Regulation of the Melanoma Cell Adhesion Molecule Gene in Melanoma: Modulation of mRNA Synthesis by Cyclic Adenosine Monophosphate, Phorbol Ester, and Stem Cell Factor/c-Kit Signaling
1999; Elsevier BV; Volume: 113; Issue: 5 Linguagem: Inglês
10.1046/j.1523-1747.1999.00746.x
ISSN1523-1747
AutoresStéphane Karlen, Lasse R. Braathen,
Tópico(s)Mast cells and histamine
ResumoThe melanoma cell adhesion molecule was identified as a human melanoma-associated antigen that increases in expression as tumors increase in thickness and begin to acquire metastatic potential. Clinical and experimental evidences suggest that the development of metastatic capacity might be the consequence of increased melanoma cell adhesion molecule expression. The mechanisms for upregulation of the melanoma cell adhesion molecule during melanoma progression are, however, still poorly understood. In this study, we show that melanoma cell adhesion molecule expression is tightly regulated at the transcriptional level. Using a combination of CAT reporter assays and semiquantitative reverse transcriptase–polymerase chain reaction, we observed that cyclic adenosine monophosphate significantly increases transcription of the melanoma cell adhesion molecule in nonmetastatic melanoma cells. In metastatic cells, transcription of the gene was constitutive and could not be further increased by cyclic adenosine monophosphate. On the other hand, melanoma cell adhesion molecule promoter activity was impeded upon treatment with phorbol esters or in the presence of stem cell factor, a phenomenon which was protein kinase C-dependent. Promoter-deletion studies demonstrated that the first 196 nt of the melanoma cell adhesion molecule promoter region are sufficient to get full expression in metastatic melanoma cells. This fragment contains five binding sites for the transcription factor Sp1 and DNA mobility shift experiments showed direct binding of Sp1 to the promoter. In conclusion, our results indicate that Sp1 is sufficient to drive constitutive melanoma cell adhesion molecule expression in metastatic melanoma cells. In nonmetastatic cells, however, melanoma cell adhesion molecule expression is repressed and we speculate that stem cell factor/c-Kit signaling might be responsible for the control of melanoma cell adhesion molecule synthesis, and thus, perhaps, of melanoma progression and metastasis. The melanoma cell adhesion molecule was identified as a human melanoma-associated antigen that increases in expression as tumors increase in thickness and begin to acquire metastatic potential. Clinical and experimental evidences suggest that the development of metastatic capacity might be the consequence of increased melanoma cell adhesion molecule expression. The mechanisms for upregulation of the melanoma cell adhesion molecule during melanoma progression are, however, still poorly understood. In this study, we show that melanoma cell adhesion molecule expression is tightly regulated at the transcriptional level. Using a combination of CAT reporter assays and semiquantitative reverse transcriptase–polymerase chain reaction, we observed that cyclic adenosine monophosphate significantly increases transcription of the melanoma cell adhesion molecule in nonmetastatic melanoma cells. In metastatic cells, transcription of the gene was constitutive and could not be further increased by cyclic adenosine monophosphate. On the other hand, melanoma cell adhesion molecule promoter activity was impeded upon treatment with phorbol esters or in the presence of stem cell factor, a phenomenon which was protein kinase C-dependent. Promoter-deletion studies demonstrated that the first 196 nt of the melanoma cell adhesion molecule promoter region are