Leukocyte β7 Integrin Targeted by Krüppel-like Factors Melanie Alles, Gleb Turchinovich, Pumin Zhang, Wolfgang Schuh, Fabien Agenès and Jörg Kirberg This information is current as of September 29, 2021. J Immunol 2014; 193:1737-1746; Prepublished online 11 July 2014; doi: 10.4049/jimmunol.1302613 http://www.jimmunol.org/content/193/4/1737 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2014/07/11/jimmunol.130261 Material 3.DCSupplemental References This article cites 69 articles, 32 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/193/4/1737.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Leukocyte b7 Integrin Targeted by Kruppel-like€ Factors Melanie Alles,* Gleb Turchinovich,†,‡ Pumin Zhang,x Wolfgang Schuh,{ Fabien Agene`s,‖,#,1 and Jo¨rg Kirberg* Constitutive expression of Kruppel-like€ factor 3 (KLF3, BKLF) increases marginal zone (MZ) B cell numbers, a phenotype shared with mice lacking KLF2. Ablation of KLF3, known to interact with serum response factor (SRF), or SRF itself, results in fewer MZ B cells. It is unknown how these functional equivalences result. In this study, it is shown that KLF3 acts as transcriptional repressor for the leukocyte-specific integrin b7 (Itgb7, Ly69) by binding to the b7 promoter, as revealed by chromatin immuno- precipitation. KLF2 overexpression antagonizes this repression and also binds the b7 promoter, indicating that these factors may compete for target sequence(s). Whereas b7 is identified as direct KLF target, its repression by KLF3 is not connected to the MZ B cell increase because b7-deficient mice have a normal complement of these and the KLF3-driven increase still occurs when b7 is deleted. Despite this, KLF3 overexpression abolishes lymphocyte homing to Peyer’s patches, much like b7 deficiency does. Furthermore, KLF3 expression alone overcomes the MZ B cell deficiency when SRF is absent. SRF is also dispensable for the Downloaded from KLF3-mediated repression of b7. Thus, despite the shared phenotype of KLF3 and SRF-deficient mice, cooperation of these factors appears neither relevant for the formation of MZ B cells nor for the regulation of b7. Finally, a potent negative regulatory feedback loop limiting KLF3 expression is shown in this study, mediated by KLF3 directly repressing its own gene promoter. In summary, KLFs use regulatory circuits to steer lymphocyte maturation and homing and directly control leukocyte integrin expression. The Journal of Immunology, 2014, 193: 1737–1746. http://www.jimmunol.org/ ruppel-like€ factors (KLFs) are zinc finger family tran- KLF3 may act exclusively in concert with other transcriptional scription factors with 17 members described for mam- regulators. K malian cells (1). KLFs have a highly conserved DNA In lymphocytes, KLFs play important roles in their development binding domain comprised of three C2H2 zinc fingers near or at the and function (7, 9, 13–20). When constitutively expressed under C terminus and share similarity to the transcription factors of the the B cell–specific CD19 promoter, KLF3 increases maturation specificity protein family (2–5). The KLF zinc fingers are thought toward marginal zone (MZ) B cells, resulting in a 5- to 10-fold to mediate binding to CACCC elements and GC-rich DNA increase of this subset, whereas, accordingly, MZ B cells were sequences in target genes. reduced in KLF3-deficient mice (7, 9). Interestingly, absence of by guest on September 29, 2021 In Drosophila Schneider cells, KLF3 (BKLF) had been shown KLF2 in B cells results in an increase of MZ B cells, mimicking to act as a weak transcriptional activator (6). However, more re- the effect of constitutive KLF3 expression (15, 16, 19). Although cent data in murine cells implicate KLF3 as a repressor of tran- the actual target genes mediating this effect are unknown, it has scription (7–9). Indeed, KLF3 has been shown to interact with the been demonstrated that the lymphocyte phenotypes of KLF2- common transcriptional corepressors C-terminal binding protein 2 deficient mice result from effects on the positioning, recircula- (CtBP2) and four and a half LIM domain protein 3 (FHL3) (8, 10, tion, and/or tissue-homing ability of these cells (13, 14, 19, 21– 11). A further KLF3-interacting transcription factor is serum 24). However, not all outcomes of KLF2 deficiency resulted response factor (SRF), as identified in muscle cells (12). Thus, from cell-autonomous processes (18). Integrins perform crucial functions for the positioning of leu- kocytes by acting both as signaling and signaling-responsive ad- *Division of