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In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular calcium ion concentration [Ca2+]i and inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) formation in the human renal carcinoma cell line CaKi-1. We also examined the possible role of a Ca(2+)-dependent mechanism during IFN-gamma-induced intercellular adhesion molecule 1 (ICAM-1) antigen expression. IFN-gamma caused a rapid concentration-dependent rise in [Ca2+]i, which was partly inhibited by diltiazem, a calcium channel blocker, TMB-8, an inhibitor of intracellular calcium redistribution, and in calcium-free medium. IFN-gamma caused a fourfold increase in Ins 1,4,5-P3 formation. The induction of ICAM-1 antigen expression was synergistically enhanced by 4-bromocalcium ionophore A23187. Finally, the calcium antagonists diltiazem. TMB-8 and EGTA, as well as two potent inhibitors of Ca(2+)-dependent kinases, calmidazolium (R24571) and W7, had no or only a minor inhibitory effect on IFN-gamma induction. Our data suggest that IFN-gamma increases [Ca2+]i in CaKi-1 cells by stimulating influx of Ca2+ and release of Ca2+ from intracellular stores, probably via Ins 1,4,5-P3 formation. IFN-gamma signal transduction in our model may not be limited to an increase in [Ca2+]i and Ins 1,4,5-P3, since IFN-gamma-induced ICAM-1 antigen expression was abrogated to a minor degree by calcium antagonists and not coupled to Ins 1,4,5-P3 formation.

Incubation of the human renal carcinoma cell line CaKi-1 with interferon-gamma (IFN-gamma) or the phorbol ester, phorbol-12-myristate 13-acetate (PMA) strongly stimulated the immunocytochemical expression of the intercellular adhesion molecule-1 (ICAM-1) in a dose-dependent manner. Since PMA is capable of activating the Ca2+/phospholipid-dependent protein kinase C (PKC), we investigated the role of this kinase during IFN-gamma signal transduction. Calcium ionophore A23187 significantly enhanced IFN-gamma- and PMA-induced ICAM-1 staining. While staurosporine, H7 and sphingosine, three known PKC inhibitors, blocked the PMA effect, only staurosporine abrogated the action of IFN-gamma. Finally, 24 h of PMA pretreatment with subsequent IFN-gamma stimulation enhanced ICAM-1 staining above values from cultures where IFN-gamma was omitted. This occurred despite the fact that 24 h of PMA pretreatment abolished the effect of IFN-gamma on PKC activation, as determined by acetylated myelin basic protein 4-14 phosphorylation. In conclusion, these results suggest that additional events other than PKC activation are required for complete regulation of ICAM-1 antigen by IFN-gamma in the whole cell population. Hence, other Ca(2+)-dependent signalling pathway(s) mediated by IFN-gamma receptors must act. Further studies are needed to elucidate these specific pathway(s) activated during IFN-gamma stimulation in our model.

We have recently shown that interferon-gamma (IFN-gamma) stimulated immunocytochemical staining of the intercellular adhesion molecule ICAM-1 may be dependent on inositol phosphate formation in the human renal carcinoma cell line CaKi-1. In the present study we investigated the possible role of GTP-binding proteins (G-proteins) during IFN-gamma signalling. Preincubation of CaKi-1 cells for 24 h with increasing amounts of pertussis toxin (PT) or cholera toxin (CT), two regulators of G-protein activity, inhibited IFN-gamma induced ICAM-1 staining. Preincubation with PT or CT for 24 h also inhibited IFN-gamma induced inositol 1-monophosphate (Ins 1-P), inositol 1,4 bisphosphate (Ins 1,4-P2) and inositol 1,4,5 trisphosphate (Ins 1,4,5-P3) formation. Our findings suggest that IFN-gamma induced ICAM-1 staining and inositol phosphate formation in CaKi-1 cells is dependent on a PT and CT sensitive signalling pathway. This may reflect a role for G-proteins in the coupling of IFN-gamma receptor activation and phospholipase C catalyzed phosphoinositide hydrolysis.