Endothelin-1 Upregulates MCAM in Melanocytes
2004; Elsevier BV; Volume: 123; Issue: 6 Linguagem: Inglês
10.1111/j.0022-202x.2004.23480.x
ISSN1523-1747
AutoresCatherine R. Mangahas, Gelo de la Cruz, Robert J. Schneider, Sumayah Jamal,
Tópico(s)Cancer Cells and Metastasis
ResumoMelanoma cell adhesion molecule (MCAM) is a cell-surface adhesion molecule expressed on over 70% of metastatic melanoma cells but not expressed in normal melanocytes invivo. Protein levels of MCAM correlate with aggressive invasive behavior of melanoma cells in vitro and invivo. Here we demonstrate that endothelin-1 (ET-1) upregulates MCAM protein in primary human melanocytes. MCAM upregulation by ET-1 occurs irrespective of degree of melanocyte pigmentation and is dose-responsive. The drug BQ788 is an endothelin-B (ETB) receptor antagonist and inhibits upregulation of MCAM by ET-1. In addition, endothelin-3 (ET-3) and N-succinyl-[Glu9, Ala11, 15]-ET-1-1620, both selective ETB agonists, are potent upregulators of MCAM. These demonstrate a critical role for the ETB receptor in the upregulation of MCAM by ET-1 and related isoforms. MCAM mRNA abundance is also increased by ET-1 stimulation, thus the mechanism of MCAM protein upregulation may occur at the level of transcription. Our previous studies have demonstrated that ET-1 downregulates E-cadherin in melanocytes and melanoma cells. Since E-cadherin is a melanoma invasion suppressor, and MCAM is a melanoma invasion promoter, ET-1 may promote melanoma invasion and metastasis through the regulation of adhesion molecule expression. Melanoma cell adhesion molecule (MCAM) is a cell-surface adhesion molecule expressed on over 70% of metastatic melanoma cells but not expressed in normal melanocytes invivo. Protein levels of MCAM correlate with aggressive invasive behavior of melanoma cells in vitro and invivo. Here we demonstrate that endothelin-1 (ET-1) upregulates MCAM protein in primary human melanocytes. MCAM upregulation by ET-1 occurs irrespective of degree of melanocyte pigmentation and is dose-responsive. The drug BQ788 is an endothelin-B (ETB) receptor antagonist and inhibits upregulation of MCAM by ET-1. In addition, endothelin-3 (ET-3) and N-succinyl-[Glu9, Ala11, 15]-ET-1-1620, both selective ETB agonists, are potent upregulators of MCAM. These demonstrate a critical role for the ETB receptor in the upregulation of MCAM by ET-1 and related isoforms. MCAM mRNA abundance is also increased by ET-1 stimulation, thus the mechanism of MCAM protein upregulation may occur at the level of transcription. Our previous studies have demonstrated that ET-1 downregulates E-cadherin in melanocytes and melanoma cells. Since E-cadherin is a melanoma invasion suppressor, and MCAM is a melanoma invasion promoter, ET-1 may promote melanoma invasion and metastasis through the regulation of adhesion molecule expression. endothelin-1 endothelin-3 endothelin-A endothelin-B melanoma cell adhesion molecule Melanoma cell adhesion molecule (MCAM) is a 113 kDa cell-surface adhesion molecule expressed by smooth muscle cells and vascular endothelium in normal adult tissues but is rarely expressed in carcinoma cells (Mintz-Weber and Johnson, 2000Mintz-Weber C.S. Johnson J.P. Identification of the elements regulating the expression of the cell adhesion molecule MCAM/MUC18. Loss of AP-2 is not required for MCAM expression in melanoma cell lines.J Biol Chem. 2000; 275: 34672-34680https://doi.org/10.1074/jbc.M003812200Crossref PubMed Scopus (25) Google Scholar). MCAM, however, is expressed in over 70% of metastatic melanomas and levels of expression increase with increasing vertical thickness of tumors and aggressive invasive behavior (Bar-Eli, 1999Bar-Eli M. Role of AP-2 in tumor growth and metastasis of human melanoma.Cancer Metastasis Rev. 1999; 18: 377-385Crossref PubMed Scopus (74) Google Scholar). Nevus cells (cells from skin "moles" demonstrating cytologic atypia) express low levels of MCAM (Bar-Eli, 