Transforming growth factor β1 (TGF-β1) enhances expression of profibrotic genes through a novel signaling cascade and microRNAs in renal mesangial cells - PubMed (original) (raw)

Transforming growth factor β1 (TGF-β1) enhances expression of profibrotic genes through a novel signaling cascade and microRNAs in renal mesangial cells

Nancy E Castro et al. J Biol Chem. 2014.

Abstract

Increased expression of transforming growth factor-β1 (TGF-β1) 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-β1 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-β1. In parallel, there was a down-regulation in miR-130b host gene 2610318N02RIK (RIK), suggesting host gene-dependent expression of this miRNA. TGF-β receptor 1 (TGF-βR1) 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-β1, suggesting that a signaling cascade, involving TGF-β1-induced decreases in NF-YC, RIK, and miR-130b, may up-regulate TGF-βR1 to augment expression of TGF-β1 target fibrotic genes. miR-130b was down-regulated, whereas TGF-βR1, 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-β1 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-β1 that results in the up-regulation of profibrotic factors, such as TGF-βR1 and collagens associated with the progression of DN.

Keywords: Collagen; Diabetes; Kidney; MicroRNA (miRNA); Signaling.

© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

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Figures

FIGURE 1.

FIGURE 1.

Decreased expression of miR-130b and let-7b in TGF-β1-treated MMC. A–C, quantitative PCR analysis of miR-130b, let-7b, and miR-30e* expression levels in SD MMC treated without or with 10 ng/ml TGF-β1 for 6 and 24 h. D, schematic representation of miR-30e* seed sequence alignment and Col12a1 3′-UTR. E–H, _TGF-β_R1, Col12a1, PAI-1, and CTGF mRNA levels were measured in MMC at 6 and 24 h of TGF-β1 treatment. _TGF-β_R1 and Col12a1 expression levels were significantly increased at 24 h of TGF-β1 treatment, whereas PAI-1 and CTGF mRNA levels were increased at both time periods. Results for miRNAs were normalized with internal control 18 S. *, **, and ***, p < 0.05, p < 0.005, and p < 0.0005, respectively, versus SD control condition. Expression of other genes were normalized to CypA internal control. Results shown are mean ± S.E. (error bars) (n = 3). *, **, and ***, p < 0.05, p < 0.005, and p < 0.0005, respectively, versus SD control condition.

FIGURE 2.

FIGURE 2.

TGF-β1 induces TGF-βR1 expression via down-regulation of miR-130b and let-7b. A, schematic diagram showing several 3′-UTR luciferase reporter constructs, which include WT-TGF-βR1 3′-UTR (with WT intact let-7b and miR-130b target sites), Mut-miR-130b binding site, Mut-let-7b binding site, and double mutant (DM; where both miR-130b and let-7b binding sites have been mutated). B, schematic representation of miR-130b seed sequence alignment to the TGF-βR1 3′-UTR. C, luciferase reporter assays using WT-TGF-βR1 3′-UTR in MMC transfected with miR-130b mimic (M) or miR-130b inhibitor (I). Transfection with miR-130b-M decreased WT-TGF-βR1 3′-UTR activity; conversely, WT-TGF-βR1 3′-UTR reporter activity was increased with miR-130b-I relative to negative control (NC). D, luciferase reporter assay in cells co-transfected with WT-TGF-βR1 3′-UTR or Mut-let-7b with either negative control mimic (NC-M) or let-7b mimic (M) in the presence or absence of 10 ng/ml TGF-β1 for 24 h in MMC. E, luciferase reporter assays in MMC transfected with WT-TGF-βR1 3′-UTR, Mut-let7b, Mut-130b, or double mutant in the presence or absence of TGF-β1 for 24 h. F, MMC transfected with miR-130del mutant elicited higher basal activity and greater TGF-β1-induced activity compared with WT- TGF-βR1. G, luciferase reporter assay in cells co-transfected with WT-TGF-βR1 3′-UTR and either NC-M, let-7b-M, 130b-M, or double mimic (D-M; where both let-7b-M and 130b-M were transfected) and in cells with Mut-let-7b co-transfected with NC-M or 130b-M. H, luciferase reporter assays showing increased basal and TGF-β1-induced TGF-βR1 3′-UTR activity in MMC co-transfected with the double mutant construct and either NC-M, 130b-M, or let-7b-M compared with WT control. Results shown are mean ± S.E. (error bars) (n = 3). *, **, and ***, p < 0.05, p < 0.005, and p < 0.0005, respectively, versus WT or NC-M conditions.

FIGURE 3.

FIGURE 3.

miR-130b and let-7b down-regulate endogenous TGF-βR1 and Col4a1. A and C, MMC transfected with 130b-M, let-7b-M, or double mimic (Double-M) display a TGF-β1-induced decrease of _TGF-β_R1 (A) and Col4a1 mRNA (C) expression compared with NC-M. B, similar to mRNA results, TGF-βR1 protein expression levels were significantly reduced with exogenous miR-130b-M, let-7b-M, and double-mimic conditions. D, TGF-β1-induced _TGF-β_R1 and Col4a1 mRNA levels were reduced in MMC transfected with TGF-βR1 siRNA oligonucleotides compared with NC siRNA oligonucleotides. E, MMC transfected with NC-I, 130b-I, let-7b-I, or double inhibitor (Double-I; where 130-I and let-7b-I were both transfected) resulted in increased _TGF-β_R1 mRNA expression when miR-130b alone or in combination with let-7b (double inhibitor condition) was blocked. F, schematic model depicting miR-130b and let-7b directly binding to TGF-βR1 3′-UTR to regulate _TGF-β_R1 and other downstream profibrotic genes, such as Col4a1, CTGF, and PAI-1. Results shown are mean ± S.E. (error bars) (n = 3 except Western blot (n = 2)). * and **, p < 0.05 and p < 0.005, respectively, versus NC conditions.

