Faculty Bibliography

Our previous studies suggest that replication-defective Sindbis vectors might be promising agents for specific tumor targeting and detection. However, the effects of innate and/or adaptive anti-viral immunity, in particular, the IFN-I/STAT1 signaling pathway, may impact their therapeutic potential. Using a bioluminescent imaging system, we demonstrate that although most normal cells are not permissively transduced by replication-defective Sindbis vector, transduction of liver non-sinusoidal endothelial occurs the first time IFN-I/STAT1 signaling deficient mice are inoculated with the vector. Transduction of some cells is not surprising since STAT1 knockout animals show significant delay in IFN responses such as the production of IFN-alpha/beta and transcriptional activation of several anti-viral genes (IRF7, RIG-I, PKR, TLR3, USP18, ISG15). However, beyond the initial vector transduction, which resolves rapidly, secondary inoculums of Sindbis vectors do not transduce any liver cells, suggesting that an alternative antiviral pathway may protect against further transduction. Other known signaling pathways were examined using mice lacking functional TLR3, tumor necrosis factoralphaR or nuclear factor-kappa B (p50). Surprisingly, none of those pathways seem to play a significant role in anti-Sindbis responses. Thus it appears that in vivo, in contrast to the ready transduction of tumor cells, transduction of normal cells by replication-defective Sindbis vector is limited, possibly by a novel mechanism

Nuclear receptor coregulator (NRC) is a 250-kDa nuclear protein involved in transcriptional activation of nuclear hormone receptors, nuclear factor-kappaB, c-Jun, c-Fos, and cAMP response element-binding protein. NRC is organized into a modular structure consisting of two activation domains (AD1 and AD2), two nuclear hormone receptor-interacting motifs, LxxLL-1 and LxxLL-2, and a C-terminal regulatory region rich in serines, threonines, and leucines. The LxxLL-1 motif of NRC binds to a broad spectrum of nuclear hormone receptors with high affinity whereas LxxLL-2 interacts with a very limited number of receptors. In this study we present further evidence that NRC can act as a dimer and have identified a dimerization region of 146 amino acids including LxxLL-1. Mutation of the core LxxLL-1 motif, however, indicates that it is not involved in the dimerization of NRC. AD2, just C-terminal of LxxLL-1, was found to play a central role in ligand-dependent activation by nuclear receptors even though AD1 exhibits more potent intrinsic activity. Thus, a short region of approximately 300 amino acids including and flanking LxxLL-1 plays an important role in NRC dimerization and nuclear receptor binding and transcriptional activation. In addition, consistent with its role as a cointegrator for transcriptional activation, NRC also functions as a coactivator for signal transducer and activator of transcription 2 (STAT-2) and p53. Activation of p53 by NRC appears to involve a novel mechanism where NRC interacts indirectly with p53 through Trap80, a member of the mediator complex, which binds NRC interacting factor-1 (NIF-1), which interacts with and potentiates the effect of NRC

Interleukin-21 (IL-21) is a type I cytokine that modulates functions of T, B, natural killer (NK), and myeloid cells. The IL-21 receptor (IL-21R) is closely related to the IL-2 receptor beta chain and is capable of transducing signals through its dimerization with the common cytokine receptor gamma chain (gamma(c)), the protein whose expression is defective in humans with X-linked severe combined immunodeficiency. To clarify the molecular basis of IL-21 actions, we investigated the role of tyrosine residues in the IL-21R cytoplasmic domain. Simultaneous mutation of all 6 tyrosines greatly diminished IL-21-mediated proliferation, whereas retention of tyrosine 510 (Y510) allowed full proliferation. Y510 efficiently mediated IL-21-induced phosphorylation of Stat1 and Stat3, but not of Stat5, and CD8(+) T cells from Stat1/Stat3 double knock-out mice exhibited decreased proliferation in response to IL-21 + IL-15. In addition, IL-21 weakly induced phosphorylation of Shc and Akt, and consistent with this, specific inhibitors of the MAPK and PI3K pathways inhibited IL-21-mediated proliferation. Collectively, these data indicate the involvement of the Jak-STAT, MAPK, and PI3K pathways in IL-21 signaling

