Requirement for Stat5 in Thymic Stromal Lymphopoietin-Mediated Signal Transduction

This information is current as Deborah E. Isaksen, Heinz Baumann, Patty A. Trobridge, of September 28, 2021. Andrew G. Farr, Steven D. Levin and Steven F. Ziegler J Immunol 1999; 163:5971-5977; ; http://www.jimmunol.org/content/163/11/5971 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 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Requirement for Stat5 in Thymic Stromal Lymphopoietin-Mediated Signal Transduction1

Deborah E. Isaksen,* Heinz Baumann,† Patty A. Trobridge,‡ Andrew G. Farr,‡§ Steven D. Levin,‡ and Steven F. Ziegler2*

Thymic stromal lymphopoietin (TSLP) is a newly identified cytokine that uniquely promotes B lymphopoiesis to the B220؉/IgM؉ immature B cell stage. In addition, TSLP shares many biological properties with the related cytokine IL-7. This can be explained by the finding that the receptor complexes for TSLP and IL-7 both contain the IL-7R ␣-chain; IL-7R␣ is paired with the common ␥-chain (␥c) in the IL-7 receptor complex and the unique TSLP-R chain in the TSLP receptor complex. Although TSLP and IL-7 both induce tyrosine phosphorylation of the transcription factor Stat5, only IL-7-mediated signal transduction could be associated with activation of Janus family kinases (Jaks). Because Stat5 phosphorylation following cytokine stimulation is generally mediated by Jaks, the lack of Jak activation after TSLP treatment suggested the possibility that tyrosine-phosphorylated Stat5 may be Downloaded from nonfunctional. Herein, we demonstrate that TSLP induces a functional Stat5 transcription factor in that TSLP stimulation results in Stat5-DNA complex formation and transcription of the Stat5-responsive gene CIS. We also show that the TSLP receptor complex is functionally reconstituted using TSLP-R and IL-7R␣ and that TSLP-mediated signal transduction requires Stat5. Moreover, TSLP-mediated signaling is inhibited by suppressor of cytokine signaling (SOCS)-1 and a kinase-deficient version of Tec but not by kinase-deficient forms of Jak1 and Jak2. The Journal of Immunology, 1999, 163: 5971–5977. http://www.jimmunol.org/ he generation of mature, functional B lymphocytes in- the IL-2R ␥-chain (also known as the common ␥-chain or ␥c; Refs. volves the complex interplay between developing B cells 6 and 7). The TSLP receptor complex also employs IL-7R␣, but and stromal cells in the fetal liver and adult bone marrow here IL-7R␣ associates with a novel receptor chain designated T 4 microenvironments. This interplay includes intimate cell-cell con- TSLP-R (5). The sharing of cytokine receptor subunits by differ- tact as well as production of secreted proteins, or cytokines, that ent cytokines is well documented. IL-2R␤ is shared by IL-2 and play important, yet poorly understood, roles in lymphocyte devel- IL-15 (8); IL-4R is shared by IL-4 and IL-13 (9); and common opment. For example, the cytokine IL-7 is essential for B lympho- ␤-chain is shared by IL-3, IL-5, and GM-CSF (10). Moreover, poiesis, in that mice lacking either IL-7 or the IL-7 receptor common ␥-chain is shared by the receptors for IL-2, IL-4, IL-7, ␣-chain (IL-7R␣) have developmentally arrested B cells (1, 2). We IL-9, and IL-15 (11) but is not a component of the TSLP receptor by guest on September 28, 2021 have recently identified a novel cytokine designated thymic stro- complex (5). mal lymphopoietin (TSLP)3. TSLP is similar to IL-7 in that both A prevalent feature of cytokine-mediated signal transduction is cytokines can costimulate thymocytes and mature T cells, support activation of the Janus family kinase (Jak)/Stat pathway. Seven B lymphopoiesis in long-term cultures of fetal liver cells, and sus- mammalian Stat proteins (Stat1 to -4, -5a, -5b, and -6) and four tain the factor-dependent, fetal liver-derived NAG8/7 pre-B cell Janus protein kinases (Jak1 to -3 and Tyk2) have been identified line (3–5). But, while TSLP facilitates B cell development to the (for review see Ref. 11). Cytokine-induced receptor aggregation B220ϩ/IgMϩ immature B cell stage, IL-7 supports development activates the Jak/Stat pathway by bringing the receptor-associated only to the less mature B220ϩ/IgMϪ pre-B cell stage (5). Janus kinases (Jaks) into mutual proximity, thus allowing for The similarity between TSLP and IL-7 extends to their receptor transphosphorylation and activation. The activated Jaks then phos- complexes. The IL-7 receptor complex is composed of IL-7R␣ and phorylate the cytokine receptor, thereby providing a docking site for SH2 containing proteins, including members of the Stat transcription factor family. Upon binding to the phosphorylated cyto- *Virginia Mason Research Center, Seattle, WA 98101; †Department of Molecular and kine receptor, Stats themselves become tyrosine phosphorylated, Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY 14263; and Depart- are released from the receptor complex, dimerize, and translocate ments of ‡Immunology and §Biological Structure, University of Washington, Seattle, WA 98195 to the nucleus where they bind DNA with sequence specificity to Received for publication May 28, 1999. Accepted for publication September initiate transcription of target genes. 