IFN-α Activates Stat6 and Leads to the Formation of Stat2:Stat6 Complexes in B Cells This information is current as Sanjay Gupta, Man Jiang and Alessandra B. Pernis of October 5, 2021. J Immunol 1999; 163:3834-3841; ; http://www.jimmunol.org/content/163/7/3834 References This article cites 55 articles, 31 of which you can access for free at: Downloaded from http://www.jimmunol.org/content/163/7/3834.full#ref-list-1 Why The JI? Submit online. http://www.jimmunol.org/ • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: by guest on October 5, 2021 http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. IFN-a Activates Stat6 and Leads to the Formation of Stat2: Stat6 Complexes in B Cells1 Sanjay Gupta, Man Jiang, and Alessandra B. Pernis2 IFN-a consists of a family of highly homologous proteins, which exert pleiotropic effects on a wide variety of cell types. The biologic activities of IFN-a are mediated by its binding to a multicomponent receptor complex resulting in the activation of the Janus kinase-STAT signaling pathway. In most cell types, activation of Stat1 and Stat2 by IFN-a leads to the formation of either STAT homo-/heterodimers or of the IFN-stimulated gene factor 3 complex composed of Stat1, Stat2, and p48, a non-STAT protein. These distinct transcriptional complexes then target two different sets of cis-elements, g-activated sites and IFN-stimulated response elements. Here, we report that IFN-a can activate complexes containing Stat6, which, until now, has been primarily associated with signaling by two cytokines with biologic overlap, IL-4 and IL-13. Induction of Stat6 complexes by IFN-a appears to be cell type specific, given that tyrosine phosphorylation of Stat6 in response to IFN-a is predominantly detected in B cells. Activation of Stat6 by IFN-a in B cells is accompanied by the formation of novel Stat2:Stat6 complexes, including an IFN-stimulated gene Downloaded from factor 3-like complex containing Stat2, Stat6, and p48. B cell lines resistant to the antiproliferative effects of IFN-a display a decrease in the IFN-a-mediated activation of Stat6. Activation of Stat6 as well as of Stat2:Stat6 complexes by IFN-a in B cells may allow modulation of target genes in a cell type-specific manner. The Journal of Immunology, 1999, 163: 3834–3841. nterferon-a consists of a family of highly homologous pro- Stat1, and of Stat2 in most cell types (9–11), although activation teins that exert pleiotropic actions, including immunomodu- of Stat3, Stat4, and Stat5 has also been reported (12–14). http://www.jimmunol.org/ I latory, antiviral, and antiproliferative effects (1–3). These bi- STAT homo-/heterodimers activated in response to IFN-a can ologic activities are mediated by the ability of the IFN-a species to drive the expression of a subset of IFN-a-inducible genes, e.g., the bind to a multicomponent receptor complex. Two distinct human gene encoding the transcription factor IFN regulatory factor 1 receptor chains have been cloned so far: the IFNaR1 and the (IRF-1), by binding to specific promoter elements termed g-acti- IFNaR2 chains (4). However, additional receptor components may vated sites (GAS).3 Unlike other cytokines, IFN-a activation of exist, given that transfection of both human receptor chains in STATs also leads to the formation of an additional transcriptional murine cells does not fully reconstitute biologic responses to all complex termed IFN-stimulated gene factor 3 (ISGF3), which is human IFN-a subspecies (5). Cell type-specific differences in re- composed of a Stat1:Stat2 heterodimer and a non-STAT protein, ceptor composition may also occur, as suggested by the finding that p48. The ISGF3 complex can then induce the expression of a dis- by guest on October 5, 2021 IFN-a interacts differently with lymphoid vs epithelial cells (6). tinct subset of IFN-responsive genes by binding to a different sub- The early intracellular events triggered by IFN-a have recently set of cis-acting elements, termed IFN-stimulated response ele- been elucidated and have served as a paradigm for cytokine sig- ments (ISREs) (15). naling (7, 8). Binding of IFN-a to its receptor leads to the activa- IFN-a has been widely used as an antiviral as well as an anti- tion, via two receptor-associated tyrosine kinases, Tyk2 and Jak1, tumor agent because of its growth-inhibitory effects (16–18). of latent cytoplasmic factors belonging to the STAT (signal trans- Up-regulation of IRF-1 is believed to mediate some of the anti- ducers and activators of transcription) family