RESUMO
Anterior pituitary cell turnover depends on a tight balance between proliferation and apoptosis. We have previously shown that estrogens sensitize anterior pituitary cells to pro-apoptotic stimuli. c-FLIP (cellular-FLICE-inhibitory-protein) isoforms are regulatory proteins of apoptosis triggered by death receptors. c-FLIPshort isoform competes with procaspase-8 inhibiting its activation. However, c-FLIPlong isoform may have a pro- or anti-apoptotic function depending on its expression level. In the present study, we explored whether estrogens modulate c-FLIP expression in anterior pituitary cells from ovariectomized (OVX) rats and in GH3 cells, a somatolactotrope cell line. Acute administration of 17ß-estradiol to OVX rats increased c-FLIPlong expression in the anterior pituitary gland without changing c-FLIPshort expression as assessed by Western blot. Estradiol in vitro also increased c-FLIPlong expression in anterior pituitary cells but not in GH3 cells. As determined by flow cytometry, the percentage of anterior pituitary cells expressing c-FLIP was higher than in GH3 cells. However, c-FLIP fluorescence intensity in GH3 cells was higher than in anterior pituitary cells. FasL increased the percentage of TUNEL-positive GH3 cells incubated either with or without estradiol suggesting that the pro-apoptotic action of Fas activation is estrogen-independent. Our results show that unlike what happens in nontumoral pituitary cells, estrogens do not modulate either c-FLIPlong expression or FasL-induced apoptosis in GH3 cells. The stimulatory effect of estradiol on c-FLIPlong expression could be involved in the sensitizing effect of this steroid to apoptosis in anterior pituitary cells. The absence of this estrogenic action in tumor pituitary cells could be involved in their tumor-like behavior.
Assuntos
Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/genética , Estradiol/metabolismo , Adeno-Hipófise/metabolismo , Animais , Apoptose , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Células Cultivadas , Estrogênios/metabolismo , Feminino , Adeno-Hipófise/citologia , Ratos , Ratos Wistar , Regulação para CimaRESUMO
Activation of nuclear factor (NF)-κB promotes cell proliferation and inhibits apoptosis. We have previously shown that oestrogens sensitise normal anterior pituitary cells to the apoptotic effect of tumour necrosis factor (TNF)-α by inhibiting NF-κB nuclear translocation. In the present study, we examined whether oestrogens also modulate the NF-κB signalling pathway and apoptosis in GH3 cells, a rat somatolactotroph tumour cell line. As determined by Western blotting, 17ß-oestradiol (E2 ) (10(-9) m) increased the nuclear concentration of NF-κB/p105, p65 and p50 in GH3 cells. However, E2 did not modify the expression of Bcl-xL, a NF-κB target gene. TNF-α induced apoptosis of GH3 cells incubated in either the presence or absence of E2 . Inhibition of the NF-kB pathway using BAY 11-7082 (BAY) (5 µm) decreased the viability of GH3 cells and increased the percentage of terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL)-positive GH3 cells. BAY also increased TNF-α-induced apoptosis of GH3 cells, an effect that was further increased by an inhibitor of the c-Jun N-terminal protein kinase pathway, SP600125 (10 µm). We also analysed the role of the NF-κB signalling pathway on proliferation and apoptosis of GH3 tumours in vivo. The administration of BAY to nude mice bearing GH3 tumours increased the number of TUNEL-positive cells and decreased the number of proliferating GH3 cells. These findings suggest that GH3 cells lose their oestrogenic inhibitory action on the NF-κB pathway and that the pro-apoptotic effect of TNF-α on these tumour pituitary cells does not require sensitisation by oestrogens as occurs in normal pituitary cells. NF-κB was required for the survival of GH3 cells, suggesting that pharmacological inhibition of the NF-κB pathway could interfere with pituitary tumour progression.
