RESUMO
INTRODUCTION: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. METHODS: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. RESULTS: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. CONCLUSIONS: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.
Assuntos
Macroautofagia , Neoplasias Hipofisárias , Humanos , Ratos , Animais , Proteína Beclina-1 , Ratos Endogâmicos F344 , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Mamíferos/metabolismoRESUMO
To investigate the putative stem cell/tumor stem cell (SC/TSC) niche contribution to hyperplasic/adenomatous pituitary lesions, we analyzed variation in the pituitary stem cell population during the development of experimental pituitary tumors. Pituitary tumors were induced in female F344 rats with estradiol benzoate for 5, 10, 20 and 30 days. Cells positive for GFRa2, Sox2, Sox9, Nestin, CD133 and CD44 were identified in the marginal zone and in the adenoparenchyma in both control and 30D groups, with predominant adenoparenchyma localization of GRFa2 and SOX9 found in tumoral pituitaries. GFRa2, Nestin, CD133 and CD44 were upregulated at the initial stages of tumor growth, whereas Sox9 significantly decreased at 5D, with Sox2 remaining invariable during the hyperplasic/adenomatous development. In addition, isolated pituispheres from normal and tumoral pituitary glands enriched in SC/TSC were characterized. Pituispheres from the 30D glands were positive for the above-mentioned markers and showed a significant increase in the proliferation. In conclusion, our data revealed pituitary SC pool fluctuations during hyperplastic/adenomatous development, with differential localization of the SC/TSC niche in this process. These findings may help to provide a better understanding of these cell populations, which is crucial for achieving advancements in the field of pituitary tumor biology.
Assuntos
Adenoma/patologia , Neoplasias Hipofisárias/patologia , Nicho de Células-Tronco/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Feminino , Células-Tronco Neoplásicas/patologia , Células-Tronco Neoplásicas/fisiologia , Hipófise/patologia , Hipófise/fisiologia , Ratos , Ratos Endogâmicos F344 , Microambiente Tumoral/fisiologiaRESUMO
The molecular mechanisms underlying the capability of pituitary tumours to avoid unregulated cell proliferation are still not well understood. However, the NF-κB transcription factor, which is able to modulate not only cellular senescence but also tumour progression, has emerged as a targeted candidate. This work was focused on the NF-κB role in cellular senescence during the progression of experimental pituitary tumours. Also, the contribution of the signalling pathways in senescence-associated NF-κB activation and the senescence-associated secretory phenotype (SASP) and pro-survival-NF-κB target genes transcription were analysed. A robust NF-κB activation was seen at E20-E40 of tumour development accompanied by a marked SA-ß-Gal co-reactivity in the tumour pituitary parenchyma. The induction of TNFα and IL1-ß as specific SASP-related NF-κB target genes as well as Bcl-2 and Bcl-xl pro-survival genes was shown to be accompanied by increases in the p-p38 MAPK protein levels, starting at the E20 stage and strengthening from 40 to 60 days of tumour growth. It is noteworthy that p-JNK displayed a similar pattern of activation during pituitary tumour development, while p-AKT and p-ERK1/2 were downregulated. By employing a pharmacological strategy to abrogate NF-κB activity, we demonstrated a marked reduction in SA-ß-Gal activity and a slight decrease in Ki67 immunopositive cells after NF-κB blockade. These results suggest a central role for NF-κB in the regulation of the cellular senescence programme, leading to the strikingly benign intrinsic nature of pituitary adenomas.
