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• W1973417966 abstract "Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Gαq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment. Overexpression of cAMP-response element (CRE)-binding protein (CREB) and activating transcription factor (ATF) 1 contributes to melanoma progression and metastasis at least in part by promoting tumor cell survival and stimulating matrix metalloproteinase (MMP) 2 expression. However, little is known about the regulation of CREB and ATF-1 activities and their phosphorylation within the tumor microenvironment. We analyzed the effect of platelet-activating factor (PAF), a potent phospholipid mediator of inflammation, for its ability to activate CREB and ATF-1 in eight cultured human melanoma cell lines, and we found that PAF receptor (PAFR) was expressed in all eight lines. In metastatic melanoma cell lines, PAF induced CREB and ATF-1 phosphorylation via a PAFR-mediated signal transduction mechanism that required pertussis toxin-insensitive Gαq protein and adenylate cyclase activity and was antagonized by a cAMP-dependent protein kinase A and p38 MAPK inhibitors. Addition of PAF to metastatic A375SM cells stimulated CRE-dependent transcription, as observed in a luciferase reporter assay, without increasing the CRE DNA binding capacity of CREB. Furthermore, PAF stimulated the gelatinase activity of MMP-2 by activating transcription and MMP-2 expression. MMP-2 activation correlated with the PAF-induced increase in the expression of an MMP-2 activator, membrane type 1 MMP. PAF-induced expression of pro-MMP-2 was causally related to PAF-induced CREB and ATF-1 phosphorylation; it was prevented by PAFR antagonist and inhibitors of p38 MAPK and protein kinase A and was abrogated upon quenching of CREB and ATF-1 activities by forced overexpression of a dominant-negative form of CREB. PAF-induced MMP-2 activation was also down-regulated by p38 MAPK and protein kinase A inhibitors. Finally, PAFR antagonist PCA4248 inhibited the development of A375SM lung metastasis in nude mice. This result indicated that PAF acts as a promoter of melanoma metastasis in vivo. We proposed that metastatic melanoma cells overexpressing CREB/ATF-1 are better equipped than nonmetastatic cells to respond to PAF within the tumor microenvironment. cAMP-response element (CRE) 2The abbreviations used are: CRE, cAMP-response element; ATF-1, activating transcription factor 1; CREB, cAMP-response element-binding protein; CBP, CREB-binding protein; PAF, platelet-activating factor; PAFR, PAF receptor; MMP, matrix metalloproteinase; MT1-MMP, membrane-type 1 MMP; Ptx, pertussis toxin; AC, adenylate cyclase; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; TIMP, tissue inhibitor of metalloproteinases; Me2SO, dimethyl sulfoxide; HBSS, Hanks' balanced salt solution; cPAF, carbamyl-PAF; EMSA, electrophoretic mobility shift assay; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidium iodide; IL, interleukin. -binding protein (CREB), activating transcription factor (ATF) 1, and CRE modulators are members of the large basic region leucine zipper superfamily of transcription factors that regulate gene expression in response to cAMP, intracellular Ca2+ mobilization, and cell stimulation with growth factors (1Mayr B. Montminy M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 599-609Crossref PubMed Scopus (2066) Google Scholar). The ubiquitously expressed CREB and ATF-1 and the preferentially expressed (in neuroendocrine tissues) CRE modulator bind to complete and partial palindromic CRE DNA consensus sites 5′-TGACGTCA-3′ and 5′-CGTCA-3′. These transcription factors induce genes involved in cellular metabolism and respiration, gene transcription, cell cycle regulation and cell survival, as well as growth factor and cytokine genes (reviewed in Ref. 1Mayr B. Montminy M. Nat. Rev. Mol. Cell. Biol. 2001; 2: 599-609Crossref PubMed Scopus (2066) Google Scholar). The transcriptional activity of the DNA-bound CREB protein dimers is regulated by phosphorylation of the serine 133 site in the kinase-inducible domain, which promotes recruitment of the transcriptional co-activator CREB-binding protein (CBP) and p300 (2Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2055) Google Scholar, 3Chrivia J.C. Kwok R.P. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 365: 855-859Crossref PubMed Scopus (1768) Google Scholar). In response to elevated levels of cAMP caused by activation of guanine nucleotide-binding (G) protein-coupled receptors, CREB phosphorylation occurs as a result of activation and nuclear translocation of cAMP-dependent protein kinase A (PKA) (2Gonzalez G.A. Montminy M.R. Cell. 1989; 59: 675-680Abstract Full Text PDF PubMed Scopus (2055) Google Scholar). Several growth factors and stress signals have been shown to induce CREB phosphorylation via mechanisms mediated by various other protein kinases, including protein kinase C, pp90RSK, AKT, MSK-1, MAP-KAP-2, calcium-calmodulin kinases II and IV, and p38 mitogen-activated protein kinase (p38 MAPK) (4Bohm M. Moellmann G. Cheng E. Alvarez-Franco M. Wagner S. Sassone-Corsi P. Halaban R. Cell Growth Differ. 1995; 6: 291-302PubMed Google Scholar, 5Deak M. Clifton A.D. Lucocq L.M. Alessi D.R. EMBO J. 1998; 17: 4426-4441Crossref PubMed Scopus (845) Google Scholar, 6Du K. Montminy M. J. Biol. Chem. 1998; 273: 32377-32379Abstract Full Text Full Text PDF PubMed Scopus (822) Google Scholar, 7Matthews R.P. Guthrie C.R. Wailes L.M. Zhao X. Means A.R. McKnight G.S. Mol. Cell. Biol. 1994; 14: 6107-6116Crossref PubMed Scopus (497) Google Scholar, 8Tan Y. Rouse J. Zhang A. Cariati S. Cohen P. Comb M.J. EMBO J. 1996; 15: 4629-4642Crossref PubMed Scopus (566) Google Scholar). CREB and ATF-1 expression levels correlate directly with the transition of human melanoma cells from the radial to the vertical growth phase and with their metastatic potential in an experimental nude mice model (4Bohm M. Moellmann G. Cheng E. Alvarez-Franco M. Wagner S. Sassone-Corsi P. Halaban R. Cell Growth Differ. 1995; 6: 291-302PubMed Google Scholar, 9Rutberg S.E. Goldstein I.M. Yang Y.M. Stackpole C.W. Ronai Z. Mol. Carcinog. 1994; 10: 82-87Crossref PubMed Scopus (44) Google Scholar). We showed previously that quenching CREB and ATF-1 activities in metastatic melanoma cells by means of overexpression of a dominant-negative form of CREB, KCREB, which has a single base pair substitution in the DNA-binding domain (10Walton K.M. Rehfuss R.P. Chrivia J.C. Lochner J.E. Goodman R.H. Mol. Endocrinol. 1992; 6: 647-655PubMed Google Scholar), decreased melanoma tumorigenicity and metastatic potential in nude mice (11Xie S. Price J.E. Luca M. Jean D. Ronai Z. Bar-Eli M. Oncogene. 1997; 15: 2069-2075Crossref PubMed Scopus (109) Google Scholar). Furthermore, we have shown that intracellular expression of an inhibitory anti-ATF-1 single-chain antibody fragment in MeWo human melanoma cells suppressed their tumorigenicity and metastatic potential in vivo (12Jean D. Tellez C. Huang S. Davis D.W. Bruns C.J. McConkey D.J. Hinrichs S.H. Bar-Eli M. Oncogene. 2000; 19: 2721-2730Crossref PubMed Scopus (59) Google Scholar). At least two mechanisms explain how CREB and ATF-1 overexpression contributes to the metastatic phenotype. (i) CREB and ATF-1 play essential roles in regulating the expression of the type IV matrix metalloproteinase 2 (gelatinase A), a key enzyme in melanoma invasion and metastasis, and of the adhesion molecule MCAM/MUC18 genes (11Xie S. Price J.E. Luca M. Jean D. Ronai Z. Bar-Eli M. Oncogene. 1997; 15: 2069-2075Crossref PubMed Scopus (109) Google Scholar). (ii) CREB and ATF-1 act as survival factors that promote melanoma cell survival in vitro in response to apoptosis-inducing agents (e.g. thapsigargin and ionizing radiation) and in in vivo experimental tumors (12Jean D. Tellez C. Huang S. Davis D.W. Bruns C.J. McConkey D.J. Hinrichs S.H. Bar-Eli M. Oncogene. 