Assuntos
Apoptose/fisiologia , Estrogênios/metabolismo , Lactotrofos/metabolismo , NF-kappa B/metabolismo , Neoplasias Hipofisárias/metabolismo , Transdução de Sinais/fisiologia , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Camundongos , Camundongos Nus , Ratos , Ratos WistarRESUMO
Nuclear factor-kappa B (NF-κB), an important pro-inflammatory factor, is a crucial regulator of cell survival. Both lipopolysaccharide (LPS) and tumour necrosis factor (TNF)-α activate NF-κB signalling. Oestrogens were shown to suppress NF-κB activation. Oestrogens exert a sensitising action to pro-apoptotic stimuli such as LPS and TNF-α in anterior pituitary cells. In the present study, we show by western blotting that 17ß-oestradiol (E(2)) decreases TNF-α-induced NF-κB/p65 and p50 nuclear translocation in primary cultures of anterior pituitary cells from ovariectomised (OVX) rats. Also, the in vivo administration of E(2) decreases LPS-induced NF-κB/p65 and p50 nuclear translocation. To investigate whether the inhibition of NF-κB pathway sensitises anterior pituitary cells to pro-apoptotic stimuli, we used an inhibitor of NF-κB activity, BAY 11-7082 (BAY). BAY, at a concentration that fails to induce apoptosis, has permissive action on TNF-α-induced apoptosis of lactotrophs and somatotrophs from OVX rats, as assessed by terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL). Pharmacological inhibition of NF-κB signalling enhances E(2)-sensitising effect to TNF-α-induced apoptosis in lactotrophs but not in somatotrophs. In vivo administration of BAY allowed LPS-induced apoptosis in anterior pituitary cells from OVX rats (determined by fluorescence activated cell sorting). Furthermore, LPS-induced expression of Bcl-xL in pituitaries of OVX rats is decreased by E(2) administration. Our results show that inhibition of the NF-κB signalling pathway sensitises anterior pituitary cells to the pro-apoptotic action of LPS and TNF-α. Because E(2) inhibits LPS- and TNF-α-activated NF-κB nuclear translocation, the present study suggests that E(2) sensitises anterior pituitary cells to TNF-α- and LPS-induced apoptosis by inhibiting NF-κB activity.
Assuntos
Apoptose/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , NF-kappa B/antagonistas & inibidores , Adeno-Hipófise/citologia , Fator de Necrose Tumoral alfa/farmacologia , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Células Cultivadas , Estradiol/farmacologia , Feminino , NF-kappa B/metabolismo , Nitrilas/farmacologia , Ovariectomia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sulfonas/farmacologiaRESUMO
It is now accepted that estrogens not only stimulate lactotrope proliferation but also sensitize anterior pituitary cells to proapoptotic stimuli. In addition to their classical mechanism of action through binding to intracellular estrogen receptors (ERs), there is increasing evidence that estrogens exert rapid actions mediated by cell membrane-localized ERs (mERs). In the present study, we examined the involvement of membrane-initiated steroid signaling in the proapoptotic action of estradiol in primary cultures of anterior pituitary cells from ovariectomized rats by using estren, a synthetic estrogen with no effect on classical transcription and a cell-impermeable 17beta-estradiol conjugate (E2-BSA). Both compounds induced cell death of anterior pituitary cells after 60 min of incubation as assessed by flow cytometry and the [3-(4,5-dimethylthiazol-2-yl)]-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Estren, E2, and E2-BSA induced apoptosis of lactotropes and somatotropes as evaluated by the deoxynucleotidyltransferase-mediated dUTP nick end-labeling assay and immunodetection of prolactin (PRL) and growth hormone (GH). The proapoptotic effect of E2-BSA was abrogated by ICI-182,780, an antagonist of ERs. The expression of membrane-associated ERalpha was observed in PRL- and GH-bearing cells. Our results indicate that estradiol is able to exert a rapid apoptotic action in anterior pituitary cells, especially lactotropes and somatotropes, by a mechanism triggered by mERs. This mechanism could be involved in anterior pituitary cell turnover.