1999Bar-Eli M. Role of AP-2 in tumor growth and metastasis of human melanoma.Cancer Metastasis Rev. 1999; 18: 377-385Crossref PubMed Scopus (74) Google Scholar), whereas normal melanocytes rarely express MCAM invivo (Bar-Eli, 1999Bar-Eli M. Role of AP-2 in tumor growth and metastasis of human melanoma.Cancer Metastasis Rev. 1999; 18: 377-385Crossref PubMed Scopus (74) Google Scholar). Enforced expression of MCAM in MCAM-negative melanoma cells confers invasiveness and metastatic potential invivo and in vitro (Bar-Eli, 1999Bar-Eli M. Role of AP-2 in tumor growth and metastasis of human melanoma.Cancer Metastasis Rev. 1999; 18: 377-385Crossref PubMed Scopus (74) Google Scholar;Mintz-Weber and Johnson, 2000Mintz-Weber C.S. Johnson J.P. Identification of the elements regulating the expression of the cell adhesion molecule MCAM/MUC18. Loss of AP-2 is not required for MCAM expression in melanoma cell lines.J Biol Chem. 2000; 275: 34672-34680https://doi.org/10.1074/jbc.M003812200Crossref PubMed Scopus (25) Google Scholar). MCAM also confers an increase in adhesion to human endothelial cells, laminin as well as homotypic cell: cell adhesion (Bar-Eli, 1999Bar-Eli M. Role of AP-2 in tumor growth and metastasis of human melanoma.Cancer Metastasis Rev. 1999; 18: 377-385Crossref PubMed Scopus (74) Google Scholar). The mechanism for MCAM upregulation during melanoma progression is unknown (Karlen and Braathen, 1999Karlen S. Braathen L.R. Regulation of the melanoma cell adhesion molecule gene in melanoma: Modulation of mRNA synthesis by cyclic adenosine monophosphate, phorbol ester, and stem cell fFactor/c-kKit signaling.J Invest Dermatol. 1999; 113: 711-719https://doi.org/10.1046/j.1523-1747.1999.00746.xCrossref PubMed Scopus (11) Google Scholar). There is no evidence that MCAM undergoes chromosomal translocation, mutation or amplification thus, it has been postulated that MCAM upregulation occurs secondary to an aberrant cytokine network present in the epidermal microenvironment (Karlen and Braathen, 1999Karlen S. Braathen L.R. Regulation of the melanoma cell adhesion molecule gene in melanoma: Modulation of mRNA synthesis by cyclic adenosine monophosphate, phorbol ester, and stem cell fFactor/c-kKit signaling.J Invest Dermatol. 1999; 113: 711-719https://doi.org/10.1046/j.1523-1747.1999.00746.xCrossref PubMed Scopus (11) Google Scholar). Endothelin-1 (ET-1) is a 21-amino acid peptide secreted by epidermal keratinocytes that may participate in such a network modulating MCAM expression invivo. ET-1 secretion by epidermal keratinocytes is induced by ultraviolet (UV) irradiation (Imokawa et al., 1997Imokawa G. Kobayashi T. Miyagishi M. Higashi K. Yada Y. The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis.Pigment Cell Res. 1997; 10: 218-228Crossref PubMed Scopus (152) Google Scholar). Once secreted, ET-1 can then bind to high-affinity cell-surface ET receptors present on melanocytes and melanoma cells present in the epidermis of the skin (Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748https://doi.org/10.1111/1523-1747.ep12325522Crossref PubMed Scopus (116) Google Scholar;Imokawa et al., 1996Imokawa G. Yada Y. Kimura M. Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.Biochem J. 1996; 314: 305-312Crossref PubMed Scopus (150) Google Scholar,Imokawa et al., 1997Imokawa G. Kobayashi T. Miyagishi M. Higashi K. Yada Y. The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis.Pigment Cell Res. 1997; 10: 218-228Crossref PubMed Scopus (152) Google Scholar). ET-1 stimulates a variety of physiologic responses in melanocytes and melanoma cells including an increase in dendricity, mitogenesis, chemotaxis, and pigment production (Yohn et al., 1994Yohn J.J. Smith C. Stevens T. et al.Human melanoma cells express functional endothelin-1 receptors.Biochem Biophys Res Commun. 