Klotho attenuates glucocorticoid-induced osteoblast cytotoxicity via Wnt signaling pathway modulation

Klotho attenuates glucocorticoid-induced osteoblast cytotoxicity via Wnt signaling pathway modulation | 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 Research Article Klotho attenuates glucocorticoid-induced osteoblast cytotoxicity via Wnt signaling pathway modulation Sen Wang, Miao He, Xiao Liang, Baoshan Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5854318/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Background Glucocorticoids are commonly prescribed in clinical settings; however, their prolonged use at high doses can adversely affect human health. One significant complication following glucocorticoid therapy is glucocorticoid-induced osteoporosis (GIO), which is second in incidence only to senile osteoporosis. Objective Based on previous research indicating that Klotho alleviates dexamethasone-induced osteoblast cytotoxicity through the NF-kB pathway, we aimed to explore the underlying mechanisms in greater depth. Methods We assessed the impact of Lithium chloride (LiCl), a Wnt pathway activator, on glucocorticoid-induced cell cytotoxicity and viability. Cytotoxicity was specifically quantified by Annexin V/PI flow cytometry. We performed qRT-PCR and Western blotting analyses to scrutinize the expressions of genes and proteins associated with both canonical and non-canonical Wnt signaling pathways. Results Dexamethasone treatment induced an upregulation of the non-canonical Wnt ligand, Wnt5a, and a downregulation of the canonical ligand, Wnt3a, along with its downstream marker, β-catenin. Transfection with Klotho counteracted these effects. Conclusion Klotho has the potential to modulate both canonical and non-canonical Wnt signaling pathways, thereby counteracting osteoblast cytotoxicity induced by glucocorticoids. Klotho Wnt signaling pathway Glucocorticoid Osteoporosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction First identified by Nusse in 1982, Wnt genes have since emerged as central regulators within the Wnt signaling pathway, governing pivotal cellular processes such as cell cycle progression and tumor differentiation 1 . This pathway, extending its influence beyond embryogenesis, organ formation, and tumorigenesis, also critically modulates bone formation and osteoblast activity 2,3 . The Wnt pathway can be categorized into the classic Wnt/β-catenin pathway and the non-classical Wnt signaling pathway, both of which necessitate the presence of Frizzled (FZD), a transmembrane protein 4 . Within the canonical pathway, the Wnt3a ligand's interaction with FZD impedes the action of Glycogen Synthase Kinase 3 (GSK3), enabling the accumulation and nuclear translocation of β-catenin. This cascade culminates in the transcriptional activation of osteogenic genes, steering osteoblast differentiation 5 . Conversely, in the absence of Wnt ligands, GSK3 phosphorylates β-catenin, thwarting its nuclear translocation and thereby influencing osteoblast differentiation and bone maturation 6 . A salient feature is that bone formation is amplified upon activation of the canonical Wnt/β-catenin pathway and diminished when inhibited. Parallelly, the non-canonical pathway, with ligands such as Wnt4, Wnt5a, and Wnt16, orchestrates bone homeostasis through an intricate interplay with a broader cellular ensemble and multifaceted mechanisms 6 . Chronic glucocorticoid exposure promotes osteoblast cytotoxicity and disrupts bone remodeling, as demonstrated in both preclinical and clinical studies 7,8 . Research indicates glucocorticoids (GC) can inhibit bone formation by suppressing Wnt pathway-related molecules 9 . During the early stages of GC treatment, it may hinder the co-receptors of the Wnt signal, whereas, in the later stages, they might inhibit the ligands themselves 10,11 . Prolonged and excessive use of GC therapy is also associated with the upregulation of Dickkopf-related protein 1 (DKK1), an inhibitor of the Wnt pathway, suggesting its potential involvement in GC-induced osteoporosis 12,13 . Recent evidence further suggests that GCs disrupt the balance between canonical and non-canonical Wnt signaling: dexamethasone (DEX) downregulates Wnt3a and β-catenin while elevating Wnt5a, thereby shifting osteoblasts toward apoptosis 11,14 . Glucocorticoids can also suppress osteoblastogenesis by downregulating critical transcription factors such as Runx2 and Osterix. 15 These alterations correlate with reduced bone formation and increased fracture risk in glucocorticoid-induced osteoporosis (GIO) 16 . Klotho (KL), originally identified as an anti-aging protein, demonstrates significant associations with various health conditions in mice lacking the KL gene, including atherosclerosis, glucose metabolism abnormalities, and emphysema 17,18 . Subsequent research has further unveiled its close ties to conditions such as diabetes, lipid metabolism disorders, renal damage, coronary heart disease, hypertension, and brain injury 19,20 . Importantly, the expression of the KL gene also plays a role in the development of osteoporosis. Studies have indicated that KL can mitigate microstructural damage in bone, although the underlying mechanisms remain partially understood 21 . Additionally, KL gene expression in osteoblasts can inhibit osteoblast apoptosis and regulate bone metabolism 22 . For instance, Komaba et al demonstrated that Klotho deficiency in osteocytes disrupts bone mineralization, leading to reduced bone mass and increased fragility 23 . These findings underscore KL's multifaceted role in maintaining skeletal integrity. Disruptions in KL gene expression can significantly influence osteoblast and osteoclast differentiation, leading to a decrease in bone mass 23,24 . Our prior investigations have demonstrated Klotho's ability to inhibit glucocorticoid-induced apoptosis in MC3T3-E1 osteoblasts by reducing levels of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2) and suppressing the expression of pro-apoptotic proteins, such as BCL2-associated X protein (Bax) and Nuclear Factor-Kappa B (NF-kB) 25 . Recent studies have revealed that the extracellular domain of the Klotho protein can act on various Wnt ligands, thus diminishing their capacity to activate the Wnt signaling pathway 26–28 . Consequently, Klotho may modulate the normal transmission of the Wnt signaling pathway through its interaction with Wnt ligands 29 . Based on these findings, we hypothesize that KL gene expression plays a pivotal role in the development of glucocorticoid-induced osteoporosis (GIO) through its association with the Wnt signaling pathway. To investigate this hypothesis, we have established a dexamethasone-induced osteoblast apoptosis model to elucidate further the impacts of the Wnt pathway and KL gene expression on glucocorticoid-induced osteoblast cytotoxicity. Materials and methods The reagents and chemicals used in this study are as follows: MC3T3-E1 cells (Shanghai ZhongqiaoXinzhou); Dexamethasone (DEX) (Shanghai Yeyuan Biotechnology); Recombinant adenovirus (AD-GFP and AD-KL; Shanghai Jikai Company); qPCR reagents (Beijing Qingen Biological); Cell Counting Kit-8 (CCK-8; Japan Tohjin Research Institute); High-glucose DMEM, 0.25% trypsin (Wuhan Cellbiological Technology); Fetal bovine serum (FBS; PAN Biotech); Penicillin-streptomycin solution (Biosharp); Antibodies: Anti-Klotho (Zhengneng Bio), Anti-Wnt5a, Anti-Wnt3a, Anti-β-catenin (Wanlei Biology), Anti-β-actin (Sanying Bio). 1.1 Cell culture MC3T3-E1 cells were cultured in a humidified incubator at 37°C with 5% CO 2 . The culture medium consisted of 89% high-glucose DMEM, 10% FBS, and 1% penicillin-streptomycin. Cells were passaged at a 1:3 ratio upon reaching 80–90% confluence. 1.2 Experimental Design and Treatments Cells were divided into seven groups: a. Control: Untreated cells. b. Ad-GFP: Cells transfected with GFP adenovirus (MOI = 100) for 12 h, then cultured in standard medium. c. Ad-KL + DEX: Ad-KL-transfected cells (MOI = 100, 12 h) treated with 2 mM DEX for 24 h. d. DEX: Cells treated with 2 mM DEX for 24 h. e. Ad-KL: Ad-KL-transfected cells (MOI = 100, 12 h). LiCl: Cells treated with 5 mM LiCl for 24 h. f. DEX + LiCl: Co-treated with 2 mM DEX and 5 mM LiCl for 24 h. 1.3 Optimization of DEX Concentration for Apoptosis Induction Cells were seeded in 96-well plates (10⁵ cells/mL) and allowed to adhere for 24 h. Subsequently, they were treated with DEX (0, 0.5, 1, 2, or 4 mM) for 24 h. Cell viability was quantified using the CCK-8 assay (n = 4 replicates per concentration). 1.4 Adenoviral Transfection Cells were transfected with Ad-GFP or Ad-KL (MOI = 100) for 12 h. After replacing the medium, transfection efficiency was assessed via fluorescence microscopy at 24, 48, and 72 h. Klotho mRNA and protein levels were analyzed by qPCR and Western blot. 1.5 Cell Viability and Apoptosis Assessment: Viability: Cells were seeded in 96-well plates (1×10 5 cells/mL) and treated as per experimental groups. After 24 h, 10μL CCK-8 reagent (Japan Tohjin Research Institute) was added to each well, incubated for 2 h, and absorbance was measured at 450 nm (n = 4 replicates). Apoptosis: Cells (5×10 5 cells/mL) were harvested, washed with PBS, and submitted to the Flow Cytometry Platform at the School of Basic Medical Sciences, Chongqing Medical University for apoptosis analysis. Cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer’s protocol. Flow cytometry was performed on a BD FACSCanto II system, and data were analyzed using FlowJo v10.8.1. Apoptotic cells were defined as Annexin V + /PI - (early apoptosis) and Annexin V + /PI + (late apoptosis). 