Faculty Bibliography

Multiple signaling pathways are activated during engagement of the Type I interferon (IFN) receptor to mediate biological responses, including the Jak-Stat and Rac1/p38 Map kinase signaling cascades. In the present study we sought to determine the functional relevance of the p38alpha isoform in IFN signaling, using cells from mouse embryos with targeted disruption of the p38alpha gene. Our data demonstrate that p38alpha activation is essential for Type I IFN-dependent transcriptional regulation via ISRE or GAS elements. On the other hand, the function of p38alpha is not required for IFN-dependent Ser727 or Tyr701 phosphorylation of Stat1 and does not impact on the formation of ISGF3 or SIF nuclear binding complexes. In efforts to identify downstream effectors of p38 that may mediate IFN-dependent transcriptional responses, we found that IFNalpha activates the kinase Msk1, a known regulator of histone phosphorylation and chromatin remodeling. In other studies, we demonstrate that Type I IFN-dependent activation of the kinases MapKapK-2 and MapKapK-3 is defective in the absence of p38alpha, while Type I IFN-dependent antiviral properties are decreased in cells with targeted disruption of the MapKapK-2 gene. Altogether, our data establish that the p38alpha Map kinase pathway regulates activation of downstream effectors that participate in the induction of IFN-dependent gene transcription, to mediate IFN-responses

A large number of extracellular polypeptides bound to their cognate receptors activate the transcription factor STAT3 by phosphorylation of tyrosine 705. Supplemental activation occurs when serine 727 is also phosphorylated. STAT3 deletion in mice leads to embryonic lethality. We have produced mice with alanine substituted for serine 727 in STAT3 (the SA allele) to examine the function of serine 727 phosphorylation in vivo. Embryonic fibroblasts from SA/SA mice had approximately 50% of the transcriptional response of wild-type cells. However, SA/SA mice were viable and grossly normal. STAT3 wild-type/null (+/-) animals were also normal and were interbred with SA/SA mice to study SA/- mice. The SA/- mice progressed through gestation, showing 10 to 15% reduced birth weight, three-fourths died soon after birth, and the SA/- survivors reached only 50 to 60% of normal size at 1 week of age. The lethality and decreased growth were accompanied by altered insulin-like growth factor 1 (IGF-1) levels in serum, establishing a role for the STAT3 serine phosphorylation acting through IGF-1 in embryonic and perinatal growth. The SA/- survivors have decreased thymocyte number associated with increased apoptosis, but unexpectedly normal STAT3-dependent liver acute phase response. These animals offer the opportunity to study defined reductions in the transcriptional capacity of a widely used signaling pathway

We had previously demonstrated that in mice acute toxoplasmosis leads to systemic inhibition of angiogenesis and, consequently, strong suppression of neoplastic growth. Here we investigated the role of Th1 cytokines, in particular interferon gamma (IFN-gamma), in this phenomenon. Besides toxoplasma, neoplastic growth was readily blocked during acute infection with other Th1 response-inducing pathogens such as Listeria monocytogenes and lymphocytic choriomeningitis virus (LCMV). In contrast, chronic infection with LCMV (when Th1 responses were strongly suppressed) and acute infection with Schistosoma mansoni (when Th2 responses predominated) afforded no anti-tumor protection. To corroborate the involvement of Th1 cytokines in infection-mediated suppression of neoplastic growth, we utilized mice deficient in interleukin-10 (IL10), a suppressor of Th1 responses. When challenged with B16 cells concomitantly with toxoplasma infection, both IL10-null and wild type mice exhibited resistance to neoplastic growth. However, tumors borne by IL10-null animals were even smaller than those borne by their wild type counterparts. This enhanced resistance correlated with dramatically elevated levels of circulating IFN-gamma, a principal Th1 cytokine. Furthermore, while interleukin-12 and tumor necrosis factor a were dispensable for tumor suppression, in animals deficient in IFN-gamma production or signaling, tumor growth and neovascularization were markedly enhanced. Interestingly, the enhancement was also apparent in uninfected animals suggesting that IFN-gamma and its anti-angiogenic effects underlie both infection-dependent and -independent tumor surveillance

The WD repeat-containing protein receptor for activated protein kinase C (RACK)-1 has been linked to a variety of signaling systems including protein kinase C, growth factors, and IFNs. In the IFN system, RACK-1 functions as an adaptor recruiting the transcription factor STAT1 to the receptor complex. However, RACK-1 should play a broader role in type I IFN signaling because mutation of the RACK-1 binding site in the IFN-alpha receptor 2/beta subunit of the type I IFN receptor abrogates not only STAT1, but also STAT2, activation. In this study, we demonstrate that RACK-1 serves as a scaffold protein for a multiprotein complex that includes the IFN-alpha receptor 2/beta-chain of the receptor, STAT1, Janus kinase 1, and tyrosine kinase 2. In vitro data further suggest that within this complex tyrosine kinase 2 is the tyrosine kinase responsible for the phosphorylation of STAT1. Finally, we provide evidence that RACK-1 may also serve as a scaffold protein in other cytokine systems such as IL-2, IL-4, and erythropoietin

Toll-like receptor-4 activation by lipopolysaccharide (LPS) induces the expression of interferon-beta (IFN-beta) in a MyD88-independent manner. Here we report that mice devoid of the JAK protein tyrosine kinase family member, Tyk2, were resistant to shock induced by high doses of LPS. Basal and LPS-induced expression of IFN-beta and IFN-alpha4 mRNA in Tyk2-null macrophages were diminished. However, Tyk2-null mice showed normal systemic production of nitric oxide and proinflammatory cytokines and the in vivo response to tumor necrosis factor (TNF) was unperturbed. IFN-beta-null but not STAT1-null mice were also resistant to high dose LPS treatment. Together, these data suggest that Tyk2 and IFN-beta are essential effectors in LPS induced lethality.

Type I IFNs (IFN-alpha/beta) modulate innate immune responses. Here we show activation of transcription factor IFN regulatory factor 3, the synthesis of large amounts of IFN-beta mRNA, and type I IFN signal transduction in macrophages infected with Listeria monocytogenes. Expression of the bacterial virulence protein listeriolysin O was necessary, but not sufficient, for efficient IFN-beta production. Signaling through a pathway involving the type I IFN receptor and Stat1 sensitized macrophages to L. monocytogenes-induced cell death in a manner not requiring inducible NO synthase (nitric oxide synthase 2) or protein kinase R, potential effectors of type I IFN action during microbial infections. The data stress the importance of type I IFN for the course of infections with intracellular bacteria and suggest that factors other than listeriolysin O contribute to macrophage death during Listeria infection

Extracellular proteins bound to cell-surface receptors can change nuclear gene expression patterns in minutes, with far-reaching consequences for development, cell growth and homeostasis. The signal transducer and activator of transcription (STAT) proteins are among the most well studied of the latent cytoplasmic signal-dependent transcription-factor pathways. In addition to several roles in normal cell decisions, dysregulation of STAT function contributes to human disease, making the study of these proteins an important topic of current research