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The 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

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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 activation results in the release of stored and newly synthesized inflammatory mediators. We found that Zeb2 (also named Sip1, Zfhx1b), a 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 -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 TNF-a, IL-13, and CCL-4. This suggests that low Zeb2 expression differentially regulates signaling pathways in mast cells. Multiple 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 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 . 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 ) 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 and 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 Syk is recruited to the receptor and a domain that binds the CtBP. Binding with its and activated. This then results, directly or indirectly, in the phos- zinc finger to regulatory elements of causes Zeb2 to act phorylation of a number of downstream substrates, including PI3K, mainly as a transcriptional , although there have been phospholipase Cg (PLCg), Vav1, LAT, SLP76, Btk, and Gab2 (4– reports that it can also be an of 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 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 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 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 (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 ; PLCg, phospholipase Cg;SCF, NFAT and NF-kB but in a substantial increase in the LPS- 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 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. After centrifugation, the pellet Downloaded from (Santa Cruz, CA); anti–phospho-Akt (Ser473), anti-Akt, anti–phospho- was resuspended in 100 U RNase and incubated at 37˚C for 20 min. p38, anti-p38 (Thr180/Tyr182), anti–phospho-p44/42 MAPK (ERK Propidium iodide solution (500 mlof50mg/ml in DPBS) was then added, Thr202/Tyr204), anti-p44/42 MAPK, anti–phospho-Syk (Tyr525/Tyr526), and samples were further incubated for 30 min at 4˚C. Percent of events anti–phospho-PLCg2 (Tyr1217), and anti–phospho-LAT (Tyr191) were gated in the cell cycle phases (G0–G1, S, G2–M) was determined by from Technology (Beverly, MA); anti-SNAI1 (N-term R8) a FACScan. was from Abgent (San Diego, CA). The Abs for PLCg1, Btk, and PI3K p85 were from Upstate/Millipore (Lake Placid, NY). All other materials