1.6 qPCR Analysis: Total RNA was extracted with TRIzol (Thermo Fisher) and reverse-transcribed using a HiScript III RT SuperMix (Vazyme). qPCR was performed on a QuantStudio 5 system (Applied Biosystems) with SYBR Green (Takara). Primer sequences are provided in Table 1. Relative mRNA levels were calculated using the 2^-ΔΔCt method. 1.7 Western Blotting: Cells were lysed in RIPA buffer containing protease inhibitors. Proteins (20μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution), followed by HRP-conjugated secondary antibodies (1:5000). Signals were detected using ECL (Bio-Rad) and quantified by ImageLab. 1.8 Statistical Analysis: Data from three independent experiments are expressed as mean ± SD. Statistical significance (p < 0.05) was determined by one-way ANOVA followed by Tukey’s test (GraphPad Prism 8). Results 2.1 The Relationship between Different Concentrations of DEX and Osteoblast Cell Viability The impact of varying concentrations of dexamethasone (DEX) on osteoblast cell viability was evaluated through the application of the CCK-8 assay. Notably, a marked reduction in cell viability (p < 0.01) was observed subsequent to the administration of 0.5 mmol/L DEX. As the concentration of DEX was elevated to 2 mmol/L, a progressive decline in cellular viability was noted, culminating in a viability rate that was approximately half that of the control group（Figure 1）. Consequently, a DEX concentration of 2 mmol/L was determined to be the optimal level for triggering osteoblast apoptosis in subsequent experiments. 2.2 LiCl Mitigates DEX-Induced Cytotoxicity and Apoptosis Cell Viability: DEX treatment reduced cell viability to 52.3±3.1% of the control (p < 0.01, CCK-8 assay), while LiCl co-treatment restored viability to 78.5±4.2% (p < 0.01 vs. DEX group) (Figure 2A). Apoptosis Rate: Flow cytometry analysis revealed that DEX significantly increased the total apoptotic cell population (Annexin V + ) from 4.8±0.5% (Control) to 34.7±2.9% (p < 0.001). LiCl co-treatment reduced apoptosis to 14.2±1.7% (p < 0.01 vs. DEX group) (Figure 2B). 2.3 Adenovirus Transfection and Expression of the Klotho Gene and Protein In accordance with findings from previous studies, a viral solution carrying the KL gene and GFP gene was introduced at an MOI value of 100 to the respective experimental groups. Following a 12-hour transfection period, the culture medium was replaced with a standard culture medium. Minimal fluorescence expression was observed in both the AD-KL and AD-GFP groups 24 hours post-transfection. Remarkably, after 72 hours of transfection, the transfection efficiency surpassed 90% in both groups, accompanied by sustained cell viability (Figure 3). Subsequent qPCR and Western blot analyses unveiled a substantial elevation in Klotho mRNA and protein expression in the AD-KL group (P < 0.01) (Figure 4). 2.4 Expression of Wnt5a, Wnt3a, and β-Catenin mRNA and Protein in Each Cellular Group Following the application of specific interventions to the cells in each research group, total cellular RNA and protein were extracted to assess the expression of the Wnt signaling pathway. The experimental findings indicated that in the DEX group, the levels of Wnt5a mRNA and protein significantly increased compared to the Control group (P < 0.01), while the levels of Wnt3a, β-catenin mRNA, and protein markedly decreased (P < 0.05). In contrast to the Dex group, the AD-KL+DEX group exhibited a significant reduction in the levels of Wnt5a mRNA and protein (P < 0.01), coupled with a notable increase in Wnt3a levels (P < 0.01) (Figure 5). Discussion Glucocorticoid drugs exhibit commendable therapeutic efficacy in immune disorders and inflammatory diseases. However, their prolonged and high-dose usage raises concerns about potential impacts on human health. Among the complications arising from glucocorticoid drug use, glucocorticoid-induced osteoporosis (GIO) stands out as a prevalent issue, ranking second only to senile osteoporosis in terms of prevalence. Research has underscored that the osteoblasts, which possess glucocorticoid receptors, are a critical target for the impact of glucocorticoids on bone structure 16 . In healthy adults, normal glucocorticoid levels positively stimulate osteoblast function. Conversely, elevated glucocorticoid levels may impede osteoblast differentiation and proliferation while concurrently activating osteoclast function 30 . A noteworthy histomorphological alteration in GIO is the direct induction of osteoblast cytotoxicity by glucocorticoids. In the intricate process of bone metabolism, osteoclasts primarily engage in the resorption of old bone, while osteoblasts generate an equivalent amount of new bone. Their collaborative efforts ensure the completion of bone remodeling. Consequently, a reduction in osteoblasts may lead to diminished bone formation. At the same time, an increase in osteoclasts can result in elevated old bone mass, reduced bone density, and ultimately compromised bone stability, substantially heightening the risk of fractures 30 . Comprehending these complexities is essential for grasping the multifaceted effects of glucocorticoids on bone health and for developing effective preventive strategies against glucocorticoid-induced osteoporosis (GIO). The Wnt signaling pathway delicately orchestrates the differentiation, maturation, and apoptosis of osteoblasts. Notably, glucocorticoids have been identified as promoters of osteoblast apoptosis and inhibitors of osteoblast synthesis—processes tightly linked to the Wnt signaling pathway 31 . Osteoblasts emerge as pivotal target cells in the synthesis and metabolism of the classical Wnt/β-catenin signaling pathway within bone. Treatment of osteoblasts with dexamethasone results in a significant increase in the expression of Wnt pathway inhibitors, including DKK-1 and sclerosteosis protein (SOST) 14 . Within the Wnt signaling pathway, the ligands Wnt3a and Wnt5a assume critical roles as regulators of osteoblast function. Wnt3a has been found to induce phosphorylation of LRP6 to activate the mTORC1/β-catenin axis, thus promoting osteoblast differentiation 32 . Moreover, the reduction of the ratio of Wnt3a to Wnt inhibitors, secreted frizzled-related protein 1 (sFRP-1) and Wnt inhibitory factor 1 (Wif-1), suppresses Wnt signaling, which may result in impaired bone formation 9 .Wnt5a, a well-studied ligand in the non-canonical Wnt pathway, activates non-canonical Wnt signal transduction through receptor tyrosine kinase-like orphan receptor protein (ROR) 33 . Research on Wnt5a-knockout mature osteoclasts indicates that deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation, highlighting Wnt5a's role in normal bone remodeling through the classical Wnt signaling pathway 34 . Lithium chloride (LiCl), a compound known to promote the Wnt/β-catenin pathway by inhibiting GSK-3β activity, was employed in this experiment. The inhibition allows β-catenin to accumulate inside cells, entering the nucleus and activating downstream Wnt/β-catenin signaling. The successful establishment of an osteoblast apoptosis model using a 2mmol/L concentration of dexamethasone revealed that the addition of LiCl, a Wnt pathway activator, significantly increased cell survival and reduced apoptosis compared to the DEX group. This suggests that activation of the classical Wnt pathway can counteract glucocorticoid-induced cytotoxicity in osteoblasts. Thus, it implies that DEX-induced pathological processes in osteoporosis are, in part, mediated through the Wnt pathway. This finding underscores the potential therapeutic relevance of modulating the Wnt signaling pathway to mitigate the adverse effects of glucocorticoids on osteoblasts and bone health. Klotho, recognized as an aging-related protein, impacts the bone mineralization process by reducing the number and function of osteoblasts 23 . In vivo experiments have further demonstrated that the upregulation of endogenous Klotho inhibits classical Wnt pathway transduction 27 . Carrillo et al. have elucidated that, under the influence of Klotho protein, FGF23 induces the generation of DKK1, thereby inhibiting the Wnt/β-catenin pathway. This inhibition subsequently affects osteoblast differentiation and mineralization 35 . Hence, the interaction between FGF23-induced DKK1 expression and Klotho suggests that Klotho may indirectly influence Wnt signal transduction and osteoblast function. This intricate interplay highlights the multifaceted role of Klotho in bone homeostasis and emphasizes its potential as a target for interventions aimed at modulating bone health and aging-related processes. In our prior investigations, we achieved successful transfection of MC3T3-E1 cells with recombinant adenovirus carrying the KL gene. Subsequently, we observed that Klotho effectively mitigated cytotoxicity in MC3T3-E1 cells induced by dexamethasone (DEX) by inhibiting the NF-κB signal 25 . Building upon this groundwork, we sought to delve deeper into the intricate mechanisms underlying Klotho's impact on glucocorticoid-induced osteoblast cytotoxicity. Experimental cells were transfected with recombinant adenovirus carrying the KL gene at an MOI value of 100. After 72 hours of transfection, robust fluorescence was evident under a fluorescence microscope. In the Klotho group, both KL mRNA and protein expression witnessed a substantial increase, confirming the successful construction of MC3T3-E1 osteoblasts with heightened KL gene expression levels. The observed downregulation of Wnt3a and β-catenin under DEX treatment (Figure 5) aligns with prior evidence that glucocorticoids suppress canonical Wnt signaling, while non-canonical Wnt5a signaling is enhanced 11 . This imbalance likely contributes to impaired osteoblast survival and differentiation. This suggests that DEX ay influence the Wnt pathway by modulating the expression of both classical