Vitamin K-dependent carboxylation in osteoblasts regulates bone resorption through GAS6 in male mice

Abstract

Studies in humans suggest that vitamin K is involved in the regulation of bone remodeling, but the precise mechanism at play remains unknown. In cells, vitamin K functions as a co-factor for the γ-glutamyl carboxylase (GGCX), an enzyme responsible for the conversion of glutamic acid residues (Glu) into γ-carboxyglutamic acid (Gla) residues in secreted proteins. We aim here at determining the role of γ-carboxylation in bone remodeling and at identifying the Gla protein(s) involved. We show that male mice lacking γ-carboxylation specifically in osteoblasts (Ggcx^flox/flox;OCN-Cre) have increased bone mass at 6 months of age due to a reduced number of multinucleated bone resorbing osteoclasts. In co-culture experiments, Ggcx-deficient osteoblasts were less effective than control osteoblasts at supporting osteoclast formation. Among known Gla proteins, we identify GAS6 as an osteoblast-secreted γ-carboxylated factor which signals to differentiating osteoclasts. The GAS6 receptors MerTK and AXL are expressed in pre-osteoclasts and pharmacological inhibitors of AXL and MerTK block osteoclast generation in co-culture. Conversely, recombinant γ-carboxylated GAS6 dose-dependently increases the size of osteoclasts and the number of nuclei per osteoclast in culture. GAS6 marginally affected the induction of osteoclast-specific genes during osteoclast differentiation but significantly increased pre-osteoclast fusion. Finally, increasing bone marrow GAS6 level in transgenic male mice was sufficient to increase the number and size of osteoclasts and to decrease bone mass. This work identifies GAS6 as a novel osteoblast-derived vitamin K-dependent protein regulating osteoclast maturation.

References

[1] Morris JA, et al.. An atlas of genetic influences on osteoporosis in humans and mice. Nat. Genet, 2019, 51: 258-266

[2] Zhu X, Bai W, Zheng H. Twelve years of GWAS discoveries for osteoporosis and related traits: advances, challenges and applications. Bone Res, 2021, 9: 23

[3] Moller AMJ, et al.. Aging and menopause reprogram osteoclast precursors for aggressive bone resorption. Bone Res., 2020, 8: 27

[4] Wilson-Barnes SL, Lanham-New SA, Lambert H. Modifiable risk factors for bone health & fragility fractures. Best. Pr. Res. Clin. Rheumatol., 2022, 36: 101758

[5] Nieves JW. Osteoporosis: the role of micronutrients. Am. J. Clin. Nutr., 2005, 81: 1232S-1239S

[6] Veldurthy V, et al.. Vitamin D, calcium homeostasis and aging. Bone Res, 2016, 4 Article ID: 16041

[7] Brzezinska O, Lukasik Z, Makowska J, Walczak K. Role of vitamin C in osteoporosis development and treatment-A literature review. Nutrients, 2020, 12: 2394

[8] Clements M, et al.. A 2-year randomized controlled trial with low-dose B-vitamin supplementation shows benefits on bone mineral density in adults with lower B12 status. J. Bone Min. Res., 2022, 37: 2443-2455

[9] Tucker KL, et al.. Low plasma vitamin B12 is associated with lower BMD: The Framingham Osteoporosis Study. J. Bone Min. Res, 2005, 20: 152-158

[10] Booth SL, et al.. Vitamin K intake and bone mineral density in women and men. Am. J. Clin. Nutr., 2003, 77: 512-516

[11] Booth SL, et al.. Associations between vitamin K biochemical measures and bone mineral density in men and women. J. Clin. Endocrinol. Metab., 2004, 89: 4904-4909

[12] Booth SL, et al.. Dietary vitamin K intakes are associated with hip fracture but not with bone mineral density in elderly men and women. Am. J. Clin. Nutr., 2000, 71: 1201-1208

[13] Feskanich D, et al.. Vitamin K intake and hip fractures in women: a prospective study. Am. J. Clin. Nutr., 1999, 69: 74-79

[14] Hao G, et al.. Vitamin K intake and the risk of fractures: A meta-analysis. Med. (Baltim.), 2017, 96: e6725

[15] Hamidi MS, Gajic-Veljanoski O, Cheung AM. Vitamin K and bone health. J. Clin. Densitom. J. Int. Soc. Clin. Densitom., 2013, 16: 409-413

[16] Thijssen HH, Drittij-Reijnders MJ. Vitamin K distribution in rat tissues: dietary phylloquinone is a source of tissue menaquinone-4. Br. J. Nutr., 1994, 72: 415-425

[17] Ronden JE, Thijssen HH, Vermeer C. Tissue distribution of K-vitamers under different nutritional regimens in the rat. Biochim Biophys. Acta, 1998, 1379: 16-22

[18] Fang Y, Hu C, Tao X, Wan Y, Tao F. Effect of vitamin K on bone mineral density: a meta-analysis of randomized controlled trials. J. Bone Min. Metab., 2012, 30: 60-68

[19] Cockayne S, et al.. Vitamin K and the prevention of fractures: systematic review and meta-analysis of randomized controlled trials. Arch. Intern. Med., 2006, 166: 1256-1261

[20] Inoue T, et al.. Randomized controlled study on the prevention of osteoporotic fractures (OF study): a phase IV clinical study of 15-mg menatetrenone capsules. J. Bone Min. Metab., 2009, 27: 66-75

[21] Cheung AM, et al.. Vitamin K supplementation in postmenopausal women with osteopenia (ECKO trial): a randomized controlled trial. PLoS Med., 2008, 5: e196

[22] Dupuy M, et al.. Vitamin K: Metabolism, genetic influences, and chronic disease outcomes. Food Sci. Nutr., 2025, 13 Article ID: e70431

[23] Zhou M, Han S, Zhang W, Wu D. Efficacy and safety of vitamin K2 for postmenopausal women with osteoporosis at a long-term follow-up: meta-analysis and systematic review. J. Bone Min. Metab., 2022, 40: 763-772

[24] Salma et al. Effect of vitamin K on bone mineral density and fracture risk in adults: Systematic review and meta-analysis. Biomedicines10 (2022).

[25] Lacombe J, Ferron M. Vitamin K-dependent carboxylation in beta-cells and diabetes. Trends Endocrinol. Metab., 2024, 35: 661-673

[26] Berkner KL, Runge KW. Vitamin K-Dependent Protein Activation: Normal Gamma-Glutamyl Carboxylation and Disruption in Disease. Int. J. Mol. Sci., 2022, 23: 5759

[27] Hauschka PV, Lian JB, Gallop PM. Direct identification of the calcium-binding amino acid, gamma-carboxyglutamate, in mineralized tissue. Proc. Natl. Acad. Sci. USA, 1975, 72: 3925-3929

[28] Lambert LJ, et al.. Increased trabecular bone and improved biomechanics in an osteocalcin-null rat model created by CRISPR/Cas9 technology. Dis. Models Mech., 2016, 9: 1169-1179

[29] Ducy P, et al.. Increased bone formation in osteocalcin-deficient mice. Nature, 1996, 382: 448-452

[30] Zhang M, et al.. Osteoblast-specific knockout of the insulin-like growth factor (IGF) receptor gene reveals an essential role of IGF signaling in bone matrix mineralization. J. Biol. Chem., 2002, 277: 44005-44012

[31] Ferron M, Lacombe J, Germain A, Oury F, Karsenty G. GGCX and VKORC1 inhibit osteocalcin endocrine functions. J. Cell Biol., 2015, 208: 761-776

[32] Coutu DL, et al.. Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells. J. Biol. Chem., 2008, 283: 17991-18001

[33] Barone LM, et al.. Developmental expression and hormonal regulation of the rat matrix Gla protein (MGP) gene in chondrogenesis and osteogenesis. J. Cell Biochem, 1991, 46: 351-365

[34] Ducy P, Karsenty G. Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene. Mol. Cell Biol., 1995, 15: 1858-1869

[35] Gerbaix M, Vico L, Ferrari SL, Bonnet N. Periostin expression contributes to cortical bone loss during unloading. Bone, 2015, 71: 94-100

[36] Marulanda J, Gao C, Roman H, Henderson JE, Murshed M. Prevention of arterial calcification corrects the low bone mass phenotype in MGP-deficient mice. Bone, 2013, 57: 499-508

[37] Lemke G. Biology of the TAM receptors. Cold Spring Harb. Perspect. Biol., 2013, 5: a009076

[38] Schott C, et al.. GAS6 and AXL promote insulin resistance by rewiring insulin signaling and increasing insulin receptor trafficking to endosomes. Diabetes, 2024, 73: 1648-1661

[39] Goruppi S, Ruaro E, Varnum B, Schneider C. Requirement of phosphatidylinositol 3-kinase-dependent pathway and Src for Gas6-Axl mitogenic and survival activities in NIH 3T3 fibroblasts. Mol. Cell Biol., 1997, 17: 4442-4453

