Characterization of a Gene Encoding Two Isoforms of a Mitochondrial Protein Up-regulated by Cyclosporin A in Activated T Cells

Abstract

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.

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

“…7). Accumulation of the Slp2-a, -b, -e, and -f isoforms is observed in presence of CSA or FK506, but not of rapamycin suggesting a role of calcineurin in gene activation, as already reported (Mascarell et al, , 2004, and in the choice of alternative splicing sites.…”

“…We have previously reported that activation of T-cells in presence of CSA promotes the transcription of many genes among which we identified the Slp2 gene (Mascarell et al, 2004). The alternative splicing of Slp2 mRNA gives rise to four species of mRNAs sharing common exons, on one hand, and an exclusive set of individual exons on the other .…”

“…We have recently identified by representational differential analyses (RDA) some of the corresponding genes (Mascarell et al, 2004). Among these, we have detected the expression of the synaptotagmin-like 2 gene (Slp2) (Mascarell et al, 2004).…”

“…The sequencing of the Slp2 clone originating from the RDA experiments (Mascarell et al, 2004) showed that the sequence is located between exon 8 and exon 9 and, therefore, it is not associated to any of the four Slp2 isoforms previously detected in brain . In order to analyze whether the transcript extended to the flanking exons, RT-PCR were carried out using different couples of primers located in intron 8 (group 1), exon 8 and intron 8 (group 2) and intron 8 and exon 9 (group 3), respectively ( Fig.…”

“…Several studies have revealed that activation of T-cells in the presence of CSA induces the synthesis of IL6 (Williamson et al, 1994), TGF-␤ (Prashar et al, 1995), LAT (linker for T-cell activation) (Peters et al, 2000) and of hundreds of other proteins in vitro and in vivo Mascarell and Truffa-Bachi, 2002;Mascarell and Truffa-Bachi, 2003). We have recently identified by representational differential analyses (RDA) some of the corresponding genes (Mascarell et al, 2004). Among these, we have detected the expression of the synaptotagmin-like 2 gene (Slp2) (Mascarell et al, 2004).…”