Title: The Cyclosporin A-sensitive Nuclear Factor of Activated T Cells (NFAT) Proteins Are Expressed in Vascular Smooth Muscle Cells
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 Gαq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Gαq 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. 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 Gαq-coupled receptors, are significantly weaker inducers of NFAT-mediated luciferase expression than is PDGF-BB. However, co-stimulation studies show that Gαq 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. Transcription mediated by the nuclear factor of activated T lymphocytes (NFAT) 1The abbreviations used are: NFAT, nuclear factor of activated T cells; PLC, phospholipase C; CsA, cyclosporin A; VSMC, vascular smooth muscle cells; PMA, phorbol myristate acetate; PDGF-BB, platelet-derived growth factor BB chain; IL-2, the cytokine interleukin-2; HRP, horseradish peroxidase; PKA, protein kinase A; NFRE, NFAT response element; CREB, cAMP response element-binding protein; ATF, activating transcription factor; HBSS, Hank's balanced salt solution; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; LTR, long terminal repeat; GM, growth medium. is regulated tightly in response to elevations of both intracellular calcium ion (Ca2+) and diacylglycerol second messengers following activation of phospholipase C (PLC) (1Crabtree G.R. Clipstone N.A. Annu. Rev. Biochem. 1994; 63: 1045-1083Crossref PubMed Scopus (627) Google Scholar). Increased intracellular Ca2+ stimulates calcineurin-dependent dephosphorylation of cytoplasmic NFAT, leading to its nuclear translocation (2Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar, 3Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (429) Google Scholar, 4Jain J. Miner Z. Rao A. J. Immunol. 1993; 151: 837-848PubMed Google Scholar, 5Shaw K.T. Ho A.M. Raghavan A. Kim J. Jain J. Park J. Sharma S. Rao A. Hogan P.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11205-11209Crossref PubMed Scopus (318) Google Scholar). Macrolide immunosuppressive agents, such as cyclosporin A (CsA) and FK506 (6Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1971) Google Scholar), block this step. In the nucleus, NFAT forms a heteromeric transcriptional co-activator complex with AP1 proto-oncogene partners that are co-induced downstream of receptor activation (3Jain J. McCaffrey P.G. Valge-Archer V.E. Rao A. Nature. 1992; 356: 801-804Crossref PubMed Scopus (429) Google Scholar). This multimeric NFAT complex participates in the transactivation of several cytokine genes by interactions with purine-rich genomic NFAT-responsive enhancer elements (NFRE) (7Rao A. Immunol. Today. 1994; 15: 274-281Abstract Full Text PDF PubMed Scopus (490) Google Scholar). The first evidence for the existence of NFAT was derived from analysis of antigen-responsive enhancer elements in the cytokine IL-2 gene promoter in lymphocytes (8Durand D.B. Shaw J.-P. Bush M.R. Replogle R.E. Belagaje R. Crabtree G.R. Mol. Cell. Biol. 1988; 8: 1715-1724Crossref PubMed Scopus (375) Google Scholar). A growing body of data now suggests that NFAT is expressed more widely but the picture is far from complete. To date, NFAT expression or function has been described in several types of non-lymphoid cells, including mast (9Weiss D.H. Hural J. Tara D. Timmerman L.A. Henkel G. Brown M.A. Mol. Cell. Biol. 1996; 16: 228-235Crossref PubMed Scopus (69) Google Scholar), endothelial (10Cockerill G.W. Bert A.G. Ryan G.R. Gamble J.R. Vadas M.A. Cockerill P.N. Blood. 1995; 86: 2689-2698Crossref PubMed Google Scholar), and neuronal cells (11Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar). Each of the mRNAs for the known NFAT isoforms are expressed in distinct tissue-specific patterns (12Hoey T. Sun Y.