FIGURE 4.

FIGURE 4.

NF-YC regulates the promoter of the miR-130b host gene. A, quantitative PCR analysis of RIK expression levels in SD MMC treated without or with 10 ng/ml TGF-β1 for 6 and 24 h. RIK mRNA levels were significantly decreased throughout the time course. miR-130b gene location reveals that it forms a cluster with miR-301b between exon 1 and exon 2 of the RIK host gene. B, WT-RIK luciferase promoter construct depicting three NF-Y sites located −200, −240, and −285 bp upstream of the transcription start site of the RIK gene. The RIK-Del construct lacks these three NF-Y sites. MMC were transfected with WT-RIK or RIK-Del constructs and treated with or without TGF-β1 for 24 h. C, MMC were co-transfected with WT-RIK or RIK-Del and either NC siRNA (20 n

m

) or NF-YC siRNA (20 n

m

) oligonucleotides. WT-RIK promoter activity was significantly reduced with NF-YC siRNA compared with the NC siRNA condition. The RIK-Del construct showed very little activity with or without NF-YC siRNA. Mean ± S.E. (error bars) (n = 3). ** and ***, p < 0.005 and p < 0.0005, respectively, versus WT, NC, or SD conditions.

FIGURE 5.

FIGURE 5.

NF-YC regulates RIK and other downstream components. A–C, MMC transfected with NF-YC siRNA showed a significant decrease in basal and TGF-β1-induced mRNA expression of NF-YC and other downstream components (namely RIK and miR-130b) compared with the NC siRNA condition. D, conversely, basal levels of _TGF-β_R1 mRNA expression were up-regulated in the presence of NF-YC siRNA. E, schematic diagram depicting how decreased expression of NF-YC in response to TGF-β1 leads to reduced binding of NF-YC to the CCAAT domains (NF-Y sites) within the RIK promoter, thereby decreasing RIK transcriptional activity and consequently decreasing the expression of miR-130b located within the RIK gene, ultimately resulting in increased TGF-βR1 expression. Mean ± S.E. (error bars) (n = 3). *, **, and ***, p < 0.05, p < 0.005, and p < 0.0005, respectively, versus NC conditions.

FIGURE 6.

FIGURE 6.

Expression of miR-130b and key parameters of DN in glomeruli of diabetic mice. A and B, glomeruli were isolated from kidney cortical tissues from six mice in the control group and nine mice in the STZ-diabetic group. miR-130b, let-7b, and RIK mRNA expression levels were significantly decreased in glomeruli of STZ-injected diabetic mice (STZ) (4 weeks of diabetes) compared with vehicle-injected (Control) mice. C–E, conversely, _TGF-β_R1, Col4a1, Col12a1, PAI-1, and CTGF mRNA expression levels were significantly increased in glomeruli of these diabetic mice compared with control non-diabetic mice. Results shown are mean ± S.E. (error bars) (n = 3). *, **, and ***, p < 0.05, p < 0.005, and p < 0.0005, respectively, versus control conditions. Results were normalized to CypA internal control for regular genes and 18 S internal control for miRNAs. F, protein lysates were made from renal cortical tissues of STZ mice and control mice. TGF-βR1 protein expression levels were increased under diabetic conditions compared with control. β-Actin was used as a loading control for protein lysates. G, representative NF-YC immunostaining images and quantification using NF-YC antibody in cortical tissue from STZ-injected diabetic mice and control mice. NF-YC protein expression was significantly reduced in the STZ mice glomeruli compared with control mice. H, representative PAS staining images and quantification using cortical tissue from STZ-injected diabetic mice and control mice. Significantly increased PAS staining was observed in diabetic mice compared with control mice. Results shown are mean ± S.E. * and ***, p < 0.05 and p < 0.0005 (n = 50 glomeruli counted/condition). Scale bar, 20 μm.

FIGURE 7.

FIGURE 7.

Schematic model. Under diabetic conditions where TGF-β1 levels are increased, NF-YC expression is down-regulated (possibly via up-regulated miR-216a), resulting in reduced binding of NF-YC to the NF-Y sites (CCAAT) within the RIK promoter. Alternatively, miR-216a can target and down-regulate Ybx1, which can result in reduced binding of Ybx1 to Y-box (CCAAT) within the RIK promoter. Decreased RIK promoter activity leads to reduced transcriptional activity of the RIK gene, thereby attenuating miR-130b expression, because miR-130b expression is dependent upon transcriptional activity of the host gene, RIK. This feed-forward amplifying cascade results in elevated expression of TGF-βR1 and subsequently other downstream profibrotic and ECM genes involved in mediating the progression of diabetic nephropathy, such as Col4a1, CTGF, and PAI-1. Down-regulation of miR-30e* might be a consequence of reduced NF-YC, a putative host gene for miR-30e* that can potentially target Col12a1.

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