Inhibition of Cyclic AMP Response Element-Binding Protein/Cyclic AMP Response Element-Mediated Transcription by the Immunosuppressive Drugs Cyclosporin A and FK506 Depends on the Promoter Context GERO SIEMANN, ROLAND BLUME, DANIELA GRAPENTIN, ELKE OETJEN, MARKUS SCHWANINGER 1 , and WILLHART KNEPEL Department of Molecular Pharmacology, University of Go ¨ ttingen, Go ¨ ttingen, Germany Received October 26, 1998; accepted February 22, 1999 This paper is available online at http://www.molpharm.org ABSTRACT The immunosuppressants cyclosporin A and FK506 (tacroli- mus) can block the phosphatase calcineurin, thereby inhibiting gene transcription directed by the cyclic AMP (cAMP)- and calcium-responsive transcription factor, cAMP response ele- ment (CRE)-binding protein, and its binding site, CRE, in vari- ous cell lines. This action is a novel molecular mechanism of cyclosporin A and FK506 action. Because inhibition of CREB/ CRE-directed transcription by cyclosporin A and FK506 has previously been observed by using synthetic minienhancers, reporter fusion genes were constructed to examine the effect of cyclosporin A and FK506 on the transcriptional activity of CRE- containing natural promoters. In transient transfection experi- ments, cyclosporin A and FK506 inhibited the transcriptional activation by cAMP and the membrane depolarization of three CRE-containing promoters. However, cyclosporin A and FK506 failed to inhibit the activation by cAMP of another promoter, the rat insulin I gene promoter. The lack of cyclosporin A/FK506 sensitivity is not intrinsic to the insulin CRE because cyclo- sporin A and FK506 inhibited the activation by cAMP of the insulin CRE when isolated and used as a synthetic minien- hancer. Rather, cyclosporin A/FK506 resistance may be con- ferred by specific promoter interactions because a mutational analysis of the insulin promoter revealed that inside this pro- moter, CRE activity depends on an adjacent control element. These data show that cyclosporin A and FK506 can inhibit CRE activity when the CRE resides in its natural promoter. However, the cyclosporin A/FK506 sensitivity depends on the specific promoter context. The results suggest that cyclosporin A and FK506 may alter target tissue function through the regulation of a subset of CRE-containing genes. Cyclosporin A and FK506 (tacrolimus) are clinically impor- tant immunosuppressive drugs that are widely used to pre- vent graft rejection after organ transplantation. The intro- duction of cyclosporin A in the field of organ transplantation in the early 1980s resulted in extraordinary improvements of graft survival, and cyclosporin A has become a first-choice drug for patients with allograft organs. In addition, cyclo- sporin A is used in the therapy of an increasing number of autoimmune diseases. However, the therapeutic application of cyclosporin A and FK506 is limited by untoward effects that are shared by both drugs, including nephrotoxicity, hy- pertension, neurotoxicity, and impaired glucose tolerance (European FK506 Multicenter Liver Study Group, 1994; U.S. Multicenter FK506 Liver Study Group, 1994). The structur- ally unrelated drugs bind to their respective intracellular receptors, the immunophilins. These drug/immunophilin complexes directly target the calcium/calmodulin-dependent phosphatase calcineurin, thereby blocking its activity (Ho et al., 1996). To date, all of the therapeutic effects, as well as the toxic effects, of these drugs have been shown to be due to inhibition of calcineurin. Inhibition of calcineurin blocks the translocation of the cytosolic component of the nuclear factor of activated T cells (NFAT) into the nucleus, resulting in a failure to activate the genes regulated by the NFAT tran- scription factor, including those necessary for T cell prolifer- ation, such as interleukin 2 (Ho et al., 1996; Rao et al., 1997; Ru ¨ hlmann and Nordheim, 1997). Inhibition of NFAT-di- rected transcription may be important for the repression of early steps in T cell activation and, thus, immunosuppression induced by cyclosporin A and FK506 (Ho et al., 1996; Rao et al., 1997; Ru ¨ hlmann and Nordheim, 1997). However, other targets of calcineurin are likely to play a role in this process. This work was supported by Deutsche Forschungsgemeinschaft Grant SFB402/A3. 1 Present address: Department of Neurology, University of Heidelberg, Hei- delberg, Germany. ABBREVIATIONS: CAT, chloramphenicol acetyltransferase; CRE, cAMP response element; CREB, CRE-binding protein; NFAT, nuclear factor of activated T cells; bp, base pair. 0026-895X/99/061094-07$3.00/0 Copyright © The American Society for Pharmacology and Experimental Therapeutics All rights of reproduction in any form reserved. MOLECULAR PHARMACOLOGY, 55:1094 –1100 (1999). 1094 at ASPET Journals on December 20, 2015 molpharm.aspetjournals.org Downloaded from