Faculty Bibliography

Several genetic forms of human dwarfism have been linked to activating mutations in FGF receptor 3, indicating that FGF signaling has a critical role in chondrocyte maturation and skeletal development. However, the mechanisms through which FGFs affect chondrocyte proliferation and differentiation remain poorly understood. We show here that activation of FGF signaling inhibits chondrocyte proliferation both in a rat chondrosarcoma (RCS) cell line and in primary murine chondrocytes. FGF treatment of RCS cells induces phosphorylation of STAT-1, its translocation to the nucleus, and an increase in the expression of the cell-cycle inhibitor p21WAF1/CIP1. We have used primary chondrocytes from STAT-1 knock-out mice to provide genetic evidence that STAT-1 function is required for the FGF mediated growth inhibition. Furthermore, FGF treatment of metatarsal rudiments from wild-type and STAT-1(-/-) murine embryos produces a drastic impairment of chondrocyte proliferation and bone development in wild-type, but not in STAT-1(-/-) rudiments. We propose that STAT-1 mediated down regulation of chondrocyte proliferation by FGF signaling is an homeostatic mechanism which ensures harmonious bone development and morphogenesis

Propagation of mouse embryonic stem (ES) cells in vitro requires exogenous leukemia inhibitory factor (LIF) or related cytokines. Potential downstream effectors of the LIF signal in ES cells include kinases of the Src, Jak, and mitogen-activated protein families and the signal transducer and transcriptional activator STAT3. Activation of nuclear STAT3 and the ability of ES cells to grow as undifferentiated clones were monitored during LIF withdrawal. A correlation was found between levels of STAT3 activity and maintenance of an undifferentiated phenotype at clonal density. In contrast, variation in STAT3 activity did not affect cell proliferation. The requirement for STAT3 was analyzed by targeted mutagenesis in ES cell lines exhibiting different degrees of LIF dependency. An insertional mutation was devised that abrogated Stat3 gene expression but could be reversed by Cre recombination-mediated excision. ES cells heterozygous for the Stat3 mutation could be isolated only from E14 cells, the line least dependent on LIF for self-renewal. Targeted clones isolated from other ES cell lines were invariably trisomic for chromosome 11, which carries the Stat3 locus, and retained normal levels of activated STAT3. Cre-regulated reduction of Stat3 gene copy number in targeted, euploid E14 clones resulted in dose-dependent losses of STAT3 activity and the efficiency of self-renewal without commensurate changes in cell cycle progression. These results demonstrate an essential role for a critical amount of STAT3 in the maintenance of an undifferentiated ES cell phenotype

The precise role of the different subunits (alpha/IFNAR1 and betaL/IFNAR2) of the type I interferon receptor (IFN-R) in the activation of signal transducer and activator of transcription (Stat) 1, Stat2, and Stat3 has not yet been established. In this report we demonstrate that there are functionally redundant phosphotyrosine-dependent and -independent binding sites for Stat2 in the alpha and beta subunits of the type I IFN-R. Expression of a type I IFN-R containing only the constitutive Stat2 site or the proximal tyrosines of betaL, but not the docking site on the alpha chain (Tyr466 and Tyr481), supported low levels of Stat2 activation. However, the presence of only one intact Stat2 site did not lead to induction of interferon-stimulated gene factor 3 (ISGF3) or an antiviral state. Normal levels of Stat2 tyrosine phosphorylation, induction of ISGF3, and an antiviral effect always required the proximal tyrosines of betaL and at least one of the other Stat2 sites (Tyralpha466, 481 or betaL404-462). These data suggest that a threshold of Stat2 tyrosine phosphorylation is required for complete activation of ISGF3. Interestingly, a receptor in which all tyrosines were mutated to phenylalanine shows normal Stat3 phosphorylation and low levels of activation of Stat1

The NS1 protein is the only nonstructural protein encoded by influenza A virus. It has been proposed that the NS1 performs several regulatory functions during the viral replication cycle, including the regulation of synthesis, transport, splicing, and translation of mRNAs. Through the use of reverse genetics, a viable transfectant influenza A virus (delNS1) which lacks the NS1 gene has been generated. Our results indicate that the NS1 of influenza A virus is an auxiliary (virulence) factor which plays a crucial role in inhibiting interferon-mediated antiviral responses of the host

