Regulation of the laminin C1 promoter in cultured mesangial cells

Expression of the genes encoding several matrix proteins, including the lam inin gamma 1and beta 1subunits, is increased in glomeruli or renal cortex f rom diabetic animals or in mesangial cells cultured in high concentrations of glucose. Transforming growth factor (TGF)-beta 1 and IGF-1 have been imp licated as mediators of this response. In the present study, me assessed th e influence of high glucose concentrations and the roles of TGF-beta 1 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 mou se mesangial(MES-13) cells in 500 mg/dl D-glucose for 2 days to 3 weeks sig nificantly 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-beta 1 was without effect. The influence o f 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 meek increased laminin C1 promoter a ctivity compared with cells maintained in 100 mg/dl glucose. In contrast, c ulture 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 prom oter 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-beta 1 had no influence on promoter activi ty in stable integrants. Whereas IGF-1 transiently increased promoter activ ity in stable integrants, the increase was not sustained (6 h). Moreover, n eutralizing antibody to TGF-beta or to IGF-1 receptor did not suppress incr eases in laminin C1 promoter activity induced by culture of stable integran ts in high glucose. Several inhibitors of protein kinase C, including bisin dolylmaleimide (GFX), myristoylated PKC inhibitor peptide, and LY333531, we re also without effect on increases in laminin C1 promoter activity induced by culture in high glucose. Exposure to the NO donor (+/-)-s-nitroso-n-ace typenicillamine (SNAP) blocked increases in laminin C1 promoter activity in duced 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 gl ucose actions by the NO donor SNAP, provide potential mechanisms whereby th e synthesis of the laminin gamma 1 chain may be regulated in the glomerulus in diabetes. Of note, the mechanism by which high glucose increases lamini n C1 promoter activity appears to differ from mechanisms previously describ ed for some other glucose actions on matrix protein synthesis. In this rega rd, TGF-beta and protein kinase C were not implicated as mediators of the e ffect of high glucose on laminin C1 promoter activity.