and non-canonical Wnt pathway ligands, thereby contributing to the induction of osteoblast cytotoxicity. Interestingly, even in MC3T3-E1 osteoblasts that overexpress Klotho and are exposed to the identical concentration of DEX, we noted a consistent decrease in the expression of Wnt3a and β-catenin at both the mRNA and protein levels, coupled with a persistent elevation in Wnt5a mRNA and protein, as opposed to the Control group. However, compared to the DEX group, a reversal in this pattern was evident. This indicated that Klotho overexpression reversed DEX-induced Wnt5a upregulation and restored Wnt3a/β-catenin levels, suggesting its dual capacity to suppress non-canonical pro-apoptotic signals while reactivating canonical pro-survival pathways. This rebalancing mechanism may underlie Klotho’s protective effects on osteoblastogenesis.In contrast, these findings suggest the involvement of the Wnt signaling pathway in Klotho-mediated improvement of glucocorticoid-induced osteoblast apoptosis, the potential interplay between the classical and non-canonical Wnt signaling pathways, as well as their collective impact on the observed processes, warrants further investigation. The identified insights open new avenues for understanding and exploring the use of Klotho in the prevention and treatment of glucocorticoid-induced osteoporosis in the future. Declarations The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. The authors have no relevant financial or non-financial interests to disclose. All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Sen Wang, Miao He, Xiao Liang and Baoshan Li . The first draft of the manusc ript was written by Sen Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. The MC3T3-E1 cells used in this study were commercially purchased and are commonly utilized in basic research settings. Ethical approval was not required for the use of these cells in our experiments, as they are de-identified and sourced from commercial vendors, thereby not involving direct identifiable human material or requiring informed consent. The research was conducted in accordance with internationally recognized ethical guidelines for the use of cell lines in scientific research. This study did not involve any human samples, animal experiments, or biological materials requiring ethical review. The MC3T3-E1 osteoblast cell line used in this study was commercially purchased from Shanghai ZhongqiaoXinzhou Company, a widely recognised bone biology cell line. According to the ethical guidelines of international scientific research and our institution's policies, using such commercialized, established cell lines for in vitro research does not require additional ethical review or approval. Therefore, this study has adhered to all applicable ethical standards. Author Contribution All authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Sen Wang, Miao He, Xiao Liang, and Baoshan Li. The first draft of the manuscript was written by Sen Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. References Nusse R, Varmus HE (1982) Many Tumors Induced by the Mouse Mammary Tumor Virus Contain a Provirus Integrated in the Same Region of the Host Genome. Cell 31(1):99–109. https://doi.org/10.1016/0092-8674(82)90409-3 Zhang L, Adu IK, Zhang H, Wang J (2023) The WNT/β-Catenin System in Chronic Kidney Disease-Mineral Bone Disorder Syndrome. Int Urol Nephrol 55(10):2527–2538. https://doi.org/10.1007/s11255-023-03569-2 Zhang D-H, Shao J (2025) Research Progress of Basing on Wnt/β-Catenin Pathway in the Treatment of Bone Tissue Diseases. Tissue Eng Part B Rev. https://doi.org/10.1089/ten.teb.2024.0170 Duchartre Y, Kim Y-M, Kahn M (2016) The Wnt Signaling Pathway in Cancer. Crit Rev Oncol Hematol 99:141–149. https://doi.org/10.1016/j.critrevonc.2015.12.005 Schoeman MAE, Moester MJC, Oostlander AE, Kaijzel EL, Valstar ER, Nelissen RGHH, Löwik CWGM, de Rooij KE (2015) Inhibition of GSK3β Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation. J Cell Biochem 116(12):2938–2946. https://doi.org/10.1002/jcb.25241 Lojk J, Marc J (2021) Roles of Non-Canonical Wnt Signalling Pathways in Bone Biology. Int J Mol Sci 22(19):10840. https://doi.org/10.3390/ijms221910840 Komori T (2016) Glucocorticoid Signaling and Bone Biology. Horm Metab Res 48(11):755–763. https://doi.org/10.1055/s-0042-110571 Weinstein RS, Nicholas RW, Manolagas SC (2000) Apoptosis of Osteocytes in Glucocorticoid-Induced Osteonecrosis of the Hip. J Clin Endocrinol Metab 85(8):2907–2912. https://doi.org/10.1210/jcem.85.8.6714 The molecular etiology and treatment of glucocorticoid-induced osteoporosis - PubMed . https://pubmed.ncbi.nlm.nih.gov/34386357/ (accessed 2025-01-12) Ohlsson C, Nilsson KH, Henning P, Wu J, Gustafsson KL, Poutanen M, Lerner UH, Movérare-Skrtic S (2018) WNT16 Overexpression Partly Protects against Glucocorticoid-Induced Bone Loss. Am J Physiol Endocrinol Metab 314(6):E597–E604. https://doi.org/10.1152/ajpendo.00292.2017 Kawazoe M, Kaneko K, Nanki T (2021) Glucocorticoid Therapy Suppresses Wnt Signaling by Reducing the Ratio of Serum Wnt3a to Wnt Inhibitors, sFRP-1 and Wif-1. Clin Rheumatol 40(7):2947–2954. https://doi.org/10.1007/s10067-020-05554-x Elevated Expression of Dkk-1 by Glucocorticoid Treatment Impairs Bone Regenerative Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells - PubMed. https://pubmed.ncbi.nlm.nih.gov/29084466/ (accessed 2025-01-12). Mutation of the mouse klotho gene leads to a syndrome resembling ageing - PubMed . https://pubmed.ncbi.nlm.nih.gov/9363890/ (accessed 2025-01-12) Gao Y, Chen N, Fu Z, Zhang Q (2023) Progress of Wnt Signaling Pathway in Osteoporosis. Biomolecules 13(3):483. https://doi.org/10.3390/biom13030483 Koromila T, Baniwal SK, Song YS, Martin A, Xiong J, Frenkel B (2014) Glucocorticoids Antagonize RUNX2 during Osteoblast Differentiation in Cultures of ST2 Pluripotent Mesenchymal Cells. J Cell Biochem 115(1):27–33. https://doi.org/10.1002/jcb.24646 Gado M, Baschant U, Hofbauer LC, Henneicke H (2022) Bad to the Bone: The Effects of Therapeutic Glucocorticoids on Osteoblasts and Osteocytes. Front Endocrinol (Lausanne) 13:835720. https://doi.org/10.3389/fendo.2022.835720 Ajay AK, Zhu L-J, Zhao L, Liu Q, Ding Y, Chang Y-C, Shah SI, Hsiao L-L (2024) Local Vascular Klotho Mediates Diabetes-Induced Atherosclerosis via ERK1/2 and PI3-Kinase-Dependent Signaling Pathways. Atherosclerosis 396:118531. https://doi.org/10.1016/j.atherosclerosis.2024.118531 Decreased ADAM17 expression in the lungs of α-Klotho reduced mouse - PubMed . https://pubmed.ncbi.nlm.nih.gov/31951006/ (accessed 2025-01-12) Liu J, Wang H, Liu Q, Long S, Wu Y, Wang N, Lin W, Chen G, Lin M, Wen J (2024) Klotho Exerts Protection in Chronic Kidney Disease Associated with Regulating Inflammatory Response and Lipid Metabolism. Cell Biosci 14(1):46. https://doi.org/10.1186/s13578-024-01226-4 Xu J-P, Zeng R-X, He M-H, Lin S-S, Guo L-H, Zhang M-Z (2022) Associations Between Serum Soluble α-Klotho and the Prevalence of Specific Cardiovascular Disease. Front Cardiovasc Med 9:899307. https://doi.org/10.3389/fcvm.2022.899307 Roig-Soriano J, Edo Á, Verdés S, Martín-Alonso C, Sánchez-de-Diego C, Rodriguez-Estevez L, Serrano AL, Abraham CR, Bosch A, Ventura F, Jordan BA, Muñoz-Cánoves P, Chillón M (2025) Long-Term Effects of s-KL Treatment in Wild-Type Mice: Enhancing Longevity, Physical Well-Being, and Neurological Resilience. Mol Ther S1525–0016(25):00120–00120. https://doi.org/10.1016/j.ymthe.2025.02.030 Agoro R, Ni P, Noonan ML, White KE (2020) Osteocytic FGF23 and Its Kidney Function. Front Endocrinol (Lausanne) 11:592. https://doi.org/10.3389/fendo.2020.00592 Komaba H, Kaludjerovic J, Hu DZ, Nagano K, Amano K, Ide N, Sato T, Densmore MJ, Hanai J-I, Olauson H, Bellido T, Larsson TE, Baron R, Lanske B (2017) Klotho Expression in Osteocytes Regulates Bone Metabolism and Controls Bone Formation. Kidney Int 92(3):599–611. https://doi.org/10.1016/j.kint.2017.02.014 Fan Y, Cui C, Rosen CJ, Sato T, Xu R, Li P, Wei X, Bi R, Yuan Q, Zhou C (2022) Klotho in Osx+-Mesenchymal Progenitors Exerts pro-Osteogenic and Anti-Inflammatory Effects during Mandibular Alveolar Bone Formation and Repair. Signal Transduct Target Ther 7(1):155. https://doi.org/10.1038/s41392-022-00957-5 Liang X, Li B, Huang Q, Liu D, Ma H, Klotho Prevents (2018) DEX-Induced Apoptosis in MC3T3-E1 Osteoblasts through the NF-κB Signaling Pathway. Biochem Biophys Res Commun 507(1–4):355–361. https://doi.org/10.1016/j.bbrc.2018.11.040 Chen Y-X, Huang C, Duan Z-B, Xu C-Y, Chen Y (2019) Klotho/FGF23 Axis Mediates High Phosphate-Induced Vascular Calcification in Vascular Smooth Muscle Cells via Wnt7b/β-Catenin Pathway. Kaohsiung J Med Sci 35(7):393–400. https://doi.org/10.1002/kjm2.12072 Mohanty SK, Mohanty AK, Kumar MS, Suchiang K (2024) Triiodothyronine Enhances Various Forms of Kidney-Specific Klotho Protein and Suppresses the Wnt/β-Catenin Pathway: Insights from in-Vitro, in-Vivo and in-Silico Investigations. Cell Signal 120:111214. https://doi.org/10.1016/j.cellsig.2024.111214 Wu Q, Jiang L, Wu J, Dong H, Zhao Y (2021) Klotho Inhibits Proliferation in a RET Fusion Model of Papillary Thyroid Cancer by Regulating the Wnt/β-Catenin Pathway. Cancer Manag Res 13:4791–4802. https://doi.org/10.2147/CMAR.S295086 Loss of Klotho contributes to cartilage damage by derepression of canonical Wnt/β-catenin signaling in osteoarthritis mice - PubMed . https://pubmed.ncbi.nlm.nih.gov/31895692/ (accessed 2025-01-12) Li S, Jiang H, Gu X (2018) Echinacoside Suppresses Dexamethasone-Induced Growth Inhibition and Apoptosis in Osteoblastic MC3T3-E1 Cells. Exp Ther Med 16(2):643–648. https://doi.org/10.3892/etm.2018.6199 Wang T, Yu X, He C, Pro-Inflammatory, Cytokines (2019) Cellular and Molecular Drug Targets for