Loss of lysyl hydroxylase 2 activates CRP-mediated cancer invasion through MEK/ERK/MMP9 signaling in the bone-microenvironment

Loss of lysyl hydroxylase 2 activates CRP-mediated cancer invasion through MEK/ERK/MMP9 signaling in the bone-microenvironment | 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 Loss of lysyl hydroxylase 2 activates CRP-mediated cancer invasion through MEK/ERK/MMP9 signaling in the bone-microenvironment Saori Tomiku, Atsushi Kasamatsu, Tomoaki Saito, Reo Fukushima, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7989174/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 16 You are reading this latest preprint version Abstract Lysyl hydroxylase 2 (LH2), encoded by procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 ( Plod2 ) gene, plays critical roles in collagen cross-linking and bone matrix organization. Recent studies have also shown that aberrant LH2 expression is promotes tumor progression, but its specific role in modulating cancer behavior within the bone-microenvironment remains unclear. Given the clinical importance of bone metastasis and the involvement of inflammatory mediators in tumor-stromal interactions, understanding the LH2 function in bone-tumor dynamics is of particular interest. Here, we used bone-specific LH2 conditional knockout (bsLH2-cKO) mice to investigate the impact of LH2 deficiency on cancer progression in bone by proteomic analysis and bone invasion models. Protein expression profiling revealed that C-reactive protein (CRP) was markedly upregulated in bone marrow stromal cells isolated from bsLH2-cKO femurs. Our in vitro and in vivo analyses demonstrated that CRP significantly enhanced cancer proliferation and exacerbated bone destruction in bsLH2-cKO mice following tumor cell injection. Pathway analysis further indicated that CRP induced by LH2 deficiency promoted overexpression of matrix metalloproteinase-9 through activation of the MEK/ERK signaling pathway. These findings identify LH2 as a critical regulator of CRP-mediated cancer progression in the bone-microenvironment, suggesting that targeting the LH2-CRP axis may represent a promising therapeutic strategy for bone-invasive cancers. Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Molecular biology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Lysine hydroxylation of fibrillar collagens catalyzed by lysyl hydroxylases 1–3 (LH1–3) is a critical posttranslational modification for proper connective tissue development and biomechanical stability [ 1 , 2 ]. LH2 specifically hydroxylates lysine (Lys) residues in the telopeptide domain of type I collagen, which is essential for forming hydroxylysine (Hyl) aldehyde-derived cross-links. LH1 hydroxylates Lys residues in the helical domain and LH3 primarily functions as a glucosyltransferase [ 1 , 3 ]. Mutations in the LH2-encoding gene, procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 ( Plod2 ), are causative of connective tissue disorders such as Bruck syndrome, a rare form of osteogenesis imperfecta characterized by joint contractures, reduced bone mineralization, and skeletal bone fragility [ 4 , 5 ]. To investigate the outcome of LH2 deficiency in vivo , we initially generated LH2-deficient mice using CRISPR-Cas9 technology. However, these mice died during early embryogenesis due to cardiac defects [ 6 ]. LH2 heterozygous mice exhibited altered biomechanical and biochemical properties in femurs [ 7 ]. We therefore generated bone-specific LH2 conditional knockout (bsLH2-cKO) mice using the osteocalcin-Cre/loxP system. Biochemical and mechanical analyses of these mice revealed reduced telopeptide Lys hydroxylation, abnormal collagen cross-link formation, and increased bone fragility, demonstrating that LH2 is indispensable for maintaining the structural integrity of bone collagen [ 8 ]. In cancer biology, aberrant LH2 activity has been shown to affect the tumor microenvironment by altering collagen cross-linking, thereby enhancing tumor stiffness, invasion, and metastatic potential [ 3 , 9 – 13 ]. Especially in head and neck cancers, LH2 overexpression has been associated with increased tumor aggressiveness [ 14 ]. Bone is one of the most common metastatic sites for several malignancies, including breast, prostate, and head and neck cancers [ 15 , 16 ]. Once tumor cells invade the bone-microenvironment, they exploit the native extracellular matrix (ECM) to promote colonization, osteolysis, and systemic skeletal complications. Inflammatory pathways are intimately involved in this process. Notably, elevated circulating levels of C-reactive protein (CRP), a widely used biomarker of systemic inflammation, have been linked to poor prognosis and increased bone metastatic burden in cancer patients [ 17 , 18 ]. However, the molecular mechanisms by which matrix-modifying enzymes like LH2 modulate both inflammation and tumor progression within the bone-microenvironment remain largely undefined. During the course of characterizing the protein expression profile of bone marrow stromal cells (BMSCs) from bsLH2-cKO mice, we found a marked upregulation of CRP, a key inflammatory mediator previously associated with cancer aggressiveness and poor prognosis. Based on this finding, we hypothesized that loss of LH2 promotes cancer progression in bone via CRP-mediated activation of downstream signaling pathways. In the present study, we demonstrate that CRP upregulation enhances cancer cell proliferation and bone invasion through activation of the MEK/ERK pathway and subsequent induction of matrix metalloproteinase-9 (MMP-9). These findings identify a novel LH2–CRP–MMP9 signaling axis that mechanistically links ECM disorganization to inflammation-driven tumor invasion in bone, providing a rationale for its therapeutic targeting. Results LH2 expression in bone marrow stromal cells (BMSCs) of bsLH2-cKO mice. To establish the bsLH2-cKO strain of mice, we used Osteocalcin-Cre transgenic (OC-Cre) mice. Although previous studies confirmed LH2 deficiency in the cortical bones of LH2 F/F OC-Cre mice [ 8 ], LH2 expression in BMSCs had not been examined. Here, we evaluated the mRNA and protein levels of LH2 in BMSCs isolated from femurs in LH2 F/F OC-Cre mice. The Plod2 mRNA and LH2 protein expression levels in BMSCs from LH2 F/F OC-Cre mice were significantly lower than those in control mice (LH2 F/F ) (Fig. 1 A, B) (The corresponding full-length blots are provided in Supplementary Fig. S1 ). Data-independent acquisition (DIA) proteomic analysis in BMSCs of bsLH2-cKO mice. We performed DIA proteomic analysis to assess the protein expression patterns in the BMSCs of LH2 F/F OC-Cre and LH2 F/F mice. Volcano plots and heat mapping revealed that 395 proteins were significantly upregulated and 257 proteins downregulated more than 2-fold in LH2 F/F OC-Cre femurs (Fig. 1 C, D). To further narrow down LH2 deficiency-induced proteins, 395 overexpressed proteins and 44 predicted extracellularly secreted proteins were extracted from the DIA dataset. Among the 32 overlapping proteins, CRP level was by far > 10-fold higher than the rest of the proteins, thus, selected for further analysis. CRP expression in BMSCs of bsLH2-cKO mice. To confirm expression levels of mRNA and secretion levels of CRP, we performed a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA assays. The CRP mRNA and protein levels in BMSCs of LH2 F/F OC-Cre were both significantly greater than those of LH2 F/F (10.48- and 3.13-fold, respectively) (Fig. 2 A, B). CRP promotes proliferation of B16F10 and MOC1 cells. To assess the effect of CRP on cancer cell proliferation, B16F10 (mouse malignant melanoma) and MOC1 (mouse oral squamous cell carcinoma) cells were cultured with/without 25 µg/mL of recombinant CRP protein for 72 h, followed by crystal violet staining. CRP-treated cells exhibited significantly greater stained areas compared to controls, indicating CRP enhanced cancer cell proliferation (Fig. 2 C-D). Bone invasion models in bsLH2-cKO mice. To evaluate cancer progression in vivo , B16F10 or MOC1 cells were injected into the femurs of LH2 F/F and LH2 F/F OC-Cre mice. After 14 days, the femurs were collected and evaluated (Fig. 3 A). In LH2 F/F OC-Cre femurs, bone hypertrophy was visibly apparent compared to LH2 F/F mice (Fig. 3 B). Micro computed tomography (µCT) analysis also demonstrated that in LH2 F/F OC-Cre femurs, cortical bone mineral density (BMD) was significantly decreased, while cortical bone area and total bone volume significantly increased, after injection with both cancer cell types (Fig. 3 C-D). There were no evident differences between the LH2 F/F and LH2 F/F OC-Cre femurs in terms of weight and length, while the width of the femurs was slightly wider in LH2 F/F OC-Cre, but the difference did not reach the significance level (Fig. 3 B, Supplementary Fig. S2 , Supplementary Table 1). Individual body weight data are provided in Supplementary Table 2. There was no significant difference in body weight after cancer cell injection between LH2 F/F OC-Cre and LH2 F/F (Supplementary Fig. S3 ). Histological characterization of bone invasion in bsLH2-cKO mice. Histological examination with hematoxylin and eosin (H&E) staining revealed that extensive destruction of cortical bone was observed in LH2 F/F OC-Cre mice, whereas cortical bone structure in LH2 F/F mice was preserved well, suggesting that LH2 deficiency enhances tumor-induced bone destruction (Fig. 4 A). Picrosirius Red (PSR) staining under polarized light exhibited patches of disorganized nonlamellar, woven bone-like structure of collagen (green) in LH2 F/F OC-Cre mice compared with well-organized lamellar bone structure (yellow) in LH2 F/F mice. Quantitative image