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

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 {gamma}-glutamyl carboxylase (GGCX), an enzyme responsible for the conversion of glutamic acid residues (Glu) into {gamma}-carboxyglutamic acid (Gla) residues in secreted proteins. We aim here at determining the role of {gamma}-carboxylation in bone remodeling and at identifying the Gla protein(s) involved. We show that male mice lacking {gamma}-carboxylation specifically in osteoblasts (Ggcxflox/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 {gamma}-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 {gamma}-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.