Glucocorticoid-Induced-Osteoporosis via Osteocyte. Curr Drug Targets 20(1):1–15. https://doi.org/10.2174/1389450119666180405094046 Shen X, Feng S, Chen S, Gong B, Wang S, Wang H, Song D, Ni J (2024) Wnt3a-Induced LRP6 Phosphorylation Enhances Osteoblast Differentiation to Alleviate Osteoporosis through Activation of mTORC1/β-Catenin Signaling. Arch Biochem Biophys 761:110169. https://doi.org/10.1016/j.abb.2024.110169 Non-canonical WNT5A-ROR signaling: New perspectives on an ancient developmental pathway - PubMed . https://pubmed.ncbi.nlm.nih.gov/36967195/ (accessed 2025-01-17) Deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation - PubMed . https://pubmed.ncbi.nlm.nih.gov/31919867/ (accessed 2025-01-17) Carrillo-López N, Panizo S, Alonso-Montes C, Román-García P, Rodríguez I, Martínez-Salgado C, Dusso AS, Naves M, Cannata-Andía JB (2016) Direct Inhibition of Osteoblastic Wnt Pathway by Fibroblast Growth Factor 23 Contributes to Bone Loss in Chronic Kidney Disease. Kidney Int 90(1):77–89. https://doi.org/10.1016/j.kint.2016.01.024 Tables Table 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Table1.docx Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 25 Jul, 2025 Reviews received at journal 23 Jul, 2025 Reviewers agreed at journal 05 Jul, 2025 Reviews received at journal 18 Jun, 2025 Reviewers agreed at journal 04 Jun, 2025 Reviews received at journal 09 Apr, 2025 Reviewers agreed at journal 09 Apr, 2025 Reviewers invited by journal 09 Apr, 2025 Submission checks completed at journal 02 Apr, 2025 First submitted to journal 20 Mar, 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. 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. 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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-5854318","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":447559791,"identity":"e54957f8-7003-4090-b41d-9e351c1357aa","order_by":0,"name":"Sen Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1ElEQVRIiWNgGAWjYBADHn75wwcfkKRFTnIGW7IBSVqMDWbwmEkQpdTg+NnDL39U3EncIN1gVvGmjEGeX+wAAS1n8tKsec48S9wucyDt5pxzDIYzZyfg12J2IMfMmLHtcOLOhoRjt3nbGBIMbhPScv6NmeHPf4cTNxxIbCsmTsuNHOMHvA2HjQ1uJLMxE6XF/sYbM2aeY4flJHuOMUvOOSdB2C+S/TnGH3/UHObhZ+//+OFNmY08vzQBLUDAhogOHjbioob5A5IWonSMglEwCkbBCAMAym5I4EA/xjAAAAAASUVORK5CYII=","orcid":"","institution":"Capital Medical University \u0026 Nanchong Central Hospital","correspondingAuthor":true,"prefix":"","firstName":"Sen","middleName":"","lastName":"Wang","suffix":""},{"id":447559792,"identity":"fe5ab42c-a310-451b-91a5-34c45858a6ba","order_by":1,"name":"Miao He","email":"","orcid":"","institution":"Affiliated Hospital of North Sichuan Medical College","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"He","suffix":""},{"id":447559793,"identity":"ba17d5f4-1092-44bb-b2fa-7b564cf160f0","order_by":2,"name":"Xiao Liang","email":"","orcid":"","institution":"University-Town Hospital of Chongqing Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiao","middleName":"","lastName":"Liang","suffix":""},{"id":447559794,"identity":"8a0dfa06-9400-492b-919e-ae13a6ba185b","order_by":3,"name":"Baoshan Li","email":"","orcid":"","institution":"Chongqing University Central Hospital, Chongqing Emergency Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Baoshan","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-01-18 09:38:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5854318/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5854318/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":81584298,"identity":"3dfed03e-6402-411c-95c4-dc7f3f4e5080","added_by":"auto","created_at":"2025-04-28 20:13:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":241695,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability of MC3T3-E1 osteoblasts treated with DEX (0–4 mM) for 24 h, measured by CCK-8 assay. ****p \u0026lt; 0.0001 (0 vs. 0.5, 0 vs. 2, 0 vs. 4 mM), **p \u0026lt; 0.01 (0.5 vs. 2 mM), ns: Not significant (p\u0026gt;0.05; 0.5 vs. 1 mM). (one-way ANOVA with Tukey’s test). Data presented as mean ± SD(n=4).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/38df82f36a97f48e8ca34f78.png"},{"id":81584480,"identity":"8f55debb-6222-429b-9673-2eb977141d15","added_by":"auto","created_at":"2025-04-28 20:21:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3922813,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of dexamethasone (DEX) and LiCl on MC3T3-E1 cell viability and apoptosis. A: Cell viability (CCK-8 assay); B: Apoptosis rate (Flow cytometry); C-F: Representative flow cytometry plots: (C) Compare group; (D) DEX group; (E)DEX+LiCl group; (F) LiCl group. p-values: **p \u0026lt; 0.01 vs. Control; ##p \u0026lt; 0.01 vs. DEX (one-way ANOVA with Tukey’s test). Data presented as mean ± SD (n=4).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/1472521aaad28215ffcb147f.png"},{"id":81584303,"identity":"69abb193-7e40-4480-904c-361948c41fc8","added_by":"auto","created_at":"2025-04-28 20:13:36","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6111702,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTransfection of MC3T3-E1 cells under fluorescence microscope\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/69c3cf94d949a3861858e8b6.png"},{"id":81584300,"identity":"44295597-3467-4f4d-940c-1ace5d12a24a","added_by":"auto","created_at":"2025-04-28 20:13:36","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1615207,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression level of mRNA and protein for Klotho was detected by qPCR and Western blot. ** P\u0026lt;0.01, vs control group (one-way ANOVA with Tukey’s test). Data presented as mean ± SD (n=4).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/f00925b0e8d0ef7dccfaf924.png"},{"id":81584481,"identity":"77e744a2-1f77-493c-902f-db54df7200db","added_by":"auto","created_at":"2025-04-28 20:21:36","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1440384,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative expression level of mRNA and protein of Wnt5a, Wnt3a, and β-catenin were detected by qPCR and Western blot. A: The relative expression level of mRNA and protein of Wnt5a; B: The relative expression level of mRNA and protein of Wnt3a; C: The relative expression level of mRNA and protein of β-catenin; D: Protein expression of each group. **P\u0026lt;0.01, vs control group; * P\u0026lt;0.05, vs control group; ## P\u0026lt;0.01, vs DEX group; # P\u0026lt;0.05, vs DEX group (one-way ANOVA with Tukey’s test). Data presented as mean ± SD (n=4).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/16221571ba99b0577af2a76f.png"},{"id":81585873,"identity":"57efce62-16b9-4cd7-8fe5-82da2c5270c8","added_by":"auto","created_at":"2025-04-28 20:45:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":13162232,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/3cbfd66b-b390-4957-9a25-b959d659b9fc.pdf"},{"id":81585181,"identity":"47ae1946-b68a-48a9-bc33-ae32c3f805be","added_by":"auto","created_at":"2025-04-28 20:37:36","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":17027,"visible":true,"origin":"","legend":"","description":"","filename":"Table1.docx","url":"https://assets-eu.researchsquare.com/files/rs-5854318/v1/411fa144ec1c338941615194.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Klotho attenuates glucocorticoid-induced osteoblast cytotoxicity via Wnt signaling pathway modulation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eFirst identified by Nusse in 1982, Wnt genes have since emerged as central regulators within the Wnt signaling pathway, governing pivotal cellular processes such as cell cycle progression and tumor differentiation\u003csup\u003e1\u003c/sup\u003e. This pathway, extending its influence beyond embryogenesis, organ formation, and tumorigenesis, also critically modulates bone formation and osteoblast activity\u003csup\u003e2,3\u003c/sup\u003e. The Wnt pathway can be categorized into the classic Wnt/β-catenin pathway and the non-classical Wnt signaling pathway, both of which necessitate the presence of Frizzled (FZD), a transmembrane protein\u003csup\u003e4\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWithin the canonical pathway, the Wnt3a ligand's interaction with FZD impedes the action of Glycogen Synthase Kinase 3 (GSK3), enabling the accumulation and nuclear translocation of β-catenin. This cascade culminates in the transcriptional activation of osteogenic genes, steering osteoblast differentiation\u003csup\u003e5\u003c/sup\u003e. Conversely, in the absence of Wnt ligands, GSK3 phosphorylates β-catenin, thwarting its nuclear translocation and thereby influencing osteoblast differentiation and bone maturation\u003csup\u003e6\u003c/sup\u003e. A salient feature is that bone formation is amplified upon activation of the canonical Wnt/β-catenin pathway and diminished when inhibited. Parallelly, the non-canonical pathway, with ligands such as Wnt4, Wnt5a, and Wnt16, orchestrates bone homeostasis through an intricate interplay with a broader cellular ensemble and multifaceted mechanisms\u003csup\u003e6\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eChronic glucocorticoid exposure promotes osteoblast\u0026nbsp;cytotoxicity\u0026nbsp;and disrupts bone remodeling, as demonstrated in both preclinical and clinical studies\u003csup\u003e7,8\u003c/sup\u003e.\u0026nbsp;Research indicates glucocorticoids (GC) can inhibit bone formation by suppressing Wnt pathway-related molecules\u003csup\u003e9\u003c/sup\u003e. During the early stages of GC treatment, it may hinder the co-receptors of the Wnt signal, whereas, in the later stages, they might inhibit the ligands themselves\u003csup\u003e10,11\u003c/sup\u003e. Prolonged and excessive use of GC therapy is also associated with the upregulation of Dickkopf-related protein 1 (DKK1), an inhibitor of the Wnt pathway, suggesting its potential involvement in GC-induced osteoporosis\u003csup\u003e12,13\u003c/sup\u003e. Recent evidence further suggests that GCs disrupt the balance between canonical and non-canonical Wnt signaling: dexamethasone (DEX) downregulates Wnt3a and\u0026nbsp;β-catenin while elevating Wnt5a, thereby shifting osteoblasts toward apoptosis\u003csup\u003e11,14\u003c/sup\u003e.