Immunology (3/3), Paul-Ehrlich-Institut, 63225 Langen, Germany; hesion molecules (25). The functions of the leukocyte-specific b2 †Department of Biomedicine, Laboratory of Developmental Immunology, 4058 Basel, x b Switzerland; ‡Basel University Children’s Hospital, 4031 Basel, Switzerland; Department (CD18) and 7 (Ly69) integrin subunits have been elucidated of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX initially by blocking studies and subsequently by the respective { 77030; Division of Molecular Immunology, Department of Internal Medicine III, gene-deficient mice. This led to a mouse model of leukocyte ad- Nikolaus-Fiebiger-Center, University of Erlangen-Nurnberg,€ 91054 Erlangen, Germany; ‖INSERM U743, Montreal, Quebec H2X 1P1, Canada; and #INSERM ADR Paris V Saint hesion deficiency (LFA-A immunodeficiency, OMIM 116920) and Anne, 75014 Paris, France specific defects in the migration of leukocytes to tissues such as 1Current address: Office for Science and Technology, Consulate General of France, skin and gut, respectively (26–31). Indeed, whereas leukocyte Los Angeles, CA. migration via the afferent lymph and within lymph nodes is not Received for publication September 30, 2013. Accepted for publication June 6, 2014. affected in the absence of all integrins, their extravasation is Address correspondence and reprint requests to Dr. Jo¨rg Kirberg, Paul-Ehrlich- crucially dependent on them (32). With respect to b7 integrin, this Institut, Division of Immunology (3/3), D-63225 Langen, Germany. E-mail address: subunit can either pair with a (CD49d) to form lymphocyte [email protected] 4 Peyer’s patch (PP) adhesion molecule-1 (a /b ), mediating bind- The online version of this article contains supplemental material. 4 7 ing to VCAM-1, mucosal addressin cell adhesion molecule 1 Abbreviations used in this article: ChIP, chromatin immunoprecipitation; CtBP2, a C-terminal binding protein 2; KLF, Kruppel-like€ factor; MAdCAM-1, mucosal (MAdCAM-1; addressin), and fibronectin, or pair with E (CD103), addressin cell adhesion molecule 1; MLn, mesenteric lymph node; MZ, marginal a heterodimer found particularly among intestinal intraepithelial zone; PP, Peyer’s patch; QRT-PCR, quantitative real-time PCR; RIPA, radioimmu- lymphocytes mediating binding to E-cadherin. Thus, b expres- noprecipitation assay; SRF, serum response factor. 7 sion is considered a hallmark of mucosal lymphocytes because Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 a4/b7 allows for binding to the high endothelial venules of gut www.jimmunol.org/cgi/doi/10.4049/jimmunol.1302613 1738 KLF2/3-REGULATING LEUKOCYTE b7 INTEGRIN PP and mesenteric lymph nodes (MLn), and endothelial cells of streptomycin (Life Technologies), b-ME (50 mM), Gln (4 mM), 0.03% the lamina propria, all specifically expressing MAdCAM-1 (33), (w/v) Primatone RL (Quest, 5x59051, 10-kDa ultrafiltrate) (52), 5 mg/ml insulin (Sigma-Aldrich I5500), and 2% FCS (unless indicated differently) and aE/b7 mediating the retention within the tissue by binding to at 5–7.5% CO2, 37˚C. E-cadherin, expressed in the gut epithelial layer. Whereas aE (CD103) only pairs with b7, a4 (CD49d) can pair with either b7 or Retroviral transduction b a b 1 (CD29), the latter termed VLA-4 ( 4/ 1, CD49d/CD29), medi- Retroviral vector particles were obtained by transient transfection of ret- ating binding to VCAM-1, MAdCAM-1, and fibronectin. Whereas roviral vector plasmid DNA into Plat-E producer cells (53). Briefly, the day 3 6 binding specificities thus overlap, the ratio of a4/b7 and a4/b1 de- prior to transfection, 5 10 Plat-E cells were plated in a T75 flask in 8– 10 ml medium (10% FCS). The next afternoon, chloroquine (25 mM final) termines lymphocyte-homing specificity because a4/b7 preferentially (54) was added, and 1 h later cells were transfected using calcium- binds to MAdCAM-1 and a4/b1 has higher affinity for VCAM-1 phosphate [to 1 ml 20 mg plasmid DNA in 0.295 M CaCl2, 1 ml 50 mM (34–36). The relative expression of these integrins is set transcrip- N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 280 mM tionally but also depends on intracellular pairing preferences. Ap- NaCl, 1.5 mM Na2HPO4 (pH 7.16) was added, gassed with air for 20 s, and parently, under nonstimulated conditions, the expression of a4 and, immediately the mixture was added to the cells] (55). The next morning, medium was exchanged. Virus particle-containing supernatants were taken particularly, b1 is limited, such that the preferentially associating a4/b1 a b 42, 50, 66, and 72 h after transfection.