Abrogation of Cbl-PI3K Interaction Increases Bone Volume and Osteoblast Proliferation

Abstract

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 (not 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 the rate of the proliferation for the YF osteoblasts. This result was further substantiated by colony forming unit assay using bone marrow stromal cells. To establish the role of extracellular factors on osteoblast increased proliferation, various growth factors were assessed (EGF, FGF, IGF, PDGF). Treatment with the growth factors has no differential effects on the WT versus YF osteoblasts. We next used conditioned media from differentiated osteoclasts and bone marrow cells to treat MC3T3-E1, preosteoblast cell line. The osteoclast media from YF osteoclasts did not increase osteoblast proliferation. However, YF bone marrow conditioned media increased proliferation of the MC3T3-E1. Cytokine assays were done to determine the factor(s) that were increased in the YF conditioned media compared to the WT conditioned media. SDF-1 was found to be increased in the YF conditioned media compared to the WT conditioned media. Taken together, this suggests that the abrogation of Cbl-PI3K interaction leads to increased bone formation due to osteoclast resorption deficiency and increased osteoblast proliferation, which may be caused in part by increased SDF-1 expression in the bone marrow niche.