2000; 19: 2721-2730Crossref PubMed Scopus (59) Google Scholar, 13Jean D. Harbison M. McConkey D.J. Ronai Z. Bar-Eli M. J. Biol. Chem. 1998; 273: 24884-24890Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Although it has been shown that melanocyte proliferation and differentiation can be positively regulated by agents that increase cAMP levels (14Halaban R. Ghosh S. Baird A. In Vitro Cell. Dev. Biol. 1987; 23: 47-52Crossref PubMed Scopus (211) Google Scholar), little is known about factors that induce CREB and ATF-1 activation in melanoma. Meanwhile, melanoma is strongly associated with the inflammatory process (15Goulet A.C. Einsphar J.G. Alberts D.S. Beas A. Burk C. Bhattacharyya A. Bangert J. Harmon J.M. Fujiwara H. Koki A. Nelson M.A. Cancer Biol. Ther. 2003; 2: 713-718Crossref PubMed Scopus (94) Google Scholar, 16Amiri K.I. Richmond A. Cancer Metastasis Rev. 2005; 24: 301-313Crossref PubMed Scopus (159) Google Scholar, 17Leslie M.C. Bar-Eli M. J. Cell. Biochem. 2005; 94: 25-38Crossref PubMed Scopus (53) Google Scholar), and the inflammatory cells are known to be polarized within the tumor microenvironment to release growth and survival factors, extracellular proteases, proangiogenic factors, and chemokines, which contribute to tumor growth and progression to malignancy (18Balkwill F. Mantovani A. Lancet. 2001; 357: 539-545Abstract Full Text Full Text PDF PubMed Scopus (6039) Google Scholar, 19Coussens L.M. Werb Z. Nature. 2002; 420: 860-867Crossref PubMed Scopus (11244) Google Scholar, 20Pikarsky E. Porat R.M. Stein I. Abramovitch R. Amit S. Kasem S. Gutkovich-Pyest E. Urieli-Shoval S. Galun E. Ben-Neriah Y. Nature. 2004; 431: 461-466Crossref PubMed Scopus (2145) Google Scholar). Among proinflammatory mediators, platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is becoming recognized as a major primary and secondary messenger involved in homotypic and heterotypic cell-to-cell communication that results in the activation of platelets, neutrophils, macrophages, lymphocytes, and endothelial cells (21Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (586) Google Scholar). At sites of acute and chronic inflammation, PAF mediates cell migration, aggregation, adhesion to endothelial cells, chemotaxis, degranulation, production of superoxide and inflammatory cytokines, and mitosis (21Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (586) Google Scholar, 22Zimmerman G.A. McIntyre T.M. Prescott S.M. Stafforini D.M. Crit. Care Med. 2002; 30: 294-301Crossref PubMed Scopus (324) Google Scholar, 23Camussi G. Montrucchio G. Lupia E. De Martino A. Perona L. Arese M. Vercellone A. Toniolo A. Bussolino F. J. Immunol. 1995; 154: 6492-6501PubMed Google Scholar). PAF is synthesized from the 1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine via a reaction catalyzed by phospholipase A2/acetyl-CoA:lyso-PAF acetyltransferase, which also yields a COX-2 substrate, arachidonic acid (21Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (586) Google Scholar). In pathologic situations, PAF and PAF-like oxidized phospholipids (21Prescott S.M. Zimmerman G.A. Stafforini D.M. McIntyre T.M. Annu. Rev. Biochem. 2000; 69: 419-445Crossref PubMed Scopus (586) Google Scholar, 24Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. Crit. Rev. Clin. Lab. Sci. 2003; 40: 643-672Crossref PubMed Scopus (184) Google Scholar) are produced in an unregulated fashion, which results in stimulation of the seven-domain, membrane-spanning G protein-coupled PAFR and the induction of various intracellular signaling cascades through arachidonate and phosphoinositide turnover, activation of protein kinase C, modulation of PKA, and increased protein-tyrosine phosphorylation (24Stafforini D.M. McIntyre T.M. Zimmerman G.A. Prescott S.M. Crit. Rev. Clin. Lab. Sci. 2003; 40: 643-672Crossref PubMed Scopus (184) Google Scholar, 25Deo D.D. Bazan N.G. Hunt J.D. J. Biol. Chem. 2004; 279: 3497-3508Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 26Honda Z. Takano T. Gotoh Y. Nishida E. Ito K. Shimizu T. J. Biol. Chem. 1994; 269: 2307-2315Abstract Full Text PDF PubMed Google Scholar, 27Kravchenko V.V. Pan Z. Han J. Herbert J.M. Ulevitch R.J. Ye R.D. J. Biol. Chem. 1995; 270: 14928-14934Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, 28Kuruvilla A. Pielop C. Shearer W.T. J. Immunol. 