Gβγ subunits inhibit Epac-induced melanoma cell migration

<p>Abstract</p> <p>Background</p> <p>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 βγ subunits (Gβγ) are known to act as an independent signaling molecule upon activation of G-protein coupled receptor. However, the role of Gβγ 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 Gβγ and Epac in melanoma, which plays a role in regulation of cell migration.</p> <p>Methods</p> <p>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.</p> <p>Results</p> <p>The effect of Gβγ on Epac-induced cell migration was first examined. Epac-induced cell migration was inhibited by mSIRK, a Gβγ -activating peptide, but not its inactive analog, L9A, in SK-Mel-2 cells. Guanosine 5', α-β-methylene triphosphate (Gp(CH2)pp), a constitutively active GTP analogue that activates Gβγ, also inhibited Epac-induced cell migration. In addition, co-overexpression of β1 and γ2, which is the major combination of Gβγ, inhibited Epac1-induced cell migration. By contrast, when the C-terminus of β adrenergic receptor kinase (βARK-CT), an endogenous inhibitor for Gβγ, was overexpressed, mSIRK's inhibitory effect on Epac-induced cell migration was negated, suggesting the specificity of mSIRK for Gβγ. 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 β1 and γ2 subunits inhibited 8-pMeOPT-induced Ca ²⁺elevation. Inhibition of Gβγ with βARK-CT or guanosine 5'-O-(2-thiodiphosphate) (GDPβS), a GDP analogue that inactivates Gβγ, restored 8-pMeOPT-induced Ca ²⁺elevation even in the presence of mSIRK. These data suggested that Gβγ inhibits Epac-induced Ca ²⁺elevation. Subsequently, the mechanism by which Gβγ 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.</p> <p>Conclusion</p> <p>We found the cross talk of Ca ²⁺signaling between Gβγ and Epac, which plays a major role in melanoma cell migration.</p

Epac1 increases migration of endothelial cells and melanoma cells via FGF2-mediated paracrine signaling

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

Ischemic preconditioning prevents ischemia-induced beta-adrenergic receptor sequestration

Abstract Preconditioning enables endogenous protection to repeated myocardial ischemia. However, the effect of preconditioning on beta1 adrenergic receptor (AR) signal remains controversial. We have recently developed receptor assay system using whole cells, in which overexpressed cell surface beta ARs can be readily quantitated without disrupting the cell. Using this technique, we examined the effects of chemical/metabolic ischemia on the beta1 AR sequestration and adenylyl cyclase activity. Isoproterenol treatment, but not forskolin treatment, of HEK293T cells overexpressing beta1 ARs led to a rapid decrease (within 2 hours) in the number of the cell surface receptor, which was negated in the presence of concanavalin A. Similarly, treatment of cells with potassium cyanide and 2-deoxy-D-glucose (chemical/metabolic ischemia) induced similar receptor sequestration. When isoproterenol was superimposed on chemical/metabolic ischemia, the degree of sequestration became greater. However, when cells were pre-exposed to potassium cyanide on the preceding day (chemical preconditioning), the sequestration induced by either isoproterenol or chemical/metabolic ischemia was attenuated. Adenylyl cyclase catalytic activity as assessed by stimulation with forskolin was decreased by chemical/metabolic ischemia but fully recovered after 24 hours, suggesting that chemical/metabolic ischemia treatment did not alter cell viability. Putting together, chemical/metabolic ischemia induced beta1 AR sequestration in a similar manner to isoproterenol. In addition, preconditioning prevented the beta1 AR sequestration induced by both isoproterenol and chemical/metabolic ischemia. Pre-conditioning may play a role in preserving the cell surface beta ARs by inhibiting the sequestration that is usually induced by an ischemic event or beta adrenergic stimulation

Disruption of type 5 adenylyl cyclase negates the developmental increase in Gαolf expression in the striatum

AbstractThe two stimulatory G protein α subunits, Gαs and Gαolf, activate adenylyl cyclase in a similar way. We examined whether type 5 adenylyl cyclase knockout, the major striatal isoform, can differentially and/or developmentally change the expression of these G proteins in the striatum. Gαs and Gαolf expressions at birth were unaffected in knockouts, which, however, demonstrated a blunted developmental increase in Gαolf, but not Gαs. Adenylyl cyclase activity was unaffected at birth, but subsequently became lower in knockouts. These findings suggest that type 5 adenylyl cyclase does not contribute to striatal cAMP signaling at birth. However, it may play an important role in developmental changes in the expression of Gαolf, but not Gαs

Epac increases melanoma cell migration by a heparan sulfate-related mechanism

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