analysis of PSR staining under polarized light, measuring the mean brightness intensity of the birefringent signal in the cortical bone area, showed significantly lower intensity in LH2 F/F OC-Cre mice, indicating that LH2 deficiency led to disruption of collagen architecture and resulted in disorganized bone structure (Fig. 4 B). CRP regulates MMP9 through the MEK/ERK signaling pathway in cancer cells. To investigate the effect of CRP on its downstream molecules, we performed Western blot analysis. We found increased phosphorylation of MEK1 and ERK in the cancer cell lines after treatment with CRP compared with untreated controls (Fig. 5 A) (The corresponding full-length blots are provided in Supplementary Fig. S4-6). Additionally, CRP-treatment experiments resulted in indicating that CRP controlled MMP9 expression via the MEK1/ERK pathway (The corresponding full-length blots are provided in Supplementary Fig. S7). We focused on MMP9 because it is a major collagen-degrading enzyme in bone, capable of cleaving type I collagen in the mineralized matrix and thereby contributing to ECM remodeling [ 19 ]. To assess CRP expression in bsLH2-cKO mice, we performed IHC on femur sections after injection with the cancer cells. CRP expression was notably elevated in the LH2 F/F OC-Cre group, highlighting the link between LH2 deficiency and inflammation in the bone-microenvironment. IHC scores also confirmed significantly higher CRP expression levels in the LH2 F/F OC-Cre group (Fig. 5 C). In addition, elevated MMP9 expression was demonstrated in the medullary cavity of the femurs of LH2 F/F OC-Cre mice after injection with the cancer cells (Fig. 5 D). Therefore, in addition to the in vitro study using the cancer cells, bsLH2-cKO femur samples in vivo demonstrated higher CRP expression, leading to MMP9 overexpression. Discussion It is well documented that LH2 is a key collagen-modifying enzyme that catalyzes telopeptidyl Lys hydroxylation, a post-translational modification essential for the formation of hydroxylysine aldehyde-derived, stable cross-links in type I collagen. These cross-links are critical for the mechanical strength of load-bearing tissues such as bone [ 7 , 8 ]. However, our findings in this study indicate that LH2 plays a much broader biological role, functioning as a regulator of the tumor–bone interface by modulating stromal inflammation and cancer progression. In our in vivo bone invasion models, LH2 deficiency markedly exacerbated cancer-induced cortical bone destruction and disrupted collagen architecture. These phenotypes are consistent with our previous observations that bones in bsLH2-cKO mice exhibited the diminished mature collagen cross-links and increased bone fragility [ 7 , 8 ]. While the structural vulnerability of the LH2-deficient bone matrix likely contributes to enhanced tumor invasion, our data also suggest that LH2 deficiency actively promotes a pro-tumorigenic environment via inflammatory mediators. Proteomic profiling of BMSCs from bsLH2-cKO mice revealed that CRP was among the most highly upregulated secreted proteins. CRP is an acute-phase reactant widely recognized as a clinical biomarker of systemic inflammation [ 20 , 21 ], and elevated CRP levels correlate with poor prognosis in several malignancies, including those with bone metastases [ 17 , 18 , 22 , 23 ]. While CRP has been considered a surrogate marker in the past, accumulating evidence suggests its active involvement in tumor progression through modulation of immune responses, angiogenesis, and matrix remodeling [ 24 , 25 ]. In our study, CRP was significantly upregulated in LH2-deficient BMSCs and their conditioned medium, indicating that it is a bone-intrinsic pro-inflammatory signaling. Previous studies have demonstrated that endoplasmic reticulum (ER) stress activates CRP expression via ATF4 and CHOP [ 26 ]. LH2 deficiency is known to impair collagen biosynthesis and induce ER stress responses in stromal cells [ 6 ], supporting a mechanistic link between matrix defects and inflammation. Thus, we propose that LH2 maintains stromal immune homeostasis not only through collagen integrity but also by limiting ER stress–induced CRP production in the bone-microenvironment. Functional analyses further established CRP as a key effector of cancer–bone interaction. CRP enhanced cancer cell proliferation in vitro (Fig. 2 ) and promoted bone destruction in vivo (Fig. 4 ). Mechanistically, CRP activated the MEK/ERK signaling pathway, resulting in upregulation of matrix metalloproteinase-9 (MMP9) (Fig. 5 ), a known driver of tumor-induced osteolysis and bone matrix degradation [ 19 , 27 , 28 ]. These findings align with clinical observations that MMP9 is highly expressed in bone-metastatic lesions and contributes to skeletal complications [ 29 ]. Previous studies have shown that forced LH2 overexpression in cancer cells enhances their proliferation and metastatic potential through cell-intrinsic mechanisms [ 9 , 14 ]. In contrast, our present findings reveal that LH2 deficiency in surrounding stromal cells, particularly BMSCs, promotes tumor growth indirectly by driving excessive CRP secretion, thereby creating a pro-inflammatory microenvironment. These findings identify a novel LH2–CRP–MMP9 signaling axis that links ECM disorganization to inflammation-driven tumor invasion in bone, providing a rationale for its therapeutic targeting (Fig. 6 ). We have previously developed KS122-0485428, a selective small-molecule activator of LH2 that enhances its enzymatic function [ 30 ]. We propose that pharmacological restoration of LH2 activity may suppress CRP-mediated tumor promotion and prevent cancer-induced skeletal events. Given the unmet clinical need for therapies addressing bone metastasis across multiple cancer types [ 16 ], modulation of the LH2–CRP–MMP9 pathway may offer a potential translational strategy. Materials and Methods Study approval and accordance All methods and experiments were approved by the Chiba University Review Board for Animal Care (Chiba, Japan, Protocol No. A7-013), and all procedures were performed in accordance with the relevant guidelines and regulations, including the ARRIVE guidelines and the AVMA Guidelines for the Euthanasia of Animals [31,32]. All mice were housed under diurnal lighting conditions, allowed free access to food and water, and monitored at least once daily for general health, mobility, grooming, and activity. No unexpected adverse events occurred, and no animals reached predefined humane endpoints. Mice were deeply anesthetized with isoflurane (5%) and euthanized by cervical dislocation to ensure death. All efforts were made to minimize animal suffering and to reduce the number of animals used. Generation of LH2 F/F OC-Cre mice LH2 F/F OC-Cre (bsLH2-cKO) mice were generated according to the Protocol as described previously [8,31,32]. Briefly, Heterozygous LH2 F/+ mice (Chiba University) were interbred with OC-Cre mice (Jackson Laboratory strain No. 019509) to generate LH2 F/+ OC-Cre mice. The LH2 F/+ mice were then intercrossed with LH2 F/+ OC-Cre mice to generate LH2 F/F OC-Cre (bsLH2-cKO) mice using the osteocalcin-Cre/loxP system. Mice were maintained on a C57BL/6 (Jackson Laboratory) background[8,33]. Male mice aged 8–16 weeks were used throughout the experiments. Mice were assigned to the LH2 F/F or LH2 F/F OC-Cre groups according to genotype. No animals were excluded from the study, and no predefined inclusion or exclusion criteria were applied. Cell lines and cell culture. BMSCs in 8- to 16-week-old male LH2 F/F OC-Cre and LH2 F/F mice were isolated by flushing the bone marrow from the femur and incubating with red blood cell lysis buffer (Invitrogen, Carlsbad, CA, USA). The cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich), 1% L-glutamine (200mM) (Gibco, Waltham, MA, USA), 0.1% 2-mercaptoethanol (Gibco), and 1% HEPES (1M) (Gibco) in a 5% CO 2 atmosphere at 37 °C. The BMSC cultures were rinsed 3 times with phosphate-buffered saline (PBS; Sigma-Aldrich) to remove nonadherent cells. The cell culture medium was changed 4 days after plating and cells were grown for 7 days in their respective conditions. A mouse oral squamous cell carcinoma (OSCC) cell line, MOC1, was purchased from Kerafast (Newark, CA, USA) and a mouse malignant melanoma cell line, B16F10, was purchased by RIKEN (No. RCB2630; RIKEN, Saitama, Japan). MOC1 cells were grown in a solution of 62% IMDM medium (Wako, Osaka, Japan), 31% Ham’s F-12 nutrient mix (Cytiva, Marlborough, MA, USA), 10% FBS, 100 units/mL penicillin and 100 μg/mL streptomycin, 40 μg/L hydrocortisone (Sigma-Aldrich), 5 μg/L epidermal growth factor (Merck, Darmstadt, Germany), and 5 mg/L insulin (Sigma-Aldrich). B16F10 cells were grown in RPMI-1640 supplemented with 10% FBS and 100 units/mL penicillin and 100 μg/mL streptomycin (Sigma-Aldrich) in a 5% CO 2 atmosphere at 37 °C. The cell culture medium was changed twice a week. qRT-PCR. Total RNAs of BMSCs, B16F10 cells, and MOC1 cells treated with/without 25 µg/mL CRP recombinant protein were extracted with TRIzol reagent (Invitrogen) and reverse transcribed into cDNA with ReverTra Ace qPCR RT Master Mix (TOYOBO, Tokyo, Japan). Analyses of Plod2, CRP , and MMP9 mRNA expression levels were performed by qRT-PCR with TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), with the specific probe and primers set on a Light Cycler 96 System (Roche Diagnostics GmbH, Mannheim, Germany). The specific probe and primers set for Plod2, CRP, and MMP9 were purchased from Thermo Fisher Scientific (TaqMan Gene Expression Assay; Mm00478767_m1, Mm00432680_g1, and Mm00442991_m1). The mRNA expression levels were normalized to GAPDH (Mm99999915_g1). Western blot