Ligation of immunoreceptor tyrosine-based activation motif (ITAM)-associated receptors in macrophages can initiate potent induction of negative regulators, including anti-inflammatory cytokine IL-10, signaling inhibitors SOCS3, ABIN3, A20 and transcriptional repressor Hes1 [1] . However under inflammatory conditions, the strong inhibitory pathway initiated by ITAM-bearing receptors was altered. In macrophages isolated from rheumatoid arthritis patients, as well as in blood monocytes/macrophages primed with IFN-γ, ITAM-mediated induction of IL-10 and other inhibitory molecules was markedly attenuated. Here, we investigated mechanisms underlying the suppression of IFN-γ on ITAM-mediated inhibitory pathway. We utilized primary human macrophages in this study, and compared gene expression and signaling in IFN-γ-primed versus non-primed cells, by q-PCR and western blotting respectively. Fibrinogen (Fb) was used to ligate ITAM-bearing β 2 intergrin [2] . A combination of biochemical and genetic approaches was conducted to investigate the role of GSK3, including pharmacological inhibitors of GSK3 kinase and RNA interference of GSK3α/β genes. We further analyzed the subcellular localization of GSK3, and its potential substrates, including β-catenin. Microarray experiment was performed to determine the target genes of β-catenin. We found that IFN-γ markedly increased ITAM-regulated GSK3 kinase activity and nuclear accumulation. Inhibition of GSK3 using pharmacological inhibitors or RNA interference reversed IFN-γ suppression of IL10 and HES1 , suggesting that GSK3 mediated the downregulation of inhibitory gene expression by IFN-γ. β-catenin, a major substrate of GSK3, is a transcription factor recently implicated in induction of anti-inflammatory mediator IL-10 in murine dendritic cells [3] . However, effective knockdown of β-catenin by siRNAs had little effect on ITAM-mediated gene expression, as assessed by genome-wide microarray analysis. In contrast, we found that the expression of AP-1, another target of GSK3 [4] , as well as its nuclear accumulation was significantly suppressed by IFN-γ. We provided several lines of evidence that GSK3 was the major mediator in the crosstalk between ITAM and IFN-γ Signaling. AP-1 transcription factor, but not β-catenin, was the major target of GSK3 in this scenario. These findings yield insight into mechanisms of crosstalk between ITAM-associated receptors and IFN-γ that are important for the orchestration of cytokine production and inflammation.
Read full abstract