Assuntos
Apoptose/efeitos dos fármacos , Estrogênios/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Animais , Células Cultivadas , Estradiol/farmacologia , Estrenos/farmacologia , Feminino , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/metabolismo , Adeno-Hipófise/fisiologia , Prolactina/metabolismo , Ratos , Ratos Wistar , Receptores de Estrogênio/metabolismo , Fatores de TempoRESUMO
We have isolated from rat cerebral cortex an endogenous Na(+), K(+)-ATPase inhibitor, termed endobain E, which modulates glutamatergic N-methyl-d-aspartate (NMDA) receptor. This endogenous factor allosterically decreases [(3)H]dizocilpine binding to NMDA receptor, most likely acting as a weak channel blocker. In the present study we investigated whether endobain E is present in the cerebral cortex of rats subjected to ischemia and modulates NMDA receptor exposed to the same conditions. Ischemia-reperfusion was carried out by bilateral occlusion of common carotid arteries followed by a 15-min reperfusion period. Elution profile of brain soluble fraction showed that endobain E is present in cerebral cortex of ischemia-reperfusion rats. On assaying its effect on synaptosomal membrane Na(+), K(+)-ATPase activity and [(3)H]dizocilpine binding to cerebral cortex membranes prepared from animals without treatment, it was found that the endogenous modulator isolated from ischemia-reperfusion rats was able to inhibit both enzyme activity and ligand binding. On the other hand, endobain E prepared from rats without treatment also decreased binding to cerebral cortex or hippocampal membranes obtained from animals exposed to ischemia-reperfusion. Since ischemia decreases tissue pH and NMDA receptor activity varies according to proton concentration, pH influence on endobain E effect was tested. Endobain E ( approximately 80 mg original tissue) decreased [(3)H]dizocilpine binding 25% at pH 7.4 or 8.0 but 90% at pH 6.5. These results demonstrate that endobain E is present and also able to modulate NMDA receptor in the short-term period that follows cerebral ischemia and that its effect depends on proton concentration, suggesting greater NMDA receptor modulation by endobain E at low pH, typical of ischemic tissues.
Assuntos
Isquemia Encefálica/metabolismo , Córtex Cerebral/metabolismo , Ouabaína/análogos & derivados , Receptores de N-Metil-D-Aspartato/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isquemia Encefálica/complicações , Isquemia Encefálica/enzimologia , Córtex Cerebral/enzimologia , Modelos Animais de Doenças , Maleato de Dizocilpina/metabolismo , Concentração de Íons de Hidrogênio , Membranas Intracelulares/enzimologia , Membranas Intracelulares/metabolismo , Masculino , Ouabaína/metabolismo , Ligação Proteica , Ratos , Ratos Wistar , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/etiologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Frações Subcelulares/enzimologia , Frações Subcelulares/metabolismoRESUMO
The Fas/FasL system provides the major apoptotic mechanism for many cell types, participating in cell turnover in hormone-dependent tissues. In the present study, we localized both Fas and FasL in anterior pituitary cells, mainly in lactotropes and somatotropes. The percentage of anterior pituitary cells showing immunoreactivity for Fas or FasL was higher in cells from rats killed in proestrus than in diestrus. Also, the proportion of pituitary cells from ovariectomized (OVX) rats expressing Fas or FasL increased in the presence of 17beta-estradiol (10(-9) M). This steroid increased the percentage of lactotropes with immunoreactivity for Fas or FasL and the percentage of somatotropes expressing Fas. Activation of Fas by an agonist anti-Fas antibody (Mab-Fas) decreased the vi-ability-3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT assay)-of anterior pituitary cells from OVX rats cultured in the presence of 17beta-estradiol. Also, membrane-bound FasL decreased cell viability-[3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay (MTS assay)-only when anterior pituitary cells from OVX rats were incubated with 17beta-estradiol. Moreover, FasL increased the percentage of hypodiploid anterior pituitary cells (flow cytometry). Mab-Fas increased the percentage of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL)-positive pituitary cells and lactotropes from OVX rats only when cells were incubated in the presence of 17beta-estradiol. Also, Mab-Fas triggered apoptosis of anterior pituitary cells from rats killed at proestrus but not at diestrus. Our results show that 17beta-estradiol up-regulates the expression of the Fas/FasL system in anterior pituitary cells and increases Fas-induced apoptosis in lactotropes, suggesting that Fas-induced apoptosis could be involved in the pituitary cell renewal during the estrous cycle.