Fragments of foreign antigens associated with class I molecules of the major histocompatibility complex (MHC) are presented at the cell surface to elicit an immune response. This presentation requires the coordinated expression of several genes contained in the MHC, including those encoding the MHC class I heavy chain, the proteins LMP-2 and LMP-7, which are involved in the proteasomal degradation of cytosolic antigens into peptide fragments that are destined for association with MHC class I molecules, and TAP-1 and TAP-2, which transport these fragments across the membrane of the endoplasmic reticulum at the start of their journey to the cell surface. In many virus-transformed cell lines and spontaneous tumours, these genes are simultaneously repressed. However, the key factor(s) that are essential for their expression and repression have not been identified. Here we report that the proto-oncogene product PML induces expression of LMP-2, LMP-7, TAP-1 and TAP-2 in an MHC-class I-negative, recurrent tumour, leading to the re-expression of cell-surface MHC in tumours and to rejection of the tumours. PML also regulates MHC expression in untransformed fibroblasts. We conclude that malfunction of PML may enable a tumour to evade the immune defence of its host

Interferon (IFN) genes are among the earliest transcriptional responses to virus infection of mammalian cells. Although the regulation of the IFNbeta gene has been well characterized, the induction of the large family of IFNalpha genes has remained obscure. We report that the IFNalpha genes can be divided into two groups: an immediate-early response gene (IFNalpha4) which is induced rapidly and without the need for ongoing protein synthesis; and a set of genes that display delayed induction, consisting of at least IFNalpha2, 5, 6 and 8, which are induced more slowly and require cellular protein synthesis. One protein that must be synthesized for induction of the delayed gene set is IFN itself, presumably IFNalpha4 or IFNbeta, which stimulates the Jak-Stat pathway through the IFN receptor, resulting in activation of the transcription factor interferon-stimulated gene factor 3 (ISGF3). Among the IFN-stimulated genes induced through this positive feedback loop is the IFN regulatory factor (IRF) protein, IRF7. Induction of IRF7 protein in response to IFN and its subsequent activation by phosphorylation in response to virus-specific signals, involving two C-terminal serine residues, are required for induction of the delayed IFNalpha gene set

We have studied the pathogenesis of influenza virus infection in mice that are unable to respond to type I or II interferons due to a targeted disruption of the STAT1 gene. STAT1-/- animals are 100-fold more sensitive to lethal infection with influenza A/WSN/33 virus than are their wild-type (WT) counterparts. Virus replicated only in the lungs of WT animals following intranasal (i.n.) virus inoculation, while STAT1-/- mice developed a fulminant systemic influenza virus infection following either i.n. or intraperitoneal inoculation. We investigated the mechanism underlying this altered virus tropism by comparing levels of virus replication in fibroblast cell lines and murine embryonic fibroblasts derived from WT mice, STAT-/- mice, and mice lacking gamma interferon (IFNgamma-/- mice) or the IFN-alpha receptor (IFNalphaR-/- mice). Influenza A/WSN/33 virus replicates to high titers in STAT1-/- or IFNalphaR-/- fibroblasts, while cells derived from WT or IFNgamma-/- animals are resistant to influenza virus infection. Immunofluorescence studies using WT fibroblast cell lines demonstrated that only a small subpopulation of WT cells can be infected and that in the few infected WT cells, virus replication is aborted at an early, nuclear phase. In all organs examined except the lung, influenza A WSN/33 virus infection is apparently prevented by an intact type I interferon response. Our results demonstrate that type I interferon plays an important role in determining the pathogenicity and tissue restriction of influenza A/WSN/33 virus in vivo and in vitro

Interferon mediates its biological effects of antiviral, antiproliferative, and immunomodulatory activities through induction of specific gene expression. Transcription of target genes is regulated through a set of transcription factors that bind to specific cis-acting regulatory sequences in target promoters and enhancers. The activity or abundance of these transcription factors is modulated by interferon treatment. Because of their DNA binding activity, regulatory factors can be recognized and characterized using methods to detect protein-DNA complexes. The availability of antibodies directed against common interferon regulatory proteins coupled with analysis of binding-site specificity provides comprehensive analysis.

A bispecific Ab (BsAb) that binds the TCR on T cells and the G protein of the vesicular stomatitis virus (VSV) can redirect staphylococcal enterotoxin B (SEB)-activated T cells to kill VSV-infected cells and to inhibit VSV replication in vitro. Inhibition of virus replication in our system is dependent upon the specificity of the Ab for the viral protein. IFN-gamma does not play a very important role in this phenomenon, which is mainly mediated by the release of Pfp from CD8+ T cells. We have used a Stat1 knockout mouse model in which VSV infection is lethal. Infusion of staphylococcal enterotoxin-activated B T cells and bispecific Ab significantly slowed virus progression and prolonged the survival of VSV-infected Stat1 knockout mice in vivo