-L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (354) Google Scholar). However, the precise non-lymphoid cell types that express NFAT isoforms in vivo are largely unknown. For instance, it remains unclear whether NFAT is expressed to some degree in all cells, or if its expression in non-lymphoid tissues is restricted to certain subpopulations of cells within tissues. The presentation of foreign antigen on lymphocytes provides the best understood physiological inducer of NFAT-mediated transcription. The existence of NFAT in non-immune cells suggests that it can be directed to regulate gene expression by other physiologic stimuli. Physiological agonists for receptors that activate PLC signaling, such as hormones, neurotransmitters, and growth factors, provide obvious candidates for this function (11Ho A.M. Jain J. Rao A. Hogan P.G. J. Biol. Chem. 1994; 269: 28181-28186Abstract Full Text PDF PubMed Google Scholar, 13Boss V. Talpade D.J. Murphy T.J. J. Biol. Chem. 1996; 271: 10429-10432Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). The extent to which such stimuli can trigger NFAT-mediated transcription in non-lymphoid cells that express endogenous NFAT isoforms needs to be determined. Additionally, it is not known if NFAT transactivation properties in non-lymphoid cells differs from that in the better understood lymphocyte context. Vascular smooth muscle cells (VSMC) respond to a diverse group of extracellular signals to progress through phenotypic modulation, a process that occurs in the development and progression of cardiovascular diseases such as hypertension and atherosclerosis (14Jackson C.L. Schwartz S.M. Hypertension. 1992; 20: 713-736Crossref PubMed Scopus (236) Google Scholar). Changes in gene expression patterns underlie this transition, which serves to regulate processes involved in extracellular matrix remodeling, VSMC growth, and migration. Cultured VSMC serve as a useful system to understand the molecular and cellular consequences of VSMC activation, and are helpful in elucidating the genes, gene expression control pathways, and the extracellular signals associated with VSMC activation. We speculated that NFAT might be expressed in cultured VSMC and may participate in facilitating the VSMC activated state in response to mitogenic signals. The experiments described herein represent the first demonstration that NFAT is expressed in VSMC and provide evidence that PDGF-BB, which activates a tyrosine kinase-linked receptor, can serve as a physiological inducer of NFAT-mediated transcription in VSMC. This response is synergized by co-stimulation of VSMC with thrombin and angiotensin II, which activate PLCβ activity by coupling through the Gαq class of heterotrimeric GTP-binding proteins. However, these latter agonists are weaker inducers of NFAT-mediated transcription than is PDGF-BB. The following plasmids used in this study were received as gifts: the retroviral plasmid pLNCX, from A. D. Miller (Seattle, WA); plasmids pSH210 and pSH250, encoding human NFATc1 and NFATc2, from G. Crabtree (Stanford, CA); and plasmid pNFAT/luc, from R. Bram (Memphis, TN). A mouse monoclonal antibody (IgG1, clone 7A6) against NFATc1 was purchased from Affinity Bioreagents, Inc. (Golden, CO). The monoclonal antibody (GH8-G1-G9) against NFATc2 was a gift from G. Crabtree. Mouse IgG1 in clarified ascites was purchased from Sigma. Secondary antibodies were bought from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). The tyramide-green immunochemical reporter was purchased from NEN Life Science Products). Except where noted all cell culture media, antibiotics and molecular biology supplies were purchased from Life Technologies, Inc. Fetal bovine and calf serum were obtained from Atlanta Biologicals (Norcross, GA). Luciferin and additional salts and buffers used were purchased from Sigma. The retroviral plasmids used in this study were subcloned and maintained in Escherichia coliTop10F′ (Invitrogen, Inc., Carlsbad, CA) in 100 μg/ml ampicillin plus 12.5 μg/ml tetracycline. The VSMC used in this study were derived from dissociated rat thoracic aorta (passages 10 to 25) and are maintained as a continuous primary cell line in DMEM, 4.5 μg/ml glucose, 10% heat-inactivated calf serum with 100 units/ml each penicillin and streptomycin (15Nickenig G. Murphy T.J. Mol. Pharmacol. 