Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center This information is current as of September 28, 2021. Zeljka Korade Mirnics, Eva Caudell, YanHua Gao, Kazuhiko Kuwahara, Nobuo Sakaguchi, Tomohiro Kurosaki, Joan Burnside, Károly Mirnics and Seth J. Corey J Immunol 2004; 172:4133-4141; ; doi: 10.4049/jimmunol.172.7.4133 Downloaded from http://www.jimmunol.org/content/172/7/4133 References This article cites 56 articles, 31 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/172/7/4133.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 Microarray Analysis of Lyn-Deficient B Cells Reveals Germinal Center-Associated Nuclear Protein and Other Genes Associated with the Lymphoid Germinal Center1 Zeljka Korade Mirnics,2* Eva Caudell,† YanHua Gao,* Kazuhiko Kuwahara,‡§ Nobuo Sakaguchi,‡ Tomohiro Kurosaki,¶ Joan Burnside,ʈ Ka´roly Mirnics,2# and Seth J. Corey2*† Lyn is the only member of the Src family expressed in DT40 B cells, which provide a unique model to study the singular contribution of this protein tyrosine kinase (PTK) family to cell signaling. In these cells, gene ablation of Lyn leads to defective B cell receptor signaling. Complementary DNA array analysis of Lyn-deficient DT40 cells shows that the absence of Lyn leads to down-regulation of numerous genes encoding proteins involved in B cell receptor signaling, proliferation, control of transcription, Downloaded from immunity/inflammation response, and cytoskeletal organization. Most of these expression changes have not been previously as- sociated with Lyn PTK signaling. They include alterations in mRNA levels of germinal center-associated nuclear protein (germinal center-associated DNA primase) (GANP), CD74, CD22, NF-␬B, elongation factor 1␣, CD79b, octamer binding factor 1, Ig H chain, stathmin, and ␥-actin. Changes in GANP expression were also confirmed in Lyn-deficient mice, suggesting that Lyn PTK has a unique function not compensated for by other Src kinases. Because Lyn-deficient mice have impaired development of germinal http://www.jimmunol.org/ centers in spleen, the decreased expression of GANP in the Lyn-deficient DT40 cell line and Lyn-deficient mice suggests that Lyn controls the formation and proliferation of germinal centers via GANP. GANP promoter activity was higher in wild-type vs Lyn-deficient cells. Mutation of the PU.1 binding site reduced activity in wild-type cells and had no effect in Lyn-deficient cells. The presence of Lyn enhanced PU.1 expression in a Northern blot. Thus, the following new signaling pathway has been described: Lyn3PU.13GANP. The Journal of Immunology, 2004, 172: 4133–4141. timulation of the B cell receptor (BCR)3 activates signal- Tec, and Csk families (3–6). BCR evoked tyrosine phosphoryla- ing cascades that lead to proliferation, differentiation, and tion of Lyn, and Syk triggers a biochemical cascade in which a S effector functions (1–3). BCR activation recruits members variety of cellular proteins become tyrosine phosphorylated (3, 7). by guest on September 28, 2021 of nonreceptor protein tyrosine kinases (PTKs) of the Src, Syk, Although native B cell express several Src family PTKs (Lyn and Blk), the chicken B cell DT40 expresses only Lyn (8). Src PTKs are believed to have overlapping expression and redundant *Department of Pediatrics, University of Pittsburgh, School of Medicine, Children’s function (9). However, because Lyn is the only Src PTK family Hospital of Pittsburgh, Pittsburgh, PA 15213; †Division of Pediatrics, University of Texas-M. D. Anderson Cancer Center, Houston, TX 77030; ‡Department of Immu- member expressed in DT40 cells, this cell line provides a unique nology, Graduate School of Medical Sciences, Kumamoto University School of Med- experimental system to study Lyn’s role in B cell signaling. A icine, Honjo, Kumamoto, Japan; §PRESTO, Japan Science and Technology Corpo- unique feature of DT40 cells is their ability to undergo efficient ration, Kawaguchi, Japan; ¶Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan; ʈDelaware Biotechnology homologous recombination, so that more than several dozen genes Institute, University of Delaware, Newark, DE 19711; and #Departments of Psychi- have been