Expression and Function of the Nuclear Factor of Activated T Cells in Colon Carcinoma Cells
2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês
10.1074/jbc.m413076200
ISSN1083-351X
AutoresJavier Duque, Manuel Fresno, Miguel A. Íñiguez,
Tópico(s)NF-κB Signaling Pathways
ResumoIncreasing evidence shows a crucial role of the Ca2+/ calcineurin-mediated activation of the nuclear factor of activated T cells (NFAT) in the regulation of a variety of processes in nonimmune cells. Here we provide evidence that NFATc1 and NFATc2 are expressed in human colon carcinoma cell lines. These proteins are translocated from the cytoplasm to the nucleus upon treatment with a combination of phorbol 12-myristate 13-acetate plus the calcium ionophore A23187. Subsequent to translocation to the nucleus, NFATc1 and NFATc2 were able to bind to a NFAT response element in the DNA, regulating transcriptional activation of genes containing a NFAT-responsive element such as cyclooxygenase-2 (COX-2). COX-2 expression and prostaglandin E2 (PGE2) production were induced upon pharmacological stimuli leading to NFAT activation and blunted by inhibition of calcineurin phosphatase with cyclosporin A or tacrolimus (FK506). Expression of NFAT wild type protein or the active catalytic subunit of calcineurin transactivates COX-2 promoter activity, whereas a dominant negative mutant of NFAT inhibited COX-2 induction in colon carcinoma cell lines. Furthermore, mutation or deletion of NFAT binding sites in the human COX-2 promoter greatly diminished its induction by phorbol 12-myristate 13-acetate/calcium ionophore A23187. These findings demonstrate the presence and activation of NFAT in human colon carcinoma cells, with important implications in the regulation of genes involved in the transformed phenotype as COX-2. Increasing evidence shows a crucial role of the Ca2+/ calcineurin-mediated activation of the nuclear factor of activated T cells (NFAT) in the regulation of a variety of processes in nonimmune cells. Here we provide evidence that NFATc1 and NFATc2 are expressed in human colon carcinoma cell lines. These proteins are translocated from the cytoplasm to the nucleus upon treatment with a combination of phorbol 12-myristate 13-acetate plus the calcium ionophore A23187. Subsequent to translocation to the nucleus, NFATc1 and NFATc2 were able to bind to a NFAT response element in the DNA, regulating transcriptional activation of genes containing a NFAT-responsive element such as cyclooxygenase-2 (COX-2). COX-2 expression and prostaglandin E2 (PGE2) production were induced upon pharmacological stimuli leading to NFAT activation and blunted by inhibition of calcineurin phosphatase with cyclosporin A or tacrolimus (FK506). Expression of NFAT wild type protein or the active catalytic subunit of calcineurin transactivates COX-2 promoter activity, whereas a dominant negative mutant of NFAT inhibited COX-2 induction in colon carcinoma cell lines. Furthermore, mutation or deletion of NFAT binding sites in the human COX-2 promoter greatly diminished its induction by phorbol 12-myristate 13-acetate/calcium ionophore A23187. These findings demonstrate the presence and activation of NFAT in human colon carcinoma cells, with important implications in the regulation of genes involved in the transformed phenotype as COX-2. The nuclear factor of activated T cells (NFAT) 1The abbreviations used are: NFAT, nuclear factor of activated T cells; Cn, calcineurin; COX, cyclooxygenase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Io, A23187 calcium ionophore; PG, prostaglandin; PMA, phorbol 12-myristate 13-acetate; CsA, cyclosporin A; IL, interleukin; dNFAT, distal NFAT site; pNFAT, proximal NFAT site. family of transcription factors was originally involved in the transcriptional regulation of a large number of inducible genes encoding cytokines and cell-surface receptors that are essential for a productive immune response (for review, see Refs. 1Serfling E. Berberich-Siebelt F. Chuvpilo S. Jankevics E. Klein-Hessling S. Twardzik T. Avots A. Biochim. Biophys. Acta. 2000; 