Rama Natarajan

MicroRNA-192 in diabetic kidney glomeruli and its function in TGF-beta-induced collagen expression via inhibition of E-box repressors.

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.

TGF-beta activates Akt kinase through a microRNA-dependent amplifying circuit targeting PTEN.

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 cells. 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 previously. 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 function, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

Epigenetic histone H3 lysine 9 methylation in metabolic memory and inflammatory phenotype of vascular smooth muscle cells in diabetes

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.

Role of the Histone H3 Lysine 4 Methyltransferase, SET7/9, in the Regulation of NF-κB-dependent Inflammatory Genes RELEVANCE TO DIABETES AND INFLAMMATION

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.

The role of epigenetics in the pathology of diabetic complications

Diabetes is associated with significantly accelerated rates of several debilitating microvascular complications such as nephropathy, retinopathy, and neuropathy, and macrovascular complications such as atherosclerosis and stroke. While several studies have been devoted to the evaluation of genetic factors related to type 1 and type 2 diabetes and associated complications, much less is known about epigenetic changes that occur without alterations in the DNA sequence. Environmental factors and nutrition have been implicated in diabetes and can also affect epigenetic states. Exciting research has shown that epigenetic changes in chromatin can affect gene transcription in response to environmental stimuli, and changes in key chromatin histone methylation patterns have been noted under diabetic conditions. Reports also suggest that epigenetics may be involved in the phenomenon of metabolic memory observed in clinic trials and animal studies. Further exploration into epigenetic mechanisms can yield new insights into the pathogenesis of diabetes and its complications and uncover potential therapeutic targets and treatment options to prevent the continued development of diabetic complications even after glucose control has been achieved.

Magnesium deficiency produces insulin resistance and increased thromboxane synthesis.

Evidence suggests that magnesium deficiency may play an important role in cardiovascular disease. In this study, we evaluated the effects of a magnesium infusion and dietary-induced isolated magnesium deficiency on the production of thromboxane and on angiotensin II-mediated aldosterone synthesis in normal human subjects. Because insulin resistance may be associated with altered blood pressure, we also measured insulin sensitivity using an intravenous glucose tolerance test with minimal model analysis in six subjects. The magnesium infusion reduced urinary thromboxane concentration and angiotensin II-induced plasma aldosterone levels. The low magnesium diet reduced both serum magnesium and intracellular free magnesium in red blood cells as determined by nuclear magnetic resonance (186 +/- 10 [SEM] to 127 +/- 9 mM, p < 0.01). Urinary thromboxane concentration measured by radioimmunoassay increased after magnesium deficiency. Similarly, angiotensin II-induced plasma aldosterone concentration increased after magnesium deficiency. Analysis showed that all subjects studied had a decrease in insulin sensitivity after magnesium deficiency (3.69 +/- 0.6 to 2.75 +/- 0.5 min-1 per microunit per milliliter x 10(-4), p < 0.03). We conclude that dietary-induced magnesium deficiency 1) increases thromboxane urinary concentration and 2) enhances angiotensin-induced aldosterone synthesis. These effects are associated with a decrease in insulin action, suggesting that magnesium deficiency may be a common factor associated with insulin resistance and vascular disease.

Inhibiting MicroRNA-192 Ameliorates Renal Fibrosis in Diabetic Nephropathy

TGF-β1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)-modified inhibitor of miR-192, designated LNA-anti-miR-192, in mouse models of diabetic nephropathy. LNA-anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-β, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA-anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA-based translational approach to the treatment of diabetic nephropathy.

Lymphocytes From Patients With Type 1 Diabetes Display a Distinct Profile of Chromatin Histone H3 Lysine 9 Dimethylation: An Epigenetic Study in Diabetes