analysis. Proteins were extracted with RIPA lysis buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing a cocktail of protease inhibitors (Santa Cruz Biotechnology, Dallas, TX, USA). Lysates were centrifuged at 10,000g and the supernatants were collected. Extracted proteins (20 µg) were separated on 4-12 % NuPAGE Tris-Acetate Pre-Cast Gels (Invitrogen) under denaturing and reducing conditions, and the proteins were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) by electroblotting. The membranes were blocked with Blocking One (Nacalai Tesque, Inc.) for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti‑mouse LH2 polyclonal antibody, 1:500; (21214-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), rabbit anti-mouse ERK1/2, 1:1000; (11257-1-AP, Proteintech Group, Inc.), rabbit anti-mouse phospho-ERK1/2, 1:500; (28733-1-AP, Proteintech Group, Inc.), rabbit anti-mouse MEK1, 1:2000; (GTX134234, GeneTex, Irvine, CA, USA), rabbit anti-mouse phospho-MEK1, 1:2000; (28930-1-AP, Proteintech Group, Inc.), rabbit anti-mouse MMP9, 1:1000; (10375-2-AP, Proteintech Group, Inc.), and rabbit anti-mouse α-Tubulin, 1:2500; (11224-1-AP, Proteintech Group, Inc.). After washing the membranes with 0.1% Tween-20 Tris-buffered saline 3 times, the membranes were incubated with horseradish peroxidase-conjugated IgG as an anti-rabbit secondary antibody (Promega Corporation, Madison, WI, USA) at room temperature. Finally, the membranes were visualized using enhanced chemiluminescence (ECL) with the ECL Prime Western Blotting Detection Reagent (Cytiva) and imaged using a ChemiDoc XRS Plus System with Image Lab software (Bio-Rad Laboratories). DIA analysis. DIA proteomic analysis was performed to characterize and evaluate protein levels in the bone marrow of LH2 F/F and LH2 F/F OC-Cre mice ( n = 3), according to the protocol described previously[34]. Volcano plots and heat map were evaluated for protein expression variation between the LH2 F/F and LH2 F/F OC-Cre mice. ELISA of culture medium. CRP concentrations in the culture medium were determined using a mouse C-reactive protein/CRP Sandwich ELISA Kit (Proteintech) according to the manufacturer’s protocol and read at 450 nm using a Benchmark Plus Microplate Reader (Bio-Rad Laboratories). Briefly, cell suspension was centrifuged at 500 g and the supernatants were collected. Each assay was repeated three times independently. Standard curves using CRP recombinant protein and individual well concentrations were determined using the ELISA calculator (Proteintech). To keep experimental values within the linear region of the standard curves, samples were diluted to 1:2 as necessary for stimulated culture samples. Crystal Violet Staining. B16F10 and MOC1 cells were seeded in 96-well plates at a density of 1×10³ cells per well with/without 25 µg/mL CRP recombinant protein for 72 h. At this point, the culture medium was removed and the cells were fixed with 100% methanol for 10 min, followed by washing with PBS. Cells were then stained with 0.1% crystal violet solution for 20 min. Excess dye was thoroughly washed off with PBS, and the plates were air dried. Stained cell-covered areas were imaged using a digital microscope (Keyence Corporation, Osaka, Japan), and the stained area was quantified using analysis software (BZ-X810 Analyzer, Keyence Corporation) [35]. Bone invasion models. B16F10 and MOC1 cells were washed with PBS and the cells (2.5 × 10⁶ / 10 μL in PBS) were injected into the femur of the LH2 F/F or LH2 F/F OC-Cre mice using a 1 mL tuberculin syringe with a 27G x 1/2 inch needle (Fig. 3A). The procedures were performed under mixed anesthesia with medetomidine, midazolam, and butorphanol to minimize distress. Mice were monitored daily for pain, distress, or impaired mobility. No animals met humane endpoint criteria. Fourteen days after cancer cell injection, mice were deeply anesthetized with isoflurane (5%) and euthanized by cervical dislocation, in accordance with the AVMA Guidelines for the Euthanasia of Animals [32]. Sample sizes were determined based on our previous studies using similar bone invasion models, which demonstrated that n = 5 animals per group was sufficient to detect biologically meaningful differences in bone destruction and molecular changes. No a priori statistical power calculation was performed. Histology and IHC. Femurs were fixed with 4% paraformaldehyde and demineralized with 0.5 M EDTA at 4 °C for 2 weeks [7] and embedded in paraffin before sectioning and staining. To evaluate the general structures of bone and the levels of CRP and MMP-9 proteins, 4 µm sections of paraffin-embedded femurs were cut and subjected to H&E staining, PSR staining, and IHC, as described previously [7]. The IHC scores for the protein levels were calculated using the IHC Profiler (https://sourceforge.net/projects/ihcprofiler/, Source Forge). As a negative control, sections were also stained without the addition of primary antibodies. The antibodies and respective dilutions were as follows: rabbit anti-mouse MMP9, 1:200; (10375-2A-P, Proteintech Group, Inc.) and rabbit anti-mouse CRP, 1:200; (10375-2-AP, Proteintech Group, Inc.). Radiological assessment. To assess the morphology of femurs, mice were scanned using a μCT system (LaTheta LCT-200; Hitachi Aloka Medical, Tokyo, Japan). Parameters for the CT scans were set as follows: tube voltage, 50 kVp; tube current, 145 μA; integration time, 3.6 ms; axial field of view, 48 mm, with an isotropic voxel size of 48 × 24 μm, according to the manufacturer’s instructions. Regions of interest were defined as the central portion of the femurs. Using LaTheta software (version 3.22), cortical bone mineral density, cortical bone area, and total bone volume were measured in the central portion of the femurs. Statistical analysis. Welch’s t -test was used to analyze statistical differences, and P values < 0.05 were considered statistically significant. All experiments were independently performed in triplicate. Randomization was not performed; animals were allocated to experimental groups based on their genotype. Because this study used genetically defined mouse lines, group allocation could not be concealed. Blinding was not performed because genotypes could be visually confirmed during tissue preparation, making complete masking impractical. However, image-based quantitative analyses were conducted using automated software to minimise measurement bias. The data are expressed as the mean ± the standard deviation (SD). Declarations Data availability statement Data availability All data supporting the findings of this study are available within the article and its supplementary information files, including Supplementary Fig. S1–7 and Supplementary Table 1-2. Additional datasets generated during the current study are available from the corresponding author upon reasonable request. Funding We thank JAM Post (https://www.jamp.com/svcs/) for proofreading this paper. This work was supported by JSPS KAKENHI, Grant-in-Aid for Scientific Research (B) (Grant Number 24K02640) and International Collaborative Research (Grant Number 24KK0166). JM was supported in part by the Intramural Research Program of the National Institutes of Health (NIH). Contributions by JM were made as part of his official duties as an NIH federal employee, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services. Author Contributions S.T. designed and performed experiments, analyzed data, prepared figures, and wrote the draft manuscript. A.K. supervised the project, contributed to experimental design, and edited the manuscript. T.S., R.F., M.Y. and D.N. contributed to experimental design and manuscript editing. Y.N. contributed to data analysis. T.N. contributed to animal experiments and immunohistochemical analysis. A.T. performed Western blot analysis and contributed to image quantification. M.I. conducted qRT-PCR analysis. Y.S. performed Western blot analysis. M.Y. and J.M provided conceptual advice, review, and editing. K.U. provided conceptual advice, secured funding and contributed to writing, review and editing. All authors read and approved the final version of the manuscript. 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Supplementary Files supplementalinformation1201.pdf supplementarytable1.pdf supplementarytable2.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 27 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviews received at journal 07 Feb, 2026 Reviews received at journal 25 Jan, 2026 Reviewers agreed at journal 24 Jan, 2026 Reviewers agreed at journal 24 Jan, 2026 Reviews received at journal 21 Jan, 2026 Reviews received at journal 20 Jan, 2026 Reviewers agreed at journal 19 Jan, 2026 Reviewers agreed at journal 18 Jan, 2026 Reviewers agreed at journal 06 Jan, 2026 Reviewers invited by journal 06 Jan, 2026 Editor invited by journal 18 Dec, 2025 Editor assigned by journal 13 Dec, 2025 Submission checks completed at journal 10 Dec, 2025 First submitted to journal 10 Dec, 2025 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. 