Role of miR-379 in high-fat diet-induced kidney injury and dysfunction.

Obesity is associated with increased risk for diabetes and damage to the kidneys. Evidence suggests that miR-379 plays a role in the pathogenesis of diabetic kidney disease. However, its involvement in obesity-induced kidney injury is not known and was therefore investigated in this study by comparing renal phenotypes of high-fat diet (HFD)-fed wild-type (WT) and miR-379 knockout (KO) mice. Male and female WT mice on the HFD for 10 or 24 wk developed obesity, hyperinsulinemia, and kidney dysfunction manifested by albuminuria and glomerular injuries. However, these adverse alterations in HFD-fed WT mice were significantly ameliorated in HFD-fed miR-379 KO mice. HFD feeding increased glomerular expression of miR-379 and decreased its target gene, endoplasmic reticulum (ER) degradation enhancing α-mannosidase-like protein 3 (Edem3), a negative regulator of ER stress. Relative to the standard chow diet-fed controls, expression of profibrotic transforming growth factor-ß1 (Tgf-ß1) was significantly increased, whereas Zeb2, which encodes ZEB2, a negative regulator of Tgf-ß1, was decreased in the glomeruli in HFD-fed WT mice. Notably, these changes as well as HFD-induced increased expression of other profibrotic genes, glomerular hypertrophy, and interstitial fibrosis in HFD-fed WT mice were attenuated in HFD-fed miR-379 KO mice. In cultured primary glomerular mouse mesangial cells (MMCs) isolated from WT mice, treatment with high insulin (mimicking hyperinsulinemia) increased miR-379 expression and decreased its target, Edem3. Moreover, insulin also upregulated Tgf-ß1 and downregulated Zeb2 in WT MMCs, but these changes were significantly attenuated in MMCs from miR-379 KO mice. Together, these experiments revealed that miR-379 deletion protects mice from HFD- and hyperinsulinemia-induced kidney injury at least in part through reduced ER stress.NEW & NOTEWORTHY miR-379 knockout mice are protected from high-fat diet (HFD)-induced kidney damage through key miR-379 targets associated with ER stress (Edem3). Mechanistically, treatment of mesangial cells with insulin (mimicking hyperinsulinemia) increased expression of miR-379, Tgf-ß1, miR-200, and Chop and decreases Edem3. Furthermore, TGF-ß1-induced fibrotic genes are attenuated by a GapmeR targeting miR-379. The results implicate a miR-379/EDEM3/ER stress/miR-200c/Zeb2 signaling pathway in HFD/obesity/insulin resistance-induced renal dysfunction. Targeting miR-379 with GapmeRs can aid in the treatment of obesity-induced kidney disease.

A Novel Angiotensin II-Induced Long Noncoding RNA Giver Regulates Oxidative Stress, Inflammation, and Proliferation in Vascular Smooth Muscle Cells.

RATIONALE: AngII (angiotensin II)-mediated vascular smooth muscle cell (VSMC) dysfunction plays a major role in hypertension. Long noncoding RNAs have elicited much interest, but their molecular roles in AngII actions and hypertension are unclear. OBJECTIVE: To investigate the regulation and functions of a novel long noncoding RNA growth factor- and proinflammatory cytokine-induced vascular cell-expressed RNA ( Giver), in AngII-mediated VSMC dysfunction. METHODS AND RESULTS: RNA-sequencing and real-time quantitative polymerase chain reactions revealed that treatment of rat VSMC with AngII increased the expression of Giver and Nr4a3, an adjacent gene encoding a nuclear receptor. Similar changes were observed in rat and mouse aortas treated ex vivo with AngII. RNA-FISH (fluorescence in situ hybridization) and subcellular fractionation showed predominantly nuclear localization of Giver. AngII increased Giver expression via recruitment of Nr4a3 to Giver promoter. Microarray profiling and real-time quantitative polymerase chain reaction validation in VSMC showed that Giver knockdown attenuated the expression of genes involved in oxidative stress ( Nox1) and inflammation ( Il6, Ccl2, Tnf) but increased Nr4a3. Conversely, endogenous Giver overexpression showed opposite effects supporting its role in oxidative stress and inflammation. Chromatin immunoprecipitation assays showed Giver overexpression also increased Pol II (RNA polymerase II) enrichment and decreased repressive histone modification histone H3 trimethylation on lysine 27 at Nox1 and inflammatory gene promoters. Accordingly, Giver knockdown inhibited AngII-induced oxidative stress and proliferation in rat VSMC. RNA-pulldown combined with mass spectrometry showed Giver interacts with nuclear and chromatin remodeling proteins and corepressors, including NONO (non-pou domain-containing octamer-binding protein). Moreover, NONO knockdown elicited similar effects as Giver knockdown on the expression of key Giver-regulated genes. Notably, GIVER and NR4A3 were increased in AngII-treated human VSMC and in arteries from hypertensive patients but attenuated in hypertensive patients treated with ACE (angiotensin-converting enzyme) inhibitors or angiotensin receptor blockers. Furthermore, human GIVER also exhibits partial functional conservation with rat Giver. CONCLUSIONS: Giver and its regulator Nr4a3 are important players in AngII-mediated VSMC dysfunction and could be novel targets for antihypertensive therapy.

