Eighty percent of patients newly infected with the hepatitis C virus (HCV) develop chronic infection, suggesting that HCV can develop effective strategies to escape the unspecific and specific immune response of the host. Because SOCS molecules have been recognized to be powerful inhibitors of cytokine signaling via the Jak/STAT pathway, virus-induced expression of these molecules should be an efficient instrument to counteract the cellular response toward interferons (IFNs), an essential part of first line antiviral immune response. This study shows that overexpression of HCV core protein inhibits IFN-alpha-induced tyrosine phosphorylation and activation of STAT1 in hepatic cells. With the use of a STAT1-YFP fusion protein, further evidence is given that HCV core is capable to inhibit nuclear translocation of STAT1. Inhibition of STAT1-tyrosine phosphorylation was accompanied by the induction of SOCS3-mRNA expression, suggesting that the HCV core protein impairs IFN-alpha-induced signal transduction via induction of SOCS3 expression. HCV core protein was competent to partially rescue growth of a genetically engineered influenza A virus lacking its own IFN antagonist. These IFN-antagonistic properties of the HCV core protein may be part of the molecular basis of IFN-alpha unresponsiveness in about one-half of chronically infected HCV-patients.

The potential of some proinflammatory mediators to inhibit gp130-dependent STAT3 activation by enhancing suppressor of cytokine signaling (SOCS) 3 expression represents an important molecular mechanism admitting the modulation of the cellular response toward gp130-mediated signals. Thus, it is necessary to understand the mechanisms involved in the regulation of SOCS3 expression by proinflammatory mediators. In this study, we investigate SOCS3 expression initiated by the proinflammatory cytokine TNF-α. In contrast to IL-6, TNF-α increases SOCS3 expression by stabilizing SOCS3 mRNA. Activation of the MAPK kinase 6 (MKK6)/p38MAPK-cascade is required for TNF-α-mediated stabilization of SOCS3 mRNA and results in enhanced SOCS3 protein expression. In fibroblasts or macrophages deficient for MAPK-activated protein kinase 2 (MK2), a downstream target of the MKK6/p38MAPK cascade, basal SOCS3-expression is strongly reduced and TNF-α-induced SOCS3-mRNA stabilization is impaired, indicating that MK2 is crucial for the control of SOCS3 expression by p38MAPK-dependent signals. As a target for SOCS3 mRNA stability-regulating signals, a region containing three copies of a pentameric AUUUA motif in close proximity to a U-rich region located between positions 2422 and 2541 of the 3′ untranslated region of SOCS3 is identified. One factor that could target this region is the zinc finger protein tristetraprolin (TTP), which is shown to be capable of destabilizing SOCS3 mRNA via this region. However, data from TTP-deficient cells suggest that TTP does not play an irreplaceable role in the regulation of SOCS3 mRNA stability by TNF-α. In summary, these data indicate that TNF-α regulates SOCS3 expression on the level of mRNA stability via activation of the MKK6/p38MAPK cascade and that the activation of MK2, a downstream target of p38MAPK, is important for the regulation of SOCS3 expression.

Nickel compounds are prime inducers of contact allergy reactions in humans. To identify the signal transduction pathways mediating the cellular responses to nickel and to elucidate their hierarchy, we performed Affymetrix gene profiling using human primary endothelial cells, which strongly respond to nickel stimulation. Overall, we found 258 significantly modulated transcripts, comprising 140 up-regulated and 118 down-regulated genes. The bulk of those genes were identified as targets of two distinct signaling cascades, the IKK2/NF-B pathway and a proangiogenic pathway mediated by HIF-1␣, which accumulates upon exposure to nickel. Using dominant-interfering mutants and retroviral RNA interference technology, we demonstrate that both pathways act independently to regulate expression of nonoverlapping gene pools. NF-B activation mediates most of the proinflammatory responses to nickel. Nickel-dependent HIF-1␣ activation primarily modulates expression of genes involved in proliferation, survival, metabolism, and signaling, albeit the induction of some proinflammatory nickel-response genes, most prominently IL-6, which we identified as novel bona fide HIF-1␣ target in this study, is also critically dependent on this pathway. Furthermore, we provide evidence that transactivation of both transcription factors partially depends on p38 MAPK activation that contributes to the intensity of at least some target genes. Taken E ndothelial cells (ECs),3 located between blood and tissue compartments are important components of the innate immune system. Their activation by endogenous or environmental factors is a key event in the initiation of inflammatory responses. A strong activator of endothelium is the transition metal nickel that is widely distributed in a multitude of products. Contact with nickel is potentially hazardous for health: nickel compounds not only act as carcinogens in humans and animals (1) but also represent potent allergens (haptens). In industrialized countries, nickel is the most frequent cause for contact hypersensitivity reactions (2) with an estimated rate of 39% of young women being sensitized (3). Nickel is furthermore of great relevance in the context of biocompatibility of cardiovascular stents and orthopedic and dental biomedical alloys (4 -6). Nickel ions exert proinflammatory and irritant properties, which, in addition to their sensitizing capacity, provide a "second signal" in hypersensitivity reactions (7).Studying primary ECs, we earlier demonstrated that divalent nickel ions are capable of directly inducing endothelial adhesion molecules such as ICAM-1, VCAM-1, and E-selectin (8). Further analysis revealed that transcription of these genes is mediated via the IKK2/IB␣/NF-B signal transduction pathway, which is activated in response to soluble nickel compounds but not to other divalent cations (9, 10). NF-B activation is initiated by IKK2-mediated phosphorylation and subsequent proteasomal degradation of IB proteins, which allows nuclear translocation and binding of the transcription factor...

Bacterial endotoxin (lipopolysaccharide, or LPS) has potent proinflammatory properties by acting on many cell types, including endothelial cells. Secretion of the CXC-chemokine interleukin-8 (IL-8) by LPS-activated endothelial cells contributes substantially to the inflammatory response. Using human umbilical vein endothelial cells (HUVECs), we analyzed the role of small GTP-binding Rho proteins and p38 mitogen-activated protein kinase (MAPK) for LPS-dependent IL-8 expression in endothelial cells. Specific inactivation of RhoA/Cdc42/Rac1 by Clostridium difficile toxin B-10463 (TcdB-10463) reduced LPS-induced tyrosine phosphorylation, nuclear factor (NF)-κB–dependent gene expression, IL-8 messenger RNA, and IL-8 protein accumulation but showed no effect on LPS-dependent p38 MAPK activation. Inhibition of p38 MAPK by SB 202190 also blocked LPS-induced NF-κB activation and IL-8 synthesis. Furthermore, selective activation of the p38 MAPK pathway by transient expression of a constitutively active form of MAPK kinase (MKK)6, the upstream activator of p38, was as effective as LPS with respect to IL-8 expression in HUVECs. In summary, our data suggest that LPS-induced NF-κB activation and IL-8 synthesis in HUVECs are regulated by both a Rho-dependent signaling pathway and the MKK6/p38 kinase cascade.