sufficient to get full expression in metastatic melanoma cells. This fragment contains five binding sites for the transcription factor Sp1 and DNA mobility shift experiments showed direct binding of Sp1 to the promoter. In conclusion, our results indicate that Sp1 is sufficient to drive constitutive melanoma cell adhesion molecule expression in metastatic melanoma cells. In nonmetastatic cells, however, melanoma cell adhesion molecule expression is repressed and we speculate that stem cell factor/c-Kit signaling might be responsible for the control of melanoma cell adhesion molecule synthesis, and thus, perhaps, of melanoma progression and metastasis. chloramphenicol acetyltransferase cAMP response element cAMP response element binding protein glyceraldehyde-3-phosphate-dihydrogenase melanoma cell adhesion molecule stem cell factor Malignant melanomas are invasive and highly metastatic tumors which arise from dendritic pigmented melanocytes (Mancianti and Herlyn, 1989Mancianti M.L. Herlyn M. Tumor progression in melanoma: the biology of epidermal melanocytes in vitro.Carcinog Compr Surv. 1989; 11: 369-386PubMed Google Scholar). The cell surface glycoprotein melanoma cell adhesion molecule (MCAM) (previously known as MUC18, Mel-cam, or CD146) was identified as a human melanoma-associated antigen that increases in expression as tumors increase in vertical thickness and begin to acquire metastatic potential (Lehmann et al., 1987Lehmann J.M. Holzmann B. Breitbart E.W. Schmiegelow P. Riethmuller G. Johnson J.P. Discrimination between benign and malignant cells of melanocytic lineage by two novel antigens, a glycoprotein with a molecular weight of 113,000 and a protein with a molecular weight of,. 76,000.Cancer Res. 1987; 47: 841-845PubMed Google Scholar). No expression of MCAM antigen has been detected in normal melanocytes, normal adult skin, or skin adjacent to melanocytic lesions (Shih et al., 1994Shih I.M. Elder D.E. Hsu M.Y. Herlyn M. Regulation of Mel-CAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.Am J Pathol. 1994; 145: 837-845PubMed Google Scholar,Shih et al., 1994Shih I.M. Elder D.E. Speicher D. Johnson J.P. Herlyn M. Isolation and functional characterization of the A32 melanoma-associated antigen.Cancer Res. 1994; 54: 2514-2520PubMed Google Scholar;Kraus et al., 1997Kraus A. Masat L. Johnson J.P. Analysis of the expression of intercellular adhesion molecule-1 and MUC18 on benign and malignant melanocytic lesions using monoclonal antibodies directed against distinct epitopes and recognizing denatured, non-glycosylated antigen.Melanoma Res. 1997; 7: S75-S81PubMed Google Scholar). MCAM antigen, however, can be detected on nevi, but at lower levels than on primary or metastatic melanoma (Shih et al., 1994Shih I.M. Elder D.E. Hsu M.Y. Herlyn M. Regulation of Mel-CAM/MUC18 expression on melanocytes of different stages of tumor progression by normal keratinocytes.Am J Pathol. 1994; 145: 837-845PubMed Google Scholar). The expression of MCAM by human melanoma cell lines correlates with their ability to grow and produce metastases in nude mice (Luca et al., 1993Luca M. Hunt B. Bucana C.D. Johnson J.P. Fidler I.J. Bar-Eli M. Direct correlation between MUC18 expression and metastatic potential of human melanoma cells.Melanoma Res. 1993; 3: 35-41Crossref PubMed Scopus (112) Google Scholar). Moreover, enforced expression of MCAM in low-tumorigenic, nonmetastatic melanoma cells significantly increases their tumorigenicity and metastatic potential in nude mice (Xie et al., 1997Xie S. Luca M. Huang S. Gutman M. Reich R. Johnson J.P. Bar-Eli M. Expression of MCAM/MUC18 by human melanoma cells leads to increased tumor growth and metastasis.Cancer Res. 1997; 57: 2295-2303PubMed Google