Assuntos
Senescência Celular/genética , NF-kappa B/fisiologia , Neoplasias Hipofisárias/genética , Transdução de Sinais/genética , Animais , Modelos Animais de Doenças , Regulação da Expressão Gênica , Genes bcl-2/fisiologia , Hipoxantina Fosforribosiltransferase/metabolismo , Interleucina-1beta/metabolismo , Masculino , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo , Proteína bcl-X/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In pituitary adenomas, early recurrences and resistance to conventional pharmacotherapies are common, but the mechanisms involved are still not understood. The high expression of epidermal growth factor receptor 2 (HER2)/extracellular signal-regulated kinase (ERK1/2) signal observed in human pituitary adenomas, together with the low levels of the antimitogenic transforming growth factor beta receptor 2 (TBR2), encouraged us to evaluate the effect of the specific HER2 inhibition with trastuzumab on experimental pituitary tumor cell growth and its effect on the antiproliferative response to TGFB1. Trastuzumab decreased the pituitary tumor growth as well as the expression of ERK1/2 and the cell cycle regulators CCND1 and CDK4. The HER2/ERK1/2 pathway is an attractive therapeutic target, but its intricate relations with other signaling modulators still need to be unraveled. Thus, we investigated possible cross-talk with TGFB signaling, which has not yet been studied in pituitary tumors. In tumoral GH3 cells, co-incubation with trastuzumab and TGFB1 significantly decreased cell proliferation, an effect accompanied by a reduction in ERK1/2 phosphorylation, an increase of SMAD2/3 activation. In addition, through immunoprecipitation assays, a diminution of SMAD2/3-ERK1/2 and an increase SMAD2/3-TGFBR1 interactions were observed when cells were co-incubated with trastuzumab and TGFB1. These findings indicate that blocking HER2 by trastuzumab inhibited pituitary tumor growth and modulated HER2/ERK1/2 signaling and consequently the anti-mitogenic TGFB1/TBRs/SMADs cascade. The imbalance between HER2 and TGFBRs expression observed in human adenomas and the response to trastuzumab on experimental tumor growth may make the HER2/ERK1/2 pathway an attractive target for future pituitary adenoma therapy.
Assuntos
Adenoma/metabolismo , Proliferação de Células/efeitos dos fármacos , Neoplasias Hipofisárias/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Trastuzumab/farmacologia , Adenoma/patologia , Adulto , Ciclo Celular/efeitos dos fármacos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosforilação , Neoplasias Hipofisárias/patologia , Adulto JovemRESUMO
Androgen signaling in prostate smooth muscle cells (pSMCs) is critical for the maintenance of prostate homeostasis, the alterations of which are a central aspect in the development of pathological conditions. Testosterone can act through the classic androgen receptor (AR) in the cytoplasm, eliciting genomic signaling, or through different types of receptors located at the plasma membrane for nongenomic signaling. We aimed to find evidence of nongenomic testosterone-signaling mechanisms in pSMCs and their participation in cell proliferation, differentiation, and the modulation of the response to lipopolysaccharide. We demonstrated that pSMCs can respond to testosterone by a rapid activation of ERK1/2 and Akt. Furthermore, a pool of ARs localized at the cell surface of pSMCs is responsible for a nongenomic testosterone-induced increase in cell proliferation. Through membrane receptor stimulation, testosterone favors a muscle phenotype, indicated by an increase in smooth muscle markers. We also showed that the anti-inflammatory effects of testosterone, capable of attenuating lipopolysaccharide-induced proinflammatory actions, are promoted only by receptors located inside the cell. We postulate that testosterone might perform prohomeostatic effects through intracellular-initiated mechanisms by modulating cell proliferation and inflammation, whereas some pathological, hyperproliferative actions would be induced by membrane-initiated nongenomic signaling in pSMCs.