1498: 1-18Crossref PubMed Scopus (170) Google Scholar and 2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar). However, recent evidence has confirmed that NFAT is expressed in cell types other than immune cells, regulating processes as diverse as cardiac valve formation, fiber-type specification in skeletal muscle, osteoclast differentiation, neuronal development, and angiogenesis, among others (3Crabtree G.R. Olson E.N. Cell. 2002; 109: 67-79Abstract Full Text Full Text PDF PubMed Scopus (1102) Google Scholar, 4Horsley V. Pavlath G.K. J. Cell Biol. 2002; 156: 771-774Crossref PubMed Scopus (269) Google Scholar, 5Graef I.A. Chen F. Crabtree G.R. Curr. Opin. Genet. Dev. 2001; 11: 505-512Crossref PubMed Scopus (181) Google Scholar, 6Schulz R.A. Yutzey K.E. Dev. Biol. 2004; 266: 1-16Crossref PubMed Scopus (237) Google Scholar). The NFAT family is composed of four classical calcium-responsive members named NFATc1, NFATc2, NFATc3, and NFATc4 (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar) and the recently identified calcium insensitive NFAT5 (7Lopez-Rodriguez C. Aramburu J. Rakeman A.S. Rao A. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7214-7219Crossref PubMed Scopus (319) Google Scholar). NFAT proteins are located in the cytoplasm of unstimulated cells in a highly phosphorylated form. After an increase in intracellular calcium levels, NFAT proteins are dephosphorylated by calcineurin (Cn), a serine-threonine phosphatase, and translocate to the nucleus. Once in the nucleus, they bind to specific sequences in the DNA, therefore activating transcription of NFAT-dependent genes (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 8Crabtree G.R. Cell. 1999; 96: 611-614Abstract Full Text Full Text PDF PubMed Scopus (669) Google Scholar). One of the genes that has been reported to be regulated by NFAT in non-lymphoid tissues is cyclooxygenase-2 (COX-2) (9Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (387) Google Scholar, 10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 11Sugimoto T. Haneda M. Sawano H. Isshiki K. Maeda S. Koya D. Inoki K. Yasuda H. Kashiwagi A. Kikkawa R. J. Am. Soc. Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar). Two isoforms of cyclooxygenase enzyme have been described, COX-1 and COX-2. Both isoforms catalyze the rate-limiting step in the conversion of arachidonic acid to prostaglandin H2, the common precursor of prostaglandins, prostacyclins, and thromboxanes. Whereas COX-1 is expressed constitutively in the majority of tissues, COX-2 basal level expression is low in most cells but is induced by a wide variety of mitogens, hormones, and other ligands (for review, see Ref. 12Smith W.L. DeWitt D.L. Garavito R.M. Annu. Rev. Biochem. 2000; 69: 145-182Crossref PubMed Scopus (2477) Google Scholar). The COX-2 gene contains numerous regulatory regions that bind transcription factors responsible of the inducible expression of this gene in a variety of tissues in response to several stimuli (13Tanabe T. Tohnai N. Prostaglandins Other Lipid Mediat. 2002; 68–69: 95-114Crossref PubMed Scopus (375) Google Scholar). Regarding the regulation of COX-2 expression by NFAT, two cis-acting elements have been identified in the COX-2 promoter region, named distal and proximal NFAT response elements. These sites are required for induction of COX-2 expression upon T cell receptor triggering in T lymphocytes (10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar) by vascular endothelial growth factor in vascular endothelial cells (9Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (387) Google Scholar) or by endothelin-1 in rat glomerular mesangial cells (11Sugimoto T. Haneda M. Sawano H. Isshiki K. Maeda S. Koya D. Inoki K. Yasuda H. Kashiwagi A. Kikkawa R. J. Am. Soc. Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar). There is a growing body of evidence showing a close relationship among expression of COX-2 and tumor growth and angiogenesis, making this enzyme an important therapeutic target for cancer prevention (for review, see Refs. 14Subbaramaiah K. Dannenberg A.J. Trends Pharmacol. Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar and 15Iñiguez M.A. Rodriguez A. Volpert O.V. Fresno M. Redondo J.M. Trends Mol. Med. 2003; 9: 73-78Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar)). Many human cancers display elevated COX-2 expression, and studies in COX-2 null mice have demonstrated the role of this enzyme in tumor progression and metastasis (14Subbaramaiah K. Dannenberg A.J. Trends Pharmacol. Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 16Eberhart C.E. Coffey R.J. Radhika A. Giardiello F.M. Ferrenbach S. DuBois R.N. Gastroenterology. 1994; 107: 1183-1188Abstract Full Text PDF PubMed Google Scholar, 17Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (293) Google Scholar). Moreover, epidemiological studies have revealed a role of selective COX-2 inhibitors in decreasing the risk of developing colon cancer and in suppressing tumor growth in animal models (18Huls G. Koornstra J.J. Kleibeuker J.H. Lancet. 2003; 362: 230-232Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar, 19Gupta R.A. Dubois R.N. Nat. Rev. Cancer. 2001; 1: 11-21Crossref PubMed Scopus (958) Google Scholar, 20Kawamori T. Rao C.V. Seibert K. Reddy B.S. Cancer Res. 1998; 58: 409-412PubMed Google Scholar). Recent evidence supports a role of NFAT signaling in cell growth and development (4Horsley V. Pavlath G.K. J. Cell Biol. 2002; 156: 771-774Crossref PubMed Scopus (269) Google Scholar, 5Graef I.A. Chen F. Crabtree G.R. Curr. Opin. Genet. Dev. 2001; 11: 505-512Crossref PubMed Scopus (181) Google Scholar), although little information is available on NFAT expression and function in tumor cells. These observations prompted us to examine the potential role of the NFAT signaling pathway in human colon carcinoma cells. In the present study we have analyzed the expression and function of NFAT proteins in colon carcinoma cell lines, showing that these cells express NFATc1 and NFATc2. Moreover, these proteins are efficiently translocated from the cytoplasm to the nucleus upon stimulation, where they bind to NFAT response elements in the DNA, regulating transcription of target genes. We have found that NFAT plays an essential role in the regulation of COX-2 in these cells through binding to the distal and proximal NFAT elements in the promoter region of the COX-2 gene. Inhibition of the Cn/NFAT signaling pathway with CsA or FK506 severely diminished COX-2 expression and prostaglandin production. These findings demonstrate the involvement of NFAT on gene regulation in human colon carcinoma cells, with important implications in the regulation of genes involved in the tumoral phenotype such as COX-2. Cells and Reagents—The human colon carcinoma cell lines Caco-2, HCT116, and HT29 were cultured in minimal essential medium cultured medium (Invitrogen) supplemented with 10% fetal calf serum (Sigma), 100 μg/ml streptomycin, 100 units/ml penicillin, 1 mm sodium pyruvate, 2 mm l-glutamine, and 0.1 mm nonessential amino acids. The SW620 cell line was cultured in RPMI medium (Invitrogen) supplemented with 10% fetal calf serum, 2 mm l-glutamine and antibiotics. Cells were stimulated with phorbol 12-myristate 13-acetate (PMA; Sigma) at 100 ng/ml and/or A23187 calcium ionophore (Io; Sigma) at 0.5 μm. CsA (Biomol) (1 μg/ml) and FK506 (Biomol) (100 ng/ml) were added 30 min before the addition of PMA and Io. Anti-NFAT rabbit antisera (a generous gift of Dr. J. M. Redondo) were raised against the synthetic peptides of human NFAT members coupled to carrier protein hemocyanin as described previously (21Liu F.T. Zinnecker M. Hamaoka T. Katz D.H. Biochemistry. 