Mesangial cell filamentous actin disassembly and hypocontractility in high glucose are mediated by PKC-zeta.
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 (MLC(20)) phosphorylation, or Ca(2+) 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) microm(2) in NG, P < 0.01, n = 31] with no contractile response to 0.1 microM 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 Ca(2+) signaling was not altered in HG vs. NG. Immunoblotting of phosphorylated MLC(20) showed that HG increased mono- and decreased unphosphorylated MLC(20) (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-delta and -epsilon from cytosolic to membrane and particulate fractions identically in NG and HG but did not cause PKC-zeta translocation. In HG, membrane accumulation of PKC-zeta was observed. Membrane PKC-zeta activity measured by immunoprecipitation and (32)P phosphorylation of PKC-epsilon 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 Ca(2+) signaling and normal MLC(20) phosphorylation. In HG, altered F-actin and contractile functions in MCs are mediated by PKC-zeta.
Dlugosz
John A
JA
Institute of Medical Science and Department of Medicine, University of Toronto, Toronto, Ontario, Canada.
Munk
Snezana
S
Ispanovic
Eric
E
Goldberg
Howard J
HJ
Whiteside
Catharine I
CI
D000199:Actins / Q000378:metabolism*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology; D002273:Carcinogens / Q000494:pharmacology; D048429:Cell Size; D002478:Cells, Cultured; D019332:Endothelin-1 / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology*; D015759:Ionomycin / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D018613:Microscopy, Confocal; D018994:Myosin Light Chains / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051745:Protein Kinase C-delta; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000096:biosynthesis; D012694:Serine / Q000378:metabolism; D000099221:Protein Kinase C zeta
D000199:Actins; D002273:Carcinogens; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D005947:Glucose; D015759:Ionomycin; D007476:Ionophores; D007527:Isoenzymes; D018994:Myosin Light Chains; D009842:Oligopeptides; D010703:Phorbol Esters; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D012694:Serine; D000099221:Protein Kinase C zeta; C444219:myristoylated-arginyl-arginyl-glycyl-alanyl-arginyl-arginyl-tryptophyl-arginyl-lysine