Assuntos
Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Próstata/efeitos dos fármacos , Receptores Androgênicos/metabolismo , Testosterona/farmacologia , Animais , Células Cultivadas , Masculino , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Próstata/citologia , Próstata/metabolismo , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Distribuição TecidualRESUMO
Uric acid (UA) has been associated with renal fibrosis and progression of chronic kidney disease. However, the underlying mechanisms of this process have still not been identified. Here, we studied the role of the innate imunity receptor NLRP3/ASC in UA induced epithelial-mesenchymal transition (EMT) in kidney. Wistar rats were fed with oxonic acid 2% and UA 2% (OXA + U), OXA + U plus allopurinol (ALL) or regular chow (C) for 7 weeks. We analyzed the presence of EMT markers, the expression of NLRP3, ASC, Caspase-1 and Smad 2/3 molecules and the mitochondrial morphological and functional characteristics. High UA induced renal fibrosis, mild chronic inflammation, as well as morphological and biochemical evidence of EMT. High UA also increased the expression of NLRP3/ASC with activation of both inflammasome related caspase-1 and inflammasome unrelated Smad 2/3 pathways. Ultrastructural co-localization of NLRP3 and Smad 2/3 indicated physical interaction between the two molecules. No morphological or functional changes were found between mitochondria exposed to high UA. In conclusion, kidney epithelial NLRP3/ASC expression was increased in high UA state in rats and both inflammasome related caspase-1 and non-inflammasome related P-Smad 2/3 pathways were associated with the observed EMT, inflammation and fibrosis induced by UA in the kidney.
Assuntos
Transição Epitelial-Mesenquimal/efeitos dos fármacos , Inflamassomos/metabolismo , Ácido Úrico/farmacologia , Animais , Caspase 1/metabolismo , Fibrose/induzido quimicamente , Inflamação/induzido quimicamente , Nefropatias/metabolismo , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos Wistar , Proteínas Smad/metabolismoRESUMO
Pituitary tumor cells have a poor response to the growth inhibitory effect of TGFß1, possibly resulting from the cross talk of TGFß/Smads signal with other signaling pathways, an undescribed mechanism in these tumoral cells. To address this hypothesis, we investigated whether the mitogen-activated extracellular signal-regulated kinase (MEK)/ERK1/2 and phosphoinositide-3 kinase/protein kinase B (PI3K/Akt) pathways were able to regulate the antimitogenic effect of TGFß1 on GH3B6 cells. TGFß1 treatment decreased the cell proliferation and induced an activation of mothers against decapentaplegic homolog 2/3 (Smad2/3), effects that were potentiated by MEK and PI3K inhibitors, thus indicating the existence of a cross talk between TGFß1/Smad with the MEK/ERK1/2 or PI3K/Akt pathways. In addition, through immunoprecipitation assays, a direct interaction was observed between Smad2/3-ERK1/2 and Smad2/3-Akt, which decreased when the GH3B6 cells were incubated with TGFß1 in the presence of MEK or PI3K inhibitors, thereby suggesting that the ERK1/2- and Akt-activated states were involved. These Smad2/3-ERK1/2 and Smad2/3-Akt associations were also confirmed by confocal and transmission electron microscopy. These findings indicate that the TGFß1-antimitogenic effect in GH3B6 cells was attenuated by the MEK/ERK1/2 and PI3K/Akt pathways via modulating Smad2/3 phosphorylation. This molecular mechanism could explain in part the refractory behavior of pituitary tumor cells to the inhibitory effect of TGFß1.
Assuntos
Adenoma/metabolismo , Sistema de Sinalização das MAP Quinases , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Hipofisárias/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Carcinogênese , Linhagem Celular Tumoral , Prolactina/metabolismo , Ratos , Proteínas Smad/metabolismoRESUMO
Interest in biochemistry of organoselenium compound has increased in the last decades, mainly due to their chemical and biological activities. Here, we investigated the protective effect of diphenyl diselenide (PhSe)2 (5 µmol/kg), in a mouse model of methylmercury (MeHg)-induced brain toxicity. Swiss male mice were divided into four experimental groups: control, (PhSe)2 (5 µmol/kg, subcutaneous administration), MeHg (40 mg/L, in tap water), and MeHg + (PhSe)2. After the treatment (21 days), the animals were killed and the cerebral cortex was analyzed. Electron microscopy indicated an enlarged and fused mitochondria leading to a reduced number of organelles, in the MeHg-exposed mice. Furthermore, cortical creatine kinase activity, a sensitive mitochondrial oxidative stress sensor, was almost abolished by MeHg. Subcutaneous (PhSe)2 co-treatment rescued from MeHg-induced mitochondrial alterations. (PhSe)2 also behaved as an enhancer of mitochondrial biogenesis, by increasing cortical mitochondria content in mouse-receiving (PhSe)2 alone. Mechanistically, (PhSe)2 (1 µM; 24 h) would trigger the cytoprotective Nrf-2 pathway for activating target genes, since astroglial cells exposed to the chalcogen showed increased content of hemeoxygenase type 1, a sensitive marker of the activation of this via. Thus, it is proposed that the (PhSe)2-neuroprotective effect might be linked to its mitoprotective activity.
