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.


BACKGROUND
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.


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 osteoblasts to IGF-I during denervation by increasing IRS-1 degradation and that Cbl-b-mediated modification of IGF-I signaling may contribute to decreased bone formation during denervation.
Suzue
Naoto
N
Department of Orthopedics, The University of Tokushima School of Medicine, Japan.
Nikawa
Takeshi
T
Onishi
Yuko
Y
Yamada
Chiharu
C
Hirasaka
Katsuya
K
Ogawa
Takayuki
T
Furochi
Harumi
H
Kosaka
Hirofumi
H
Ishidoh
Kazumi
K
Gu
Hua
H
Takeda
Shin'ichi
S
Ishimaru
Naozumi
N
Hayashi
Yoshio
Y
Yamamoto
Hironori
H
Kishi
Kyoichi
K
Yasui
Natsuo
N
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D001846:Bone Development / Q000502:physiology*; D002478:Cells, Cultured; D017931:DNA Primers; D003714:Denervation; D015536:Down-Regulation / Q000502:physiology*; D006868:Hydrolysis; D007150:Immunohistochemistry; D007334:Insulin-Like Growth Factor I / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D010006:Osteoblasts / Q000378:metabolism*; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000502:physiology*
D017931:DNA Primers; D025801:Ubiquitin; D007334:Insulin-Like Growth Factor I; D044767:Ubiquitin-Protein Ligases