1996; 50: 743-751PubMed Google Scholar). Amphotropic (ATCC CRL-11554) retroviral producer cells were obtained from the American Type and Culture Collection (Rockville, MD). Angiotensin II was purchased from Sigma, purified human thrombin was provided generously by S. Krishnaswamy (Dept. of Medicine, Emory University, Atlanta, GA), and PDGF-BB was purchased from Calbiochem, Inc. (San Diego, CA). pTJM9 was created by placing a new multicloning site just distal of the cytomegalovirus promoter inHindIII- and ClaI-digested pLNCX (16Miller A.D. Miller D.G. Garcia J.V. Lynch C.M. Methods Enzymol. 1993; 217: 581-599Crossref PubMed Scopus (378) Google Scholar) using the oligonucleotides 5′-AGCTTCCATTGTGCTGGTCGACGCGTCCAGCACAATGGAT and 5′-CGATCCATTGTGCTGGACGCGTCGACCAGCACAATGGA. The cytomegalovirus promoter in pTJM9 was removed by digestion with BamHI andHindIII, and the plasmid was closed by blunt-end ligation after Klenow treatment to create pTJM12. A luciferase coding sequence (17De Wet J.R. Wood K.V. DeLuca M. Helinski D.R. Subramani S. Mol. Cell. Biol. 1987; 7: 725-737Crossref PubMed Scopus (2482) Google Scholar) with upstream remnant was excised with PstI andSstI from the vector poLuc (18Braiser A.R. Tate J.E. Habener J.F. BioTechniques. 1989; 7: 1116-1122PubMed Google Scholar). After Klenow treatment, the fragment ends were ligated with non-cohesive BstXI adapters (5′-CTGGCGCG and 5′-CGCGCCAGCACA) and subsequently cloned into pTJM12 digested with BstXI. The resulting construct, pKA8, can accept promoters in the unique BamHI and HindIII (5′ to 3′) sites just proximal of the luciferase 5′ end, and transcribes luciferase off of the opposite strand as that for the viral 5′-LTR promoter. Plasmid pKA7 contains a ∼250-base pairBamHI-HindIII fragment with an NFAT enhancer triplex linked to the minimal IL-2 promoter fragment excised from pNFAT/luc (19Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar), which was cloned into the same sites in pKA8. Plasmid pKA9 contains a ∼130-base pair BamHI-HindIII fragment for the minimal IL-2 promoter, without the NFAT enhancer, amplified from pNFAT/luc by PCR using the primers 5′-CTCGGATCCTATGTAAAACATTTTGACACC and 5′-GGCAAGCTTAGGAGTTGAGGTTACTGTGA. To create a reporter plasmid bearing a mutation in the NFAT binding site (mutations shown in bold letters), the oligonucleotides F87 (5′-GATCAGGACTGATCACTGTTTCATACAGAAGGC) and F88 (5′-GATCGCCTTCTGTATGAAACAGTGATCAGTCCT) were concatenized by brief ligation before adding BamHI-digested pKA9 to the ligation reaction. pKA10 represents a clone derived from this procedure that was identified by sequencing as having an enhancer region identical that in pKA7 except for conversion of the NFAT binding site from GGAAAA to CTGATC. Retroviruses were prepared by transient transfection of helper virus-free amphotropic producer cells (20Pear W.S. Nolan G.P. Scott M.L. Baltimore D. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8392-8396Crossref PubMed Scopus (2306) Google Scholar). Bing-CAK8 cells (ATCC CRL-11554) were grown from original expanded stocks without selective antibiotics for less than 30 days in growth medium (GM) consisting of DMEM, 4.5 μg/ml glucose, 10% fetal bovine serum, and a mixture of 100 units/ml penicillin and 100 units/ml streptomycin in a humidified 5% CO2atmosphere at 37 °C. To produce infectious retroviral supernatants, the cells at 50–80% confluence in 10-cm diameter dishes were transfected with the retroviral plasmids using calcium phosphate and 25 μm chloroquine for 6–12 h before re-feeding with 25 ml of GM. Twenty-four hours after initiating the transfection, the GM was aspirated and replaced with 9 ml of fresh GM before placing the dishes in a humidified 5% CO2 atmosphere at 32 °C. Previous work has shown this lower temperature enhances retroviral titers (21Kotani H. Newton III, P.B. Zhang S. Chiang Y.L. Otto E. Weaver L. Blaese R.M. Anderson W.F. McGarrity G.J. Hum. Gene Ther. 1994; 5: 19-28Crossref PubMed Scopus (349) Google Scholar). The supernatant containing retroviral particles was harvested after 24 h, and twice more at 12–18 h intervals thereafter replenishing with 9 ml of GM each time. Each collected supernatant (9 ml) was filtered through sterile 0.45-μm syringe-tip cellulose acetate disk, aliquoted, snap-frozen in liquid nitrogen and stored at -80 °C. VSMC (∼20% confluent) grown on 6 × 35-mm multiwell plates were infected with retroviruses by adding thawed retroviral supernatant (2 ml) containing 8 μg/ml Polybrene to each well and spinning the cells at 2,500 rpm for 30 min at 32 °C in a Beckman model GS-6R refrigerated centrifuge in a swinging bucket rotor before placing in a 5% CO2 incubator at 37 °C. This infection protocol was repeated twice more at 8–12 h intervals. Forty-eight hours following the last infection, the cells were treated with 400 μg/ml G418 in normal growth VSMC medium (Life Technologies, Inc.). Within 7 days of selection, the surviving cells from each well were combined, expanded, and used subsequently for further study without subcloning and maintained in the absence of G418. Because cell death during selection of retrovirus-infected cultures was minimal compared with that in retrovirus-naı̈ve cells treated in parallel with 400 μg/ml G418, transduction efficiency was estimated in all cases to be nearly quantitative. Recombinant VSMC selected for reporter retroviruses were grown to confluence in 24-well plates for 3 days in growth medium lacking G418 and then for an additional 24 h in medium lacking serum before stimulation with drugs and agonists. The drugs were prepared as 10-fold concentrated stocks in phosphate-buffered saline, pH 7.4 (PBS), and added as aliquots directly to the cells to begin stimulation. These were performed in a humidified 5% CO2 atmosphere at 37 °C for the indicated time before the cells were lysed and extracts were prepared for luciferase determinations, as described previously (22Takeuchi K. Alexander R.W. Nakamura Y. Tsujino T. Murphy T.J. Circ. Res. 1993; 73: 612-621Crossref PubMed Scopus (94) Google Scholar). To prepare control cell extracts expressing defined NFAT isoforms, COS-7 cells were transfected by the DEAE-dextran/chloroquine approach with the vector pSH107c, encoding human NFATc1; or the vector pSH210, encoding human NFATc2 (23Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar); or pCDM8 as a sham control. Confluent VSMC or transfected COS cells were rinsed in PBS before lysis in RIPA-2 (150 mm NaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mmTris-HCl, pH 8.0) containing a mixture of protease inhibitors (90 μg/ml phenylmethylsulfonyl fluoride, 0.2 TIU/ml aprotinin, 0.1 mm leupeptin, 0.2 mm sodium orthovanadate, 5 μg/ml pepstatin A, and 5 μg/ml soybean trypsin inhibitor). Insoluble debris was removed by centrifugation at 10,000 ×g at 4 °C and discarded. Protein concentrations in the supernatants were determined using a kit purchased from Bio-Rad, and γ globulin as standard. Samples were dissolved in SDS loading buffer, heated for 5 min at 95 °C before separation on 10% SDS-polyacrylamide