1994; 153: 5433-5442PubMed Google Scholar, 29Shimizu T. Mutoh H. Kato S. Adv. Exp. Med. Biol. 1996; 416: 79-84Crossref PubMed Google Scholar). Several lines of evidence suggest that PAFR-mediated signaling may be involved in tumor growth, angiogenesis, and metastatic dissemination. PAFR-overexpressing transgenic mice displayed proliferative disorders and melanocytic tumors (30Ishii S. Nagase T. Tashiro F. Ikuta K. Sato S. Waga I. Kume K. Miyazaki J. Shimizu T. EMBO J. 1997; 16: 133-142Crossref PubMed Scopus (130) Google Scholar, 31Ishii S. Shimizu T. Prog. Lipid Res. 2000; 39: 41-82Crossref PubMed Scopus (328) Google Scholar, 32Sato S. Kume K. Ito C. Ishii S. Shimizu T. Arch. Dermatol. Res. 1999; 291: 614-621Crossref PubMed Scopus (27) Google Scholar). Inactivation of PAF by overexpression of PAF acetylhydrolase inhibited the growth and vascularization of B16F10 murine melanomas and Kaposi sarcomas (33Biancone L. Cantaluppi V. Del Sorbo L. Russo S. Tjoelker L.W. Camussi G. Clin. Cancer Res. 2003; 9: 4214-4220PubMed Google Scholar). In addition, intraperitoneally injected PAF promoted experimental lung metastasis of B16F10 cells (34Im S.Y. Ko H.M. Kim J.W. Lee H.K. Ha T.Y. Lee H.B. Oh S.J. Bai S. Chung K.C. Lee Y.B. Kang H.S. Chun S.B. Cancer Res. 1996; 56: 2662-2665PubMed Google Scholar). PAF mediated the adhesiveness of Hs294T human melanoma cells and LS180 human colon cancer cells to IL-1-stimulated endothelial cells (35Mannori G. Barletta E. Mugnai G. Ruggieri S. Clin. Exp. Metastasis. 2000; 18: 89-96Crossref PubMed Scopus (22) Google Scholar). Vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, tumor necrosis factor-α, and thrombin have been shown to induce PAF production by human breast cancer cells in vitro and to induce PAF-dependent cell proliferation (36Bussolati B. Biancone L. Cassoni P. Russo S. Rola-Pleszczynski M. Montrucchio G. Camussi G. Am. J. Pathol. 2000; 157: 1713-1725Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). In vivo, PAFR antagonists inhibited growth and neoangiogenesis in breast cancer tumors (36Bussolati B. Biancone L. Cassoni P. Russo S. Rola-Pleszczynski M. Montrucchio G. Camussi G. Am. J. Pathol. 2000; 157: 1713-1725Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). More importantly for melanoma tumorigenesis, keratinocytes secreted PAF in response to irradiation with ultraviolet light (37Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.W. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar, 38Walterscheid J.P. Ullrich S.E. Nghiem D.X. J. Exp. Med. 2002; 195: 171-179Crossref PubMed Scopus (199) Google Scholar). Moreover, the keratinocytes expressed PAFR, and PAF up-regulated the expression levels of COX-2, IL-6, IL-8, and IL-10 cells and the secretion levels of prostaglandin E2 (37Pei Y. Barber L.A. Murphy R.C. Johnson C.A. Kelley S.W. Dy L.C. Fertel R.H. Nguyen T.M. Williams D.A. Travers J.B. J. Immunol. 1998; 161: 1954-1961PubMed Google Scholar, 38Walterscheid J.P. Ullrich S.E. Nghiem D.X. J. Exp. Med. 2002; 195: 171-179Crossref PubMed Scopus (199) Google Scholar). Despite this knowledge, the role of PAF in the acquisition of the metastatic phenotype and the changes in gene expression in human melanoma cells have not been studied. In this study, we demonstrated that PAF induced CREB phosphorylation and activation in metastatic melanoma cells through the signaling cascade involving PAFR-mediated activation of pertussis toxin (Ptx)-insensitive Gαq protein, adenylate cyclase (AC), p38 MAPK, and PKA. Furthermore, PAF induced CREB-dependent expression and activation of MMP-2. Finally, we found that PAFR