Gene structure and chromosomal assignment of mouse GITR, a member of the tumor necrosis factor/nerve growth factor receptor family.

GITR is a type I transmembrane protein that belongs to the tumor necrosis factor/nerve growth factor receptor (TNF/NGFR) family. This receptor is preferentially expressed in activated T lymphocytes and may function as signaling molecule during T-cell development. In the present study, we examined the genomic organization of the entire mouse GITR (mGITR) gene. The gene spans a 2543-bp region and consists of five exons (with a length ranging from 88 bp to 395 bp) and four introns (67 bp to 778 bp). In agreement with GITR expression in activated T cells, consensus elements for transcription factors involved in T-cell development and activation were identified in the 5' flanking region, including a consensus element for NF-kappaB. Two highly significant binding sites for MyoD and one binding site for myogenin were also found, suggesting involvement of GITR in muscle development. The mGITR gene contains 17 transcription initiation sites distributed over a 76-bp region, all used with the same frequency. We localized mGITR to the murine chromosome 4 (E region), where other 4 TNF/NGFR members localize, including m4-1BB and mOX40. These results further indicate that GITR shares several features with OX40, 4-1BB, and CD27, suggesting the existence of a new subfamily of the TNFR family, as also confirmed by the similarity of their cytoplasmic domains.

Short-term dexamethasone treatment modulates the expression of the murine TCR zeta gene locus.

Glucocorticoids (GCH) are highly effective agents in controlling inflammation and immune response. We studied the effect of the synthetic GCH dexamethasone (DEX) on the expression of TCR zeta gene splicings that code for some chains belonging to the T-cell receptor (TCR)/CD3 complex. In the DEX-treated hybridoma T-cell line 3DO, TCR zeta gene splicings increase within the first 24 hr (about fourfold increase), as demonstrated by reverse transcriptase-polymerase chain reaction and RNase protection assay. This increase is due to the stimulation of TCR zeta gene locus transcription, as demonstrated by the "run-on" assay. A similar upregulation was observed in murine thymocytes following in vivo DEX treatment. As a consequence of TCR zeta gene locus modulation, the expression of the spliced mRNAs coding for TCR zeta and TCR eta subunits is increased, whereas their relative ratio is only slightly changed. Indeed, the amount of TCR zeta protein in 24-hr DEX-treated cells is fivefold more than that in the untreated cells. A similar effect was seen in 3DO cells treated with hydrocortisone but not in those treated with testosterone. TCR zeta protein increase was confined to the cytoplasm and therefore TCR/CD3 complex expression did not increase. This newly described effect of DEX may constitute an additional molecular mechanism that contributes to its immunomodulating activity.

TCR kappa, a new splicing of the murine TCR zeta gene locus, is modulated by glucocorticoid treatment.

T-cell receptor (TCR) is a multichain receptor in which the TCRzeta subunit is important for membrane assembly and signal transduction. Four alternative splicings of the murine TCRzeta gene locus have been previously described. We here describe a new alternative splicing of murine TCRzeta gene, TCRkappa, cloned by RT-PCR, that is encoded by exons 1-7, a portion of exon 9 and the whole exon 10 of TCRzeta gene. The protein encoded by TCRkappa mRNA is identical to that encoded by TCReta mRNA, because the stop codon is present in the exon 9 before splicing with exon 10. RNAse protection assays carried out on total RNA from thymocytes indicate that TCRkappa mRNA is 1 half with respect to TCReta mRNA, suggesting that TCRkappa mRNA contributes to determine the TCReta protein levels. The 3' untranslated region of TCRkappa mRNA is different from that of TCReta and this might lead to different t(1/2) for each species in vivo. We also show that dexamethasone (DEX), a synthetic glucocorticoid hormone, increases the amount of TCRkappa in the hybridoma T-cell line 3DO (about 5-fold increase), as indicated by reverse transcriptase-polymerase chain reaction (RT-PCR) and RNAse protection assays. This newly described effect of DEX may constitute a further molecular mechanism that contributes to its immunomodulating activity.

A new member of the tumor necrosis factor/nerve growth factor receptor family inhibits T cell receptor-induced apoptosis.

By comparing untreated and dexamethasone-treated murine T cell hybridoma (3DO) cells by the differential display technique, we have cloned a new gene, GITR (glucocorticoid-induced tumor necrosis factor receptor family-related gene) encoding a new member of the tumor necrosis factor/nerve growth factor receptor family. GITR is a 228-amino acids type I transmembrane protein characterized by three cysteine pseudorepeats in the extracellular domain and similar to CD27 and 4-1BB in the intracellular domain. GITR resulted to be expressed in normal T lymphocytes from thymus, spleen, and lymph nodes, although no expression was detected in other nonlymphoid tissues, including brain, kidney, and liver. Furthermore, GITR expression was induced in T lymphocytes upon activation by anti-CD3 mAb, Con A, or phorbol 12-myristate 13-acetate plus Ca-ionophore treatment. The constitutive expression of a transfected GITR gene induced resistance to anti-CD3 mAb-induced apoptosis, whereas antisense GITR mRNA expression lead to increased sensitivity. The protection toward T cell receptor-induced apoptosis was specific, because other apoptotic signals (Fas triggering, dexamethasone treatment, or UV irradiation) were not modulated by GITR transfection. Thus, GITR is a new member of tumor necrosis factor/nerve growth factor receptor family involved in the regulation of T cell receptor-mediated cell death.