21, 1999. Our previous investigations of the biochemical signals induced The costs of publication of this article were defrayed in part by the payment of page by TSLP and IL-7 revealed that TSLP and IL-7 both trigger ty- charges. This article must therefore be hereby marked advertisement in accordance rosine phosphorylation of Stat5a and Stat5b (the products of two with 18 U.S.C. Section 1734 solely to indicate this fact. distinct genes that are 95% identical). Although IL-7 stimulation 1 This work was supported by Grants AI44259 (to S.F.Z.), AI44160 (to A.G.F.), and CA26122 (to H.B.) from the National Institutes of Health and Fellowship VMRC leads to tyrosine phosphorylation and subsequent activation of 5813 from the Virginia Mason Research Center (to D.E.I.). Jak1 and Jak3, we were unable to detect activation of any of the 2 Address correspondence and reprint requests to Dr. Steven F. Ziegler, Virginia four known Jak kinases following TSLP stimulation (5). This lack Mason Research Center, 1201 9th Avenue, Seattle, WA 98101. E-mail address: [email protected] 3 Abbreviations used in this paper: TSLP, thymic stromal lymphopoietin; ␥c, common 4 L. S. Park, U. Martin, K. Garka, B. Gliniak, J. P. DiSanto, W. Muller, D. A. Lar- ␥-chain; Jak, Janus family kinase; OSM, oncostatin M; HRRE, hemopoietin receptor gaespada, N. G. Copeland, N. A. Jenkins, A. G. Farr, S. F. Ziegler, P. J. Morrissey, response element; CAT, chloramphenicol acetyl transferase; MUP, mouse major uri- R. Paxton, and J. Sims. Cloning of the murine TSLP receptor: formation of a func- nary protein; Csk, C-terminal Src kinase; SOCS, suppressor of cytokine signaling. tional heteromeric complex requires IL-7 receptor. Submitted for publication.

of Jak activation suggested the possibility that phosphorylated complete medium lacking cytokine, 10 ␮g of the CIS- or OSM-luciferase Stat5 in TSLP-treated cells may be nonfunctional. Here we show constructs were transiently transfected into 3 ϫ 107 NAG8/7 or CTLL-2 ␮ that TSLP stimulation results in a functional Stat5 transcription cells by electroporation (250V, 960 F) using a Genepulser (Bio-Rad). Following transfection, cells were washed in complete medium and divided factor. Using the TSLP and IL-7 dependent NAG8/7 pre-B cell into individual wells of a 12-well culture plate. Cells were cultured in line (3), we demonstrate that TSLP induces Stat5-DNA complex duplicate in the presence of either no cytokine, 1 ng/ml IL-7, 21 ng/ml formation, transcription of the Stat5-responsive gene CIS, and reg- TSLP, or 100 U/ml IL-2. After5hofculture, cells were washed with PBS ulation of the luciferase reporter gene when fused to the CIS pro- and lysed for 15 min at room temperature with 1ϫ passive lysis buffer (Promega, Madison, WI). Cell lysates were centrifuged, and supernatants moter. Furthermore, we show that the TSLP receptor complex is were examined for luciferase activity as determined by measuring light functionally reconstituted using TSLP-R and IL-7R␣ and that emitted for 30 s using a luminometer (Lumat LB9507, EGϩG Berthold, TSLP-mediated signal transduction requires Stat5, is blocked by Nashua, NH) and Promega’s luciferase assay reagent. overexpression of suppressor of cytokine signaling (SOCS)-1 or Transient transfection and chloramphenicol acetyl transferase kinase-deficient Tec, yet is unaffected by overexpression of kinase- (CAT) assay in HepG2 cells deficient Jak1 or Jak2. Constructs. p(8xHRRE)-CAT contains eight tandem copies of the 27 bp Materials and Methods hemopoietin receptor response element in pCAT (18). Expression vectors for the following molecules have been described: IL-7R␣ and ␥c (6), the Cell culture internal transfection control mouse major urinary protein (MUP; Ref. 19), ⌬ NAG8/7 cells (3) were maintained in RPMI 1640 complete medium (Life rat Stat5b (20), the truncated, dominant-negative mutant Stat5b 40C (21), Technologies, Grand Island, NY) supplemented with 10% FCS, 45 U/ml the murine, kinase-deficient form of Jak2 (22), and the