Mini-review: emerging roles of microRNAs in the pathophysiology of renal diseases.

MicroRNAs (miRNA) are endogenously produced short noncoding regulatory RNAs that can repress gene expression by posttranscriptional mechanisms. They can therefore influence both normal and pathological conditions in diverse biological systems. Several miRNAs have been detected in kidneys, where they have been found to be crucial for renal development and normal physiological functions as well as significant contributors to the pathogenesis of renal disorders such as diabetic nephropathy, acute kidney injury, lupus nephritis, polycystic kidney disease, and others, due to their effects on key genes involved in these disease processes. miRNAs have also emerged as novel biomarkers in these renal disorders. Due to increasing evidence of their actions in various kidney segments, in this mini-review we discuss the functional significance of altered miRNA expression during the development of renal pathologies and highlight emerging miRNA-based therapeutics and diagnostic strategies for early detection and treatment of kidney diseases.

Transforming growth factor ß1 (TGF-ß1) enhances expression of profibrotic genes through a novel signaling cascade and microRNAs in renal mesangial cells.

Increased expression of transforming growth factor-ß1 (TGF-ß1) in glomerular mesangial cells (MC) augments extracellular matrix accumulation and hypertrophy during the progression of diabetic nephropathy (DN), a debilitating renal complication of diabetes. MicroRNAs (miRNAs) play key roles in the pathogenesis of DN by modulating the actions of TGF-ß1 to enhance the expression of profibrotic genes like collagen. In this study, we found a significant decrease in the expression of miR-130b in mouse MC treated with TGF-ß1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-ß receptor 1 (TGF-ßR1) was identified as a target of miR-130b. Interestingly, the RIK promoter contains three NF-Y binding sites and was regulated by NF-YC. Furthermore, NF-YC expression was inhibited by TGF-ß1, suggesting that a signaling cascade, involving TGF-ß1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-ßR1 to augment expression of TGF-ß1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-ßR1, as well as the profibrotic genes collagen type IV α 1 (Col4a1), Col12a1, CTGF, and PAI-1 were up-regulated not only in mouse MC treated with TGF-ß1 but also in the glomeruli of streptozotocin-injected diabetic mice, supporting in vivo relevance. Together, these results demonstrate a novel miRNA- and host gene-mediated amplifying cascade initiated by TGF-ß1 that results in the up-regulation of profibrotic factors, such as TGF-ßR1 and collagens associated with the progression of DN.

FOG2 protein down-regulation by transforming growth factor-ß1-induced microRNA-200b/c leads to Akt kinase activation and glomerular mesangial hypertrophy related to diabetic nephropathy.

