SH3BP2 mutations potentiate osteoclastogenesis via PLCγ

Abstract

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.

Figure 1a

Activation of NFAT activity by over expression of SH3BP2. RAW 264.7 cells were transiently transfected with wild-type or mutant SH3BP2 and incubated with sRANKL for 48 hours. All mutant SH3BP2 proteins produced significantly greater increases in NFAT luciferase activity compared to wild-type SH3BP2 (versus wild-type, R415Q p=0.02, P418R p=0.007, D419N p=0.002, and G420E p<0.0001). This figure represents data pooled from three independent experiments.

Figure 1b

TRAP activity is increased in the presence of SH3BP2. RAW 264.7 cells were transiently transfected with mutant (R415Q, G420E, P418R or D419N) or wild-type SH3BP2 and treated with sRANKL (100ng/ml). This figure demonstrates that in cells transiently transfected with mutant SH3BP2 cDNA there is significantly increased TRAP activity compared to the wild-type control (p

Figure 1c

Mutant SH3BP2 increases the number of multinucleated giant cells (at least 3 nuclei) per high power field compared to wild-type SH3BP2. RAW 264.7 cells were transiently transfected with mutant (R415Q, G420E, P418R or D419N) or wild-type SH3BP2 and treated with sRANKL (100ng/ml). This figure demonstrates that in cells transiently transfected with mutant SH3BP2 cDNA there is significantly increased TRAP staining of multinucleated giant cells (greater than or equal to 3 nuclei) compared to the wild-type control (p< 0.05).

Figure 2

Mutant SH3BP2 increases PLCγ2 phosphorylation by immunoblot. Levels of PLCγ2 Tyr¹²¹⁷ phosphorylation and total PLCγ2 and levels of PLCγ1 Tyr⁷⁸³ phosphorylation and total PLCγ1 were examined in response to transient transfection of mutant or wild-type SH3BP2 in RAW 264.7 cells. β-actin is used as a control to ensure that equal protein was loaded in each lane. This experiment was repeated three times with the same results. WT represents cells transiently transfected with wild-type. VO represents cells transiently transfected with vector only plasmid with no SH3BP2 insert.

Figure 3

Mutant SH3BP2 does not increase Syk or Vav3 phosphorylation relative to the wild-type. Levels of Syk phosphorylation and total Syk and levels of Vav3 phosphorylation were examined in response to transient transfection of mutant or wild-type SH3BP2 in RAW 264.7 cells. β-actin is used as a control to ensure that equal protein was loaded in each lane. The mutant SH3BP2s are not more effective at Syk Tyr^525/526 or Vav3 phosphorylation compared to the wild-type. This experiment was repeated three times with the same results. WT represents cells transiently transfected with wild-type SH3BP2. VO represents cells transiently transfected with vector only plasmid with no SH3BP2 insert.