1979; 18: 690-693Crossref PubMed Scopus (331) Google Scholar, 22Lyakh L. Ghosh P. Rice N.R. Mol. Cell. Biol. 1997; 17: 2475-2484Crossref PubMed Scopus (155) Google Scholar). Anti-all NFATs 674 antiserum was raised against the synthetic peptide NH2-SDIELRKGETDIGRKNTRC (residues 542–559 of human NFATc1). Antisera against this peptide efficiently recognizes NFATc1, c2, c3, and c4 members (9Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (387) Google Scholar, 10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 22Lyakh L. Ghosh P. Rice N.R. Mol. Cell. Biol. 1997; 17: 2475-2484Crossref PubMed Scopus (155) Google Scholar). The anti-NFATc2 antiserum 672 was raised against the peptide NH2-CSPPSGPAYPDDVLDYGLK (residues 53–70 of human NFATc2) and specifically recognizes this NFAT member (22Lyakh L. Ghosh P. Rice N.R. Mol. Cell. Biol. 1997; 17: 2475-2484Crossref PubMed Scopus (155) Google Scholar, 23Lara-Pezzi E. Armesilla A.L. Majano P.L. Redondo J.M. Lopez-Cabrera M. EMBO J. 1998; 17: 7066-7077Crossref PubMed Scopus (88) Google Scholar, 24San-Antonio B. Iniguez M.A. Fresno M. J. Biol. Chem. 2002; 277: 27073-27080Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). The anti-NFATc1 antiserum 676 was raised against the peptide NH2-CVSPKTTDPEEGFPRGLGA (residues 210–227 of human NFATc1) (22Lyakh L. Ghosh P. Rice N.R. Mol. Cell. Biol. 1997; 17: 2475-2484Crossref PubMed Scopus (155) Google Scholar, 24San-Antonio B. Iniguez M.A. Fresno M. J. Biol. Chem. 2002; 277: 27073-27080Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Plasmid Constructs—Human COX-2 promoter constructs in pXP2LUC promoter plasmid have been described previously (10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). The ▵CAM-AI plasmid encodes a deletion mutant of a murine Cn catalytic subunit (25O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar). The pNFAT-LUC reporter plasmid containing three tandem copies of the distal NFAT site of the human IL-2 promoter fused to the minimal human IL-2 promoter and the dominant negative NFATc (pSH102C418) expression plasmid (26Durand 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) were generously provided by G. Crabtree. The hemagglutinin-tagged pEF-BOS-NFATc2 expression plasmid was a generous gift of Dr. J. M. Redondo. pGFP-VIVIT contains the sequence coding for VIVIT, a specific peptide inhibitor of Cn-mediated NFAT activation (27Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (524) Google Scholar) fused to the green fluorescent protein in the pEGFP.N1 plasmid (Clontech). mRNA Analysis—Total RNA was prepared from colon carcinoma cell lines by the TRIzol reagent (Invitrogen). Total RNA (1 μg) was reverse transcribed into cDNA and used for PCR amplification with human COX-2, COX-1, or GAPDH-specific primers by the RNA PCR core kit (PerkinElmer Life Sciences) as described previously (28Iniguez M.A. Punzon C. Fresno M. J. Immunol. 1999; 163: 111-119PubMed Google Scholar). The PCR reaction was amplified by 20–35 cycles of denaturation at 94 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min. Amplified cDNAs were separated by agarose gel electrophoresis, and bands are visualized by ethidium bromide staining. The data shown correspond to a number of cycles where the amount of amplified product is proportional to the abundance of starting material. For Northern blot analysis, RNA samples (20 μg/lane) were separated on formaldehyde gels and blotted onto nylon filters. The blots were hybridized with COX-2 and GAPDH cDNA probes labeled with [32P]dCTP (Amersham Biosciences) with a random primer extension kit (Stratagene, La Jolla, CA). After hybridization and washes by conventional protocols, the blots were subjected to autoradiography. Immunoblot Analysis—For whole cell extracts, cells were lysed for 30 min in ice-cold lysis buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mm phenylmethylsulfonyl fluoride, 2 μg/ml aprotinin, 2 μg/ml leupeptin, 1 μg/ml pepstatin, 1 mm sodium orthovanadate, and 1 mm sodium fluoride). Nuclear or cytoplasmic extracts were obtained essentially as described previously (29Wang Q. Wang X. Hernandez A. Kim S. Evers B.M. Gastroenterology. 