Advanced glycosylation end products stimulate collagen mRNA synthesis in mesangial cells mediated by protein kinase C and transforming growth factor–beta

Abstract

Advanced glycosylation end products (AGE) seem to be implicated in the pathogenesis of diabetic nephropathy. The present study has examined the effects of AGE on protein kinase C (PKC) activity and transforming growth factor–β1 (TGF-β1) in relation to collagen gene regulation in cultured human mesangial cells (HMCs). Quiescent HMCs were exposed to serum-free media containing bovine serum albumin (BSA), AGE-modified BSA (AGE-BSA), or glycated BSA in which AGE formation was prevented by the use of aminoguanidine (BSA-AM). AGE-BSA (200 μg/mL) induced a peak membrane-associated PKC activity, particularly PKC-a, at 4 hours. AGE-BSA stimulated α1(I) and α1(IV) collagen mRNA expression after 24-hour incubation with HMCs, which remained elevated until hour 60. HMCs incubated with AGE-BSA induced a significant inhibition of cell proliferation compared with cells incubated with BSA. AGE-BSA stimulated TGF-β mRNA and protein expression in HMCs. The TGF-β secreted by HMCs was shown by CCL-64 mink lung cell assay to be bioactive. In contrast, BSA-AM did not affect either collagen or TGF-β mRNA or protein expression in HMCs. The stimulatory effects of AGE-BSA on collagen gene regulation in HMCs could be negated by the pretreament of HMCs with GF 109203X for 30 minutes or with phorbol myristate acetate for 24 hours before AGE-BSA administration. Neutralizing antibody to TGF-β inhibited increased collagen mRNA expression by HMCs exposed to AGE-BSA. These results suggest that AGE-BSA stimulates collagen mRNA expression by activating PKC and the transcriptional upregulation of TGF-β1 in HMCs. Thus, PKC and TGF-β may function as key signaling intermediaries in the AGE–up-regulated collagen gene expression pathway in HMCs. (J Lab Clin Med 2001;138:59-68)