OxPhos in the PLD1-mTOR signal pathway, resulting in osteoclast activation, and a PLD1 inhibitor was confirmed to block that effect (Fig. 6e\u0026ndash;h). These results indicate that HSD17B7 inhibits osteoclast metabolism and activity by regulating the PLD1-mTOR pathway.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHSD17B7-ER\u0026alpha; expression is attenuated in patients with severe osteoporosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo determine the clinical relevance of our findings so far, we cultured CD11b\u003csup\u003e+\u003c/sup\u003e BMCs from human subjects with M-CSF and RANKL for 7 days and then analyzed their expression of HSD17B7 and ER\u0026alpha;. Consistent with the results from mouse studies, the expression of HSD17B7 and ER\u0026alpha; in human preosteoclasts decreased with increasing age (Fig. 7a). There was a positive correlation between the expression of HSD17B7 and ER\u0026alpha;, as well as between HSD17B7 and bone mineral density (BMD) and ER\u0026alpha; and BMD (Fig. 7a), supporting the animal data that HSD17B7 increases both ER\u0026alpha; protein levels and BMD. On the other hand, both HSD17B7 and ER\u0026alpha; correlated negatively with serum type I collagen C-terminal telopeptide (CTX), a marker of osteoclast activity (Fig. 7a). To further examine the expression of HSD17B7 in osteoclasts from patients with osteoporosis, we performed H\u0026amp;E and TRAP staining on femoral heads, revealing that trabeculae were sparser and the number of mature osteoclasts was significantly higher in osteoporosis patients than in the control group. Consistent with the results of our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003eex vivo\u003c/em\u003e experiments, HSD17B7 expression was high in osteoclasts from the control group and significantly lower in osteoclasts from patients with severe osteoporosis (Fig. 7b). Finally, we measured the expression of HSD17B7 and ER\u0026alpha; in preosteoclasts from patients with severe osteoporosis and control subjects (Fig. 7c). Compared with the control subjects, the expression of HSD17B7 and ER\u0026alpha; in preosteoclasts from osteoporosis patients, who had lower BMD, higher CTX, and older age than the control group, was significantly lower (Fig. 7d). These findings suggest that decreased expression of HSD17B7 and ER\u0026alpha; in preosteoclasts is highly associated with osteoporosis in humans. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA lack of HSD17B7 abolishes the osteoclast-inhibitory function of raloxifene\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt has been reported that SERMs bind to ER, inhibit osteoclast activity\u003csup\u003e12\u003c/sup\u003e, and block breast cancer cell proliferation because they inhibit PLD1\u003csup\u003e36,37\u003c/sup\u003e. Interestingly, our present study shows that HSD17B7 stabilizes ER\u0026alpha; to activate ER\u0026alpha; target genes and inhibits the PLD1-mTOR pathway to block osteoclast activity. In other words, we observed a significant overlap in the roles of HSD17B7 and SERMs. Thus, we hypothesized that HSD17B7 would be related to the effect of SERMs. We treated\u0026nbsp;cKO\u0026nbsp;mice and control mice with a SERM after OVX and observed the bone phenotype. For this experiment, we used AutoDock Vina to simulate molecular docking between HSD17B7 and raloxifene, with a binding energy of -8.5 kcal/mol indicating a significant potential for actual binding (Fig. S5). To determine the effect of raloxifene on\u0026nbsp;cKO\u0026nbsp;mice, we subcutaneously injected 8-week-old female\u0026nbsp;cKO\u0026nbsp;mice with raloxifene five days a week for five weeks during the OVX protocol\u003csup\u003e38,39\u003c/sup\u003e. An H\u0026amp;E staining and analysis of the distal femur showed that, following OVX treatment, raloxifene treatment increased trabecular bone in control mice but had minimal effects on\u0026nbsp;cKO\u0026nbsp;mice (Fig. 8a). Consistent with those changes, far fewer osteoclasts were formed in the presence of raloxifene in control mice, but raloxifene treatment did not lead to significant changes in the number of osteoclasts in\u0026nbsp;cKO\u0026nbsp;mice either \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003eor \u003cem\u003ein vitro\u003c/em\u003e (Fig. 8a\u0026ndash;c). These results suggest that raloxifene increases bone mass in estrogen-deficient mice, similar to the effects of estrogen on bone, but it does not reverse bone loss in HSD17B7-deficient mice. Previous results have demonstrated that 17\u0026beta;-estradiol inhibits mitochondrial function and promotes apoptosis in preosteoclasts[7]. Raloxifene also regulates mitochondrial-mediated apoptosis and inhibits mitochondrial and peroxisomal \u0026beta;-oxidation\u003csup\u003e40,41\u003c/sup\u003e. Therefore, we performed Seahorse XF mitochondrial stress tests on preosteoclasts to investigate whether raloxifene interacts with HSD17B7 in ways that affect mitochondrial function in preosteoclasts. We