2001; 120: 1381-1392Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Briefly, cells were collected by centrifugation and resuspended in 400 μl of ice-cold buffer A (10 mm HEPES, pH 7.6, 10 mm KCl, 0.1 mm EDTA, 0.1 EGTA, 0.75 mm spermidine, 0.15 mm spermine, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm Na2MoO4, 1 μg/ml pepstatin, 2 μg/ml leupeptin, and 2 μg/ml aprotinin). After 15 min on ice, Nonidet P-40 was added to a final concentration of 0.5% (v/v), and cells were vortexed and centrifuged for 20 min at 650 × g. The supernatant was used as cytosolic extract, and the nuclear pellet was extracted with 50 μl of buffer C (20 mm HEPES, pH 7.6, 0.4 m NaCl, 1 mm EDTA, 1 mmEGTA, 1 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride, 10 mm Na2MoO4, 1 μg/ml pepstatin, 2 μg/ml leupeptin, and 2 μg/ml aprotinin) for 30 min on a rocking platform and further centrifuged at 15,000 × g for 10 min. Protein concentration was determined by the Bradford assay (Bio-Rad). Both clarified whole cell and fractionated lysates were denatured and resolved by SDS-polyacrylamide gel electrophoresis. After electrophoresis, the proteins were transferred to nitrocellulose membranes. The filters were incubated with the indicated antibodies and developed by the enhanced chemiluminescence system (ECL; Amersham Biosciences). The anti-NFAT antisera used were anti-NFATc2 (672) and anti-NFATc1 (676). These antisera recognize both the dephosphorylated and phosphorylated forms of NFAT (23Lara-Pezzi E. Armesilla A.L. Majano P.L. Redondo J.M. Lopez-Cabrera M. EMBO J. 1998; 17: 7066-7077Crossref PubMed Scopus (88) Google Scholar, 24San-Antonio B. Iniguez M.A. Fresno M. J. Biol. Chem. 2002; 277: 27073-27080Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 30Jimenez J.L. Iniguez M.A. Munoz-Fernandez M.A. Fresno M. Cell. Signal. 2004; 16: 1363-1373Crossref PubMed Scopus (27) Google Scholar). Monoclonal mouse anti-COX-2 (Cayman Chemical Co. (Ann Arbor, MI)) was used at a 1:1000 dilution. β-Actin levels were determined as a control of loading in each lane with a specific antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Electrophoretic Mobility Shift Assays—For the gel retardation assay, nuclear extracts (5 μg) were incubated with 1 μg of poly(dI-dC) DNA carrier in DNA binding buffer (2% (w/v) polyvinyl ethanol, 2.5% (v/v) glycerol, 10 mm Tris, pH 8, 0.5 mm EDTA, 0.5 mm dithiothreitol) in a final volume of 13 μl for 30 min on ice. Then, 50,000 cpm of 32P-labeled double-stranded oligonucleotides were added and incubated at room temperature for 30 min. In competition experiments, a 20-fold molar excess of unlabeled oligonucleotides was added to the binding reaction mixture before the probe. The sequences of the oligonucleotides used (distal NFAT site of the human IL-2 gene, distal NFAT and proximal NFAT sites of the human COX-2 gene) have been described in Iñiguez et al. (10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Supershift assays were performed by incubating nuclear extracts with either preimmune serum or the correspondent anti-NFAT antisera before the addition of the probe. DNA•protein complexes were resolved by polyacrylamide gel electrophoresis on a 4% nondenaturing gel. Prostaglandin Measurement—Caco-2 cells were maintained in culture medium supplemented with 0.5% fetal calf serum, then pretreated with or without 500 ng/ml CsA for 1 h and further stimulated with PMA plus Io for a 1–24-h period. Levels of PGE2 in the culture supernatants were determined using a commercial PGE2 enzyme immunoassay kit (Cayman Chemical Co.) following the manufacturer's protocol. Transfection and Luciferase Assays—Caco-2 cells were transiently transfected with the Lipofectamine 2000 reagent as recommended by the manufacturer (Invitrogen). Briefly, exponentially growing cells (1.5 × 105) were incubated in complete medium for 24 h at 37 °C in 24-well plates. Then a mixture of 0.5–1 μg of the correspondent reporter plasmid and 0.5 μl of Lipofectamine 2000 in 50 μl of Opti-MEM was added to the cells. In cotransfection experiments, 0.15–1.5 μg of the correspondent expression plasmid was included. The total amount of DNA in each transfection was kept constant by using the corresponding empty expression vectors. After3hof incubation, complete medium was added, and cells were incubated at 37 °C for an additional 16 h. Transfected cells were exposed to different stimuli as indicated. Cells were harvested and lysed, and luciferase activity was determined