High Ambient Glucose Enhances Sensitivity to TGF-β1 via Extracellular Signal—Regulated Kinase and Protein Kinase Cδ Activities in Human Mesangial Cells

Abstract

Abstract. High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-␤1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-␤1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-␤1 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-␤1. Neither TGF-␤1 bioactivity nor TGF-␤ receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-␤1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-␤ 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 PKC␦-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKC␤ and ␦ kinase assay. High glucose increased expression of several PKC isozymes, but only PKC␦ showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKC␦ inhibition during glucose conditioning abrogated enhanced ␣1(I) collagen mRNA and promoter induction by TGF-␤1. Taken together, these data strongly suggest that heightened ERK and PKC␦ activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-␤1 and increased extracellular matrix production in mesangial cells.

High glucose activates PKC-ζ and NADPH oxidase through autocrine TGF-β1 signaling in mesangial cells

“…TGF-␤ 1 has been reported to enhance the activity of isolated PKC isozymes in various cell types (28,59) and, in particular, PKC-␦ in mesangial cells (19). Differences in the incubation conditions or cell lines used could explain the more widespread PKC activation by TGF-␤ 1 observed in the present experiments compared with the study by Hayashida et al (19). Conversely, high glucose-activated PKC-␤ increases TGF-␤ 1 expression (25).…”

“…Elevated expression of TGF-␤ 1 is a major stimulant for ECM accumulation by mesangial cells through regulation of genes encoding ECM, PAI-1, and other proteins through Smads and extracellular signal-regulated kinase (ERK) (13,17,18,19,29,36,44,57,68,69,71). In this study, we found for the first time that two initial effects of high ambient glucose, the generation of ROS by NADPH oxidase and activation of PKC-are dependent on signal transduction initiated by TGF-␤ 1 .…”

“…Non-Smad, TGF-␤ 1 effectors include MAPKs such as ERK, PI3 kinase, Akt, NADPH oxidase, p21-activated kinase-2, Ras, and Src (5,6,17,19,36,37,50,60). Many of these poorly understood, non-Smad TGF-␤ 1 effects may be indirectly mediated by Smads, but some appear to arise independently of Smads, as illustrated in cells lacking Smads or by mutant TGF-␤ receptors (2,6,50,60).…”

“…Hence, the nuclear accumulation of Smad2/3 has been used to monitor TGF-␤ 1 signaling (6,19,60). Immunofluorescence staining for nuclear Smad2/3 was carried out on mesangial cells cultured in 5.6 or 25 mM glucose, and stimulated with 10 nM TGF-␤ 1 or vehicle for 30 min.…”

“…In this regard, TGF-␤ 1 and its receptors are upregulated in both experimental and human diabetic nephropathy (21,56,57,68,69,65). Enhanced expression of TGF-␤ 1 , TGF-␤ receptors, TGF-␤ 1 bioactivity, and responsiveness to exogenous TGF-␤ 1 have been noted to occur in response to high glucose in glomerular mesangial cells grown in tissue culture, an in vitro model for the effects of high glucose (19,21,29,41,68,69). Furthermore, inhibiting TGF-␤ 1 attenuates the high glucoseinduced changes in gene expression in mesangial cells that lead to the accumulation of ECM both in vitro and in animal models of diabetic nephropathy (29,45,70,71).…”