Cbl is a multivalent protein that interacts with a number of signaling molecules that affect cell proliferation, migration and apoptosis. Although it is a downstream effector of growth factors, cytokines and integrin signaling all of which influence bone mass, very few studies have examined the role of Cbl in osteoblast proliferation and differentiation. To examine the role(s) of Cbl in the skeletal system we have focused specifically on phosphorylation of CblY737 since it is a unique to Cbl

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... t present on other family members) and upon phosphorylation by Src family kinases it provides a binding site for the p85 subunit of PI3K which regulates signaling events that modulate apoptosis and survival. To determine the role of tyrosine 737 we are using CblYF/YF knock-in mice (YF) where tyrosine 737 has been substituted to phenylalanine. YF mice had increased bone volume (WT 9%; YF 14%; p= 0.05 vs WT), trabecular thickness, and trabecular numbers. Although the increased bone volume is partly attributed to the decreased bone resorption, static and dynamic parameters of bone formation indicated that numbers of osteoblasts (WT 13 N.OB/BS; YF 20 N.OB/BS; p=0.05 vs WT) and bone formation rates were also upregulated in the CblYF/YF mice. To investigate the role of osteoblast differentiation in increased bone formation, we differentiated osteoblast and assessed ALP activity and Alizarin Red S staining. Both WT and YF osteoblasts had similar levels of ALP activity and mineral deposition during differentiation. To determine if the increased numbers of osteoblasts were due to increased survival and/or proliferation, we performed in vitro experiments with calvarial osteoblasts from age-matched WT and YF pups. MTT assay and TUNEL-staining, for cell viability, showed abrogation of Cbl-PI3K interaction did not affect osteoblast survival. Interestingly, inhibition of PI3K activity with LY294002 showed comparable survival between the WT and YF osteoblasts. We next examined proliferation and found that there was a 2-fold increase in t [...]