Evidence That G a q-Coupled Receptor-Induced Interleukin-6 mRNA in Vascular Smooth Muscle Cells Involves the Nuclear Factor of Activated T Cells

The immunosuppressant cyclosporin A inhibits transcription mediated by the nuclear factor of activated T-cells (NFAT), a key regulator of cytokine gene expression in lymphocytes that integrates phospholipase C signaling. NFAT is also expressed in vascular smooth muscle cells, but the genes it regulates there are unknown. Here we show that Gaq-coupled P2Y nucleotide receptor signaling in rat vascular smooth muscle cells increases NFAT-mediated luciferase reporter expression. It also induces interleukin (IL)-6 gene expression but not other cytokine mRNAs including IL-1, IL-2, IL-3, IL-4, IL-10, g-interferon, tumor necrosis factor-a, or tumor necrosis factor-b. IL-6 mRNA induction by UTP is more rapid and transient then that caused by IL-1b stimulation and is partially blocked by cyclosporin A or by expression of a trans-dominant NFAT inhibitor. Expression of recombinant NFATc1 markedly augments IL-6 mRNA induction by these and other agonists, which is partially attributable to NFAT-regulated paracrine mediators. However, trans-dominant NFkB inhibitors strongly interfere with IL-6 mRNA induction both by IL-1b and by UTP, which synergistically evoke IL-6 mRNA expression. These findings suggest that NFAT is among the cofactors involved in NFkB-dependent IL-6 gene induction by Ca-mobilizing receptors in vascular smooth muscle cells. Cardiovascular toxicity is a prominent side effect of cyclosporin A (CsA) or FK506 immunosuppressive therapy. The mechanisms underlying these drug side effects are unknown, but they occur at doses that are necessary for effective immunosuppression (Sander and Victor, 1995). This is consistent with the hypothesis that both immunosuppression and toxicity are due to a common mechanism of drug action. Inhibition of transcription mediated by the nuclear factor (NF) of activated T-cells (NFAT) is widely thought to account for immunosuppression caused by CsA and FK506 (Ho et al., 1996). The Rel-related NFAT family of transcription factors are produced from five known genes and are best understood as regulators of immune cell cytokine gene expression (Rao et al., 1997). CsA and FK506 inhibit the phosphatase calcineurin, which mediates Ca-dependent cytoplasmic to nuclear NFAT translocation after activation of phospholipase C (PLC) (Flanagan et al., 1991; Clipstone and Crabtree, 1992). In nuclei, NFAT assembles with other transcription factors on composite gene enhancer sites, the best known of which are the activator protein-1 (AP-1) basic leucine zipper proteins (e.g., c-fos and c-jun). Such NFAT partners are activated through parallel stimulation of protein kinase C and/or the mitogen-activated protein kinase cascades (Jain et al., 1992; Northrop et al., 1993). Thus, NFAT transcription reflects integration of multiple signaling pathways downstream of PLC-coupled receptors, such as those regulating responsiveness to foreign antigens. Although initially considered a lymphocyte-specific transcription factor, the five NFAT genes are expressed differentially within most tissues, and no single NFAT isoform is restricted to thymus, spleen, or peripheral blood lymphocytes (Hoey et al., 1995). Conceivably, the cardiovascular toxicity associated with immunosuppressive drug therapy might be related to disruption of NFAT-mediated transcription in nonlymphoid cells. Recent work has established that NFATmediated transcription can be induced by signaling from Supported by Grants HL52810 and HL56107 from the National Heart, Lung, and Blood Institute. T.J.M. is an Established Investigator of the American Heart Association. A.M.R. is supported by a National Institutes of Health predoctoral training grant (GM08602). 1 NFAT nomenclature is unsettled. Synonyms of the five known genes are: 1) NFATc1, NFAT2, NFATc; 2) NFATc2, NFAT1, NFATp; 3) NFATc3, NFAT4, NFATx; 4) NFATc4, NFAT3; and 5) NFAT5. ABBREVIATIONS: CsA, cyclosporin A; NFAT, nuclear factor of activated T-cells; PLC, phospholipase C; PDGF, platelet derived growth factor; IL, interleukin; VSMC, vascular smooth muscle cell; AP-1, activator protein-1; LTR, long terminal repeat; IRES, internal ribosome entry site; TNF, tumor necrosis factor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ALU, arbitrary light units; CREB, cAMP response element binding protein; AP-1, activation protein-1; RPA, ribonuclease protection assay. 0026-895X/00/050946-08$3.00/0 MOLECULAR PHARMACOLOGY Vol. 58, No. 5 Copyright © 2000 The American Society for Pharmacology and Experimental Therapeutics 105/860039 Mol Pharmacol 58:946–953, 2000 Printed in U.S.A.