The major finding of the present study is that deletion of GIT1 in mice leads to a significant increase in bone mass. Histological and bone morphometric comparisons reveal that this is largely caused by an increase in trabecular number and connectivity. The high bone mass phenotype in GIT1 KO mice is predominantly the result of a defect in OC function rather than an increase in OB mediated bone formation. Although OC numbers were not changed in GIT1 KO mice compared to WT controls, OC function was impaired as indicated by decreased bone resorption in vitro
and by the presence of increased cartilage remnants within the trabecular bone. Deletion of GIT1 did not affect OB differentiation and function in vitro
and bone formation rate in vivo
. Furthermore, we demonstrate that GIT1 functions downstream of RANK signaling in a Src kinase dependent manner. Though 60% of the GIT1 KO mice are postnatally lethal due to defective pulmonary vasculature (Pang et al., 2009
), the surviving animals develop normally and exhibit normal morphology of different tissues where GIT1 is expressed. However, GIT1 KO of 10-12 weeks old mice shows an increase in bone density. This study demonstrates for the first time that GIT1 plays a crucial role in the maintenance of bone mass in vivo.
Targeted deletion of Src in mice results in severe osteopetrosis due to complete lack of OC function (Soriano et al., 1991
). Compensation by other Src family members does not occur, signifying a unique role of Src in OC function (Lowe et al., 1993
). In naïve bone marrow derived macrophages, Src expression is absent. But upon treatment with RANKL and MCSF, its expression is induced marking the commitment of the cells to the osteoclast lineage (Kim et al., 2009
). Small molecule inhibitors of Src focused on disrupting Src interaction with its substrates, adaptor proteins or proteins that determine its localization are being developed (Metcalf et al., 2002
; Sawyer et al., 2002
). But, whether the kinase activity of Src or its scaffolding function is important for OC function remains unresolved. Overexpression of kinase defective Src mutant K295M in Src KO mice reduces the osteopetrotic phenotype due to partial rescue of the OC cytoskeletal defect (Schwartzberg et al., 1997
). In contrast to these findings, several studies show that Src K295M failed to restore podosome belt and bone resorbing activity in Src KO OC, implying that its kinase activity is critical for OC function (Destaing et al., 2008
; Miyazaki et al., 2004
). In this study we demonstrate that deletion of GIT1, a substrate of Src, results in a osteopetrosis-like phenotype. Our finding suggests that the kinase activity of Src that is required for GIT1 phosphorylation is important for OC function.
Both Src and GIT1 KO OC show loss of podosome belt formation and defective OC function. However, compared to Src KO mice that completely lack OC function (Boyce et al., 1992
), G1T1 KO OCs have a 65% decrease in their OC function as observed by their decreased resorbtion on dentine slices. This suggests that there might be a compensatory mechanism involved. GIT2, an ortholog of GIT1, has recently been shown to be important for sealing zone formation in OC cultured from RAW 264.7 cells (Heckel et al., 2009
). The authors showed that knockdown of GIT2 by siRNA perturbed sealing zone formation while analogous knockdown of GIT1 had no effect. Our in vitro
and in vivo
results are in contrast with these findings. This disparity could be due to the differences in the model system used in these studies. In our study, we used OCs cultured from the BM cells of GIT1 KO mice, which are physiologically relevant. Though we did not observe up-regulation of GIT2 protein expression in GIT1 KO OC (Fig. S6
), it is possible that expression of GIT2 might be sufficient to functionally compensate for some of the functions of GIT1. We do not dispute the fact that GIT2 is important for podosome belt formation in OC, but we believe that GIT1 in combination with GIT2 regulate podosome belt formation in OC. The bone phenotype in GIT2 KO mice has not yet been described. However even in the presence of GIT2, GIT1 KO mice show increase in bone mass indicating that GIT1 is important for OC function and bone homeostasis.
RANK signaling is known to be important for cytoskeletal organization in OC, though the exact mechanism is not clearly defined (Armstrong et al., 2002
). Wong et al (Wong et al., 1999
) showed that Src is activated in a multi-step process downstream of RANK signaling. Src is constitutively bound to the RANK receptor and its basal activity is enhanced by the recruitment of the adaptor protein Traf6 to the receptor complex. Activated Src then phosphorylates and regulates several signaling proteins that mediate cell polarization, podosome belt formation and ruffle border formation. Intriguingly, we observe that GIT1 is phosphorylated in a Src dependent manner upon RANK stimulation. Loss of GIT1 did not affect all RANK signaling pathways. This suggests that GIT1 couples RANK to specific Src signaling cascades through its scaffolding action. Indeed, GIT1 KO OC show decreased phosphorylation of PLCγ2 that is tyrosine phosphorylated in Src dependent manner upon RANK stimulation (Mao et al., 2006
). Whether GIT1 can directly associate with RANK and regulate specific signaling cascades or whether this interaction is mediated by Trafs remains to be determined. Additionally, whether GIT1 can regulate immunoreceptor tyrosine based activation motif [ITAM] signaling by regulating PLCγ2 phosphorylation requires further investigation. In addition to RANK signaling, it is known that Src regulates actin cytoskeleton through Src-Syk-αvβ3-Vav3 complex (Zou et al., 2007
). Since GIT1 regulates outside-in integrin signaling in platelets (Sato et al., 2008
), it is possible that GIT1 functions downstream of RANK and integrin signaling pathways and regulates podosome belt formation.
In conclusion, our data shows that genetic deletion of GIT1 in mice leads to increases in bone mass. Our findings suggest that GIT1 maintains OC cytoskeleton integrity and function. These data identify for the first time GIT1 as a key mediator of bone homeostasis. It is possible that strategies to inhibit GIT1 function specifically in the bone can block bone degradation observed in several pathological conditions, thus suggesting GIT1 as a potential therapeutic target.