RESUMO
Fibroblast growth factor (FGF2) regulates endothelial and melanoma cell migration. The binding of FGF2 to its receptor requires N-sulfated heparan sulfate (HS) glycosamine. We have previously reported that Epac1, an exchange protein activated by cAMP, increases N-sulfation of HS in melanoma. Therefore, we examined whether Epac1 regulates FGF2-mediated cell-cell communication. Conditioned medium (CM) of melanoma cells with abundant expression of Epac1 increased migration of human umbilical endothelial cells (HUVEC) and melanoma cells with poor expression of Epac1. CM-induced increase in migration was inhibited by antagonizing FGF2, by the removal of HS and by the knockdown of Epac1. In addition, knockdown of Epac1 suppressed the binding of FGF2 to FGF receptor in HUVEC, and in vivo angiogenesis in melanoma. Furthermore, knockdown of Epac1 reduced N-sulfation of HS chains attached to perlecan, a major secreted type of HS proteoglycan that mediates the binding of FGF2 to FGF receptor. These data suggested that Epac1 in melanoma cells regulates melanoma progression via the HS-FGF2-mediated cell-cell communication.
Assuntos
Movimento Celular , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Células Endoteliais da Veia Umbilical Humana/metabolismo , Melanoma/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica/metabolismo , Comunicação Parácrina , Linhagem Celular Tumoral , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica/genética , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Células Endoteliais da Veia Umbilical Humana/patologia , Humanos , Melanoma/irrigação sanguínea , Melanoma/genética , Melanoma/patologia , Proteínas de Neoplasias/genética , Neovascularização Patológica/genética , Neovascularização Patológica/patologiaRESUMO
Store-operated Ca(2+) entry (SOCE) is a major mechanism of Ca(2) (+) import from extracellular to intracellular space, involving detection of Ca(2+) store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca(2+) channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.
Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Melanócitos/metabolismo , Melanoma/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Canais de Cálcio/metabolismo , Linhagem Celular Tumoral , Retículo Endoplasmático/metabolismo , Humanos , Melanoma/patologia , Camundongos , Neoplasias Cutâneas/patologiaRESUMO
Despite numerous discoveries from genetically engineered mice, relatively few have been translated to the bedside, mainly because it is difficult to translate from genes to drugs. This investigation examines an antiviral drug, which also has an action to selectively inhibit type 5 adenylyl cyclase (AC5), a pharmaceutical correlate of the AC5 knockout (KO) model, which exhibits longevity and stress resistance. Our objective was to examine the extent to which pretreatment with this drug, adenine 9-ß-d-arabinofuranoside (Ara-A), favorably ameliorates the development of heart failure (HF). Ara-A exhibited selective inhibition for AC5 compared with the other major cardiac AC isoform, AC6, i.e., it reduced AC activity significantly in AC5 transgenic (Tg) mice, but not in AC5KO mice and had little effect in either wild-type or AC6Tg mice. Permanent coronary artery occlusion for 3 wk in C57Bl/6 mice increased mortality and induced HF in survivors, as reflected by reduced cardiac function, while increasing cardiac fibrosis. The AC5 inhibitor Ara-A significantly improved all of these end points and also ameliorated chronic isoproterenol-induced cardiomyopathy. As with the AC5KO mice, Ara-A increased mitogen/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (ERK) phosphorylation. A MEK inhibitor abolished the beneficial effects of the AC5 inhibitor in the HF model, indicating the involvement of the downstream MEK-ERK pathway of AC5. Our data suggest that pharmacological AC5 inhibition may serve as a new therapeutic approach for HF.
