DNA Microarray Gene Expression Profile of T Cells with the Splice Variants of TCRζ mRNA Observed in Systemic Lupus Erythematosus

Abstract

We have reported that the TCRζ mRNA with alternatively spliced 3′ UTR (ζ mRNA/as-3′-untranslated region (UTR)) and ζ mRNA lacking exon 7 (ζ mRNA/exon 7−) observed in systemic lupus erythematosus patient T cells can lead to down-regulation of both ζ and TCR/CD3 complexes. To determine whether these T cells expressing decreased ζ exhibit differential transcription patterns, we transfected retrovirus vectors containing wild-type ζ cDNA, ζ cDNA/as-3′ UTR, and ζ cDNA/exon 7− into murine T cell hybridoma MA5.8 cells which lack ζ expression to construct the MA5.8 mutants WT, AS3′ UTR, and EX7−, respectively. FACS analyses demonstrated reduced cell surface expression of ζ 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-β2, 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 ζ and TCR/CD3 complexes were down-regulated may help to better understand the pathogenesis of systemic lupus erythematosus.

Alterations in the expression of signal-transducing CD3ζ chain in T cells from patients with chronic inflammatory/autoimmune diseases

“…Among the up-regulated genes in T cell hybridoma MA5.8 cells transfected with CD3ζ chain mRNA splice-variant forms, the genes encoding cytotoxic molecules such as Gzma or MMP-11 were found. These proteins may be responsible for tissue damage [100]. Moreover, several molecules which account for lipid transport and metabolism, such as Pmm1, Lpn2, and Fads2, were also found to be increased in T cell hybridoma MA5.8 cells transfected with CD3ζ chain mRNA splice-variant forms.…”

“…Tsuzaka et al [100] examined the gene expression profiles of T cell hybridoma MA5.8 cells expressing only either the splice variant of CD3ζ chain mRNA lacking exon 7 or CD3ζ chain mRNA with the short spliced variant of 3'UTR. The results showed that the genes encoding cytokines such as IL-2, IL-13, IL-15, IL-18, and transforming growth factor-β2, chemokines (CCL3, CCL5, CCL9, XCL1), and a chemokine receptor (CCR7) were down-regulated in these cells.…”

The Divergence, Actions, Roles, and Relatives of Sodium-Coupled Bicarbonate Transporters

“…Two mutants of the T-cell receptor chain have been linked to SLE, and overexpression of these unstable mutants in murine T-cells is associated with an eightfold increase in NDCBE transcripts in these cells (983). The cause and effect of this upregulation remains to be studied, although it has been noted that apoptosis of thymocytes, T-cell precursors, is increased by cellular alkalinization, yet is inhibited by stilbene derivatives, consistent with a proapoptotic action of NDCBE (980).…”

Down-regulation of Fas-ligand mRNA in Sjögren's syndrome patients with enlarged exocrine glands

“…To validate gene expression changes, quantitative realtime PCR was performed as described previously [9] with an Applied Biosystems Prism 7000 Sequence Detection System using TaqMan universal PCR master mix according to the manufacturer's procedure (Applied Biosystems Inc.) for the FasL gene. The TaqMan probes and primers for FasL (assay identification number Hs00181225_m1) were TaqMan Gene Expression Assays products (Applied Biosystems).…”