inactivated and resulting lines intensively studied (10). atry and Neurobiology, PittArray Microarray Core, University of Pittsburgh, Pitts- burgh, PA 15261 Lyn-deficient DT40 cells show a defective BCR cross-linking re- sponse, manifested by slow calcium mobilization and profoundly Received for publication July 7, 2003. Accepted for publication February 2, 2004. reduced phosphorylation/activation of Syk (11). Lyn-deficient The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance cells also show increased sensitivity to apoptosis; however, precise with 18 U.S.C. Section 1734 solely to indicate this fact. molecular deficiencies have not yet been associated with the Ϫ/Ϫ 1 Z.K.M. was supported by the Caligiuri Fund of the Pittsburgh Foundation, the Lupus Lyn phenotype. Foundation, the Bear Necessities Pediatric Cancer Foundation, and Children’s Hos- Lyn-deficient mice have a dramatic phenotype that resembles pital of Pittsburgh Research Advisory Committee. J.B. is supported by U.S. Depart- ment of Agriculture Grant NRICGP 98-35205-6640. K.M. is supported by National systemic lupus erythematosus in humans (12, 13). The B cells of Institutes of Health Grant R21MH/NS62760-01. S.J.C. is supported by National In- these mice show reduced response to BCR cross-linking or LPS stitutes of Health K02HL03794 and R29CA74422, and American Cancer Society Grant DHP-135. activation (14). With age, these mice develop high serum IgM levels, lymphadenopathy, and splenomegaly (15). In contrast, de- 2 Address correspondence and reprint requests to Dr. Seth J. Corey, Division of Pe- diatrics, University of Texas-M. D. Anderson Cancer Center, 1515 Holcombe Bou- ficiency of other Src PTK family members expressed predomi- levard, Houston, TX 77030; or Dr. Zeljka Korade Mirnics, Department of Pediatrics, nantly within B cells (Blk, Hck, Fgr, and Lck) (16–18) does not University of Pittsburgh, School of Medicine, Children’s Hospital of Pittsburgh, Pitts- burgh, PA 15213. Address questions regarding the microarray to Dr. Karoly Mirnics, produce a prominent phenotype (reviewed in Ref. 19). Although Departments of Psychiatry and Neurobiology, PittArray Microarray Core, University Lyn-deficient mice show normal B cell development within the of Pittsburgh, Pittsburgh, PA 15261. E-mail addresses: [email protected], bone marrow, the number of peripheral B cells in spleen, mesen- [email protected], [email protected] teric lymph nodes, and Peyer’s patches is reduced (15). Lyn defi- 3 Abbreviations used in this paper: BCR, B cell receptor; PTK, protein tyrosine ki- nase; GANP, germinal center-associated nuclear protein (germinal center-associated ciency is also associated with an increased number of immature B DNA primase); BDE, balanced differential expression; EF1␣, elongation factor 1␣. cells in the periphery (12, 15). Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 4134 Lyn-DEPENDENT EXPRESSION OF GANP Recent advances in functional genomics highlighted the advan- activity. Reused membranes did not show sensitivity differences between tages of investigating complex gene expression patterns for the subsequent hybridizations (data not shown). discovery of novel signaling and disease-related pathways (20– Image acquiring and analysis. TIF files were analyzed using GeneSight 23). To determine putative downstream effectors associated with 4.0 software (BioDiscovery, Marina Del Rey, CA). After defining the Ϫ/Ϫ metagrid and subgrids, the spot pattern was inspected and manually ad- Lyn signaling in B cells, we compared Lyn and wild-type justed for pin performance and membrane stretch. For each probe spot, avian DT40 cells using custom-made chicken cDNA membrane mean/median signal intensity and mean/median local background measure- arrays (23). The analysis revealed a set of genes involved in B cell ments were performed. Spots were considered for further analysis if they development and signaling. One of these is germinal center-asso- produced a signal from Ͼ40% of the spot area. Array measurements were imported into Excel 2000 (Microsoft, Redmond, WA) and GeneSpring 4.1 ciated nuclear protein (germinal center-associated DNA primase) (Silicon Genetics, Redwood City, CA)