Glomerular hypertrophy is a hallmark of diabetic nephropathy. Akt kinase activated by transforming growth factor-ß1 (TGF-ß) plays an important role in glomerular mesangial hypertrophy. However, the mechanisms of Akt activation by TGF-ß are not fully understood. Recently, miR-200 and its target FOG2 were reported to regulate the activity of phosphatidylinositol 3-kinase (the upstream activator of Akt) in insulin signaling. Here, we show that TGF-ß activates Akt in glomerular mesangial cells by inducing miR-200b and miR-200c, both of which target FOG2, an inhibitor of phosphatidylinositol 3-kinase activation. FOG2 expression was reduced in the glomeruli of diabetic mice as well as TGF-ß-treated mouse mesangial cells (MMC). FOG2 knockdown by siRNAs in MMC activated Akt and increased the protein content/cell ratio suggesting hypertrophy. A significant increase of miR-200b/c levels was detected in diabetic mouse glomeruli and TGF-ß-treated MMC. Transfection of MMC with miR-200b/c mimics significantly decreased the expression of FOG2. Conversely, miR-200b/c inhibitors attenuated TGF-ß-induced decrease in FOG2 expression. Furthermore, miR-200b/c mimics increased the protein content/cell ratio, whereas miR-200b/c inhibitors abrogated the TGF-ß-induced increase in protein content/cell. In addition, down-regulation of FOG2 by miR-200b/c could activate not only Akt but also ERK, which was also through PI3K activation. These data suggest a new mechanism for TGF-ß-induced Akt activation through FOG2 down-regulation by miR-200b/c, which can lead to glomerular mesangial hypertrophy in the progression of diabetic nephropathy.

Repression of let-7 by transforming growth factor-ß1-induced Lin28 upregulates collagen expression in glomerular mesangial cells under diabetic conditions.

Accumulation of mesangial extracellular matrix (ECM) proteins such as collagen type 1-α2 (Col1a2) and collagen type 4-α1 (Col4a1) is a key feature of diabetic nephropathy (DN). Transforming growth factor (TGF)-ß1 plays important roles in ECM accumulation in DN, and evidence shows a mediatory role for microRNAs. In the present study, we found that microRNA let-7 family members (let-7b/c/d/g/i) were downregulated in TGF-ß-treated mouse mesangial cells (MMCs) along with upregulation of Col1a2 and Col4a1. Ectopic expression of let-7b in TGF-ß-treated MMCs attenuated Col1a2 and Col4a1 upregulation. Conversely, let-7b inhibitors increased Col1a2 and Col4a1 levels. Cotransfection of MMCs with mouse Col1a2 or Col4a1 3'-untranslated region luciferase constructs and let-7b inhibitors increased luciferase activity. However, constructs with let-7 target site mutations were unresponsive to TGF-ß. TGF-ß-induced 3'-untranslated region activity was attenuated by let-7b mimics, suggesting that Col1a2 and Col4a1 are direct targets of let-7b. In addition, Lin28b, a negative regulator of let-7 biogenesis, was upregulated in TGF-ß-treated MMCs. Luciferase assays showed that the Lin28b promoter containing the Smad-binding element (SBE) responded to TGF-ß, which was abolished in constructs without SBE. Chromatin immunoprecipitation assays showed TGF-ß-induced enrichment of Smad2/3 at the Lin28b promoter, together suggesting that Lin28b is transcriptionally induced by TGF-ß through SBE. Furthermore, let-7b levels were decreased, whereas Lin28b, Col1a2, and Col4a1 levels were increased, in glomeruli of diabetic mice compared with nondiabetic control mice, demonstrating the in vivo relevance of this Lin28/let-7/collagen axis. These results identify Lin28 as a new TGF-ß target gene and suggest a novel role for the Lin28/let-7 pathway in controlling TGF-ß-induced collagen accumulation in DN.

Small RNA sequencing reveals microRNAs that modulate angiotensin II effects in vascular smooth muscle cells.

