To understand the effects of the immunosuppressant cyclosporin A (CsA) on Ca 2+ -mediated intracellular signalling pathways in human peripheral blood mononuclear cells (PBMCs), we investigated its effects on the activity profiles of mitogen-activated protein kinase (MAPK) cascades. PBMCs, or subpopulations thereof, were simultaneously stimulated with a phorbol ester and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. In these primary human cells, CsA significantly inhibited PMA/ionomycinmediated and ionomycin-mediated activation of the MAPK kinase MKK6, as well as its downstream kinases SAPK2a (p38a) and MAPKAP-K2. PMA/ionomycin treatment also mediated activation of SAPK1 (JNKs) which was inhibited by CsA. Treatment with ionomycin alone also resulted in CsA-sensitive activation of SAPK1. With regard to transcription factors targeted by the Ca 2+ -induced MAPK signalling network, we found CsA to inhibit the ionomycin-mediated phosphorylation of ATF2 at Thr71. We identified the heterodimeric transcription factor ATF2/CREB as constitutively binding to the essential cAMP response element (CRE) site within the Ca 2+ -regulated DNA polymerase b promoter and contributing to the activation of this promoter. Our data implicate ATF2 phosphorylation status as a nuclear sensor within PBMCs that monitors converging intracellular Ca 2+ -signalling pathways.

Alterations in gene expression may represent an underlying cause of undesired side-effects mediated by the immunosuppressant cyclosporin A (CsA). We employed the method of differential display PCR to identify new genes whose expression is modulated by CsA. Human peripheral blood mononuclear cells (PBMCs), or subpopulations thereof, were simultaneously stimulated with the phorbol ester 4b-phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. We identify the gene encoding the DNA repair enzyme DNA polymerase b (Pol b) as a novel CsA-sensitive transcription unit. Our data show that transcription of pol b mRNA is induced by Ca 2+ and that CsA significantly inhibits PMA/ionomycin-and ionomycin-mediated upregulation of both pol b mRNA and Pol b protein. The CsA-mediated inhibition of pol b upregulation is maintained for at least 21 h after gene activation and is exerted via the phosphatase calcineurin. FK506, another immunosuppressant that targets calcineurin, also inhibits pol b upregulation, while rapamycin competes with FK506 action. This work identifies Ca 2+ as an inducer of pol b gene activity in primary blood cells. The demonstrated CsA sensitivity of this process suggests a novel molecular mechanism that may contribute to the increased tumor incidence in patients receiving CsA treatment.Keywords: cyclosporin A; immunosuppression; DNA repair; Ca 2+ -mediated signal transduction.The immunosuppressant cyclosporin A (CsA) is a first choice drug for treating patients receiving allograft transplantation. The molecular mechanism by which CsA exerts its immunosuppressive function has been attributed to its inhibition of the phosphatase calcineurin [1,2]. CsA binds to a cyclophilin, forming a CsA±cyclophilin complex, which subsequently acts to inhibit the Ca 2+ -dependent protein phosphatase calcineurin. Active calcineurin dephosphorylates, for example, the cytosolic transcription factor nuclear factor of activated T-cells (NF-AT) [3], allowing its translocation into the nucleus and modulation of gene transcription. CsA affects the transcription of many genes, including interleukin-2, interleukin-3, granulocyte macropage colony stimulating factor and tumor necrosis factor-a (reviewed in [4]). We used the method of differential display [5] to find hitherto unidentified genes, whose expression in primary human cell populations (i.e. peripheral blood mononuclear cells; PBMCs) is affected by CsA.We demonstrate the ability of CsA to inhibit both the induced transcription and protein expression of the DNA polymerase b (pol b) gene. Pol b is the smallest (39 kDa, 339 amino acids) of the five known eukaryotic cellular DNA polymerases and is primarily involved in processive gap-filling syntheses associated with base excision repair of small (6 nucleotide) DNA gaps [6]. Pol b is the major enzyme in a base excision repair pathway responsible for correction of DNA damage induced by depurination [7]; formation of apurinic/apyrimidinic sites ranks ...