DNA microarray gene expression profile of T cells with the splice variants of TCRzeta mRNA observed in systemic lupus erythematosus

J Immunol. 2006 Jan 15;176(2):949-56. doi: 10.4049/jimmunol.176.2.949.

Abstract

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.

MeSH terms

  • 3' Untranslated Regions
  • Alternative Splicing*
  • Animals
  • Base Sequence
  • CD3 Complex / genetics
  • Cell Adhesion Molecules
  • DNA, Complementary / genetics
  • Down-Regulation
  • Gene Expression Profiling
  • Granzymes
  • Humans
  • Hybridomas / immunology
  • Hybridomas / metabolism
  • Lupus Erythematosus, Systemic / genetics*
  • Lupus Erythematosus, Systemic / immunology*
  • Membrane Glycoproteins / metabolism
  • Membrane Proteins / genetics*
  • Mice
  • Mutation
  • Nectins
  • Oligonucleotide Array Sequence Analysis
  • Polymerase Chain Reaction
  • Proteoglycans / metabolism
  • RNA, Messenger / genetics*
  • RNA, Messenger / metabolism*
  • Receptors, Antigen, T-Cell / genetics*
  • Serine Endopeptidases / metabolism
  • Syndecan-1
  • Syndecans
  • T-Lymphocytes / immunology*
  • Transfection

Substances

  • 3' Untranslated Regions
  • CD3 Complex
  • Cell Adhesion Molecules
  • DNA, Complementary
  • Membrane Glycoproteins
  • Membrane Proteins
  • Nectin2 protein, mouse
  • Nectins
  • Proteoglycans
  • RNA, Messenger
  • Receptors, Antigen, T-Cell
  • SDC1 protein, human
  • Sdc1 protein, mouse
  • Syndecan-1
  • Syndecans
  • antigen T cell receptor, zeta chain
  • Granzymes
  • Serine Endopeptidases
  • GZMA protein, human