human, kinase- deficient form of Tec called TecKM (23). TSLP-R4 and murine SOCS-1 penicillin, 45 ␮g/ml streptomycin, 34 mM HEPES, 3.8 mM L-glutamine, 1 Downloaded from mM sodium pyruvate, 20 ␮M 2-ME, and, unless otherwise specified, 1 (24–27) were cloned into the pcDNA3.1 expression vector (Invitrogen, ng/ml murine IL-7. The human IL-2-dependent T cell line CTLL-2 was Carlsbad, CA) using a PCR-based approach, and the sequences were ver- maintained as above with the addition of 50 U/ml human rIL-2 (Chiron, ified by DNA sequence analysis. A kinase-deficient form of murine Jak1 Emeryville, CA). Murine IL-7 and TSLP were obtained by transfection of (28, 29), in which the codon for the conserved lysine residue in the ATP- the p3XAG cell line with cDNAs to the appropriate cytokine in the BC- binding site (aa 907) was changed to an aspartic acid codon, was generated MGS expression vector (12). Cytokine concentrations were determined by using PCR-based mutagenesis. The mutant Jak1 construct was verified by comparison with purified cytokine. Human hepatoma HepG2 cells (13) DNA sequence analysis and cloned into the expression vector pDC302 were cultured in DMEM containing 10% FCS and antibiotics. (30). The human C-terminal Src kinase (Csk; Ref. 31) was expressed in http://www.jimmunol.org/ pDC302 as well (30). EMSA Transfection and analysis. HepG2 cells were transfected by the calcium phosphate method (32) using 20 ␮g/ml total plasmid DNA and 2–3 ϫ 105 NAG8/7 cells were washed three times in PBS, cultured without cytokine cells. After an overnight recovery period, cultures were released from the 6 for 4 h, and then stimulated at 10 cells/ml with either 10 ng/ml IL-7, 24 plate with trypsin and divided into six-well culture plates. After an addi- ng/ml TSLP, or left unstimulated. After 20- and 60-min incubations, nu- tional 24 h, subcultures were treated for 24 h with serum-free medium clear extracts were prepared as described (14). A probe for Stat DNA- containing 100 ng/ml IL-7, TSLP, IL-6, or OSM. Medium was then col- binding activity was generated by annealing incompletely overlapping oli- lected and subjected to immunoelectrophoresis to quantitate expression of Ј Ј Ј gonucleotides (5 -TCGAGTATTTCCCAGAAAAGGAAC-3 and 5 -AG the cotransfected control MUP plasmid. CAT activity for each culture was Ј CTGTTCCTTTTCTGGGAAATAC-3 ) corresponding to the sense and determined, normalized to the amount of MUP expression, and calculated ␥ by guest on September 28, 2021 antisense strands of the Stat-responsive DNA element from the Fc RI pro- relative to the control cultures in each experimental series (defined as 1.0). 32 moter (15). The probe was P-labeled by an end-filling T4 polymerase Data are presented as the mean of three to five trials. reaction and purified with Bio-gel P30 spin columns (Bio-Rad, Hercules, CA). Nuclear extracts (ϳ2–4 ␮g total protein) in 50 mM KCl, 15 mM Results HEPES (pH 7.9), 15% glycerol, 0.75 mM DTT, and 87.5 mg/ml poly(dI- TSLP induces Stat5-DNA complex formation dC) were incubated with the 32P-labeled probe (5000 cpm) at room temperature for 20 min. For the supershift reactions, 100 ng of Stat5 rabbit The cytokines TSLP and IL-7 both employ IL-7R␣ as part of their polyclonal Abs (Santa Cruz Biotechnology, Santa Cruz, CA) or 100 ng receptor complexes (5, 6).4 In addition, both cytokines trigger ty- rabbit polyclonal preimmune serum were added to the reaction after the first 10 min. Samples were electrophoresed at 4°C through a 4% native rosine phosphorylation of the two Stat5 isoforms, Stat5a and polyacrylamide, 0.25ϫ TBE gel followed by autoradiography. Stat5b. Yet, in the case of TSLP, Stat5 tyrosine phosphorylation occurs in the absence of detectable Jak activation (5). This finding Northern blots suggests not only that Stat5 activation may be occurring by an NAG8/7 and CTLL-2 cells were washed, starved, and stimulated as was alternate mechanism, but also that TSLP and IL-7 signal via dif- done for the EMSA experiments, except that CTLL-2 cells were stimulated ferent biochemical pathways despite sharing the IL-7R␣ receptor with 50 U/ml IL-2. After the indicated incubation times, total RNA was isolated using Trizol reagent (Life Technologies). Twenty micrograms of subunit. The fact that no detectable Jak activation occurs following total RNA from each sample was denatured for 5 min at 55°C in 50% TSLP treatment does raise the possibility, however, that, although DMSO, 0.5ϫ TBE, and 0.05% bromphenol blue and then electrophoresed TSLP induces Stat5 tyrosine phosphorylation, a functionally active through a 1% agarose, 0.5ϫ TBE gel. The size-separated