Novel blocker of NFAT activation inhibits IL-6 production in human myometrial arteries and reduces vascular smooth muscle cell proliferation

1 Department of Experimental Medical Science, Lund University, Lund, Sweden; 2 Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, Illinois; 3 Department of Pediatrics, Children's Hospital Medical Center, Cincinnati, Ohio; and 4 Department of Obstetrics and Gynecology and 5 Department of Surgery, Lund University Submitted 24 November 2005 ; accepted in final form 30 October 2006 The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway has been found to play a role in regulating growth and differentiation in several cell types. However, the functional significance of NFAT in the vasculature is largely unclear. Here we show that NFATc1, NFATc3, and NFATc4 are expressed in human myometrial arteries. Confocal immunofluorescence and Western blot analysis revealed that endothelin-1 efficiently increases NFATc3 nuclear accumulation in native arteries. Endothelin-1 also stimulates NFAT-dependent transcriptional activity, as shown by a luciferase reporter assay. Both the agonist-induced NFAT nuclear accumulation and transcriptional activity were prevented by the calcineurin inhibitor CsA and by the novel NFAT blocker A-285222. Chronic inhibition of NFAT significantly reduced IL-6 production in intact myometrial arteries and inhibited cell proliferation in vascular smooth muscle cells cultured from explants from the same arteries. Furthermore, by using small interfering RNA-mediated reduction of NFATc3, we show that this isoform is involved in the regulation of cell proliferation. Protein synthesis in intact arteries was investigated using autoradiography of [ 35 S]methionine incorporation in serum-free culture. Inhibition of NFAT signaling did not affect overall protein synthesis or specifically the synthesis rates of major proteins associated with the contractile/cytoskeletal system. An intact contractile phenotype under these conditions was also shown by unchanged force response to depolarization or agonist stimulation. Our results demonstrate NFAT expression and activation in native human vessels and point out A-285222 as a powerful pharmacological blocker of NFAT signaling in the vasculature. Ca 2+ /calcineurin; endothelin-1; cyclosporin A; contractility; differentiation Address for reprint requests and other correspondence: M. F. Gomez, Dept. of Experimental Medical Science, Lund Univ., BMC D12, 22184 Lund, Sweden (e-mail: maria.gomez{at}med.lu.se )