Assuntos
Inibidores de Adenilil Ciclases , Antivirais/uso terapêutico , Insuficiência Cardíaca/prevenção & controle , Coração/efeitos dos fármacos , Vidarabina/uso terapêutico , Adenilil Ciclases/metabolismo , Animais , Antivirais/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Coração/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Miocárdio/metabolismo , Vidarabina/farmacologiaRESUMO
BACKGROUND: Recently we reported that activation of Epac1, an exchange protein activated by cAMP, increases melanoma cell migration via Ca 2+ release from the endoplasmic reticulum (ER). G-protein ßγ subunits (Gßγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gßγ in cell migration and Ca 2+ signaling in melanoma has not been well studied. Here we report that there is crosstalk of Ca 2+ signaling between Gßγ and Epac in melanoma, which plays a role in regulation of cell migration. METHODS: SK-Mel-2 cells, a human metastatic melanoma cell line, were mainly used in this study. Intracellular Ca 2+ was measured with Fluo-4AM fluorescent dyes. Cell migration was examined using the Boyden chambers. RESULTS: The effect of Gßγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gßγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-ß-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gßγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of ß1 and γ2, which is the major combination of Gßγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of ß adrenergic receptor kinase (ßARK-CT), an endogenous inhibitor for Gßγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gßγ. We next examined the effect of mSIRK on Epac-induced Ca 2+ response. When cells were pretreated with mSIRK, but not with L9A, 8-(4-Methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT), an Epac-specific agonist, failed to increase Ca 2+ signal. Co-overexpression of ß1 and γ2 subunits inhibited 8-pMeOPT-induced Ca 2+ elevation. Inhibition of Gßγ with ßARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPßS), a GDP analogue that inactivates Gßγ, restored 8-pMeOPT-induced Ca 2+ elevation even in the presence of mSIRK. These data suggested that Gßγ inhibits Epac-induced Ca 2+ elevation. Subsequently, the mechanism by which Gßγ inhibits Epac-induced Ca 2+ elevation was explored. mSIRK activates Ca 2+ influx from the extracellular space. In addition, W-5, an inhibitor of calmodulin, abolished mSIRK's inhibitory effects on Epac-induced Ca 2+ elevation, and cell migration. These data suggest that, the mSIRK-induced Ca 2+ from the extracellular space inhibits the Epac-induced Ca 2+ release from the ER, resulting suppression of cell migration. CONCLUSION: We found the cross talk of Ca 2+ signaling between Gßγ and Epac, which plays a major role in melanoma cell migration.
Assuntos
Sinalização do Cálcio/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Melanoma/patologia , Proteínas de Neoplasias/fisiologia , Sequência de Aminoácidos , Bloqueadores dos Canais de Cálcio/farmacologia , Calmodulina/fisiologia , Linhagem Celular Tumoral/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Subunidades beta da Proteína de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/genética , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Humanos , Melanoma/secundário , Dados de Sequência Molecular , Proteínas de Neoplasias/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Peptídeos/farmacologia , Proteínas Recombinantes de Fusão/fisiologia , Proteínas Recombinantes/farmacologia , Tionucleotídeos/farmacologia , Quinases de Receptores Adrenérgicos beta/antagonistas & inibidoresRESUMO
Our previous report suggested the potential role of the exchange protein directly activated by cyclic AMP (Epac) in melanoma metastasis via heparan sulfate (HS)-mediated cell migration. In order to obtain conclusive evidence that Epac1 plays a critical role in modification of HS and melanoma metastasis, we extensively investigated expression and function of Epac1 in human melanoma samples and cell lines. We have found that, in human melanoma tissue microarray, protein expression of Epac1 was higher in metastatic melanoma than in primary melanoma. In addition, expression of Epac1 positively correlated with that of N-sulfated HS, and N-deacetylase/N-sulfotransferase-1 (NDST-1), an enzyme that increases N-sulfation of HS. Further, an Epac agonist increased, but ablation of Epac1 decreased, expressions of NDST-1, N-sulfated HS, and cell migration in various melanoma cell lines. Finally, C8161 cells with stable knockdown of Epac1 showed a decrease in cell migration, and metastasis in mice. These data suggest that Epac1 plays a critical role in melanoma metastasis presumably because of modification of HS.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Heparitina Sulfato/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Animais , Linhagem Celular Tumoral , Movimento Celular , Deleção de Genes , Humanos , Melanoma/enzimologia , Camundongos , Metástase Neoplásica , Coloração e Rotulagem , Sulfotransferases/metabolismoRESUMO
Melanoma has a poor prognosis due to its strong metastatic ability. Although Ca(2+) plays a major role in cell migration, little is known about the role of Ca(2+) in melanoma cell migration. We recently found that the exchange protein directly activated by cyclic AMP (Epac) increases melanoma cell migration via a heparan sulfate-related mechanism. In addition to this mechanism, we also found that Epac regulates melanoma cell migration by a Ca(2+)-dependent mechanism. An Epac agonist increased Ca(2+) in several different melanoma cell lines but not in melanocytes. Ablation of Epac1 with short hairpin RNA inhibited the Epac agonist-induced Ca(2+) elevation, suggesting the critical role of Epac1 in Ca(2+) homeostasis in melanoma cells. Epac-induced Ca(2+) elevation was negated by the inhibition of phospholipase C (PLC) and inositol triphosphate (IP(3)) receptor. Furthermore, Epac-induced cell migration was reduced by the inhibition of PLC or IP(3) receptor. These data suggest that Epac activates Ca(2+) release from the endoplasmic reticulum via the PLC/IP(3) receptor pathway, and this Ca(2+) elevation is involved in Epac-induced cell migration. Actin assembly was increased by Epac-induced Ca(2+), suggesting the involvement of actin in Epac-induced cell migration. In human melanoma specimens, mRNA expression of Epac1 was higher in metastatic melanoma than in primary melanoma, suggesting a role for Epac1 in melanoma metastasis. In conclusion, our findings reveal that Epac is a potential target for the suppression of melanoma cell migration, and, thus, the development of metastasis.
Assuntos
Cálcio/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Melanoma/patologia , Actinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Retículo Endoplasmático/metabolismo , Fatores de Troca do Nucleotídeo Guanina/biossíntese , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Melanoma/genética , Melanoma/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma. Epac activation, by either specific agonist or overexpression of Epac, increased melanoma cell migration. Deletion of endogenous Epac with small interfering RNA decreased basal melanoma cell migration. These data suggested a major role of Epac in melanoma cell migration. Epac-induced cell migration was mediated by translocation of syndecan-2, a cell-surface heparan sulfate proteoglycan, to lipid rafts. This syndecan-2 translocation was regulated by tubulin polymerization via the Epac/phosphoinositol-3 kinase pathway. Epac-induced cell migration was also regulated by the production of heparan sulfate, a major extracellular matrix. Epac-induced heparan sulfate production was attributable to the increased expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) accompanied by an increased NDST-1 translation rate. Finally, Epac overexpression enhanced lung colonization of melanoma cells in mice. Taken together, these data indicate that Epac regulates melanoma cell migration/metastasis mostly via syndecan-2 translocation and heparan sulfate production.
Assuntos
Movimento Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Heparitina Sulfato/biossíntese , Melanoma/metabolismo , Sulfotransferases/metabolismo , Sindecana-2/metabolismo , Animais , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/fisiologia , Transporte Proteico , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Sevelamer improves hyperphosphatemia without increasing the calcium load. However, it remains unknown whether sevelamer restores bone metabolism in hemodialysis patients with low bone turnover osteodystrophy and hypoparathyroidism. We investigated the changes in serum intact parathyroid hormone (iPTH) and bone metabolic marker levels after replacing calcium carbonate with sevelamer in these patients. We also conducted stratified analysis based on patient background and multivariate analysis to determine the factors affecting these parameters. During sevelamer replacement therapy, serum calcium and phosphate concentrations, and the calcium phosphate product were measured at 0, 1, 3, and 6 months. Serum iPTH, bone alkaline phosphatase and osteocalcin concentrations were measured at 0 and 6 months. In hemodialysis patients (71 men and 46 women, 63 +/- 12 years old) serum calcium levels and the calcium phosphate product decreased significantly at 1 month. Serum iPTH, bone alkaline phosphatase and osteocalcin levels increased significantly at 6 months. Increases in serum iPTH concentrations were observed in all stratified groups. Significant increases in serum bone alkaline phosphatase and osteocalcin concentrations were found only in the relative hypoparathyroidism group (iPTH levels > or =51.5 pg/mL, the median pretreatment level). Multivariate analysis showed that the factors affecting change in serum iPTH level are baseline serum iPTH, baseline calcium level (> or =9.5 mg/dL), and dialysis duration of 10 years or longer. Sevelamer appears useful for the treatment of hyperphosphatemia in these patients. Particularly, in the relative hypoparathyroidism group, the iPTH secretory response is probably enhanced and bone turnover may have been improved as a result of reducing the calcium load.