Angiotensin II (Ang II)-mediated vascular smooth muscle cell dysfunction plays a critical role in cardiovascular diseases. However, the role of microRNAs (miRNAs) in this process is unclear. We used small RNA deep sequencing to profile Ang II-regulated miRNAs in rat vascular smooth muscle cells (VSMC) and evaluated their role in VSMC dysfunction. Sequencing results revealed several Ang II-responsive miRNAs, and bioinformatics analysis showed that their predicted targets can modulate biological processes relevant to cardiovascular diseases. Further studies with the most highly induced miR-132 and miR-212 cluster (miR-132/212) showed time- and dose-dependent up-regulation of miR-132/212 by Ang II through the Ang II Type 1 receptor. We identified phosphatase and tensin homolog (PTEN) as a novel target of miR-132 and demonstrated that miR-132 induces monocyte chemoattractant protein-1 at least in part via PTEN repression in rat VSMC. Moreover, miR-132 overexpression enhanced cyclic AMP-response element-binding protein (CREB) phosphorylation via RASA1 (p120 Ras GTPase-activating protein 1) down-regulation, whereas miR-132 inhibition attenuated Ang II-induced CREB activation. Furthermore, miR-132 up-regulation by Ang II required CREB activation, demonstrating a positive feedback loop. Notably, aortas from Ang II-infused mice displayed similar up-regulation of miR-132/212 and monocyte chemoattractant protein-1, supporting in vivo relevance. In addition, microarray analysis and reverse transcriptase-quantitative PCR validation revealed additional novel miR-132 targets among Ang II-down-regulated genes implicated in cell cycle, motility, and cardiovascular functions. These results suggest that miR132/212 can serve as a novel cellular node to fine-tune and amplify Ang II actions in VSMC.

Involvement of p300/CBP and epigenetic histone acetylation in TGF-ß1-mediated gene transcription in mesangial cells.

Transforming growth factor-ß1 (TGF-ß1)-induced expression of plasminogen activator inhibitor-1 (PAI-1) and p21 in renal mesangial cells (MCs) plays a major role in glomerulosclerosis and hypertrophy, key events in the pathogenesis of diabetic nephropathy. However, the involvement of histone acetyl transferases (HATs) and histone deacetylases (HDACs) that regulate epigenetic histone lysine acetylation, and their interaction with TGF-ß1-responsive transcription factors, are not clear. We evaluated the roles of histone acetylation, specific HATs, and HDACs in TGF-ß1-induced gene expression in rat mesangial cells (RMCs) and in glomeruli from diabetic mice. Overexpression of HATs CREB binding protein (CBP) or p300, but not p300/CBP-activating factor, significantly enhanced TGF-ß1-induced PAI-1 and p21 mRNA levels as well as transactivation of their promoters in RMCs. Conversely, they were significantly attenuated by HAT domain mutants of CBP and p300 or overexpression of HDAC-1 and HDAC-5. Chromatin immunoprecipitation assays showed that TGF-ß1 treatment led to a time-dependent enrichment of histone H3-lysine9/14-acetylation (H3K9/14Ac) and p300/CBP occupancies around Smad and Sp1 binding sites at the PAI-1 and p21 promoters. TGF-ß1 also enhanced the interaction of p300 with Smad2/3 and Sp1 and increased Smad2/3 acetylation. High glucose-treated RMCs exhibited increased PAI-1 and p21 levels, and promoter H3K9/14Ac, which were blocked by TGF-ß1 antibodies. Furthermore, increased PAI-1 and p21 expression was associated with elevated promoter H3K9/14Ac levels in glomeruli from diabetic mice. Thus TGF-ß1-induced PAI-1 and p21 expression involves interaction of p300/CBP with Smads and Sp1, and increased promoter access via p300/CBP-induced H3K9/14Ac. This in turn can augment glomerular dysfunction linked to diabetic nephropathy.

TGF-ß-induced signaling circuit loops mediated by microRNAs as new therapeutic targets for renal fibrosis?

MicroRNAs (miRNAs) are emerging molecules in the pathogenesis of human diseases. Identification of miRNAs related to renal fibrosis provides clues to find new signaling pathways to fill the gaps between signaling molecules. Li et al. report another new pathway mediated by miR-433 that is induced by transforming growth factor-ß1 in mouse models of renal fibrosis. The signaling also makes a positive-feedback circuit loop, which could be translated into new therapeutic targets.