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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-7989174","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":571896397,"identity":"09f73907-e74d-477d-92da-68ff38f04521","order_by":0,"name":"Saori Tomiku","email":"","orcid":"","institution":"Chiba University","correspondingAuthor":false,"prefix":"","firstName":"Saori","middleName":"","lastName":"Tomiku","suffix":""},{"id":571896401,"identity":"97f9895e-9741-4b05-87c9-a636605685bd","order_by":1,"name":"Atsushi Kasamatsu","email":"","orcid":"","institution":"Chiba 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1","display":"","copyAsset":false,"role":"figure","size":575110,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDIA analysis in BMSCs of bsLH2-cKO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003ePlod\u003c/em\u003e2 mRNA level in BMSCs of bsLH2-cKO mice by qRT-PCR. \u003cem\u003ePlod2\u003c/em\u003e mRNA levels in BMSCs of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre are lower than those from LH2\u003csup\u003eF/F\u003c/sup\u003e. \u003cem\u003ePlod\u003c/em\u003e2 mRNA levels are calculated by normalizing to GAPDH (\u003cem\u003en \u003c/em\u003e= 3; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Welch's t-test). \u003cstrong\u003e(B)\u003c/strong\u003e Western blot analysis of LH2 protein level in BMSCs. The LH2 protein level in BMSCs of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre is significantly lower than that of LH2\u003csup\u003eF/F\u003c/sup\u003e (\u003cem\u003en \u003c/em\u003e= 3). Full-length blots are presented in Supplementary Fig. S1.\u003cstrong\u003e (C, D)\u003c/strong\u003e Volcano plots and heat mapping reveal that 395 and 257 proteins are significantly up and down-regulated more than 2-fold, respectively, in bone marrow of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre when compared to that of LH2\u003csup\u003eF/F\u003c/sup\u003e. \u003cstrong\u003e(E)\u003c/strong\u003e To further narrow down LH2 deficiency-related proteins, 395 overexpressed proteins and 44 predicted extracellularly secreted proteins were extracted from DIA data.\u003cstrong\u003e (F)\u003c/strong\u003e Among 32 overlapping proteins, CRP was selected for further analyses.\u003c/p\u003e\n\u003cp\u003eBMSC, bone marrow stromal cells; CRP, C-reactive protein; DIA, data-independent acquisition; LH2, lysyl hydroxylase 2\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/4f4d6d0f8efcdc51ee5f9828.png"},{"id":100070839,"identity":"bffa98e2-76b9-4e5a-a2c4-c85516968ae6","added_by":"auto","created_at":"2026-01-12 16:18:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1032461,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRP expression in BMSCs of bsLH2-cKO mice and effect of CRP treatment on B16F10 and MOC1 cell proliferation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e \u003cem\u003eCRP\u003c/em\u003e mRNA level in BMSCs of bsLH2-cKO mice by qRT-PCR. The \u003cem\u003eCRP\u003c/em\u003e mRNA level in BMSCs of LH2\u003csup\u003ef/f\u003c/sup\u003e OC-Cre was significantly higher than that from LH2\u003csup\u003eF/F\u003c/sup\u003e. Relative \u003cem\u003eCRP\u003c/em\u003e mRNA levels were calculated by normalizing to GAPDH (\u003cem\u003en \u003c/em\u003e= 3; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; Welch's \u003cem\u003et\u003c/em\u003e-test). \u003cstrong\u003e(B)\u003c/strong\u003e Measurement of the secreted CRP protein levels in the BMSC culture medium. The CRP protein levels in the culture medium of LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre BMSCs were significantly higher than those of LH2\u003csup\u003eF/F\u003c/sup\u003e (\u003cem\u003en \u003c/em\u003e= 3; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). \u003cstrong\u003e(C, D)\u003c/strong\u003e Cell proliferation analysis after treatment with CRP. To assess the impact of CRP on cell proliferation, crystal violet staining was performed after culturing B16F10 and MOC1 cells with/without 25 µg/mL CRP recombinant protein for 72 h. Statistical analysis of the stained area with crystal violet revealed that CRP-treated cells exhibited a significantly larger stained area than untreated controls in both B16F10 and MOC1 cells (\u003cem\u003en \u003c/em\u003e= 3; *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05).\u003c/p\u003e\n\u003cp\u003eBMSC, bone marrow stromal cells; CRP, C-reactive protein; LH2, lysyl hydroxylase 2\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/32bc7080b77df205e35d6e2e.png"},{"id":100070919,"identity":"b5380ee3-0f7a-40c2-a12f-ecbd40bc5ec7","added_by":"auto","created_at":"2026-01-12 16:18:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2869932,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBone invasion models in bsLH2-cKO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e A schematic illustration of bone invasion models into the femur. \u003cstrong\u003e(B)\u003c/strong\u003e B16F10 or MOC1 cells were injected into the femurs of LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre mice. The femurs were collected 14 days after the injection. Representative pictures of the femurs are shown. \u003cstrong\u003e(C)\u003c/strong\u003e µCT analyses were performed to quantitatively evaluate bone destruction caused by tumors. Only the LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre femurs showed significant cortical bone destruction. µCT analysis also demonstrated a decrease in cortical BMD and increases in Ct. Ar and total BV in the LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre mice (\u003cem\u003en \u003c/em\u003e= 5; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eBMD, bone mineral density; BV, bone volume; Ct. Ar, cortical bone area; LH, lysyl hydroxylase 2; µCT, micro computed tomography\u003c/p\u003e","description":"","filename":"figure.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/317b97309a2f025711471bf4.png"},{"id":100070916,"identity":"c2d8758e-ad04-4d97-80c0-5f25f809c056","added_by":"auto","created_at":"2026-01-12 16:18:44","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1868536,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistochemical analysis of bone invasion models in bsLH2-cKO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Representative H\u0026amp;E images of femurs from LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice after the injection with B16F10 or MOC1 cells. B16F10 or MOC1 cells are injected into the femur (\u003cem\u003en \u003c/em\u003e= 5). The mice are euthanized after 14 days and the femurs were collected. These analyses reveal that only the LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre femur shows significant cortical bone destruction. \u003cstrong\u003e(B)\u003c/strong\u003e Representative PSR staining of femurs from LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice after the injection with B16F10 or MOC1 cells. To further evaluate changes in collagen tissue associated with bone invasion, PSR staining is performed and analyzed under a polarizing microscope. When subjected to PSR staining and observed under the polarized light, LH2\u003csup\u003eF/F\u003c/sup\u003e shows a well-organized and lamellar structure with yellow colors, while LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre a poorly organized and nonlamellar structure with green colors. Quantitative analysis of PSR-stained sections under polarized light reveals that the intensity of yellow birefringence is significantly lower in LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre mice compared with LH2\u003csup\u003eF/F\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003eLH2, lysyl hydroxylase 2; PSR, Picrosirius Red\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/a6d8104da19a6da543542b2c.png"},{"id":100070914,"identity":"41b8a643-e44b-4f86-be98-467dc41d2f3f","added_by":"auto","created_at":"2026-01-12 16:18:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2324175,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRP regulates MMP9 upregulation through MEK1/ERK signaling pathway in cancer cells.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Western blot analysis of MEK1/ERK signaling pathway. MEK1 and ERK in CRP-treated cancer cells show greater phosphorylation compared to untreated controls. α-Tubulin is used as a loading control. Full-length blots are presented in Supplementary Fig. S2-4. \u003cstrong\u003e(B)\u003c/strong\u003e qRT-PCR and Western blot analyses of MMP9 expression in cancer cells treated with CRP. Both mRNA and protein levels of MMP9 are significantly upregulated in CRP-treated cells compared to untreated controls (\u003cem\u003en \u003c/em\u003e= 5; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). Full-length blots are presented in Supplementary Fig. S5. \u003cstrong\u003e(C)\u003c/strong\u003e Representative IHC staining images for CRP in the cancellous and cortical regions of femurs from LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice. CRP expression is more intense in both cancellous and cortical bone regions of LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre mice compared to LH2\u003csup\u003eF/F\u003c/sup\u003e mice. Quantification using IHC scores shows significantly higher CRP levels in LH2\u003csup\u003eF/F \u003c/sup\u003eOC-Cre femurs (\u003cem\u003en\u003c/em\u003e = 5; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01). (D) Representative IHC staining for MMP9 in femurs collected from LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice. IHC demonstrates elevated MMP9 expression in tumors from LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice (\u003cem\u003en\u003c/em\u003e = 5; **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003eCRP, C-reactive protein\u003c/p\u003e","description":"","filename":"figure51.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/5a970a4da8bb19283ebe5547.png"},{"id":100070915,"identity":"2e110530-742b-4a0f-a44e-ea5d22cb10c7","added_by":"auto","created_at":"2026-01-12 16:18:44","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1945180,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic summary of CRP-mediated bone invasion in bsLH2-cKO mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe schematic illustration and clinical perspective summarizes the proposed mechanism in which LH2 deficiency in BMSCs leads to increased CRP expression. Elevated CRP promotes upregulation of MMP9 through the MEK1/ERK pathway in cancer cells.