Expression of constitutively active cGMP-dependent protein kinase prevents glucose stimulation of thrombospondin 1 expression and TGF-beta activity.
Hyperglycemia is a crucial factor in the development of diabetic nephropathy. We previously showed that high glucose upregulates thrombospondin 1 (TSP1)-dependent transforming growth factor (TGF)-beta activation by altering cGMP-dependent protein kinase (PKG) activity as a result of decreased nitric oxide signaling. In the present study, we showed that high glucose concentrations significantly reduced endogenous PKG activity. To further examine the mechanisms by which PKG regulates TSP1 expression and TSP1-dependent TGF-beta activation, we generated stably transfected rat mesangial cells (RMCs) with inducible expression tetracycline-induced gene expression of the catalytic domain of PKG. After tetracycline induction, the catalytic domain of PKG is expressed as a cGMP-independent active kinase. Expression of the catalytic domain prevented high glucose-mediated increases in transcription of the TSP1 gene with no alteration in TSP1 mRNA stability. Glucose stimulation of TSP1 protein expression and TGF-beta bioactivity were also downregulated. TGF-beta-dependent fibronectin and type IV collagen expression under high glucose conditions were significantly reduced upon catalytic domain expression in transfected RMCs. These results show that constitutively active PKG inhibits the fibrogenic potential of high glucose through repression of TSP1-dependent TGF-beta bioactivity, suggesting that gene transfer of the catalytic domain of PKG might provide a new strategy for treatment of diabetic renal fibrosis.
Wang
Shuxia
S
Department of Pathology, Division of Molecular and Cellular Pathology, Cell Adhesion and Matrix Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
Wu
Xing
X
Lincoln
Thomas M
TM
Murphy-Ullrich
Joanne E
JE
D000818:Animals; D000900:Anti-Bacterial Agents; D020134:Catalytic Domain / Q000235:genetics; D002460:Cell Line; D003094:Collagen / Q000378:metabolism; D017869:Cyclic GMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism*; D003928:Diabetic Nephropathies / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005109:Extracellular Matrix / Q000201:enzymology; D005353:Fibronectins / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005920:Glomerular Mesangium / Q000166:cytology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism*; D051381:Rats; D013752:Tetracycline; D019700:Thrombospondin 1 / Q000235:genetics*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D005353:Fibronectins; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; D003094:Collagen; D017869:Cyclic GMP-Dependent Protein Kinases; D013752:Tetracycline; D005947:Glucose