Studies on interferon-gamma signalling and the regulation of gene expression in macrophages

Interferon (IFN)-γ plays a central role in the pathogenesis of atherosclerosis. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway is the most widely used mechanism for IFN-γ signalling and STAT1 Ser727 phosphorylation is known to be required for its full transcriptional activity. Agonists for peroxisome proliferator-activated receptors (PPARs) and liver X receptors (LXRs) as well as statins have been shown to exert profound anti-inflammatory actions and protective effects in atherosclerosis. However, the molecular mechanisms involved in the IFN-γ-stimulated STAT1 Ser727 phosphorylation and the anti-inflammatory actions of these therapeutic agents, particularly in relation to IFN-γ responses are yet to be fully elucidated. Studying such mechanisms may lead to the identification of new avenues for the treatment of this disease, and was therefore the main focus of studies. Inhibitors for extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), casein kinase 2 (CK2), phosphoinostide 3'-kinase (PI3K), protein kinase C-8 (PKC-8) and calcium-calmodulin-dependent protein kinase II (CaMKII) individually inhibited the IFN-γ-stimulated STAT1 Ser727 phosphorylation. The most extensive inhibition was observed for ERK and JNK and hence their roles in IFN-γ signalling pathway were investigated in more detail. The ERK inhibitor, PD98059 attenuated the IFN-γ-induced expression of a range of genes implicated in atherosclerosis. A role for the ERK- and JNK-cascades in the regulation of STAT1 transactivation and MCP-1 gene expression by IFN-γ was further confirmed through co-transfection assays with plasmids specifying for inactive mutant forms of Ras, Raf-1, ERK1, ERK2, MEK1 and JNK. The important roles of ERK and JNK in IFN-γ signalling were further supported by kinase assays that showed that the activity of ERK and JNK was induced approximately 3- and 1.5-fold respectively in response to IFN-γ. RT-PCR analysis also revealed an inhibitory effect of agonists for PPARs and LXRs, as well as statins, on the IFN-γ-induced expression of several genes implicated in atherosclerosis with agent-specific actions being observed. These agents were also found to inhibit the IFN-γ-stimulated STAT1 transactivation and the activation of MCP-1 gene promoter. Endogenous ligands for LXRs, but not agonists for PPARs or statins, attenuated the IFN-γ-induced STAT1 phosphorylation, DNA binding activity and nuclear level of this transcription factor. Co-transfection assays revealed that the inhibitory effects on STAT1 activity by the LXR endogenous ligands were dependent on the corresponding receptor and that the repressive actions of agonists for PPARs and LXRs were not dependent on a mechanism involving competing for a limiting amount of shared co-activators in the cells. These studies together reveal novel roles for ERK and JNK, via modulation of STAT1 Ser727 phosphorylation, in the regulation of gene expression by IFN-γ. In addition, the work has demonstrated the inhibitory effects of agonists for PPARs and LXRs as well as statins on the IFN-γ-induced inflammatory gene expression and potential mechanisms underlying such regulation