The molecular and functional interplay between the osteopetrosis-associated proteins SNX10, OSTM1, and CLC-7 during...

Bone-resorbing osteoclasts (OCLs) are large, multi-nucleated cells that are formed through well-regulated differentiation and cell fusion of monocyte-macrophage precursors. Abnormally increased or decreased OCL-mediated bone resorption perturbs bone structure and homeostasis and may lead to severe illnesses, such as osteoporosis and autosomal recessive osteopetrosis (ARO), respectively. Mutations in the intracellular trafficking-associated protein sorting nexin 10 (SNX10) lead to "OCL-rich" ARO, in which OCLs are inactive. Mature, SNX10-deficient murine OCLs fuse continuously to generate gigantic cells, in vitro and in vivo, unlike WT OCLs that stop fusing with each other upon maturation, indicating that SNX10 is required for both the resorptive activity of OCLs and the arrest of cell fusion upon maturation. Mutations in CLC-7 and OSTM1, which comprise the lysosomal voltage-gated Cl-/H+ exchanger, also induce OCL-rich ARO in humans and in mouse models, and are associated with the presence of large OCLs. In this study, we explored the molecular interplay between SNX10, CLC-7, and OSTM1 by comparing the phenotypes of cultured mouse OCLs lacking one of these proteins. We show that loss of each protein leads to the formation of similarly-gigantic OCLs in culture, due to deregulated fusion between mature OCLs that proceeds with similar kinetics. All 3 proteins co-localize in LAMP1-positive lysosomes, located at both perinuclear and peripheral regions of mature WT OCLs. SNX10-KO OCLs exhibit few peripheral lysosomes containing CLC-7 and OSTM1, indicating that SNX10 is required for regulating their trafficking to the cell periphery. CLC-7 and SNX10 physically interact with each other and loss of CLC-7 depletes peripheral OSTM1-containing lysosomes, indicating that CLC-7 is also required for this transport. Taken together, these findings indicate that SNX10 and CLC-7 regulate the subcellular distribution of lysosomes containing CLC-7 and OSTM1, thereby establishing a functional link between these 3 proteins that controls both the fusion and functionality of mature OCLs.