Immunoblotting and densitometry http://www.jimmunol.org/ were as previously described (21, 22). Cell pellets from transfections with control or experimental siRNA were Cell culture used for immunoblotting to determine changes in the level of expression of proteins. Phosphorylation of Akt was determined in cells starved of SCF as The -dependent MC9 mouse mast cell lines stably expressing described previously (23). Proteins were resolved under reducing con- NFAT binding sites fused to enhanced GFP or NF-kB binding sites fused to ditions on precast minigels, electrophoretically transferred onto polyvi- GFP were described previously (23). Bone marrow cells were collected nylidene fluoride Immobilon membranes, probed with specific Abs, and from 2- to 4-mo-old C57BL/6 females and cultured for up to 8 wk in detected using ECL. The blots were scanned and processed on a Kodak DMEM supplemented with 20% heat-inactivated FBS, 2 mM L-glutamine, Image Station 4000MM Digital Imaging System (Carestream Molecular 3 25 5 10 M 2-mercaptoethanol, 10% NCTC 109 medium, 0.1 mM non- Imaging, Rochester, NY). The densitometry of the bands was used to essential amino acids, 1 mM sodium pyruvate, antibiotics, 30 ng/ml IL-3, calculate the changes as a fraction of that in control cells transfected with by guest on October 2, 2021 and 25 ng/ml SCF. Bone marrow-derived mast cells (BMMC) were used nontargeting siRNA using the values of the anti-actin or specific protein to for experiments starting with the fourth week of culture. correct for gel loading. Transient transfection with siRNA Data analysis An initial screen was with a library containing pools of four siRNA duplexes Statistical analysis was by nonparametric t tests using GraphPad Prism 5, per gene that target 198 mouse enzymes with known or predicted phos- and the p value is represented in the figures as *p , 0.1, **p , 0.01, and phatase activity purchased from Dharmacon (Lafayette, CO). The char- ***p , 0.005. The number of repeats for each set of experiments is acteristics of this library are described in our previous publication where it specified in the methods or figure legends. was used to study degranulation in mast cells (22). The siRNA targeting Zeb2 (NM_015753) containing either a pool of four siRNA duplexes or the single duplexes was from Dharmacon. For negative controls, a pool con- Results taining four scrambled siRNAs with minimal effect on known mouse genes Zeb2 regulated Fc«RI-induced activation of NFAT and NF-kB (ON-TARGET plus siCONTROL nontargeting pool, cat. no. D-001810- 10-20) was also from Dharmacon. Mouse Syk (NM_011518) siGENOME in mast cells SMARTpool (Dharmacon) was used in some transfections as a positive To screen for new molecules involved in FcεRI-induced gene control. Transfection was by use of Amaxa Nucleofector 96-well Shuttle System as previously described (23). After transfection, cells were trans- expression, an siRNA screen was developed using NFAT and ferred into 96-well plates and assayed for 3 d. Cell pellets in each ex- NF-kB reporter-expressing mast cells. These reporter cells were periment were analyzed by immunoblotting to determine changes in the derived from MC9 growth factor-dependent mouse mast cells in level of expression of proteins. which expression of GFP was regulated either by NFAT or NF-kB ε Flow cytometric measurement of NF-kB and NFAT activation activation. After Fc RI stimulation, there was expression of GFP that was determined by FACS analysis. Previously, a screen using Fluorescence of the GFP reporter was used as a marker of FcεRI-induced an siRNA library targeting known or predicted mouse phosphatase NF-kB or NFAT activation. Cells were sensitized for 24 h with 0.3 mg/ml Ag-specific IgE (anti–TNP-142 mAb) and then stimulated with Ag for 5 h genes in a mast cell degranulation assay identified several po- (for NF-kB activity) or 18 h (for NFAT activity) at which times there was tential new targets (22). When the same siRNA library was used maximal GFP expression. The mean fluorescence intensity of treated and with the NFAT and NF-kB reporter cells, one of the strongest control cells was determined by a FACScan three-color analysis cytometer positives was for the transcription factor Zeb2, which, although and processed by CellQuest software (BD Biosciences, San Jose, CA). The included in this library, is not a phosphatase. In this assay, the activation in the treated cells is presented as a percentage or fraction of that in cells transfected with control siRNA. reporter cells were transfected with siRNA duplex pools com- posed of four distinct siRNA species targeting Zeb2 and then Degranulation measurements sensitized with IgE and challenged with Ag. The Ag-induced The b-hexosaminidase release was determined in transfected BMMC that activation of NFAT and NF-kB was determined 1, 2, or 3 d af- were sensitized for 24 h with 0.3 mg/ml Ag-specific IgE (anti-TNP mAb ter siRNA treatment. Compared to cells transfected with control The Journal of Immunology 3 nontargeting siRNAs, the Zeb2 siRNA resulted in a decrease of changes in the FcεRI-induced NFAT and NF-kB activation dem- .40% in FcεRI-induced NFAT and NF-kBresponses(NFAT241% onstrating that these were related events. This strongly suggested on day 2 and 239% on day 3; NF-kB 242% on day 2 and 230% that Zeb2 was involved in the regulation of the signaling pathway on day 3). To exclude the possibility that these effects were due leading to synthesis and release of cytokines by stimulated mast to off-target effects of the siRNA, the four independent siRNAs cells. to Zeb2 that were in the original pool were separately tested (Fig. « 1). For both the NFAT and NF-kB response, at least two of the Zeb2 regulated Fc RI- and ionophore-mediated cellular single siRNAs induced the phenotypic changes confirming that responses in primary mast cells these were true-positive hits. Zeb2-3 was the most efficient of the Because cell lines such as MC9, although growth factor depen- siRNA duplexes and decreased Ag-induced activation of both dent, could still have , Zeb2 function was validated in transcription factors at all 3 d posttransfection; maximum decrease primary mast cells. Mouse BMMC were treated with the pool of was at day 3, with 252% in NFAT activity, whereas NF-kB ac- Zeb2 siRNA, and IgE–FcεRI–mediated degranulation and cyto- tivation decreased by 233%. For NFAT, all the singles induced kine release were determined for 3 d posttransfection. The re- some decrease by day 3, whereas for NF-kB, Zeb2-3 and Zeb2-4 lease of b-hexosaminidase was used to measure degranulation, were the strongest. Notably, Zeb2 knockdown caused an increase and TNF-a, IL-13, and CCL-4 release was used to evaluate changes in NF-kB activation induced through the LPS–TLR4 pathway, in cytokine responses (Fig. 2A). Knockdown of Zeb2 decreased having the opposite effect to that with Ag. Thus, at day 3 post- b-hexosaminidase release at all 3 d posttransfection with the transfection, LPS-stimulated NF-kB activity increased by 213% in maximum reduction of 265 6 7.5% (n = 7) on day 3. However, cells treated with Zeb2-3, by 80% in cells treated with Zeb2-4, and the effect of decreased Zeb2 expression on release of the three by 166% in cells treated with the pooled siRNA (Fig. 1A). These cytokines was more variable; release was enhanced at the early Downloaded from results indicate that siRNAs to Zeb2 have profound effects on time points, but by day 3 it was inhibited. On all 3 d after trans- signaling in mast cells. fection there was 90% decrease in Zeb2 protein expression (Fig. After FACS analysis, the remaining cells were used for immu- 2B). In Ag dose-response curves, the results were similar with noblotting to confirm that the observed functional changes were more dramatic effects on degranulation than on cytokine release due to siRNA-induced decrease in protein expression (Fig. 1B–D). (Supplemental Fig.1). Therefore, decrease in Zeb2 expression The pooled siRNA decreased Zeb2 expression by .90% on all results in reduction in degranulation and release of cytokines. http://www.jimmunol.org/ 3 d of testing. All the single siRNAs decreased Zeb2 expression to Mast cells can also be activated by calcium ionophores and some extent; the most active was Zeb2-3, which was also the most PMA, which bypasses the early steps in the FcεRI–Ag pathway. active by phenotypic effects, whereas Zeb2-2 had minimal effects Treatment with ionomycin induces an increase in intracellular on protein expression and phenotype. The other two singles, Zeb2- calcium, which results in degranulation. This ionomycin-induced 1 and Zeb2-4, had an intermediate effect on protein expression. degranulation was inhibited similar to that with Ag by the de- In general, there was correlation between the level of protein crease in Zeb2 expression (Fig. 2C). BMMC stimulated with expression and the phenotype; although when there was an in- ionomycin together with PMA result in the release of cytokines; termediate level of Zeb2 expression, changes in phenotype were again, the changes in TNF-a and IL-13 release with Ag were by guest on October 2, 2021 more variable. Notably, whereas there was already a dramatic similar to that with ionomycin–PMA in the treated cells. These decrease in Zeb2 expression by day 1, the phenotypic changes results indicate that decreased Zeb2 levels have effects at late were greater at day 2 and 3 suggesting that the change in Zeb2 stages of the BMMC response. levels required time to have effects on cell signaling. To validate these results further, the FcεRI-mediated degranu- These results indicate that the expression of Zeb2 can be spe- lation and cytokine responses were measured in BMMC treated cifically and efficiently reduced by siRNA treatment in mouse mast with four single siRNAs from the original pool (Fig. 3). Syk- cells. The decrease in Zeb2 expression was accompanied with specific siRNA was used as a control that efficiently inhibits

FIGURE 1. Zeb2 regulates FcεRI-induced acti- vation of NFAT and NF-kB transcription factors in mast cells. (A) MC9-NFAT-GFP or MC9-NF-kB- GFP cells were treated with individual and pooled siRNA specific for Zeb2 (indicated by 1 to 4 and P) or with control nontargeting siRNA. FcεRI- or LPS- induced activation of the transcription factors was determined by FACS analysis of GFP expression on days 1, 2, and 3 posttransfection. The results are expressed as a percentage of that in controls and are the average from three experiments. Cell lysates were analyzed by immunoblotting for expression of Zeb2 at day 1 (B), day 2 (C), and day 3 (D) in MC9- NFAT-GFP treated with control siRNA (lane C)or individual and pooled siRNAs for Zeb2 (lanes 1 to 4 and P). Anti-actin was used as loading control. Fraction of protein still expressed in cells is shown under each lane. Similar immunoblots were ob- served in six experiments (three each for NFAT and NF-kB). 4 Zeb2 REGULATES FcεRI SIGNALING