Scholar) and the production of tumorigenic variants from a nontumorigenic melanoma cell line is accompanied by MCAM upregulation (Bani et al., 1996Bani M.R. Rak J. Adachi D. Wiltshire R. Trent J.M. Kerbel R.S. Ben-David Y. Multiple features of advanced melanoma recapitulated in tumorigenic variants of early stage (radial growth phase) human melanoma cell lines: evidence for a dominant phenotype.Cancer Res. 1996; 56: 3075-3086PubMed Google Scholar). Finally, transfection of AP-2 regulatory factors into highly metastasic melanoma cells inhibits their tumor growth and metastatic potential in nude mice through downregulation of MCAM expression (Jean et al., 1998Jean D. Gershenwald J.E. Huang S. Luca M. Hudson M.J. Tainsky M.A. Bar-Eli M. Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells.J Biol Chem. 1998; 273: 16501-16508Crossref PubMed Scopus (134) Google Scholar). These various lines of evidence support the hypothesis that the development of metastatic capacity in melanoma may result from increased MCAM expression. The mechanisms for upregulation of MCAM expression during melanoma progression are unknown. There is no evidence that the expression of MCAM by malignant tumors is associated either with chromosomal translocation, mutation, or gene amplification (Luca et al., 1993Luca M. Hunt B. Bucana C.D. Johnson J.P. Fidler I.J. Bar-Eli M. Direct correlation between MUC18 expression and metastatic potential of human melanoma cells.Melanoma Res. 1993; 3: 35-41Crossref PubMed Scopus (112) Google Scholar). MCAM expression is essentially restricted to tumors of the neuroectodermal lineage and in cutaneous melanoma it appears to increase with tumor thickness and progression. These two observations indicate that the regulation of MCAM synthesis is transcriptional (Grimm and Johnson, 1995Grimm T. Johnson J.P. Ectopic expression of carcinoembryonic antigen by a melanoma cell leads to changes in the transcription of two additional cell adhesion molecules.Cancer Res. 1995; 55: 3254-3257PubMed Google Scholar). An increase in intracellular cyclic adenosine monophosphate (cAMP) levels leads to an upregulation of cell surface MCAM due to increased mRNA synthesis (Rummel et al., 1996Rummel M.M. Sers C. Johnson J.P. Phorbol ester and cyclic AMP-mediated regulation of the melanoma-associated cell adhesion molecule MUC18/MCAM.Cancer Res. 1996; 56: 2218-2223PubMed Google Scholar). In contrast, exposure of the cells to phorbol esters reduces expression to background levels by 24 h. This downregulation is associated with a significant decrease in mRNA levels. These later observations point to a protein kinase C (PKC)-activated pathway that could repress MCAM expression. In malignant tumors this pathway would be no longer functional. The proto-oncogene c-Kit encodes a transmembrane tyrosine-protein kinase receptor. Whereas normal function of c-Kit is required for human melanocyte development, its expression progressively decreases during local tumor growth and invasion of human melanoma (Lassam and Bickford, 1992Lassam N. Bickford S. Loss of c-kit expression in cultured melanoma cells.Oncogene. 1992; 7: 51-56PubMed Google Scholar;Natali et al., 1992Natali P.G. Nicotra M.R. Sures I. Santoro E. Bigotti A. Ullrich A. Expression of c-kit receptor in normal and transformed human nonlymphoid tissues.Cancer Res. 1992; 52: 6139-6143PubMed Google Scholar). The ligand for c-Kit, stem cell factor (SCF), has a growth inhibitory effect on c-Kit expressing melanoma cells (Funasaka et al., 1992Funasaka Y. Boulton T. Cobb M. et al.c-Kit-kinase induces a cascade of protein tyrosine phosphorylation in normal human melanocytes in response to mast cell growth factor and stimulates mitogen-activated protein kinase but is down-regulated in melanomas.Mol Biol Cell. 1992; 3: 197-209Crossref PubMed Scopus (178) Google Scholar;Zakut et al., 1993Zakut R. Perlis R. Eliyahu S. Yarden Y. Givol D. Lyman S.D. Halaban R. KIT ligand (mast cell growth factor) inhibits the growth of KIT-expressing melanoma cells.Oncogene. 