Pro-inflammatory role of microrna-200 in vascular smooth muscle cells from diabetic mice.

OBJECTIVE: Vascular smooth muscle cells (VSMC) from type 2 diabetic db/db mice exhibit enhanced proinflammatory responses implicated in accelerated vascular complications. We examined the role of microRNA(miR)-200 family members and their target Zeb1, an E-box binding transcriptional repressor, in these events. METHODS AND RESULTS: The expression levels of miR-200b, miR-200c, and miR-429 were increased, although protein levels of Zeb1 were decreased in VSMC and aortas from db/db mice relative to control db/+ mice. Transfection of miR-200 mimics into VSMC downregulated Zeb1 by targeting its 3'-UTR, upregulated the inflammatory genes cyclooxygenase-2 and monocyte chemoattractant protein-1, and promoted monocyte binding in db/+VSMC. In contrast, miR-200 inhibitors reversed the enhanced monocyte binding of db/dbVSMC. Zeb1 gene silencing with siRNAs also increased these proinflammatory responses in db/+VSMC confirming negative regulatory role of Zeb1. Both miR-200 mimics and Zeb1 siRNAs increased cyclooxygenase-2 promoter transcriptional activity. Chromatin immunoprecipitation showed that Zeb1 occupancy at inflammatory gene promoters was reduced in VSMC from type 2 diabetic db/db mice. Furthermore, Zeb1 knockdown increased miR-200 levels demonstrating a feedback regulatory loop. CONCLUSION: Disruption of the reciprocal negative regulatory loop between miR-200 and Zeb1 under diabetic conditions enhances proinflammatory responses of VSMC implicated in vascular complications.

MicroRNAs and the glomerulus.

MicroRNAs (miRNAs) are short non-coding RNAs regulating gene expression at the post-transcriptional level by blocking translation or promoting cleavage of their target mRNAs. Increasing evidence shows that miRNAs play central roles in gene transcription, signal transduction and pathogenesis of human diseases. Diabetic nephropathy (DN) is a severe microvascular complication that can lead to end-stage renal disease. Increased expansion (hypertrophy) and accumulation of extracellular matrix (ECM) proteins such as collagen (fibrosis) in the glomerular mesangium along with glomerular podocyte dysfunction are major features of DN. Profiling of miRNAs and study\ of their functions in renal glomeruli can provide critical new information to advance our knowledge of DN as well as other kidney diseases and thereby uncover much needed new therapeutic targets. In this review, we summarize the biogenesis of miRNAs and their functions in the glomerulus, with particular emphasis on glomerular mesangial cells and podocytes related to the pathogenesis of DN.

Inhibiting microRNA-192 ameliorates renal fibrosis in diabetic nephropathy.

TGF-ß1 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-ß, 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.

Identify MicroRNA Targets Using AGO2-CLASH (Cross-linking, Ligation, and Sequencing of Hybrids) and AGO2-CLIP (Cross-Linking and Immuno-Precipitation) in Cells with or Without the MicroRNA of Interest Depleted.

MicroRNAs (miRNAs) are short noncoding RNAs and important players in the regulation of gene expression through post-transcriptional mechanisms. MicroRNAs regulate many cellular processes and are involved in disease progression. Identification of novel miRNA-to-target RNA connections can fill the gaps in the signaling pathways and suggest new therapeutic targets. MiRNA targets are often predicted by base-complementarity of their seed and flanking sequences with target sequences. Direct targets can also be identified by the physical interaction between the miRNA and the target RNA using immunoprecipitation of the Argonaute (AGO) protein, a component of the RNA-induced silencing complex, followed by ligation of AGO-associated miRNA and target RNA and next generation sequencing (CLASH). Databases describing these miRNA-RNA interactions have been generated from cells commonly studied or used. However, because the regulation by miRNAs varies among organs, tissues, cell types and species, identifying relevant targets in specific cells under conditions of interest may not be available. Here, the author describes simplified methods of AGO2-CLASH and AGO2-CLIP to identify miRNA targets by comparing primary cells derived from wild-type mice and those from specific miRNA knockout mice.

