Interferon-gamma increases inositol phosphate formation and cellular calcium ion concentration independent of ICAM-1 antigen enhancement in renal tubular cells.

In the present study, we investigated the effect of interferon-gamma (IFN-gamma) on cellular inositol phosphate formation and cellular calcium ion concentration [Ca2+]i in human renal proximal tubular (HRPT) cells. We also examined the possible role of the inositol phosphate-Ca2+ signalling pathway during IFN-gamma-induced intercellular adhesion molecule-1 (ICAM-1) antigen expression. IFN-gamma caused an increase in the formation of inositol 1-monophosphate (Ins 1-P), inositol 1,4-bisphosphate (Ins 1,4-P2), inositol 1,4,5-trisphosphate (Ins 1,4,5-P3) and inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5-P4). A rapid time-dependent rise in [Ca2+]i was observed upon IFN-gamma stimulation, with maximal levels reached after 1 min. A lower rise in [Ca2+]i was observed when cells were stimulated in Ca2+-free medium. This correlated with the generation of Ins 1,4,5-P3 by IFN-gamma, a well-known secondary messenger capable of releasing Ca2+ from intracellular stores. The induction of ICAM-1 antigen expression was enhanced by IFN-gamma, 4-bromocalcium ionophore A23187 (Bromo-A23187), and their combinations. However, the calcium antagonist diltiazem and calcium chelator EGTA had no effect on IFN-gamma antigen induction. In conclusion, our data suggest that IFN-gamma stimulation of HRPT cells results in the cleavage of phosphatidylinositol bisphosphate by phospholipase C, generating inositol phosphates, of which Ins 1,4,5-P3 probably releases Ca2+ from intracellular stores. A further increase in [Ca2+]i upon IFN-gamma stimulation results from influx of extracellular Ca2+. IFN-gamma signal transduction in HRPT cells may not be limited to the inositol phosphate-Ca2+ pathway since IFN-gamma-induced ICAM-1 antigen expression was unaffected by calcium antagonist/chelator.

Role of protein kinase C during interferon-gamma- and phorbol ester-stimulated immunocytochemical expression of ICAM-1 in human renal carcinoma cells.

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.