RNA was cap- Stat5 transcription factor may not result. illary transferred to Transfer-IT plus nylon membrane (CPG, Lincoln Park, To determine whether TSLP-activated Stat5 enters the nucleus NJ) and cross-linked to the membrane by UV irradiation. Blots were pre- hybridized at 42°C in a heat-sealed pouch with hybridization buffer (50% and binds a Stat-responsive DNA element, we performed EMSA. formamide, 5ϫ SSC, 1ϫ Denhart’s solution, 20 mM mono and dibasic Nuclear extracts were generated from NAG8/7 cells that were sodium phosphate, 100 ␮g/ml salmon sperm DNA, 10% dextran sulfate, treated with TSLP or IL-7 for 60 min (or 20 min, data not shown), 32 and 1% SDS). DNA probes were P-labeled using the high-prime labeling and these extracts were tested for DNA-binding activity using the kit (Boehringer Mannheim, Indianapolis, IN) and purified with Bio-gel P30 ␥ spin columns (Bio-Rad). After an overnight hybridization at 42°C, blots Fc RI Stat-responsive DNA element (15). Both TSLP- and IL-7- were washed several times, each time with increasing stringency. The final stimulated cell extracts showed specific DNA-binding activity wash, before autoradiography, consisted of 0.1ϫ SSC, 0.1% SDS at am- whereas extracts from unstimulated cells did not (Fig. 1). Further- bient temperature (CIS probe) or 65°C (oncostatin M (OSM) and GAPDH more, addition of an antisera to Stat5 (which recognizes both probes). Before the blots were reprobed, they were stripped by immersion Stat5a and Stat5b isoforms) supershifted these complexes. The in boiling water for 5 min. lower level of complexed Stat5 seen in TSLP-treated cells, as com- Transient transfections and luciferase assay in NAG8/7 cells pared with IL-7-treated cells, is consistent with our previous report The Ϫ404 CIS promoter-luciferase construct and Ϫ694 OSM promoter- that TSLP induced tyrosine phosphorylation of Stat5 less effi- luciferase construct have been described (16, 17). After a 5-h incubation in ciently and with delayed kinetics relative to IL-7 (5). Thus, TSLP The Journal of Immunology 5973

FIGURE 3. TSLP and IL-7 both activate the CIS, but not the OSM, promoter. NAG8/7 cells were transfected with the CIS or OSM promoter FIGURE 1. TSLP stimulation induces a Stat5-DNA-binding complex. fused to the luciferase reporter gene. After stimulation with cytokine-free NAG8/7 cells were factor depleted and then stimulated for 60 min with media, IL-7 (1 ng/ml) or TSLP (21 ng/ml) luciferase activity was mea- either medium alone (unstim.), TSLP, or IL-7. Nuclear extracts were in- sured. Data are presented as the average activity (ϫ103) from duplicate cubated with a 32P-labeled, double-stranded, oligonucleotide correspond- cultures. ing to the Stat-responsive element from the Fc␥RI promoter, in the absence or presence of either rabbit preimmune serum (control Ig) or rabbit polyclonal Stat5 serum, and analyzed by EMSA. The supershifted complex occurs only when Stat5 Ab is included, conﬁrming the presence of Stat5 in sis, stimulation with either cytokine induced luciferase activity the DNA-protein complex following TSLP and IL-7 stimulation. when the CIS-luciferase construct was used but not when the Downloaded from OSM-luciferase construct was used (Fig. 3). However, luciferase activity was induced in IL-2-treated CTLL-2 cells after transfec- induces changes in Stat5 that result in nuclear translocation and tion with the OSM-luciferase construct, indicating that this re- DNA-binding activity, despite the lack of concomitant Jak porter gene is functional (data not shown). In NAG8/7 cells there activation. was less activity induced from the CIS reporter construct following

TSLP induces expression of the Stat5-responsive gene CIS but TSLP stimulation than following IL-7 stimulation, and this reduc- http://www.jimmunol.org/ not OSM tion was proportional to the levels of Stat5 phosphorylation previously seen (5). We do not attribute the lower luciferase activity A functional Stat5 transcription factor must do more than translo- seen after TSLP stimulation to limiting amounts of cytokine be- cate to the nucleus and bind DNA. It must be able to initiate tran- cause using 10 ng/ml, instead of 21 ng/ml, of TSLP gave identical scription of Stat5 target genes. The promoters of both the CIS and results, suggesting that both levels were saturating (data not OSM genes contain Stat5 target sequences, and both genes are shown). Together, the Northern blot and luciferase assay results induced upon Stat5 activation by IL-2, IL-3, and erythropoietin reveal that TSLP- and IL-7-mediated signal transduction include (17, 33, 34). Therefore, we