1994; 201: 449-457https://doi.org/10.1006/bbrc.1994.1722Crossref PubMed Scopus (54) Google Scholar;Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748https://doi.org/10.1111/1523-1747.ep12325522Crossref PubMed Scopus (116) Google Scholar;Imokawa et al., 1996Imokawa G. Yada Y. Kimura M. Signalling mechanisms of endothelin-induced mitogenesis and melanogenesis in human melanocytes.Biochem J. 1996; 314: 305-312Crossref PubMed Scopus (150) Google Scholar;Imokawa et al., 1997Imokawa G. Kobayashi T. Miyagishi M. Higashi K. Yada Y. The role of endothelin-1 in epidermal hyperpigmentation and signaling mechanisms of mitogenesis and melanogenesis.Pigment Cell Res. 1997; 10: 218-228Crossref PubMed Scopus (152) Google Scholar). The endothelin-B (ETB) receptor is a melanoma tumor progression marker and ETB blockade induces melanoma cell death and growth arrest invivo and in vitro (Lahav et al., 1999Lahav R. Heffner G. Patterson P.H. An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and invivo.Proc Natl Acad Sci USA. 1999; 96: 11496-11500https://doi.org/10.1073/pnas.96.20.11496Crossref PubMed Scopus (127) Google Scholar;Demunter et al., 2001Demunter A. De Wolf-Peeters C. Degreef H. Stas M. van den Oord J.J. Expression of the endothelin-B receptor in pigment cell lesions of the skin. Evidence for its role as tumor progression marker in malignant melanoma.Virchows Arch. 2001; 438: 485-491https://doi.org/10.1007/s004280000362Crossref PubMed Scopus (91) Google Scholar). Thus, the endothelin pathway plays an important role in melanoma development and progression. The exact nature of this role has yet to be elucidated, however. An alteration in adhesive properties is commonly seen during tumor progression and is critical to the development of aggressive metastatic behavior of cancer cells. It is therefore important to identify those factors promoting changes in adhesion during the early transformation of normal cells to cancer cells. E-cadherin expression is important for maintenance of the normal melanocyte phenotype and is commonly downregulated during melanoma development (Hsu et al., 1996Hsu M.Y. Wheelock M.J. Johnson K.R. Herlyn M. Shifts in cadherin profiles between human normal melanocytes and melanomas.J Investig Dermatol Symp Proc. 1996; 1: 188-194PubMed Google Scholar,Hsu et al., 2000Hsu M.Y. Meier F.E. Nesbit M. Hsu J.Y. Van Belle P. Elder D.E. Herlyn M. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.Am J Pathol. 2000; 156: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). E-cadherin suppresses melanoma invasion invivo and in vitro (Hsu et al., 1996Hsu M.Y. Wheelock M.J. Johnson K.R. Herlyn M. Shifts in cadherin profiles between human normal melanocytes and melanomas.J Investig Dermatol Symp Proc. 1996; 1: 188-194PubMed Google Scholar,Hsu et al., 2000Hsu M.Y. Meier F.E. Nesbit M. Hsu J.Y. Van Belle P. Elder D.E. Herlyn M. E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.Am J Pathol. 2000; 156: 1515-1525Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar). We have previously demonstrated that ET-1 is a potent downregulator of E-cadherin in melanocytes and melanoma cells (Jamal, 2000Jamal S. Endothelin-1 down-regulates E-cadherin in melanocytic cells by apoptosis-independent activation of caspase-8.J Am Acad Dermatol. 2000; 43: 703-704https://doi.org/10.1067/mjd.2000.109275cCrossref PubMed Google Scholar;Jamal and Schneider, 2002Jamal S. Schneider R.J. UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells.J Clin Invest. 2002; 110: 443-452https://doi.org/10.1172/JCI200213729Crossref PubMed Scopus (71) Google Scholar). Here, we demonstrate that ET-1 is also a potent upregulator of MCAM, a known promoter of melanoma invasion. This provides further evidence that ET-1 plays a critical role in altering the adhesive properties of melanocytes during tumor progression to promote metastasis and invasion. To determine whether ET-1 altered protein levels of MCAM, four different primary human melanocyte cultures were stimulated with 10 nM ET-1 over a 7-d time course. We selected two cultures derived from lightly pigmented individuals and two from darkly pigmented individuals, in case degree of pigmentation had an impact on the observed response. The prolonged time course was chosen since ET-1 is known to elicit delayed responses in melanocytes (Hara et al., 1995Hara M. Yaar M. Gilchrest B.A. Endothelin-1 of keratinocyte origin is a mediator of melanocyte dendricity.J Invest Dermatol. 