[40] Pao-Chun L, Chan PM, Chan W, Manser E. Cytoplasmic ACK1 interaction with multiple receptor tyrosine kinases is mediated by Grb2: an analysis of ACK1 effects on Axl signaling. J. Biol. Chem., 2009, 284: 34954-34963

[41] Paolino M, et al.. The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells. Nature, 2014, 507: 508-512

[42] Holland SJ, et al.. R428, a selective small molecule inhibitor of Axl kinase, blocks tumor spread and prolongs survival in models of metastatic breast cancer. Cancer Res., 2010, 70: 1544-1554

[43] Lew ED, et al.. Differential TAM receptor-ligand-phospholipid interactions delimit differential TAM bioactivities. eLife, 2014, 3: e03385

[44] Schindeler A, McDonald MM, Bokko P, Little DG. Bone remodeling during fracture repair: The cellular picture. Semin Cell Dev. Biol., 2008, 19: 459-466

[45] Xie X, et al.. Fracture risks in patients with atrial fibrillation treated with different oral anticoagulants: A meta-analysis and systematic review. Age Ageing, 2022, 51: afab264

[46] Shiozawa Y, et al.. GAS6/AXL axis regulates prostate cancer invasion, proliferation, and survival in the bone marrow niche. Neoplasia, 2010, 12: 116-127

[47] Engelmann J, et al.. Regulation of bone homeostasis by MERTK and TYRO3. Nat. Commun., 2022, 13 Article ID: 7689

[48] Ryu KY, et al.. Mer tyrosine kinase regulates bone metabolism, and its deficiency partially ameliorates periodontitis- and ovariectomy-induced bone loss in mice. JBMR Plus, 2024, 8: ziad014

[49] Maillard C, Berruyer M, Serre CM, Dechavanne M, Delmas PD. Protein-S, a vitamin K-dependent protein, is a bone matrix component synthesized and secreted by osteoblasts. Endocrinology, 1992, 130: 1599-1604

[50] Lemke G. Phosphatidylserine is the signal for TAM receptors and their ligands. Trends Biochem. Sci., 2017, 42: 738-748

[51] Krishnacoumar B, et al.. Caspase-8 promotes scramblase-mediated phosphatidylserine exposure and fusion of osteoclast precursors. Bone Res, 2024, 12: 40

[52] Verma SK, et al.. Cell-surface phosphatidylserine regulates osteoclast precursor fusion. J. Biol. Chem., 2018, 293: 254-270

[53] Whitlock JM, et al.. Cell surface-bound La protein regulates the cell fusion stage of osteoclastogenesis. Nat. Commun., 2023, 14 Article ID: 616

[54] Nakamura T, et al.. Estrogen prevents bone loss via estrogen receptor alpha and induction of Fas ligand in osteoclasts. Cell, 2007, 130: 811-823

[55] Ferron M, Wei J, Yoshizawa T, Ducy P, Karsenty G. An ELISA-based method to quantify osteocalcin carboxylation in mice. Biochem Biophys. Res Commun., 2010, 397: 691-696

[56] Chappard D, Palle S, Alexandre C, Vico L, Riffat G. Bone embedding in pure methyl methacrylate at low temperature preserves enzyme activities. Acta Histochem, 1987, 81: 183-190

[57] Bouxsein ML, et al.. Guidelines for assessment of bone microstructure in rodents using micro-computed tomography. J. Bone Min. Res, 2010, 25: 1468-1486

[58] Ferron M, et al.. Inositol polyphosphate 4-phosphatase B as a regulator of bone mass in mice and humans. Cell Metab., 2011, 14: 466-477

[59] Chomczynski P, Sacchi N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: Twenty-something years on. Nat. Protoc., 2006, 1: 581-585

[60] Lacombe J, et al.. Vitamin K-dependent carboxylation regulates Ca(2+) flux and adaptation to metabolic stress in beta cells. Cell Rep., 2023, 42 Article ID: 112500

Funding

Gouvernement du Canada | Canadian Institutes of Health Research (Instituts de Recherche en Santé du Canada)(PJT-159534)

Canada Research Chairs (Chaires de recherche du Canada)(NA)

Fonds de Recherche du Québec - Santé (Fonds de la recherche en sante du Quebec)(NA)

RIGHTS & PERMISSIONS

The Author(s)