used Northern blot analysis to test transcription of the Stat5 target gene CIS, but not OSM, and un- whether CIS and OSM were transcribed following TSLP stimula- cover another similarity in the signaling pathways of these cyto- by guest on September 28, 2021 tion. As shown in Fig. 2, CIS transcription was induced in NAG8/7 kines. Furthermore, these results highlight the fact that, although cells by both TSLP and IL-7, although to a lesser extent by TSLP. CIS and OSM are both regulated by Stat5, they are regulated dif- When the Northern blots were stripped and reprobed for OSM ferently, and Stat5 activation is not sufficient for OSM induction. expression, OSM transcripts were detected in neither the TSLP- nor the IL-7-stimulated lanes. OSM transcripts were readily ap- Functional reconstitution of the TSLP receptor complex requires parent, however, in control lanes containing comparable amounts TSLP-R and Stat5 of RNA isolated from IL-2-stimulated CTLL-2 cells (data not To further address the role of Stat5 in TSLP-mediated signal trans- shown). These data suggest that the OSM gene is either not active duction, we used reconstitution of defined receptor combinations or not regulated by these cytokines in NAG8/7 cells. in a cell culture system. This system involves cotransfection of the To confirm the induction of CIS, but not OSM, following TSLP human hepatoma cell line HepG2 with cDNA clones encoding and IL-7 stimulation, we employed a luciferase expression assay. receptor subunits, as well as a CAT-reporter plasmid containing NAG8/7 cells were transiently transfected with constructs contain- eight copies of the cytokine-inducible hemopoietin receptor re- ing the CIS or OSM promoter driving the expression of a lucif- sponse element (HRRE-CAT; Refs. 6 and 18). The ability of the erase reporter gene and were stimulated with no cytokine, IL-7, or transfected cells to signal when treated with the appropriate cyto- TSLP. Consistent with what was seen in the Northern blot analy- kine is measured by CAT activity. As shown in Fig. 4A, cells cotransfected with TSLP-R and IL- 7R␣ generated a TSLP-specific signal only when cDNAs encoding Stat5b (or Stat5a, data not shown) were included in the cotransfection. On the other hand, cells cotransfected with TSLP-R and ␥c were unable to activate the cytokine-inducible promoter irrespective of which cytokine was used and whether or not Stat5b was included in the cotransfection. Moreover, HepG2 cells cotransfected with IL-7R␣ and ␥c responded to IL-7 in the absence of cotransfected Stat5b (consistent with earlier findings by Ziegler et al.; Ref. 6), but the response was enhanced if Stat5b cDNAs were included. These results suggest several things. First, they demon- FIGURE 2. Northern blot analysis showing induction of CIS mRNA in strate that a functional TSLP receptor complex can be reconsti- NAG8/7 cells after IL-7 and TSLP stimulation for the indicated number of tuted with TSLP-R and IL-7R␣. Second, HepG2 cells express hours. The blot was stripped and reprobed for GAPDH expression to dem- abundant amounts of Stat1 and Stat3, but only very low levels of onstrate even loading. Stat5. Therefore, these results suggest that TSLP-R is unable to 5974 REQUIREMENT FOR Stat5 IN TSLP-MEDIATED SIGNAL TRANSDUCTION Downloaded from

FIGURE 4. Stat5 is necessary for TSLP-mediated signal transduction. HepG2 cells were transiently cotransfected with a CAT-reporter plasmid containing the HRRE cytokine-inducible promoter, plasmids expressing the receptor subunits (as indicated at the top of each panel), plus plasmids encoding either Stat5b or vector alone. For B, cells were also transfected with increasing plasmid amounts, as indicated (micrograms per milliliter of transfection http://www.jimmunol.org/ solution), of a dominant-negative version of Stat5b (Stat5b⌬40C). Cells were treated with no cytokine (Control), TSLP, or IL-7 and analyzed for CAT activity. A, Activation of the cytokine-inducible promoter via the TSLP receptor complex (TSLP-R ϩ IL-7␣) occurred only upon TSLP treatment when plasmids encoding Stat5b were included (left panel). Cells cotransfected with the IL-7 receptor complex (IL-7␣ ϩ ␥c) responded to IL-7 in the absence of Stat5b, although the addition of Stat5b resulted in a substantial increase in CAT activity (middle panel). The TSLP-R and ␥c receptor subunits are insufﬁcient to form a functional TSLP or IL-7 receptor complex irrespective of whether Stat5b is added (right panel). B, Cotransfection of a dominant- negative version of Stat5b (Stat5b⌬40C) inhibits TSLP-mediated signal transduction in a dose-dependent manner.

