Dysregulation of histone H3 lysine 27 trimethylation in transforming growth factor-β1–induced gene expression in mesangial cells and diabetic kidney

Sadhan Das,Maryam Abdollahi,Erli Zhang,Linda Lanting,Marpadga A Reddy,Hang Yuan,Ye Jia,Hyung Jung Oh,Mei Wang,Rama Natarajan

doi:10.1074/jbc.ra119.007575

Abstract

Transforming growth factor-β1 (TGF-β)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN). TGF-β regulates gene expression in MCs by altering key chromatin histone modifications at target gene promoters. However, the role of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification is unclear. Here we show that TGF-β reduces H3K27me3 at the Ctgf, Serpine1, and Ccl2 gene promoters in rat MCs (RMCs) and reciprocally up-regulates the expression of these pro-fibrotic and inflammatory genes. In parallel, TGF-β down-regulates Enhancer of Zeste homolog 2 (Ezh2), an H3K27me3 methyltransferase, and decreases its recruitment at Ctgf and Ccl2 but not Serpine1 promoters. Ezh2 knockdown with siRNAs enhances TGF-β-induced expression of these genes, supporting its repressive function. Mechanistically, Ezh2 down-regulation is mediated by TGF-β-induced microRNA, miR-101b, which targets Ezh2 3'-UTR. TGF-β also up-regulates Jmjd3 and Utx in RMCs, suggesting a key role for these H3K27me3 demethylases in H3K27me3 inhibition. In RMCs, Utx knockdown inhibits hypertrophy, a key event in glomerular dysfunction. The H3K27me3 regulators are similarly altered in human and mouse MCs. High glucose inhibits Ezh2 and increases miR-101b in a TGF-β-dependent manner. Furthermore, in kidneys from rodent models of DN, fibrotic genes, miR-101b, and H3K27me3 demethylases are up-regulated, whereas Ezh2 protein levels as well as enrichment of Ezh2 and H3K27me3 at target genes are decreased, demonstrating in vivo relevance. These results suggest that H3K27me3 inhibition by TGF-β via dysregulation of related histone-modifying enzymes and miRNAs augments pathological genes mediating glomerular mesangial dysfunction and DN.

Highlights

Transforming growth factor-␤1 (TGF-␤)-induced fibrotic and inflammatory genes in renal mesangial cells (MCs) play important roles in glomerular dysfunction associated with diabetic nephropathy (DN)
Immunofluorescence staining with Enhancer of Zeste homolog 2 (Ezh2) antibodies showed that nuclear Ezh2 levels were reduced by TGF-␤ treatment versus control (Fig. 1, D and E)
We observed that TGF-␤ can induce pathologic genes in MCs via reducing enrichment of the epigenetic repressive mark H3K27me3 at gene promoters

Summary

Results

To evaluate whether Ezh modulates TGF-␤–induced fibrotic and inflammatory genes related to glomerular MC dysfunction, we first examined the expression and function of Ezh in rat MCs (RMCs). Examination of expression of Ezh and other TGF-␤ target genes 72 h post-transfection showed that siEzh treatment resulted in significant inhibition of Ezh mRNA (Fig. 3A) and protein levels (Fig. 3, B and C) relative to siNTC in both control and TGF-␤–treated RMCs. Fig. Ezh knockdown significantly enhanced TGF-␤–induced expression of all three genes, Ctgf, Serpine, and Ccl, compared with siNTC-transfected RMCs (Fig. 3, D–F). HG (72 h) significantly reduced Ezh protein in RMCs pretreated with IgG, but this effect of HG was ameliorated in RMCs pretreated with TGF-␤–neutralizing antibodies compared with control IgG (Fig. 7, E and F) These results demonstrate that HG down-regulates Ezh in a TGF-␤– dependent manner in RMCs. we examined the role of Ezh and Utx on mesangial cell hypertrophy, a key event in renal dysfunction in DN.

E SERPINE1

Discussion

Experimental procedures