\u003c/p\u003e\n\u003cp\u003eBMSCs, bone marrow stromal cells\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/61257d0ab3bf12ccd7b3e77d.png"},{"id":100071161,"identity":"2f65739f-7c63-4af4-9390-a3db9d5e36f7","added_by":"auto","created_at":"2026-01-12 16:19:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11341827,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/d5064a01-5ee3-40fe-acc4-d88217d58c3a.pdf"},{"id":100070931,"identity":"56e45325-d566-46d1-a5ce-99814ea3031d","added_by":"auto","created_at":"2026-01-12 16:18:47","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1756993,"visible":true,"origin":"","legend":"","description":"","filename":"supplementalinformation1201.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/ace91f6920ad8e00652af2bc.pdf"},{"id":100070910,"identity":"61fce781-02b7-431e-aceb-3e7f6a7647b9","added_by":"auto","created_at":"2026-01-12 16:18:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":51998,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/36dd9f453adac5cc453216fd.pdf"},{"id":100070738,"identity":"8f73e1ca-121b-4e91-8115-0c1f5e11cd55","added_by":"auto","created_at":"2026-01-12 16:18:23","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":79928,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarytable2.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7989174/v1/0a7cce72432a3d87cc97de80.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Loss of lysyl hydroxylase 2 activates CRP-mediated cancer invasion through MEK/ERK/MMP9 signaling in the bone-microenvironment","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLysine hydroxylation of fibrillar collagens catalyzed by lysyl hydroxylases 1\u0026ndash;3 (LH1\u0026ndash;3) is a critical posttranslational modification for proper connective tissue development and biomechanical stability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. LH2 specifically hydroxylates lysine (Lys) residues in the telopeptide domain of type I collagen, which is essential for forming hydroxylysine (Hyl) aldehyde-derived cross-links. LH1 hydroxylates Lys residues in the helical domain and LH3 primarily functions as a glucosyltransferase [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Mutations in the LH2-encoding gene, \u003cem\u003eprocollagen-lysine, 2-oxoglutarate 5-dioxygenase\u003c/em\u003e 2 (\u003cem\u003ePlod2\u003c/em\u003e), are causative of connective tissue disorders such as Bruck syndrome, a rare form of osteogenesis imperfecta characterized by joint contractures, reduced bone mineralization, and skeletal bone fragility [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo investigate the outcome of LH2 deficiency \u003cem\u003ein vivo\u003c/em\u003e, we initially generated LH2-deficient mice using CRISPR-Cas9 technology. However, these mice died during early embryogenesis due to cardiac defects [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. LH2 heterozygous mice exhibited altered biomechanical and biochemical properties in femurs [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. We therefore generated bone-specific LH2 conditional knockout (bsLH2-cKO) mice using the osteocalcin-Cre/loxP system. Biochemical and mechanical analyses of these mice revealed reduced telopeptide Lys hydroxylation, abnormal collagen cross-link formation, and increased bone fragility, demonstrating that LH2 is indispensable for maintaining the structural integrity of bone collagen [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn cancer biology, aberrant LH2 activity has been shown to affect the tumor microenvironment by altering collagen cross-linking, thereby enhancing tumor stiffness, invasion, and metastatic potential [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan additionalcitationids=\"CR10 CR11 CR12\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Especially in head and neck cancers, LH2 overexpression has been associated with increased tumor aggressiveness [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Bone is one of the most common metastatic sites for several malignancies, including breast, prostate, and head and neck cancers [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Once tumor cells invade the bone-microenvironment, they exploit the native extracellular matrix (ECM) to promote colonization, osteolysis, and systemic skeletal complications. Inflammatory pathways are intimately involved in this process. Notably, elevated circulating levels of C-reactive protein (CRP), a widely used biomarker of systemic inflammation, have been linked to poor prognosis and increased bone metastatic burden in cancer patients [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the molecular mechanisms by which matrix-modifying enzymes like LH2 modulate both inflammation and tumor progression within the bone-microenvironment remain largely undefined.\u003c/p\u003e \u003cp\u003eDuring the course of characterizing the protein expression profile of bone marrow stromal cells (BMSCs) from bsLH2-cKO mice, we found a marked upregulation of CRP, a key inflammatory mediator previously associated with cancer aggressiveness and poor prognosis. Based on this finding, we hypothesized that loss of LH2 promotes cancer progression in bone via CRP-mediated activation of downstream signaling pathways. In the present study, we demonstrate that CRP upregulation enhances cancer cell proliferation and bone invasion through activation of the MEK/ERK pathway and subsequent induction of matrix metalloproteinase-9 (MMP-9). These findings identify a novel LH2\u0026ndash;CRP\u0026ndash;MMP9 signaling axis that mechanistically links ECM disorganization to inflammation-driven tumor invasion in bone, providing a rationale for its therapeutic targeting.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eLH2 expression in bone marrow stromal cells (BMSCs) of bsLH2-cKO mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo establish the bsLH2-cKO strain of mice, we used Osteocalcin-Cre transgenic (OC-Cre) mice. Although previous studies confirmed LH2 deficiency in the cortical bones of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], LH2 expression in BMSCs had not been examined. Here, we evaluated the mRNA and protein levels of LH2 in BMSCs isolated from femurs in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice. The \u003cem\u003ePlod2\u003c/em\u003e mRNA and LH2 protein expression levels in BMSCs from LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice were significantly lower than those in control mice (LH2\u003csup\u003eF/F\u003c/sup\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, B) (The corresponding full-length blots are provided in Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eData-independent acquisition (DIA) proteomic analysis in BMSCs of bsLH2-cKO mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe performed DIA proteomic analysis to assess the protein expression patterns in the BMSCs of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre and LH2\u003csup\u003eF/F\u003c/sup\u003e mice. Volcano plots and heat mapping revealed that 395 proteins were significantly upregulated and 257 proteins downregulated more than 2-fold in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre femurs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D). To further narrow down LH2 deficiency-induced proteins, 395 overexpressed proteins and 44 predicted extracellularly secreted proteins were extracted from the DIA dataset. Among the 32 overlapping proteins, CRP level was by far\u0026thinsp;\u0026gt;\u0026thinsp;10-fold higher than the rest of the proteins, thus, selected for further analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCRP expression in BMSCs of bsLH2-cKO mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo confirm expression levels of mRNA and secretion levels of CRP, we performed a quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and ELISA assays. The CRP mRNA and protein levels in BMSCs of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre were both significantly greater than those of LH2\u003csup\u003eF/F\u003c/sup\u003e (10.48- and 3.13-fold, respectively) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCRP promotes proliferation of B16F10 and MOC1 cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo assess the effect of CRP on cancer cell proliferation, B16F10 (mouse malignant melanoma) and MOC1 (mouse oral squamous cell carcinoma) cells were cultured with/without 25 \u0026micro;g/mL of recombinant CRP protein for 72 h, followed by crystal violet staining. CRP-treated cells exhibited significantly greater stained areas compared to controls, indicating CRP enhanced cancer cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-D).