Hop2 interacts with the transcription factor CEBPα and suppresses adipocyte differentiation

Tonghui Lin,Yang Zhang,Tingting Zhang,Rita A Steckler,Xiangli Yang

doi:10.1016/j.jbc.2021.101264

Abstract

CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2−/−) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet–containing colonies formed in Hop2−/− adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator–activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes.

Highlights

The endogenous expression of homologous chromosome pairing protein 2 (Hop2) in preadipocytes and adipocytes was validated by quantitative RT–PCR (qRT–PCR) and Western blot with validated antibody (Fig. S1) analyses of differentiating primary adipocyte-derived mesenchymal stem cells and 3T3-L1 preadipocytes, and interestingly, Hop2 mRNA and protein levels are higher in undifferentiated 3T3-L1 preadipocytes than in fully differentiated adipocytes (Fig. 1, B–G)
QRT–PCR of total visceral adipose tissue (VAT) RNA revealed an overall increase ranging twofold to fourfold in the level of late adipogenic marker genes, including CEBPα, peroxisome proliferator–activated receptor γ (PPARγ), adipocyte protein 2 (aP2), diglyceride acyltransferase (Dgat), and cluster of differentiation 36 (CD36) known to be required for triglyceride storage and energy homeostasis, in Hop2−/− mice compared with controls
The same amounts of Terminal adipocyte differentiation is a process in which lineage-committed preadipocytes acquire characteristics such as the machinery for lipid transport and synthesis, insulin action, and secretion of adipocyte-specific proteins

Summary

Introduction

Ablation of Hop2 in mice leads to increased adiposity compared with wt or het (+/−) littermates, whereas, in cells, forced expression of Hop2 in preadipocytes suppresses adipocyte differentiation. To further explore at which stage(s) of adipocyte differentiation that the interaction between Hop2 and CEBPs takes place, we analyzed the temporal and dynamic expression pattern of the three CEBP bZip TFs. Western blot found that the protein expression levels of CEBPβ and CEBP-δ in 3T3-L1 cells are low in undifferentiated cultures (day 0), increased in 2-day cultures, and reduced to undetectable in 4-day cultures upon adipocyte induction (Fig. S2C).

Results

Conclusion