1993; 8: 2221-2229PubMed Google Scholar) by inducing apoptosis (Huang et al., 1996Huang S. Luca M. Gutman M. McConkey D.J. Langley K.E. Lyman S.D. Bar-Eli M. Enforced c-KIT expression renders highly metastatic human melanoma cells susceptible to stem cell factor-induced apoptosis and inhibits their tumorigenic and metastatic potential.Oncogene. 1996; 13: 2339-2347PubMed Google Scholar). As SCF has been shown to be a regulator of PKC activity (Blume-Jensen et al., 1993Blume-Jensen P. Siegbahn A. Stabel S. Heldin C.H. Ronnstrand L. Increased Kit/SCF receptor induced mitogenicity but abolished cell motility after inhibition of protein kinase C.EMBO J. 1993; 12: 4199-4209Crossref PubMed Scopus (88) Google Scholar,Blume-Jensen et al., 1995Blume-Jensen P. Wernstedt C. Heldin C.H. Ronnstrand L. Identification of the major phosphorylation sites for protein kinase C in kit/stem cell factor receptor in vitro and in intact cells.J Biol Chem. 1995; 270: 14192-14200Crossref PubMed Scopus (80) Google Scholar), it seems possible that MCAM expression may be controlled by signaling through the c-Kit receptor. Sequence analysis on the MCAM promoter region reveals several interesting properties (Sers et al., 1993Sers C. Kirsch K. Rothbacher U. Riethmuller G. Johnson J.P. Genomic organization of the melanoma-associated glycoprotein MUC18: implications for the evolution of the immunoglobulin domains.Proc Natl Acad Sci USA. 1993; 90: 8514-8518Crossref PubMed Scopus (128) Google Scholar). The 150 bp fragment found immediately upstream of the transcription site is 88% (G + C)-rich. No TATA or CAAT boxes can be identified, but the pyrimidine-rich initiator sequence (Inr) CTCACTT (Smale and Baltimore, 1989Smale S.T. Baltimore D. The “initiator” as a transcription control element.Cell. 1989; 57: 103-113Abstract Full Text PDF PubMed Scopus (1148) Google Scholar) is found overlapping the RNA start site. Five GC boxes which bind the transcription factor Sp1 (Dynan and Tjian, 1983Dynan W.S. Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter.Cell. 1983; 35: 79-87Abstract Full Text PDF PubMed Scopus (911) Google Scholar) are clustered within 130 bp from the Inr. The MCAM promoter also contains four putative AP-2 binding elements. AP-2 plays an important part in the control of gene expression in melanomas. Upregulation of MCAM (Jean et al., 1998Jean D. Gershenwald J.E. Huang S. Luca M. Hudson M.J. Tainsky M.A. Bar-Eli M. Loss of AP-2 results in up-regulation of MCAM/MUC18 and an increase in tumor growth and metastasis of human melanoma cells.J Biol Chem. 1998; 273: 16501-16508Crossref PubMed Scopus (134) Google Scholar) and downregulation of c-Kit expression (Huang et al., 1998Huang S. Jean D. Luca M. Tainsky M.A. Bar-Eli M. Loss of AP-2 results in downregulation of c-KIT and enhancement of melanoma tumorigenicity and metastasis.EMBO J. 1998; 17: 4358-4369Crossref PubMed Scopus (228) Google Scholar) in highly metastatic cells correlate with loss of expression of the transcription factor AP-2. A consensus motif for the cAMP-response element (CRE) is found at position –32 in the control region. The CRE-binding factor, CREB, has been recently found to be involved in the malignant transformation of melanoma cells (Jean et al., 1998Jean D. Harbison M. McConkey D.J. Ronai Z. Bar-Eli M. CREB and its associated proteins act as survival factors for human melanoma cells.J Biol Chem. 1998; 273: 24884-24890Crossref PubMed Scopus (144) Google Scholar). As mentioned above, MCAM expression can be modulated by cAMP, thus this particular CRE motif could account for MCAM upregulation in response to cAMP-inducing agents. In this study, we developed a transient transfection assay for measuring the activity of MCAM-CAT reporter gene constructs in human melanoma cells. We demonstrated that MCAM expression is tightly regulated at the transcriptional level. We also provide evidence that MCAM mRNA synthesis can be downregulated through activation of the SCF/c-Kit regulatory pathway. The human melanoma cell line SK-Mel2 was purchased from ATCC (catalog no. HTB68) and is highly metastatic in nude mice (Fogh et al., 1977Fogh J. Fogh J.M. Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.J Natl Cancer Inst. 1977; 59: 221-226Crossref PubMed Scopus (1300) Google Scholar). The SB2 cell line was isolated from a primary cutaneous lesion and was a gift from Dr B. Giovanella (The Stehlin Foundation for Cancer Research, Houston, TX). In nude mice the SB2 cell line is poorly tumorigenic and nonmetastatic (Verschraegen et al., 1991Verschraegen C.F. Giovanella B.C. Mendoza J.T. Kozielski A.J. Stehlin J.S.J. Specific organ metastases of human melanoma cells injected into the arterial circulation of nude mice.Anticancer Res. 1991; 11: 529-535PubMed Google Scholar;Luca et al., 1993Luca M. Hunt B. Bucana C.D. Johnson J.P. Fidler I.J. Bar-Eli M. Direct correlation between MUC18 expression and metastatic potential of human melanoma cells.Melanoma Res. 1993; 3: 35-41Crossref PubMed Scopus (112) Google Scholar;Singh et al., 1995Singh R.K. Gutman M. Reich R. Bar-Eli M. Ultraviolet B irradiation promotes tumorigenic and metastatic properties in primary cutaneous melanoma via induction of interleukin 8.Cancer Res. 1995; 55: 3669-3674PubMed Google Scholar). The SK-Mel2 cell line was maintained in culture as adherent monolayers in Earle’s minimal essential medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, 2-fold vitamin solution, and penicillin–streptomycin (Gibco-Life Technologies, Basel, Switzerland). The SB2 cells were maintained in MCDB153 medium (Sigma, St Louis, MO) supplemented with LB15 medium (four parts MCDB and one part LB15), 2% fetal bovine serum, L-glutamine, and penicillin–streptomycin. For stimulation studies, phorbol 12-myristate 13-acetate (PMA) and forskolin (both purchased from Sigma) were used at concentrations of 10 ng per ml and 20 μM, respectively. The human recombinant SCF was obtained from Roche Diagnostics (Basel, Switzerland) and was used at a final concentration of 50 ng per ml. The cAMP-dependent protein kinase inhibitor H89 and the PKC inhibitor GF109203X were purchased from Calbiochem (La Jolla, CA) and used at a concentration of 5 μM. To generate the MCAM promoter CAT construct (pMCAM-IV-CAT; Figure 1), the following strategy was used. The 5′-flanking region of the MCAM gene (nt +5 to –527) was amplified by PCR, using the Clontech Advantage-GC genomic PCR system (Palo Alto, CA). The RNA initiation site was designated +1. The PCR product was subcloned upstream of the CAT gene into the pCAT3 basic vector (Promega, Madison, WI) by using the SacI and BglII sites. The sequence of the promoter was verified by sequencing. The construct pMCAM-III-CAT was obtained by cutting pMCAM-IV-CAT at the SacI site (site in the polylinker) and TthIII1 (nt –392), filling the ends with T4 DNA, and religating. Similarly, pMCAM-II-CAT, pMCAM-I-CAT and pMCAM-0-CAT were made by cutting the pMCAM-IV-CAT construct at the SacI site and the BstXI (nt –195), SrfI (nt –65), or XhoI (nt –11), respectively, making the ends blunt and religating. pMCAM-del1-CAT, pMCAM-del2-CAT, and pMCAM-del3-CAT were obtained by removing from