\u003c/p\u003e \u003cp\u003e \u003cb\u003eBone invasion models in bsLH2-cKO mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate cancer progression \u003cem\u003ein vivo\u003c/em\u003e, B16F10 or MOC1 cells were injected into the femurs of LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice. After 14 days, the femurs were collected and evaluated (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). In LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre femurs, bone hypertrophy was visibly apparent compared to LH2\u003csup\u003eF/F\u003c/sup\u003e mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Micro computed tomography (\u0026micro;CT) analysis also demonstrated that in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre femurs, cortical bone mineral density (BMD) was significantly decreased, while cortical bone area and total bone volume significantly increased, after injection with both cancer cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-D). There were no evident differences between the LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre femurs in terms of weight and length, while the width of the femurs was slightly wider in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre, but the difference did not reach the significance level (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, Supplementary Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e, Supplementary Table\u0026nbsp;1). Individual body weight data are provided in Supplementary Table\u0026nbsp;2. There was no significant difference in body weight after cancer cell injection between LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre and LH2\u003csup\u003eF/F\u003c/sup\u003e (Supplementary Fig. \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eHistological characterization of bone invasion in bsLH2-cKO mice.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHistological examination with hematoxylin and eosin (H\u0026amp;E) staining revealed that extensive destruction of cortical bone was observed in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice, whereas cortical bone structure in LH2\u003csup\u003eF/F\u003c/sup\u003e mice was preserved well, suggesting that LH2 deficiency enhances tumor-induced bone destruction (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Picrosirius Red (PSR) staining under polarized light exhibited patches of disorganized nonlamellar, woven bone-like structure of collagen (green) in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice compared with well-organized lamellar bone structure (yellow) in LH2\u003csup\u003eF/F\u003c/sup\u003e mice. Quantitative image analysis of PSR staining under polarized light, measuring the mean brightness intensity of the birefringent signal in the cortical bone area, showed significantly lower intensity in LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice, indicating that LH2 deficiency led to disruption of collagen architecture and resulted in disorganized bone structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eCRP regulates MMP9 through the MEK/ERK signaling pathway in cancer cells.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effect of CRP on its downstream molecules, we performed Western blot analysis. We found increased phosphorylation of MEK1 and ERK in the cancer cell lines after treatment with CRP compared with untreated controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) (The corresponding full-length blots are provided in Supplementary Fig. S4-6). Additionally, CRP-treatment experiments resulted in\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eindicating that CRP controlled MMP9 expression via the MEK1/ERK pathway (The corresponding full-length blots are provided in Supplementary Fig. S7). We focused on MMP9 because it is a major collagen-degrading enzyme in bone, capable of cleaving type I collagen in the mineralized matrix and thereby contributing to ECM remodeling [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To assess CRP expression in bsLH2-cKO mice, we performed IHC on femur sections after injection with the cancer cells. CRP expression was notably elevated in the LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre group, highlighting the link between LH2 deficiency and inflammation in the bone-microenvironment. IHC scores also confirmed significantly higher CRP expression levels in the LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). In addition, elevated MMP9 expression was demonstrated in the medullary cavity of the femurs of LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre mice after injection with the cancer cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Therefore, in addition to the \u003cem\u003ein vitro\u003c/em\u003e study using the cancer cells, bsLH2-cKO femur samples \u003cem\u003ein vivo\u003c/em\u003e demonstrated higher CRP expression, leading to MMP9 overexpression.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIt is well documented that LH2 is a key collagen-modifying enzyme that catalyzes telopeptidyl Lys hydroxylation, a post-translational modification essential for the formation of hydroxylysine aldehyde-derived, stable cross-links in type I collagen. These cross-links are critical for the mechanical strength of load-bearing tissues such as bone [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, our findings in this study indicate that LH2 plays a much broader biological role, functioning as a regulator of the tumor\u0026ndash;bone interface by modulating stromal inflammation and cancer progression.\u003c/p\u003e \u003cp\u003eIn our \u003cem\u003ein vivo\u003c/em\u003e bone invasion models, LH2 deficiency markedly exacerbated cancer-induced cortical bone destruction and disrupted collagen architecture. These phenotypes are consistent with our previous observations that bones in bsLH2-cKO mice exhibited the diminished mature collagen cross-links and increased bone fragility [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While the structural vulnerability of the LH2-deficient bone matrix likely contributes to enhanced tumor invasion, our data also suggest that LH2 deficiency actively promotes a pro-tumorigenic environment via inflammatory mediators.\u003c/p\u003e \u003cp\u003eProteomic profiling of BMSCs from bsLH2-cKO mice revealed that CRP was among the most highly upregulated secreted proteins. CRP is an acute-phase reactant widely recognized as a clinical biomarker of systemic inflammation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], and elevated CRP levels correlate with poor prognosis in several malignancies, including those with bone metastases [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. While CRP has been considered a surrogate marker in the past, accumulating evidence suggests its active involvement in tumor progression through modulation of immune responses, angiogenesis, and matrix remodeling [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In our study, CRP was significantly upregulated in LH2-deficient BMSCs and their conditioned medium, indicating that it is a bone-intrinsic pro-inflammatory signaling.\u003c/p\u003e \u003cp\u003ePrevious studies have demonstrated that endoplasmic reticulum (ER) stress activates CRP expression via ATF4 and CHOP [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. LH2 deficiency is known to impair collagen biosynthesis and induce ER stress responses in stromal cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], supporting a mechanistic link between matrix defects and inflammation. Thus, we propose that LH2 maintains stromal immune homeostasis not only through collagen integrity but also by limiting ER stress\u0026ndash;induced CRP production in the bone-microenvironment.\u003c/p\u003e \u003cp\u003eFunctional analyses further established CRP as a key effector of cancer\u0026ndash;bone interaction. CRP enhanced cancer cell proliferation \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and promoted bone destruction \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Mechanistically, CRP activated the MEK/ERK signaling pathway, resulting in upregulation of matrix metalloproteinase-9 (MMP9) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), a known driver of tumor-induced osteolysis and bone matrix degradation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings align with clinical observations that MMP9 is highly expressed in bone-metastatic lesions and contributes to skeletal complications [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have shown that forced LH2 overexpression in cancer cells enhances their proliferation and metastatic potential through cell-intrinsic mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In contrast, our present findings reveal that LH2 deficiency in surrounding stromal cells, particularly BMSCs, promotes tumor growth indirectly by driving excessive CRP secretion, thereby creating a pro-inflammatory microenvironment. These findings identify a novel LH2\u0026ndash;CRP\u0026ndash;MMP9 signaling axis that links ECM disorganization to inflammation-driven tumor invasion in bone, providing a rationale for its therapeutic targeting (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). We have previously developed KS122-0485428, a selective small-molecule activator of LH2 that enhances its enzymatic function [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. We propose that pharmacological restoration of LH2 activity may suppress CRP-mediated tumor promotion and prevent cancer-induced skeletal events. Given the unmet clinical need for therapies addressing bone metastasis across multiple cancer types [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], modulation of the LH2\u0026ndash;CRP\u0026ndash;MMP9 pathway may offer a potential translational strategy.