Assuntos
Apoptose/fisiologia , Estrogênios/fisiologia , Glicoproteínas de Membrana/metabolismo , Adeno-Hipófise/fisiologia , Prolactina/metabolismo , Fatores de Necrose Tumoral/metabolismo , Receptor fas/metabolismo , Animais , Diestro , Estradiol/farmacologia , Proteína Ligante Fas , Feminino , Ovariectomia , Adeno-Hipófise/citologia , Adeno-Hipófise/metabolismo , Proestro , Ratos , Ratos Wistar , Regulação para CimaRESUMO
We previously reported that TNF-alpha-induced apoptosis of lactotropes is estrogen dependent and predominant at proestrus. Here we observed that TNF-alpha (50 ng/ml) failed to induce apoptosis of anterior pituitary cells from ovariectomized rats cultured in the presence of progesterone (10(-6) m). However, progesterone blocked the apoptotic effect of TNF-alpha in anterior pituitary cells and lactotropes cultured with 17beta-estradiol (10(-9) m). In addition, 17beta-estradiol induced apoptosis of somatotropes and triggered the proapoptotic action of TNF-alpha in these cells, effects completely blocked by ICI 182 780 (10(-6) m), an estrogen receptor antagonist. Progesterone reverted the permissive effect of 17beta-estradiol on TNF-alpha-induced apoptosis of somatotropes. TNF-alpha induced apoptosis of somatotropes from rats killed at proestrus but not at diestrus. The antiprogestine ZK 98,299 (10(-6) m) completely inhibited the protective action of progesterone on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Although progesterone can interact with glucocorticoid receptors, dexamethasone (10(-6) m) had no effect on TNF-alpha-induced apoptosis of anterior pituitary cells, lactotropes, and somatotropes. Our results show that progesterone, by interacting with progesterone receptors, antagonizes the permissive action of estrogens on TNF-alpha-induced apoptosis of lactotropes and somatotropes. These observations suggest that the steroid milieu may modulate the apoptotic response of anterior pituitary cells during the estrous cycle.
Assuntos
Apoptose/efeitos dos fármacos , Estradiol/farmacologia , Adeno-Hipófise/citologia , Progesterona/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Células Cultivadas , Dexametasona/farmacologia , Interações Medicamentosas , Feminino , Glucocorticoides/farmacologia , Ovariectomia , Adeno-Hipófise/efeitos dos fármacos , Ratos , Ratos Wistar , Receptores de Progesterona/fisiologiaRESUMO
A brain endogenous factor, termed endobain E, allosterically decreases [3H]dizocilpine binding to NMDA receptor. Such effect depends on receptor activation by the coagonists glutamate and glycine and is interfered by channel blockers, suggesting its interaction with the inner surface of the associated channel. To further analyze endobain E effect on NMDA receptor, in the current study competitive [3H]dizocilpine binding assays to brain membranes were performed with Zn2+ to block the associated channel, as well as with spermidine (SPD), which exerts positive allosteric modulation of NMDA receptor. Partially or nonadditive effects on [3H]dizocilpine binding were recorded, respectively, in the presence of endobain E at a concentration that inhibits binding 25% plus IC25 Zn2+ or endobain E at a concentration that inhibits binding 50% plus IC50 Zn2+. With an endobain E concentration that decreases 25% ligand binding, SPD potentiated binding over a wide concentration range but failed to modify endobain E effect. Similarly, [3H]dizocilpine binding reduction over a wide endobain E concentration range remained unaltered by high SPD concentrations. Additive effects were observed with endobain E at a concentration that decreases binding 25% plus IC25 SPD site antagonists arcaine or ifenprodil. Zn2+ experiments indicated that endobain E effect is interfered by channel blockade produced by this ion. Although endobain E effect is dependent on NMDA receptor activation by glutamate and glycine, it proves independent of the positive modulation exerted by SPD. Thus the endogenous modulator seems not to interact at NMDA receptor polyamine site, favoring the hypothesis that endobain E binds inside the associated channel.
