Loss of Cbl-PI3K interaction enhances osteoclast survival due to p21-Ras mediated PI3K activation independent of Cbl-b.

Bone remodeling is a dynamic process, which involves resorption by osteoclasts and formation by osteoblasts. Perturbation of bone remodeling process leads to diseases, including osteoporosis and osteopetrosis [Teitelbaum, 2000]. The differentiation, survival and function of osteoclasts are tightly regulated by cell signaling events. Cbl family proteins, Cbl and Cbl-b, are E3 ubiquitin ligases with adaptor function that are abundantly expressed in osteoclasts and osteoblasts [Horne et al., 2005; Severe et al., 2013]. E3 ubiquitin ligase function of Cbl proteins facilitates negative regulation of cell signaling by targeting the phosphorylated receptor and non-receptor kinases and associated signaling molecules for proteasomal degradation by ubiquitylation system. The adaptor function of Cbl proteins facilitates signaling from several receptors including c-Fms, RANK and integrins, by forming molecular complexes via protein–protein interactions [Thien and Langdon, 2005; Swaminathan and Tsygankov, 2006]. In the skeletal system, Cbl and Cbl-b have been demonstrated to perform distinct functions in regulating bone formation and bone resorption [Horne et al., 2005; Severe et al., 2013]. In mice, the absence of both proteins resulted in embryonic lethality before day 10.5, suggesting overlapping functions during embryonic development [Naramura et al., 2002]. However, the absence of either protein resulted in distinct outcomes in skeletal tissue. Lack of Cbl protein resulted in decreased osteoclast migration and a developmental delay but adult mice had no overt skeletal phenotype [Chiusaroli et al., 2003]. In contrast, adult Cbl-b−/− mice had decreased bone volume because of hyper-resorptive osteoclasts [Nakajima et al., 2009]. The structural differences in their C-terminal half have been attributed to distinct functional roles of Cbl and Cbl-b, since the N-terminal half, which harbors the tyrosine kinase binding (TKB) domain and the RING finger domain, are highly conserved [Swaminathan and Tsygankov, 2006]. Both Cbl and Cbl-b undergo phosphorylation down stream of tyrosine kinase signaling pathways. However, the tyrosine phosphorylation site Y737 (Y731 in human) is unique to Cbl (not present on Cbl-b) and has a downstream sequence (EAM) that provides a docking site for the SH2 domain of the p85 regulatory subunit of phosphatidylinositol-3-kinase (PI3K) [Songyang and Cantley, 1995; Songyang and Cantley, 2004]. PI3Ks are lipid kinases that phosphorylate phosphoinositides at 3′ position. Members of the class IA PI3K are made up of a heterodimeric p110 catalytic subunit and a p85 regulatory subunit. The p110 catalytic subunit binds to the p85 regulatory subunit with high affinity preventing the activation of PI3K. Upon engagement of the SH2 domain of the p85 subunit through binding to phosphorylated tyrosines of signaling proteins, the inhibitory effect of p85 on p110 is released resulting in increased lipid kinase activity [Foukas and Okkenhaug, 2003]. PI3K has been demonstrated to be important for osteoclast differentiation, survival and bone resorption due to its involvement downstream of RANK, c-Fms, and avb3 integrin signaling [Golden and Insogna, 2004]. p85−/− mice are osteopetrotic due to defective osteoclast function [Munugalavadla et al., 2008]. In addition, inhibition of PI3K activity decreased the bone resorbing activity of osteoclasts [Nakamura et al., 1995; Lakkakorpi et al., 1997]. Recent work by Shinohara et al. 2012 showed that osteoclast-specific deletion of the p85 genes results in an osteopetrotic phenotype caused by a defect in the bone-resorbing activity of osteoclasts [Shinohara et al., 2012]. Studies in a number of cell types have shown that stimulation through tyrosine kinase coupled receptors induces prominent association between Cbl and the p85 subunit of PI3K. In turn, a significant proportion of PI3K activity is attributable to Cbl–PI3K interaction following receptor stimulation [Ueno et al., 1998; Feshchenko et al., 1999; Hunter et al., 1999]. In contrast to this phosphorylation-dependent interaction of Cbl, the proline rich region of Cbl-b provides constitutive association with the p85 subunit of PI3K [Fang et al., 2001]. In osteoclasts, PI3K is activated and translocated to the actin-rich cytoskeletal fraction upon adhesion to matrix proteins [Lakkakorpi et al., 1997]. In macrophages, Src family kinase-mediated phosphorylation of Cbl is required for Cbl–PI3K association and for the transfer of this complex to the actin rich-cytoskeleton [Meng and Lowell, 1998]. Cbl–PI3K interaction is also critical for the spreading and motility of transformed fibroblasts [Feshchenko et al., 1999]. In osteoclasts, over-expression of CblY737F protein, which does not interact with PI3K, reduced in vitro bone resorbing activity of osteoclasts by 80% [Miyazaki et al., 2004]. In agreement with these observations, our recent work demonstrated that Cbl knock-in mice with a tyrosine to phenylalanine substitution at position 737, which prevents Cbl–PI3K interaction (YF mice), showed decreased osteoclast function and increased bone volume [Adapala et al., 2010a,b; Brennan et al., 2011]. Increased bone volume in the YF mice was in contrast to adult Cbl−/− mice, which do not show an overt bone phenotype, probably due to compensation by Cbl-b protein [Chiusaroli et al., 2003]. Characterization of YF osteoclasts also highlighted a negative role for the Cbl–PI3K interaction in osteoclastogenesis and survival. Loss of Cbl–PI3K interaction resulted in a two-fold increase in the number of osteoclasts in YF mice. Furthermore, the ex vivo differentiation of precursors into osteoclasts and the survival of mature osteoclasts was enhanced. At the molecular level, these effects were attributed to perturbed RANKL-mediated signaling [Adapala et al., 2010a]. In this report we evaluated: (1) whether Cbl-b influences the enhanced osteoclast survival due to increased PI3K activity observed in the absence of Cbl–PI3K interaction; (2) the molecular mechanism responsible for the increased PI3K activity in the absence of Cbl–PI3K interaction and (3) whether the YF mutation on Cbl has a dominant-negative role in osteoclast function for both Cbl and Cbl-b proteins. Our results demonstrate that irrespective of the presence of Cbl-b, (1) loss of Cbl–PI3K interaction resulted in up regulation of RANK signaling and increased PI3K activity in osteoclasts. (2) The survival of osteoclasts is enhanced due to increased Ras activity in the absence of Cbl–PI3K interaction; inhibition of Ras activation attenuated the enhanced PI3K signaling and abrogated enhanced survival resulted from the loss of Cbl–PI3K interaction. (3) The role of Cbl–PI3K interaction in bone resorption by osteoclasts is distinct from Cbl-b since loss of Cbl–PI3K interaction in Cbl-b−/− mice (YF/YF;Cbl-b−/−) did not prevent osteopenia due to hyperactivity of osteoclasts observed in Cbl-b−/− mice. Thus, we provide direct evidence that Cbl and Cbl-b play distinct and mostly independent roles in osteoclast-mediated bone remodeling in which Cbl-b negatively regulates bone resorption, while Cbl–PI3K interaction regulates survival of osteoclasts mediated by RANK signaling.