engage endogenous signal transduction pathways in these cells, required component of the TSLP signaling pathway. On the other by guest on September 28, 2021 including those mechanisms used by the IL-7 receptor and other hand, overexpression of kinase-deficient Jak1 did reduce IL-7- and hemopoietin receptors, unless Stat5 is also provided. In contrast, OSM-mediated CAT activation, corroborating previous findings IL-7-mediated signal transduction is able to utilize the endogenous that Jak1 is necessary for IL-7- and OSM-mediated signaling (37– signaling pathways, which may include Stat1 and/or Stat3, in ad- 39). Similarly, overexpression of kinase-deficient Jak2 had no ap- dition to experimentally enhanced Stat5. This finding is consistent preciable effect on TSLP, IL-7, IL-6, and OSM signaling but did with previous reports of IL-7-mediated activation of Stat1, Stat3, decrease thrombopoietin-induced CAT activity in c-mpl-trans- and Stat5 (35, 36). fected cells (data not shown and Ref. 22). To confirm and extend the findings presented in Fig. 4A,we We have also used the HepG2 receptor reconstitution system to tested whether cotransfection of a dominant-negative version of begin to identify and characterize components of the TSLP-medi- ⌬ Stat5b (Stat5b 40C) would inhibit reconstituted TSLP signal ated signaling cascade that may be involved in activating Stat5. transduction in the HepG2 system (20). For these experiments, SOCS-1 (also known as SSI-1, JAB, and TIP3) has been shown to HepG2 cells were cotransfected with cDNAs encoding TSLP-R, inhibit members of both the Jak and Tec families of protein ty- IL-7R␣, and Stat5b. In addition, increasing amounts of the dom- rosine kinases and is involved in the negative feedback regulation inant-negative Stat5b construct, or vector alone, were included. As of the Jak/Stat pathway (24–27). As shown in Fig. 5B, expression shown in Fig. 4B, expression of dominant-negative Stat5b inhib- of SOCS-1 abolished TSLP-mediated induction of CAT activity in ited TSLP-mediated signaling in a dose-dependent manner. To- ␣ gether, these data support the premise that the TSLP receptor com- HepG2 cells cotransfected with TSLP-R, IL-7R , Stat5b, and plex is composed of the TSLP-R and IL-7R␣ subunits and that SOCS-1. This result, combined with our findings on the lack of Jak TSLP requires Stat5 for effective gene induction. kinase involvement in TSLP-mediated signaling (Ref. 5 and Fig. 5A), implicates a kinase that is inhibited by SOCS-1, such as a Tec Protein tyrosine kinase involvement in TSLP-mediated signal family kinase and/or a novel Jak kinase, in the TSLP-signaling transduction pathway. Conversely, overexpression of Csk, a negative regulator TSLP receptor engagement results in tyrosine phosphorylation, of the Src family kinases (31, 40), had no appreciable effect on and subsequent activation, of Stat5 in the absence of concomitant TSLP receptor signaling, suggesting that in this assay Src family Jak kinase activation (5). To confirm and extend these findings, we kinases are not involved (Fig. 5B). To further test for the involve- used the HepG2 receptor reconstitution system to overexpress ki- ment of a Tec family kinase, we cotransfected a kinase-deficient nase-deficient versions of Jak1 or Jak2 (two Jaks that are endog- version (TecKM) of the founding family member Tec (23). Over- enously expressed in HepG2 cells; Ref. 22). As shown in Fig. 5A, expression of TecKM decreased TSLP-mediated signaling, thereby overexpression of inactive Jak1 was unable to inhibit TSLP-me- further implicating Tec or a similar protein tyrosine kinase in the diated signaling, thus providing further evidence that Jak1 is not a TSLP signaling pathway (Fig. 5C). The Journal of Immunology 5975 Downloaded from

FIGURE 5. Overexpression of SOCS-1 or a kinase-deficient version of Tec (TecKM) inhibited TSLP-mediated signal transduction whereas overexpression of kinase-deficient Jak1 (dnJAK1) or of Csk had no appreciable effect on TSLP-mediated signaling. HepG2 cells were transiently cotransfected with plasmids encoding the required components for TSLP-mediated induction of the cytokine-inducible HRRE (TSLP-R, IL-7R␣, and Stat5b). At the same time, cells were cotransfected with vector alone or plasmids encoding dnJAK1, the Jak and Tec family protein tyrosine kinase inhibitor SOCS-1, the Src http://www.jimmunol.org/ family kinase inhibitor Csk, or an inactive form of the protein tyrosine kinase Tec. The DNA concentrations (micrograms per milliliter of transfection solution) used for each construct are indicated. A, Overexpression of kinase-deficient Jak1 had no effect on TSLP-mediated signaling although it was able to partially inhibit OSM- and IL-7-mediated signaling. B, In TSLP-treated cells, SOCS-1 was able to inhibit CAT activity in a dose-dependent manner and therefore activation of the HRRE. Conversely, overexpression of the Src family kinase inhibitor Csk had no appreciable effect on TSLP-induced CAT activity. C, Overexpression of TecKM partially inhibited TSLP-mediated signaling but had no effect on OSM signal transduction.

