SH3BP2 mutations potentiate osteoclastogenesis via PLCγ.
To determine the mechanism for the increased osteoclastogenesis in the jaw of cherubism patients with SH3BP2 mutations we evaluated the effect of mutant compared to wild-type SH3BP2 on activation of osteoclast signaling pathways. Indeed mutant forms of SH3BP2 do induce greater osteoclastogenesis. Heterozygous activating mutations in exon 9 of SH3BP2 have been found in most patients with cherubism, an unusual genetic syndrome characterized by excessive remodeling of the mandible and maxilla due to spontaneous and excessive osteoclastic bone resorption. Here we have investigated the functional consequences of SH3BP2 mutations on sRANKL-induced osteoclastogenesis in RAW 264.7 pre-osteoclast cells. sRANKL-stimulated RAW 264.7 cells were transfected with wild-type or mutant SH3BP2 plasmids. NFAT-luciferase and tartrate resistant acid phosphatase (TRAP), a marker of osteoclastic differentiation, levels were evaluated. Western immunoblots were also performed to determine phosphorylation of key proteins involved in the PI-PLC pathway leading to NFATc1 translocation. Our results indicate that forced expression of mutant forms of SH3BP2, found in cherubism patients, in RAW 264.7 cells induce greater NFAT activity and greater expression of TRAP than forced expression of wild-type SH3BP2. These findings indicate that missense SH3BP2 mutations cause a gain of protein function. Moreover, over expression of SH3BP2 in RAW 264.7 cells potentiates sRANKL-stimulated phosphorylation of PLCγ1 and PLCγ2. Our studies demonstrate that cherubism is due to gain-of-function mutations in SH3BP2 that stimulate RANKL-induced activation of PLCγ. The consequent activation of calcineurin and NFAT proteins induces the excessive osteoclastic phenotype of cherubism.
Lietman
Steven A
SA
Department of Orthopaedic Surgery, Cleveland Clinic Lerner Research Institute, Cleveland, Ohio 44195, USA. lietmas@ccf.org
Yin
Lihong
L
Levine
Michael A
MA
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics* / Q000502:physiology; D000818:Animals; D002478:Cells, Cultured; D051379:Mice; D009154:Mutation; D050778:NFATC Transcription Factors / Q000378:metabolism; D010010:Osteoclasts / Q000502:physiology*; D051966:Phospholipase C gamma / Q000502:physiology*; D053245:RANK Ligand / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D050778:NFATC Transcription Factors; D053245:RANK Ligand; C484644:Sh3bp2 protein, mouse; D051966:Phospholipase C gamma