observed that raloxifene significantly reduced OCR in control preosteoclasts, but it did not affect cKO preosteoclasts (Fig. 8d). Similarly, raloxifene decreased mitochondrial membrane potential in control preosteoclasts but not in cKO preosteoclasts (Fig. 8e), suggesting that HSD17B7 might be an essential mediator of the mitochondrial function\u0026ndash;regulating effects of raloxifene in preosteoclasts. Next, we evaluated the expression of the PLD1 pathway in preosteoclasts after raloxifene treatment. Interestingly, RAL-stimulated control preosteoclasts showed significantly reduced expression of PLD1-related protein and PLD activity, along with increased expression of HSD17B7 and ER\u0026alpha;, compared with untreated control cells. However, that effect was absent in cKO preosteoclasts (Fig. 8f, g). Through co-immunoprecipitation studies after raloxifene treatment of control and cKO preosteoclasts, we observed an increase in the physical interaction between ER\u0026alpha; and HSD17B7 in control mice but not cKO mice (Fig. 8h). These findings suggest that raloxifene exerts its effects via HSD17B7 in preosteoclasts and suppresses bone loss in estrogen-deficient osteoporosis by regulating the expression of HSD17B7-ER\u0026alpha;.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified a previously unrecognized function of HSD17B7 as an ERα-binding protein that serves as a coactivator to influence trabecular bone mass in estrogen deficiency. Specifically, deleting HSD17B7 in myeloid cells enhanced the number of osteoclasts and decreased trabecular bone mass post-OVX, compared with control OVX, mirroring observations in myeloid ERα KO mice\u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. During increased bone resorption, HSD17B7 expression levels paralleled those of ERα in preosteoclasts. However, HSD17B7 ablation accelerated bone resorption, as evidenced by elevated mitochondrial content and oxidative capacity in preosteoclasts. A transcriptome analysis combined with physiological data pinpointed PLD1 as the target through which HSD17B7 modulates mitochondrial OxPhos, aligning with previous research that highlighted the crucial role of the PLD1 and mTOR signaling pathway in metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. An analysis of human subjects revealed that preosteoclasts from severely osteoporotic patients exhibited lower expression levels of HSD17B7 and ERα than those from controls, suggesting that HSD17B7 acts as a positive regulator in human osteoporosis. Lastly, we demonstrated that the selective ER modulator raloxifene had no therapeutic effect on estrogen deficiency\u0026ndash;induced osteoporosis in cKO mice. Therefore, raloxifene's inhibitory effect on osteoclast activation is mediated through HSD17B7, which emphasizes the importance of the HSD17B7\u0026ndash;ERα regulatory mechanism in bone homeostasis.\u003c/p\u003e \u003cp\u003eIn this study, we used LC-MS/MS to find that HSD17B7 binds to ERα and reconfirmed that finding by \u003cem\u003ein vitro\u003c/em\u003e co-immunoprecipitation. This binding occurred at 119\u0026ndash;172 on HSD17B7, which includes the substrate binding site and the docking prediction site, rather than the steroid and NADP binding sites of HSD17B7\u003csup\u003e20\u003c/sup\u003e. The binding of the two proteins enhanced the stability of ERα by inhibiting its ubiquitin-proteasomal degradation. Evidence from diverse expression patterns supports this mechanism of ERα regulation by HSD17B7. First, when young mice are rendered estrogen-deficient, a complementary increase in ERα is observed in myeloid cells\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e, at which time the expression of HSD17B7 is significantly increased. Second, in aged rodents, ERα expression is markedly reduced by non-ligand-dependent ubiquitination\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e, and HSD17B7 is also reduced in the same manner. Third, ERα expression is consistently reduced in HSD17B7 KO preosteoclasts, compared with control cells. Lastly, the expression of HSD17B7 and ERα in patient-derived preosteoclasts showed a significant positive correlation. Because ERα has various post-translational modification sites that regulate its ubiquitin-proteasomal degradation\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e, binding to HSD17B7 is thought to affect stability by inducing changes in those lysine and serine residues. On the other hand, it is also possible that ERα regulates HSD17B7. ERα bound to the ligand was recruited to the promoter of HSD17B7 and stimulated its expression\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Furthermore, we found that the expression of HSD17B7 increased when control preosteoclasts were treated with raloxifene. Thus, the regulatory mechanism of HSD17B7 and ERα is thought to be a feed-forward loop.