Regulation of the laminin C1 promoter in cultured mesangial cells.

Abstract

Expression of the genes encoding several matrix proteins, including the laminin gamma1 and beta1 subunits, is increased in glomeruli or renal cortex from diabetic animals or in mesangial cells cultured in high concentrations of glucose. Transforming growth factor (TGF)-beta1 and IGF-1 have been implicated as mediators of this response. In the present study, we assessed the influence of high glucose concentrations and the roles of TGF-beta1 and IGF-1 in the regulation of laminin C1 gene expression in cultured mesangial cells. Culture of normal rat mesangial cells (RMC) or SV40-transformed mouse mesangial (MES-13) cells in 500 mg/dl D-glucose for 2 days to 3 weeks significantly increased laminin C1 mRNA abundance compared with cells cultured in 100 mg/dl D-glucose. IGF-1 also increased laminin C1 mRNA abundance in RMC or MES-13 cells, whereas TGF-beta1 was without effect. The influence of raising the medium glucose concentration on laminin C1 promoter activity was further studied in MES-13 cells that had been stably transfected with a reporter gene containing the promoter linked to luciferase. Culture in 500 mg/dl D-glucose for 4 h to at least 1 week increased laminin C1 promoter activity compared with cells maintained in 100 mg/dl glucose. In contrast, culture of cells in medium that contained 400 mg/dl mannitol or 400 mg/dl L-glucose in addition to 100 mg/dl D-glucose did not increase laminin C1 promoter activity. The ability of high glucose to increase laminin C1 promoter activity was absolutely dependent on the presence of serum. Consistent with results obtained with mRNA, TGF-beta1 had no influence on promoter activity in stable integrants. Whereas IGF-1 transiently increased promoter activity in stable integrants, the increase was not sustained (6 h). Moreover, neutralizing antibody to TGF-beta or to IGF-1 receptor did not suppress increases in laminin C1 promoter activity induced by culture of stable integrants in high glucose. Several inhibitors of protein kinase C, including bisindolylmaleimide (GFX), myristoylated PKC inhibitor peptide, and LY333531, were also without effect on increases in laminin C1 promoter activity induced by culture in high glucose. Exposure to the NO donor (+/-)-s-nitroso-n-acetylpenicillamine (SNAP) blocked increases in laminin C1 promoter activity induced by serum and by culture in high glucose without influencing promoter activity in cells cultured in the absence of serum and in 100 mg/dl glucose. The ability of high glucose concentrations and IGF-1 to increase laminin C1 promoter activity in cultured mesangial cells, and the suppression of glucose actions by the NO donor SNAP, provide potential mechanisms whereby the synthesis of the laminin gamma1 chain may be regulated in the glomerulus in diabetes. Of note, the mechanism by which high glucose increases laminin C1 promoter activity appears to differ from mechanisms previously described for some other glucose actions on matrix protein synthesis. In this regard, TGF-beta and protein kinase C were not implicated as mediators of the effect of high glucose on laminin C1 promoter activity.