RESUMO
Although the lipid mediator sphingosine 1-phosphate (S1P) has been identified to induce cell growth arrest of human keratinocytes, the sphingolipid effectively protects these epidermal cells from apoptosis. The molecular mechanism of the anti-apoptotic action induced by S1P is less characterized. Apart from S1P, endogenously produced nitric oxide (NOâ¢) has been recognized as a potent modulator of apoptosis in keratinocytes. Therefore, it was of great interest to elucidate whether S1P protects human keratinocytes via a NOâ¢-dependent signalling pathway. Indeed, S1P induced an activation of endothelial nitric oxide synthase (eNOS) in human keratinocytes leading to an enhanced formation of NOâ¢. Most interestingly, the cell protective effect of S1P was almost completely abolished in the presence of the eNOS inhibitor L-NAME as well as in eNOS-deficient keratinocytes indicating that the sphingolipid metabolite S1P protects human keratinocytes from apoptosis via eNOS activation and subsequent production of protective amounts of NOâ¢. It is well established that most of the known actions of S1P are mediated by a family of five specific G protein-coupled receptors. Therefore, the involvement of S1P-receptor subtypes in S1P-mediated eNOS activation has been examined. Indeed, this study clearly shows that the S1P(3) is the exclusive receptor subtype in human keratinocytes which mediates eNOS activation and NO⢠formation in response to S1P. In congruence, when the S1P(3) receptor subtype is abrogated, S1P almost completely lost its ability to protect human keratinocytes from apoptosis.
Assuntos
Apoptose , Queratinócitos/metabolismo , Lisofosfolipídeos/metabolismo , Óxido Nítrico/biossíntese , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Óxido Nítrico Sintase Tipo III/metabolismo , Transdução de Sinais , Esfingosina/metabolismo , Esfingosina/farmacologiaRESUMO
BACKGROUND: FTY720, a synthetic compound produced by modification of a metabolite from Isaria sinclairii, is known as a unique immunosuppressive agent that exerts its activity by inhibiting lymphocyte egress from secondary lymphoid tissues. FTY720 is phosphorylated in vivo by sphingosine kinase 2 to FTY720-phosphate (FTY720-P), which acts as a potent sphingosine-1-phosphate (S1P) receptor agonist. Despite its homology to S1P, which exerts antiapoptotic actions in different cells, FTY720 has also been reported to be able to induce apoptosis in a variety of cells. METHODS: Therefore, we investigated the action of both, FTY720 and its phosphorylated version FTY720-P, on apoptosis. Moreover, signalling pathways of apoptosis in response to FTY720 and FTY720-P were examined. RESULTS AND CONCLUSIONS: Although FTY720 acts apoptotic at micromolar concentrations in human fibroblasts the phosphorylated compound FTY720-P possesses a pronounced antiapoptotic effect counteracting FTY720-induced programmed cell death. Interestingly, none of the classical antiapoptotic pathways like MAP kinases, Akt or mTOR play a role in the protective role of FTY720-P. Most important, we identified that the S1P(3) receptor subtype is involved in the antiapoptotic action of FTY720-P leading to an increased phosphorylation of Bcl-2 and changes in the mitochondrial membrane potential.
Assuntos
Apoptose/efeitos dos fármacos , Fibroblastos/metabolismo , Imunossupressores/farmacologia , Organofosfatos/farmacologia , Propilenoglicóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Cloridrato de Fingolimode , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Esfingosina/farmacologia , Serina-Treonina Quinases TORRESUMO
The preprotachykinin gene Tac4 expressed in murine uterus and placenta is thought to encode a peptide RSRTRQFYGLM-NH(2), mouse hemokinin 1. We have examined the uterotonic effects of mouse hemokinin 1 and its N-terminally truncated analogue, mouse hemokinin 1(2-11) on mouse uterus. Mouse hemokinin 1(2-11) was equieffective with but slightly less potent than substance P in tissues from non-pregnant Swiss mice. On myometrium from Balb C mice primed with oestrogen the positions of concentration-response curves to substance P and the mouse hemokinins were similar to those of neurokinin A, but the maximum responses were lower. The tachykinin NK(1) receptor antagonist, 1-{2-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl}-4phenyl-1-azonia-bicyclo[2.2.2]octane (SR 140333), reduced the effects of the agonists in tissues from both groups of mice. In myometria from late pregnant (Days 17-18) Balb C mice the responses to mouse hemokinin 1(2-11) were less potent than in those from oestrogen-primed mice. Human hemokinin 1, the human orthologue of mouse hemokinin 1, was more effective than mouse hemokinin 1(2-11), while endokinin D was inactive. Mouse hemokinin 1 effects were blocked by SR 140333 alone and in combination with ((S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48968) but not by SR 48968 alone. Thus the mouse hemokinins are tachykinin NK(1) receptor-preferring uterotonic agonists in non-pregnant mice but lack action at the myometrial tachykinin NK(2) receptors present in late pregnant mice.
Assuntos
Precursores de Proteínas/metabolismo , Receptores da Neurocinina-1/metabolismo , Receptores da Neurocinina-2/metabolismo , Taquicininas/metabolismo , Útero/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Miométrio/metabolismo , Neurocinina A/farmacologia , Gravidez , Prenhez , Precursores de Proteínas/farmacologia , Receptores da Neurocinina-1/efeitos dos fármacos , Receptores da Neurocinina-2/efeitos dos fármacos , Substância P/farmacologia , Taquicininas/farmacologiaRESUMO
Breast cancer development and breast cancer progression involves the deregulation of growth factors leading to uncontrolled cellular proliferation, invasion and metastasis. Transforming growth factor (TGF)-beta plays a crucial role in breast cancer because it has the potential to act as either a tumor suppressor or a pro-oncogenic chemokine. A cross-communication between the TGF-beta signaling network and estrogens has been postulated, which is important for breast tumorigenesis. Here, we provide evidence that inhibition of TGF-beta signaling is associated with a rapid estrogen-dependent nongenomic action. Moreover, we were able to demonstrate that estrogens disrupt the TGF-beta signaling network as well as TGF-beta functions in breast cancer cells via the G protein-coupled receptor 30 (GPR30). Silencing of GPR30 in MCF-7 cells completely reduced the ability of 17-beta-estradiol (E2) to inhibit the TGF-beta pathway. Likewise, in GPR30-deficient MDA-MB-231 breast cancer cells, E2 achieved the ability to suppress TGF-beta signaling only after transfection with GPR30-encoding plasmids. It is most interesting that the antiestrogen fulvestrant (ICI 182,780), which possesses agonistic activity at the GPR30, also diminished TGF-beta signaling. Further experiments attempted to characterize the molecular mechanism by which activated GPR30 inhibits the TGF-beta pathway. Our results indicate that GPR30 induces the stimulation of the mitogen-activated protein kinases (MAPKs), which interferes with the activation of Smad proteins. Inhibition of MAPK activity prevented the ability of E2 from suppressing TGF-beta signaling. These findings are of great clinical relevance, because down-regulation of TGF-beta signaling is associated with the development of breast cancer resistance in response to antiestrogens.