Impact of all-trans retinoic acid on COX-2 expression and Smad pathway in rat glomerular mesangial cells induced by TGF-β1

转化生长因子β1(TGF-β1)可能是致组织纤维化的核心因子,其经典信号通路为Smad通路.环氧化酶2(COX-2)是一种膜结合蛋白,在炎性反应中起重要作用.局部浸润的炎性细胞、肾小球的巨噬细胞、系膜细胞都是COX-2的来源[1].维甲酸能抑制肾脏纤维化,保护肾功能[2],其主要包括全反式维甲酸(atRA),92顺式维甲酸和132顺式维甲酸.本研究探讨atRA对肾小球系膜细胞TGF-β-Smad信号通路中COX-2表达的影响.     

Effect of all-trans retinoic acid on the expression of Sp1 and c-met in glomerular mesangial cells induced by TGF-β

肝细胞生长因子(HGF)是一种多效性的细胞因子,c-met是HGF现已知的唯一的受体,HGF与c-met结合后,发挥相应的生物学效应[1].Sp1蛋白是众多基因的基本转录因子,能与c-met基因启动子区域的多重GC盒结合,是调节肾脏细胞表达c-met的重要转录因子[2].全反式维甲酸(ATRA)能抑制肾脏纤维化,保护肾功能.本研究探讨ATRA对肾小球系膜细胞Sp1、c-met表达的影响,以进一步了解ATRA对肾脏的保护机制.     

Effect of all-trans retinoic acid on the expression of Sp1 and c-met in glomerular mesangial cells induced by TGF-β

肝细胞生长因子(HGF)是一种多效性的细胞因子,c-met是HGF现已知的唯一的受体,HGF与c-met结合后,发挥相应的生物学效应[1].Sp1蛋白是众多基因的基本转录因子,能与c-met基因启动子区域的多重GC盒结合,是调节肾脏细胞表达c-met的重要转录因子[2].全反式维甲酸(ATRA)能抑制肾脏纤维化,保护肾功能.本研究探讨ATRA对肾小球系膜细胞Sp1、c-met表达的影响,以进一步了解ATRA对肾脏的保护机制.     

Impact of PCI-neo-HGF on the expression of TGF-β1 and proliferation of glomerular mesangial cells in rats

肾小球系膜细胞(GMC)的过度增生是导致肾小球硬化及肾间质纤维化的重要机制之一[1].肝细胞生长因子(HGF)是一种多效性的细胞因子,其可通过加速细胞外基质降解,阻断小管上皮细胞转分化等实现对肾脏的保护[2-4].目前HGF对正常及增生的GMC是否有抑制作用尚不明确.本研究采用可在体内持续平稳表达的PCI-neo-HGF质粒进行研究[5],主要探讨HGF是否能抑制正常及脂多糖(LPS)刺激后的大鼠GMC的增生,以及这种作用是否与抑制转化生长因子β1(TGF-β1)的表达相关.     

Impact of PCI-neo-HGF on the expression of TGF-β1 and proliferation of glomerular mesangial cells in rats

肾小球系膜细胞(GMC)的过度增生是导致肾小球硬化及肾间质纤维化的重要机制之一[1].肝细胞生长因子(HGF)是一种多效性的细胞因子,其可通过加速细胞外基质降解,阻断小管上皮细胞转分化等实现对肾脏的保护[2-4].目前HGF对正常及增生的GMC是否有抑制作用尚不明确.本研究采用可在体内持续平稳表达的PCI-neo-HGF质粒进行研究[5],主要探讨HGF是否能抑制正常及脂多糖(LPS)刺激后的大鼠GMC的增生,以及这种作用是否与抑制转化生长因子β1(TGF-β1)的表达相关.     

