Ubiquitin-specific protease 26 facilitates endochondral ossification by driving chondrocyte hypertrophy and mineralization

Abstract

Chondrocyte hypertrophy and mineralization are essential for endochondral ossification; however, the mechanisms underlying these processes remain incompletely understood. In this study, we have identified the facilitated role of ubiquitin-specific protease 26 (USP26) in endochondral ossification by stimulating chondrocyte hypertrophy and mineralization. Ultimately, this promotes skeletal development, bone fracture healing, and the occurrence of osteoarthritis. Mechanistically, USP26 decreases FBP2 undergoing K63-linked ubiquitination, leading to a reduction in the protein level of FBP2. This reduction promotes mitochondrial biogenesis and oxidative phosphorylation, thus facilitating chondrocyte hypertrophy and mineralization and aiding in the process of endochondral ossification. Furthermore, our study found that compression loading induces USP26 to initiate chondrocyte hypertrophy and mineralization through the phosphorylation of estrogen receptor-α at serine 118. These findings suggest that USP26, acting as a mechanosensor, facilitates chondrocyte hypertrophy and mineralization by maintaining mitochondrial biogenesis through the reduction of FBP2. Identifying USP26 as a potential therapeutic target for physiological skeletal growth, bone fracture healing, and osteoarthritis.

References

[1] Kronenberg HM. Developmental regulation of the growth plate. Nature, 2003, 423: 332-336

[2] Kawaguchi H. Endochondral ossification signals in cartilage degradation during osteoarthritis progression in experimental mouse models. Mol. Cells, 2008, 25,: 1-6

[3] Saito T, et al. . Transcriptional regulation of endochondral ossification by HIF-2alpha during skeletal growth and osteoarthritis development. Nat. Med., 2010, 16,: 678-686

[4] Hosaka Y, et al. . Notch signaling in chondrocytes modulates endochondral ossification and osteoarthritis development. Proc. Natl. Acad. Sci. USA, 2013, 110,: 1875-1880

[5] Morita K, et al. . Reactive oxygen species induce chondrocyte hypertrophy in endochondral ossification. J. Exp. Med., 2007, 204,: 1613-1623

[6] Stickens D, et al. . Altered endochondral bone development in matrix metalloproteinase 13-deficient mice. Development, 2004, 131,: 5883-5895

[7] Zelzer E, et al. . VEGFA is necessary for chondrocyte survival during bone development. Development, 2004, 131: 2161-2171

[8] Wang X, et al. . Inhibition of overactive TGF-beta attenuates progression of heterotopic ossification in mice. Nat. Commun., 2018, 9 551

[9] Yoshida M, et al. . The transcription factor Foxc1 is necessary for Ihh-Gli2-regulated endochondral ossification. Nat. Commun., 2015, 6 6653

[10] Lanske B, et al. . PTH/PTHrP receptor in early development and Indian hedgehog-regulated bone growth. Science, 1996, 273: 663-666

[11] Mak KK, Chen MH, Day TF, Chuang PT, Yang Y. Wnt/beta-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation. Development, 2006, 133: 3695-3707

[12] Li C, et al. . The osteoprotective role of USP26 in coordinating bone formation and resorption. Cell Death Differ., 2022, 29: 1123-1136

[13] Xu Y, et al. . USP26 combats age-related declines in self-renewal and multipotent differentiation of BMSC by maintaining mitochondrial homeostasis. Adv. Sci., 2024, 11 e2406428

[14] Tang J, et al. . Activating the osteoblastic USP26 pathway alleviates multi-organ fibrosis by decreasing insulin resistance. Adv. Sci., 2025, 13: e12424

[15] Sun J, et al. . Histone demethylase LSD1 is critical for endochondral ossification during bone fracture healing. Sci. Adv., 2020, 6: eaaz1410

[16] Gibson G. Active role of chondrocyte apoptosis in endochondral ossification. Microsc. Res. Tech., 1998, 43: 191-204

[17] Atkins GJ, et al. . Sclerostin is a locally acting regulator of late-osteoblast/preosteocyte differentiation and regulates mineralization through a MEPE-ASARM-dependent mechanism. J. Bone Miner. Res., 2011, 26: 1425-1436

[18] Peng Z, et al. . MiR-20a: a mechanosensitive microRNA that regulates fluid shear stress-mediated osteogenic differentiation via the BMP2 signaling pathway by targeting BAMBI and SMAD6. Ann. Transl. Med., 2022, 10: 683

[19] Zhuang X, et al. . Differential effects on lung and bone metastasis of breast cancer by Wnt signalling inhibitor DKK1. Nat. Cell Biol., 2017, 19: 1274-1285

[20] Hendrickx G, et al. . Piezo1 inactivation in chondrocytes impairs trabecular bone formation. J. Bone Miner. Res., 2021, 36: 369-384

[21] Qiu M, et al. . Black phosphorus accelerates bone regeneration based on immunoregulation. Adv. Sci., 2024, 11 e2304824

[22] Jia S, et al. . Insufficient mechanical loading downregulates Piezo1 in chondrocytes and impairs fracture healing through ApoE-induced senescence. Adv. Sci., 2024, 11 e2400502

[23] Kamekura S, et al. . Osteoarthritis development in novel experimental mouse models induced by knee joint instability. Osteoarthr. Cartil., 2005, 13: 632-641

[24] Stegen S, et al. . HIF-1alpha metabolically controls collagen synthesis and modification in chondrocytes. Nature, 2019, 565: 511-515

[25] Sherratt HS. Mitochondria: structure and function. Rev. Neurol., 1991, 147: 417-430

[26] Li X, et al. . Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease. Cell Metab., 2023, 35: 200-211.e209

[27] Xue K, et al. . The mitochondrial calcium uniporter engages UCP1 to form a thermoporter that promotes thermogenesis. Cell Metab., 2022, 34: 1325-1341.e1326

[28] Huangyang P, et al. . Fructose-1,6-bisphosphatase 2 inhibits sarcoma progression by restraining mitochondrial biogenesis. Cell Metab., 2020, 31: 174-188e177

[29] Pluska L, et al. . The UBA domain of conjugating enzyme Ubc1/Ube2K facilitates assembly of K48/K63-branched ubiquitin chains. EMBO J., 2021, 40 e106094

[30] Iskratsch T, Wolfenson H, Sheetz MP. Appreciating force and shape-the rise of mechanotransduction in cell biology. Nat. Rev. Mol. Cell Biol., 2014, 15: 825-833

[31] Wang L, et al. . Mechanical sensing protein PIEZO1 regulates bone homeostasis via osteoblast-osteoclast crosstalk. Nat. Commun., 2020, 11 282

[32] Li C, et al. . Double-stranded RNA released from damaged articular chondrocytes promotes cartilage degeneration via Toll-like receptor 3-interleukin-33 pathway. Cell Death Dis., 2017, 8 e3165

[33] Zhang H, et al. . Mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7. Ann. Rheum. Dis., 2022, 81: 676-686

[34] Yamagishi K, et al. . Activation of the renin-angiotensin system in mice aggravates mechanical loading-induced knee osteoarthritis. Eur. J. Histochem., 2018, 62: 2930

[35] Lee YJ, et al. . Evaluation of osteoarthritis induced by treadmill-running exercise using the modified Mankin and the new OARSI assessment system. Rheumatol. Int., 2011, 31: 1571-1576

[36] Siebelt M, et al. . Hsp90 inhibition protects against biomechanically induced osteoarthritis in rats. Arthritis Rheum., 2013, 65: 2102-2112

[37] Yan X, et al. . ERα/β/DMP1 axis promotes trans-differentiation of chondrocytes to bone cells through GSK-3β/β-catenin pathway. J. Anat., 2022, 240: 1152-1161

[38] Kato S, et al. . Activation of the estrogen receptor through phosphorylation by mitogen-activated protein kinase. Science, 1995, 270: 1491-1494

[39] Du Z, et al. . The sequence-structure-function relationship of intrinsic ERalpha disorder. Nature, 2025, 638: 1130-1138

