Down-regulation of interferon g-activated STAT1 by UV light

STAT1 is a cytoplasmic transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon-g (IFNg). Phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFNg-inducible genes. Here, we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby hindering IFNg from exerting its biological effects. This effect is not due to a down-regulation of the IFNg receptor because phosphorylation of upstream-located Jak1 and Jak2 was not suppressed by UV light. In contrast, UV light had no effect on the phosphorylation of STAT3, which is activated by the proinf lammatory cytokine interleukin 6. The UV light effect on STAT1 phosphorylation could be antagonized by vanadate, indicating at least partial involvement of a protein tyrosine phosphatase. Therefore, this study indicates a mechanism by which UV light can inhibit gene activation and suggests STAT1 as a new extranuclear UV target closely located to the membrane. Interferon g (IFNg) is a pleiotropic cytokine with antiproliferative and immunomodulatory activities that are crucial for the regulation of immune responses (1). IFNg signals through a multimeric receptor complex consisting of a ligand binding chain and a transmembrane accessory factor (2, 3). IFNg signaling is initiated when IFNg binds to its receptor, thereby inducing its dimerization. Upon this event, the receptorassociated Janus kinases Jak1 and Jak2 start to transphosphorylate each other, resulting in kinase activation. The cytoplasmic domains of the receptors then become tyrosine phosphorylated, enabling interaction with the signal transducer and activator of transcription protein STAT1 (4–6). Finally, STAT1 is activated by tyrosine phosphorylation, leading to its dimerization, and translocates to the nucleus, where it activates transcription of IFNg-responsive genes by binding to the IFNg-activated sequences (GAS) (7, 8). UV light is known for its immunosuppressive properties, which are best demonstrated by the inhibition of cellular immune reactions and by the exacerbation of infectious diseases (9, 10). UV light induces the release of immunosuppressive cytokines (11) but obviously also can interfere with the biological effects of cytokines; we recently found that UV light counteracts biological activities of IFNg (12). Specifically, we observed that stimulation of the murine keratinocyte line Pam212 with IFNg induced mRNA expression of the cytokine interleukin 7 (IL-7) whereas IL-7 transcripts were suppressed in cells that were UV light-exposed before IFNg treatment. The inhibitory effect of UV light does not, as initially anticipated, target the IL-7 promoter in a negative regulatory way but hinders IFNg from exerting its biological activity. IFNg induces IL-7 mRNA expression via activation of an IFNstimulated response element located in the 59 upstream region of the IL-7 gene (13) by inducing the transcription factor IFN regulatory factor 1 (IRF-1), which binds to the IFN-stimulated response element (14). Electrophoretic mobility-shift assays (EMSA) revealed that IFNg significantly induced IRF-1 binding whereas UV treatment remarkably decreased IFNginduced IRF-1 binding. Northern blot analysis indicated that UV light reduced IFNg-induced IRF-1 mRNA (12). Because IFNg is an important immunomodulatory cytokine, inhibition of its activity by UV light may contribute to its immunosuppressive properties. Therefore and based on the fact that inhibition of cytokine activities may represent a new biological activity of UV light, we were interested to clarify the molecular mechanisms underlying the inhibition of IRF-1 mRNA expression by UV light. Here we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby preventing binding of STAT1 to the GAS element in the IRF-1 promoter. This study both indicates a mechanism by which UV light inhibits gene activation and suggests STAT1 as a further extranuclear, closely membrane-located UV light target. MATERIALS AND METHODS Cell Culture and Treatment of Cells. The murine keratinocyte cell line Pam212 was cultured in supplemented RPMI 1640 medium. UV irradiation was performed as described (15) using FS-40 bulbs (Westinghouse Electric, Pittsburgh), which emit most of the energy in the UVB range (290–320 nm). A dose of 250 Jym2 was used throughout the study because this dose yielded the optimal effect without significantly affecting cell viability. Thereafter, cells were medium supplemented with or without recombinant murine IFNg (Genzyme) added. Unless otherwise stated, experiments were performed at least three times, and data shown are representative of one of those. Chloramphenicol Acetyltransferase (CAT) Assay. Using a calciumyphosphate coprecipitation method, cells were transfected with the GAS oligonucleotide (59-AGCCTGATTTCCCCGAAATGATGAGGCCGAGTGC-39) and cloned just upstream of the thymidine kinase promoter; CAT coding sequencing (pGAScat) followed. Thirty six hours after transfection, cells were treated with IFNg and UV light, respectively; 18 h later protein fractions were extracted. Enzymatic activity of CAT was determined using [3H]-chloramphenicol, and CAT activities obtained were normalized by bgalactosidase activity derived from cotransfection of a cytomegalovirus promoter-driven b-galactosidase expression vector. EMSA. Fifteen minutes after stimulation, cells were swelled in hypotonic buffer as described (12). After addition of Nonidet P-40, cell suspensions were minifuged, and supernatants were kept for Western blot analysis as cytosolic protein fractions. To extract nuclear proteins, pellets were resusThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1997 by The National Academy of Sciences 0027-8424y97y9411490-6$2.00y0 PNAS is available online at http:yywww.pnas.org. This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: IL, interleukin; Ab, antibody; EMSA, electrophoretic mobility-shift assay; IFNg, interferon g; IRF-1, IFN regulatory factor-1; CAT, chloramphenicol acetyltransferase. *To whom reprint requests should be addressed. e-mail: schwtho@ uni-muenster.de.