\u0026nbsp;Glucocorticoids can also suppress osteoblastogenesis by downregulating critical transcription factors such as Runx2 and Osterix.\u003csup\u003e15\u003c/sup\u003e These alterations correlate with reduced bone formation and increased fracture risk in glucocorticoid-induced osteoporosis (GIO)\u0026nbsp;\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eKlotho (KL), originally identified as an anti-aging protein, demonstrates significant associations with various health conditions in mice lacking the KL gene, including atherosclerosis, glucose metabolism abnormalities, and emphysema\u003csup\u003e17,18\u003c/sup\u003e. Subsequent research has further unveiled its close ties to conditions such as diabetes, lipid metabolism disorders, renal damage, coronary heart disease, hypertension, and brain injury\u003csup\u003e19,20\u003c/sup\u003e. Importantly, the expression of the KL gene also plays a role in the development of osteoporosis. Studies have indicated that KL can mitigate microstructural damage in bone, although the underlying mechanisms remain partially understood\u003csup\u003e21\u003c/sup\u003e. Additionally, KL gene expression in osteoblasts can inhibit osteoblast apoptosis and regulate bone metabolism\u003csup\u003e22\u003c/sup\u003e. For instance, Komaba et al demonstrated that Klotho deficiency in osteocytes disrupts bone mineralization, leading to reduced bone mass and increased fragility\u003csup\u003e23\u003c/sup\u003e. These findings underscore KL's multifaceted role in maintaining skeletal integrity.\u003c/p\u003e\n\u003cp\u003eDisruptions in KL gene expression can significantly influence osteoblast and osteoclast differentiation, leading to a decrease in bone mass\u003csup\u003e23,24\u003c/sup\u003e. Our prior investigations have demonstrated Klotho's ability to inhibit glucocorticoid-induced apoptosis in MC3T3-E1 osteoblasts by reducing levels of the anti-apoptotic protein B-cell lymphoma-2 (BCL-2) and suppressing the expression of pro-apoptotic proteins, such as BCL2-associated X protein (Bax) and Nuclear Factor-Kappa B (NF-kB)\u003csup\u003e25\u003c/sup\u003e. Recent studies have revealed that the extracellular domain of the Klotho protein can act on various Wnt ligands, thus diminishing their capacity to activate the Wnt signaling pathway\u003csup\u003e26–28\u003c/sup\u003e. Consequently, Klotho may modulate the normal transmission of the Wnt signaling pathway through its interaction with Wnt ligands\u003csup\u003e29\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eBased on these findings, we hypothesize that KL gene expression plays a pivotal role in the development of glucocorticoid-induced osteoporosis (GIO) through its association with the Wnt signaling pathway. To investigate this hypothesis, we have established a dexamethasone-induced osteoblast apoptosis model to elucidate further the impacts of the Wnt pathway and KL gene expression on glucocorticoid-induced osteoblast cytotoxicity.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eThe reagents and chemicals used in this study are as follows: MC3T3-E1 cells (Shanghai ZhongqiaoXinzhou); Dexamethasone (DEX) (Shanghai Yeyuan Biotechnology); Recombinant adenovirus (AD-GFP and AD-KL; Shanghai Jikai Company); qPCR reagents (Beijing Qingen Biological); Cell Counting Kit-8 (CCK-8; Japan Tohjin Research Institute); High-glucose DMEM, 0.25% trypsin (Wuhan Cellbiological Technology); Fetal bovine serum (FBS; PAN Biotech); Penicillin-streptomycin solution (Biosharp); Antibodies: Anti-Klotho (Zhengneng Bio), Anti-Wnt5a, Anti-Wnt3a, Anti-β-catenin (Wanlei Biology), Anti-β-actin (Sanying Bio).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.1 Cell culture\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMC3T3-E1 cells were cultured in a humidified incubator at 37°C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The culture medium consisted of 89% high-glucose DMEM, 10% FBS, and 1% penicillin-streptomycin. Cells were passaged at a 1:3 ratio upon reaching 80–90% confluence.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.2\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExperimental Design and Treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were divided into seven groups:\u003c/p\u003e\n\u003cp\u003ea. Control: Untreated cells.\u003c/p\u003e\n\u003cp\u003eb. Ad-GFP: Cells transfected with GFP adenovirus (MOI = 100) for 12 h, then cultured in standard medium.\u003c/p\u003e\n\u003cp\u003ec. Ad-KL + DEX: Ad-KL-transfected cells (MOI = 100, 12 h) treated with 2 mM DEX for 24 h.\u003c/p\u003e\n\u003cp\u003ed. DEX: Cells treated with 2 mM DEX for 24 h.\u003c/p\u003e\n\u003cp\u003ee. Ad-KL: Ad-KL-transfected cells (MOI = 100, 12 h).\u003c/p\u003e\n\u003cp\u003eLiCl: Cells treated with 5 mM LiCl for 24 h.\u003c/p\u003e\n\u003cp\u003ef. DEX + LiCl: Co-treated with 2 mM DEX and 5 mM LiCl for 24 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.3 Optimization of DEX Concentration for Apoptosis Induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded in 96-well plates (10⁵\u0026nbsp;cells/mL) and allowed to adhere for 24 h. Subsequently, they were treated with DEX (0, 0.5, 1, 2, or 4 mM) for 24 h. Cell viability was quantified using the CCK-8 assay (n = 4 replicates per concentration).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.4 Adenoviral Transfection\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were transfected with Ad-GFP or Ad-KL (MOI = 100) for 12 h. After replacing the medium, transfection efficiency was assessed via fluorescence microscopy at 24, 48, and 72 h. Klotho mRNA and protein levels were analyzed by qPCR and Western blot.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.5 Cell Viability and Apoptosis Assessment:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eViability: Cells were seeded in 96-well plates (1×10\u003csup\u003e5\u003c/sup\u003e cells/mL) and treated as per experimental groups. After 24 h, 10μL CCK-8 reagent (Japan Tohjin Research Institute) was added to each well, incubated for 2 h, and absorbance was measured at 450 nm (n = 4 replicates).\u003c/p\u003e\n\u003cp\u003eApoptosis: Cells (5×10\u003csup\u003e5\u003c/sup\u003e cells/mL) were harvested, washed with PBS, and submitted to the Flow Cytometry Platform at the School of Basic Medical Sciences, Chongqing Medical University for apoptosis analysis. Cells were stained with Annexin V-FITC/PI Apoptosis Detection Kit according to the manufacturer’s protocol. Flow cytometry was performed on a BD FACSCanto II system, and data were analyzed using FlowJo v10.8.1. Apoptotic cells were defined as Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e-\u003c/sup\u003e (early apoptosis) and Annexin V\u003csup\u003e+\u003c/sup\u003e/PI\u003csup\u003e+\u003c/sup\u003e (late apoptosis).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.6 qPCR Analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted with TRIzol (Thermo Fisher) and reverse-transcribed using a HiScript III RT SuperMix (Vazyme). qPCR was performed on a QuantStudio 5 system (Applied Biosystems) with SYBR Green (Takara). Primer sequences are provided in Table 1. Relative mRNA levels were calculated using the 2^-ΔΔCt method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.7 Western Blotting:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were lysed in RIPA buffer containing protease inhibitors. Proteins (20μg/lane) were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% skim milk, membranes were incubated overnight at 4°C with primary antibodies (1:1000 dilution), followed by HRP-conjugated secondary antibodies (1:5000). Signals were detected using ECL (Bio-Rad) and quantified by ImageLab.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1.8 Statistical Analysis:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData from three independent experiments are expressed as mean ± SD. Statistical significance (p \u0026lt; 0.05) was determined by one-way ANOVA followed by Tukey’s test (GraphPad Prism 8).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e2.1 The Relationship between Different Concentrations of DEX and Osteoblast Cell Viability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe impact of varying concentrations of dexamethasone (DEX) on osteoblast cell viability was evaluated through the application of the CCK-8 assay. Notably, a marked reduction in cell viability (p \u0026lt; 0.01) was observed subsequent to the administration of 0.5 mmol/L DEX. As the concentration of DEX was elevated to 2 mmol/L, a progressive decline in cellular viability was noted, culminating in a viability rate that was approximately half that of the control group（Figure 1）. Consequently, a DEX concentration of 2 mmol/L was determined to be the optimal level for triggering osteoblast apoptosis in subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 LiCl Mitigates DEX-Induced Cytotoxicity and Apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCell Viability: DEX treatment reduced cell viability to 52.3\u0026plusmn;3.1% of the control (p \u0026lt; 0.01, CCK-8 assay), while LiCl co-treatment restored viability to 78.5\u0026plusmn;4.2% (p \u0026lt; 0.01 vs. DEX group) (Figure 2A). Apoptosis Rate: Flow cytometry analysis revealed that DEX significantly increased the total apoptotic cell population (Annexin V\u003csup\u003e+\u003c/sup\u003e) from 4.8\u0026plusmn;0.5% (Control) to 34.7\u0026plusmn;2.9% (p \u0026lt; 0.001). LiCl co-treatment reduced apoptosis to 14.2\u0026plusmn;1.7% (p \u0026lt; 0.01 vs. DEX group) (Figure 2B).