High glucose-enhanced mesangial cell extracellular signal-regulated protein kinase activation and alpha 1 (IV) collagen expression in response to endothelin-1 - Role of specific protein kinase C isozymes

High glucose (HG) stimulates glomerular mesangial cell (MC) expression of e xtracellular matrix, a process involving protein kinase C (PKC) isozymes an d 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 al(IV) collagen expression in response to ET-1. HG (30 mm ol/l for 72 h) enhanced ET-1-stimulated alpha1(IV) collagen mRNA expression from 1.2 +/- 0.1-fold to 1.9 +/- 0.2-fold (P < 0.05 vs. normal glucose [NG ] + ET-I), and the effect was significantly reduced by Calphostin C or the MEK (mitogen-activated protein kinase kinase) inhibitor PD98059. In transie ntly transfected MCs, dominant-negative (DN)-PKC-delta, -epsilon, or -zeta inhibited ET-1 activation of ERK1/2. Likewise, downstream of ERK1/2, ET-1 s timulated Elk-1-driven GAL4 luciferase activity to 11 +/- 1-fold (P < 0.002 vs. NG + ET-1) in HG, and DN-PKC-delta, -epsilon, or -zeta attenuated this response to NG levels. HG enhanced ET-1-stimulated intracellular alpha1(IV ) collagen protein expression, assessed by confocal immunofluorescence imag ing, showed that individual DN-PKC-delta, -epsilon, -zeta, as well as DN-PK C-alpha and -beta, attenuated the response. Thus, HG-enhanced ET-1 stimulat ion of alpha1(IV) collagen expression requires PKC-delta, -epsilon, and -ze ta to act through an ERK1/2-dependent pathway and via PKC-alpha and -beta, which are independent of ERK1/2.