Phospholipase Cγ2 Modulates Integrin Signaling in the Osteoclast by Affecting the Localization and Activation of Src Kinase

Abstract

Integrin engagement induces a cascade of signaling pathways that include tyrosine phosphorylation of numerous proteins that lead to modulation of the actin cytoskeleton. Src is a major intracellular mediator of integrin-dependent functions, but the mechanism(s) by which Src is regulated in response to integrin signals is not fully understood. Here, we demonstrate an important role for phospholipase C gamma 2 (PLC␥2) in Src activation in the osteoclast. Through analysis of primary cells from PLC␥2 ؊/؊ mice, PLC␥2 was found to be an important regulator of ␣ v ␤ 3 integrin-mediated bone osteoclast cell adhesion, migration, and bone resorption. Adhesion-induced PYK2 and Src phosphorylation is decreased in the absence of PLC␥2, and the interaction of Src with ␤ 3 integrin and PYK2 is dramatically reduced. Importantly, PLC␥2 was found to be required for proper localization of Src to the sealing actin ring, and this function required both its catalytic activity and adapter domains. Based on these results, we propose that PLC␥2 influences Src activation by mediating the localization of Src to the integrin complex and thereby regulating integrin-mediated functions in the osteoclast.

Type I Phosphotidylinosotol 4-Phosphate 5-Kinase γ Regulates Osteoclasts in a Bifunctional Manner*

“…These observations raised the possibility that excess PI(4,5)P 2 synthesis may, similar to its deficiency, arrest osteoclastogenesis. This hypothesis is in keeping with the anti-osteoclastogenic effects of a likely cause of PI(4,5)P 2 accumulation, namely absence of phospholipase C␥2, which also disrupts organization of the cytoskeleton of the cell (7,8). In fact, while the magnitude of the 18:0/20:4-PIP 2 peak in empty vector-transduced BMMs is similar to that of WT, overexpressed PIP5KI␥1 and PIP5KI␥2 increases it by ϳ2 orders of magnitude (supplemental Table 1) and markedly alters the facade of PIP5KI␥ Ϫ/Ϫ osteoclasts.…”

“…Additionally, PI(4,5)P 2 and PIP 3 interact with gelsolin, thereby regulating podosome assembly and signaling in the bone resorptive cell (3,25). Finally, phospholipase C␥2, which metabolizes PI(4,5)P 2 to diacylglycerol and PIP 3 , modulates osteoclast formation and function (7,8).…”

“…PI(4,5)P 2 also regulates osteoclast cytoskeletal organization by interacting with gelsolin and WASP to control podosome signaling and formation of actin rings which isolate the resorptive milieu from the general extracellular space (3). However, phospholipase C␥2 deletion, which presumably increases PI(4,5)P 2 by preventing its metabolism, blunts the capacity of the cell to organize its cytoskeleton via the ␣v␤3 integrin and thus degrades bone (8,9). PI(4,5)P 2 is principally synthesized by phosphorylation of phosphatidylinositol-4 phosphate at the 5-position of the inositol ring via type I phosphatidylinositol 4-phosphate 5 kinase (PIP5KI).…”

Targeted Inhibition of Phospholipase C γ2 Adaptor Function Blocks Osteoclastogenesis and Protects from Pathological Osteolysis

“…Bone Resorption-Analysis of bone resorption was completed as described previously (18). Briefly, BMMs were plated on bovine bone slices and cultured with 0.01 CMG14-12 and 100 ng/ml GST-RANKL for 10 days.…”

“…Cells with a point mutation in the SH2 domain of PLC␥2 are incapable of in vitro OCG despite intact catalytic function (18). Thus, we hypothesized that the scaffolding function of endogenous PLC␥2 could be disrupted through a dominant-negative effect by a molecule encompassing the adaptor domains of PLC␥2.…”