ABSTRACT: Mesangial cells (MC) respond to various insults, such as hyperglycemia, with expressive phenotypic changes. Modifications in the patterns of MC proliferation, death, and the balance between matrix production and degradation affect directly glomerular function and ultimately lead to glomerulosclerosis. MC activation by growth factors (GF) has been demonstrated to be a trigger to changes in cell phenotype, and blockade of specific systems tend to attenuate glomerular damage. The identification of elements activated following GF stimulation of MC may facilitate the comprehension of the pathophysiology of glomerular diseases. By means of DNA microarray technology we identified that three inhibitors of DNA binding (ID1, ID3, and ID4) were among the most significantly regulated genes in serum-treated MC. We extended these studies and demonstrated that ID gene mRNA expression is markedly and rapidly affected by the pro-fibrotic cytokine TGF-beta1. Of interest, bone-morphogenetic proteins (BMPs), members of the TGF-beta superfamily with anti-fibrotic effects, also enhanced the expression of ID genes. Additional experiments indicate that these genes, classically described as dominant negative inhibitors of E-proteins, are regulated by TGF-beta1 and BMPs through distinct pathways and may act as immediate-early genes. Our results suggest that ID genes may play an active role in MC fate determination through undefined mechanisms. Keywords: comparative gene regulation by serum treatment The GE HealthCare CodeLink Human UniSet I Bioarray system, containing 10 000 gene probes, was used to generate global transcription profiles of human Mesangial Cells. All reagents were provided in the CodeLink Expression Assay Kit (GE HEALTHCARE, Piscataway, NJ, USA), except where noted. cRNA synthesis was performed according to the manufacturerâs instructions from 10 µg of pooled total RNA, purified on RNeasy columns (QIAGEN GmbH, Hilden, Germany), and quantified by UV spectrophotometry. cRNA was fragmented by heating at 94°C for 20 min in the presence of magnesium and hybridized overnight at 37°C in an appropriate buffer to a CodeLink slide under continuous shaking in an Innova 4080 incubator (New Brunswick, Edison, USA) at 300 rpm. After the hybridization, arrays were washed in 0.75x TNT (1x TNT: 100 mM Tris-HCl pH 7.6, 150 mM NaCl, and 0.05% Tween 20) at 46°C for 1 h followed by incubation with streptavidin-Cy5 (GE HealthCare) at room temperature for 30 min in the dark. Arrays were then washed in 1x TNT twice for 5 min and rinsed in 0.05% Tween-20. The slides were dried by centrifugation and kept in the dark until scanning. Images were captured on an GenePix 4000B Scanner (AXON INSTRUMENTS, Arlington, TX) using CodeLink Expression Scanning Software and were analyzed using CodeLink Expression Analysis Software (GE HEALTHCARE). This software generates normalized intensity values for each gene in each microarray, using the overall mean intensity of the array as the normalization standard.