OBJECTIVE—The complexity of interactions between genes and the environment is a major challenge for type 1 diabetes studies. Nuclear chromatin is the interface between genetics and environment and the principal carrier of epigenetic information. Because histone tail modifications in chromatin are linked to gene transcription, we hypothesized that histone methylation patterns in cells from type 1 diabetic patients can provide novel epigenetic insights into type 1 diabetes and its complications. RESEARCH DESIGN AND METHODS—We used chromatin immunoprecipitation (ChIP) linked to microarray (ChIP-chip) approach to compare genome-wide histone H3 lysine 9 dimethylation (H3K9me2) patterns in blood lymphocytes and monocytes from type 1 diabetic patients versus healthy control subjects. Bioinformatics evaluation of methylated candidates was performed by Ingenuity Pathway Analysis (IPA) tools. RESULTS—A subset of genes in the type 1 diabetic cohort showed significant increase in H3K9me2 in lymphocytes but not in monocytes. CLTA4, a type 1 diabetes susceptibility gene, was one of the candidates displaying increased promoter H3K9me2 in type 1 diabetes. IPA identified two high-scoring networks that encompassed genes showing altered H3K9me2. Many of them were associated with autoimmune and inflammation-related pathways, such as transforming growth factor-β, nuclear factor-κB, p38 mitogen-activated protein kinase, toll-like receptor, and interleukin-6. IPA also revealed biological relationships between these networks and known type 1 diabetes candidate genes. CONCLUSIONS—The concerted and synergistic alteration of histone methylation within the identified network in lymphocytes might have an effect on the etiology of type 1 diabetes and its complications. These studies provide evidence of a novel association between type 1 diabetes and altered histone methylation of key genes that are components of type 1 diabetes–related biological pathways and also a new understanding of the pathology of type 1 diabetes.

Specific down-regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes

Diabetic nephropathy (DN) remains a major complication in both type 1 and type 2 diabetes. Systemic administration of antitransforming growth factor‐β (TGF‐β) antibody has shown some promise in mouse models of DN. However, chronic blockade of the multifunctional TGB‐β could be problematic. Several downstream effects of TGF‐β are mediated by connective tissue growth factor (CTGF), which is up‐regulated in several renal cells and secreted in the urine in the diabetic state. Using murine models of DN (type 1 and type 2) and a CTGF antisense oligonucleotide (ASO) of novel chimeric chemistry, we evaluated the specific role of this target in DN. In the type 1 model of DN, C57BL6 mice were made diabetic using streptozo‐tocin injections and hyperglycemic animals were treated with CTGF ASOs (20 mg/kg/2 qw) for 4 months. ASO, but not mismatch control oligonucleo‐tide, ‐treated animals showed significant reduction in target CTGF expression in the kidney with a concomitant decrease in proteinuria and albuminuria. Treatment with the CTGF ASO for 8 wk reduced serum creatinine and attenuated urinary albuminuria and pro‐teinuria in diabetic db/db mice, a model of type 2 DN. The ASO also reduced expression of genes involved in matrix expansion such as fibronectin and collagen (I and IV) and an inhibitor of matrix degradation, PAI‐1, in the renal cortex, contributing to significant reversal of mesangial expansion in both models of DN. Pathway analyses demonstrated that diabetes‐induced phosphory‐lation of p38 MAPK and its downstream target CREB was also inhibited by the ASO. Our results strongly suggest that blocking CTGF using a chimeric ASO holds substantial promise for the treatment of DN.—Guha, M., Xu, Z.‐G., Tung, D., Lanting, L., Natarajan, R. Specific down‐regulation of connective tissue growth factor attenuates progression of nephropathy in mouse models of type 1 and type 2 diabetes. FASEB J. 21, 3355–3368 (2007)

A microRNA circuit mediates transforming growth factor-β1 autoregulation in renal glomerular mesangial cells

Enhanced transforming growth factor-β1 (TGF-β1) expression in renal cells promotes fibrosis and hypertrophy during the progression of diabetic nephropathy. The TGF-β1 promoter is positively controlled by the E-box regulators, upstream stimulatory factors (USFs), in response to diabetic (high glucose) conditions; however, it is not clear whether TGF-β1 is autoregulated by itself. As changes in microRNAs (miRNAs) have been implicated in kidney disease, we tested their involvement in this process. TGF-β1 levels were found to be upregulated by microRNA-192 (miR-192) or miR-200b/c in mouse mesangial cells. Amounts of miR-200b/c were increased in glomeruli from type 1 (streptozotocin) and type 2 (db/db) diabetic mice, and in mouse mesangial cells treated with TGF-β1 in vitro. Levels of miR-200b/c were also upregulated by miR-192 in the mesangial cells, suggesting that miR-200b/c are downstream of miR-192. Activity of the TGF-β1 promoter was upregulated by TGF-β1 or miR-192, demonstrating that the miR-192-miR-200 cascade induces TGF-β1 expression. TGF-β1 increased the occupancy of activators USF1 and Tfe3, and decreased that of the repressor Zeb1 on the TGF-β1 promoter E-box binding sites. Inhibitors of miR-192 decreased the expression of miR-200b/c, Col1a2, Col4a1, and TGF-β1 in mouse mesangial cells, and in mouse kidney cortex. Thus, miRNA-regulated circuits may amplify TGF-β1 signaling, accelerating chronic fibrotic diseases such as diabetic nephropathy.