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Adenovirus Transfection and Expression of the Klotho Gene and Protein\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with findings from previous studies, a viral solution carrying the KL gene and GFP gene was introduced at an MOI value of 100 to the respective experimental groups. Following a 12-hour transfection period, the culture medium was replaced with a standard culture medium. Minimal fluorescence expression was observed in both the AD-KL and AD-GFP groups 24 hours post-transfection. Remarkably, after 72 hours of transfection, the transfection efficiency surpassed 90% in both groups, accompanied by sustained cell viability (Figure 3). Subsequent qPCR and Western blot analyses unveiled a substantial elevation in Klotho mRNA and protein expression in the AD-KL group (P \u0026lt; 0.01) (Figure 4).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Expression of Wnt5a, Wnt3a, and \u0026beta;-Catenin mRNA and Protein in Each Cellular Group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFollowing the application of specific interventions to the cells in each research group, total cellular RNA and protein were extracted to assess the expression of the Wnt signaling pathway. The experimental findings indicated that in the DEX group, the levels of Wnt5a mRNA and protein significantly increased compared to the Control group (P \u0026lt; 0.01), while the levels of Wnt3a, \u0026beta;-catenin mRNA, and protein markedly decreased (P \u0026lt; 0.05). In contrast to the Dex group, the AD-KL+DEX group exhibited a significant reduction in the levels of Wnt5a mRNA and protein (P \u0026lt; 0.01), coupled with a notable increase in Wnt3a levels (P \u0026lt; 0.01) (Figure 5).\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eGlucocorticoid drugs exhibit commendable therapeutic efficacy in immune disorders and inflammatory diseases. However, their prolonged and high-dose usage raises concerns about potential impacts on human health. Among the complications arising from glucocorticoid drug use, glucocorticoid-induced osteoporosis (GIO) stands out as a prevalent issue, ranking second only to senile osteoporosis in terms of prevalence. Research has underscored that the osteoblasts, which possess glucocorticoid receptors, are a critical target for the impact of glucocorticoids on bone structure\u003csup\u003e16\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eIn healthy adults, normal glucocorticoid levels positively stimulate osteoblast function. Conversely, elevated glucocorticoid levels may impede osteoblast differentiation and proliferation while concurrently activating osteoclast function\u003csup\u003e30\u003c/sup\u003e. A noteworthy histomorphological alteration in GIO is the direct induction of osteoblast cytotoxicity by glucocorticoids. In the intricate process of bone metabolism, osteoclasts primarily engage in the resorption of old bone, while osteoblasts generate an equivalent amount of new bone. Their collaborative efforts ensure the completion of bone remodeling.\u0026nbsp;Consequently, a reduction in osteoblasts may lead to diminished bone formation. At the same time, an increase in osteoclasts can result in elevated old bone mass, reduced bone density, and ultimately compromised bone stability, substantially heightening the risk of fractures\u003csup\u003e30\u003c/sup\u003e. Comprehending these complexities is essential for grasping the multifaceted effects of glucocorticoids on bone health and for developing effective preventive strategies against glucocorticoid-induced osteoporosis (GIO).\u003c/p\u003e\n\u003cp\u003eThe Wnt signaling pathway delicately orchestrates the differentiation, maturation, and apoptosis of osteoblasts. Notably, glucocorticoids have been identified as promoters of osteoblast apoptosis and inhibitors of osteoblast synthesis—processes tightly linked to the Wnt signaling pathway\u003csup\u003e31\u003c/sup\u003e. Osteoblasts emerge as pivotal target cells in the synthesis and metabolism of the classical Wnt/β-catenin signaling pathway within bone. Treatment of osteoblasts with dexamethasone results in a significant increase in the expression of Wnt pathway inhibitors, including DKK-1 and sclerosteosis protein (SOST)\u003csup\u003e14\u003c/sup\u003e. Within the Wnt signaling pathway, the ligands Wnt3a and Wnt5a assume critical roles as regulators of osteoblast function. Wnt3a has been found to induce phosphorylation of LRP6 to activate the mTORC1/β-catenin axis, thus promoting osteoblast differentiation\u003csup\u003e32\u003c/sup\u003e. Moreover, the reduction of the ratio of Wnt3a to Wnt inhibitors, secreted frizzled-related protein 1 (sFRP-1) and Wnt inhibitory factor 1 (Wif-1), suppresses Wnt signaling, which may result in impaired bone formation\u003csup\u003e9\u003c/sup\u003e.Wnt5a, a well-studied ligand in the non-canonical Wnt pathway, activates non-canonical Wnt signal transduction through receptor tyrosine kinase-like orphan receptor protein (ROR)\u003csup\u003e33\u003c/sup\u003e. Research on Wnt5a-knockout mature osteoclasts indicates that deletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation, highlighting Wnt5a's role in normal bone remodeling through the classical Wnt signaling pathway\u003csup\u003e34\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eLithium chloride (LiCl), a compound known to promote the Wnt/β-catenin pathway by inhibiting GSK-3β activity, was employed in this experiment. The inhibition allows β-catenin to accumulate inside cells, entering the nucleus and activating downstream Wnt/β-catenin signaling. The successful establishment of an osteoblast apoptosis model using a 2mmol/L concentration of dexamethasone revealed that the addition of LiCl, a Wnt pathway activator, significantly increased cell survival and reduced apoptosis compared to the DEX group. This suggests that activation of the classical Wnt pathway can counteract glucocorticoid-induced cytotoxicity in osteoblasts. Thus, it implies that DEX-induced pathological processes in osteoporosis are, in part, mediated through the Wnt pathway. This finding underscores the potential therapeutic relevance of modulating the Wnt signaling pathway to mitigate the adverse effects of glucocorticoids on osteoblasts and bone health.\u003c/p\u003e\n\u003cp\u003eKlotho, recognized as an aging-related protein, impacts the bone mineralization process by reducing the number and function of osteoblasts\u003csup\u003e23\u003c/sup\u003e. In vivo experiments have further demonstrated that the upregulation of endogenous Klotho inhibits classical Wnt pathway transduction\u003csup\u003e27\u003c/sup\u003e.\u0026nbsp;Carrillo et al. have elucidated that, under the influence of Klotho protein, FGF23 induces the generation of DKK1, thereby inhibiting the Wnt/β-catenin pathway. This inhibition subsequently affects osteoblast differentiation and mineralization\u003csup\u003e35\u003c/sup\u003e. Hence, the interaction between FGF23-induced DKK1 expression and Klotho suggests that Klotho may indirectly influence Wnt signal transduction and osteoblast function. This intricate interplay highlights the multifaceted role of Klotho in bone homeostasis and emphasizes its potential as a target for interventions aimed at modulating bone health and aging-related processes.\u003c/p\u003e\n\u003cp\u003eIn our prior investigations, we achieved successful transfection of MC3T3-E1 cells with recombinant adenovirus carrying the KL gene. Subsequently, we observed that Klotho effectively mitigated cytotoxicity in MC3T3-E1 cells induced by dexamethasone (DEX) by inhibiting the NF-κB signal\u003csup\u003e25\u003c/sup\u003e. Building upon this groundwork, we sought to delve deeper into the intricate mechanisms underlying Klotho's impact on glucocorticoid-induced osteoblast cytotoxicity. Experimental cells were transfected with recombinant adenovirus carrying the KL gene at an MOI value of 100. After 72 hours of transfection, robust fluorescence was evident under a fluorescence microscope. In the Klotho group, both KL mRNA and protein expression witnessed a substantial increase, confirming the successful construction of MC3T3-E1 osteoblasts with heightened KL gene expression levels. The observed downregulation of Wnt3a and\u0026nbsp;β-catenin under DEX treatment (Figure 5) aligns with prior evidence that glucocorticoids suppress canonical Wnt signaling, while non-canonical Wnt5a signaling is enhanced\u003csup\u003e11\u003c/sup\u003e. This imbalance likely contributes to impaired osteoblast survival and differentiation.\u003c/p\u003e\n\u003cp\u003eThis suggests that DEX ay influence the Wnt pathway by modulating the expression of both classical and non-canonical Wnt pathway ligands, thereby contributing to the induction of osteoblast cytotoxicity. Interestingly, even in MC3T3-E1 osteoblasts that overexpress Klotho and are exposed to the identical concentration of DEX, we noted a consistent decrease in the expression of Wnt3a and β-catenin at both the mRNA and protein levels, coupled with a persistent elevation in Wnt5a mRNA and protein, as opposed to the Control group.