FIGURE 2. Zeb2 regulates FcεRI- and ionophore- mediated cellular responses in primary mast cells. (A) Mouse BMMC were transfected with control non- targeting siRNA or the pool of four siRNAs specific for Zeb2. The Ag-induced release of b-hexosaminidase, TNF-a, IL-13, and CCL-4 were determined at days 1, 2, and 3 posttransfection (n = 2). The changes in the cellular responses are presented as a percentage of that in control-transfected cells. (B) Cell lysates from BMMC transfected with nontargeting siRNA (“C”) or the Zeb2-specific (“T”) were analyzed by immuno- blotting with Abs to Zeb2 or actin. Fraction of protein still expressed in cells is shown under each lane. (C) Comparison of the IgE-Ag and ionomycin–PMA re- sponses in siRNA-treated cells; changes in cellular re- sponses are presented as a percentage of that in control transfected cells. *p , 0.1, **p , 0.01, ***p , 0.005. Downloaded from

IgE-mediated responses (22). With the Zeb2-treated cells, the Knockdown of Zeb2 decreased Fc«RI-induced largest decrease in b-hexosaminidase release was at day 2 and 3 http://www.jimmunol.org/ with all four siRNAs, with Zeb2-3 being the most effective (Fig. Protein phosphorylation was then examined to understand the 3A). There was less inhibition of degranulation when the cells mechanism by which decrease in Zeb2 impaired FcεRI-mediated were tested 24 or 48 h after transfection. In the cytokine re- degranulation and cytokine release (Fig. 4). Activation of Syk is ε sponse, there was a decrease in Fc RI-induced release of TNF-a, critical for signaling in mast cells; a marker for this is the phos- IL-13, and CCL-4 by day 3 with Zeb2-3, whereas the other single phorylation of the two adjacent tyrosines in the activation loop siRNAs had much less or little effect. There was enhancement or (24–28). There was some minor decrease in this phosphorylation minimal inhibition when the cells were tested at earlier time of the activation loop tyrosines of Syk in the Zeb2 siRNA-treated points. The changes in CCL-4 release were minimal. On immu- cells (Fig. 4B). Downstream of Syk activation is the phosphory- noblotting, changes in Zeb2 expression paralleled the phenotype lation of several proteins including LAT and PLCg. There was by guest on October 2, 2021 changes; the greatest decrease in protein levels was with Zeb2-3 a dramatic decrease in phosphorylation of LAT-Tyr191, which is with more modest effects with Zeb2-4 or Zeb2-1 and less effects partly responsible for phosphoinositide 3 kinase/phospholipase with Zeb2-2 (Fig. 3B–D). Notably, although the Zeb2 protein translocation to the membrane (Fig. 4B). Activation of PLCg is levels were low on day 1 posttransfection, the decrease in re- a crucial step in the cellular response pathway; there was de- sponses was progressively stronger over the 3 d with the most creased phosphorylation of PLCg2 on Tyr1217 especially at the inhibition observed on day 3. The effectiveness of the different early time points after Ag stimulation indicating reduced activa- single siRNAs in decreasing protein expression and cellular tion. The activation of PLCg is regulated, at least in part, by the responses validate a role of Zeb2 in regulating FcεRI-mediated activation of PI3K (29, 30). The phosphorylation of the serine/ responses. threonine protein kinase Akt, a PI3K substrate, was used as a

FIGURE 3. Cellular response of BMMC treated with single and pooled siRNAs targeting Zeb2.(A) BMMC were treated with individual and pooled siRNAs specific for Zeb2 (indicated by 1 to 4 and P) or with control nontargeting siRNA. Release of b-hexosaminidase, TNF-a, IL-13, and CCL-4 was determined at days 1, 2, and 3 posttransfection. The results are expressed as a percentage of that in controls. Cell lysates were analyzed by immu- noblotting for the expression of Zeb2 at day 1 (B), day 2 (C), and day 3 (D). The Journal of Immunology 5

FIGURE 4. Knockdown of Zeb2 decreases FcεRI- induced phosphorylations. BMMC transfected with control scrambled siRNA or the single Zeb2-3 siRNA (Treated) were sensitized with IgE and then activated with Ag for the indicated times (minutes). Cell lysates were immunoblotted with the anti– phospho-Abs, then stripped and probed with the specific Abs. Densitometry data were corrected for differences in loading using the actin blots and then compared with control cells treated with scrambled siRNA. In the case of PLCg2, the change in the Downloaded from expression of this protein is also shown as there was decreased expression of this protein after treatment with siRNA specific for Zeb2. The different proteins as indicated are shown in (A) and (B). The results here are from day 3 (A) and day 2 (B) posttrans- fection. Similar results were in three different ex- periments and when the cells were treated with the http://www.jimmunol.org/ pool of Zeb2 siRNA. by guest on October 2, 2021