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStudy approval and accordance\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods and experiments were approved by the Chiba University Review Board for Animal Care (Chiba, Japan, Protocol No. A7-013), and all procedures were performed in accordance with the relevant guidelines and regulations, including the ARRIVE guidelines and the AVMA Guidelines for the Euthanasia of Animals\u0026nbsp;[31,32]. All mice were housed under diurnal lighting conditions, allowed free access to food and water, and\u0026nbsp;monitored at least once daily for general health, mobility, grooming, and activity. No unexpected adverse events occurred, and no animals reached predefined humane endpoints. Mice were deeply anesthetized with isoflurane (5%) and euthanized by cervical dislocation\u0026nbsp;to ensure death. All efforts were made to minimize animal suffering and to reduce the number of animals used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eGeneration of\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003eLH2\u003csup\u003eF/F\u0026nbsp;\u003c/sup\u003eOC-Cre\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e\u0026nbsp;mice\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre (bsLH2-cKO) mice were generated according to the Protocol as described previously [8,31,32]. Briefly, Heterozygous LH2\u003csup\u003eF/+\u003c/sup\u003e mice (Chiba University) were interbred with OC-Cre mice (Jackson Laboratory strain No. 019509) to generate LH2\u003csup\u003eF/+\u003c/sup\u003e OC-Cre mice. The LH2\u003csup\u003eF/+\u003c/sup\u003e mice were then intercrossed with LH2\u003csup\u003eF/+\u003c/sup\u003e OC-Cre mice to generate LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre (bsLH2-cKO) mice using the osteocalcin-Cre/loxP system. Mice were maintained on a C57BL/6 (Jackson Laboratory) background[8,33]. Male mice aged 8–16 weeks were used throughout the experiments. Mice were assigned to the LH2\u003csup\u003eF/F\u003c/sup\u003e or LH2\u003csup\u003eF/F\u003c/sup\u003e OC-Cre groups according to genotype. No animals were excluded from the study, and no predefined inclusion or exclusion criteria were applied.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCell lines and cell culture.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBMSCs in 8- to 16-week-old male\u0026nbsp;LH2\u003csup\u003eF/F\u0026nbsp;\u003c/sup\u003eOC-Cre and LH2\u003csup\u003eF/F\u003c/sup\u003emice were isolated by flushing the bone marrow from the femur and incubating with red blood cell lysis buffer (Invitrogen, Carlsbad, CA, USA). The cells were cultured in RPMI 1640 medium (Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich), 100 units/mL penicillin and\u0026nbsp;100 μg/mL streptomycin\u0026nbsp;(Sigma-Aldrich), 1% L-glutamine (200mM) (Gibco, Waltham, MA, USA), 0.1% 2-mercaptoethanol (Gibco), and 1% HEPES (1M) (Gibco)\u0026nbsp;in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026nbsp;°C. The BMSC cultures were rinsed 3 times with phosphate-buffered saline (PBS; Sigma-Aldrich) to remove nonadherent cells. The cell culture medium was changed 4 days after plating and cells were grown for 7 days in their respective conditions.\u003c/p\u003e\n\u003cp\u003eA mouse oral squamous cell carcinoma (OSCC) cell line, MOC1, was purchased from Kerafast (Newark, CA, USA) and a mouse malignant melanoma cell line, B16F10, was purchased by RIKEN (No. RCB2630; RIKEN, Saitama, Japan). MOC1 cells were grown in a solution of 62% IMDM medium (Wako, Osaka, Japan), 31% Ham’s F-12 nutrient mix (Cytiva, Marlborough, MA, USA), 10% FBS, 100 units/mL penicillin and\u0026nbsp;100 μg/mL streptomycin,\u0026nbsp;40 μg/L hydrocortisone (Sigma-Aldrich), 5\u0026nbsp;μg/L epidermal growth factor (Merck, Darmstadt, Germany), and 5\u0026nbsp;mg/L insulin (Sigma-Aldrich).\u0026nbsp;B16F10 cells were grown in RPMI-1640\u0026nbsp;supplemented with 10% FBS and 100 units/mL penicillin and\u0026nbsp;100 μg/mL streptomycin (Sigma-Aldrich) in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37 °C. The cell culture medium was changed twice a week.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eqRT-PCR.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNAs of BMSCs, B16F10 cells, and MOC1 cells treated with/without 25 µg/mL CRP recombinant protein were extracted with TRIzol reagent (Invitrogen) and reverse transcribed into cDNA with ReverTra Ace qPCR RT Master Mix (TOYOBO, Tokyo, Japan). Analyses of \u003cem\u003ePlod2,\u003c/em\u003e \u003cem\u003eCRP\u003c/em\u003e, and \u003cem\u003eMMP9\u003c/em\u003e mRNA expression levels were performed by qRT-PCR with TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific, Waltham, MA, USA), with the specific probe and primers set on a Light Cycler 96 System (Roche Diagnostics GmbH, Mannheim, Germany). The specific probe and primers set for \u003cem\u003ePlod2, CRP,\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;MMP9\u0026nbsp;\u003c/em\u003ewere purchased from Thermo Fisher Scientific (TaqMan Gene Expression Assay; Mm00478767_m1, Mm00432680_g1, and Mm00442991_m1). The mRNA expression levels were normalized to GAPDH (Mm99999915_g1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eWestern blot analysis.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eProteins were extracted with RIPA lysis buffer (Nacalai Tesque, Inc., Kyoto, Japan) containing a cocktail of protease inhibitors (Santa Cruz Biotechnology, Dallas, TX, USA). Lysates were centrifuged at 10,000g and the supernatants were collected. Extracted proteins (20 µg) were separated on 4-12 % NuPAGE Tris-Acetate Pre-Cast Gels (Invitrogen) under denaturing and reducing conditions, and the proteins were transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA) by electroblotting. The membranes were blocked with Blocking One (Nacalai Tesque, Inc.) for 1 h at room temperature, and then incubated overnight at 4 °C with the following primary antibodies: rabbit anti‑mouse LH2 polyclonal antibody, 1:500; (21214-1-AP, Proteintech Group, Inc., Rosemont, IL, USA), rabbit anti-mouse ERK1/2, 1:1000; (11257-1-AP, Proteintech Group, Inc.), rabbit anti-mouse phospho-ERK1/2, 1:500; (28733-1-AP, Proteintech Group, Inc.), rabbit anti-mouse MEK1, 1:2000; (GTX134234, GeneTex, Irvine, CA, USA), rabbit anti-mouse phospho-MEK1, 1:2000; (28930-1-AP, Proteintech Group, Inc.), rabbit anti-mouse MMP9, 1:1000; (10375-2-AP, Proteintech Group, Inc.), and rabbit anti-mouse α-Tubulin, 1:2500; (11224-1-AP, Proteintech Group, Inc.). After washing the membranes with 0.1% Tween-20 Tris-buffered saline 3 times, the membranes were incubated with horseradish peroxidase-conjugated IgG as an anti-rabbit secondary antibody (Promega Corporation, Madison, WI, USA) at room temperature. Finally, the membranes were visualized using enhanced chemiluminescence (ECL) with the ECL Prime Western Blotting Detection Reagent (Cytiva) and imaged using a ChemiDoc XRS Plus System with Image Lab software (Bio-Rad Laboratories).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDIA analysis.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDIA proteomic analysis was performed to characterize and evaluate protein levels in the bone marrow of LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u0026nbsp;\u003c/sup\u003eOC-Cre mice (\u003cem\u003en\u0026nbsp;\u003c/em\u003e= 3), according to the protocol described previously[34]. Volcano plots and heat map were evaluated for protein expression variation between the LH2\u003csup\u003eF/F\u003c/sup\u003e and LH2\u003csup\u003eF/F\u0026nbsp;\u003c/sup\u003eOC-Cre mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eELISA of culture medium.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCRP concentrations in the culture medium were determined using a mouse C-reactive protein/CRP Sandwich ELISA Kit (Proteintech) according to the manufacturer’s protocol and\u0026nbsp;read at 450 nm using a Benchmark Plus Microplate Reader (Bio-Rad Laboratories). Briefly, cell suspension was centrifuged at 500 g and the supernatants were collected. Each assay was repeated three times independently.\u0026nbsp;Standard curves using CRP recombinant protein and individual well concentrations were determined using the\u0026nbsp;ELISA calculator (Proteintech). To keep experimental values within the linear region of the standard curves, samples were diluted to 1:2 as necessary for stimulated culture samples.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCrystal Violet Staining.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB16F10 and MOC1 cells were seeded in 96-well plates at a density of 1×10³ cells per well with/without 25 µg/mL CRP recombinant protein for 72 h. At this point, the culture medium was removed and the cells were fixed with 100% methanol for 10 min, followed by washing with PBS. Cells were then stained with 0.1% crystal violet solution for 20 min. Excess dye was thoroughly washed off with PBS, and the plates were air dried. Stained cell-covered areas were imaged using a digital microscope (Keyence Corporation, Osaka, Japan), and the stained area was quantified using analysis software (BZ-X810 Analyzer, Keyence Corporation) [35].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBone invasion models.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB16F10 and MOC1 cells were washed with PBS and the cells (2.5 × 10⁶ / 10 μL in PBS) were injected into the femur of\u0026nbsp;the LH2\u003csup\u003eF/F\u003c/sup\u003e or LH2\u003csup\u003eF/F\u0026nbsp;\u003c/sup\u003eOC-Cre mice\u0026nbsp;using a 1 mL tuberculin syringe with a 27G x 1/2 inch needle (Fig. 3A). The procedures were performed under mixed anesthesia with medetomidine, midazolam, and butorphanol to minimize distress. Mice were monitored daily for pain, distress, or impaired mobility. No animals met humane endpoint criteria. Fourteen days after cancer cell injection, mice were deeply anesthetized with isoflurane (5%) and euthanized by cervical dislocation, in accordance with the AVMA Guidelines for the Euthanasia of Animals\u0026nbsp;[32]. Sample sizes were determined based on our previous studies using similar bone invasion models, which demonstrated that n = 5 animals per group was sufficient to detect biologically meaningful differences in bone destruction and molecular changes. No a priori statistical power calculation was performed.