IL-4 Induces the Proteolytic Processing of Mast Cell STAT6 Melanie A. Sherman, Doris R. Powell and Melissa A. Brown This information is current as J Immunol 2002; 169:3811-3818; ; of September 28, 2021. doi: 10.4049/jimmunol.169.7.3811 http://www.jimmunol.org/content/169/7/3811 Downloaded from References This article cites 74 articles, 47 of which you can access for free at: http://www.jimmunol.org/content/169/7/3811.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 by guest on September 28, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: 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 © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology IL-4 Induces the Proteolytic Processing of Mast Cell STAT61 Melanie A. Sherman, Doris R. Powell, and Melissa A. Brown2 IL-4 is a potent, pleiotropic cytokine that, in general, directs cellular activation, differentiation, and rescue from apoptosis. However, in mast cells, IL-4 induces the down-regulation of activation receptors and promotes cell death. Mast cells have been shown to transduce IL-4 signals through a unique C-terminally truncated isoform of STAT6. In this study, we examine the mechanism through which STAT6 is processed to generate this isoform. We demonstrate that STAT6 processing in mast cells is initiated by IL-4-induced phosphorylation and nuclear translocation of full-length STAT6 and subsequent cleavage by a nuclear serine-family protease. The location of the protease in the nucleus ensures that the truncated STAT6 has preferential access to bind DNA. IL-4-responsive target genes in mast cells are identified by chromatin immunoprecipitation of STAT6, including the IL-4 gene itself. These results suggest a molecular explanation for the suppressive effects of IL-4 on STAT6-regulated genes in mast cells. The Journal of Immunology, 2002, 169: 3811–3818. Downloaded from ignal transducer and activator or transcription 6, a latent protein and thus acts relatively late in the STAT6 signaling cas- cytoplasmic transcription factor, is a member of a family cade to inhibit JAK1 and limit the duration of STAT6 activation S of signaling molecules that mediate responses to cyto- (10). Second, alternate isoforms of STAT proteins have been de- kines. IL-4 (and IL-13) binding to its receptor results in the acti- scribed for several STAT family members (11–15). These ␤ iso- vation of Janus kinase (JAK)3 1 and JAK3, receptor-associated forms (identified for STATs 1, 3, 5A, and 5B) lack the transacti- tyrosine kinases (1, 2). JAK1 and 3 directly phosphorylate tyrosine vation domain (TAD) but retain DNA binding ability. Therefore, http://www.jimmunol.org/ residues within STAT6 Src homology (SH)2 domains leading to by competing with the transcriptionally active form of STAT for dimerization and translocation to the nucleus where STAT6 can binding to DNA, they effectively inhibit the early transcription of associate with regulatory elements that control IL-4 (and/or IL-13) cytokine-responsive genes. Ϫ Ϫ responsive genes. Numerous studies using STAT6 / mice have The biological significance of STAT isoforms that act as dom- demonstrated the importance of this molecule in providing immu- inant negative transcription factors is clearly evident from studies nological competence (3–5). For example, the STAT6 signaling of STAT5, a signaling molecule activated in response to IL-2, pathway is of particular significance in the development of Th2 IL-3, IL-5, stem cell factor, and IL-15 (16–18). The truncated Ϫ Ϫ immunity to extracellular pathogens. STAT6 / mice are ex- STAT5␤ isoforms are expressed uniquely in myeloid progenitor tremely vulnerable to these infections, and T cells from these mice cells and are responsible for maintaining an IL-3-refractive state by guest on September 28, 2021 are unable to produce adequate amounts of the Th2 cytokines IL-4, (15). Upon differentiation, these cells lose expression of STAT5␤ IL-13, and IL-5 (3–5). B cell isotype switching, immunity to some and express only full-length STAT5, coincident with a gain of IL-3 tumors (6, 7), and development of contact hypersensitivity (8) are responsiveness (19). The truncated STAT5 proteins are generated Ϫ Ϫ also compromised in STAT6 / mice. through proteolysis of full-length STAT5 by a nuclear protease Because IL-4 plays a central role in the regulation of immune found only in immature myelocytes (20). This protease, termed responses, the magnitude and duration of STAT6-induced re- modulator of STAT activity (MSA) is a 25-kDa member of the sponses must be tightly controlled. At least two mechanisms have serine protease family. Its recognition and cleavage site in STAT5 been described to accomplish this regulation. First, suppressor of has been identified, and mutation of this site results in a STAT5 cytokine signaling (SOCS) proteins are up-regulated by cytokine- protein that resists proteolysis (19). induced STAT activation and either compete with STAT for bind- We previously identified a STAT6 isoform that shares charac- ing to receptors or bind to and inhibit JAK kinase activity (re- teristics with the ␤ forms of the other STAT family members (21). viewed by Chen et al. in Ref. 9). SOCS-1 is an IL-4-inducible In contrast to the 100-kDa full-length STAT6 molecule, STAT6␤ is truncated to ϳ65 kDa. Results from epitope mapping studies demonstrate that STAT6␤ lacks the C-terminal TAD, but it ap- Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA ␤ 30322 pears to retain a high affinity for DNA. STAT6 is expressed in a cell type-specific manner and is present in mast cells, but not in B Received for publication June 17, 2002. Accepted for publication July 31, 2002. or T cells. Interestingly, while IL-4-induced STAT6 is associated The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with an activating phenotype in B cells and T cells, it is often with 18 U.S.C. Section 1734 solely to indicate this fact. suppressive for mast cell gene expression. Mast cell receptors c-kit 1 This work was supported by National Institutes of Health Grants AR02157 (to and Fc⑀RI are down-regulated in response to IL-4 stimulation (22– M.A.S.) and CA47992 (to M.A.B.). 24) and the induction of several cytokines is inhibited upon long- 2 Address correspondence and reprint requests to Dr. Melissa A. Brown, 7311 Wood- term IL-4 exposure (23). ruff Memorial Research Building, Department of Pathology and Laboratory Medicine, Emory University, 1639 Pierce Drive, Atlanta, GA 30322. E-mail address: In this study, we set out to 1) characterize the STAT6 protein in [email protected] mast cells with regard to the mechanism of isoform generation, 3 Abbreviations used in this paper: JAK, Janus kinase; SH, Src homology; SOCS, phosphorylation status, and cellular localization, and 2) identify a suppressor of cytokine signaling; TAD, transactivation domain; MSA, modulator of role of STAT6␤ in mast cell gene transcription. We demonstrate STAT activity; BMMC, bone marrow-derived mast cells; USF, upstream stimulatory ␤ ␤ factor; IP, immunoprecipitation; ChIP, chromatin IP; SHP-1, sulfhydryl-2 domain- that analogous to STAT5 , STAT6 is also generated by proteo- containing tyrosine phosphatase-1. lytic processing. Although distinct from STAT5 MSA, the mast Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 3812 MAST CELL STAT6 PROTEASE cell STAT6 protease is a member of the serine protease family and ferred to nylon membrane. The blot was blocked in 5% milk/TBST (10 is located in the nucleus. We show that mast cell STAT6 is phos- mM Tris, pH 8.0, 150 mM NaCl, 0.5% Tween) overnight at 4°C. Abs (1 ␮ phorylated in response to IL-4 and binds in vivo to a STAT6 bind- g/ml) were added in 2% milk/TBST (the phospho-STAT6 Ab was diluted in 3% BSA/Tween 20 ϩ TBS) and incubated1hatroom temperature. ing site in the IL-4 promoter in mast cells. Mast cell STAT6 is also HRP-conjugated donkey anti-rabbit secondary Abs (Amersham, Arlington associated with the promoters of apoptosis regulator genes bax and Heights, IL) were added at a 1/5000 dilution in 2% milk/TBST and incu- bcl-2. The restricted expression and binding of this isoform to IL-4 bated1hatroom temperature. Reactive proteins were visualized by ECL responsive genes in mast cells could account for the cell-type spe- (DuPont, Boston, MA) and radiography. cific inhibitory effects of IL-4. Northern blot analysis For analysis of STAT6 expression in cell lines, 10 ␮g of total RNA was Materials and Methods electrophoresed on a 1% formaldehyde gel and transferred to nitrocellu- Cell culture and stimulation lose. The STAT6 DNA probe (bp 1–384 from STAT6 sequence, Accession CFTL-15, a murine IL-3 dependent mast cell line (C15; Ref. 25), WEHI- no. AF481809) was labeled by random hexamer priming. 3B, an immature myeloid cell line (26), and M12.4.1, a transformed B cell RNase protection analysis line (27), were cultured in complete RPMI (10% heat-inactivated FBS, 0.5% penicillin/streptomycin, 2 mM glutamine, 1.0 mM sodium pyruvate, Total RNA samples from C15 and M12 cell lines were isolated using the 50 ␮M 2-ME).