Discussion and erythropoietin activate the transcription of both genes, remains

to be determined (17, 33, 34). Recently it has become apparent that by guest on September 28, 2021 The results presented in this paper extend our ongoing analysis of a number of factors, in addition to Stat tyrosine phosphorylation, TSLP and IL-7 signal transduction. Previous work demonstrated can regulate the degree and specificity of Stat-regulated transcrip- that both cytokines can stimulate thymocytes and mature T cells, tion (reviewed in Ref. 42). These include Stat serine phosphory- sustain the NAG8/7 cell line, and support B lymphopoiesis in long lation; the number, nucleotide composition, and spacing of Stat term cultures of fetal liver cells (3, 4). However, TSLP promotes ϩ ϩ binding sequences present in the promoters of target genes; hetero- development of B220 /IgM immature B cells whereas IL-7 fa- ϩ Ϫ vs homodimerization and tetramerization of the activated Stats; cilitates development only to the less mature B220 /IgM pre-B cell stage (5). The similarities in TSLP- and IL-7-mediated re- and the possible necessity of additional proteins. sponses may reflect the fact that the receptor complexes for both We have previously shown that Stat5 tyrosine phosphorylation these cytokines contain IL-7R␣. The differences may be due to the occurs in TSLP-treated cells without concomitant tyrosine phos- presence of receptor subunits unique to each complex: ␥c for the phorylation of any of the four known Jaks (5). Here, we have used IL-7 receptor (6, 7) and TSLP-R for the TSLP receptor (Ref. 5, overexpression of kinase-deficient versions of Jak1 and Jak2 to Fig. 4A).4 The relationship between TSLP and IL-7 is suggestive confirm and extend those findings. Clearly, TSLP-mediated sig- of that between IL-4 and IL-13. The functional IL-4 receptor com- naling activates Stat5, but it still remains to be answered how Stat5 plex includes the IL-4R ␣-chain and ␥c whereas the IL-13 receptor gets phosphorylated. Although there are numerous examples of complex includes the unique IL-13R plus the IL-4R ␣-chain. IL-4 cytokine stimulation leading to Jak-mediated Stat activation (re- and IL-13 have overlapping, as well as distinct, biochemical and viewed in Ref. 11), there are only a few reports suggesting Jak- biological activities (9). A similar story is emerging for TSLP independent Stat activation. For example, Stat activation following and IL-7. stimulation with epidermal growth factor requires intrinsic kinase Here we have shown that TSLP, like IL-7, functionally activates activation of the epidermal growth factor receptor but does the Stat5 transcription factor. Specifically, TSLP or IL-7 treatment not require Jak1 phosphorylation (43). Moreover, Saharinen et al. of the NAG8/7 cell line induces Stat5 tyrosine phosphorylation (5) have shown that expression in COS cells of Bmx, a member of the and DNA binding activity (Fig. 1). Moreover, treatment with these Tec tyrosine kinase family, induces activation of endogenous cytokines results in transcription of CIS and activation of a Stat5- Stat1, Stat3, and Stat5 without the activation of endogenous Jak responsive reporter gene. Although TSLP and IL-7 both induce kinases (44). transcription of CIS, they do not induce transcription of OSM, The ability of Bmx to induce Stat activation without concomi- another Stat5-responsive gene (Fig. 2 and 3). Thus, although Stat5 tant Jak activation is of particular interest considering our findings activation results in expression of CIS and OSM in Ba/F3 cells that overexpression of SOCS-1, a negative regulator of Jak and (41), our results with TSLP and IL-7 demonstrate that Stat5 acti- Tec protein kinases (24–27), or a kinase-deficient version of the vation is not sufficient for OSM expression. Exactly why TSLP protein tyrosine kinase Tec (23) inhibited TSLP-mediated signal and IL-7 induce CIS but not OSM transcription, while IL-2, IL-3, transduction whereas overexpression of Csk, a negative regulator 5976 REQUIREMENT FOR Stat5 IN TSLP-MEDIATED SIGNAL TRANSDUCTION of Src family kinases (31, 40), had no appreciable effect on TSLP 9. Callard, R. E., D. J. Matthews, and L. Hibbert. 1996. IL-4 and IL-13 receptors: signaling. In addition, the Src family kinase inhibitor PP1 was are they one and the same? Immunol. Today 17:108. 10. Mui, A., A. Muto, K. Sakamaki, N. Sato, T. Kinoshita, S. Watanabe, T. Yokota, unable to inhibit TSLP-mediated Stat5 tyrosine phosphorylation K. Arai, and A. Miyajima. 