1995; 105: 744-748https://doi.org/10.1111/1523-1747.ep12325522Crossref PubMed Scopus (116) Google Scholar;Jamal and Schneider, 2002Jamal S. Schneider R.J. UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells.J Clin Invest. 2002; 110: 443-452https://doi.org/10.1172/JCI200213729Crossref PubMed Scopus (71) Google Scholar). As shown in Figure 1, ET-1 induced a dramatic increase in protein levels of MCAM in all four cell cultures. There was no apparent difference in the response of lightly pigmented versus darkly pigmented melanocytes. As expected, the kinetics for this response were delayed, with MCAM upregulation being evident by the 4-d time point in three of four cultures (upregulation occurred by day 5 in the remaining culture). Next, the concentration of ET-1 used to stimulate cells was titrated in order to determine whether upregulation of MCAM by ET-1 was dose-responsive. As shown in Figure 2, protein levels of MCAM increased with increasing ET-1 concentrations. This suggests that the response is occurring secondary to ET-1 addition. Although it is possible that other factors in the culture medium contribute to this response, overall these data suggest that ET-1 is a potent upregulator of MCAM. Melanocytes express two high-affinity ET-1 receptors, the endothelin-A (ETA) and the ETB subtype (Baynash et al., 1994Baynash A.G. Hosoda K. Giaid A. Richardson J.A. Emoto N. Hammer R.E. Yanagisawa M. Interaction of endothelin-3 with endothelin-B receptor is essential for development of epidermal melanocytes and enteric neurons.Cell. 1994; 79: 1277-1285Abstract Full Text PDF PubMed Scopus (798) Google Scholar). In order to determine which receptor subtype mediates upregulation of MCAM by ET-1, we pre-treated melanocytes with a selective ETA receptor antagonist (BQ610) or a selective ETB receptor antagonist (BQ788) prior to ET-1 addition. Cells were harvested at the 5-d time point and MCAM western blot analysis performed. As shown in Figure 3, ET-1 alone, once again, was a potent upregulator of MCAM. Pre-treatment of cells with ETA receptor antagonist BQ610 failed to block this upregulation. In sharp contrast, pre-treatment of cells with ETB receptor antagonist BQ788 significantly reduced MCAM upregulation by ET-1. These results suggest that activation of the ETB receptor plays a critical role in the upregulation of MCAM by ET-1. In addition, the ability of BQ788 to block this response indicates that MCAM upregulation occurs as a direct or indirect effect of ET stimulation of the ETB receptor and is not an in vitro artifact. Although ET-1 binds with equal affinity to both the ETA and ETB receptor subtypes, the endothelin isoform endothelin-3 (ET-3) demonstrates a much greater affinity for ETB than ETA and is a potent ETB agonist (Huggins and Pelton, 1997Huggins J.P. Pelton J.T. Endothelins in Biology and Medicine. CRC Press, Boca Raton1997Google Scholar). Furthermore, the synthetic peptide IRL-1620, in addition to its being a potent ETB agonist, binds exclusively to ETB and is therefore a useful tool in dissecting ETB-specific signaling pathways (Huggins and Pelton, 1997Huggins J.P. Pelton J.T. Endothelins in Biology and Medicine. CRC Press, Boca Raton1997Google Scholar). In the upper panels of Figure 4, cells were stimulated with 10 nM ET-3 or IRL-1620 over a 6-d time course. As shown, both ETB selective agonists induced a dramatic increase in MCAM protein levels. In the lower panels, melanocytes were stimulated for 2 d with ET-3 or IRL-1620 and a dramatic increase in MCAM protein levels was also observed. As shown, MCAM