Transcriptional induction of endothelial nitric oxide gene by cyclosporine A. A role for activator protein-1

7 p.-7 fig.We have previously shown that the immunosuppressant cyclosporine A (CsA) increases the activity, the protein level, and the steady-state levels of the mRNA of the endothelial nitric-oxide synthase (eNOS) gene in bovine aortic endothelial cells (BAEC). We have now investigated the mechanisms responsible for these effects. Preincubation with an inhibitor of RNA polymerase II abolished CsA-induced eNOS up-regulation. Nuclear run-on experiments demonstrated a 1.6-fold increase in the induction of eNOS gene by CsA. In agreement with these results, transient transfections showed that CsA augmented the transactivation of the eNOS promoter. Electrophoretic mobility shift assays showed an increase in the activator protein-1 (AP-1) DNA binding activity in BAEC treated with CsA. An increase in the level of c-fos mRNA and in the nuclear content of c-Fos protein was detected in BAEC treated with CsA. Site-directed mutagenesis of the AP-1 cis-regulatory element in the context of the human eNOS promoter resulted in the abrogation of the induction mediated by CsA. Hence, up-regulation ofeNOS mRNA by CsA is a transcriptional phenomenon involving the proximal AP-1 site in the 5′-regulatory region of the human eNOS gene. Furthermore, our data exemplify how immunosuppressive drugs may result in the regulation of specific genes involved in the homeostasis of endothelial function, such aseNOS. Whereas cyclosporine A (CsA)1 still stands as a cornerstone therapy for immunosuppression, its use encompasses serious side effects among which nephrotoxicity and hypertension are often encountered. The pathogenesis of their appearance has been investigated in clinical studies and animal models, but a complete understanding is still elusive. The partial reversibility of these effects suggests that the underlying disturbance has a functional component. Acute nephrotoxicity is associated with renal vasoconstriction and a reduced glomerular filtration rate. A significant amount of data support that the vasoconstrictor peptide, endothelin-1, is involved in this response (1, 2). Furthermore, transforming growth factor β (TGF-β) has been proposed as a link between CsA-enhanced endothelin synthesis and many of the pathological fibrotic lesions observed with chronic CsA toxicity (3). Although endothelin and other paracrine vasoconstrictors have been identified as mediators of the hypertensive response (1, 4, 5) associated with CsA toxicity, a less clear picture has emerged regarding the potential dysfunction of thel-arginine-NO-cGMP pathway. Several reports have described that there is a defect in the relaxation of blood vessels in response to endothelial agonists, but the precise step of diminished NO synthesis, enhanced degradation, or receptor abnormality has not been established (6-8). In previous work, using endothelial cells in culture treated with CsA, we found that NO synthesis is not only not reduced but moderately enhanced (9). These observations have been also confirmed in rats (10), in healthy volunteers, in whom venous antecubital infusion of CsA significantly decreased forearm blood flow (11), and recently in patients treated with CsA after heart transplantation (12). These data indicate that nitric oxide may constitute an important regulatory mechanism that protects against CsA-associated vasoconstriction in vivo. In support of this hypothesis we found that the expression of the eNOS gene is increased in bovine aortic endothelial cells (BAEC) treated with CsA (12-14). We have now investigated the mechanisms by whicheNOS is up-regulated by CsA.This work was supported by Grant SAF-97-0035 from the Comisión Interministerial de Ciencia y Tecnologia, Grant BMH4-CT96-0979 from European Union Program BIOMED-2, and Grant 08.4/0032/1998 from Comunidad Autonoma de Madrid.Peer reviewe