IL-17-secreting CD4(+) T cells are critically involved in inflammatory immune responses. Development of these cells is promoted in vivo and in vitro by IL-23 or TGFbeta1 plus IL-6. Despite growing interest in this inflammatory Th subset, little is known about the transcription factors that are required for their development. We demonstrate that Stat3 is required for programming the TGFbeta1 plus IL-6 and IL-23-stimulated IL-17-secreting phenotype, as well as for RORgammat expression in TGFbeta1 plus IL-6-primed cells. Moreover, retroviral transduction of a constitutively active Stat3 into differentiating T cell cultures enhances IL-17 production from these cells. We further show that Stat4 is partially required for the development of IL-23-, but not TGFbeta1 plus IL-6-primed IL-17-secreting cells, and is absolutely required for IL-17 production in response to IL-23 plus IL-18. The requirements for Stat3 and Stat4 in the development of these IL-17-secreting subsets reveal additional mechanisms in Th cell fate decisions during the generation of proinflammatory cell types

The NS1 protein of influenza A virus is a major virulence factor that is essential for pathogenesis. NS1 functions to impair innate and adaptive immunity by inhibiting host signal transduction and gene expression, but its mechanisms of action remain to be fully elucidated. We show here that NS1 forms an inhibitory complex with NXF1/TAP, p15/NXT, Rae1/mrnp41, and E1B-AP5, which are key constituents of the mRNA export machinery that interact with both mRNAs and nucleoporins to direct mRNAs through the nuclear pore complex. Increased levels of NXF1, p15, or Rae1 revert the mRNA export blockage induced by NS1. Furthermore, influenza virus down-regulates Nup98, a nucleoporin that is a docking site for mRNA export factors. Reduced expression of these mRNA export factors renders cells highly permissive to influenza virus replication, demonstrating that proper levels of key constituents of the mRNA export machinery protect against influenza virus replication. Because Nup98 and Rae1 are induced by interferons, down-regulation of this pathway is likely a viral strategy to promote viral replication. These findings demonstrate previously undescribed influenza-mediated viral-host interactions and provide insights into potential molecular therapies that may interfere with influenza infection

Transcription factors are critical for instructing the development of B lymphocytes from multipotential progenitor cells in the bone marrow (BM). Here, we show that the absence of STAT3 impaired B-cell development. Mice selectively lacking STAT3 in BM progenitor cells displayed reduced numbers of mature B cells, both in the BM and in the periphery. The reduction in the B-cell compartment included reduced percentages and numbers of pro-B, pre-B, and immature B cells in the absence of STAT3, whereas the number of pre-pro-B cells was increased. We found that pro-B and pre-B-cell populations lacking STAT3 were hyporesponsive to IL-7 because of a decreased number of IL-7-responsive cells rather than decreased expression or signaling of IL-7Ralpha. Moreover, STAT3-deficient mice displayed enhanced apoptosis in the pro-B population when deprived of survival factors, suggesting that at least 2 mechanisms (impaired differentiation and enhanced apoptosis) are involved in the mutant phenotype. Last, BM transplantation confirmed that impaired B lymphopoiesis in the absence of STAT3 was caused by a cell autonomous defect. In sum, these studies defined a specific role for STAT3 in early B-cell development, probably acting at the pre-pro-B transition by contributing to the survival of IL-7-responsive progenitors

To achieve maximal transcriptional activity in response to gp130 cytokines, Serine-727 (Ser-727) of Stat3 is phosphorylated. Ser-727 resides in the LPMSP motif, the only conserved sequence among the transcription activation domains of several STATs. We show here that in addition to Ser-727, other residues in this LPMSP motif are also required for Stat3 activity in response to cytokine signaling through regulation of Ser-727 phosphorylation and recruitment of the transcription co-activator CBP/p300 to the promoters of Stat3-target genes for transcription activation. Hence, we have demonstrated a critical role for the whole conserved LPMSP motif in JAK-STAT signaling