gels, and transferred to Immobilon P membranes that were blocked in 0.5% nonfat dried milk and incubated overnight with a 1:1000 dilution of either the anti-NFATc1 or anti-NFATc2 monoclonal antibodies. The blots were developed using a chemiluminescent detection kit (Phototope-HRP, New England Biolabs, Natick, MA) using 1:20,000 horseradish peroxidase (HRP)-labeled anti-mouse IgG secondary. VSMC (7 × 104 cells) were plated in 35-mm dishes 24 h prior to drug treatments, and changed to serum-free medium 1 h before the addition of drugs. CsA was dissolved in 2.5% ethanol in cell culture medium containing 0.1% Tween 80 to prepare a 100-fold concentrated stock solution. Ionomycin was added to the cells directly from a 100-fold stock solution. The cells received either CsA (1 μm final concentration) or its vehicle for 10 min prior to the addition of ionomycin (1 μm final concentration) or its vehicle. After 15 min, the medium was aspirated and the cells were rinsed with PBS before fixation for 10 min using cold methanol/0.3% H2O2before storing at 4 °C in PBS. Antibody staining was performed at room temperature within 1 week of these treatments. The samples were first blocked against avidin/biotin using the AVB kit (Vector Laboratories), according to directions supplied by the manufacturer. After rinsing with PBS, nonspecific protein binding was blocked by incubating the cells with PBS containing 2% horse serum (Atlanta Biologicals, Norcross, GA) and 0.5% Triton X-100 for 30 min at 22 °C. All primary antibodies were diluted in this blocking solution at a 1:250 dilution and applied to the cells for 60 min at 22 °C. Following three washes with PBS containing 0.2% Tween 20, the cells were incubated for 60 min with a 1:10,000 dilution of biotinylated anti-mouse IgG (Jackson Immunochemicals, Inc., West Grove, PA). After washing as above, antibody binding was visualized using a catalyzed reporter deposition kit (T.S.A. Direct Green, NEN Life Science Products according to the manufacturers directions but with a 1:100 dilution of the streptavidin HRP conjugate. After the final rinse, the cells were incubated for 10 min at 22 °C with a Hoechst stain (H33258) in PBS, then washed three times with PBS. After coverslipping using Vectashield, all analyses and photography were performed on a Zeiss Axiovert microscope equipped with an Optronics video camera, which was in line with a Power Macintosh 7600 computer equipped with NIH Image. Confluent 10-cm plates of VSMC were washed three times with Ca2+/Mg2+-free Hank's balanced salt solution (HBSS) and treated with 1 mg/ml type-1 collagenase (178 units/ml; Worthington Biochemical, Inc., Freehold, NJ) at 37 °C. The dispersed cells were collected, washed in Ca2+/Mg2+- replete HBSS (Ca2+/Mg2+ HBSS), counted, and then resuspended to 3–4 × 106 cells/ml in HEPES-buffered DMEM (pH 7.4) containing 0.5 mg/ml bovine serum albumin (DMEM+BSA). Fura-2/AM (1 mm stock in Me2SO) was added to a final concentration of 2 μm, and the cells were incubated for 15 min at 37 °C in the dark. The cells were brought to a final volume of 40 ml in DMEM+BSA, spun at 500 rpm for 10 min at 4 °C in a benchtop centrifuge, and the cell pellet was resuspended in 10 ml of Ca2+/Mg2+ HBSS. After counting, the cells were diluted on ice into 3-ml aliquots containing 2 × 106cells/ml. Just prior to assay, each aliquot was centrifuged briefly and the pellet resuspended gently in 1 ml of Ca2+/Mg2+ HBSS that was added to stirred cuvettes containing 2 ml of pre-warmed Ca2+/Mg2+ HBSS. Agonists were added from 100-fold concentrated stocks, and the excitation ratio (340/380 nm) was measured for emission intensity at 510 nm on a Perkin Elmer LS50 spectrometer. VSMC, or control 293 cells transfected with pSH210, were treated with drugs as described in legend to Fig. 5 before nuclei were harvested as described previously (15Nickenig G. Murphy T.J. Mol. Pharmacol. 