antagonists potently inhibited experimental melanoma lung metastasis. Taken together, our results proposed that compared with nonmetastatic cells, metastatic human melanoma cells that overexpress CREB and ATF-1 are better equipped to respond to inflammatory stimuli from the tumor microenvironment. Reagents and Antibodies—PAF, its metabolically stable analogue carbamyl-PAF (cPAF), PAFR antagonists PCA4248 and (±)-trans-2,5-bis(3,4,5-trimethoxyphenyl)-1,3-dioxolane (hereafter referred to as dioxolane), Ptx-insensitive G protein antagonist GPA-2A, AC inhibitor SQ22536, PKA inhibitor H-89, and p38 MAPK inhibitor SB202190 were obtained from Biomol (Plymouth Meeting, PA). Rabbit polyclonal anti-phospho-CREB (serine 133) antibody, rabbit polyclonal anti-CREB antibody, rabbit polyclonal anti-MMP-2 antibody, rabbit polyclonal anti-phospho-p38 MAPK (threonine 180/tyrosine 182) antibody, rabbit polyclonal anti-p38 MAPK, and horseradish peroxidase-conjugated donkey anti-rabbit antibody were from Cell Signaling Technology (Beverly, MA). Rabbit polyclonal anti-PAFR (N terminus) and rabbit polyclonal anti-MT1-MMP antibodies were purchased from Chemicon International (Temecula, CA). Fetal bovine serum was from Cambrex (Walkersville, MD). DNA Constructs—The CRE-driven luciferase reporter vector was generated by ligating one copy of the somatostatin gene promoter region (nucleotides -71 to +53), into the pGL3-Luc-Basic vector (Invitrogen). The original Somat-BgllI CAT construct reporter containing somatostatic gene promoter was obtained from Dr. Marc R. Montminy (Harvard Medical School, Boston). The MMP-2-driven luciferase reporter construct containing one copy of the human MMP-2 promoter gene region (nucleotides -393 to +290) was ligated into the pGL3-Luc-Basic reporter vector as described previously (39Mills L. Tellez C. Huang S. Baker C. McCarty M. Green L. Gudas J.M. Feng X. Bar-Eli M. Cancer Res. 2002; 62: 5106-5114PubMed Google Scholar). pRSV-KCREB was kindly provided by Dr. Richard H. Goodman (Oregon Health Sciences, Portland). pRSV-KCREB expression plasmid contained full-length CREB cDNA with a single base pair substitution in the DNA-binding domain that causes a change from Arg287 to Leu287 (10Walton K.M. Rehfuss R.P. Chrivia J.C. Lochner J.E. Goodman R.H. Mol. Endocrinol. 1992; 6: 647-655PubMed Google Scholar). pRc/RSV construct (Invitrogen) lacking CREB cDNA was used as a control. pRL-CMV-BActin reporter vector was from Promega. Cell Culture—We used the following human melanoma cell lines with various metastatic potentials in the experimental nude mice model: nonmetastatic (SB2, DX3, and TXM40), moderately metastatic (TXM13, TXM18, and MeWo), and highly metastatic (WM2664 and A375SM); these eight cell lines are described elsewhere (40Luca M. Hunt B. Bucana C.D. Johnson J.P. Fidler I.J. Bar-Eli M. Melanoma Res. 1993; 3: 35-41Crossref PubMed Scopus (112) Google Scholar). The highly metastatic A375SM human melanoma cell line, which was used for most of our experiments, was established from pooled lung metastases produced by parental A375 cells injected intravenously into nude mice (41Li L. Price J.E. Fan D. Zhang R.D. Bucana C.D. Fidler I.J. J. Natl. Cancer Inst. 1989; 81: 1406-1412Crossref PubMed Scopus (126) Google Scholar). All cells were routinely cultured in minimum essential medium supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 1% HEPES buffer, and 1% penicillin and streptomycin. Cells were grown at 37 °C in a 5% CO2 atmosphere. Generation of KCREB A375SM Clones—Generation of stable A375SM cell clones expressing the dominant-negative form of CREB, KCREB, was performed as described previously for MeWo cells (11Xie S. Price J.E. Luca M. Jean D. Ronai Z. Bar-Eli M. Oncogene. 1997; 15: 2069-2075Crossref PubMed Scopus (109) Google Scholar, 13Jean D. Harbison M. McConkey D.J. Ronai Z. Bar-Eli M. J. Biol. Chem. 1998; 273: 24884-24890Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 42Yang Y.M. Dolan L.R. Ronai Z. Oncogene. 