Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-␣ 1 and -2 (Col1a1 and -2). Transforming growth factor 1 (TGF-), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF- increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, ␦EF1. TGF- treatment or short hairpin RNAs targeting ␦EF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF- also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to ␦EF1. 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- in MMC. TGF- 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 ␦EF1 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- and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF--induced Col1a2 expression by down-regulating E-box repressors.diabetic nephropathy ͉ mesangial cells ͉ small noncoding RNA ͉ transforming growth factor 1 D iabetic nephropathy (DN) is the most common cause of kidney failure in patients with diabetes mellitus. The major characteristics of DN include glomerular basement-membrane thickening, mesangial expansion and hypertrophy, and an accumulation of extracellular matrix (ECM) proteins (1). Evidence shows that transforming growth factor 1 (TGF-) levels are increased under diabetic conditions in renal cells, including mesangial cells (MC), can up-regulate ECM proteins such as collagens (2, 3), and also can promote MC survival and oxidant stress (4).To date, Smad transcription factors have been shown to be the major effectors of TGF- signaling (5, 6). Collagen 1-␣ 1 and -2 (Col1a1 and -2) and other ECM genes are regulated in MC by TGF- via Smads (7,8). The regulation of collagen by TGF- in MC also is mediated by mitogen-activated protein kinases (MAPKs) such as p38 and ERKs (9-11). However, the molecular mechanisms by which TGF- regulates ECM genes still are not understood fully. The collagen gene has E-box elements in the far upstream enhancer region (12, 13). An E-box repressor, ␦EF1, is a key inhibitor of E-cadherin (14) and E2-box transcription factors such as Nkx2.5 (12). Moreover, it is a known repressor of collagen type 1 and type 2 genes in other cells (12, 13), but its role in MC is ...
Akt kinase is activated by transforming growth factor-beta1 (TGF-β) in diabetic kidneys and plays important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells (MC)1–11. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in MC by inducing microRNA-216a (miR-216a) and miR-217, both of which target phosphatase and tensin homologue (PTEN). Both these miRs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and also by miR-192 via E-box-regulated mechanisms as shown previously3. Akt activation by these miRs also led to MC survival and hypertrophy similar to TGF-β. These studies reveal a mechanism of Akt activation via PTEN downregulation by two miRs regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function12, 13, this miR amplifying circuit may play key roles not only in kidney disorders, but also 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-␣ inflammatory stimulus, which induced dramatic and sustained decreases in promoter H3K9me3 and Suv39h1 occupancy. Recruitment of corepressor HP1␣ 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.
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 , 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-b, 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. Diabetic nephropathy (DN) is a major microvascular complication of diabetes and the leading cause of ESRD, which can manifest despite tight glycemic control and various therapeutic interventions. 1 There is thus an imperative need to identify additional biomarkers and novel targets for better management of DN, which is clinically manifested as microalbuminuria, proteinuria, and progressive glomerular dysfunction. The key pathologic features of DN include podocyte loss, mesangial cell (MC) hypertrophy, glomerular basement membrane thickening, and tubulointerstitial fibrosis due to the increased deposition of extracellular matrix (ECM) proteins such as collagens and fibronectin (FN). 2-4 TGF-b1 is increased in several renal cells in diabetes, including MCs, and mediates these profibrotic events, hypertrophy, and cell survival. 3,5,6 Therefore, TGF-b has been evaluated as a major target for DN treatment. However, this approach could have drawbacks due to the multifunctional role of TGF-b. Hence, further evaluation of the subtle molecular mechanisms by which TGF-b regulates fibrotic events in renal cells can lead to more effective translational approaches for DN treatment.MicroRNAs (miRNAs) are small noncoding RNAs that are increasingly recognized as critical players in gene regulation and various diseases. [7][8][9][10] Interestingly, a cluster of miRNAs is reported to be highly expressed in the kidney, and recent studies show that key miRNAs are upregulated in the kidneys of diabetic mice.
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. Since 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.
The transcription factor NF-B (NF-B) plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators including histone acetyltransferases (HATs) such as CBP/p300 and p/CAF. Evidence shows that high glucose (HG) conditions mimicking diabetes can activate the transcription of NF-B-regulated inflammatory genes. However, the underlying in vivo transcription and nuclear chromatin remodeling events are unknown. We therefore carried out chromatin immunoprecipitation (ChIP) assays in monocytes to identify 1) chromatin factors bound to the promoters of tumor necrosis factor-␣ (TNF-␣) and related NF-B-regulated genes under HG or diabetic conditions, 2) specific lysine (Lys (K)) residues on histone H3 (HH3) and HH4 acetylated in this process. HG treatment of THP-1 monocytes increased the transcriptional activity of NF-B p65, which was augmented by CBP/ p300 and p/CAF. ChIP assays showed that HG increased the recruitment of NF-B p65, CPB, and p/CAF to the TNF-␣ and COX-2 promoters. Interestingly, ChIP assays also demonstrated concomitant acetylation of HH3 at Lys 9 and Lys 14 , and HH4 at Lys 5 , Lys 8 , and Lys 12 at the TNF-␣ and COX-2 promoters. Overexpression of histone deacetylase (HDAC) isoforms inhibited p65-mediated TNF-␣ transcription. In contrast, a HDAC inhibitor stimulated gene transcription and histone acetylation. Finally, we demonstrated increased HH3 acetylation at TNF-␣ and COX-2 promoters in human blood monocytes from type 1 and type 2 diabetic subjects relative to nondiabetic. These results show for the first time that diabetic conditions can increase in vivo recruitment of NF-B and HATs, as well as histone acetylation at the promoters of inflammatory genes, leading to chromatin remodeling and transcription.
It is important to find better treatments for diabetic nephropathy (DN), a debilitating renal complication. Targeting early features of DN, including renal extracellular matrix accumulation (ECM) and glomerular hypertrophy, can prevent disease progression. Here we show that a megacluster of nearly 40 microRNAs and their host long non-coding RNA transcript (lnc-MGC) are coordinately increased in the glomeruli of mouse models of DN, and mesangial cells treated with transforming growth factor-β1 (TGF- β1) or high glucose. Lnc-MGC is regulated by an endoplasmic reticulum (ER) stress-related transcription factor, CHOP. Cluster microRNAs and lnc-MGC are decreased in diabetic Chop−/− mice that showed protection from DN. Target genes of megacluster microRNAs have functions related to protein synthesis and ER stress. A chemically modified oligonucleotide targeting lnc-MGC inhibits cluster microRNAs, glomerular ECM and hypertrophy in diabetic mice. Relevance to human DN is also demonstrated. These results demonstrate the translational implications of targeting lnc-MGC for controlling DN progression.
TGF-1-induced expression of extracellular matrix (ECM) genes plays a major role in the development of chronic renal diseases such as diabetic nephropathy. Although many key transcription factors are known, mechanisms involving the nuclear chromatin that modulate ECM gene expression remain unclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation (H3Kme) in TGF-1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-␣1 [⌱], and plasminogen activator inhibitor-1. Increased levels of chromatin marks associated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks (H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-1-induced ECM gene expression. Furthermore, a TGF-1 antibody not only blocked HG-induced ECM gene expression but also reversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together, these results show the functional role of epigenetic chromatin histone H3Kme in TGF-1-mediated ECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and other therapies that reverse these modifications could have potential renoprotective effects for diabetic nephropathy.
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