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A new ubiquitin ligase involved in p57KIP2 proteolysis regulates osteoblast cell differentiation.

ABSTRACT: Transforming growth factor-beta1 (TGF-beta1) has many physiological functions and inhibits the differentiation of osteoblasts. Previously, we reported that TGF-beta1 stimulation induces the degradation of p57(KIP2) in osteoblasts. p57(KIP2) proteolysis depends on the ubiquitin-proteasome pathway and SMAD-mediated transcription; however, the molecular mechanism underlying p57(KIP2) degradation has been largely unknown. Here, we show that FBL12, a new F-box protein expressed in the limb bud of developing embryos, is involved in TGF-beta1-induced degradation of p57(KIP2). FBL12 formed an SCF(FBL12) complex and directly ubiquitinated p57(KIP2) in a phosphorylation-dependent manner. Inhibition of FBL12 by RNA interference suppressed the degradation of p57(KIP2) and a dominant-negative mutant of FBL12 (FBL12DeltaF) increased the steady-state level of p57(KIP2). Furthermore, wild-type FBL12 inhibited and FBL12DeltaF promoted the differentiation of primary osteoblasts. As overexpression of p57(KIP2) promoted osteoblast differentiation, these results indicate the importance of FBL12 and the degradation of p57(KIP2) in the regulation of osteoblast cell differentiation.

SUBMITTER: Kim M

PROVIDER: S-EPMC2529345 | biostudies-literature | 2008 Sep

REPOSITORIES: biostudies-literature

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A new ubiquitin ligase involved in p57KIP2 proteolysis regulates osteoblast cell differentiation.

Kim Minsoo M Nakamoto Takashi T Nishimori Shigeki S Tanaka Keiji K Chiba Tomoki T

EMBO reports 20080725 9

Transforming growth factor-beta1 (TGF-beta1) has many physiological functions and inhibits the differentiation of osteoblasts. Previously, we reported that TGF-beta1 stimulation induces the degradation of p57(KIP2) in osteoblasts. p57(KIP2) proteolysis depends on the ubiquitin-proteasome pathway and SMAD-mediated transcription; however, the molecular mechanism underlying p57(KIP2) degradation has been largely unknown. Here, we show that FBL12, a new F-box protein expressed in the limb bud of dev ...[more]

PMID: 18660753

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Project description:OBJECTIVES:Ubiquitin E3 ligase-mediated protein degradation regulates osteoblast function. Itch, an E3 ligase, affects numerous cell functions by regulating ubiquitination and proteasomal degradation of related proteins. However, the Itch-related cellular and molecular mechanisms by which osteoblast differentiation and function are elevated during bone fracture repair are as yet unknown. METHODS:We examined the expression levels of E3 ligases and NF-?B members in callus samples during bone fracture repair by quantitative polymerase chain reaction (qPCR) and the total amount of ubiquitinated proteins by Western blot analysis in wild-type (WT) mice. The expression levels of osteoblast-associated genes in fracture callus from Itch knockout (KO) mice and their WT littermates were examined by qPCR. The effect of NF-?B on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay. RESULTS:The expression levels of WW Domain Containing E3 Ubiquitin Protein Ligase 1 (Wwp1), SMAD Specific E3 Ubiquitin Protein Ligase 1 (Smurf1), SMAD Specific E3 Ubiquitin Protein Ligase 2 (Smurf2) and Itch were all significantly increased in the fracture callus of WT mice, which was associated with elevated expression of NF-?B members and total ubiquitinated proteins. Callus tissue isolated from Itch KO mice expressed higher levels of osteoblast-associated genes, including Runx2, a positive regulator of osteoblast differentiation, but osteoclast-associated genes were not increased. Both NF-?B RelA and RelB proteins were found to bind to the NF-?B binding site in the mouse Itch promoter. CONCLUSIONS:Our findings indicate that Itch depletion may have a strong positive effect on osteoblast differentiation in fracture callus. Thus, ubiquitin E3 ligase Itch could be a potential target for enhancing bone fracture healing.Cite this article: J. Liu, X. Li, H. Zhang, R. Gu, Z. Wang, Z. Gao, L. Xing. Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins. Bone Joint Res 2017;6:154-161. DOI: 10.1302/2046-3758.63.BJR-2016-0237.R1.

Dataset Information

A new ubiquitin ligase involved in p57KIP2 proteolysis regulates osteoblast cell differentiation.

Publications

A new ubiquitin ligase involved in p57KIP2 proteolysis regulates osteoblast cell differentiation.

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