\u003c/p\u003e\n\u003cp\u003eHowever, compared to the DEX group, a reversal in this pattern was evident. This indicated that Klotho overexpression reversed DEX-induced Wnt5a upregulation and restored Wnt3a/β-catenin levels, suggesting its dual capacity to suppress non-canonical pro-apoptotic signals while reactivating canonical pro-survival pathways. This rebalancing mechanism may underlie Klotho’s protective effects on osteoblastogenesis.In contrast, these findings suggest the involvement of the Wnt signaling pathway in Klotho-mediated improvement of glucocorticoid-induced osteoblast apoptosis, the potential interplay between the classical and non-canonical Wnt signaling pathways, as well as their collective impact on the observed processes, warrants further investigation. The identified insights open new avenues for understanding and exploring the use of Klotho in the prevention and treatment of glucocorticoid-induced osteoporosis in the future.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cem\u003eThe authors declare that no funds, grants, or other support were received during the preparation of this manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAll authors contributed to the study\u0026apos;s conception and design. Material preparation, data collection, and analysis were performed by Sen Wang, Miao He, Xiao Liang\u003c/em\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003eand Baoshan Li\u003c/em\u003e\u003cem\u003e.\u0026nbsp;\u003c/em\u003e\u003cem\u003eThe first draft of the manusc\u003c/em\u003e\u003cem\u003eript was written by\u0026nbsp;\u003c/em\u003e\u003cem\u003eSen Wang\u003c/em\u003e\u003cem\u003e\u0026nbsp;and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eThe MC3T3-E1 cells used in this study were commercially purchased and are commonly utilized in basic research settings. Ethical approval was not required for the use of these cells in our experiments, as they are de-identified and sourced from commercial vendors, thereby not involving direct identifiable human material or requiring informed consent. The research was conducted in accordance with internationally recognized ethical guidelines for the use of cell lines in scientific research.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eThis study did not involve any human samples, animal experiments, or biological materials requiring ethical review. The MC3T3-E1 osteoblast cell line used in this study was commercially purchased from Shanghai ZhongqiaoXinzhou Company, a widely recognised bone biology cell line. According to the ethical guidelines of international scientific research and our institution\u0026apos;s policies, using such commercialized, established cell lines for in vitro research does not require additional ethical review or approval. Therefore, this study has adhered to all applicable ethical standards.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAll authors contributed to the study's conception and design. Material preparation, data collection, and analysis were performed by Sen Wang, Miao He, Xiao Liang, and Baoshan Li. The first draft of the manuscript was written by Sen Wang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eNusse R, Varmus HE (1982) Many Tumors Induced by the Mouse Mammary Tumor Virus Contain a Provirus Integrated in the Same Region of the Host Genome. Cell 31(1):99\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0092-8674(82)90409-3\u003c/span\u003e\u003cspan address=\"10.1016/0092-8674(82)90409-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang L, Adu IK, Zhang H, Wang J (2023) The WNT/β-Catenin System in Chronic Kidney Disease-Mineral Bone Disorder Syndrome. Int Urol Nephrol 55(10):2527\u0026ndash;2538. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11255-023-03569-2\u003c/span\u003e\u003cspan address=\"10.1007/s11255-023-03569-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang D-H, Shao J (2025) Research Progress of Basing on Wnt/β-Catenin Pathway in the Treatment of Bone Tissue Diseases. Tissue Eng Part B Rev. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1089/ten.teb.2024.0170\u003c/span\u003e\u003cspan address=\"10.1089/ten.teb.2024.0170\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDuchartre Y, Kim Y-M, Kahn M (2016) The Wnt Signaling Pathway in Cancer. Crit Rev Oncol Hematol 99:141\u0026ndash;149. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.critrevonc.2015.12.005\u003c/span\u003e\u003cspan address=\"10.1016/j.critrevonc.2015.12.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchoeman MAE, Moester MJC, Oostlander AE, Kaijzel EL, Valstar ER, Nelissen RGHH, L\u0026ouml;wik CWGM, de Rooij KE (2015) Inhibition of GSK3β Stimulates BMP Signaling and Decreases SOST Expression Which Results in Enhanced Osteoblast Differentiation. J Cell Biochem 116(12):2938\u0026ndash;2946. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcb.25241\u003c/span\u003e\u003cspan address=\"10.1002/jcb.25241\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLojk J, Marc J (2021) Roles of Non-Canonical Wnt Signalling Pathways in Bone Biology. Int J Mol Sci 22(19):10840. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms221910840\u003c/span\u003e\u003cspan address=\"10.3390/ijms221910840\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomori T (2016) Glucocorticoid Signaling and Bone Biology. Horm Metab Res 48(11):755\u0026ndash;763. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-0042-110571\u003c/span\u003e\u003cspan address=\"10.1055/s-0042-110571\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeinstein RS, Nicholas RW, Manolagas SC (2000) Apoptosis of Osteocytes in Glucocorticoid-Induced Osteonecrosis of the Hip. J Clin Endocrinol Metab 85(8):2907\u0026ndash;2912. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1210/jcem.85.8.6714\u003c/span\u003e\u003cspan address=\"10.1210/jcem.85.8.6714\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eThe molecular etiology and treatment of glucocorticoid-induced osteoporosis - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/34386357/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/34386357/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhlsson C, Nilsson KH, Henning P, Wu J, Gustafsson KL, Poutanen M, Lerner UH, Mov\u0026eacute;rare-Skrtic S (2018) WNT16 Overexpression Partly Protects against Glucocorticoid-Induced Bone Loss. Am J Physiol Endocrinol Metab 314(6):E597\u0026ndash;E604. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpendo.00292.2017\u003c/span\u003e\u003cspan address=\"10.1152/ajpendo.00292.2017\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawazoe M, Kaneko K, Nanki T (2021) Glucocorticoid Therapy Suppresses Wnt Signaling by Reducing the Ratio of Serum Wnt3a to Wnt Inhibitors, sFRP-1 and Wif-1. Clin Rheumatol 40(7):2947\u0026ndash;2954. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10067-020-05554-x\u003c/span\u003e\u003cspan address=\"10.1007/s10067-020-05554-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eElevated Expression of Dkk-1 by Glucocorticoid Treatment Impairs Bone Regenerative Capacity of Adipose Tissue-Derived Mesenchymal Stem Cells - PubMed. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/29084466/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/29084466/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eMutation of the mouse klotho gene leads to a syndrome resembling ageing - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/9363890/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/9363890/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Y, Chen N, Fu Z, Zhang Q (2023) Progress of Wnt Signaling Pathway in Osteoporosis. Biomolecules 13(3):483. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom13030483\u003c/span\u003e\u003cspan address=\"10.3390/biom13030483\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoromila T, Baniwal SK, Song YS, Martin A, Xiong J, Frenkel B (2014) Glucocorticoids Antagonize RUNX2 during Osteoblast Differentiation in Cultures of ST2 Pluripotent Mesenchymal Cells. J Cell Biochem 115(1):27\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcb.24646\u003c/span\u003e\u003cspan address=\"10.1002/jcb.24646\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGado M, Baschant U, Hofbauer LC, Henneicke H (2022) Bad to the Bone: The Effects of Therapeutic Glucocorticoids on Osteoblasts and Osteocytes. Front Endocrinol (Lausanne) 13:835720. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fendo.2022.835720\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2022.835720\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAjay AK, Zhu L-J, Zhao L, Liu Q, Ding Y, Chang Y-C, Shah SI, Hsiao L-L (2024) Local Vascular Klotho Mediates Diabetes-Induced Atherosclerosis via ERK1/2 and PI3-Kinase-Dependent Signaling Pathways. Atherosclerosis 396:118531. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.atherosclerosis.2024.118531\u003c/span\u003e\u003cspan address=\"10.1016/j.atherosclerosis.2024.118531\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eDecreased ADAM17 expression in the lungs of α-Klotho reduced mouse - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/31951006/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/31951006/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu J, Wang H, Liu Q, Long S, Wu Y, Wang N, Lin W, Chen G, Lin M, Wen J (2024) Klotho Exerts Protection in Chronic Kidney Disease Associated with Regulating Inflammatory Response and Lipid Metabolism. Cell Biosci 14(1):46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13578-024-01226-4\u003c/span\u003e\u003cspan address=\"10.1186/s13578-024-01226-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu J-P, Zeng R-X, He M-H, Lin S-S, Guo L-H, Zhang M-Z (2022) Associations Between Serum Soluble α-Klotho and the Prevalence of Specific Cardiovascular Disease. Front Cardiovasc Med 9:899307. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fcvm.2022.899307\u003c/span\u003e\u003cspan address=\"10.3389/fcvm.2022.899307\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRoig-Soriano J, Edo \u0026Aacute;, Verd\u0026eacute;s S, Mart\u0026iacute;n-Alonso C, S\u0026aacute;nchez-de-Diego C, Rodriguez-Estevez L, Serrano AL, Abraham CR, Bosch A, Ventura F, Jordan BA, Mu\u0026ntilde;oz-C\u0026aacute;noves P, Chill\u0026oacute;n M (2025) Long-Term Effects of s-KL Treatment in Wild-Type Mice: Enhancing Longevity, Physical Well-Being, and Neurological Resilience. Mol Ther S1525\u0026ndash;0016(25):00120\u0026ndash;00120. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ymthe.2025.02.030\u003c/span\u003e\u003cspan address=\"10.1016/j.ymthe.2025.02.030\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAgoro R, Ni P, Noonan ML, White KE (2020) Osteocytic FGF23 and Its Kidney Function. Front Endocrinol (Lausanne) 11:592. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fendo.2020.00592\u003c/span\u003e\u003cspan address=\"10.3389/fendo.2020.00592\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKomaba H, Kaludjerovic J, Hu DZ, Nagano K, Amano K, Ide N, Sato T, Densmore MJ, Hanai J-I, Olauson H, Bellido T, Larsson TE, Baron R, Lanske B (2017) Klotho Expression in Osteocytes Regulates Bone Metabolism and Controls Bone Formation. Kidney Int 92(3):599\u0026ndash;611. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.kint.2017.02.014\u003c/span\u003e\u003cspan address=\"10.1016/j.kint.2017.02.014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFan Y, Cui C, Rosen CJ, Sato T, Xu R, Li P, Wei X, Bi R, Yuan Q, Zhou C (2022) Klotho in Osx+-Mesenchymal Progenitors Exerts pro-Osteogenic and Anti-Inflammatory Effects during Mandibular Alveolar Bone Formation and Repair. Signal Transduct Target Ther 7(1):155. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41392-022-00957-5\u003c/span\u003e\u003cspan address=\"10.1038/s41392-022-00957-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiang X, Li B, Huang Q, Liu D, Ma H, Klotho Prevents (2018) DEX-Induced Apoptosis in MC3T3-E1 Osteoblasts through the NF-κB Signaling Pathway. Biochem Biophys Res Commun 507(1\u0026ndash;4):355\u0026ndash;361. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2018.11.040\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2018.11.040\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Y-X, Huang C, Duan Z-B, Xu C-Y, Chen Y (2019) Klotho/FGF23 Axis Mediates High Phosphate-Induced Vascular Calcification in Vascular Smooth Muscle Cells via Wnt7b/β-Catenin Pathway. Kaohsiung J Med Sci 35(7):393\u0026ndash;400. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/kjm2.12072\u003c/span\u003e\u003cspan address=\"10.1002/kjm2.12072\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMohanty SK, Mohanty AK, Kumar MS, Suchiang K (2024) Triiodothyronine Enhances Various Forms of Kidney-Specific Klotho Protein and Suppresses the Wnt/β-Catenin Pathway: Insights from in-Vitro, in-Vivo and in-Silico Investigations. Cell Signal 120:111214. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.cellsig.2024.111214\u003c/span\u003e\u003cspan address=\"10.1016/j.cellsig.2024.111214\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Q, Jiang L, Wu J, Dong H, Zhao Y (2021) Klotho Inhibits Proliferation in a RET Fusion Model of Papillary Thyroid Cancer by Regulating the Wnt/β-Catenin Pathway. Cancer Manag Res 13:4791\u0026ndash;4802. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2147/CMAR.S295086\u003c/span\u003e\u003cspan address=\"10.2147/CMAR.S295086\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eLoss of Klotho contributes to cartilage damage by derepression of canonical Wnt/β-catenin signaling in osteoarthritis mice - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/31895692/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/31895692/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-12)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi S, Jiang H, Gu X (2018) Echinacoside Suppresses Dexamethasone-Induced Growth Inhibition and Apoptosis in Osteoblastic MC3T3-E1 Cells. Exp Ther Med 16(2):643\u0026ndash;648. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3892/etm.2018.6199\u003c/span\u003e\u003cspan address=\"10.3892/etm.2018.6199\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang T, Yu X, He C, Pro-Inflammatory, Cytokines (2019) Cellular and Molecular Drug Targets for Glucocorticoid-Induced-Osteoporosis via Osteocyte. Curr Drug Targets 20(1):1\u0026ndash;15. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2174/1389450119666180405094046\u003c/span\u003e\u003cspan address=\"10.2174/1389450119666180405094046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShen X, Feng S, Chen S, Gong B, Wang S, Wang H, Song D, Ni J (2024) Wnt3a-Induced LRP6 Phosphorylation Enhances Osteoblast Differentiation to Alleviate Osteoporosis through Activation of mTORC1/β-Catenin Signaling. Arch Biochem Biophys 761:110169. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.abb.2024.110169\u003c/span\u003e\u003cspan address=\"10.1016/j.abb.2024.110169\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eNon-canonical WNT5A-ROR signaling: New perspectives on an ancient developmental pathway - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/36967195/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/36967195/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-17)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e\u003cem\u003eDeletion of Wnt5a in osteoclasts results in bone loss through decreased bone formation - PubMed\u003c/em\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://pubmed.ncbi.nlm.nih.gov/31919867/\u003c/span\u003e\u003cspan address=\"https://pubmed.ncbi.nlm.nih.gov/31919867/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e (accessed 2025-01-17)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarrillo-L\u0026oacute;pez N, Panizo S, Alonso-Montes C, Rom\u0026aacute;n-Garc\u0026iacute;a P, Rodr\u0026iacute;guez I, Mart\u0026iacute;nez-Salgado C, Dusso AS, Naves M, Cannata-And\u0026iacute;a JB (2016) Direct Inhibition of Osteoblastic Wnt Pathway by Fibroblast Growth Factor 23 Contributes to Bone Loss in Chronic Kidney Disease. Kidney Int 90(1):77\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.kint.2016.01.024\u003c/span\u003e\u003cspan address=\"10.1016/j.kint.2016.01.024\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Klotho, Wnt signaling pathway, Glucocorticoid, Osteoporosis","lastPublishedDoi":"10.21203/rs.3.rs-5854318/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5854318/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGlucocorticoids are commonly prescribed in clinical settings; however, their prolonged use at high doses can adversely affect human health. One significant complication following glucocorticoid therapy is glucocorticoid-induced osteoporosis (GIO), which is second in incidence only to senile osteoporosis.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eBased on previous research indicating that Klotho alleviates dexamethasone-induced osteoblast cytotoxicity through the NF-kB pathway, we aimed to explore the underlying mechanisms in greater depth.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWe assessed the impact of Lithium chloride (LiCl), a Wnt pathway activator, on glucocorticoid-induced cell cytotoxicity and viability. Cytotoxicity was specifically quantified by Annexin V/PI flow cytometry. We performed qRT-PCR and Western blotting analyses to scrutinize the expressions of genes and proteins associated with both canonical and non-canonical Wnt signaling pathways.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eDexamethasone treatment induced an upregulation of the non-canonical Wnt ligand, Wnt5a, and a downregulation of the canonical ligand, Wnt3a, along with its downstream marker, β-catenin. Transfection with Klotho counteracted these effects.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eKlotho has the potential to modulate both canonical and non-canonical Wnt signaling pathways, thereby counteracting osteoblast cytotoxicity induced by glucocorticoids.\u003c/p\u003e","manuscriptTitle":"Klotho attenuates glucocorticoid-induced osteoblast cytotoxicity via Wnt signaling pathway modulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-28 20:13:31","doi":"10.21203/rs.3.rs-5854318/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-07-26T03:15:18+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-23T20:25:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"155773869139902388583527760195173960496","date":"2025-07-06T03:19:39+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-06-18T14:36:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315477575964511263482035330122064795890","date":"2025-06-04T14:44:11+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-10T01:14:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"60719418300502104627299080977006834387","date":"2025-04-09T18:02:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-09T14:11:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-02T07:07:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biochemical Genetics","date":"2025-03-20T17:01:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biochemical-genetics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bigi","sideBox":"Learn more about [Biochemical Genetics](http://link.springer.com/journal/10528)","snPcode":"10528","submissionUrl":"https://submission.nature.com/new-submission/10528/3","title":"Biochemical Genetics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"cb20a029-09a4-4ea4-8f77-6bad1585603d","owner":[],"postedDate":"April 28th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-22T10:40:15+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-28 20:13:31","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5854318","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5854318","identity":"rs-5854318","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}