measure of the activation of the PI3K pathway. There was up to controls signaling in mast cells, we investigated whether its knock- 80% decrease in the phosphorylation of Akt Ser473 after the down induced changes in cell-surface FcεRI expression. Com- Zeb2-3–specific siRNA treatment compared with the control non- pared to the controls or cells transfected with siRNA for Syk, there targeting siRNA. This indicated a less active PI3K pathway after was decreased FcεRI expression in BMMC treated with the most Zeb2 knockdown. Later events in the pathway of cell activation active single or the pool of Zeb2 siRNA (Fig. 5). The greatest include the phosphorylation of MAPKs. Zeb2 knockdown de- decrease was with Zeb2-3 and the least with Zeb2-2, similar to the creased phosphorylation of both ERK and p38; these changes effect of these siRNAs on Zeb2 protein expression and cellular were more striking in p38 (Fig. 4A, Supplemental Fig. 2A). The responses. Lower cell-surface FcεRIa expression would result results for all these phosphorylations were similar at days 2 and 3 in decreased Ag-induced clustering, and thus diminished cellular posttransfection. response. However, increasing the concentration of Ag did not The decreased response in the Zeb2 siRNA-treated cells in reverse the reduced cellular response in Zeb2 siRNA-treated cells. degranulation and cytokine release with ionophore/PMA sug- These results suggest that the decreased receptor levels had a mi- gested that there were defects in the late steps of these pathways. nor role in changes in degranulation or cytokine release. However, these phosphorylation results indicate that there are As Zeb2 regulates critical cellular functions such as prolifera- multiple defects in signaling including early defects such as those tion, differentiation, and survival (18, 31, 32), we investigated that involve the PI3K, PLCg, and LAT besides late events such as whether there were changes in cell cycle progression in the treated those in the MAPKs, which together can explain the changes in cells (Fig. 6). FACS analysis was used to determine the percen- degranulation and release of cytokines. tages of BMMC undergoing each of the three phases of the cell cycle, G1, S, and G2/M, after treatment with the most efficient Zeb2 regulated the expression level of several proteins in mast individual siRNA (Zeb2-3) and the pooled mix (Zeb2-P). Treat- cells ment with Zeb2 siRNA resulted in alteration in cell cycle pro- During these phosphorylation studies, it was noted that there was gression with the more easily detectable changes in the S or G2/M decreased expression of PLCg2 in the Zeb2 siRNA-transfected phase. In the treated cells, there was significant decrease in the cells (Fig. 4A). These observations raised the possibility that number of cells in S phase, which was most prominent on day 2 Zeb2 could, as a transcription repressor, regulate the expression (Fig. 6B). There was a similar reduction in treated cells in the G2/ of other signaling molecules. To begin to understand how Zeb2 M phase (Fig. 6C). There were minor increases in cells in the G1 6 Zeb2 REGULATES FcεRI SIGNALING

FIGURE 5. Knockdown of Zeb2 decreased cell-surface FcεRI expres- sion. The expression of FcεRI was determined in BMMC treated with nontargeting siRNA or the single and the pooled siRNAs specific for Zeb or Syk. The change in FcεRI expression was calculated from the mean fluorescence intensity as determined by FACS (n = 4) using the non- targeting siRNA-transfected cells as controls. *p , 0.1, **p , 0.01. phase, however this was statistically significant only at one time point (Fig. 6A). These results indicate that Zeb2 is a positive Downloaded from regulator of G1–S and S–G2/M transitions in mast cells, but even in the treated cells most were still in the G1 phase. Therefore, there was only a minor shift in the cell cycle, which could not explain the dramatic change in cell responses. Previous reports have shown that Zeb2 regulates the protein level of the adhesion molecule E-cadherin and the transcription http://www.jimmunol.org/ factor Snai1 (33, 34). At 3 d after Zeb2 siRNA treatment, there was decrease in the level of these two proteins (Fig. 7). We then tested for proteins involved in FcεRI-mediated signaling (Fig. 7A and Supplemental Fig. 3 show representative experiments, and Fig. 7B is the expression level of these proteins from multiple FIGURE 7. Zeb2 regulates the expression of several proteins in mast cells. BMMC were treated with nontargeting siRNA (“Control”) or the single siRNA specific for Zeb2 (“Zeb2-3”) and the pooled siRNAs (“Zeb2- P”). (A) Lysates from day 3 posttransfection were analyzed by immuno-

blotting with the indicated Abs. Similar results were in four to seven by guest on October 2, 2021 different experiments. (B) Densitometry data of the changes induced by decreased expression of Zeb2 as a percentage of controls; data are with both Zeb2-3 and Zeb2-pool siRNA from 6 to 15 determinations (mean 6 SD). *p , 0.1, **p , 0.01, ***p , 0.005.

experiments). There were decreases in the level of several proteins that are required for signaling; these included the b subunit of FcεRI, the two isoforms of PLCg, and Gab2, which are all im- portant for FcεRI signaling. However, there were minimal or no changes in the level of some of the other proteins such as Btk and the p85 subunit of PI3K (Fig. 7). Among the inhibitory regulators, decreased expression of which would enhance signaling, there was reduction in the levels of SHIP-1 and PTEN. There was much less change in the expression of these proteins at earlier time points after transfection (Supplemental Fig. 4). As noted in Fig. 4, Zeb2 siRNA had no effect on the expression level of Syk, LAT, Akt, and the MAPKs. Therefore, in mast cells the knockdown of Zeb2 resulted in reduced expression of FcεRI and of several signaling molecules with decreased signal transduction as measured by re- duced degranulation and release of cytokines indicating effects on early and late mast cell responses (Fig. 8).

Discussion Zeb2 interacts with Smad proteins, which are involved in TGF-b FIGURE 6. Decreased Zeb2 expression alters cell cycle progression. BMMC were transfected with nontargeting siRNA (“Control”) or the most signaling; a pathway important for and differentiation. efficient single siRNA (Zeb2-3) and the pooled siRNA specific for Zeb2 The current experiments suggest a role for Zeb2 in mast cell (“Target”). Changes in the cell cycle were monitored by FACS. Percent of differentiation and development, as knockdown of Zeb2 decreased cells in G1 phase (A), S phase (B), and G2/M phase (C) at days 1, 2, and 3 the rate of cell cycle progression. This also reduced the expression posttransfection is presented (n = 4). *p , 0.1, **p , 0.01, ***p , 0.005. of FcεRI, as well as that of several other downstream intracellular The Journal of Immunology 7