\u003c/p\u003e \u003cp\u003eThrough a transcriptome analysis, we found that the phospholipase D signaling pathway was a significant mechanism in the increase in the number of osteoclasts in HSD17B7-deficient preosteoclasts. PLD1 is an upstream factor of the mitogenic activation of mTOR signaling and regulates cell growth through the PLD1-mTOR-S6K1 axis\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Consistent with these results, the osteoclastic activity of HSD17B7 knockout preosteoclasts accompanied the activation of the PLD1-mTOR-S6K1 axis, and that osteoclastic activity was suppressed by a PLD1 inhibitor. PLD1 is involved in synoviocyte activation and the expression of various cytokines in rheumatoid arthritis\u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e, and PLD1 inhibitors have been reported to alleviate the symptoms of collagen-induced arthritis and inhibit osteoclast activity\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e, supporting our findings in this study. Meanwhile, PLD1-mTOR is a central regulator of not only cell growth but also cellular metabolism. PLD1 or mTOR inhibition suppresses oxygen consumption and mitochondrial capacity and increases ROS levels, leading to cell death\u003csup\u003e\u003cspan additionalcitationids=\"CR50 CR51\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. HSD17B7 KO preosteoclasts showed increased OCR, enhanced mitochondrial biogenesis, and decreased ROS, which reduced the number of apoptotic cells. All of these metabolic-related preosteoclast phenotypes were reversed by a PLD1 inhibitor, suggesting that metabolic events associated with HSD17B7 are regulated by PLD1-mTOR signaling. Although we could not provide direct evidence that the phenotypes of HSD17B7 KO preosteoclasts were entirely related to ERα, we can speculate that the effects of HSD17B7 are highly associated with ERα based on our result that raloxifene, which binds to ERα and acts on preosteoclasts, did not improve the bone phenotype in HSD17B7 KO mice and on reports that increased metabolic activity in ERα KO preosteoclasts\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e and the overexpression of PLD1 were observed in ER-negative breast cancer cells\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRaloxifene, a SERM, exploits the positive effects of ERα on bone to alleviate osteoporosis caused by estrogen deficiency. It has been used extensively clinically and has shown various pharmacological properties, including anti-osteoporotic, antiviral, immunomodulatory, and anticancer activities\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e,\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e\u003c/sup\u003e. We introduced raloxifene to investigate the role of HSD17B7 in estrogen-deficiency osteoporosis for three reasons. First, in cKO and control OVX models, the RANKL-to-OPG ratio remained the same between the OVX genotypes, and estrogen was reduced by the same amount. However, the degree of bone resorption was different, suggesting that ERα, which is highly related to HSD17B7 in preosteoclasts, might have estrogen-independent bone protective effects. Second, although the exact mechanism has not yet been fully elucidated, raloxifene is a PLD1 inhibitor\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Because HSD17B7 regulated osteoclast activity through the PLD1 signaling pathway, we expected raloxifene to be highly related to HSD17B7. Third, the \u003cem\u003ein silico\u003c/em\u003e structure prediction and molecular docking analysis predicted that HSD17B7 and raloxifene would have a high binding affinity. We have demonstrated that raloxifene did not affect bone phenotypes in OVX mice lacking HSD17B7 in their preosteoclasts, indicating that the osteoprotective effect of raloxifene occurs through HSD17B7. The expression of HSD17B7 in preosteoclasts decreased with increasing age, which could explain the clinical decline seen in the effect of raloxifene on BMD with increasing age after menopause\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e,\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e. Thus, boosting the protein level of HSD17B7 could be one treatment method for improving the effect of raloxifene on osteoporosis.\u003c/p\u003e \u003cp\u003eIn summary, this study has demonstrated that HSD17B7 upregulates and increases ERα expression in preosteoclasts, inhibiting their metabolic activity and controlling bone resorption. Therefore, HSD17B7 regulation could be a novel therapeutic approach to alleviating osteoporosis in postmenopausal patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a grant from the National Research Foundation (NRF-2022R1C1C1006721, 2022R1A2C2005734), a grant from the Korea Health Technology R\u0026amp;D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health \u0026amp; Welfare (HR22C1832), by Fund of Biomedical Research Institute, Jeonbuk National University Hospital. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJ.Z., Y.S., J.K.,\u0026nbsp;and\u0026nbsp;S.C.\u0026nbsp;performed\u0026nbsp;experiments and analyzed the data. Y.J.M. designed experiment.