High glucose levels enhance TGF-β1–thrombospondin-1 pathway in cultured human mesangial cells via mechanisms dependent on glucose-induced PKC activation

Abstract

We have previously demonstrated that thrombospondin-1 (TSP-1) promotes activation of latent TGF-β1 in cultured human mesangial cells (MCs) [Nephron 79 (1998) 38.]. This study was performed to clarify the relationship between protein kinase C (PKC) activity and activation of TGF-β1 and TSP-1 production in cultured human MCs exposed to high glucose levels. MCs grown in 33 mmol/l glucose demonstrated a significant ( P<.01) increase in activation of TGF-β1 compared with those in 5 mmol/l glucose, which were evaluated by both an ELISA and a bioassay. High glucose-induced increase in active TGF-β1 was completely inhibited by coincubation with 5 μmol/l GFX, a PKC inhibitor, and was mimicked by the addition of 0.1 μmol/l phorbol 12,13-dibutyrate (PDBu). On the other hand, increased TSP-1 production from MCs stimulated by 0.1 nmol/l recombinant TGF-β1 and 0.1 μmol/l PDBu were significantly ( P<.01) reduced by the addition of 10 nmol/l latency-associated peptide (LAP), a specific inhibitor of TGF-β1 activity. The amount of TSP-1 secreted from MCs increased by a high ambient glucose. The glucose-induced increase in TSP-1 production was markedly attenuated by the treatment with GFX and LAP, while those agents did not affect TSP-1 production in low-glucose concentrations. Taken together, our results suggest that glucose-induced activation of TGF-β1 is dependent on PKC activity, leading to a sequential increase in TSP-1 synthesis in cultured human MCs. Thus, we propose that high glucose conditions induce an increase in PKC–TGF-β1 activity–TSP-1 pathway, and that glucose-induced increase in TSP-1 may synergistically facilitate TGF-β1 activation in an autocrine manner in MCs.