Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival This information is current as of September 25, 2021. Hui-Chen Chen, John C. Byrd and Natarajan Muthusamy J Immunol 2006; 176:2208-2218; ; doi: 10.4049/jimmunol.176.4.2208 http://www.jimmunol.org/content/176/4/2208 Downloaded from References This article cites 66 articles, 36 of which you can access for free at: http://www.jimmunol.org/content/176/4/2208.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 25, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Differential Role for Cyclic AMP Response Element Binding Protein-1 in Multiple Stages of B Cell Development, Differentiation, and Survival1 Hui-Chen Chen,† John C. Byrd,*§¶ and Natarajan Muthusamy2*†‡¶ CREB-1 is expressed in the bone marrow and in developing B cells. To determine the role of CREB-1 in developing B cells in the bone marrow, several lines of transgenic (Tg) mice overexpressing a dominant-negative Ser119-ala phosphomutant CREB-1 in the bone marrow were generated. Analysis of RNA and protein revealed expression of the transgene in the bone marrow. Flow cytometric analysis of bone marrow cells from Tg mice revealed ϳ70% increase in pre-B1 (CD43؉B220؉CD24؉(int)) and ϳ60% decreased pre-BII (CD43؉B220؉CD24؉؉(high)) cells, indicating a developmental block in pre-BI to pre-BII transition. Consistent with this, the Tg mice showed ϳ4-fold decrease in immature and mature B cells in the bone marrow. RT-PCR analysis of RNA from Tg mice revealed increased JunB and c-Jun in pre-BII cells associated with decreased S-phase entry. Adoptive transfer of bone marrow cells into Downloaded from -RAG-2؊/؊ mice resulted in reconstitution of non-Tg but not Tg bone marrow-derived CD43؉B220؉CD24high population that is nor mally absent in RAG-2؊/؊ mice. In the periphery, the Tg mice exhibited decreased CD21dimCD23highIgM؉ follicular B cells in the spleen and increased B1a and B1b B cells in the peritoneum. While exhibiting normal Ab responses to T-independent Ags and primary response to the T-dependent Ag DNP-keyhole limpet hemocyanin, the Tg mice exhibited severely impaired secondary Ab responses. These studies provide the first evidence for a differential role for CRE-binding proteins in multiple stages of B cell development, functional maturation, and B1 and B2 B cells. The Journal of Immunology, 2006, 176: 2208–2218. http://www.jimmunol.org/ yclic AMP response element binding protein-1 belongs Activation of resting murine B and T cells through antigen re- to the basic leucine zipper family of transcription factors. ceptors leads to the phosphorylation of CREB-1 at critical Ser133, C CREB-1 binds to CRE, which is composed of which is essential for the transcriptional activation function of the TGANNTCA sequences as homo- and heterodimers in association protein (22–24). Stimulation through the BCR resulted in a dose- with members of the CREB/activating transcription factor (ATF)3 dependent induction of CREB-1-binding activity that is subjected family of proteins, including ATF-1 and cAMP response element to negative regulation by IFN-␥ in a STAT-1-dependent manner modulator, as well as AP-1 family members such as c-Jun (1–5). (25). Consistent with a role for CREB-1 in the regulation of cell 133 The activation-induced phosphorylation of CREB-1 at Ser (or growth, the expression of immediate early growth response genes, by guest on September 25, 2021 Ser119) in an alternatively spliced form increased the DNA-binding such as c-fos and junB, and cell cycle regulatory genes, such as the activity of the protein in some but not other cell types (4–7). Phos- proliferating cell nuclear Ag (PCNA), is regulated by Ser133-phos- phorylation at Ser133 activates CREB-1, at least in part, by facil- phorylated CREB-1 through direct binding and activation of the itating its binding to CREB-binding protein (CBP). This, in turn, promoter region of these genes (26–30). interacts with and activates the components of basal transcriptional A requirement for CREB-1 in mature B cell survival is impli- machinery (8–13). Multiple signaling pathways, including cAMP- cated by the CREB-1-dependent induction of the bcl-2 gene in responsive protein kinase A, protein kinase C, calcium/calmodu- human B cell lines and transgenic (Tg) mice (31–33). Ser133-phos- lin-dependent CaM kinases II and IV, and a Ras-dependent serine/ phorylated CREB-1 binds to the CRE element