upregulation by ET-3 and IRL-1620 occurred with faster kinetics than those observed using ET-1. This upregulation was strongly evident by the 2-d time point (no upregulation of MCAM was observed prior to this time point, data not shown). These results reinforce our earlier data indicating that the ETB signaling pathway mediates MCAM upregulation by ET-1. We next tested five human melanoma cell lines to determine whether ET-1 induced MCAM expression in a manner similar to that observed in melanocytes. SKMEL28, M20, WM2664, WM1232, and SB2 cells were stimulated with ET-1, ET-3, and IRL-1620 as described previously. We were unable to detect upregulation of MCAM in these melanoma cells lines using these agents. As shown in Figure 5, ET-3 did not upregulate MCAM in either SKMEL28 or SB2 cells. It is apparent, however, that SKMEL28 cells express constitutive levels of MCAM that are significantly higher than those expressed by D15' human melanocytes whether at baseline or when induced by ET-3. In addition it is also clear in Figure 5 that SB2 human melanoma cells express far lower levels of MCAM than either SKMEL28 cells or human melanocytes. We were unable to detect any significant MCAM protein in these cells even when the total amount of protein was titrated to a 30-fold excess of that used in this figure. All other melanoma cell lines expressed MCAM constitutively at levels similar to those observed in SKMEL28 cells (data not shown). This data suggests that ET-1 is unable to induce MCAM in melanoma cells expressing high constitutive levels of MCAM. In order to investigate the mechanism of MCAM upregulation by ET-1, we performed northern blot analysis. Cells were stimulated over a 7-d time course with 10 nM ET-1 and then harvested for RNA at different time points. As shown in Figure 6, MCAM mRNA abundance gradually increased over the duration of the assay and peaked at the 7-d time point. This gradual increase in mRNA abundance is consistent with the gradual increase in MCAM protein levels observed in response to ET-1 stimulation. These data suggest that the mechanism of MCAM upregulation by ET-1 is either transcriptional or affects MCAM mRNA stability. We have clearly demonstrated that ET-1 is a potent upregulator of MCAM in normal human melanocytes and that the ETB receptor, which is a melanoma tumor progression marker (Demunter et al., 2001Demunter A. De Wolf-Peeters C. Degreef H. Stas M. van den Oord J.J. Expression of the endothelin-B receptor in pigment cell lesions of the skin. Evidence for its role as tumor progression marker in malignant melanoma.Virchows Arch. 2001; 438: 485-491https://doi.org/10.1007/s004280000362Crossref PubMed Scopus (91) Google Scholar), is critical for this response. The ETB receptor antagonist BQ788 substantially reduced the ability of ET-1 to upregulate MCAM. The failure of BQ788 to completely block this response may reflect the fact that although BQ788 receptor binding is competitive and reversible, ET-1 binding is irreversible (Huggins and Pelton, 1997Huggins J.P. Pelton J.T. Endothelins in Biology and Medicine. CRC Press, Boca Raton1997Google Scholar). Thus, there may have been a small amount of ET-1 signaling occurring even in the presence of the antagonist. ET-1 activates both the ETA and ETB receptors whereas ET-3 and IRL-1620 are selective ETB agonists (Huggins and Pelton, 1997Huggins J.P. Pelton J.T. Endothelins in Biology and Medicine. CRC Press, Boca Raton1997Google Scholar). Since upregulation of MCAM by ET-3 and IRL-1620 occurs with faster kinetics than those observed with ET-1, it is possible that co-activation of the ETA and ETB receptors by ET-1 results in cross-talk between the two pathways that antagonizes ETB-dependent signaling. Prolonged ET-1 stimulation of cells may cause a selective desensitization of the ETA pathway, after which time, ETB signaling would predominate and result in a delayed upregulation of MCAM. Given our results, it is likely