\u0026nbsp;S.Y.\u0026nbsp;provided\u0026nbsp;the\u0026nbsp;clinical\u0026nbsp;samples.\u0026nbsp;J.Z\u0026nbsp;and\u0026nbsp;Y.J.M\u0026nbsp;wrote\u0026nbsp;the\u0026nbsp;manuscript.\u0026nbsp;K.Y.J.,\u0026nbsp;and\u0026nbsp;J.R.K.\u0026nbsp;supervised and conceived the project. All authors critically revised and approved the manuscript.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData abailability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the article and its supplementary files. RNA-seq data will be deposited in NCBI Gene Expression Omnibus database (GSE274826) and will be publicly available from the date of publication.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLeBoff, M. S. et al. The clinician\u0026rsquo;s guide to prevention and treatment of osteoporosis. 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Mechanistic Target of Rapamycin Complex 1 Promotes the Expression of Genes Encoding Electron Transport Chain Proteins and Stimulates Oxidative Phosphorylation in Primary Human Trophoblast Cells by Regulating Mitochondrial Biogenesis. Sci Rep 9, 1\u0026ndash;14 (2019).\u003c/li\u003e\n\u003cli\u003ePag\u0026aacute;n, A. J. et al. mTOR-regulated mitochondrial metabolism limits mycobacterium-induced cytotoxicity. Cell 185, 3720-3738.e13 (2022).\u003c/li\u003e\n\u003cli\u003eGadiya, M. et al. Phospholipase D1 and choline kinase-\u0026alpha; are interactive targets in breast cancer. Cancer Biol Ther 15, 593\u0026ndash;601 (2014).\u003c/li\u003e\n\u003cli\u003eGozgit, J. M. et al. PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas. Br J Cancer 97, 809\u0026ndash;817 (2007).\u003c/li\u003e\n\u003cli\u003eAllegretti, M. et al. Repurposing the estrogen receptor modulator raloxifene to treat SARS-CoV-2 infection. Cell Death Differ 29, 156\u0026ndash;166 (2022).\u003c/li\u003e\n\u003cli\u003eTong, D. Selective estrogen receptor modulators contribute to prostate cancer treatment by regulating the tumor immune microenvironment. J Immunother Cancer 10, 1\u0026ndash;9 (2022).\u003c/li\u003e\n\u003cli\u003eJohnston, C. C. et al. Long-term effects of raloxifene on bone mineral density, bone turnover, and serum lipid levels in early postmenopausal women: Three-year data from 2 double-blind, randomized, placebo-controlled trials. Arch Intern Med 160, 3444\u0026ndash;3450 (2000).\u003c/li\u003e\n\u003cli\u003eJacobsen, D. E., Melis, R. J. F., Verhaar, H. J. J. \u0026amp; Olde Rikkert, M. G. M. Raloxifene and tibolone in elderly women: A randomized, double-blind, double-dummy, placebo-controlled trial. J Am Med Dir Assoc 13, 189.e1-189.e7 (2012).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HSD17B7, Estrogen Receptor alpha, Osteoporosis, Mitochondria, Raloxifene","lastPublishedDoi":"10.21203/rs.3.rs-6158228/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6158228/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEstrogen receptor (ER) α is a key regulator of osteoclasts in osteoporosis induced by estrogen deficiency. Although ERα is regulated through interactions with various coactivators, the precise mechanisms remain unknown. We used LC-MS/MS to screen proteins that bind to ERα and identified a physical interaction between HSD17B7 and ERα, specifically that ERα binds to the 119\u0026ndash;172 domain of HSD17B7. This interaction blocked ubiquitin-proteasomal degradation of ERα and increased ERE activity. Estrogen-deficient mice lacking HSD17B7 in their preosteoclasts showed more severe bone loss than control mice. This was attributed to increased mitochondrial biogenesis through the activation of PLD1-mTOR signaling. Additionally, in preosteoclasts derived from patients with severe osteoporosis, HSD17B7 and ERα expressions were significantly reduced, compared with control subjects. Finally, raloxifene, which boosts ERα, did not inhibit bone loss without HSD17B7, confirming the modulation of ERα through HSD17B7. Therefore, HSD17B7 regulation is a novel therapeutic approach for alleviating estrogen-deficient osteoporosis.\u003c/p\u003e","manuscriptTitle":"HSD17B7 counters bone loss in estrogen deficiency via estrogen receptor stabilization and mediates the effect of raloxifene","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-26 09:48:13","doi":"10.21203/rs.3.rs-6158228/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d075121-3009-4f9d-b889-35e9d734c7ce","owner":[],"postedDate":"March 26th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":46102074,"name":"Health sciences/Endocrinology/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis"},{"id":46102075,"name":"Health sciences/Diseases/Endocrine system and metabolic diseases/Metabolic bone disease/Osteoporosis"}],"tags":[],"updatedAt":"2025-05-20T05:05:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-26 09:48:13","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6158228","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6158228","identity":"rs-6158228","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}