Psoriatic keratinocytes show reduced IRF-1 and STAT-1alpha activation in response to gamma-IFN.
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Psoriasis is a chronic inflammatory dermatosis characterized by hyperproliferative keratinocytes (KC). The skin lesions are infiltrated by T cells, which secrete gamma interferon (gamma-IFN) and are believed to be necessary to maintain the psoriatic phenotype. In normal KC, gamma-IFN is a potent inhibitor of proliferation, but proliferation of KC persists in psoriatic plaques despite the presence of gamma-IFN. Immunostaining of interferon regulatory factor-1 (IRF-1) revealed that IRF-1 was localized to the basal cells of the epidermis in normal and in nonlesional psoriatic skin, but was suprabasal or completely absent in lesional psoriatic skin. This finding led to the hypothesis that abnormal signaling in the gamma-IFN pathway may occur in psoriatic KC. To test this hypothesis, we measured activation of IRF-1 and signal transducer and activator of transcription (STAT)-1alpha transcription factors in KC after stimulation with gamma-IFN. Primary cultures of KC from normal and nonlesional psoriatic skin were stimulated with gamma-IFN and subsequent transcription factor activation was measured by electrophoretic mobility shift assay. Psoriatic KC showed a reduced induction of IRF-1 and STAT-1alpha activation after stimulation with gamma-IFN, compared with normal KC. Reduced activation of IRF-1 and STAT-1alpha in response to gamma-IFN indicates a fundamental defect in the growth and differentiation control of psoriatic KC in the absence of the influence of other cell types. (+info)
Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins.
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STAT proteins (signal transducers and activators of transcription) are a family of transcription factors which are used by many cytokines and cell growth factors for initiating gene expression. They are activated by tyrosine phosphorylation through the cytoplasmic domain of stimulated receptors. Upon phosphorylation STAT proteins dimerize, translocate to the nucleus and activate transcription by binding to specific recognition sites. Different cytokines activate different subsets of STATs and other signaling proteins. We have made use of green fluoresencent protein (GFP) fusion proteins to visualize the subcellular localization and trafficking of STAT1, STAT2 and p48 during interferon (IFN) stimulation and have analysed in detail STAT1-GFP trafficking in living cells. Analysis of GFP fusion proteins allowed the determination of time kinetics of subcellular trafficking in individual living cells. STAT1-GFP is indistinguishable from its wild-type protein displaying strong activity as transcriptional activator as well as the same time kinetics of transport to the nucleus and retreat to the cytoplasm. After prolonged exposure to IFN, STAT1-GFP is no longer retained in the nucleus and relocation to the cytoplasm is observed. Restimulation with the same type of IFN does not lead to repeated nuclear translocation of STAT1-GFP. STAT1 is not subject of inhibition, as restimulation with another type of IFN allows immediate reuse of previously activated STAT1-GFP. However, restimulation with the same type of IFN can be achieved when the primary stimulus is removed after a short induction period. This method of visualizing signal transduction reveals a considerable inhomogeneity with respect to the extent of STAT1-GFP shuttling within a clonal cell population, indicating that competence for full-blasted IFN response is restricted to a cellular subpopulation whereas other cells respond incompletely, retarded or not at all. (+info)
IFN-gamma regulation of the type IV class II transactivator promoter in astrocytes.
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The transcriptional activation of class II MHC genes requires the class II transactivator (CIITA) protein, a regulator that is essential for both constitutive and IFN-gamma-inducible class II MHC expression. The CIITA gene is controlled by multiple independent promoters; two promoters direct constitutive expression, while another, the type IV CIITA promoter, mediates IFN-gamma-induced expression. We investigated the molecular regulation of IFN-gamma-induced type IV CIITA promoter activity in astrocytes. IFN-gamma inducibility of the type IV CIITA promoter is dependent on three cis-acting elements contained within a 154-bp fragment of the promoter; the proximal IFN-gamma activation sequence (GAS) element, the E box, and the proximal IFN regulatory factor (IRF) element. Two IFN-gamma-activated transcription factors, STAT-1alpha and IRF-1, bind the proximal GAS and IRF elements, respectively. The E box binds upstream stimulating factor-1 (USF-1), a constitutively expressed transcription factor. Furthermore, STAT-1alpha binding to the proximal GAS element is dependent on the binding of USF-1 to the adjacent E box. Functionally, the proximal IRF element is essential for IFN-gamma induction of type IV CIITA promoter activity, while the proximal GAS and E box elements contribute to the IFN-gamma inducibility of this promoter. In astrocytes, TNF-alpha enhances IFN-gamma-induced class II MHC transcription. Our results demonstrate that TNF-alpha does not enhance IFN-gamma-induced transcriptional activation of the type IV CIITA promoter, indicating that the enhancing effect of TNF-alpha is mediated downstream of CIITA transcription. These results define the molecular basis of IFN-gamma activation of the type IV CIITA promoter in astrocytes. (+info)
Human cytomegalovirus inhibits IFN-alpha-stimulated antiviral and immunoregulatory responses by blocking multiple levels of IFN-alpha signal transduction.
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The type I IFNs represent a primordial, tightly regulated defense system against acute viral infection. IFN-alpha confers resistance to viral infection by activating a conserved signal transduction pathway that up-regulates direct antiviral effectors and induces immunomodulatory activities. Given the critical role of IFN-alpha in anti-human cytomegalovirus (HCMV) immunity and the profound ability of HCMV to escape the host immune response, we hypothesized that HCMV blocks IFN-alpha-stimulated responses by disrupting multiple levels of the IFN-alpha signal transduction pathway. We demonstrate that HCMV inhibits IFN-alpha-stimulated MHC class I, IFN regulatory factor-1, MxA and 2',5-oligoadenylate synthetase gene expression, transcription factor activation, and signaling in infected fibroblasts and endothelial cells by decreasing the expression of Janus kinase 1 and p48, two essential components of the IFN-alpha signal transduction pathway. This investigation is the first to report inhibition of type I IFN signaling by a herpesvirus. We propose that this novel immune escape mechanism is a major means by which HCMV is capable of escaping host immunity and establishing persistence. (+info)
STAT protein recruitment and activation in c-Kit deletion mutants.
