Identification of novel targets of immunosuppressive agents by cDNA-based microarray analysis.

Anthony D Cristillo,Barbara E Bierer

doi:10.1074/jbc.m108598200

Abstract

The immunosuppressive agents cyclosporin A (CsA) and tacrolimus (FK506) bind to unrelated intracellular immunophilin receptors, cyclophilin (CyP) and FK506-binding protein (FKBP), respectively. The complexes of CsA-CyP and of FK506-FKBP both bind to and inhibit the activity of the calcium/calmodulin-dependent serine/threonine phosphatase calcineurin. We used cDNA microarray analysis to characterize early human peripheral blood T cell transcriptional responses following antigen receptor stimulation in the absence or presence of CsA or FK506, hoping to identify novel targets dependent upon calcineurin or immunophilins or, perhaps, specific targets of either CyP or FKBP inhibitable by one drug alone. The array data failed to identify genes uniquely sensitive to only one drug, suggesting that transcriptionally regulated, immunophilin-dependent but calcineurin-independent targets fell below the limits of detection in this system. In contrast, transcript profiling identified and mRNA and protein analysis confirmed novel as well as known genes reproducibly induced or inhibited by both immunosuppressive agents. In this context, we show that transcriptional activation of Stat5a and repression of the cytokine interleukin-16 are regulated by T cell receptor engagement and dependent upon drug-immunophilin complexes and, presumably, calcineurin activity.

Highlights

Hydrolysis of inositol 3,4,5-trisphosphate that leads inter alia to calcium (Ca2ϩ) mobilization (9 –11)
An increase in intracellular Ca2ϩ, together with calmodulin, activates calcineurin (phosphatase 2B or PP2B [12]), a serine/threonine phosphatase that is required for the activation and nuclear translocation of a number of transcription factors [13,14,15,16] including nuclear factor of activated T cells (NFAT) [17, 18]; NFAT activity regulates the transactivation of a number of cytokine and other genes, including interleukin (IL)-2, IL-3, IL-4, IL-12, inflammatory mediators (e.g. TNF␣), and growth factors (19 –22)
Inhibition of Gene Expression in Activated Human Peripheral Blood T Cells by Immunosuppressive Agents—To identify transcriptional events regulated coordinately or differentially by the immunosuppressive agents cyclosporin A (CsA) and FK506, we compared resting and activated human peripheral blood T cells cultured in the presence or absence of either vehicle or drug

Summary

The abbreviations used are

TcR, T cell receptor; NFAT, nuclear factor of activated T cells; IL, interleukin; CsA, cyclosporin A; FK506, tacrolimus; CyP, cyclophilin; FKBP, FK506-binding protein; TNF␣, tumor necrosis factor ␣; PBL, peripheral blood T lymphocytes; PBS, phosphate-buffered saline; mAb, monoclonal antibody; PMA, phorbol 12-myristate 13-acetate; RT-PCR, reverse transcriptase-PCR; STAT, signal transducers and activators of transcription; ActD, actinomycin D; EST, expressed sequence tag. We failed to identify transcripts regulated by only one immunosuppressive agent, suggesting that immunophilin-dependent, calcineurin-independent gene expression was below the limits of detection of our analysis. Analysis of mRNA transcription and protein in both CD4ϩ and CD8ϩ T cell subpopulations, used to verify and extend these cDNA microarray results, demonstrated both induction of Stat5a and inhibition of IL-16, genetic elements not previously appreciated to be affected by either CsA or FK506. Hierarchy profiles of these target genes suggested relative specificity of these drugs

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION