High ambient glucose enhances sensitivity to TGF-β1 via extracellular signal-regulated kinase and protein kinase Cδ activities in human mesangial cells

High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-β1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-β1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-β1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-β1. Neither TGF-β1 bioactivity nor TGF-β receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-β1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-β expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCδ-specific inhibitor rottlerin, whereas G56976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCβ and δ kinase assay. High glucose increased expression of several PKC isozymes, but only PKCδ showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCδ inhibition during glucose conditioning abrogated enhanced α1(I) collagen mRNA and promoter induction by TGF-β1. Taken together, these data strongly suggest that heightened ERK and PKCδ activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-β1 and increased extracellular matrix production in mesangial cells.