Ubiquitin Ligase Cbl‐b Downregulates Bone Formation Through Suppression of IGF‐I Signaling in Osteoblasts During Denervation

Unloading can prevent bone formation by osteoblasts. To study this mechanism, we focused on a ubiquitin ligase, Cbl‐b, which was highly expressed in osteoblastic cells during denervation. Our results suggest that Cbl‐b may mediate denervation‐induced osteopenia by inhibiting IGF‐I signaling in osteoblasts. Introduction: Unloading, such as denervation (sciatic neurectomy) and spaceflight, suppresses bone formation by osteoblasts, leading to osteopenia. The resistance of osteoblasts to growth factors contributes to such unloading‐mediated osteopenia. However, a detailed mechanism of this resistance is unknown. We first found that a RING‐type ubiquitin ligase, Cbl‐b, was highly expressed in osteoblastic cells after sciatic neurectomy in mice. In this study, we reasoned that Cbl‐b played an important role in the resistance of osteoblasts to IGF‐I. Materials and Methods: Cbl‐b–deficient (Cbl‐b–/–) or wildtype (Cbl‐b+/+) mice were subjected to sciatic neurectomy. Bone formation in these mice was assessed by calcein labeling and histomorphometric analyses. We examined IGF‐I signaling molecules in femora of these mice by Western blot and immunohistochemical analyses. We also examined the mitogenic response of Cbl‐b–overexpressing or –deficient osteoblastic cells to various growth factors. Results: In Cbl‐b+/+ mice, denervation decreased femur mass and bone formation, whereas it increased the expression of Cbl‐b protein in osteoprogenitor cells and in osteocalcin‐positive cells (osteoblastic cells) in hindlimb bone. In contrast, in Cbl‐b−/− mice, bone mass and bone formation were sustained during denervation. Denervation inhibited the mitogenic response of osteoprogenitor cells most significantly to IGF‐I. Therefore, we focused on Cbl‐b–mediated modification of IGF‐I signaling. Denervation decreased the amounts of insulin receptor substrate‐1 (IRS‐1), phosphatidly inositol 3‐phosphate kinase (PI3K), and Akt‐1 proteins in femora of Cbl‐b+/+ mice, whereas the amounts of these IGF‐I signaling molecules in femora of Cbl‐b–/– mice were constant after denervation. On a cellular level, primary osteoblastic cells from Cbl‐b–/– mice were more stimulated to proliferate by IGF‐I treatment compared with those from Cbl‐b+/+ mice. Furthermore, overexpression of Cbl‐b increased ubiquitination and degradation of IRS‐1 in primary Cbl‐b–/– osteoblastic cells, leading to their impaired mitogenic response to IGF‐I. Conclusions: These results suggest that Cbl‐b induces resistance of os