PLCγ2 regulates osteoclastogenesis via its interaction with ITAM proteins and GAB2

Abstract

Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cγ (PLCγ) isoforms are known modulators of T and B lymphocyte-mediated immune responses, we found that blockade of PLCγ enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCγ2, independent of PLCγ1, was required for receptor activator of NF-κB ligand-induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator protein-1 (AP1), and NF-κB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCγ2 downstream of Dap12/Fc receptor γ (Dap12/FcRγ) receptors and is blocked by the PLCγ inhibitor U73122. In contrast, activation of JNK and NF-κB was not affected by U73122 or Dap12/FcRγ deletion. Interestingly, we found that in osteoclasts, PLCγ2 formed a complex with the regulatory adapter molecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCγ2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy.

Early estrogen-induced gene 1, a novel RANK signaling component, is essential for osteoclastogenesis

“…It has been reported that RANK associates with Gab2 and PLCγ2 upon RANKL stimulation [10,11,20]. As our data indicate that EEIG1 physically interacts with RANK, we asked whether endogenous EEIG1, Gab2 and PLCγ2 form complexes with RANK.…”

“…It has become clear that RANK transduces key signals by directly interacting with TRAF6 [24,25]. In addition, it has been reported that PLCγ2 binds to Gab2 in response to RANKL, mediates Gab2 recruitment to RANK, and is required for osteoclast formation [10,20]. Moreover, PLCγ2 and Tec/ Btk kinases are activated by RANK and ITAM signals, thereby activating calcium signaling required for the induction of NFATc1, the key transcription factor for osteoclast differentiation [11,16,26].…”

“…Given that PLCγ2 is required for the activation of osteoclastogenic signaling pathways downstream of RANK [10,16], we investigated whether EEIG1 is potentially involved in regulating the tyrosine phosphorylation of PLCγ2 induced upon RANKL stimulation. We found that overexpression of EEIG1 increased PLCγ2 phosphoryla- Figure 7B).…”

“…Recent findings indicate that the activation of JNK and NF-κB pathways requires the phosphorylation of growth factor receptor-bound protein 2 (Grb2)-binding adaptor protein (Gab2), and its subsequent recruitment to RANK. Moreover, phospholipase Cγ2 (PLCγ2) forms a complex with Gab2, is required for Gab2 phosphorylation and modulates Gab2 recruitment to RANK [10,11].…”

“…These adaptors associate with osteoclast-associated receptor (OSCAR), an immunoglobulin-like receptor [12][13][14]. Phosphorylation of ITAM-containing adaptors results in the recruitment of spleen tyrosine kinase (Syk), leading to the activation of PLCγ and subsequent calcium mobilization [10,12,13,15]. Therefore, osteoclastogenesis is regulated by both RANK and ITAM signalings.…”