Assuntos
Derivados de Benzeno/administração & dosagem , Encéfalo/metabolismo , Heme Oxigenase-1/biossíntese , Mitocôndrias/metabolismo , Compostos Organosselênicos/administração & dosagem , Animais , Encéfalo/patologia , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Masculino , Intoxicação do Sistema Nervoso por Mercúrio/metabolismo , Intoxicação do Sistema Nervoso por Mercúrio/patologia , Compostos de Metilmercúrio/toxicidade , Camundongos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacosRESUMO
Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17ß-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17ß-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.
Assuntos
Proliferação de Células/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptor 4 Toll-Like/metabolismo , Animais , Células Cultivadas , Hiperplasia/metabolismo , Interleucina-6/metabolismo , Masculino , NF-kappa B/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Hipófise/ultraestrutura , Neoplasias Hipofisárias/imunologia , Neoplasias Hipofisárias/ultraestrutura , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: In this report, we explored the role of PKCalpha and PKCe as mediators of phorbol 12-myristate13-acetate (PMA)-induced proliferation in pituitary tumor GH3B6 cells, and determined if the ERK1/2 and Akt pathways were activated. METHODS: The GH3B6 cell proliferation was estimated by BrdU incorporation and the cell cycle progression by flow cytometric cell cycle analysis. We determined the expression of PKCalpha and PKCe in membrane and cytosolic fractions by western blotting. The subcellular redistribution of both PKC isozymes was analyzed by confocal microscopy. RESULTS: Incubation with PMA for 15 min stimulated PKCalpha and PKCe activation, which was correlated with the phosphorylation of ERK1/2 but not Akt. The activation of both these PKC isozymes was closely associated with the stimulation of proliferation and the cell cycle progression induced by PMA in GH3B6 cells, an effect that was blocked by the inhibitors of PKCalpha (Gö6976) and PKCe (eV1-2). In addition, the pretreatment with the inhibitor of ERK1/2 (PD98059) prevented the mitogenic activity induced by treatment with PMA for 15 min. CONCLUSION: We demonstrated that the activation of PKCalpha and PKCe by phorbol ester in tumor pituitary GH3B6 cells led to cell proliferation and cell cycle progression, effects that involved ERK1/2 activation.
