High ambient glucose enhances sensitivity to TGF-beta1 via extracellular signal--regulated kinase and protein kinase Cdelta activities in human mesangial cells.
High ambient glucose activates intracellular signaling pathways to induce cytokines such as TGF-beta1 in the extracellular matrix accumulation of diabetic nephropathy. These same pathways also may directly modulate TGF-beta1 signaling. R-Smad phosphorylation, association with Smad4, and nuclear accumulation after TGF-beta1 treatment (1.0 ng/ml) were significantly higher in mesangial cells that were conditioned to 20 mM glucose for 72 h than mesangial cells in 6.5 mM glucose, suggesting that high glucose enhanced responsiveness to TGF-beta1. Neither TGF-beta1 bioactivity nor TGF-beta receptor binding was significantly different between in 6.5 and 20 mM glucose-conditioned cultures. Furthermore, adding a neutralizing anti-TGF-beta1 antibody during glucose conditioning did not affect the enhanced Smad responsiveness, indicating that enhancement likely did not result from increased TGF-beta expression. In contrast, a mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)/ERK inhibitor, PD98059, completely abrogated the effect of high glucose. Glucose stimulation of ERK was inhibited by the general protein kinase C (PKC) inhibitor calphostin C and by the PKCdelta-specific inhibitor rottlerin, whereas Gö6976, an inhibitor of conventional PKC, had no effect on ERK activity. Specificity of the PKC inhibitors was further verified by PKCbeta and delta kinase assay. High glucose increased expression of several PKC isozymes, but only PKCdelta showed proportionally increased membrane translocation and kinase activity in cells that were conditioned to 20 mM glucose. Finally, both ERK and PKCdelta inhibition during glucose conditioning abrogated enhanced alpha1(I) collagen mRNA and promoter induction by TGF-beta1. Taken together, these data strongly suggest that heightened ERK and PKCdelta activity in high ambient glucose conditions interact with the Smad pathway, leading to enhanced responsiveness to TGF-beta1 and increased extracellular matrix production in mesangial cells.
Department of Pediatrics, Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA. hayashida@northwestern.edu
Tomoko
T
Hayashida
H William
HW
Schnaper
D002478:Cells, Cultured; D003094:Collagen / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism; D005109:Extracellular Matrix / Q000378:metabolism; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D005947:Glucose / Q000494:pharmacology*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D051899:Smad2 Protein; D051900:Smad3 Protein; D015534:Trans-Activators / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000378:metabolism / Q000494:pharmacology*; D053773:Transforming Growth Factor beta1
D004268:DNA-Binding Proteins; D018125:Receptors, Transforming Growth Factor beta; C497782:SMAD2 protein, human; C497790:SMAD3 protein, human; D051899:Smad2 Protein; D051900:Smad3 Protein; C508960:TGFB1 protein, human; D015534:Trans-Activators; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D003094:Collagen; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose