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Signaling in Mast Cells Regulates Zeb2 the Transcription Factor The Transcription Factor Zeb2 Regulates Signaling in Mast Cells Emilia Alina Barbu, Juan Zhang, Elsa H. Berenstein, Jacqueline R. Groves, Lauren M. Parks and Reuben P. This information is current as Siraganian of October 2, 2021. J Immunol published online 4 May 2012 http://www.jimmunol.org/content/early/2012/05/04/jimmun ol.1102660 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2012/05/04/jimmunol.110266 Material 0.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • 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 October 2, 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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published May 4, 2012, doi:10.4049/jimmunol.1102660 The Journal of Immunology The Transcription Factor Zeb2 Regulates Signaling in Mast Cells Emilia Alina Barbu, Juan Zhang, Elsa H. Berenstein, Jacqueline R. Groves, Lauren M. Parks, and Reuben P. Siraganian Mast cell activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a zinc finger transcription factor, regulates both early and late mast cell responses. Transfection with small inter- fering RNA (siRNA) reduced Zeb2 expression and resulted in decreased Fc«RI-mediated degranulation, with a parallel reduction in receptor-induced activation of NFAT and NF-kB transcription factors, but an enhanced response to the LPS-mediated activa- tion of NF-kB. There was variable and less of a decrease in the Ag-mediated release of the cytokines TNF-a, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple phosphorylation events were impaired that affected molecules both at early and late events in the signaling pathway. The Zeb2 siRNA-treated mast cells Downloaded from had altered cell cycle progression, as well as decreased expression of several molecules including cell surface Fc«RI and its b subunit, Gab2, phospholipase-Cg1, and phospholipase-Cg2, all of which are required for receptor-induced signal transduction. The results indicate that the transcription factor Zeb2 controls the expression of molecules thereby regulating signaling in mast cells. The Journal of Immunology, 2012, 188: 000–000. ast cells play a key role in immunological reactions by We report in this study that the zinc finger transcription factor http://www.jimmunol.org/ releasing many mediators. A major mechanism for Zeb2 (also called Sip1 for Smad-interacting protein) regulates M activation of these cells is by the aggregation of the FcεRI-mediated responses in mast cells. Zeb2 was initially iden- high-affinity IgE receptor (FcεRI), which leads to the rapid se- tified as a protein that interacted with receptor-activated Smads in cretion of preformed/stored mediators and synthesis of products, the pathway associated with signaling of activin and other mem- such as leukotrienes and cytokines (1–3). The signaling pathway bers of the TGF-b superfamily (16). Zeb2 is now recognized as leading from receptor activation to release involves molecules that a zinc finger DNA-binding protein that functions as a transcrip- directly function in these responses and others that regulate these tional regulator (17, 18). Structurally, Zeb2 has two separate reactions. Phosphorylation of proteins and lipids plays an impor- clusters of zinc fingers, one at the N terminus, the other at the C by guest on October 2, 2021 tant role in these events: after phosphorylation of the immuno- terminus, and a central region with a homeodomain, thought to be receptor tyrosine-based motifs of the g and b signaling subunits of involved in protein–protein interactions, a SMAD binding domain, FcεRI, the protein tyrosine kinase Syk is recruited to the receptor and a domain that binds the corepressor CtBP. Binding with its and activated. This then results, directly or indirectly, in the phos- zinc finger to regulatory elements of genes causes Zeb2 to act phorylation of a number of downstream substrates, including PI3K, mainly as a transcriptional repressor, although there have been phospholipase Cg (PLCg), Vav1, LAT, SLP76, Btk, and Gab2 (4– reports that it can also be an activator of gene transcription. Zeb2 7). The phosphorylation of lipids also is critical in the steps that functions to regulate epithelial to mesenchymal transition pro- lead to calcium mobilization and degranulation. The increase in cesses in cells; its expression in epithelial cells causes these po- intracellular calcium concentration leads to activation of MAPKs larized cells to change morphology by losing their cell–cell and the transcription factors NF-kB and NFAT, all of which play contacts and to acquire the motile, migratory properties of mes- important roles in the control of IgE-mediated cytokine synthesis enchymal cells. Zeb2 therefore is essential in embryogenesis: (8–12). Among the molecules that have been found to regulate this knockout results in embryonic lethality by day 9.5 due to multiple pathway are the lipid phosphatases SHIP-1/2 and PTEN (13–15). defects (18). Haploinsufficiency of Zeb2 in humans results in the Mowat–Wilson syndrome with typical facial features and variable symptoms depending on the mutation but generally with mental Receptors and Signal Transduction Section, Oral Infection and Immunity Branch, retardation and neurologic defects (19). Zeb2 is overexpressed in National Institute of Dental and Craniofacial Research, National Institutes of Health, a number of cancers in which there is a epithelial to mesenchymal Bethesda, MD 20892 shift resulting in a more aggressive phenotype (20). Received for publication September 14, 2011. Accepted for publication April 6, 2012. Zeb2 was identified during a small interfering RNA (siRNA) library screen for molecules that regulate Ag-induced NFAT or This work was supported by the Intramural Research Program of the National In- stitutes of Health, National Institute of Dental and Craniofacial Research. NF-kB activation in mast cells. In this study, we used an RNA Address correspondence and reprint requests to Dr. Reuben P. Siraganian, National interference approach to investigate the function of Zeb2 in mast Institute of Dental and Craniofacial Research, National Institutes of Health, 9000 cells. Cells were transfected with siRNA, and changes in the Rockville Pike, MSC 4410, Building 49, Room 1A-16, Bethesda, MD 20892. E-mail cellular responses and signaling events were monitored for 3 d address: [email protected] posttransfection. A decrease in Zeb2 expression resulted in re- The online version of this article contains supplemental material. duced FcεRI-mediated activation of the transcription factors Abbreviations used in this article: BMMC, bone marrow-derived mast cell; miRNA, microRNA; MRE, microRNA response element; PLCg, phospholipase Cg;SCF, NFAT and NF-kB but in a substantial increase in the LPS- stem cell factor; siRNA, small interfering RNA. mediated NF-kB activation. Zeb2 knockdown also decreased de- www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102660 2 Zeb2 REGULATES FcεRI SIGNALING granulation at all 3 d of testing; however, it resulted in increased TIB-142) as described previously (23). In some experiments, cells were release of TNF-a, IL-13, and CCL-4 at day 1 but decreased stimulated with 1 mM ionomycin. Degranulation of cells treated with the responses by day 2 or 3. Decreased expression of Zeb2 resulted in experimental siRNA is presented as percent or fraction of that in cells ε transfected with siCONTROL. Ag-induced release in control transfected cells changes in IgE–Fc RI–mediated signaling events with reduced was 27 6 8% on day 1, 27 6 7% on day 2, and 23 6 11% on day 3 (n =7). phosphorylation of several proteins. There were also changes in ionomycin-mediated degranulation and cytokine release indicat- Cytokine release measurements ing that Zeb2 knockdown altered signaling events both upstream BMMC transfected with siRNA were sensitized overnight with Ag-specific and downstream of the calcium response. There were also effects IgE and then challenged for 3 h in complete culture medium with Ag or on the expression of proteins implicated in signal transduction. a combination of 20 nM PMA and 1 mM ionomycin. Quantitative meas- a b These results indicate that Zeb2 by regulating the expression of urements of TNF- , IL-13, and CCL-4 (MIP-1 ) in the supernatants were performed with cytokine-specific Quantikine ELISA Kits (R&D Systems) several proteins controls mast cell responses. according to the manufacturer’s instructions. Ag-induced release in cells transfected with experimental siRNA is expressed as a fraction of that in Materials and Methods control-treated cells. Abs and reagents Other cellular analyses Mouse IL-3, stem cell factor (SCF), and propidium iodide were purchased For FcεRI expression, transfected BMMC (7 3 105) were cultured over- from Invitrogen (Carlsbad, CA). Ionomycin and PMA were from Calbio- night with 0.6 mg/ml Ag-specific IgE. After washing, the cells were in- chem (La Jolla, CA). The anti-mouse IgE PE was from eBioscience (San cubated for 1 h at 4˚C with 2 mg FITC-conjugated anti-mouse IgE. The Diego, CA). RNase was from Sigma Aldrich (St. Louis, MO). The Abs for cells were then washed, and the fluorescence was read by FACScan. SHIP-1 (P1C1), Syk (N-19), Gab2 (M-19), PLCg2 (Q-20), Zeb2 (SIP1, For cell cycle analysis, BMMC (7 3 105) were washed with PBS and H-260), and E-cadherin (H-108) were from Santa Cruz Biotechnology fixed overnight with ice-cold 70% ethanol.
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