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 isola ted from streptozotocin-induced diabetic rats provide evidence that st imulation of matrix synthesis is important in early phases of diabetic glomerulopathy. We and others have demonstrated that high glucose sti mulates collagen mRNA levels in short-term mesangial cell culture, To test whether transcriptional activation is operative and to gain insig hts 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, wit h optimal stimulation (two-fold) achieved after 48 h in 450 mg/dL gluc ose (G450) versus 100 mg/dL (G100). We next tested the involvement of protein kinase C (PKC) because high glucose has been shown to stimulat e de novo synthesis of diacylglycerol (DAG), Increasing PKC activity b y treatment with a DAG analogue or active phorbol ester stimulated luc iferase activity preferentially in G100; addition of the PKC inhibitor s staurosporine or calphostin C markedly inhibited luciferase activity preferentially in G450. Thus high glucose promotes transcriptional ac tivity 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 si gnal-transduction mechanisms underlying the transcriptional activation of alpha 1(IV) gene in mesangial cells by high glucose are mediated b y pathways involving the PKC system and possibly the cAMP/PKA system