creased levels of both Zeb2 and Snai1 would result in changes in the expression of proteins that reflect less the mesenchymal phe- notype and cause the BMMC to revert to a less differentiated phenotype. Zeb2 knockdown resulted in decreased IgE–FcεRI–induced signal transduction most likely due to changes in the expression of several signaling molecules. The response of mast cells is the result of a sequence of biochemical events that begins with the clustering of the IgE–FcεRI complexes by a multivalent Ag. The FcεRI on the cell surface binds IgE with high affinity through the a subunit and transduces intracellular signals through the bg2 FIGURE 8. Zeb2 regulation of mast cell responses. The decrease in the subunits. Therefore, strength of the signal depends on the number expression of Zeb2 causes reduction in the level of other molecules in mast of surface receptors, the amount of IgE bound to the a subunit, cells. Some of these are known to be part of the FcεRI signaling network. and the amount of Ag. We found that with low Zeb2 expression, ε The decrease in cell surface Fc RI together with reduced amounts of other there was decreased cell-surface expression of the receptor with signaling proteins results in decreased degranulation and cytokine release. a more dramatic reduction of the b subunit, which is important for signal transduction (Fig. 7). These changes would contribute to transducers. The decreased expression of Zeb2 affected multiple a decrease in the initiation step of the FcεRI-induced signal trans- sites in the FcεRI-induced signaling pathway with decreased duction pathway. phosphorylation of proteins at multiple sites in the pathway. This The downstream progression of signaling from the receptor Downloaded from results in a dramatic decrease in degranulation and reduced cy- depends on several key molecules; among these is the protein tokine release. Syk, which is essential for progression of signal The Zeb2 protein has two zinc finger clusters each of which toward both degranulation and cytokines synthesis (23, 26). Syk binds to the CACCT(G) sequence located in gene promoters (17, directly or indirectly controls the activity of other key substrates, 18). Zeb2 functions as a repressor of transcription; this action may such as PI3K and LAT. We found that Zeb2 knockdown induced also depend on the binding of a corepressor, CtBP. A decrease in changes in signal transduction from these molecules. Although the http://www.jimmunol.org/ Zeb2 levels induced by siRNA treatment should therefore result in expressions of Syk, LAT, and the p85 subunit of PI3K were not an increase in the level of a protein if the region of its changed, the extent of the FcεRI-induced gene has a binding site for Zeb2. As we observed a decrease in and activation of these molecules was reduced. There was de- expression of these signaling molecules, binding of Zeb2 to their creased activation of Syk as indicated by the level of phosphor- promoter region cannot therefore be the mechanism for their de- ylation of the activation loop tyrosines, which are important for crease. downstream propagation of signals (27, 28). The reduction in the (miRNAs) are endogenous, noncoding, ∼22 nu- initiation of signals from FcεRI could be the explanation for the cleotide short that pair to microRNA response elements decrease in the activation of Syk. Impaired signaling downstream by guest on October 2, 2021 (MREs) in the mRNA of protein-coding genes and result in their of Syk was evident by the reduced phosphorylation of Tyr191 on suppression by either inhibiting and/or by inducing LAT and Ser473 on Akt. Phosphorylated tyrosine sites on LAT degradation of the target mRNAs (35). Most mRNAs have mul- bind Grb2, Gads/SLP-76, PLCg1, and PI3K to propagate the tiple MREs and therefore can be regulated by several miRNAs, signal further (40, 41). The reduced PI3K activation was appar- and miRNAs are known to target many mRNA transcripts (36, ent from the decreased phosphorylation of its substrate Akt, the 37). It was recently proposed that competitive endogenous RNAs phosphorylation of which reflects PI3K activity. There was also sequester miRNAs to regulate mRNA transcripts containing decreased expression of Gab2, a regulator of the PI3K pathway. common miRNA recognition elements (38). Notably, Zeb2 tran- Decreased Gab2 levels, combined with reduced activation of Syk, script was identified in a study to find such competitive inhibitor which is the major kinase that phosphorylates Gab2, could also decoys for PTEN, and it was shown that siRNA-induced knock- contribute to the decreased PI3K activation (42). The decrease in down of Zeb2 results in a decrease in the expression of PTEN PI3K activity would depress the membrane recruitment and acti- protein levels in a miRNA-dependent, protein coding-independent vation of Btk and PLCg, which are essential through the genera- manner (39). Interrogation of the TargetScanMouse site for pre- tion of inositol 1,4,5-triphosphate and diacylglycerol for the in- dicted miRNA targets (www.targetscan.org/mmu_50/) showed crease in intracellular calcium and activation of protein kinase C that the proteins whose level changed in the current experiments (6, 43, 44). Further contributing to the decreased activation of this have multiple targets for the same miRNA as Zeb2. Therefore, the pathway was the reduced levels of both Btk and PLCg2 in the disruption of the Zeb2 mRNA by treatment with siRNA would Zeb2 siRNA-treated cells. Zeb2 knockdown therefore results in free these miRNAs to bind other mRNA leading to the repression reduction in the early signaling events, which precede activation of that message with a decrease in protein expression levels (35). of protein kinase C, LAT, MAPKs, NFAT, and NF-kB (23, 45). Zeb2 suppress the epithelial phenotype by regulating transcrip- FcεRI-mediated activation of the NFAT and NF-kB transcrip- tion especially of genes that are important for epithelial mesen- tion factors is pivotal for initiation of transcription of cytokines chymal transitions. Zeb2 decreases the expression of E-cadherin, genes. Activation of these transcription factors depends on up- which is a component of the adherens junctions in epithelial cells stream signals that were clearly inhibited in the Zeb2 siRNA- and upregulates genes normally expressed in mesenchymal cells. treated cells. NFAT controls diverse cellular processes and tran- It is also essential for hematopoietic cell differentiation (31). Zeb2 scription of ILs (IL-2, IL-3, IL-4, IL-5, IL-13), GM-CSF, IFN overexpression correlates with aggressive phenotype in various (IFN-g), and TNF-a (10, 46). NFAT also regulates the induction cancers where it may be involved in malignant transformation. In of other transcription factors and is also crucial for the FcεRI BMMC, we found that when Zeb2 expression was reduced, there activation-induced survival of mast cells (47, 48). Similarly NF- was also decrease in the level of the transcription factor Snai1, kB regulates the transcription of proinflammatory cytokines (12). which also regulates epithelial mesenchymal transitions. The de- Therefore, the reduced FcεRI-induced activation of NFAT and 8 Zeb2 REGULATES FcεRI SIGNALING