Assuntos
Ouabaína/análogos & derivados , Ouabaína/farmacologia , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Espermidina/fisiologia , Zinco/farmacologia , Animais , Maleato de Dizocilpina/metabolismo , Masculino , Ensaio Radioligante , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , TrítioRESUMO
The entry of rotaviruses into epithelial cells seems to be a multistep process. Infection competition experiments have suggested that at least three different interactions between the virus and cell surface molecules take place during the early events of infection, and glycolipids as well as glycoproteins have been suggested to be primary attachment receptors for rotaviruses. The infectivity of some rotavirus strains depends on the presence of sialic acid on the cell surface, however, it has been shown that this interaction is not essential, and it has been suggested that there exists a neuraminidase-resistant cell surface molecule with which most rotaviruses interact. The comparative characterization of the sialic acid-dependent rotavirus strain RRV (G3P5[3]), its neuraminidase-resistant variant nar3, and the human rotavirus strain Wa (G1P1A[8]) has allowed us to show that alpha 2 beta 1 integrin is used by nar3 as its primary cell attachment site, and by RRV in a second interaction, subsequent to its initial contact with a sialic acid-containing cell receptor. We have also shown that integrin alpha V beta 3 is used by all three rotavirus strains as a co-receptor, subsequent to their initial attachment to the cell. We propose that the functional rotavirus receptor is a complex of several cell molecules most likely immersed in glycosphingolipid-enriched plasma membrane microdomains.
Assuntos
Receptores Virais/metabolismo , Rotavirus/metabolismo , Animais , Capsídeo/genética , Capsídeo/metabolismo , Genes Virais , Humanos , Integrinas/metabolismo , Modelos Biológicos , Rotavirus/genética , Proteínas Estruturais Virais/genética , Replicação ViralRESUMO
Rotavirus strains differ in their need for sialic acid (SA) for initial binding to the cell surface; however, the existence of a postattachment cell receptor, common to most, if not all, rotavirus strains, has been proposed. In the present study, antibodies to the alpha(v) and beta(3) integrin subunits, and the alpha(v)beta(3) ligand, vitronectin, efficiently blocked the infectivity of the SA-dependent rhesus rotavirus RRV, its SA-independent variant nar3, and the neuraminidase-resistant human rotavirus strain Wa. Vitronectin and anti-beta(3) antibodies, however, did not block the binding of virus to cells, indicating that rotaviruses interact with alpha(v)beta(3) at a postbinding step, probably penetration. This interaction was shown to be independent of the tripeptide motif arginine-glycine-aspartic acid present in the natural ligands of this integrin. Transfection of CHO cells with alpha(v)beta(3) genes significantly increased their permissiveness to all three rotavirus strains, and the increment of virus infectivity was reverted by incubation of these cells either with antibodies to beta(3) or with vitronectin. These findings implicate alpha(v)beta(3) integrin as a cellular receptor common to neuraminidase-sensitive and neuraminidase-resistant rotaviruses, and support the hypothesis that this integrin could determine, at least in part, the cellular susceptibility to rotaviruses.
