RESUMO
We have demonstrated that T-cell receptor zeta (zeta) mRNA with a 562-bp deleted alternatively spliced 3'-untranslated region (3'UTR) observed in T cells of patients with systemic lupus erythematosus (SLE) can lead to a reduction in zeta and TCR/CD3 (J. Immunol., 2003 & 2005). To determine the region in zeta mRNA 3'UTR for the regulation of zeta, zeta mRNA with 3'UTR truncations ligated into pDON-AI was used to infect murine T-cell hybridoma MA5.8 cells, which do not contain zeta. As a Western blot analysis demonstrated the importance of the regions from +871 to +950, containing conservative sequence 1 (CS1), and +1070 to +1136, containing CS2, for the production of zeta, we constructed MA5.8 mutants carrying zeta mRNA 3'UTR with deletions of these regions (DeltaCS1 and DeltaCS2 mutants). Western blot and FACS analyses showed significant reduction in the cell surface zeta and TCR/CD3 in both these mutants, and IL-2 production was decreased, compared with MA5.8 cells transfected with wild-type zeta mRNA. Furthermore, real-time PCR demonstrated the instability of zeta mRNA with 3'UTR deletions in these MA5.8 mutants. In conclusion, CS1 and CS2 may be responsible for the regulation of zeta and TCR/CD3 through the stability of zeta mRNA in SLE T cells.
Assuntos
Regiões 3' não Traduzidas/genética , Sequência Conservada/genética , Proteínas de Membrana/genética , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/fisiologia , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , CamundongosRESUMO
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown etiology. Tyrosine phosphorylation and protein expression of the T-cell receptor zeta chain (zeta) have been reported to be significantly decreased in SLE T cells. In addition, zeta mRNA with alternatively spliced 3' untranslated region (zetamRNA/as-3'UTR) is detected predominantly in SLE T cells, and aberrant zeta mRNA accompanied by the mutations in the open reading frame including zeta mRNA lacking exon7 (zetamRNA/exon7-) is observed in SLE T cells. These zeta mRNA splice variant forms exhibit a reduction in the expression of TCR/CD3 complex and zeta protein on their cell surface due to the instability of zeta mRNA splice variant forms as well as the reduction in interleukin (IL)-2 production after stimulating with anti-CD3 antibody. Data from cDNA microarray showed that 36 genes encoding cytokines and chemokines, including IL-2, IL-15, IL-18, and TGF-beta2, were down-regulated in the MA5.8 cells transfected with the zeta mRNA splice variant forms. Another 16 genes were up-regulated and included genes associated with membranous proteins and cell damage granules, including the genes encoding poliovirus-receptor-related 2, syndecan-1, and granzyme A.
Assuntos
Processamento Alternativo/genética , Lúpus Eritematoso Sistêmico/genética , Mutação , RNA Mensageiro/genética , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T , Regiões 3' não Traduzidas/genética , Regiões 3' não Traduzidas/imunologia , Processamento Alternativo/imunologia , Animais , Linhagem Celular , Citocinas/genética , Citocinas/imunologia , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Éxons/genética , Éxons/imunologia , Humanos , Lúpus Eritematoso Sistêmico/imunologia , Lúpus Eritematoso Sistêmico/patologia , RNA Mensageiro/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
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.
Assuntos
Processamento Alternativo , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/imunologia , Proteínas de Membrana/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Regiões 3' não Traduzidas , Animais , Sequência de Bases , Complexo CD3/genética , Moléculas de Adesão Celular , DNA Complementar/genética , Regulação para Baixo , Perfilação da Expressão Gênica , Granzimas , Humanos , Hibridomas/imunologia , Hibridomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos , Mutação , Nectinas , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Proteoglicanas/metabolismo , Serina Endopeptidases/metabolismo , Sindecana-1 , Sindecanas , TransfecçãoRESUMO
The integrin alpha(E)beta(7) is expressed on intestinal intraepithelial T lymphocytes and CD8(+) T lymphocytes in inflammatory lesions near epithelial cells. Adhesion between alpha(E)beta(7)(+) T and epithelial cells is mediated by the adhesive interaction of alpha(E)beta(7) and E-cadherin; this interaction plays a key role in the damage of target epithelia. To explore the structure-function relationship of the heterophilic adhesive interaction between E-cadherin and alpha(E)beta(7), we performed cell aggregation assays using L cells transfected with an extracellular domain-deletion mutant of E-cadherin. In homophilic adhesion assays, L cells transfected with wild-type or a domain 5-deficient mutant formed aggregates, whereas transfectants with domain 1-, 2-, 3-, or 4-deficient mutants did not. These results indicate that not only domain 1, but domains 2, 3, and 4 are involved in homophilic adhesion. When alpha(E)beta(7)(+) K562 cells were incubated with L cells expressing the wild type, 23% of the resulting cell aggregates consisted of alpha(E)beta(7)(+) K562 cells. In contrast, the binding of alpha(E)beta(7)(+) K562 cells to L cells expressing a domain 5-deficient mutant was significantly decreased, with alpha(E)beta(7)(+) K562 cells accounting for only 4% of the cell aggregates, while homophilic adhesion was completely preserved. These results suggest that domain 5 is involved in heterophilic adhesion with alpha(E)beta(7), but not in homophilic adhesion, leading to the hypothesis that the fifth domain of E-cadherin may play a critical role in the regulation of heterophilic adhesion to alpha(E)beta(7) and may be a potential target for treatments altering the adhesion of alpha(E)beta(7)(+) T cells to epithelial cells in inflammatory epithelial diseases.