SNX10 regulates osteoclastogenic cell fusion and osteoclast size in mice.

ABSTRACT

Bone-resorbing osteoclasts (OCLs) are formed by differentiation and fusion of monocyte precursor cells, generating large multinucleated cells. Tightly regulated cell fusion during osteoclastogenesis leads to formation of resorption-competent OCLs, whose sizes fall within a predictable physiological range. The molecular mechanisms that regulate the onset of OCL fusion and its subsequent arrest are, however, largely unknown. We have previously shown that OCLs cultured from mice homozygous for the R51Q mutation in the vesicle trafficking-associated protein sorting nexin 10, a mutation that induces autosomal recessive osteopetrosis in humans and in mice, display deregulated and continuous fusion that generates gigantic, inactive OCLs. Fusion of mature OCLs is therefore arrested by an active, genetically encoded, cell-autonomous, and SNX10-dependent mechanism. To directly examine whether SNX10 performs a similar role in vivo, we generated SNX10-deficient (SKO) mice and demonstrated that they display massive osteopetrosis and that their OCLs fuse uncontrollably in culture, as do homozygous R51Q SNX10 (RQ/RQ) mice. OCLs that lack SNX10 exhibit persistent presence of DC-STAMP protein at their periphery, which may contribute to their uncontrolled fusion. To visualize endogenous SNX10-mutant OCLs in their native bone environment, we genetically labeled the OCLs of WT, SKO, and RQ/RQ mice with enhanced Green Fluorescent Protein (EGFP), and then visualized the 3D organization of resident OCLs and the pericellular bone matrix by 2-photon, confocal, and second harmonics generation microscopy. We show that the volumes, surface areas and, in particular, the numbers of nuclei in the OCLs of both mutant strains were on average 2-6-fold larger than those of OCLs from WT mice, indicating that deregulated, excessive fusion occurs in the mutant mice. We conclude that the fusion of OCLs, and consequently their size, is regulated in vivo by SNX10-dependent arrest of fusion of mature OCLs.

Osteoclasts (OCLs) are cells that degrade bone. These cells are generated by fusion of monocyte precursor cells, but the mechanisms that regulate this process and eventually arrest it are unknown. We had previously shown that OCLs cultured from mice carrying the R51Q mutation in the protein sorting nexin 10 (SNX10) lose their resorptive capacity and become gigantic due to uncontrolled fusion. To examine whether SNX10 is required for OCL fusion arrest also in vivo, we inactivated the Snx10 gene in mice and fluorescently labeled their OCLs and OCLs of R51Q SNX10 mice, isolated their femurs, and used advanced 3D microscopy methods to visualize OCLs within the bone matrix. As expected, mice lacking SNX10 exhibited excessive bone mass, indicating that their OCLs are inactive. OCLs within bones of both mutant mouse strains were on average 2­6-fold larger than in control mice and contained proportionally more nuclei. We conclude that OCL fusion is arrested in control, but not SNX10 mutant, mice, indicating that the sizes of mature OCLs are limited in vivo by an active, SNX10-dependent mechanism that suppresses cell fusion.