1996; 50: 743-751PubMed Google Scholar). Nuclear extracts were prepared by gently resuspending the nuclei in 0.5 volumes of low salt buffer (20 mm HEPES, pH 7.9 at 4 °C, 1.5 mm MgCl2, 25% glycerol, 0.2 mmEDTA, 20 mm KCl). An equivalent volume of high salt buffer (low salt buffer except with 0.8 m KCl) was next added dropwise with gentle mixing, and the nuclei were incubated on ice for 30 min. After centrifugation in a Beckman TLS55 rotor at 25,000 ×g for 30 min at 4 °C, the supernatant was dialyzed against 500 volumes of a buffer identical to the low salt buffer above except containing 100 mm KCl. Aliquots of the dialyzed sample were frozen at –80 °C until use. Binding assays were performed at 22 °C for 30 min in a 20-μl volume composed of nuclear extract (containing 10 μg of VSMC nuclear protein or 1 μg of 293/NFAT nuclear protein), 4.5 μg of bovine serum albumin, 2 μg of poly(dI-dC), 12 mm Hepes (pH 7.9), 4 mmTris-HCl (pH 7.9), 80 mm KCl, 13% glycerol, 1 mm EDTA, 1 mm dithiothreitol, and 1 mm phenylmethylsulfonyl fluoride. The probe used in binding assays (wtNFRE) was derived from the distal NFRE in the human IL-2 gene and formed by the oligonucleotides D79 (5′-GAAAGGAGGAAAAACTGTTTCATACAGAAGGCGT) and D80 (5′-GAACGCCTTCTGTATGAAACAGTTTTTCCTCCTT), which were end-labeled with 32P using T4 polynucleotide kinase. Unlabeled competitor probes used in binding assays include mutNFRE, which was formed by annealing oligonucleotides F87 and F88 (see above), and the wtNFRE composed of oligonucleotides D79 and D80. After the binding incubation period, the samples were loaded onto Tris-glycine polyacrylamide gels, resolved by electrophoresis, and subjected to autoradiography. Western blot analysis indicates that a protein in VSMC reacts with monoclonal antibody 7A6 (23Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), which was prepared against a polypeptide from the NFATc1 isoform (Fig. 1). The antibody specificity for the NFATc1 isoform is indicated by immunoreactivity in extracts of COS-7 cells transfected with a human cDNA for NFATc1, but not in extracts transfected with sham plasmid or with a plasmid encoding the NFATc2 isoform (23Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). The heterogeneous mobility of the immunoreactive VSMC protein is typically seen in NFAT-expressing cells, and might result from either alternate exon usage or various post-translational modifications, including phosphorylation (24Ruff V.A. Leach K.L. J. Biol. Chem. 1995; 270: 22602-22607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). No other currently available antibody directed against the three other NFAT isoforms (12Hoey T. Sun Y.-L. Williamson K. Xu X. Immunity. 1995; 2: 461-472Abstract Full Text PDF PubMed Scopus (354) Google Scholar, 23Ho S.N. Thomas D.J. Timmerman L.A. Li X. Francke U. Crabtree G.R. J. Biol. Chem. 1995; 270: 19898-19907Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 25McCaffrey P.G. Luo C. Kerpolla T.K. Jain J. Badalian T.M. Ho A.M. Burgeon E. Lane W.S. Lambert J.N. Curran T. Verdine G.L. Rao A. Hogan P.G. Science. 1993; 262: 750-754Crossref PubMed Scopus (379) Google Scholar) is compatible with our Western blot protocol, including the GH8-G1-G9 monoclonal antibody directed against NFATc2 that is described below (data not shown). Immunohistochemical analysis of cultured VSMC provides evidence that, in addition to NFATc1, NFATc2 is not only expressed in these cells, but has a subcellular pattern of expression distinct from that of NFATc1. In unstimulated cells, NFATc1 immunoreactivity is predominantly cytoplasmic, but occurs in a clustered pattern, consistent with vesicular localization (Fig.2 a). This basal pattern contrasts sharply with the more pronounced nuclear localization observed after a 10-min exposure to 1.0 μm ionomycin (Fig. 2 c). Treatment of the cells with 1 μmCsA before and during challenge with ionomycin prevents ionomycin-induced nuclear translocation of NFATc1 (Fig. 2 e). This calcium-dependent, CsA-sensitive nuclear transport is a characteristic property of NFAT (2Clipstone N.A. Crabtree G.R. Nature. 