Thrombin signal transduction mechanisms in human glomerular epithelial cells

Abstract

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.

Long‐term effects of thrombin require sustained activation of the functional thrombin receptor

“…[Ca2+]i in HGEC was measured using fura-2 acetoxymethylester (4 pM) exactly as described [9]. Fluorescence was monitored continuously in a Perkin-Elmer model LS-5 spectrofluorometer using 339 nm excitation/500 nm emission wavelengths.…”

“…Thrombin is a potent platelet agonist [4], chemotactic for monocytes [5], mitogenic for mesenchymal cells [6] and activates endothelial cells [7]. The effects of thrombin on HGEC and signal transduction mechanisms involved have previously been described [8,9].…”

Urokinase receptor is a multifunctional protein: influence of receptor occupancy on macrophage gene expression.

“…Potentially, uPAR occupancy may directly transduce signals, as has been documented for some GPI-anchored proteins (24,25), and influence the phenotype of macrophages. In fact, uPAR bound uPA has been demonstrated to induce cellular responses both dependent on (26,27) and independent of (28)(29)(30) its catalytic activity.…”

Unexpected Role of TRPC6 Channel in Familial Nephrotic Syndrome

“…The effects of vasoactive agents are typically mediated by binding to cell surface receptors that belong to the large superfamily of seven transmembrane-spanning receptor proteins (40,41). As shown in Table 1, podocytes express numerous heptahelical receptor systems (43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56)(57)(58)(59)(60)(61). The majority of these receptors activate intracellular effector pathways (Table 1) by coupling to G proteins (41,62).…”