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Transforming Growth Factor 1-Mediated mRNA Expression And Oxidative Stress in Renal Mesangial Cells:Comparison With High Glucose And Hexosamine-Induced Gene Expression Profiles

Lalit P.Singh, Davis W.Cheng, Yan Jiang, Errol D.Crook

Recently, we demonstrated high glucose and glucosamine induced expression of thioredoxin interacting protein(TXNIP) and several genes involved in oxidative stress and endoplasmic reticular stress in mesangial cells in a microarray study. Transforming growth factor-beta(TGF-) 1 is considered to be a mediator of renal hypertrophy and ECM gene expression in mesangial cells and the development of diabetic glomerulopathy. Here, we further investigate the effects of TGF-1 on mRNA expression of a mouse mesangial cell line MES-13 using Affymetrix murine expression U430 2.0 Oligochips. The data obtained are further compared with the previously described high glucose (HG) and glucosamine (GlcN)-induced gene expression profile. We identified 264 genes (0.7% of the total 34,000 genes present in the microarray) as TGF-1-regulated genes at 1.5-fold differential expression of which 161 genes are up-regulated and 85-genes are down-regulated. Of the 264 genes, 63 were also targeted by HG and 38 genes by GlcN while 13 genes are commonly regulated by all three and others are unique to TGF-1. Biological process-based ontological analysis of differentially expressed genes reveal that a majority of them are involved in (i) regulation of physiological processes, (ii) metabolism, (iii) transcription and DNA binding, (iv) cell cycle control and (v) differentiation. A number of genes identified in the microarray experiment are further validated by quantitative real time-PCR. TGF-1 induces the expression of various cellular hypertrophic and fibrotic factors such as connective tissue growth factor, platelet-derived growth factor B and fibroblast growth factor 21. Furthermore, TGF-1 increases reactive oxygen species (ROS) generation, TXNIP expression and apoptosis of mesangial cells as revealed by TUNEL. Extracellular matrix related genes targeted by TGF- 1 are osteopontin, tenascin C, fibronectin, laminin 3, plaminogen activator inhibitor (PAI)-1 and cell surface glycoprotein modifying I-type antigen enzyme, glucosaminyl (N-acetyl) transferase 2. There also exist cross-talks between TGF-1 and IFN-  signaling pathways as revealed by cis-acting reporter activity assay for STAT3, -activated sequence (GAS) and interferon response sequence element (ISRE). The common targets of HG, GlcN and TGF-1 induced genes include TXNIP, osteopontin, PAI-1 and others. The results reveal new signaling pathways and gene expression patterns that were not previously understood and reinforces the hypothesis that TGF-1 mediates several harmful effects of chronic hyperglycemia and hexosamines to generate ROS, ECM accumulation and apoptosis of renal mesangial cells in diabetic glomerulopathy.