NF-kB after Zeb2 knockdown resulted in the decreased release of 6. Kawakami, Y., J. Kitaura, A. B. Satterthwaite, R. M. Kato, K. Asai, S. E. Hartman, M. Maeda-Yamamoto, C. A. Lowell, D. J. Rawlings, O. N. Witte, cytokines. and T. Kawakami. 2000. Redundant and opposing functions of two tyrosine There were also changes in molecules that are known to neg- , Btk and Lyn, in mast cell activation. J. Immunol. 165: 1210–1219. atively regulate signaling from FcεRI. Knockdown of SHIP-1 and 7. Siraganian, R. P. 2003. Mast cell signal transduction from the high-affinity IgE receptor. Curr. Opin. Immunol. 15: 639–646. PTEN is known to enhance signaling by the PI3K pathway leading 8. Vig, M., and J. P. Kinet. 2009. in immune cells. Nat. Immunol. to increased degranulation and cytokine release. There was mod- 10: 21–27. est to considerable decrease in these two proteins in the Zeb2 9. Perkins, N. D. 2007. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat. Rev. Mol. Cell Biol. 8: 49–62. siRNA-treated cells although there was inhibition of the mast cell 10. Klein, M., S. Klein-Hessling, A. Palmetshofer, E. Serfling, C. Tertilt, T. Bopp, responses. The reduced cellular responses indicate that the de- V. Heib, M. Becker, C. Taube, H. Schild, et al. 2006. Specific and redundant crease in molecules essential for signaling are on balance more roles for NFAT transcription factors in the expression of mast cell-derived cytokines. J. Immunol. 177: 6667–6674. important than the changes in these two regulatory proteins. 11. Klemm, S., J. Gutermuth, L. Hu¨ltner, T. Sparwasser, H. Behrendt, C. Peschel, In Zeb2 siRNA-treated cells, there was rapid reduction in the T. W. Mak, T. Jakob, and J. Ruland. 2006. The Bcl10-Malt1 complex segregates Fc epsilon RI-mediated nuclear factor kappa B activation and cytokine pro- expression of the Zeb2 protein that was apparent within 24 h, duction from mast cell degranulation. J. Exp. Med. 203: 337–347. whereas the phenotypic changes in degranulation and cytokine 12. Klemm, S., and J. Ruland. 2006. Inflammatory signal transduction from the Fc release were more prominent at later time points. In fact, at early epsilon RI to NF-kappa B. Immunobiology 211: 815–820. 13. Kalesnikoff, J., V. Lam, and G. Krystal. 2002. SHIP represses mast cell acti- time points after transfection, there was enhancement in the re- vation and reveals that IgE alone triggers signaling pathways which enhance lease of cytokines. There was also much greater inhibition of de- normal mast cell survival. Mol. Immunol. 38: 1201–1206. granulation than cytokine release by reduced Zeb2 expression. As 14. Leung, W. H., and S. Bolland. 2007. The inositol 59-phosphatase SHIP-2 neg- atively regulates IgE-induced mast cell degranulation and cytokine production. Zeb2 is known to regulate the transcription of other proteins, the J. Immunol. 179: 95–102. Downloaded from results suggest that the phenotypic effects are due to changes in 15. Furumoto, Y., S. Brooks, A. Olivera, Y. Takagi, M. Miyagishi, K. Taira, the expression of these other molecules (31, 49, 50). The differ- R. Casellas, M. A. Beaven, A. M. Gilfillan, and J. Rivera. 2006. Cutting edge: lentiviral short hairpin RNA silencing of PTEN in human mast cells reveals ence in the extent of inhibition in degranulation compared with constitutive signals that promote cytokine secretion and cell survival. J. Immu- the cytokine response with decreased Zeb2 levels is likely due to nol. 176: 5167–5171. 16. Verschueren, K., J. E. Remacle, C. Collart, H. Kraft, B. S. Baker, different rates at which there are changes in the expression of P. Tylzanowski, L. Nelles, G. Wuytens, M. T. Su, R. Bodmer, et al. 1999. SIP1, a signaling proteins. novel zinc finger/homeodomain repressor, interacts with Smad proteins and Notably, when Zeb2 expression was decreased, the LPS-medi- binds to 59-CACCT sequences in candidate target genes. J. Biol. Chem. 274: http://www.jimmunol.org/ ε 20489–20498. ated NF-kB response was enhanced although Fc RI-induced 17. Postigo, A. A., and D. C. Dean. 2000. Differential expression and function of NF-kB activation was inhibited (Fig. 1). Therefore, not all the members of the zfh-1 family of zinc finger/homeodomain . Proc. Natl. effects of Zeb2 siRNA treatment are inhibition of signaling path- Acad. Sci. USA 97: 6391–6396. 18. Vandewalle, C., F. Van Roy, and G. Berx. 2009. The role of the ZEB family of ways. The mechanism of this enhancement was not a focus of this transcription factors in development and disease. Cell.Mol.LifeSci.66: 773–787. study, but it is possible that it was due to changes in the PI3K 19. Zweier, C., C. T. Thiel, A. Dufke, Y. J. Crow, P. Meinecke, M. Suri, S. Ala- pathway, which is known to play a role in signaling from TLRs Mello, F. Beemer, S. Bernasconi, P. Bianchi, et al. 2005. Clinical and mutational spectrum of Mowat-Wilson syndrome. Eur. J. Med. Genet. 