DNA microarray gene expression profile of T cells with the splice variants of TCRzeta mRNA observed in systemic lupus erythematosus.
We have reported that the TCRzeta mRNA with alternatively spliced 3' UTR (zeta mRNA/as-3'-untranslated region (UTR)) and zeta mRNA lacking exon 7 (zeta mRNA/exon 7-) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both zeta and TCR/CD3 complexes. To determine whether these T cells expressing decreased zeta exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type zeta cDNA, zeta cDNA/as-3' UTR, and zeta cDNA/exon 7- into murine T cell hybridoma MA5.8 cells which lack zeta expression to construct the MA5.8 mutants WT, AS3' UTR, and EX7-, respectively. FACS analyses demonstrated reduced cell surface expression of zeta and TCR/CD3 complexes on the AS3' UTR mutant and the EX7- mutant in comparison to that on the WT mutant. Total RNA was collected after stimulating the MA5.8 mutants with anti-CD3 Ab. Reverse-transcribed cDNA was applied to the mouse cDNA microarray containing 8691 genes, and the results were confirmed by real-time PCR. The results showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-beta2, were down-regulated in both the AS3' UTR mutant and the EX7- mutant. Another 16 genes were up-regulated in both, and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus receptor-related 2, syndecan-1, and granzyme A. Increased protein expression of these genes was confirmed by Western blot and FACS analyses. Identification of these responsive genes in T cells in which the zeta and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.
Tsuzaka
Kensei
K
Division of Rheumatology, Department of Internal Medicine, Saitama Medical Center, Saitama Medical School, Saitama, Japan. kentsu@saitama-med.ac.jp
Nozaki
Kyoko
K
Kumazawa
Chika
C
Shiraishi
Kiyono
K
Setoyama
Yumiko
Y
Yoshimoto
Keiko
K
Suzuki
Katsuya
K
Abe
Tohru
T
Takeuchi
Tsutomu
T
D020413:3' Untranslated Regions; D017398:Alternative Splicing; D000818:Animals; D001483:Base Sequence; D017252:CD3 Complex / Q000235:genetics; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary / Q000235:genetics; D015536:Down-Regulation; D020869:Gene Expression Profiling; D053804:Granzymes; D006801:Humans; D006825:Hybridomas / Q000276:immunology / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000235:genetics* / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D009154:Mutation; D000075983:Nectins; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D011509:Proteoglycans / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012697:Serine Endopeptidases / Q000378:metabolism; D053668:Syndecan-1; D053667:Syndecans; D013601:T-Lymphocytes / Q000276:immunology*; D014162:Transfection
D020413:3' Untranslated Regions; D017252:CD3 Complex; D015815:Cell Adhesion Molecules; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C000619016:Nectin2 protein, mouse; D000075983:Nectins; D011509:Proteoglycans; D012333:RNA, Messenger; D011948:Receptors, Antigen, T-Cell; C508241:SDC1 protein, human; C508242:Sdc1 protein, mouse; D053668:Syndecan-1; D053667:Syndecans; C054979:antigen T cell receptor, zeta chain; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human