Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-ζ

Abstract

In high glucose (HG), mesangial cells (MCs) lose their contractile response to endothelin-1 (ET-1) coincidently with filamentous (F)-actin disassembly. We postulated that these MC phenotypic changes are mediated by altered protein kinase C (PKC) isozyme activity, myosin light chain (MLC20) phosphorylation, or Ca2+ signaling. MCs were growth arrested for 24 h in 0.5% fetal bovine serum (FBS)-DMEM in 5.6 (normal glucose; NG) or 30 mM glucose (high glucose; HG). In HG, the planar area was reduced [2,608 ± 135 vs. 3,952 ± 225 (SE) μm2 in NG, P < 0.01, n = 31] with no contractile response to 0.1 μM ET-1. Mannitol did not affect cell size or ET-1 response. Confocal imaging of fluo 3- loaded cells revealed that the peak intensity of ET-1-induced Ca2+signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC20 showed that HG increased mono- and decreased unphosphorylated MLC20 (42 ± 16 and 49 ± 15 vs. 13 ± 3 and 80 ± 4% of total in NG, P < 0.05, n = 3), but the peak phosphorylation responses to ET-1 were identical in NG and HG. ET-1 stimulated translocation of PKC-δ and -ε from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-ζ translocation. In HG, membrane accumulation of PKC-ζ was observed. Membrane PKC-ζ activity measured by immunoprecipitation and32P phosphorylation of PKC-ε pseudosubstrate peptide was 190 ± 18% of NG ( P < 0.01, n = 4), which was completely inhibited by pretreatment with a myristoylated peptide inhibitor (ZI). In HG, pretreatment with ZI for 24 h restored normal MC size and contractile and F-actin disassembly responses to ET-1. In conclusion, in HG, decreased MC size is due to decreased F-actin assembly, and loss of contractile response to ET-1 occurs in the presence of normal Ca2+ signaling and normal MLC20 phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-ζ.