Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-α 1 and -2 (Col1a1 and -2). Transforming growth factor β1 (TGF-β), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-β increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, δEF1. TGF-β treatment or short hairpin RNAs targeting δEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-β also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to δEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-β in MMC. TGF-β treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3′ UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with δEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-β and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-β-induced Col1a2 expression by down-regulating E-box repressors.

How TGF-β activates Akt in disease situations is unclear. By acting on the promotor of RP23, a non-coding RNA which encodes two microRNAs, TGF-β signalling downregulates the phospatase PTEN, a target of these microRNAs and an inhibitor of Akt signalling. Akt kinase is activated by transforming growth factor-β1 (TGF-β) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells1,2,3,4,5,6,7,8,9,10,11. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and miR-192 through E-box-regulated mechanisms, as shown previously3. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function12,13, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Diabetic patients continue to develop inflammation and vascular complications even after achieving glycemic control. This poorly understood “metabolic memory” phenomenon poses major challenges in treating diabetes. Recent studies demonstrate a link between epigenetic changes such as chromatin histone lysine methylation and gene expression. We hypothesized that H3 lysine-9 tri-methylation (H3K9me3), a key repressive and relatively stable epigenetic chromatin mark, may be involved in metabolic memory. This was tested in vascular smooth muscle cells (VSMC) derived from type 2 diabetic db / db mice. These cells exhibit a persistent atherogenic and inflammatory phenotype even after culture in vitro . ChIP assays showed that H3K9me3 levels were significantly decreased at the promoters of key inflammatory genes in cultured db / db VSMC relative to control db /+ cells. Immunoblotting demonstrated that protein levels of the H3K9me3 methyltransferase Suv39h1 were also reduced in db / db VSMC. Furthermore, db / db VSMC were hypersensitive to TNF-α inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1α was also reduced under these conditions in db / db cells. Overexpression of SUV39H1 in db / db VSMC reversed this diabetic phenotype. Conversely, gene silencing of SUV39H1 with shRNAs in normal human VSMC (HVSMC) increased inflammatory genes. HVSMC cultured in high glucose also showed increased inflammatory gene expression and decreased H3K9me3 at their promoters. These results demonstrate protective roles for H3K9me3 and Suv39h1 against the preactivated state of diabetic VSMC. Dysregulation of epigenetic histone modifications may be a major underlying mechanism for metabolic memory and sustained proinflammatory phenotype of diabetic cells.

Nuclear factor κ-B (NF-κB)-regulated inflammatory genes, such as TNF-α (tumor necrosis factor-α), play key roles in the pathogenesis of inflammatory diseases, including diabetes and the metabolic syndrome. However, the nuclear chromatin mechanisms are unclear. We report here that the chromatin histone H3-lysine 4 methyltransferase, SET7/9, is a novel coactivator of NF-κB. Gene silencing of SET7/9 with small interfering RNAs in monocytes significantly inhibited TNF-α-induced inflammatory genes and histone H3-lysine 4 methylation on these promoters, as well as monocyte adhesion to endothelial or smooth muscle cells. Chromatin immunoprecipitation revealed that SET7/9 small interfering RNA could reduce TNF-α-induced recruitment of NF-κB p65 to inflammatory gene promoters. Inflammatory gene induction by ligands of the receptor for advanced glycation end products was also attenuated in SET7/9 knockdown monocytes. In addition, we also observed increased inflammatory gene expression and SET7/9 recruitment in macrophages from diabetic mice. Microarray profiling revealed that, in TNF-α-stimulated monocytes, the induction of 25% NF-κB downstream genes, including the histone H3-lysine 27 demethylase JMJD3, was attenuated by SET7/9 depletion. These results demonstrate a novel role for SET7/9 in inflammation and diabetes.