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Stem cell factor (SCF) and its tyrosine kinase receptor, c-Kit, play a crucial role in regulating migration and proliferation of melanoblasts, germ cells, and hemopoietic cell progenitors by activating a number of intracellular signaling molecules. Here we report that SCF stimulation of myeloid cells or fibroblasts ectopically expressing c-Kit induces physical association with and tyrosine phosphorylation of three signal transducers and activators of transcription (STATs) as follows: STAT1alpha, STAT5A, and STAT5B. Other STAT proteins are not recruited upon SCF stimulation. Recruitment of STATs leads to their dimerization, nuclear translocation, and binding to specific promoter-responsive elements. Whereas STAT1alpha, possibly in the form of homodimers, binds to the sis-inducible DNA element, STAT5 proteins, either as STAT5A/STAT5B or STAT5/STAT1alpha heterodimers, bind to the prolactin-inducible element of the beta-casein promoter. The tyrosine kinase activity of Kit appears essential for STAT activation since a kinase-defective mutant lacking a kinase insert domain was inactive in STAT signaling. However, another mutant that lacked the carboxyl-terminal region retained STAT1alpha activation and nuclear translocation but was unable to fully activate STAT5 proteins, although it mediated their transient phosphorylation. These results indicate that different intracellular domains of c-Kit are involved in activation of the various STAT proteins. (+info)
Interferon regulatory transcription factors are constitutively expressed and spatially regulated in the mouse lens.
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Interferon regulatory factors (IRFs) are a family of transcription factors involved in regulation of cell growth and immunological responses. Nine IRFs have been described and they are expressed in a variety of cells, except for ICSBP and LSIRF/Pip, which are thought to be expressed exclusively in immune cells. Here, we show that IRF-1, IRF-2, ICSBP, and LSIRF/Pip are constitutively expressed in the mouse lens. These IRFs are present in both the cytoplasm and the nuclei of lens cells. However, the nuclear and cytoplasmic proteins exhibit distinct mobilities on SDS/PAGE. We further show that in the developing mouse lens, IRF-1 and IRF-2 are expressed at high levels in differentiated lens fiber cells with very low and barely detectable levels in undifferentiated lens epithelial cells. Although the level of ICSBP expression is very low in the normal mouse lens, in transgenic mice with constitutive expression of interferon gamma in the lens, its level is markedly elevated and ICSBP expression is detected exclusively in the nuclei of undifferentiated lens cells. Taken together, our data suggest that expression of IRF transcription factors is spatially regulated in the lens and that distinct IRFs may contribute to differential gene regulation in the epithelial and fiber compartments of the vertebrate lens. (+info)
The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-alpha.
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Greater than 95% of all cervical carcinomas have been found to be associated with "high-risk" human papillomavirus (mainly types 16 and 18) infections, with the viral E6 and E7 oncoproteins essential for neoplastic development and maintenance. Interferon-alpha (IFNalpha) is used in the treatment of HPV infections yet both in vivo and in vitro data suggest that the virus has developed mechanisms to avoid the effects of interferon. Here we show that the HPV16 E7 oncoprotein is able to inhibit the induction of IFNalpha-inducible genes but has no effect of IFNgamma-inducible genes. Expression of E7 correlates with the loss of formation of the interferon-stimulated gene factor 3 (ISGF3) transcription complex. Moreover, in the presence of E7, p48, the DNA-binding component of ISGF3, was unable to translocate to the nucleus upon IFNalpha stimulation. A direct protein-protein interaction was identified between E7 and p48 with the site of interaction within E7 defined as the region between amino acids 17-37, a domain that includes the binding site for the retinoblastoma protein, pRb. These results suggest that HPV, via E7, targets p48, resulting in the loss of IFNalpha-mediated signal transduction and may provide a means by which HPV can avoid the innate immune system. (+info)
Retinoic acid resistance in NB4 APL cells is associated with lack of interferon alpha synthesis Stat1 and p48 induction.
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In the t(15;17) acute promyelocytic leukaemia (APL), all trans-retinoic (RA) treatment induces maturation leading to clinically complete but not durable remission, as RA resistance develops in the treated patients as well as in vitro. RA and interferons (IFNs) are known inhibitors of proliferation in various cells including those from APL. In this report, we show that they can act cooperatively to inhibit growth and to induce differentiation of NB4 cells but not of two RA-resistant NB4 derived cell lines, NB4-R1 and NB4-R2. However, the resistant cell lines respond to IFN. In NB4 cells, RA increases the expression of Stat1, p48 and IRF-1, three transcription factors playing a central role in the IFN response and induces the synthesis and the secretion of IFN alpha. RA-induced IFN alpha seems to play a role in inhibition of NB4 cell growth but not in their differentiation. In the resistant cells, NB4-R1 and NB4-R2, both the induction of IFN and the increase of Statl and p48 expression by RA are completely blocked. In contrast, IRF-1 mRNA and protein expressions are induced in the three cell lines. This suggests that increase of IRF-1 expression is not sufficient for IFN induction. Our results identify some defects linked to RA-resistance in APL and support the hypothesis that RA-induced Stat1 expression and IFN secretion may be one of the mechanisms mediating growth inhibition by RA. (+info)