High Glucose–Enhanced Mesangial Cell Extracellular Signal–Regulated Protein Kinase Activation and α1(IV) Collagen Expression in Response to Endothelin-1

Abstract

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of extracellular matrix, a process involving protein kinase C (PKC) isozymes and enhanced signaling by autocrine peptides such as endothelin-1 (ET-1). The purpose of this study was to identify the specific PKC isozymes mediating the effects of HG on MC extracellular signal–regulated protein kinase (ERK1/2) signaling and α1(IV) collagen expression in response to ET-1. HG (30 mmol/l for 72 h) enhanced ET-1–stimulated α1(IV) collagen mRNA expression from 1.2 ± 0.1–fold to 1.9 ± 0.2–fold (P < 0.05 vs. normal glucose [NG] + ET-1), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transiently transfected MCs, dominant-negative (DN)–PKC-δ, -ε, or -ζ inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 stimulated Elk-1–driven GAL4 luciferase activity to 11 ± 1–fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC–δ, –ε, or –ζ attenuated this response to NG levels. HG enhanced ET-1–stimulated intracellular α1(IV) collagen protein expression, assessed by confocal immunofluorescence imaging, showed that individual DN–PKC-δ, -ε, -ζ, as well as DN–PKC-α and -β, attenuated the response. Thus, HG-enhanced ET-1 stimulation of α1(IV) collagen expression requires PKC-δ, -ε, and -ζ to act through an ERK1/2-dependent pathway and via PKC-α and -β, which are independent of ERK1/2.