1996; 12: 2223-2233PubMed Google Scholar). Briefly, 1 × 105 cells were transfected with 1 μg of the pRSV-KCREB or pRc/RSV construct by using 2 μl of Lipofectamine 2000 reagent (Invitrogen). Six hours later, the transfection medium was replaced with serum-containing growth medium. To select for stable transfectants, growth medium was supplemented with 2 mg/ml G418 (Invitrogen). Expression of KCREB was verified by dual luciferase assay using pGL3-Luc construct driven by a somatostatin gene promoter containing four CREs. Transient Transfections and Luciferase Activity Assays—Transient transfections were performed using Lipofectamine 2000. A total of 25 × 103 cells/well in a 24-well plate were transfected with 0.5 μg of the basic pGL3 expression vector with no promoter or enhancer sequence (pGL3-Basic) or with 0.5 μg of the pGL3/CRE-Luc or pGL3/MMP2-Luc expression construct. After 6 h, the transfection medium was replaced with serum-containing growth medium. PAF was added 24 h later, and after the relevant incubation period, the cells were harvested and lysed, and luciferase activity was assayed using a dual luciferase reporter assay system (Promega, Madison, WI) as instructed by the manufacturer. For each transfection, 30 ng of Renilla luciferase reporter pRL-CMV-BActin (Promega) was included to normalize for differences in transfection efficiency. Zymography—Zymographic assays were performed as described previously (11Xie S. Price J.E. Luca M. Jean D. Ronai Z. Bar-Eli M. Oncogene. 1997; 15: 2069-2075Crossref PubMed Scopus (109) Google Scholar, 39Mills L. Tellez C. Huang S. Baker C. McCarty M. Green L. Gudas J.M. Feng X. Bar-Eli M. Cancer Res. 2002; 62: 5106-5114PubMed Google Scholar). Briefly, after cells were incubated with PAF for 16 h, the medium was replaced with serum-free minimum essential medium. The conditioned medium was collected 16 h later and separated by electrophoresis in a polyacrylamide gel containing 1 mg/ml gelatin. The volume of supernatants loaded on the gel was normalized to the cell number. The gel was then washed at room temperature for 2 h with 2.5% Triton X-100 and, subsequently, at 37 °C overnight in a buffer containing 10 mm CaCl2, 150 mm NaCl, and 50 mm Tris-HCl, pH 7.5. The gel was stained with 0.5% Coomassie Blue and photographed on a light box. Proteolysis was detected as a white zone in a dark blue field. MMP-2 Activity Assay—Cells were treated as described for the zymography experiments except that the medium without phenol red was used in the last step of collecting the conditioned supernatants, which were analyzed for their ability to cleave a fluorogenic peptide substrate (7-methoxycoumarin-4-yl-acetyl-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl-l-2,3-diaminopropionyl)-Ala-Arg-NH2) as described by the manufacturer (R & D Systems, Minneapolis, MN). Briefly, the volume of supernatants was normalized to the cell number, and 100-μl aliquots were added to 10 μg of a substrate diluted in TCNB reaction buffer (50 mm Tris, pH 7.5, 150 mm CaCl2, 10 mm NaCl, 0.05% Brij). The fluorescence of the cleaved substrate was measured 16 h later (excitation wavelength 320 nm and emission wavelength 405 nm). A specific concentration of active MMP-2 was recalculated based on a standard curve obtained with recombinant human MMP-2 activated by 1 μm p-aminophenylmercuric acetate for 1 h at 37 °C in TCNB buffer. Western Blotting—Proteins of total cell extract (typically 15 μg) were separated by 10% SDS-PAGE and transferred to Immobilon P transfer membrane (Millipore, Bedford, MA). The membranes were washed in Tris-buffered saline with Tween (10 mm Tris-HCl, pH 8, 150 mm NaCl, and 0.05% Tween 20) and blocked with 5% nonfat milk in Tris-buffered saline with Tween for 2 h at room temperature. The blots were then probed overnight with relevant antibodies at dilution of 1:1000 except for the anti-CREB antibody (1:2000) and anti-phospho-p38 MAPK anti-body (1:500). After 2 h of incubation with horseradish peroxide-conjugated secondary antibody, immunoreactive proteins were detected by enhanced chemiluminescence per the manufacturer's instructions (ECL detection system; Amersham Biosciences). Electrophoretic Mobility Shift Assay (EMSA)—The EMSA probe consisted of annealed synthetic complementary oligonucleotides containing the cAMP-binding consensus site (TGACGCTA) and was obtained from Promega. 