1992; 357: 695-697Crossref PubMed Scopus (1477) Google Scholar, 24Ruff V.A. Leach K.L. J. Biol. Chem. 1995; 270: 22602-22607Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 26Timmerman L.A. Clipstone N.A. Ho S.N. Northrop J.P. Crabtree G.R. Nature. 1996; 383: 837-840Crossref PubMed Scopus (472) Google Scholar). In striking contrast, NFATc2 immunoreactivity is predominantly nuclear in unstimulated cells (Fig. 2 g), and this localization is unaffected by ionomycin treatment (Fig. 2 i) or by ionomycin treatment in the presence of CsA (Fig. 2 k). 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. Furthermore, there is no detectable staining in VSMC incubated with non-immune IgG as a negative control (data not shown). From these controls and the staining shown in VSMC, these data indicate that localization of various endogenously expressed NFAT isoforms differs in VSMC, suggesting they may be under control of different regulatory processes. In order to study control of NFAT-mediated transcription in VSMC, a retroviral-based luciferase transcriptional reporter system was developed to take advantage of the higher efficiency of retroviral gene transfer than is possible in VSMC using plasmid transfection approaches. The retroviral NFAT/luciferase reporter vector pKA7 is depicted in Fig. 3 A. This vector contains a luciferase coding sequence under the control of a minimal human IL-2 promoter with an upstream triplex of the distal IL-2 gene NFRE. These elements were derived from a plasmid NFAT-responsive reporter vector whose fidelity has been characterized extensively, by ourselves and others (13Boss V. Talpade D.J. Murphy T.J. J. Biol. Chem. 1996; 271: 10429-10432Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 19Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Abstract Full Text PDF PubMed Google Scholar). A neomycin resistance gene is expressed from the viral 5′-LTR promoter, and selection for cells resistant to this antibiotic ensures that all cells in the culture have been infected with a retrovirus, which is important for comparing responses among the various control reporter vectors. In preliminary experiments, we found that NFAT-luciferase reporter vectors are substantially more responsive to stimuli when the orientation of luciferase transcription is directed toward the retroviral 5′-LTR, rather than when transcription of the two promoters are driven off of the same strand (data not shown). The control reporter vectors are shown in Fig. 3 B and include a promoterless luciferase (pKA8), a luciferase driven by a minimal IL-2 promoter but lacking an NFRE (pKA9), and a minimal promoter with a NFRE possessing mutations that disrupt the NFAT-binding site within the NFRE (pKA10). The data shown in Fig. 3 C indicate that highly inducible and synergistic luciferase expression consistent with NFAT-mediated transcription occurred only in VSMC infected with retrovirus prepared from plasmid pKA7, and that no responses were observed in cells infected with any of the other reporter viruses. Stimulation for 4 h with 100 nm PMA alone yielded a response that was 28 ± 1-fold over basal (mean ± S.E., n = 3), whereas the response to 1 μm ionomycin (1.3 ± 0.2-fold; mean ± S.E., n = 3) was no different from that of vehicle. Co-stimulation of VSMC with ionomycin and PMA resulted in a synergistic response that was 86 ± 10-fold over basal (mean ± S.E., n = 3), and was c