its metabolic role still remains to be elucidated. A function related to the production or utilization of genetic message is suggested by the presence of poly(A) in messenger RNA.
Culture of mesangial cells (MCs) in 5.6 vs. 30.0 mmol/l glucose for 3 weeks induced a sustained increase in protein kinase C (PKC) activity, transforming growth factor (TGF)-beta1 mRNA, bioactive TGF-beta, and collagen synthesis. Nitric oxide (NO), generated exogenously by the NO donor S-nitroso-N-acetyl, D,L-penicillamine (SNAP) or endogenously after the exposure of MC to interleukin-1beta (IL-1beta), suppressed bioactive TGF-beta in MCs cultured in 5.6 or 30.0 mmol/l glucose and suppressed or abolished increases in TGF-beta1 mRNA and collagen synthesis induced by high concentrations of glucose or phorbol 12,13-dibutyrate without altering values obtained with normal glucose concentrations. SNAP had a transient suppressive effect on PKC activity, which may explain at least in part some of the actions of SNAP. The selective inhibitor of PKC, bisindolylmaleimide (GFX), mimicked NO action. The ability of SNAP and IL-1beta to suppress TGF-beta and collagen synthesis was not mediated by cGMP, since the cGMP analog, 8-Br-PET-cGMP, did not mimic NO action and an antagonist of cGMP-dependent protein kinase, Rp-8-pCPT-cGMPs, did not prevent the inhibitory actions of SNAP. N-omega-L-arginine methyl ester (NMMA) increased TGF-beta in glomerular capillary endothelial cells (GCECs) and stimulated collagen synthesis by MC in a co-culture with GCECs. Captopril inhibited TGF-beta and collagen synthesis and increased cGMP in co-cultures of GCECs and MCs. These effects of captopril were abolished by NMMA, implying mediation by NO. Thus, endogenous NO produced by GCECs may modulate TGF-beta production by both GCECs and MCs and act to suppress matrix protein synthesis by MCs.
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 t...
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