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eHistology and IHC.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemurs were fixed with 4% paraformaldehyde and demineralized with 0.5 M EDTA at 4 °C for 2 weeks [7] and embedded in paraffin before sectioning and staining. To evaluate the general structures of bone and the levels of CRP and MMP-9 proteins, 4 µm sections of paraffin-embedded femurs were cut and subjected to H\u0026amp;E staining, PSR staining, and IHC, as described previously [7]. The IHC scores for the protein levels were calculated using the IHC Profiler (https://sourceforge.net/projects/ihcprofiler/, Source Forge). As a negative control, sections were also stained without the addition of primary antibodies. The antibodies and respective dilutions were as follows: rabbit anti-mouse MMP9, 1:200; (10375-2A-P, Proteintech Group, Inc.) and rabbit anti-mouse CRP, 1:200; (10375-2-AP, Proteintech Group, Inc.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eRadiological assessment.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the morphology of femurs, mice were scanned using a μCT system (LaTheta LCT-200; Hitachi Aloka Medical, Tokyo, Japan). Parameters for the CT scans were set as follows: tube voltage, 50 kVp; tube current, 145 μA; integration time, 3.6 ms; axial field of view, 48 mm, with an isotropic voxel size of 48 × 24 μm, according to the manufacturer’s instructions. Regions of interest were defined as the central portion of the femurs. Using LaTheta software (version 3.22), cortical bone mineral density, cortical bone area, and total bone volume were measured in the central portion of the femurs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eStatistical analysis.\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWelch’s \u003cem\u003et\u003c/em\u003e-test was used to analyze statistical differences, and \u003cem\u003eP\u003c/em\u003e values \u0026lt; 0.05 were considered statistically significant. All experiments were independently performed in triplicate. Randomization was not performed; animals were allocated to experimental groups based on their genotype. Because this study used genetically defined mouse lines, group allocation could not be concealed. Blinding was not performed because genotypes could be visually confirmed during tissue preparation, making complete masking impractical. However, image-based quantitative analyses were conducted using automated software to minimise measurement bias. The data are expressed as the mean ± the standard deviation (SD).\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003eData availability All data supporting the findings of this study are available within the article and its supplementary information files, including Supplementary Fig. S1–7 and Supplementary Table 1-2. Additional datasets generated during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank JAM Post (https://www.jamp.com/svcs/) for proofreading this paper. This work was supported by JSPS KAKENHI, Grant-in-Aid for Scientific Research (B) (Grant Number 24K02640) and International Collaborative Research (Grant Number 24KK0166). JM was supported in part by the Intramural Research Program of the National Institutes of Health (NIH). Contributions by JM were made as part of his official duties as an NIH federal employee, are in compliance with agency policy requirements, and are considered Works of the United States Government. However, the findings and conclusions presented in this paper are those of the author(s) and do not necessarily reflect the views of the NIH or the U.S. Department of Health and Human Services.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eS.T. designed and performed experiments, analyzed data, prepared figures, and wrote the draft manuscript. A.K. supervised the project, contributed to experimental design, and edited the manuscript. T.S., R.F., M.Y. and D.N. contributed to experimental design and manuscript editing. Y.N. contributed to data analysis. T.N. contributed to animal experiments and immunohistochemical analysis. A.T. performed Western blot analysis and contributed to image quantification. M.I. conducted qRT-PCR analysis. Y.S. performed Western blot analysis. M.Y. and J.M provided conceptual advice, review, and editing. K.U. provided conceptual advice, secured funding and contributed to writing, review and editing. All authors read and approved the final version of the manuscript.\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\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eYamauchi, M. \u0026amp; Sricholpech, M. 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L. \u003cem\u003eAVMA GUIDELINES FOR THE EUTHANASIA OF ANIMALS: 2020 EDITION AVMA Guidelines for the Euthanasia of Animals: 2020 Edition* Members of the Panel on Euthanasia AVMA Staff Consultants\u003c/em\u003e. (2020).\u003c/li\u003e\n \u003cli\u003eNozaki, R., Kasamatsu, A., Moss, J. \u0026amp; Uzawa, K. Lysyl hydroxylase 2 deficiency promotes filopodia formation and fibroblast migration. \u003cem\u003eBiochem Biophys Res Commun\u003c/em\u003e \u003cstrong\u003e587\u003c/strong\u003e, 146\u0026ndash;152 (2022).\u003c/li\u003e\n \u003cli\u003eRose, J. P. \u003cem\u003eet al.\u003c/em\u003e Deep coverage and quantification of the bone proteome provides enhanced opportunities for new discoveries in skeletal biology and disease. \u003cem\u003ePLoS One\u003c/em\u003e \u003cstrong\u003e18\u003c/strong\u003e, (2023).\u003c/li\u003e\n \u003cli\u003eKosaka, K. \u003cem\u003eet al.\u003c/em\u003e iPSC-derived megakaryocytes and platelets accelerate wound healing and angiogenesis. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e \u003cstrong\u003e15\u003c/strong\u003e, 364 (2024).\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7989174/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7989174/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eLysyl hydroxylase 2 (LH2), encoded by \u003cem\u003eprocollagen-lysine, 2-oxoglutarate 5-dioxygenase 2\u003c/em\u003e (\u003cem\u003ePlod2\u003c/em\u003e) gene, plays critical roles in collagen cross-linking and bone matrix organization. Recent studies have also shown that aberrant LH2 expression is promotes tumor progression, but its specific role in modulating cancer behavior within the bone-microenvironment remains unclear. Given the clinical importance of bone metastasis and the involvement of inflammatory mediators in tumor-stromal interactions, understanding the LH2 function in bone-tumor dynamics is of particular interest. Here, we used bone-specific LH2 conditional knockout (bsLH2-cKO) mice to investigate the impact of LH2 deficiency on cancer progression in bone by proteomic analysis and bone invasion models. Protein expression profiling revealed that C-reactive protein (CRP) was markedly upregulated in bone marrow stromal cells isolated from bsLH2-cKO femurs. Our \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e analyses demonstrated that CRP significantly enhanced cancer proliferation and exacerbated bone destruction in bsLH2-cKO mice following tumor cell injection. Pathway analysis further indicated that CRP induced by LH2 deficiency promoted overexpression of matrix metalloproteinase-9 through activation of the MEK/ERK signaling pathway. These findings identify LH2 as a critical regulator of CRP-mediated cancer progression in the bone-microenvironment, suggesting that targeting the LH2-CRP axis may represent a promising therapeutic strategy for bone-invasive cancers.\u003c/p\u003e","manuscriptTitle":"Loss of lysyl hydroxylase 2 activates CRP-mediated cancer invasion through MEK/ERK/MMP9 signaling in the bone-microenvironment","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 16:11:07","doi":"10.21203/rs.3.rs-7989174/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-02-27T12:38:12+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T19:47:40+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-07T05:36:32+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-25T16:01:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"98469902579996725539054835930801999244","date":"2026-01-24T13:02:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"185359836202985631447957277650738609194","date":"2026-01-24T09:44:09+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-21T15:53:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-01-20T20:01:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"320144443149002340024188631167490742073","date":"2026-01-19T07:09:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"301909585657334614467450774360063367830","date":"2026-01-18T23:07:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"3542969550781593645961490673715720420","date":"2026-01-06T21:11:01+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-06T21:04:29+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-12-18T06:57:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-13T12:21:37+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-10T13:03:44+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-10T12:59:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af965d2e-e2ce-4cea-b0db-95a660fe26e7","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":60861215,"name":"Biological sciences/Cancer"},{"id":60861216,"name":"Biological sciences/Cell biology"},{"id":60861217,"name":"Biological sciences/Molecular biology"}],"tags":[],"updatedAt":"2026-02-27T12:53:46+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 16:11:07","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7989174","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7989174","identity":"rs-7989174","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}