A Splice Variant of the TCR ζ mRNA Lacking Exon 7 Leads to the Down-Regulation of TCR ζ, the TCR/CD3 Complex, and IL-2 Production in Systemic Lupus Erythematosus T Cells

Abstract

The reduction or absence of TCR ζ-chain (ζ) expression in patients with systemic lupus erythematosus (SLE) is thought to be a factor in the pathogenesis of SLE. We previously reported a splice variant of ζ mRNA that lacks the 36-bp exon 7 (ζ mRNA/exon 7(−)) and is accompanied by the down-regulation of ζ protein in T cells from SLE patients. In this study, we show that EX7− mutants (MA5.8 cells deficient in ζ protein that have been transfected with ζ mRNA/exon 7(−)) exhibit a reduction in the expression of TCR/CD3 complex and ζ protein on their cell surface as well as a reduction in the production of IL-2 after stimulation with anti-CD3 Ab, compared with that in wild-type (WT) mutants (MA5.8 cells transfected with the WT ζ mRNA). Furthermore, real-time PCR analyses demonstrated that ζ mRNA/exon 7(−) in EX7− mutants was easily degraded compared with ζ mRNA by the WT mutants. Pulse-chase experiment showed ζ protein produced by this EX7− mutants was more rapidly decreased compared with the WT mutants. Thus, the lower stability of ζ mRNA/exon 7(−) might also be responsible for the reduced expression of the TCR/CD3 complex, including ζ protein, in SLE T cells.

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

“…Also the decreased expression of certain genes could simply reflect reduced Ag-receptor mediated signaling intensity. We have already reported significantly reduced IL-2 production after anti-CD3 Ab treatment in both of these AS3Ј UTR and EX7Ϫ mutants (11,12). Our results in the study presented here showed downregulation not only of IL-2 but also of other ILs (IL-13, IL-15, and IL-18), chemokines (CCL3, CCL5, CCL9, XCL1), and a chemokine receptor (CCR7) in both the AS3Ј UTR and EX7Ϫ mutants, indicating that down-regulation of may lead to aberrant signal transduction followed by reduced expression of these cytokines and chemokines.…”

“…Expression of in the PBT of cancer patients or in tumor-infiltrating T cells has also been reported to be decreased (20 -23), and, interestingly, cytokine expression of IL-2 and IL-4 in these T cells has been observed to be down-regulated. DNA microarray analyses of the gene expression profiles of SLE patient T cells has revealed down-regulation of several TCRs, such as TCR␣␤, TCR␦, and TCR␥ (24 -26), indicating reduced expression of the TCR/CD3 complex in SLE patient T cells, the same as observed in the AS3Ј UTR and EX7Ϫ mutants (11)(12).…”

“…Moreover, we and other groups have demonstrated that an aberrant form of the mRNA 3Ј-untranslated region (3Ј UTR), which is alternatively spliced and 562 bp shorter than the WT 3Ј UTR, is predominantly expressed in SLE T cells ( mRNA/as-3Ј UTR) (9,10). Predominant expression of these two mRNA splice variant forms ( mRNA/as-3Ј UTR and mRNA/exon 7Ϫ) leads to down-regulation not only of but of the other TCR/CD3 components because of the instability of these mRNA splice variant forms (11)(12)(13). Several molecules corresponding to the TCR costimulatory pathway have been reported to be up-regulated in SLE T cells and play crucial roles in the pathogenesis of SLE.…”

“…Several molecules corresponding to the TCR costimulatory pathway have been reported to be up-regulated in SLE T cells and play crucial roles in the pathogenesis of SLE. To identify the genes encoding membranous proteins or TCR costimulatory molecules that are induced secondarily by the expression of the mRNA splice variant forms, we used a cDNA microarray analysis to compare the gene expression profile of an AS3Ј UTR mutant (MA5.8 cells (murine T cell hybridoma lacking of ) transfected with mRNA/as-3Ј UTR) (11) and an EX7Ϫ mutant (MA5.8 cells transfected with mRNA/exon 7Ϫ) (12) with a WT mutant (MA5.8 cells transfected with wild-form mRNA) (11,12). The results showed up-regulation of membrane proteins, including syndecan-1 (Sdc1) and poliovirus receptor-related 2 (Pvrl2), and down-regulation of ILs and chemokines, in both the EX7Ϫ and AS3Ј UTR mutants.…”

Novel molecular targets in the treatment of systemic lupus erythematosus

“…The reduction of the ζ chain levels, along with the reciprocal increase in the expression of FcRγ, is directly responsible for some of the phenotypic abnormalities characteristic of lupus T cells [10]. Accordingly, replenishment of the ζ chain leads to the down regulation of the FcRγ chain and subsequently to the correction of the TCR/CD3-induced increased calcium response and the abnormal phosphorylation of cellular substrates [11].…”