Assuntos
Receptores Virais/metabolismo , Receptores de Vitronectina/metabolismo , Rotavirus/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Sítios de Ligação , Células CHO , Cricetinae , Fibronectinas/metabolismo , Humanos , Integrinas/metabolismo , Ligantes , Oligopeptídeos/metabolismo , Receptores de Colágeno , Receptores de Vitronectina/imunologia , Rotavirus/fisiologia , Vitronectina/metabolismoRESUMO
It was previously reported that integrins alpha2beta1, alpha4beta1, and alphaXbeta2 are involved in rotavirus cell infection. In this work we studied the role of integrin subunits alpha2, alpha4, and beta2 on the attachment of rotaviruses RRV and nar3 to MA104 cells. Integrin alpha2beta1 was found to serve as the binding receptor for the neuraminidase-resistant virus nar3, whereas the neuraminidase-sensitive strain RRV interacted with this integrin at a postattachment step. It was shown that nar3 binds alpha2beta1 through the DGE integrin-recognition motif located in the virus surface protein VP5. Integrin subunits alpha4 and beta2 do not seem to be involved in the initial cell binding of either virus.
Assuntos
Integrinas/fisiologia , Neuraminidase/farmacologia , Rotavirus/fisiologia , Anticorpos Monoclonais/farmacologia , Antígenos CD/imunologia , Antígenos CD/fisiologia , Sítios de Ligação , Antígenos CD18/imunologia , Antígenos CD18/fisiologia , Capsídeo/química , Capsídeo/metabolismo , Proteínas do Capsídeo , Linhagem Celular , Resistência Microbiana a Medicamentos , Ensaio de Imunoadsorção Enzimática , Integrina alfa2 , Integrina alfa4 , Integrinas/química , Integrinas/metabolismo , Receptores de Colágeno , Rotavirus/patogenicidadeRESUMO
We have tested the effect of metabolic inhibitors, membrane cholesterol depletion, and detergent extraction of cell surface molecules on the susceptibility of MA104 cells to infection by rotaviruses. Treatment of cells with tunicamycin, an inhibitor of protein N glycosylation, blocked the infectivity of the SA-dependent rotavirus RRV and its SA-independent variant nar3 by about 50%, while the inhibition of O glycosylation had no effect. The inhibitor of glycolipid biosynthesis d, l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) blocked the infectivity of RRV, nar3, and the human rotavirus strain Wa by about 70%. Sequestration of cholesterol from the cell membrane with beta-cyclodextrin reduced the infectivity of the three viruses by more than 90%. The involvement of N-glycoproteins, glycolipids, and cholesterol in rotavirus infection suggests that the virus receptor(s) might be forming part of lipid microdomains in the cell membrane. MA104 cells incubated with the nonionic detergent octyl-beta-glucoside (OG) showed a ca. 60% reduction in their ability to bind rotaviruses, the same degree to which they became refractory to infection, suggesting that OG extracts the potential virus receptor(s) from the cell surface. Accordingly, when preincubated with the viruses, the OG extract inhibited the virus infectivity by more than 95%. This inhibition was abolished when the extract was treated with either proteases or heat but not when it was treated with neuraminidase, indicating the protein nature of the inhibitor. Two protein fractions of around 57 and 75 kDa were isolated from the extract, and these fractions were shown to have rotavirus-blocking activity. Also, antibodies to these fractions efficiently inhibited the infectivity of the viruses in untreated as well as in neuraminidase-treated cells. Five individual protein bands of 30, 45, 57, 75, and 110 kDa, which exhibited virus-blocking activity, were finally isolated from the OG extract. These proteins are good candidates to function as rotavirus receptors.
Assuntos
Receptores Virais/isolamento & purificação , Rotavirus/fisiologia , Colesterol/fisiologia , Glucosídeos/farmacologia , Glicolipídeos/biossíntese , Glicosilação , Humanos , Peso Molecular , Receptores Virais/fisiologiaAssuntos
Humanos , Criança , Peso-Estatura , Estado Nutricional , Congressos como Assunto , CongressoAssuntos
Criança , Adolescente , Adulto , Pessoa de Meia-Idade , Humanos , Masculino , Feminino , Halotano , Histamina , Basófilos , Fatores Inibidores da Migração de LeucócitosRESUMO
The authors refers the actual status of medical and dermatological administration in Cuba.