SIGNAL TRANSDUCTION BY INTERFERON

Abstract

Interferons (IFNs) are a family of proteins that can be divided into 3 antigenically distinct types, namely, alpha, beta and gamma IFNs. IFNs induce a spectrum of biological effects in cells. Although much is known of the chemical nature of IFNs and the biological responses they elicit, the signalling mechanism(s) by which IFNs bring about these biological responses are not well understood. A study was carried out to elucidate the signal transduction pathway(s) of IFN. It was found that alpha and beta IFNs induce an increase in diacylglycerol (DAG) concentration in a number of cell types including normal human fibroblasts, Daudi cells and WISH cells. In addition, a DAG increase was also observed in normal human fibroblasts and WISH cells treated with gamma IFN. The DAG increase induced by IFN was found to be rapid and transient. The magnitude of the DAG increase was found to be variable. The DAG increase induced by IFN required the presence of the appropriate receptor on the cell surface and was found to be related to the number of receptors. Furthermore, DAG increases required the specific binding of IFN to its receptor. These findings suggest that the increase in DAG induced by IFN is a receptor-mediated event. The increase in DAG was not accompanied by changes in intracellular calcium and inositol phosphates. The magnitude of the IFN-induced DAG increase was found to correlate in a dose-dependent manner to the antiproliferative effect of IFN in Daudi cells and the antiviral effect of IFN in normal human fibroblasts, suggesting that the DAG increase may be relevant to these two effects. With the knowledge that DAG is a physiological activator of protein kinase C (PKC), efforts were directed to determine the role of PKC in beta IFN signalling. Using assay methods that involve measurement of the subcellular redistribution of PKC and phosphorylation of PKC-specific substrates, the activation of PKC in beta IFN-treated human fibroblasts could not be demonstrated. However, the possibility that IFN activates a small population of calcium-independent subspecies of PKC(s) cannot be excluded, since the sensitivities of these assay methods will not necessarily allow detection of activation of minor PKC subspecies. Consistent with this possibility are reports by others that specific subspecies of PKC are activated in alpha IFN signal transduction. Although activation of PKC was difficult to demonstrate, indirect evidence for the involvement of PKC in beta IFN signalling was obtained from studies on the inhibition of PKC. In several experiments involving the use of anti-PKC antibodies and inhibitors of PKC such as H-7 and pseudosubstrate inhibitor peptides, it was found that inhibition of PKC inhibited the beta IFN-induced antiviral state. Furthermore, down-regulation of PKC by prolonged treatment of normal human fibroblasts with phorbol dibutyrate also inhibited the ability of beta IFN to induce an antiviral state. However, the treatment of normal human fibroblasts with phorbol myristate acetate (PMA) and oleoyl-acetylglycerol, which are known activators of PKC, failed to mimic the effect of IFN in inducing an antiviral state. These findings suggest that a functional PKC is relevant to, but not sufficient for, the antiviral state induced by beta IFN in normal human fibroblasts. Furthermore, these data also suggest that PKC may not be the only pathway involved in beta IFN signal transduction. High resolution two-dimensional gel electrophoresis was used to investigate the IFN-induced changes in the phosphorylation of cellular proteins. In human fibroblasts, beta IFN stimulated the rapid phosphorylation of an 18.4 kDa protein and reduced the phosphorylation of 3 proteins with molecular weights of 19.5, 24 and 35 kDa. In contrast, PMA, an activator of PKC, stimulated the phosphorylation of at least 14 proteins, none of which are in common with the 18.4 kDa protein phosphorylated by beta IFN. These findings suggest that the phosphorylation of the 18.4 kDa protein by beta IFN is mediated by kinase(s) other than PKC. Unlike beta IFN, no detectable changes in protein phosphorylation were observed upon treatment of human fibroblasts with gamma IFN. However, in WISH cells, gamma IFN stimulated the phosphorylation of 16 proteins, 10 of which are in common with proteins phosphorylated by PMA. Out of 9 proteins phosphorylated by alpha IFN, 7 proteins were in common with those phosphorylated by PMA. Although the PKC-mediated phosphorylation of proteins is not as evident in IFN-treated human fibroblasts, results obtained with WISH cells suggest a role for PKC in protein phosphorylation during IFN signalling. The isolation and characterization of these phosphoproteins will be a major step in the elucidation of the signalling mechanism(s) of IFN. The identification of DAG as a putative IFN-induced second messenger, in conjunction with the demonstrated involvement of the PKC in IFN action, has contributed to an understanding of some of the earlier cellular events which may be critical steps in the IFN signal transduction pathway.