that in order for MCAM to be upregulated in melanocytes by ET-1 invivo, a prolonged exposure to ET-1 would be required. Since ET-1 is normally secreted by the skin in response to UV irradiation (Imokawa et al., 1992Imokawa G. Yada Y. Miyagishi M. Endothelins secreted from human keratinocytes are intrinsic mitogens for human melanocytes.J Biol Chem. 1992; 267: 24675-24680Abstract Full Text PDF PubMed Google Scholar;Ahn et al., 1998Ahn G.Y. Butt K.I. Jindo T. Yaguchi H. Tsuboi R. Ogawa H. The expression of endothelin-1 and its binding sites in mouse skin increased after ultraviolet B irradiation or local injection of tumor necrosis factor alpha.J Dermatol. 1998; 25: 78-84Crossref PubMed Scopus (41) Google Scholar), there must exist a mechanism to prevent the upregulation of MCAM by ET-1 secreted in response to physiologic doses of sunlight. After UV-irradiation, ET-1 levels peak at 2 d, gradually falling back to baseline several days later (Ahn et al., 1998Ahn G.Y. Butt K.I. Jindo T. Yaguchi H. Tsuboi R. Ogawa H. The expression of endothelin-1 and its binding sites in mouse skin increased after ultraviolet B irradiation or local injection of tumor necrosis factor alpha.J Dermatol. 1998; 25: 78-84Crossref PubMed Scopus (41) Google Scholar). It is therefore reasonable to expect that melanocytes would be resistant to MCAM upregulation by ET-1 for a period of several days and this is what our data demonstrate. An aberrant or constitutive exposure to ET-1, however, may eventually lead to MCAM upregulation in these cells. Early in melanoma progression, atypical melanocytes secrete factors such as interleukin-1-α, and tumor necrosis factor-α (Moretti et al., 1999Moretti S. Pinzi C. Spallanzani A. et al.Immunohistochemical evidence of cytokine networks during progression of human melanocytic lesions.Int J Cancer. 1999; 84: 160-168https://doi.org/10.1002/(SICI)1097-0215(19990420)84:2 3.3.CO;2-ICrossref PubMed Scopus (0) Google Scholar), which are known to stimulate ET-1 secretion by keratinocytes. Keratinocyte-derived ET-1, would upregulate MCAM in the atypical melanocyte and promote invasive behavior. Thus it can be seen that signals from the atypical melanocyte may trigger an aberrant signaling loop involving adjacent keratinocytes with potentially disastrous effects within the epidermal microenvironment. Interestingly, constitutive ET-1 secretion is seen in the epidermis of skin samples obtained from lentiginous proliferations of melanocytes (Kadono et al., 2001Kadono S. Manaka I. Kawashima M. Kobayashi T. Imokawa G. The role of epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis.J Invest Dermatol. 2001; 114: 571-577https://doi.org/10.1046/j.1523-1747.2001.01296.xCrossref Scopus (121) Google Scholar) and 70% of melanomas arise in association with such proliferations (Kadono et al., 2001Kadono S. Manaka I. Kawashima M. Kobayashi T. Imokawa G. The role of epidermal endothelin cascade in the hyperpigmentation mechanism of lentigo senilis.J Invest Dermatol. 2001; 114: 571-577https://doi.org/10.1046/j.1523-1747.2001.01296.xCrossref Scopus (121) Google Scholar). ET-1 did not upregulate MCAM in any of the five melanoma cell lines tested. It is likely that ET-1 cannot upregulate MCAM when it is being expressed maximally. Previous studies have shown that MCAM is uninducible when expressed at constitutively high levels in melanoma cells (Karlen and Braathen, 1999Karlen S. Braathen L.R. Regulation of the melanoma cell adhesion molecule gene in melanoma: Modulation of mRNA synthesis by cyclic adenosine monophosphate, phorbol ester, and stem cell fFactor/c-kKit signaling.J Invest Dermatol. 