Thrombin signal transduction mechanisms in human glomerular epithelial cells.
We have previously shown that alpha-thrombin exerted a mitogenic effect on human glomerular epithelial cells and stimulated the synthesis of urokinase-type (u-PA) and tissue-type plasminogen activator (t-PA) and of their inhibitor, plasminogen activator inhibitor 1 (PAI-1). In the present study, we investigate the signal transduction mechanisms of thrombin in these cultured cells. Thrombin induced an increase in intracellular free calcium concentrations ([Ca2+]i) in a dose-dependent manner, a plateau being reached at 1 U/ml thrombin. A 60% inhibition of this effect was produced by 300 nM nicardipine, a dihydroperidine agent, or by 4 mM EGTA, indicating that increase in [Ca2+]i was due in part to extracellular Ca2+ entry through L-type voltage-sensitive calcium channels. Thrombin also induced an increase in inositol trisphosphate (IP3), suggesting that phospholipase C activation and phosphatidylinositides breakdown were stimulated. Interestingly thrombin-stimulated cell proliferation measured by 3H thymidine incorporation was inhibited by 300 nM nicardipine, and restored by addition of 10(-8) M ionomycin, indicating that calcium entry was critical for the mitogenic signal of thrombin. Conversely, nicardipine did not modify thrombin-stimulated synthesis of u-PA, t-PA, and PAI-1. Both thrombin-stimulated cell proliferation and protein synthesis required protein kinase C activation since these effects were blocked by 10 microM H7, an inhibitor of protein kinases, and by desensitization of protein kinase C by phorbol ester pretreatment of the cells. Interestingly, DFP-inactivated thrombin which binds the thrombin receptor and gamma-thrombin, which has some enzymatic activity but does not bind to thrombin receptor, had no effect when used alone. Simultaneous addition of these two thrombin derivatives had no effect on [Ca2+]i, and 3H thymidine incorporation but stimulated u-PA, t-PA, and PAI-1 synthesis although to a lesser extent than alpha-thrombin. This effect also required protein kinase C activation to occur, presumably by a pathway distinct from phosphoinositoside turnover since it was not associated with IP3 generation. In conclusion, multiple signalling pathways can be activated by alpha-thrombin in glomerular epithelial cells: 1) Ca2+ influx through a dihydroperidine-sensitive calcium channel, which seems critical for mitogenesis; 2) protein kinase C activation by phosphoinositide breakdown, which stimulates both mitogenesis and synthesis of u-PA, t-PA, and PAI-1; 3) protein kinase C activation by other phospholipid breakdown can stimulate u-PA, t-PA, and PAI-1 synthesis but not mitogenesis.
He
C J
CJ
INSERM U 64, Hôpital Tenon, Paris, France.
Peraldi
M N
MN
Adida
C
C
Rebibou
J M
JM
Meulders
Q
Q
Sraer
J D
JD
Rondeau
E
E
D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division; D004789:Enzyme Activation; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D009529:Nicardipine / Q000494:pharmacology; D015849:Plasminogen Inactivators / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D013917:Thrombin / Q000502:physiology*; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis; D014568:Urokinase-Type Plasminogen Activator / Q000096:biosynthesis
D002121:Calcium Channel Blockers; D015849:Plasminogen Inactivators; D015759:Ionomycin; D015544:Inositol 1,4,5-Trisphosphate; D009529:Nicardipine; D011493:Protein Kinase C; D013917:Thrombin; D010959:Tissue Plasminogen Activator; D014568:Urokinase-Type Plasminogen Activator; D002118:Calcium