32P-End-labeled oligonucleotides (20,000 cpm) were incubated for 30 min at 30 °C with 1 μg of nuclear extract in 20 μl of binding buffer containing 25 mm HEPES, pH 7.9, 0.5 mm EDTA, 0.5 mm dithiothreitol, 4% glycerol, 50 mm NaCl, and 0.5 μg of poly(dI-dC). Competition reactions were performed with a 100-fold molar excess of unlabeled double-stranded CRE competitor DNA (Promega). For supershift analysis, the nuclear extracts were incubated with 1 μg of anti-CREB antibody for 1 h prior to the binding reaction with labeled probe. The DNA-protein complexes were separated on 4% native polyacrylamide gel in 0.5× Tris borate-EDTA buffer. Detection of Apoptosis by Propidium Iodide (PI) Staining and Flow Cytometry—Cells at 90% confluency were exposed to the PAFR antagonist PCA4248, collected at various time points, washed once with cold phosphate-buffered saline, and fixed in cold 70% ethanol overnight. Cells were then rehydrated using PBS, resuspended in PBS solution containing 50 μg/ml PI (Sigma) and 20 μg/ml RNase A, and incubated at 37 °C for 20 min. Cell cycle analysis was performed on a flow cytometer (Epics XL-MCL; Beckman Coulter, Brea, CA) using MultiCycle software (Phoenix Flow Systems, San Diego, CA). Confocal Microscopy—Cells were plated onto chamber slides and allowed to attach. After three brief PBS washes, cells were fixed with 4% paraformaldehyde/PBS, permeabilized with 0.1% Triton X-100/PBS, blocked in 5% normal horse serum, 1% normal goat serum/PBS, exposed to an anti-PAFR antibody (1:100) in blocking solution overnight at 4 °C and then exposed to a secondary goat anti-rabbit-Cy5 antibody (1:1200) for 1 h at room temperature, washed with PBS, exposed to 10 nmol/liter Sytox green for 10 min to stain nuclei, washed, and covered with propylgallate and coverslips for microscopic evaluation. Confocal microscopy was done with a Zeiss LSM 510 confocal microscope and LSM 510 Image Brower software (Carl Zeiss). MTT Assays—A375SM cells were plated onto 96-well plates (1 × 104 cells/well). Complete medium was supplemented with cPAF or PCA4248 at different concentrations. For the MTT analysis, cells were incubated with 1 mg/ml methyltetrazolium bromide for 3 h, after which the medium was replaced with dimethyl sulfoxide (Me2SO), and resuspended. Absorbance was read at 570 nm using a Ceres UV900C microplate reader (Bio-Tek Instruments, Inc., Winooski, VT). Animals and Experimental Lung Metastasis Assays—Male athymic BALB/c nude mice were purchased from the NCI-Frederick Cancer Research Facility (Frederick, MD). The mice were housed in laminar flow cabinets under specific pathogen-free conditions and used at 8 weeks of age. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the Department of Agriculture, Department of Health and Human Services, and the National Institutes of Health. For cell injection, A375SM cells in the exponential growth phase were harvested by brief exposure to 0.25% trypsin, 0.2% EDTA solution (w/v). Cell viability was determined by trypan blue exclusion, and only single-cell suspensions of more than 90% viability were used. Cells were wa" @default.
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• W1973417966 title "Platelet-activating Factor Mediates MMP-2 Expression and Activation via Phosphorylation of cAMP-response Element-binding Protein and Contributes to Melanoma Metastasis" @default.
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• W1973417966 doi "https://doi.org/10.1074/jbc.m508683200" @default.
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