48: 97–111. (51–53). Alternatively, the enhanced NF-kB response may be 20. Sayan, A. E., T. R. Griffiths, R. Pal, G. J. Browne, A. Ruddick, T. Yagci, related to the interaction of Zeb2 with Smad proteins, as the LPS- R. Edwards, N. J. Mayer, H. Qazi, S. Goyal, et al. 2009. SIP1 protein protects by guest on October 2, 2021 induced cytokine response is enhanced in mice that are deficient cells from DNA damage-induced and has independent prognostic value in bladder cancer. Proc. Natl. Acad. Sci. USA 106: 14884–14889. in Smad3 (54). 21. Xie, Z. H., I. Ambudkar, and R. P. Siraganian. 2002. The adapter molecule Gab2 Stimulated mast cells release a wide range of cytokines and regulates Fc epsilon RI-mediated signal transduction in mast cells. J. Immunol. 168: 4682–4691. chemokines, many with inflammatory potential. Besides their di- 22. Zhang, J., M. Mendoza, M. F. Guiraldelli, E. A. Barbu, and R. P. Siraganian. rect effects, these products also recruit and activate other cells such 2010. Small interfering RNA screen for phosphatases involved in IgE-mediated as neutrophils and lymphocytes that influence the intensity of the mast cell degranulation. J. Immunol. 184: 7178–7185. 23. Barbu, E. A., J. Zhang, and R. P. Siraganian. 2010. The limited contribution of inflammatory response and also regulate immune and autoimmune Fyn and Gab2 to the high affinity IgE receptor signaling in mast cells. J. Biol. conditions (55). As Zeb2 knockdown results in a decrease in the Chem. 285: 15761–15768. release of these mediators, it is possible that control of Zeb2 may 24. Siraganian, R. P., J. Zhang, K. Suzuki, and K. Sada. 2002. Protein tyrosine kinase Syk in mast cell signaling. Mol. Immunol. 38: 1229–1233. be another approach to regulate the inflammatory response. 25. Benhamou, M., N. J. P. Ryba, H. Kihara, H. Nishikata, and R. P. Siraganian. In summary, these studies show that Zeb2 regulates the ex- 1993. Protein-tyrosine kinase p72syk in high affinity IgE receptor signaling. pression of several molecules in mast cells that are involved in Identification as a component of pp72 and association with the receptor gamma chain after receptor aggregation. J. Biol. Chem. 268: 23318–23324. and signal transduction. As a regulator of mast cell 26. Zhang, J., E. H. Berenstein, R. L. Evans, and R. P. Siraganian. 1996. Transfection responses, Zeb2 may prove a possible target for pharmacological of Syk protein tyrosine kinase reconstitutes high affinity IgE receptor-mediated degranulation in a Syk-negative variant of rat basophilic leukemia RBL-2H3 agents that would control the inflammatory response. cells. J. Exp. Med. 184: 71–79. 27. Zhang, J., T. Kimura, and R. P. Siraganian. 1998. Mutations in the activation loop tyrosines of protein tyrosine kinase Syk abrogate intracellular signaling but Disclosures not kinase activity. J. Immunol. 161: 4366–4374. The authors have no financial conflicts of interest. 28. Zhang, J., M. L. Billingsley, R. L. Kincaid, and R. P. Siraganian. 2000. Phos- phorylation of Syk activation loop tyrosines is essential for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine . References J. Biol. Chem. 275: 35442–35447. 29. Falasca, M., S. K. Logan, V. P. Lehto, G. Baccante, M. A. Lemmon, and 1. Kalesnikoff, J., and S. J. Galli. 2008. New developments in mast cell . J. Schlessinger. 1998. Activation of phospholipase C gamma by PI 3-kinase- Nat. Immunol. 9: 1215–1223. induced PH domain-mediated membrane targeting. EMBO J. 17: 414–422. 2. Alvarez-Errico, D., E. Lessmann, and J. Rivera. 2009. Adapters in the organi- 30. Bae, Y. S., L. G. Cantley, C. S. Chen, S. R. Kim, K. S. Kwon, and S. G. Rhee. zation of mast cell signaling. Immunol. Rev. 232: 195–217. 1998. Activation of phospholipase C-gamma by phosphatidylinositol 3,4,5- 3. Kambayashi, T., D. F. Larosa, M. A. Silverman, and G. A. Koretzky. 2009. trisphosphate. J. Biol. Chem. 273: 4465–4469. Cooperation of adapter molecules in proximal signaling cascades during allergic 31. Goossens, S., V. Janzen, S. Bartunkova, T. Yokomizo, B. Drogat, M. Crisan, inflammation. Immunol. Rev. 232: 99–114. K. Haigh, E. Seuntjens, L. Umans, T. Riedt, et al. 2011. The EMT regulator 4. Siraganian, R. P., R. O. de Castro, E. A. Barbu, and J. Zhang. 2010. Mast cell Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell signaling: the role of protein tyrosine kinase Syk, its activation and screening differentiation and mobilization. Blood 117: 5620–5630. methods for new pathway participants. FEBS Lett. 584: 4933–4940. 32. Browne, G., A. E. Sayan, and E. Tulchinsky. 2010. ZEB proteins link cell 5. Kim, M. S., M. Ra˚dinger, and A. M. Gilfillan. 2008. The multiple roles of motility with cell cycle control and cell survival in cancer. Cell Cycle 9: 886– phosphoinositide 3-kinase in mast cell biology. Trends Immunol. 29: 493–501. 891. The Journal of Immunology 9

