Role of protein kinase C and cyclic AMP/protein kinase A in high glucose-stimulated transcriptional activation of collagen alpha 1 (IV) in glomerular mesangial cells.
The elevated mRNA levels encoding matrix components in glomeruli isolated from streptozotocin-induced diabetic rats provide evidence that stimulation of matrix synthesis is important in early phases of diabetic glomerulopathy. We and others have demonstrated that high glucose stimulates collagen mRNA levels in short-term mesangial cell culture. To test whether transcriptional activation is operative and to gain insights into the underlying mechanisms, we studied a murine mesangial cell line stably transfected with a minigene expressing luciferase driven by 5'-flanking and first-intron regions of the alpha 1(IV) gene. High glucose stimulated luciferase activity dose and time dependently, with optimal stimulation (two-fold) achieved after 48 h in 450 mg/dL glucose (G450) versus 100 mg/dL (G100). We next tested the involvement of protein kinase C (PKC) because high glucose has been shown to stimulate de novo synthesis of diacylglycerol (DAG). Increasing PKC activity by treatment with a DAG analogue or active phorbol ester stimulated luciferase activity preferentially in G100; addition of the PKC inhibitors staurosporine or calphostin C markedly inhibited luciferase activity preferentially in G450. Thus high glucose promotes transcriptional activity of alpha 1(IV) gene through PKC activation. We also tested the involvement of protein kinase A (PKA). Intracellular cyclic AMP levels were increased two fold after 48 h in G450 versus G100, and addition of 8-Br-cAMP (0.1 mM) preferentially stimulated luciferase activity by almost three fold in G100 versus only 1.2-fold in G450. Hence, the signal-transduction mechanisms underlying the transcriptional activation of alpha 1(IV) gene in mesangial cells by high glucose are mediated by pathways involving the PKC system and possibly the cAMP/PKA system.
Ziyadeh
F N
FN
Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6144, USA.
Fumo
P
P
Rodenberger
C H
CH
Kuncio
G S
GS
Neilson
E G
EG
D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics*; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003921:Diabetes Mellitus, Experimental / Q000378:metabolism*; D015232:Dinoprostone / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression; D005920:Glomerular Mesangium / Q000187:drug effects / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis; D051379:Mice; D011493:Protein Kinase C / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D019311:Staurosporine; D015533:Transcriptional Activation / Q000187:drug effects* / Q000502:physiology; D014162:Transfection
D000470:Alkaloids; D004791:Enzyme Inhibitors; D007328:Insulin; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003094:Collagen; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D005947:Glucose; D015232:Dinoprostone