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

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 theγ-glutamyl carboxylase(GGCX),an enzyme responsible for the conversion of glutamic acid residues(Glu)intoγ-carboxyglutamic acid(Gla)residues in secreted proteins.We aim here at determining the role ofγ-carboxylation in bone remodeling and at identifying the Gla protein(s)involved.We show that male mice lackingγ-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-secretedγ-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,recombinantγ-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.