by using a luciferase assay kit (Promega) in a luminometer Monolight 2010 (Analytical Luminescence Laboratory, San Diego, CA). Transfection experiments were performed in triplicate. The data presented are expressed as the mean of the determinations in relative luciferase units (RLU) ± S.D. or as fold induction (observed experimental RLU/basal RLU in absence of any stimulus). A representative experiment from the several performed is shown in all cases. Activation of NFAT Transcription Factor in Caco-2 Colon Carcinoma Cells—The presence of NFATc1 or NFATc2 isoforms in the cytosol of unstimulated Caco-2 cells was shown by Western blot analysis with specific antisera against these proteins (Fig. 1, A and B). Treatment with Io and the phorbol ester PMA, pharmacological agents that activate NFAT in other cell types (31Truneh A. Albert F. Golstein P. Schmitt-Verhulst A.M. Nature. 1985; 313: 318-320Crossref PubMed Scopus (610) Google Scholar), promoted a rapid NFAT dephosphorylation and translocation to the nucleus. Furthermore, inhibition of the calcium-induced Cn phosphatase activity by CsA prevented NFATc1 and NFATc2 dephosphorylation and entry into the nucleus. Once NFAT is activated and translocated into the nucleus, it binds to specific sequences in the promoter region of target genes (2Rao A. Luo C. Hogan P.G. Annu. Rev. Immunol. 1997; 15: 707-747Crossref PubMed Scopus (2227) Google Scholar, 32Hogan P.G. Chen L. Nardone J. Rao A. Genes Dev. 2003; 17: 2205-2232Crossref PubMed Scopus (1572) Google Scholar). To test this we carried out electrophoretic mobility shift assays with nuclear extracts obtained from Caco-2 cells treated for 30 min with PMA, Io, or PMA/Io using an oligonucleotide corresponding to the distal NFAT site in the IL-2 gene as the probe. As shown in Fig. 1C, although a DNA-protein complex was present in the absence of stimulus, a strong increase in the binding was observed after Io or PMA/Io treatment. As expected, the formation of this complex was abolished in extracts from cells pretreated with CsA. The specificity of the complex was determined using a 30-molar excess of unlabeled IL-2 probe as a competitor. The presence of NFAT proteins in the retarded complexes was confirmed by EMSA with nuclear extracts from Caco-2 cells and NFAT probes in the presence of a polyclonal antiserum directed against a common epitope located in the DNA binding domain of NFAT-c1, -c2, -c3, and -c4 isoforms. As shown in Fig. 1D, whereas the preimmune serum did not substantially alter the retarded complexes in samples with nuclear extracts from PMA plus Io-treated cells, incubation with the anti-all NFATs 674 antisera efficiently prevented the binding. These results along with similar data obtained in SW620 and HT-29 cell lines (data not shown) demonstrate the presence of functional NFAT proteins in colon carcinoma cells that could be activated by stimuli leading calcium mobilization and Cn activation. To address whether NFAT activation in these cells was able to mediate NFAT-dependent transcription, Caco-2 cells were transiently transfected with a reporter vector containing the luciferase gene under the control of three tandem copies of the distal NFAT site of the IL-2 gene (NFAT-LUC). The behavior of this reporter upon stimulation of Caco-2 cells resembled that described in T cells (33Jain J. Lom C. Rao A. Curr. Opin. Immunol. 1995; 7: 333-342Crossref PubMed Scopus (501) Google Scholar). Thus, whereas single treatment with PMA or Io led a 2–3-fold induction, combined treatment with PMA plus Io was required to get the maximal induction. On the other hand, blockade of NFAT activation by inhibition of Cn phosphatase activity with CsA attenuated the induction of the transcriptional activation of this reporter by PMA/Io (Fig. 2A). The role of Cn in the activation of NFAT proteins in carcinoma cells was further confirmed by cotransfection of the NFAT reporter construct along with an expression plasmid encoding a deletion mutant of a murine constitutively active Cn catalytic subunit (▵CAM-AI). This mutant has been described previously to efficiently substitute the calcium signal for activation of NFAT-driven transcription (25O'Keefe S.J. Tamura J. Kincaid R.L. Tocci M.J. O'Neill E.A. Nature. 