of proteins. Tyrosine proliferative effects of the IFNs (19). The effectiveness of the phosphorylation of the STATs allows them to homodimerize or growth-inhibitory actions of IFNs is, however, limited by the heterodimerize and to translocate into the nucleus, where they emergence of IFN resistance. In B cells, IFN resistance character- modulate gene transcription. So far, seven distinct members of the izes EBV transformation of B cells as well as certain subclones of STAT family have been identified in eukaryotes. Some STATs Daudi, a Burkitt’s lymphoma cell line, which is normally exquis- display a restricted pattern of activation. For instance, Stat6 com- itely sensitive to growth inhibition by IFN-a. IFN resistance in plexes are induced primarily in response to two cytokines with EBV-transformed B cells has been attributed to the presence of the overlapping biologic functions, IL-4 and IL-13. In contrast, other EBV nuclear Ag 2 (EBNA2) (20). However, a distinct mechanism STATs can be activated in response to a variety of cytokines (9). is believed to be responsible for the emergence of IFN resistance IFN-a has been shown to lead to the tyrosine phosphorylation of in Daudi variants, because the EBV genome present in Daudi cells is defective and carries a deletion of the entire coding region of EBNA2 (21). In both of these systems, IFN resistance leads to the Department of Medicine, Columbia University, New York, NY 10032 selective extinction of the antiproliferative pathways triggered by Received for publication September 22, 1998. Accepted for publication July 23, 1999. IFN-a, whereas the induction of other IFN-a responses is main- The costs of publication of this article were defrayed in part by the payment of page tained at normal levels (20, 22). Studies of the molecular mecha- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nisms underlying IFN resistance in both Daudi as well as EBV- 1 This work was supported by a Grant-in-Aid from the American Heart Association, transformed B cells have failed to demonstrate changes in either the Silverberg Award, and the Saydman Trust Fund for Research in Septicemia in honor of Dr. Harold C. Neu at Columbia University. 2 Address correspondence and reprint requests to Dr. Alessandra Pernis, Department 3 Abbreviations used in this paper: GAS, g-activated site; IRF, IFN regulatory factor; of Medicine, Columbia University, 630 West 168th Street, New York, NY 10032. ISRE, IFN-stimulated response element; ISGF3, IFN-stimulated gene factor 3; E-mail address: [email protected] EBNA2, EBV nuclear Ag 2; WCE, whole-cell extract. Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 3835 the affinity or number of IFN-a receptors. A variety of defects in Antibodies STAT activation has been reported in Daudi resistant clones, in- Rabbit polyclonal antisera against Stat1, Stat2, and p48 were a generous cluding lack of Stat3 activation (12), premature loss of Stat1 phos- gift of Dr. Chris Schindler. Rabbit polyclonal Abs against human Stat3, phorylation (23), and an inability to activate an ISRE binding com- Stat5, and Stat6 were purchased from Santa Cruz Biotechnology (Santa plex (24). A similar defect in ISGF3 activation has not been Cruz, CA). The antiserum against IFN-aRI was a generous gift of Dr. detected in EBV-transformed B cells (21), while the ability of Oscar Colamonici (University of Tennessee). these cells to activate other STAT complexes has not been thor- Immunoprecipitations oughly investigated. In this study, we have found that exposure of B cells to IFN-a Extracts were immunoprecipitated with anti-STAT antisera as described leads to activation of Stat6-containing complexes, which are in- previously (31). The immunoprecipitates were fractionated by 7% SDS- PAGE before immunoblotting with either an anti-phosphotyrosine Ab distinguishable from those induced by IL-4 (25–28). Stat6 activa- (4G10, Upstate Biotechnology, Lake Placid, NY) or anti-STAT antisera. tion in response to IFN-a is not observed in non-B cell lines. The Bands were detected by enhanced chemiluminescence (Amersham, Arling- ability of IFN-a to activate Stat6 in addition to previously char- ton Heights, IL). acterized STATs allows for the formation of novel Stat2:Stat6 complexes in B cells, including an ISGF3-like complex containing Northern analysis a Stat2, Stat6, and p48. IFN- -mediated activation of Stat6 is mark- Total RNA was extracted by utilizing the ULTRA-SPEC II kit (Bioteck edly reduced in both EBV-transformed B cells and D-R cells.