The usage of alternative splice sites in Mus musculus synaptotagmin-like 2 gene is modulated by cyclosporin A and FK506 in T-lymphocytes.

Cyclosporin-A and FK506 block the calcineurin activity preventing the transcription of genes sharing NFAT-like binding sequences in their promoter region. We presently show that activation of murine T-cells in presence of these immunosuppressors results in the up-regulation of the synaptotagmin-like 2 gene. However, of the four known isoforms, only mRNAs encoding the a and b isoforms accumulate. Two previously undected isoforms, each characterized by the retention of an intron, were found. The first, Slp2-e, includes exon 8, intron 8 and exon 9. The second, Slp2-f, is composed of exon 7, intron 7 and exon 8. Slp2-f has an open reading frame coding for a putative protein of 1229 amino acids sharing 47% identities with the human breast-associated antigen, SGA-72 M. In addition to the well-documented modulation of gene transcription, the two immunosuppressors also play a role in the choice of alternative splice sites on murine Slp2 pre-mRNA.

T lymphocyte activation initiates the degradation of the CD62L encoding mRNA and increases the transcription of the corresponding gene.

Following T-cell activation, CD62L, a member of the selectin family of cell adhesion molecules, is proteolytically cleaved by a constitutive endoprotease and subsequently re-expressed. To define whether the cleavage regulates CD62L gene transcription, we have analyzed the outcome of T-cell activation on the level of CD62L gene transcription and mRNA stability. Here, we report that CD62L shedding correlates with the concomitant upregulation of CD62L gene transcription and the rapid degradation of the corresponding mRNA. Novel protein synthesis is not required for CD62L gene upregulation, mRNA degradation or protein shedding. The three events are insensitive to cyclosporin A (CSA) and, thus, do not depend on the calcineurin signaling pathway. Activation of T cells in presence of a metallo-protease inhibitor, that protects CD62L shedding, does not prevent CD62L gene upregulation or mRNA degradation. In contrast induction of CD62L shedding by the chemically-induced dissociation of calmodulin from the CD62L cytosolic tail, in absence of T-cell activation, has no consequences on the levels of CD62L gene transcription or mRNA accumulation. These data demonstrate that the transcriptional and post-transcriptional events are exclusively regulated by T-cell activation and not by the CD62L density on cell membrane.

Characterization of a gene encoding two isoforms of a mitochondrial protein up-regulated by cyclosporin A in activated T cells.

Cyclosporin A (CSA) is an immunosuppressor used in organ transplantation. A recent proteomic analysis has revealed that activation of T cells in the presence of CSA induces the synthesis of hundreds of new proteins. Here we used representational difference analysis to characterize some of the corresponding induced genes. After cDNA bank screening we focused on one of these genes, which we named CSA-conditional, T cell activation-dependent (CSTAD) gene. This gene produces two mRNAs resulting from alternative splicing events. They encode two proteins of 104 and 141 amino acids, CSTADp-S and CSTADp-L, for the short and long forms, respectively. FK506 had the same effect as CSA, whereas rapamycin did not affect the level of CSTAD gene expression, demonstrating that inhibition of the calcineurin activation pathway is involved in CSTAD gene up-regulation. CSA also led to overexpression of CSTAD in mice immunized in the presence of CSA, confirming the in vitro analysis. Microscopic and cytofluorimetric analysis of cells expressing green fluorescent protein-tagged CSTADp-L and CSTADp-S showed that both proteins colocalize with mitochondrial markers and depolarize the mitochondrial transmembrane potential without causing release of cytochrome c, apoptosis, or necrosis. Both CSTADp isoforms are sensitive to proteinase K, implying that they are located in the mitochondrial outer membrane. These data reveal a new mechanism of action for CSA, which involves up-regulation of a gene whose products are sorted to mitochondria and depolarize the mitochondrial membrane.

New aspects of cyclosporin a mode of action: from gene silencing to gene up-regulation.

Cyclosporin A (CSA) has transformed clinical transplantation, both in term of success and of quality-of-life of the patient. Studies aimed to unfold the site of CSA action have shown that this molecule binds to cytosolic proteins of the cyclophilin family. CSA:cyclophilin complexes have a high affinity for calcineurin, a key enzyme in T-cell activation. By blocking the calcineurin activity, CSA prevents the induction of genes encoding for cytokines and their receptors. Thus, humoral and cellular immune responses are abolished, this resulting in the successful graft acceptance. Disappointingly, CSA and the other molecules as FK506, sharing the capacity to inhibit calcineurin, should be administered for all patient life, as tolerance to alloantigens is not achieved by these molecules. The long term utilization of this class of immunosuppressors increases the incidence of different tumors. The finding that CSA does not interfere with various biochemical pathways has prompted different groups to analyze a possible effect of CSA on molecules that might be involved in different functions of the immune response and/or in tumorogenesis. A new picture of CSA mode of action is emerging in which the immunosuppressor prevents the transcription of a group of genes, concomitantly inducing the transcription of another set. Here, we review the data and discuss the consequences of these new findings in term of T-cell activation mechanisms.

Cyclosporin A therapy differently affects immunological-relevant gene expression following immunization.

We have studied the effect of cyclosporin A (CSA) on the expression of genes involved in the immune response in mice bearing a transgenic T cell receptor specific for the peptide 88-104 of the pigeon cytochrome c. Immunization of mice treated with CSA resulted in the blockade of the IL2, IFN-gamma, CXCR-5, CCR-5 and CD25 gene transcription. CSA decreased the density of CD69 on T-cells but did not interfere with the induction of the chemokine receptors CCR-1, CCR-4, CXCR2 and CXCR-4. Finally, CSA accelerated the kinetics of CD44 and CD62L expression or re-expression and increased the density of both markers on T-cell membrane. The present data show that priming in presence of CSA resulted in the acquisition of a particular phenotype by the activated T-cells.