1992; 357: 692-694Crossref PubMed Scopus (788) Google Scholar). As shown in Fig. 2B, cotransfection of ▵CAM-AI slightly induced NFAT-LUC reporter activity but strongly cooperated with PMA/Io to activate NFAT-dependent transcription. Both PMA/Io- and ▵CAM-AI-mediated induction of the NFAT-driven transcription was inhibited by CsA. Further analysis of the role of endogenous NFAT proteins in Caco-2 cells was performed with cotransfection of these cells with a dominant negative version of NFAT, described previously to abolish NFAT-driven promoter activity (26Durand 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). Expression of a dominant negative form of NFAT resulted in a dose-dependent inhibition of the induced activity of the NFAT-LUC reporter (Fig. 2C). Additional evidence of the role of NFAT and Cn in the transcriptional activation of genes in these cells came from the fact that PMA/Io induction was blunted by cotransfection with a green fluorescent protein fused to the VIVIT peptide (GFP-VIVIT) (Fig. 2D). This peptide is based on the sequence of the highly conserved Cn binding site in the regulatory domain of NFATs and competes for binding to Cn phosphatase, inhibiting Cn-sensitive NFAT-dependent inducible expression of target genes (27Aramburu J. Yaffe M.B. Lopez-Rodriguez C. Cantley L.C. Hogan P.G. Rao A. Science. 1999; 285: 2129-2133Crossref PubMed Scopus (524) Google Scholar). CsA Inhibits Induction of COX-2 Expression by PMA/Io in Caco-2 Cells—Once the role of NFAT activation in the regulation of NFAT-dependent transcription in colon carcinoma cells was established, we next evaluated its functional relevance in the expression of target genes. We examined the expression of COX-2, a gene with important implications in colon carcinoma progression (14Subbaramaiah K. Dannenberg A.J. Trends Pharmacol. Sci. 2003; 24: 96-102Abstract Full Text Full Text PDF PubMed Scopus (614) Google Scholar, 17Marnett L.J. DuBois R.N. Annu. Rev. Pharmacol. Toxicol. 2002; 42: 55-80Crossref PubMed Scopus (293) Google Scholar), which has been reported to be regulated by NFAT in several cell types (9Hernandez G.L. Volpert O.V. Iniguez M.A. Lorenzo E. Martinez-Martinez S. Grau R. Fresno M. Redondo J.M. J. Exp. Med. 2001; 193: 607-620Crossref PubMed Scopus (387) Google Scholar, 10Iñiguez M.A. Martinez-Martinez S. Punzon C. Redondo J.M. Fresno M. J. Biol. Chem. 2000; 275: 23627-23635Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 11Sugimoto T. Haneda M. Sawano H. Isshiki K. Maeda S. Koya D. Inoki K. Yasuda H. Kashiwagi A. Kikkawa R. J. Am. Soc. Nephrol. 2001; 12: 1359-1368Crossref PubMed Google Scholar). Reverse transcription-PCR analysis of COX-2 expression showed low levels of COX-2 mRNA in serum-starved Caco-2 cells. Both PMA or Io treatments were able to induce COX-2 mRNA, although maximal induction was observed with the combined treatment PMA/Io (Fig. 3A). Kinetics experiments showed that COX-2 mRNA induction by PMA/Io was already evident at 2 h after PMA/Io treatment (Fig. 3B). Indeed, COX-2 has been identified as an inducible early gene in response to several stimuli in different cell types (34Maier J.A. Hla T. Maciag T. J. Biol. Chem. 1990; 265: 10805-10808Abstract Full Text PDF PubMed Google Scholar, 35Herschman H.R. Fletcher B.S. Kujubu D.A. J. Lipid Mediators. 1993; 6: 89-99PubMed Google Scholar). Moreover, induction of COX-2 expression by PMA/Io was completely suppressed by actinomycin D (ActD), an inhibitor of transcription, indicating that the increase in mRNA levels occurs mainly at the transcriptional level, requiring new RNA synthesis (Fig. 3C). On the other hand, inhibition of translation by cycloheximide (CHX), although stimulating the steady state levels of COX-2 mRNA, did not affect the stimulation of COX-2 transcription. These results support the hypothesis that COX-2 behaves as an early gene induce
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