1999; 113: 711-719https://doi.org/10.1046/j.1523-1747.1999.00746.xCrossref PubMed Scopus (11) Google Scholar). Since SB2 melanoma cells express less MCAM than unstimulated melanocytes, it is likely that endogenous MCAM is not expressed to any appreciable degree in these cells and is not readily inducible. MCAM mRNA abundance as determined by northern blot, increases gradually in response to ET stimulation, and this may be due to an ET-dependent increase in transcription. The MCAM promoter contains a Ca2+/cAMP responsive element binding protein (CREB) binding motif and factors such as forskolin, that increase intracellular cyclic adenosine monophosphate (cAMP) concentrations in cells, have been shown to upregulate MCAM (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;Karlen and Braathen, 1999Karlen S. Braathen L.R. Regulation of the melanoma cell adhesion molecule gene in melanoma: Modulation of mRNA synthesis by cyclic adenosine monophosphate, phorbol ester, and stem cell fFactor/c-kKit signaling.J Invest Dermatol. 1999; 113: 711-719https://doi.org/10.1046/j.1523-1747.1999.00746.xCrossref PubMed Scopus (11) Google Scholar). Interestingly, ET-1 is a known activator of CREB and induces an ET-1-dependent phosphorylation of this important transcription factor (Tada et al., 2002Tada A. Pereira E. Beitner-Johnson D. Kavanagh R. Abdel-Malek Z.A. Mitogen- and ultraviolet-B-induced signaling pathways in normal human melanocytes.J Invest Dermatol. 2002; 118: 316-322https://doi.org/10.1046/j.0022-202x.2001.01694.xCrossref PubMed Scopus (79) Google Scholar;Harrison et al., 2004Harrison J.G. Sugden P.H. Clerk A. Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: Implications for the regulation of c-jun expression.Biochim Biophys Acta. 2004; 1644: 17-25https://doi.org/10.1016/j.bbamcr.2003.10.008Crossref PubMed Scopus (24) Google Scholar). MCAM upregulation by forskolin stimulation of cells results in maximal upregulation of MCAM protein levels at 48 h (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), and thus the kinetics of this response are identical to those observed for MCAM upregulation in melanocytes in response to ET-3 or IRL-1620. Our future studies will investigate the role of CREB in MCAM upregulation by ET-1. The endothelins play an important role in tumor progression in many different types of carcinomas and blockade of the endothelin signaling cascades using ET receptor antagonists is showing promise in the treatment of advanced prostate cancer (Carducci et al., 2002Carducci M.A. Nelson J.B. Bowling M.K. et al.Atrasentan, an endothelin-receptor antagonist for refractory adenocarcinomas: Safety and pharmacokinetics.J Clin Oncol. 2002; 20: 2171-2180Crossref PubMed Scopus (158) Google Scholar,Carducci et al., 2003Carducci M.A. Padley R.J. Breul J. et al.Effect of endothelin-A receptor blockade with atrasentan on tumor progression in men with hormone-refractory prostate cancer: A randomized, phase II, placebo-controlled trial.J Clin Oncol. 2003; 21: 679-689Crossref PubMed Scopus (349) Google Scholar). It is now clear from numerous studies that the ET-1/ETB pathway also plays a vital role in melanoma progression and viability (Lahav et al., 1999Lahav R. Heffner G. Patterson P.H. An endothelin receptor B antagonist inhibits growth and induces cell death in human melanoma cells in vitro and invivo.Proc Natl Acad Sci USA. 1999; 96: 11496-11500https://doi.org/10.1073/pnas.96.20.11496Crossref PubMed Scopus (127) Google Scholar;Demunter et al., 2001Demunter A. De Wolf-Peeters C. Degreef H. Stas M. van den Oord J.J. Expression of the endothelin-B receptor in pigment cell lesions of the skin. Evidence for its role as tumor progression marker in malignant melanoma.Virchows Arch. 2001; 438: 485-491https://doi.org/10.1007/s004280000362Crossref PubMed Scopus (91) Google Scholar). Our studies provide further clues as to the role of this pathway in altering the adhesive properties of melanocytes and melanoma cells such that invasion and metastasis are favored. Overall the ET-1/ETB pathway represents an excellent target for future therapeutic intervention in the treatment of melanoma. Human melanoma cells were cultured as described previously (Jamal and Schneider, 2002Jamal S. Schneider R.J. UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells.J Clin Invest. 