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Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

ABSTRACT: 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.

SUBMITTER: Liu J

PROVIDER: S-EPMC5376659 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

Liu J J Li X X Zhang H H Gu R R Wang Z Z Gao Z Z Xing L L

Bone & joint research 20170301 3

<h4>Objectives</h4>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.<h4>Methods</h4>We examined the expression levels of E3 ligases and NF-κB members in callus samples du ...[more]

PMID: 28298321

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Project description:CCAAT/enhancer-binding protein alpha (C/EBP?) is an important transcription factor involved in granulocytic differentiation. Here, for the first time we demonstrate that E6-associated protein (E6AP), an E3 ubiquitin ligase targets C/EBP? for ubiquitin-mediated proteasome degradation and thereby negatively modulates its functions. Wild-type E6AP promotes ubiquitin dependent proteasome degradation of C/EBP?, while catalytically inactive E6-associated protein having cysteine replaced with alanine at amino-acid position 843 (E6AP-C843A) rather stabilizes it. Further, these two proteins physically associate both in non-myeloid (overexpressed human embryonic kidney epithelium) and myeloid cells. We show that E6AP-mediated degradation of C/EBP? protein expression curtails its transactivation potential on its target genes. Noticeably, E6AP degrades both wild-type 42?kDa CCAAT-enhancer-binding protein alpha (p42C/EBP?) and mutant isoform 30?kDa CCAAT-enhancer-binding protein alpha (p30C/EBP?), this may explain perturbed p42C/EBP?/p30C/EBP? ratio often observed in acute myeloid leukemia (AML). We show that overexpression of catalytically inactive E6AP-C843A in C/EBP? inducible K562- p42C/EBP?-estrogen receptor (ER) cells inhibits ?-estradiol (E2)-induced C/EBP? degradation leading to enhanced granulocytic differentiation. This enhanced granulocytic differentiation upon E2-induced activation of C/EBP? in C/EBP? stably transfected cells (?-estradiol inducible K562 cells stably expressing p42C/EBP?-ER (K562-C/EBP?-p42-ER)) was further substantiated by siE6AP-mediated knockdown of E6AP in both K562-C/EBP?-p42-ER and 32dcl3 (32D clone 3, a cell line widely used model for in vitro study of hematopoietic cell proliferation, differentiation, and apoptosis) cells. Taken together, our data suggest that E6AP targeted C/EBP? protein degradation may provide a possible explanation for both loss of expression and/or functional inactivation of C/EBP? often experienced in myeloid leukemia.

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Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

Publications

Ubiquitin E3 ligase Itch negatively regulates osteoblast function by promoting proteasome degradation of osteogenic proteins.

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