IL-17, hallmark cytokine of the newly-described “Th17” population, signals through a novel subclass of receptors, yet surprisingly little is known about mechanisms of IL-17 receptor signaling. IL-17 activates target gene expression via NF-κB and two members of the C/EBP transcription factor family, C/EBPδ and C/EBPβ. It has been shown that TRAF6 and Act1 are upstream of NF-κB and C/EBPδ, but the signaling pathways regulating C/EBPβ remain entirely undefined. In this report, we demonstrate that IL-17 signaling leads to phosphorylation of two sites in the C/EBPβ “regulatory 2 domain” in a sequential, interdependent fashion. First rapid phosphorylation of Thr188 occurs within 15 minutes and is ERK-dependent. A second phosphorylation event targets Thr179, which requires prior Thr188 phosphorylation and GSK3β activity. We further show that the pathways leading to C/EBPβ phosphorylation are mediated by distinct subdomains within IL-17RA. Whereas phosphorylation of Thr188 is mediated by the previously-identified SEFIR/TILL domain, activation of Thr179 occurs through a motif located in the IL-17RA distal tail. Functionally, C/EBPβ phosphorylation mediates a negative signal, as blocking ERK and GSK3β upregulates IL-17-induced genes, and a C/EBPβ-Thr188 mutant enhances activation of a C/EBP-dependent reporter. Consistently, GSK3β overexpression inhibits IL-17 signaling to a C/EBP-dependent reporter, and GSK3β-deficient cells fail to induce phosphorylation at T179. Thus, IL-17 triggers dual phosphorylation of C/EBPβ, which down-modulates expression of inflammatory genes. This is the first detailed dissection of the IL-17-mediated C/EBP pathway and is also the first known example of a negative signal mediated by this unique receptor.

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The IFN family of cytokines operates a frontline defense against pathogens and neoplastic cells in vivo by controlling the expression of several genes. The death-associated protein kinase 1 (DAPK1), an IFN-γ-induced enzyme, controls cell cycle, apoptosis, autophagy, and tumor metastasis, and its expression is frequently down-regulated in a number of human tumors. Although the biochemical action of DAPK1 is well understood, mechanisms that regulate its expression are unclear. Previously, we have shown that transcription factor C/EBP-β is required for the basal and IFN-γ-induced expression of DAPK1. Here, we show that ATF6, an ER stress-induced transcription factor, interacts with C/EBP-β in an IFN-stimulated manner and is obligatory for Dapk1 expression. IFN-stimulated proteolytic processing of ATF6 and ERK1/2-mediated phosphorylation of C/EBP-β are necessary for these interactions. More importantly, IFN-γ failed to activate autophagic response in cells lacking either ATF6 or C/EBP-β. Consistent with these observations, the Atf6 −/− mice were highly susceptible to lethal bacterial infections compared with the wild-type mice. These studies not only unravel an IFN signaling pathway that controls cell growth and antibacterial defense, but also expand the role of ATF6 beyond ER stress.

Studies suggest that tunicamycin may work as a therapeutic drug to cancer cells by inducing stress in the endoplasmic reticulum (ER) through unfolded protein response (UPR) and thereby promoting apoptosis. However, mechanisms of the prolonged activation of the UPR under sustained ER stress in the regulation of cell apoptosis are largely unknown. To delineate the role of candidate genes in the apoptotic process under ER stress and to search for new therapeutic strategies to treat metastatic castration resistant prostate cancer, we performed whole genome expression microarray analysis in tunicamycin treated metastatic androgen-insensitive prostate cancer cells, PC-3. Among several induced genes, the expression of eNOS (NOS3) gene was remarkably high. The increased expression of eNOS activates mTORC1 through RagC. This results into an accumulation of p62 (SQSTM1) which facilitates aggregation of ubiquitinated protein thus compromising clearance of misfolded toxic protein aggregates. Lastly, association of p62 proteins and misfolded proteins promote reactive oxygen species (ROS) mediated mitochondrial apoptosis. Overall, our data demonstrate that tunicamycin induced ER stress promotes prostate cancer cell death by activating mTORC1 through eNOS-RagC pathway.

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