[40] Wang PJ, McCarrey JR, Yang F, Page DC. An abundance of X-linked genes expressed in spermatogonia. Nat. Genet., 2001, 27: 422-426

[41] Wosnitzer MS, et al. . Ubiquitin Specific Protease 26 (USP26) expression analysis in human testicular and extragonadal tissues indicates diverse action of USP26 in cell differentiation and tumorigenesis. PLoS One, 2014, 9: e98638

[42] Stouffs K, Lissens W, Tournaye H, Van Steirteghem A, Liebaers I. Possible role of USP26 in patients with severely impaired spermatogenesis. Eur. J. Hum. Genet., 2005, 13: 336-340

[43] Tian H, et al. . Disruption of ubiquitin specific protease 26 gene causes male subfertility associated with spermatogenesis defects in mice†. Biol. Reprod., 2019, 100: 1118-1128

[44] Sakai K, et al. . Usp26 mutation in mice leads to defective spermatogenesis depending on genetic background. Sci. Rep., 2019, 9 13757

[45] Li L, Zhou H, Zhu R, Liu Z. USP26 promotes esophageal squamous cell carcinoma metastasis through stabilizing Snail. Cancer Lett., 2019, 448: 52-60

[46] Wei Y, et al. . Aerobic glycolysis is the predominant means of glucose metabolism in neuronal somata, which protects against oxidative damage. Nat. Neurosci., 2023, 26: 2081-2089

[47] Osipova E, et al. . Loss of a gluconeogenic muscle enzyme contributed to adaptive metabolic traits in hummingbirds. Science, 2023, 379: 185-190

[48] Park HJ, et al. . The essential role of fructose-1,6-bisphosphatase 2 enzyme in thermal homeostasis upon cold stress. Exp. Mol. Med., 2020, 52: 485-496

[49] Pietras L, Stefanik E, Rakus D, Gizak A. FBP2-a new player in regulation of motility of mitochondria and stability of microtubules in cardiomyocytes. Cells, 2022, 11: 1170

[50] Skedros JG, Hunt KJ, Hughes PE, Winet H. Ontogenetic and regional morphologic variations in the turkey ulna diaphysis: implications for functional adaptation of cortical bone. Anat. Rec. A Discov. Mol. Cell Evol. Biol., 2003, 273: 609-629

[51] Talts JF, et al. . Endochondral ossification is dependent on the mechanical properties of cartilage tissue and on intracellular signals in chondrocytes. Ann. N. Y. Acad. Sci., 1998, 857: 74-85

[52] Moon EH, et al. . TMEM100 is a key factor for specification of lymphatic endothelial progenitors. Angiogenesis, 2020, 23: 339-355

[53] Chen K, et al. . Osteocytic HIF-1alpha pathway manipulates bone micro-structure and remodeling via regulating osteocyte terminal differentiation. Front. Cell Dev. Biol., 2021, 9 721561

[54] Qiu M, et al. . 3D biomimetic calcified cartilaginous callus that induces type H vessels formation and osteoclastogenesis. Adv. Sci., 2023, 10 e2207089

[55] Zhao S, et al. . Desferrioxamine alleviates UHMWPE particle-induced osteoclastic osteolysis by inhibiting caspase-1-dependent pyroptosis in osteocytes. J. Biol. Eng., 2022, 16 34

[56] Gosset M, Berenbaum F, Thirion S, Jacques C. Primary culture and phenotyping of murine chondrocytes. Nat. Protoc., 2008, 3: 1253-1260

[57] Pritzker KP, et al. . Osteoarthritis cartilage histopathology: grading and staging. Osteoarthr. Cartil., 2006, 14: 13-29

[58] Deng R, et al. . Periosteal CD68(+) F4/80(+) macrophages are mechanosensitive for cortical bone formation by secretion and activation of TGF-beta1. Adv. Sci., 2022, 9 e2103343

Funding

National Natural Science Foundation of China (National Science Foundation of China)(81802119)

Science and Technology Bureau of Kunshan (No. KS1947)

RIGHTS & PERMISSIONS

The Author(s)