Abstract
Expression of the antigen-regulated, cyclosporin A-sensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Here, cultured vascular smooth muscle cells (VSMC) are shown to express two isoforms of the NFAT family endogenously, which are localized differentially in cells under resting conditions. Using a retroviral NFAT-specific luciferase reporter, we show that VSMC support previously unrecognized complexities in NFAT-mediated transcription, including evidence for negative regulation by Ca2+ signaling and positive regulation through co-activation of adenylyl cyclase and Ca2+ mobilization. The VSMC mitogen platelet derived growth factor-BB (PDGF-BB) induces NFAT-mediated transcription in VSMC. Thrombin and angiotensin II, which activate Galphaq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Galphaq receptor agonists augment the NFAT-mediated transcriptional response to PDGF-BB. This synergy can be explained in part by augmented intracellular Ca2+ transients elicited by multiple agonist challenges. These data indicate that agonists for phospholipase C-coupled receptors stimulate NFAT-mediated transcription in VSMC differentially, and that NFAT can function to integrate co-activating signals in the extracellular environment.
Highlights
Expression of the antigen-regulated, cyclosporin Asensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown
NFATc2 immunoreactivity is predominantly nuclear in unstimulated cells (Fig. 2g), and this localization is unaffected by ionomycin treatment (Fig. 2i) or by ionomycin treatment in the presence of cyclosporin A (CsA) (Fig. 2k)
The specificity of these antibodies was confirmed in immunohistochemical control experiments, in which 293 cells were transfected with plasmids encoding each of the four major NFAT isoforms
Summary
Expression of the antigen-regulated, cyclosporin Asensitive nuclear factor of activated T cells (NFAT) is not restricted to lymphoid cells, as thought initially, but the physiological inducers of NFAT-mediated transcription in non-lymphoid cells are unknown. Co-stimulation of VSMC with ionomycin and PMA resulted in a synergistic response that was 86 Ϯ 10-fold over basal (mean Ϯ S.E., n ϭ 3), and was consistent with the known co-dependence of NFATmediated transcription upon simultaneous Ca2ϩ and protein kinase C signaling [27].
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