2002; 110: 443-452https://doi.org/10.1172/JCI200213729Crossref PubMed Scopus (71) Google Scholar). Human neonatal melanocytes were isolated from foreskin and culture conditions as described previously (Tada et al., 1998Tada A. Suzuki I. Im S. et al.Endothelin-1 is a paracrine growth factor that modulates melanogenesis of human melanocytes and participates in their responses to ultraviolet radiation.Cell Growth Differ. 1998; 9: 575-584PubMed Google Scholar) (Jamal and Schneider, 2002Jamal S. Schneider R.J. UV-induction of keratinocyte endothelin-1 downregulates E-cadherin in melanocytes and melanoma cells.J Clin Invest. 2002; 110: 443-452https://doi.org/10.1172/JCI200213729Crossref PubMed Scopus (71) Google Scholar). Briefly, melanocyte growth medium consisted of MCDB153, 4% heat-inactivated fetal bovine serum (Cellgro, Mediatech, Harndon, Virginia), 5 μg per mL insulin, 13 μg per mL bovine pituitary extract, 0.6 ng per mL basic fibroblast growth factor, 10−8 M 12-O-tetradecanoyl phorbol-13-acetate (TPA), and 1 μg per mL α-tocopherol (Invitrogen, Carlsbad, California). Two days prior to stimulation with ET-1, ET-3, or IRL-1620 (Bachem, King of Prussia, Pennsylvania), TPA was removed from the medium. During ET stimulations, TPA-free culture medium was changed every other day with fresh ET added. BQ610 and BQ788 (Bachem) at a final concentration of 1 μM were added to cells 1 hour prior to ET addition. All culture reagents were purchased from Sigma (St Louis, Missouri) unless otherwise indicated. All experimental protocols were approved by our institutional review board. Cells were lysed in RIPA buffer (1% NP40, 0.5% deoxycholic acid, 10 mM Tris pH 8.0, 150 mM NaCl, 50 mM NaF, 0.2 mM sodium vanadate, 1 mM dithiothreitol, 1 × protease inhibitors—Complete-mini (Boehringer-Ingelheim, Ridgefield, Connecticut)). Lysates were quantitated using protein assay reagent (Biorad, Hercules, California) and 10 μg of protein per sample was used for 8% SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Proteins were electrophoretically transferred and immobilized on nitrocellulose membranes (Schleicher and Schuell, Keene, New Hampshire). The antibodies used were as follows: anti-MCAM (BD-Biosciences Pharmingen, San Diego, California), anti-Actin (Santa Cruz Biotechnologies SC-8432, Santa Cruz, California), and anti-mouse IgG-horseradish peroxidase (Santa Cruz Biotechnologies, SC-2005). The ECL chemiluminescent system (Amersham, Piscataway, New Jersey) was used for signal detection. Total RNA was isolated from cells using Trizol reagent (Invitrogen) and Rneasy columns (Qiagen, Valencia, California). RNA at 10 μg per sample were separated by agarose gel electrophoresis and transferred by capillary action and immobilized onto nylon membranes (Genescreen, Perkin-Elmer Life & Analytical Sciences, Boston, Massachusetts). An MCAM-specific cDNA probe was generated by reverse transcriptase polymerase chain reaction (RT-PCR) using the Access RT-PCR system (Promega, Madison, Wisconsin). The primer sequences were as follows: sense (nt 185–206) 5′-CCA AGG CAA CCT CAG CCA TGT C-3′, antisense (nt 598–622) 5′-CTC GAC TCC ACA GTC TGG GAC GAC T-3′. GAPDH (glyceraldehyde-3-phosphate dehydrogenase) probes were similarly generated using the following primers: sense (nt 85–106) 5′-AAC GGA TTT GGT CGT ATT GGG C-3′, antisense (nt 663–684) 5′-AGG GAT GAT GTT CTG GAG AGC C-3′. The sizes of the amplification products were verified by low molecular weight agarose gel electrophoresis. The probes were radiolabeled using the Rediprime random hexamer system (Amersham) and were hybridized to membranes using Perfect-Hyb solution (Sigma). We are grateful to Dr Irwin M. Freedberg for a careful reading of the manuscript. This work was supported by grants from the Dermatology Foundation, The Robert Wood Johnson Foundation, and the Lubowe Foundation.
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