in promoter regions threonine kinase RSK2, mediate phosphorylation and activation of of antiapoptotic genes such as Bcl-2 and Mcl-1 and regulate their CREB-1 in different cell types (14–22). expression. CREB-1Ϫ/Ϫ mice die shortly after birth, thus preclud- ing the analysis of the immune function of mature B cells (34). The lack of a strong phenotype in some CREB family member knock- *Division of Hematology and Oncology, Department of Internal Medicine, †Molec- ular Virology, Immunology and Medical Genetics, ‡Veterinary BioSciences, §Divi- out studies suggested the functional redundancy among the mem- sion of Medicinal Chemistry, College of Pharmacy, and ¶OSU Comprehensive Can- bers of this family (35). To determine the in vivo role of CRE- cer Center, The Ohio State University, Columbus, OH 43210 binding proteins in early B cell development and functional Received for publication April 28, 2005. Accepted for publication October 18, 2005. maturation, we generated four independent Tg mice lines overex- The costs of publication of this article were defrayed in part by the payment of page pressing a dominant-negative phosphomutant CREB-1 in devel- charges. This article must therefore be hereby marked advertisement in accordance oping B cells in the bone marrow. Characterization of these Tg with 18 U.S.C. Section 1734 solely to indicate this fact. mice revealed a critical role for CRE binding proteins in different 1 This work was supported by grants from the American Cancer Society (to N.M.), Leukemia and Lymphoma Society of America (to J.C.B.), and The D. Warren Brown stages of B cell development and functional maturation. Foundation (to J.C.B. and N.M.). H.-C.C. is a recipient of a Raymon E. Mason Foundation Fellowship for graduate research. 2 Address correspondence and reprint requests to Dr. Natarajan Muthusamy, Chronic Materials and Methods Lymphocytic Leukemia, Experimental Therapeutics Laboratory, Division of Hema- Animals tology and Oncology, 1132C, James Cancer Hospital, 300 West 10th Avenue, Co- lumbus, OH 43210. E-mail address: [email protected] Mutant CREB-1-Tg animals described were generated at the OSU CCC/CCRI 3 Abbreviations used in this paper: ATF, activating transcription factor; CBP, CREB- transgenic facility. The Tg expression vector pBH was constructed by clon- binding protein; PCNA, proliferating cell nuclear Ag; Tg, transgenic; KLH, keyhole ing the EcoRI fragment of pEPB splice-neo (a gift from Drs. W. Muller and limpet hemocyanin; TNP, trinitrophenyl; NTg, nontransgenic; mCRE, mutant CRE K. Rajewsky, Institute for Genetics, University of Cologne, Cologne, Ger- oligonucleotide; HPRT, hypoxanthine phosphoribosyltransferase. many) containing the Ig H chain promoter and intronic enhancer and the Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 The Journal of Immunology 2209 SV40 splice and poly(A) into the EcoRI site of pBluescript KS II(ϩ). The moved by purification in G50 Sephadex spin columns. The binding reactions dominant-negative CREB-1 cDNA was produced by PCR-mediated mu- were conducted, at room temperature, with 3 ␮g of nuclear extract, 30,000 tagenesis of the human ⌬CREB-1 cDNA at Ser119 (Ser119 to Ala) (30). The dpm (0.1–0.5 ng) of radiolabeled oligonucleotide probe, in 5ϫ Ficoll-binding hCREB-1Ser119-Ala was cloned into the BglII site of the pBH Tg vector. buffer (10 mM Tris (pH 7.5), 1 mM DTT, 1 mM EDTA, and 4% Ficoll), 250 Four independent founder lines were identified by PCR using primers 5Ј- ng of poly(deoxyinosinic-deoxycytidylic acid) in 75 mM KCl and double- Ј Ј ␮ AAAACCACTTCTTCAAACCACAGC-3 and 5 -CTGCTGGAGAA distilled H2O to make the volume to 15 l. The DNA-protein complexes were GAAGGGACATC-3Ј corresponding to the Ig H chain intronic enhancer fractionated by electrophoresis in 4% nondenaturing polyacrylamide gel, run and the Ig H chain promoter, which amplified ϳ700 bp fragment in the Tg in 0.25ϫ Tris-borate-EDTA, at 4°C. The gel was then dried on 3M Whatman tail DNA. The lines were further confirmed by Southern blot analysis. paper and subjected to autoradiography. In vitro transcribed and translated Recombination activating gene-2-deficient (Rag-2Ϫ/Ϫ) mice were obtained CREB-1 protein generated using rabbit reticulolysate system was used as a originally from Dr. F. Alt (Harvard Medical School, Boston, MA). Female positive control (30). FVB/N mice were obtained from Harlan Sprague Dawley.