Effect of tet methylcytosine dioxygenase 2 on the regulation of transforming growth factor-β1 expression in mesangial cells induced by high glucose

Objective To investigate the role of tet methylcytosine dioxygenase 2 (TET2) in the regulation of transforming growth factor-β1 (TGF-β1) expression in human glomerular mesangial cells induced by high glucose. Methods Cultured human glomerular mesangial cells were divided into normal control group (5.5 mmol/L glucose) and high glucose group (30.0 mmol/L glucose) which was cultured for 12 h to 72 h. The gene expression of TET2 in mesangial cells were inhibited by small molecule chemical called SC1, and which were divided into high glucose group (30.0 mmol/L glucose+DMEM), DMSO group (30.0 mmol/L glucose+0.1%DMSO) and SC1 group (30.0 mmol/L glucose+3 μmol/L SC1). The mRNA and protein expression of TGF-β1, TET1 to 3 and α-smooth muscle actin (α-SMA) was detected by quantitative real-time PCR and Western blotting. Methylation of CpG islands in the regulation region of TGF-β1 was detected by bisulfite sequencing PCR (BSP). The activity of mesangial cell proliferation was assessed by colorimetry of thiazolyl blue (MTT). Results Compared with normal control group, the mRNA and protein expression of TET2 in mesangial cells induced by high glucose was increased significantly in a time-dependent manner (all P＜0.05), but the expression of TET1 and TET3 was not affected. Meanwhile methylation rate of 4 CG sites from 24 h to 72 h were decreased in the first exon of TGF-β1 (P＜0.01), but not in the promoter. Compared with high glucose group, when the expression of TET2 was inhibited by SC1, the methylation rate of TGF-β1 was recovered evidently (P＜0.05), the mRNA and protein expression of TGF-β1 and α-SMA was suppressed, and the proliferation of mesangial cells was decreased (all P＜0.05). Conclusions Demethylation of the CpG island mediated by TET2 may play an important role in the expression of TGF-β1 and mesangial cell phenotype transformation induced by high glucose.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Differentiation of human epidermal stem cells into fibroblasts induced by TGF-β1 in vitro

Objective To investigate the correlation between human epidermal stem cell (hESCs) and hypertrophic scar or keloid. Methods Improved collagen Ⅳ-coated adhesion methods was used to isolate and culture the epidermal stem cells after neutral protease selectively digested the dermo-epidermal junctions. After the cells were cultured and expanded in vitro, and passage 3 hESCs were induced by different concentrations of TGF-β1 (0.1, 5.0, and 10.0 ng/ml). Morphological fea-tures and identification of these cells were meseasured by HE, Masson, immunohistochemical staining on the days 3 and 7, respectively. Results After induced by TGF-β1 for 3 and 7 days, the morpholo-gy of the epidermal stem cell (hESCs) was changed into fusiform shape, similar to fibroblasts. 70 % ofthe cell which was induced by TGF-β1 were blue stained in the cytoplasm by Masson stain, which is the distinctive method for collagen, suggesting collagen appeared or increased in the cells. The collagen concentrations in supernatants of hESCs were 0.4150±0.0014, 0.3380±0. 0020, and 0.3870±0.0020, much higher than that in control group (0.0780±0.0025) and normal skin fibro-blast group (0.15004±0.0051) (P<0.05). Immunohistochemical staining revealed that positive rates of these cells for anti-vimentin staining were more than (95.00±1.20)% in experiments and (5.70±0.20)% in control group. Conclusion The differentiantion of hESCs induced by TGF-β1 into fibro-blasts indicates that hESCs may play a role in the pathogenesis of hypetrophic scar and keloid.     

Effects of hypoxia-inducible factor-2α on the expression of tight junction proteins and permeability in rat glomerular endothelial cells under hypoxia condition

Objective To investigate the role of hypoxia-inducible factor-2α (HIF-2α) in the expression of tight junction proteins and permeability alterations in rat glomerular endothelial cells (rGENCs) under hypoxia condition. Methods The expressions of the HIF-2α and tight junction proteins such as occludin and ZO-1 of rGENCs were examined after exposed to 5% oxygen at different treatment time periods (0 h, 12 h, 24 h and 48 h). Then lentiviral transfection was used to knock down HIF-2α expression in rGENCs. The cells were split into four groups, including i) control group where rGENCs were cultured under normal oxygen conditions, ii) hypoxia group, iii) negative control group where rGENCs were infected with a negative vector, iv) HIF-2α lentivirus transfection group. Group ii, iii and iv were kept in hypoxic chamber (5% O2, 5% CO2 and 90% N2) for 24 h. The expressions of occludin, ZO-1 and HIF-2α were assessed by Western blotting. The permeability of rGENCs was measured using trans-epithelium electrical resistant (TEER) by Millicell? ERS voltohmmeter. Results With the elongation of hypoxia time, the expression of HIF-2α was increased gradually, while the occludin expression was decreased, there was statistically significance difference in each group (all P＜0.01). The expression of ZO-1 also decreased gradually under hypoxia circumstance, but no statistically significant was found between 24 h and 48 h groups (all P＞0.05). And a dramatic decrease in TEER of hypoxia cells was detected as compare with control cells (P＜0.01). After knockdown of HIF-2α expression, both expressions of occludin and ZO-1 were increased significantly compared with hypoxia cells (P＜0.01), and TEER elevated at the same time (P＜0.01). Above indexes had no statistical difference between hypoxia cells and negative control cells (all P＞0.05). Conclusion Hypoxia may promote HIF-2α expression, which could increase the permeability of rGENCs by reducing the expression of occludin and ZO-1.     