33. Beltran, M., I. Puig, C. Pen˜a, J. M. Garcı´a, A. B. Alvarez, R. Pen˜a, F. Bonilla, PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. and A. G. de Herreros. 2008. A natural antisense transcript regulates Zeb2/Sip1 Immunity 19: 669–678. during Snail1-induced epithelial-mesenchymal transition. Genes 45. Kawakami, Y., J. Kitaura, S. E. Hartman, C. A. Lowell, R. P. Siraganian, and Dev. 22: 756–769. T. Kawakami. 2000. Regulation of protein kinase CbetaI by two protein-tyrosine 34. Gregory, P. A., A. G. Bert, E. L. Paterson, S. C. Barry, A. Tsykin, G. Farshid, kinases, Btk and Syk. Proc. Natl. Acad. Sci. USA 97: 7423–7428. M. A. Vadas, Y. Khew-Goodall, and G. J. Goodall. 2008. The miR-200 family 46. Monticelli, S., D. C. Solymar, and A. Rao. 2004. Role of NFAT proteins in IL13 and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 gene transcription in mast cells. J. Biol. Chem. 279: 36210–36218. and SIP1. Nat. Cell Biol. 10: 593–601. 47. Walczak-Drzewiecka, A., M. Ratajewski, W. Wagner, and J. Dastych. 2008. 35. Bartel, D. P. 2009. MicroRNAs: target recognition and regulatory functions. Cell HIF-1alpha is up-regulated in activated mast cells by a process that involves 136: 215–233. and NFAT. J. Immunol. 181: 1665–1672. 36. Selbach, M., B. Schwanha¨usser, N. Thierfelder, Z. Fang, R. Khanin, and 48. Ullera˚s, E., M. Karlberg, C. Mo¨ller Westerberg, J. Alfredsson, S. Gerondakis, N. Rajewsky. 2008. Widespread changes in protein synthesis induced by A. Strasser, and G. Nilsson. 2008. NFAT but not NF-kappaB is critical for microRNAs. Nature 455: 58–63. transcriptional induction of the prosurvival gene A1 after IgE receptor activation 37. Baek, D., J. Ville´n, C. Shin, F. D. Camargo, S. P. Gygi, and D. P. Bartel. 2008. in mast cells. Blood 111: 3081–3089. The impact of microRNAs on protein output. Nature 455: 64–71. 49. Vandewalle, C., J. Comijn, B. De Craene, P. Vermassen, E. Bruyneel, 38. Salmena, L., L. Poliseno, Y. Tay, L. Kats, and P. P. Pandolfi. 2011. A ceRNA H. Andersen, E. Tulchinsky, F. Van Roy, and G. Berx. 2005. SIP1/ZEB2 induces hypothesis: the Rosetta Stone of a hidden RNA language? Cell 146: 353–358. EMT by repressing genes of different epithelial cell-cell junctions. Nucleic Acids 39. Karreth, F. A., Y. Tay, D. Perna, U. Ala, S. M. Tan, A. G. Rust, G. DeNicola, Res. 33: 6566–6578. K. A. Webster, D. Weiss, P. A. Perez-Mancera, et al. 2011. In vivo identification 50. Mejlvang, J., M. Kriajevska, C. Vandewalle, T. Chernova, A. E. Sayan, G. Berx, of tumor- suppressive PTEN ceRNAs in an oncogenic BRAF-induced mouse J. K. Mellon, and E. Tulchinsky. 2007. Direct repression of cyclin D1 by SIP1 model of . Cell 147: 382–395. attenuates cell cycle progression in cells undergoing an epithelial mesenchymal 40. Zhang, W., R. P. Trible, M. Zhu, S. K. Liu, C. J. McGlade, and L. E. Samelson. transition. Mol. Biol. Cell 18: 4615–4624. 2000. Association of Grb2, Gads, and phospholipase C-gamma 1 with phos- 51.Li,X.,J.C.Tupper,D.D.Bannerman,R.K.Winn,C.J.Rhodes,and phorylated LAT tyrosine residues. Effect of LAT tyrosine mutations on J. M. Harlan. 2003. Phosphoinositide 3 kinase mediates Toll-like receptor angigen receptor-mediated signaling. J. Biol. Chem. 275: 23355–23361. 4-induced activation of NF-kappa B in endothelial cells. Infect. Immun. 71: 41. Paz, P. E., S. Wang, H. Clarke, X. Lu, D. Stokoe, and A. Abo. 2001. Mapping the 4414–4420. Zap-70 phosphorylation sites on LAT (linker for activation of T cells) required 52. Fukao, T., and S. Koyasu. 2003. PI3K and negative regulation of TLR signaling. Downloaded from for recruitment and activation of signalling proteins in T cells. Biochem. J. 356: Trends Immunol. 24: 358–363. 461–471. 53. Utsugi, M., K. Dobashi, A. Ono, T. Ishizuka, S. Matsuzaki, T. Hisada, 42. Yu, M., C. A. Lowell, B. G. Neel, and H. Gu. 2006. Scaffolding adapter Grb2- Y. Shimizu, T. Kawata, H. Aoki, Y. Kamide, and M. Mori. 2009. PI3K p110beta associated binder 2 requires Syk to transmit signals from FcepsilonRI. positively regulates -induced IL-12 production in human J. Immunol. 176: 2421–2429. macrophages and dendritic cells and JNK1 plays a novel role. J. Immunol. 182: 43. Hata, D., Y. Kawakami, N. Inagaki, C. S. Lantz, T. Kitamura, W. N. Khan, 5225–5231. M. Maeda-Yamamoto, T. Miura, W. Han, S. E. Hartman, et al. 1998. Involve- 54. Kanamaru, Y., K. Sumiyoshi, H. Ushio, H. Ogawa, K. Okumura, and A. Nakao.

ment of Bruton’s tyrosine kinase in FcepsilonRI-dependent mast cell degranu- 2005. Smad3 deficiency in mast cells provides efficient host protection against http://www.jimmunol.org/ lation and cytokine production. J. Exp. Med. 187: 1235–1247. acute septic peritonitis. J. Immunol. 174: 4193–4197. 44. Saito, K., K. F. Tolias, A. Saci, H. B. Koon, L. A. Humphries, A. Scharenberg, 55. Paul, W. E., and J. Zhu. 2010. How are T(H)2-type immune responses initiated D. J. Rawlings, J. P. Kinet, and C. L. Carpenter. 2003. BTK regulates and amplified? Nat. Rev. Immunol. 10: 225–235. by guest on October 2, 2021