Long non-coding RNA lncMGC mediates the expression of TGF-ß-induced genes in renal cells via nucleosome remodelers.

Background: MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) play key roles in diabetic kidney disease (DKD). The miR-379 megacluster of miRNAs and its host transcript lnc-megacluster (lncMGC) are regulated by transforming growth factor-ß (TGF-ß), increased in the glomeruli of diabetic mice, and promote features of early DKD. However, biochemical functions of lncMGC are unknown. Here, we identified lncMGC-interacting proteins by in vitro-transcribed lncMGC RNA pull down followed by mass spectrometry. We also created lncMGC-knockout (KO) mice by CRISPR-Cas9 editing and used primary mouse mesangial cells (MMCs) from the KO mice to examine the effects of lncMGC on the gene expression related to DKD, changes in promoter histone modifications, and chromatin remodeling. Methods: In vitro-transcribed lncMGC RNA was mixed with lysates from HK2 cells (human kidney cell line). lncMGC-interacting proteins were identified by mass spectrometry. Candidate proteins were confirmed by RNA immunoprecipitation followed by qPCR. Cas9 and guide RNAs were injected into mouse eggs to create lncMGC-KO mice. Wild-type (WT) and lncMGC-KO MMCs were treated with TGF-ß, and RNA expression (by RNA-seq and qPCR) and histone modifications (by chromatin immunoprecipitation) and chromatin remodeling/open chromatin (by Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq) were examined. Results: Several nucleosome remodeling factors including SMARCA5 and SMARCC2 were identified as lncMGC-interacting proteins by mass spectrometry, and confirmed by RNA immunoprecipitation-qPCR. MMCs from lncMGC-KO mice showed no basal or TGF-ß-induced expression of lncMGC. Enrichment of histone H3K27 acetylation and SMARCA5 at the lncMGC promoter was increased in TGF-ß-treated WT MMCs but significantly reduced in lncMGC-KO MMCs. ATAC peaks at the lncMGC promoter region and many other DKD-related loci including Col4a3 and Col4a4 were significantly lower in lncMGC-KO MMCs compared to WT MMCs in the TGF-ß-treated condition. Zinc finger (ZF), ARID, and SMAD motifs were enriched in ATAC peaks. ZF and ARID sites were also found in the lncMGC gene. Conclusion: lncMGC RNA interacts with several nucleosome remodeling factors to promote chromatin relaxation and enhance the expression of lncMGC itself and other genes including pro-fibrotic genes. The lncMGC/nucleosome remodeler complex promotes site-specific chromatin accessibility to enhance DKD-related genes in target kidney cells.

miR-379 mediates insulin resistance and obesity through impaired angiogenesis and adipogenesis regulated by ER stress.

We investigated the role of microRNA (miR-379) in the pathogenesis of obesity, adipose tissue dysfunction, and insulin resistance (IR). We used miR-379 knockout (miR-379KO) mice to test whether loss of miR-379 affects high-fat diet (HFD)-induced obesity and IR via dysregulation of key miR-379 targets in adipose tissue. Increases in body weight, hyperinsulinemia, and IR in wild-type (WT)-HFD mice were significantly attenuated in miR-379KO-HFD mice with some sex differences. Relative to control chow-fed mice, in WT-HFD mice, expression of miR-379 and C/EBP homologous protein (Chop) (pro-endoplasmic reticulum [ER] stress) and inflammation in perigonadal white adipose tissue (gWAT) were increased, whereas adipogenic genes and miR-379 target genes (Vegfb and Edem3) were decreased. These changes, as well as key parameters of brown adipose tissue dysfunction (including mitochondrial defects), were significantly attenuated in miR-379KO-HFD mice. WAT from obese human subjects with and without type 2 diabetes showed increased miR-379 and decreased miR-379 target genes. In cultured 3T3L1 pre-adipocytes, miR-379 inhibitors increased miR-379 targets and adipogenic genes. These data suggest that miR-379 plays an important role in HFD-induced obesity through increased adipose inflammation, mitochondrial dysfunction, and ER stress as well as impaired adipogenesis and angiogenesis. miR-379 inhibitors may be developed as novel therapies for obesity and associated complications.