Smad7 inhibits TGF-β1-induced MCP-1 upregulation through a MAPK/p38 pathway in rat peritoneal mesothelial cells

Purpose

The TGF-β1/Smad signaling pathway is subject to inhibition by Smad7. High expression of Smad7 in the peritoneum of rats can delay and attenuate not only peritoneal fibrosis, but also monocyte infiltration into the peritoneum. The aim of this study was to investigate the anti-inflammatory mechanism of Smad7 in peritoneal fibrosis.

Methods

Rat peritoneal mesothelial cells were stimulated with TGF-β1, and the expression of MCP-1 protein and mRNA was measured. Furthermore, the expression of MCP-1 was determined following inhibition of TGF-β/Smad or p38 signaling using Smad7 transfection or SB203580 (10 μmol/L), respectively. The effect of exogenous Smad7 and SB203580 on activation of the TGF-β/Smad and p38 signaling pathways was also studied.

Results

TGF-β1 significantly upregulated the expression of MCP-1 at both the protein and mRNA level in a time-dependent manner. Exogenous Smad7 and SB203580 markedly inhibited TGF-β1-induced MCP-1 expression. Moreover, high expression of exogenous Smad7 not only inhibited phosphorylation of Smad2 and Smad3, but also diminished the level of phosphorylated p38. However, SB203580 had no effect on the phosphorylation of Smad2 and Smad3.

Conclusions

TGF-β1 exhibits pro-inflammatory effects through the upregulation of MCP-1 in peritoneal fibrosis. Smad7 inhibits TGF-β1 induced MCP-1 upregulation through a MAPK/p38-dependent pathway.     

Type VIII collagen modulates TGF-β1-induced proliferation of mesangial cells

Mesangial cells in diabetic mice and human kidneys with diabetic nephropathy exhibit increased type VIII collagen, a nonfibrillar protein that exists as a heterodimer composed of α1(VIII) and α2(VIII), encoded by Col8a1 and Col8a2, respectively. Because TGF-β1 promotes the development of diabetic glomerulosclerosis, we studied whether type VIII collagen modulates the effects of TGF-β1 in mesangial cells. We obtained primary cultures of mesangial cells from wild-type, doubly heterozygous (Col8a1(+/-)/Col8a2(+/-)), and double-knockout (Col8a1(-/-)/Col8a2(-/-)) mice. TGF-β1 bound normally to double-knockout mesangial cells. In wild-type mesangial cells, TGF-β1 inhibited proliferation, but in double-knockout cells, it stimulated proliferation, promoted cell cycle progression, and reduced apoptosis; we could reverse this effect by reconstituting α1(VIII). Furthermore, in wild-type cells, TGF-β1 mainly stimulated the Smad pathways, whereas in double-knockout cells, it activated the MAPK and PI3K/Akt pathways and induced expression of fibroblast growth factor 21 (FGF21). Inhibiting FGF21 expression by either interfering with activation of the MAPK and PI3K/Akt pathways or by FGF21 siRNA attenuated the TGF-β1-induced proliferation of double-knockout mesangial cells. In vivo, diabetic double-knockout mice had significantly higher expression of renal FGF21 mRNA and protein compared with diabetic wild-type mice. Immunohistochemistry revealed strong expression of FGF21 in both glomerular (mesangial) and tubular cells of diabetic mice. Taken together, these data suggest that type VIII collagen significantly modulates the effect of TGF-β1 on mesangial cells and may therefore play a role in the pathogenesis of diabetic nephropathy.     

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.