pMCAM-IV-CAT the AatII–XhoI fragment (nt –11 to –30) the BssHII–AatII fragment (nt –26 to –105) and the NarI–AatII fragment (nt –26 to –134), respectively. Finally, pMCAM-del4-CAT was made by cutting pMCAM-IV-CAT with XhoI (nt –11) and BglII (site in the polylinker), filling the ends and religating. The pSV-βGal vector was purchased from Promega. Melanoma cells (8 × 105) were transfected with 2.5 μg of the various MCAM CAT constructs and 0.5 μg of pSV-βGal DNA with Fugene6 reagent (Roche Diagnostics). Two hours post-transfection, PMA, forskolin, and SCF were added, either alone or in various combinations, and the cells were incubated further for 22 h at 37°C. The cells were then washed with phosphate-buffered saline (PBS) and harvested in 1 × Reporter Lysis Buffer (Promega). MCAM promoter activity was determined by measuring CAT activity. One hundred and twenty-five microliters of cell extract (preheated for 10 min at 60°C) were incubated for 3 h at 37°C in the presence of 5 μg per ml of n-Butyril-CoA and 150 μCi of D-threo-[dichloroacetyl-1-14C] chloramphenicol (Amersham). CAT activities were visualized by thin layer chromatography assay or quantitated by liquid scintillation counting (LSC assay). Normalization of transfection efficiency was done using a cotransfected pSV-βGal vector and measuring β-galactosidase activity. The efficiency of transfection in SB2 cells was 20 times lower than in SK-Mel2 cells. Fold activation or inhibition of CAT activity were calculated relative to control cells which were given the reference value of 100. Results are the mean of three independent experiments. Error bars represent SEM. Total RNA was extracted from 106 cells using the SV total RNA isolation system from Promega. Detection of MCAM, c-Kit, and GAPDH mRNA was performed using the one tube/two enzymes Access reverse transcriptase–PCR system developed by Promega and 100 ng of total RNA for standard reactions. The following oligonucleotides were used as primers. MCAM sense: (nt 185–206) 5′-CCAAGGCAACCTCAGCCATGTC-3′ MCAM anti-sense (nt 598–622) 5′-CTCGACTCCACAGTCTGGGACGACT-3′. c-Kit sense (nt 1326–1347) 5′-GCCCACAATAGATTGGTATTT-3′ c-Kit anti-sense (nt 1875–1896) 5′-AGCATCTTTACAGCGACAGTC-3′. GAPDH sense (nt 85–106) 5′-AACGGATTTGGTCGTATTGGGC-3′ and anti-sense (nt 663–684) 5′-AGGGATGATGTTCTGGAGAGCC-3′. PCR products were analyzed by electrophoresis on 2% agarose gels and visualized by ethidium bromide staining. For semiquantitative reverse transcriptase–PCR analysis of MCAM mRNA, we used the following strategy (Yawalkar et al., 1998Yawalkar N. Karlen S. Hunger R. Brand C.U. Braathen L.R. Expression of interleukin-12 is increased in psoriatic skin.J Invest Dermatol. 1998; 111: 1053-1057Crossref PubMed Scopus (201) Google Scholar). A 1:4 serial dilution from each RNA extraction was prepared, with the concentration of the first sample being set to 100 ng per μl. An aliquot from each dilution was then subjected to 32 cycles of PCR amplification (20 s at 95°C, 20 s at 52°C and 30 s at 72°C in a Perkin Elmer 9600 thermocycler) with the appropriate primers. The resulting products were analyzed as described above. The exposed films obtained from the gels were evaluated with an optometric scanner and the integrated optical density in arbitrary units of each amplified fragment was determined. The dilution at which a 50% reduction of the signal was obtained is determined for both the MCAM and the GAPDH messengers. The amounts of MCAM transcripts, relative to the GAPDH control, was calculated and the results were expressed as the relative amount of mRNA. Results are the mean of three independent experiments. Error bars represent SEM. Melanoma nuclear cell extracts were obtained from a small number of cells (107) using the method described bySchreiber et al., 1993Schreiber E. Tobler A. Malipiero U. Schaffner W. Fontana A. cDNA cloning of human N-Oct3, a nervous-system specific POU domain transcription factor binding to the octamer DNA motif.Nucleic Acids Res. 1993; 21: 253-258Crossref PubMed Scopus (79) Google Scholar with the following modifications: Complete Protease Inhibitor Cocktail solution (Roche Diagnostics), specific for serine, cysteine, and metalloproteases, was added to the reaction buffers just before lysis. Nuclear protein extracts were assayed for protein content by the BioRad (BioRad Laboratories, Hercules, CA) protein assay reagent kit. Standard binding reaction contained 2.5 μg of nuclear protein, 60 mM KCl, 8 mM MgCl2, 12 mM HEPES buffer pH 7.9, 12% glycerol, 1 mM dithiothreitol, 3 μg poly(dI–dC), and 25 fmol end-labeled oligonucleotide probe. The binding reactions were left on ice for 20 min. Protein/DNA complexes were resolved at 4°C on a 5% nondenaturing polyacrylamide gel. The gels were dried and exposed to a phosphoimager screen and analyzed with the Storm 860 instrument (Molecular Dynamics, Sunnyvale, CA). For competition assays, unlabeled oligonucleotides were added 20 min before adding the probe. The SCA probe was a 35 bp oligonucleotide which covers the MCAM promoter region from position –12 to –46 and includes the Sp1 site at nt –41, the CRE site at nt –32, and the AP-2 site at position –24. The 23 bp ASP probe covers the promoter sequence from nt –113 to –135 and contains the AP-2 site at position –130 and the Sp1 site at position –124. Competitor oligonucleotides with high-affinity binding sequences for either Sp1, CREB/ATF, or AP-2 were purchased from Santa-Cruz Biotechnology (Santa Cruz, CA). The mutated SCA oligonucleotides used in competition studies are described in Figure 9 and the mutated ASP oligonucleotides are shown in Figure 8. Unless otherwise stated, the oligonucleotides were synthesized by Microsynth (Balgach, Switzerland). Anti-Sp1, CREB/AT-1, ATF-2 and AP-2 antibodies (Santa-Cruz) were used in supershift analyses as previously described byKarlen et al., 1996Karlen S. D’Ercole M. Sanderson C.J. Two pathways can activate the interleukin-5 gene and induce binding to the conserved lymphokine element 0.Blood. 1996; 88: 211-221Crossref PubMed Google Scholar.Figure 8Binding of Sp1 to the ASp box. DNA mobility shift experiments were done with nuclear extracts from SK-Mel2 cells and a 23-mer end-labeled oligonucleotide (nt –113 to –135) containing the ASp box. (A) Specific binding to the ASp oligonucleotide. The probe was incubated either alone (probe) or with unstimulated (SK2) or forskolin-treated SK-Mel2 (SK2 + F) extracts. For competition experiments, the probe was incubated with unstimulated extracts and unlabeled homologous (ASp) or unrelated (AP-1) oligonucleotides were added in 25- and 125-fold excess. (B) Sp1 binds to the ASp box. The ASp probe was incubated with unstimulated SK-Mel2 nuclear extracts in the presence of competing oligonucleotides. For competition, homologous (ASp), AP-2 and Sp1 consensus and mutated (ASp-mut1, -mut2, and -mut3) oligonucleotides were added in 50-fold excess. (C) DNA supershift experiments. Where indicated, reactions were carried out in the presence of specific antibody directed against Sp1, AP-2, and CREB (anti-Sp1, anti-AP-2, and ant-CREB, respectively). (D) ASp oligonucleotide and corresponding mutants.View Large Image Figure ViewerDownload (PPT) In an attempt to elucidate further the molecular mechanisms controlling MCAM transcription, the activity of the CAT reporter gene driven by the MCAM promoter (nt +5 to –527; Figure 1) was analyzed in the highly metastatic SK-Mel2 melanoma cell line. The results are summarized in Figure 2(a,b). MCAM promoter activity was strong in unstimulated SK-Mel2 cells (Figure 2b,lane 1). Upon treatment with forskolin (a potent activator of adenylate cyclase and, therefore, of cAMP), however, MCAM promoter activity was only marginally increased (lane 2). The high level of MCAM promoter activity in unstimulated cells was not surprising. Like the majority of the melanoma cell lines, SK-Mel2 is tumorigenic and metastatic in nude mice (Fogh et al., 1977Fogh J. Fogh J.M. Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice.J Natl Cancer Inst. 1977; 59: 221-226Crossref PubMed Scopus (1300) Google Scholar) and consequently might be expected to express a high amount of MCAM mRNA. Indeed, and as demonstrated in a semiquantitative reverse transcriptase–PCR assay (Figure 2c,d), MCAM transcripts were highly expressed in unstimulated SK-Mel2 cells. Similarly, MCAM mRNA synthesis could only be slightly increased in the presence of forskolin. These data indicate that MCAM production in SK-Mel2 cells is constitutive and that the high level of MCAM expression is the consequence of increased transcriptional activity. Previous experiments byRummel et al., 1996Rummel M.M. Sers C. Johnson J.P. Phorbol ester and cyclic AMP-mediated regulation of the melanoma-associated cell adhesion molecule MUC18/MCAM.Cancer Res. 1996; 56: 2218-2223PubMed Google Scholar indicated that MCAM mRNA levels were reduced in melanoma cells treated with PMA. Similarly, we observed a significant decrease in endogenous MCAM transcripts in SK-Mel2 cells in the presence of PMA (Figure 3a). The block imposed by PMA on MCAM mRNA synthesis was maintained even when cells were costimulated with forskolin. Figure 3(b) demonstrates that PMA acted at the transcriptional level. Indeed, the activity shown by the MCAM promoter in unstimulated or forskolin-treated cells was greatly reduced when the cells were costimulated with PMA. To confirm that MCAM expression is regulated at the transcriptional level, the activity of pMCAM-IV-CAT was analyzed in the nonmetastatic MCAM negative SB2 melanoma cells (Figure 4). Very low to undetectable CAT activity could be observed in unstimulated SB2 cells (Figure 4a). In contrast to that observed with SK-Mel2 cells, the MCAM promoter reacted strongly to forskolin treatment, demonstrating that the MCAM promoter can respond to cAMP elevation. MCAM transcriptional activities in stimulated SB2 cells, however, were still significantly lower than in the metastatic SK-Mel2 cells. MCAM mRNA synthesis was hardly detectable in the nonmetastatic SB2 cells when compared with the abundant production of MCAM transcripts observed in the SK-Mel2 cell line (Figure 4b). Upon forskolin treatment, however, the amounts of MCAM mRNA produced by the two cell lines were comparable (Figure 4c). The effects of forskolin could be strongly attenuated in the presence of H89, a specific inhibitor of cAMP-dependent protein kinases (Song et al., 1998Song S.-K. Choi S.Y. Kim K.T. Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes.Biochem Pharmacol. 1998; 56: 784-790Crossref Scopus (23) Google Scholar) (Figure 5a). Similarly, the production of MCAM transcripts was severely impaired by PMA at induction with forskolin. The blocking effect of PMA, however, was released upon addition of GF109203X, a specific inhibitor of PKC (Song et al., 1998Song S.-K. Choi S.Y. Kim K.T. Opposing effects of protein kinase A and C on capacitative calcium entry into HL-60 promyelocytes.Biochem Pharmacol. 1998; 56: 784-790Crossref Scopus (23) Google Scholar). Interestingly, SCF, the ligand for the c-Kit receptor, had a similar effect to PMA on MCAM RNA synthesis in c-Kit positive SB2 cells treated with fors
Referência(s)