Post-transcriptional up-regulation of Tsc-22 by Ybx1, a target of miR-216a, mediates TGF-{beta}-induced collagen expression in kidney cells.

Increased accumulation of extracellular matrix proteins and hypertrophy induced by transforming growth factor-ß1 (TGF-ß) in renal mesangial cells (MC) are hallmark features of diabetic nephropathy. Although the post-transcriptional regulation of key genes has been implicated in these events, details are not fully understood. Here we show that TGF-ß increased microRNA-216a (miR-216a) levels in mouse MC, with parallel down-regulation of Ybx1, a miR-216a target and RNA-binding protein. TGF-ß also enhanced protein levels of Tsc-22 (TGF-ß-stimulated clone 22) and collagen type I α-2 (Col1a2) expression in MC through far upstream enhancer E-boxes by interaction of Tsc-22 with an E-box regulator, Tfe3. Ybx1 colocalized with processing bodies in MC and formed a ribonucleoprotein complex with Tsc-22 mRNA, and this complex formation was reduced by TGF-ß, miR-216a mimics, or Ybx1 shRNA to increase Tsc-22 protein levels but enhanced by miR-216a inhibitor oligonucleotides. Chromatin immunoprecipitation (ChIP) assays revealed that TGF-ß could increase the occupancies of Tsc-22 and Tfe3 on enhancer E-boxes of Col1a2. Co-immunoprecipitation assays revealed that TGF-ß promoted the interaction of Tsc-22 with Tfe3. These results demonstrate that post-transcriptional regulation of Tsc-22 mediated through Ybx1, a miR-216a target, plays a key role in TGF-ß-induced Col1a2 in MC related to the pathogenesis of diabetic nephropathy.

A microRNA circuit mediates transforming growth factor-ß1 autoregulation in renal glomerular mesangial cells.

Enhanced transforming growth factor-ß1 (TGF-ß1) expression in renal cells promotes fibrosis and hypertrophy during the progression of diabetic nephropathy. The TGF-ß1 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-ß1 is autoregulated by itself. As changes in microRNAs (miRNAs) have been implicated in kidney disease, we tested their involvement in this process. TGF-ß1 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-ß1 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-ß1 promoter was upregulated by TGF-ß1 or miR-192, demonstrating that the miR-192-miR-200 cascade induces TGF-ß1 expression. TGF-ß1 increased the occupancy of activators USF1 and Tfe3, and decreased that of the repressor Zeb1 on the TGF-ß1 promoter E-box binding sites. Inhibitors of miR-192 decreased the expression of miR-200b/c, Col1a2, Col4a1, and TGF-ß1 in mouse mesangial cells, and in mouse kidney cortex. Thus, miRNA-regulated circuits may amplify TGF-ß1 signaling, accelerating chronic fibrotic diseases such as diabetic nephropathy.

Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing.

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.

Epigenetic histone methylation modulates fibrotic gene expression.

TGF-ß1-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-ß1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-ß1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-α1[Ι], 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-ß1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-ß1-induced ECM gene expression. Furthermore, a TGF-ß1 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-ß1-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.

Effects of repetitive insertion/removal cycles and simulated occlusal loads on retention of denture retainers.

The purpose of this study was to investigate the effects of repetitive insertion/removal cycle tests on denture retainers with simulated occlusal loads on the retentive force and deformation of clasp. Abutment teeth in the form of mandibular secondary premolars and clasp in the form of Akers clasps were prepared. The retentive force of the clasp on the abutment teeth were evaluated before and after undergoing repetitive insertion/removal cycle tests with or without cyclic loading. Changes in the clasp shape were monitored using a 3D scanner and scanning electron microscope. The initial retentive force was approximately 10 N and this value later decreased due to deformation of the clasp tips. In contrast to the non-load group, the load group exhibited a reduction in retentive force during earlier stages. Therefore, cyclic loading was related to a decrease in retentive forces, specifically in the early stages of repetitive insertion/removal cycles.