Dysregulated intracellular signaling impairs CTGF-stimulated responses in human mesangial cells exposed to high extracellular glucose.
High ambient glucose activates intracellular signaling pathways to induce the expression of extracellular matrix and cytokines such as connective tissue growth factor (CTGF). Cell responses to CTGF in already glucose-stressed cells may act to transform the mesangial cell phenotype leading to the development of glomerulosclerosis. We analyzed cell signaling downstream of CTGF in high glucose-stressed mesangial cells to model signaling in the diabetic milieu. The addition of CTGF to primary human mesangial cells activates cell migration which is associated with a PKC-zeta-GSK3beta signaling axis. In high ambient glucose basal PKC-zeta and GSK3beta phosphorylation levels are selectively increased and CTGF-stimulated PKC-zeta and GSK3beta phosphorylation was impaired. These effects were not induced by osmotic changes. CTGF-driven profibrotic cell signaling as determined by p42/44 MAPK and Akt phosphorylation was unaffected by high glucose. Nonresponsiveness of the PKC-zeta-GSK3beta signaling axis suppressed effective remodeling of the microtubule network necessary to support cell migration. However, interestingly the cells remain plastic: modulation of glucose-induced PKC-beta activity in human mesangial cells reversed some of the pathological effects of glucose damage in these cells. We show that inhibition of PKC-beta with LY379196 and PKC-beta siRNA reduced basal PKC-zeta and GSK3beta phosphorylation in human mesangial cells exposed to high glucose. CTGF stimulation under these conditions again resulted in PKC-zeta phosphorylation and human mesangial cell migration. Regulation of PKC-zeta by PKC-beta in this instance may establish PKC-zeta as a target for constraining the progression of mesangial cell dysfunction in the pathogenesis of diabetic nephropathy.
Furlong
Fiona
F
UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin, Belfield, Dublin, Ireland.
Crean
John
J
Thornton
Laura
L
O'Leary
Ronan
R
Murphy
Madeline
M
Martin
Finian
F
D015153:Blotting, Western; D002478:Cells, Cultured; D002633:Chemotaxis / Q000187:drug effects; D055513:Connective Tissue Growth Factor; D003928:Diabetic Nephropathies / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D005110:Extracellular Space / Q000187:drug effects / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000633:toxicity*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D017874:Immediate-Early Proteins / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects*; D013268:Stimulation, Chemical; D014947:Wounds and Injuries / Q000473:pathology; D020764:cdc42 GTP-Binding Protein / Q000096:biosynthesis
C526317:CCN2 protein, human; D017874:Immediate-Early Proteins; D036341:Intercellular Signaling Peptides and Proteins; D034741:RNA, Small Interfering; D055513:Connective Tissue Growth Factor; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D051744:Protein Kinase C-epsilon; D038362:Glycogen Synthase Kinase 3; D020764:cdc42 GTP-Binding Protein; D005947:Glucose