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The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPAR?.

ABSTRACT: Peroxisome proliferator-activated receptor gamma (PPAR?) is a ligand-dependent transcription factor that regulates adipocyte differentiation and glucose homeostasis. The transcriptional activity of PPAR? is regulated not only by ligands but also by post-translational modifications (PTMs). In this study, we demonstrate that a novel E3 ligase of PPAR?, tripartite motif-containing 25 (TRIM25), directly induced the ubiquitination of PPAR?, leading to its proteasome-dependent degradation. During adipocyte differentiation, both TRIM25 mRNA and protein expression significantly decreased and negatively correlated with the expression of PPAR?. The stable expression of TRIM25 reduced PPAR? protein levels and suppressed adipocyte differentiation in 3T3-L1 cells. In contrast, the specific knockdown of TRIM25 increased PPAR? protein levels and stimulated adipocyte differentiation. Furthermore, TRIM25-knockout mouse embryonic fibroblasts (MEFs) exhibited an increased adipocyte differentiation capability compared with wild-type MEFs. Taken together, these data indicate that TRIM25 is a novel E3 ubiquitin ligase of PPAR? and that TRIM25 is a novel target for PPAR?-associated metabolic diseases.

SUBMITTER: Lee JM

PROVIDER: S-EPMC6189217 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ.

Lee Jae Min JM Choi Sun Sil SS Lee Yo Han YH Khim Keon Woo KW Yoon Sora S Kim Byung-Gyu BG Nam Dougu D Suh Pann-Ghill PG Myung Kyungjae K Choi Jang Hyun JH

Experimental & molecular medicine 20181015 10

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-dependent transcription factor that regulates adipocyte differentiation and glucose homeostasis. The transcriptional activity of PPARγ is regulated not only by ligands but also by post-translational modifications (PTMs). In this study, we demonstrate that a novel E3 ligase of PPARγ, tripartite motif-containing 25 (TRIM25), directly induced the ubiquitination of PPARγ, leading to its proteasome-dependent degradation. During adip ...[more]

PMID: 30323259

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Project description:ObjectivesUbiquitin 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.MethodsWe examined the expression levels of E3 ligases and NF-κB 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-κB on Itch expression in C2C12 osteoblast cells was determined by a chromatin immunoprecipitation (ChIP) assay.ResultsThe 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-κB 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-κB RelA and RelB proteins were found to bind to the NF-κB binding site in the mouse Itch promoter.ConclusionsOur 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.

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.

Dataset Information

The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPAR?.

Publications

The E3 ubiquitin ligase TRIM25 regulates adipocyte differentiation via proteasome-mediated degradation of PPARγ.

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