1994. Function of the common ␤ subunit of the (S. D. Levin, unpublished observations). Together, these results GM-CSF/IL-3/IL-5 receptors. Adv. Exp. Med. Biol. 365:217. suggest the possibility that a Tec, and not a Src, family kinase is 11. Leonard, W. J., and J. J. O’Shea. 1998. JAKS and STATS: biological implica- tions. Annu. Rev. Immunol. 16:293. involved in TSLP-mediated Stat5 activation. 12. Karasuyama, H., and F. Metchers. 1988. Establishment of mouse cell lines which Using the HepG2 receptor reconstitution system, we have also constitutively secrete large quantities of interleukins 2, 3, 4, or 5, using modified shown that TSLP signaling requires Stat5 and cannot utilize Stat1 cDNA expression vectors. Eur. J. Immunol. 18:97. 13. Knowles, B. B., C. C. Howe, and D. P. Aden. 1980. Human hepatocellular car- and Stat3. Although these experiments were not performed in lym- cinoma cell lines secrete the major plasma proteins and hepatitis B surface an- phocyte or lymphocyte progenitor cells, results in HepG2 cells tigen. Science 209:497. examining IL-2, IL-4, IL-6, IL-7, and GCSF signal transduction 14. Lord, J. D., B. C. McIntosh, P. D. Greenberg, and B. H. Nelson. 1998. The IL-2 receptor promotes proliferation, bcl-2 and bcl-x induction, but not cell viability have been faithfully recapitulated in lymphocytes (6, 18, 45). In through the adapter molecule Shc. J. Immunol. 161:4627. addition, we detected TSLP-mediated CIS induction using the 15. Paquette, R. L., M. R. Minosa, M. C. Verma, S. D. Nimer, and H. P. Koeffler. HepG2 receptor reconstitution system (unpublished observations), 1995. An interferon-␥ activation sequence mediates the transcriptional regulation ␥ which is consistent with the results in NAG8/7 pre-B cells pre- of the IgG Fc receptor type IC gene by interferon- . Mol. Immunol. 32:841. 16. Verdier, F., R. Rabionet, F. Gouilleux, C. Beisenherz-Huss, P. Varlet, O. Muller, sented herein. Since our results suggest that TSLP cannot utilize P. Mayeux, C. Lacombe, S. Gisselbrecht, and S. Chretien. 1998. A sequence of Stat1 or Stat3 and since there was no detectable decrease in the the CIS gene promoter interacts preferentially with two associated STAT5A dimers: a distinct biochemical difference between STAT5A and STAT5B. Mol. number of thymocytes or peripheral B cells in mutant mice lacking Cell. Biol. 18:5852. both Stat5a and Stat5b (although the ability of IL-7 to induce bone 17. Yoshimura, A., M. Ichihara, I. Kinjyo, M. Moriyama, N. G. Copeland, Downloaded from marrow colony formation was compromised; Ref. 46), one might D. J. Gilbert, N. A. Jenkins, T. Hara, and A. Miyajima. 1996. Mouse oncostatin M: an immediate early gene induced by multiple cytokines through the JAK- predict that TSLP will be found unnecessary for B lymphopoiesis. STAT5 pathway. EMBO J. 15:1055. Alternatively, TSLP may be discovered to signal through an ad- 18. Morella, K. K., C. F. Lai, S. Kumaki, N. Kumaki, Y. Wang, E. M. Bluman, ditional, non-Stat pathway. B. A. Witthuhn, J. N. Ihle, J. Giri, D. P. Gearing, D. Cosman, S. F. Ziegler, D. J. Tweardy, S. P. Campos, and H. Baumann. 1995. The action of interleukin-2 The functional TSLP receptor complex is composed of TSLP-R receptor subunits defines a new type of signaling mechanism for hematopoietin

and IL-7R␣. Engagement of this complex activates Stat5 and in- receptors in hepatic cells and fibroblasts. J. Biol. Chem. 270:8298. http://www.jimmunol.org/ duces CIS transcription through a novel mechanism. Our data sug- 19. Prowse, K., and H. Baumann. 1988. Hepatocyte-stimulating factor, ␤ 2 interferon, and interleukin-1 enhance expression of the rat ␣ 1-acid glycoprotein gene gest Stat5 activation may be through a Tec family kinase. Identi- via a distal upstream regulatory region. Mol. Cell Biol. 8:42. fication of the kinase that phosphorylates Stat5 and identification 20. Ripperger, J. A., S. Fritz, K. Richter, G. M. Hocke, F. Lottspeich, and G. H. Fey. of the other components of TSLP-mediated signal transduction 1995. Transcription factors Stat3 and Stat5b are present in rat liver nuclei late in an acute phase response and bind interleukin-6 response elements. J. Biol. Chem. remain challenges for future studies. 270:29998. 21. Lai, C.-F., J. Ripperger, K. K. Morella, Y. Wang, D. P. Gearing, N. D. Horseman, S. P. Campos, G. H. Fey, and H. Baumann. 1995. STAT3 and STAT5B are Acknowledgments targets of two different signal pathways activated by hematopoietin receptors and control transcription via separate cytokine response elements. J. Biol. Chem 270:

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