Assuntos
Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Hipofisárias/enzimologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Animais , Bromodesoxiuridina/farmacologia , Proliferação de Células , Flavonoides/farmacologia , Citometria de Fluxo , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-épsilon/antagonistas & inibidores , Ratos , Transdução de Sinais , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
The neuropeptide EI (NEI) is derived from proMCH. It activates GnRH neurons, and has been shown to stimulate the LH release following intracerebroventricular administration in several experimental models. The aim of the present paper was to evaluate NEI actions on pituitary hormone secretion and cell morphology in vitro. Pituitary cells from female rats were treated with NEI for a wide range of concentrations (1-400x10(-8)M) and time periods (1-5h). The media were collected and LH, FSH, PRL, and GH measured by RIA. The interaction between NEI (1, 10 and 100x10(-8)M) and GnRH (0.1 and 1x10(-9)M) was also tested. Pituitary cells were harvested for electron microscopy, and the immunogold immunocytochemistry of LH was assayed after 2 and 4h of NEI incubation. NEI (100x10(-8)M) induced a significant LH secretion after 2h of stimulus, reaching a maximum response 4h later. A rapid and remarkable LH release was induced by NEI (400x10(-8)M) 1h after stimulus, attaining its highest level at 2h. However, PRL, GH and FSH were not affected. NEI provoked ultrastructural changes in the gonadotrophs, which showed accumulations of LH-immunoreactive granules near the plasma membrane and exocytotic images, while the other populations exhibited no changes. Although NEI (10x10(-8)M), caused no action when used alone, its co-incubation with GnRH (1x10(-9)M), promoted a slight but significant increase in LH. These results demonstrate that NEI acts at the pituitary level through a direct action on gonadotrophs, as well as through interaction with GnRH.
Assuntos
Oligopeptídeos/farmacologia , Hipófise/efeitos dos fármacos , Hipófise/metabolismo , Animais , Células Cultivadas , Feminino , Hormônio Foliculoestimulante/metabolismo , Gonadotrofos , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio do Crescimento/metabolismo , Imuno-Histoquímica , Hormônio Luteinizante/metabolismo , Microscopia Eletrônica de Transmissão , Hipófise/citologia , Hipófise/ultraestrutura , Prolactina/metabolismo , Radioimunoensaio , Ratos , Ratos WistarRESUMO
Bromocriptine (Bc) produces pituitary tumoral mass regression which induces the cellular death that was classically described as apoptosis. However, recent works have related that other mechanisms of cell death could also be involved in the maintenance of physiological and pathological pituitary homeostasis. The aim of this study was to evaluate and characterize the different types of cell death in the involution induced by Bc in experimental rat pituitary tumors. The current study demonstrated that Bc induced an effective regression of estrogen induced pituitary tumors by a mechanism identified as parapoptosis. This alternative cell death was ultrastructurally recognized by extensive cytoplasmic vacuolization and an increased cell electron density, represented around 25% of the total pituitary cells counted. Furthermore, the results obtained from biochemical assays did not correspond to the criteria of apoptosis or necrosis. We also investigated the participation of p38, ERK1/2 and PKC delta in the parapoptotic pathway. An important observation was the significant increase in phosphorylated forms of these MAPKs, the holoenzyme and catalytic fragments of PKC delta in nuclear fractions after Bc administration compared to control and estrogen treated rats. Furthermore, the immunolocalization at ultrastructural level of these kinases showed a similar distribution pattern, with a prevalent localization at nuclear level in lactotrophs from Bc treated rats. In summary, we determined that parapoptosis is the predominant cell death type involved in the regression of pituitary tumors in response to Bc treatment, and may cause the activation of PKC delta, ERK1/2 and p38.
Assuntos
Apoptose/efeitos dos fármacos , Bromocriptina/uso terapêutico , Neoplasias Hipofisárias/tratamento farmacológico , Neoplasias Hipofisárias/patologia , Prolactinoma/tratamento farmacológico , Prolactinoma/patologia , Animais , Apoptose/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , MAP Quinase Quinase 2/fisiologia , Masculino , Proteína Quinase 3 Ativada por Mitógeno/fisiologia , Neoplasias Hipofisárias/enzimologia , Prolactinoma/enzimologia , Proteína Quinase C-delta/fisiologia , Ratos , Ratos Wistar , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
The variations of the intracellular localization of the individual protein kinase C (PKC) isoforms are related with their different biological functions. In this study, we have investigated the precise intracellular translocation of endogenous PKCalpha and PKCepsilon in PMA-stimulated normal and tumoral lactotroph cells by using confocal and immunogold electron microscopy, which was correlated with the rate of cell proliferation of both pituitary cell phenotypes. The present results showed that the short phorbol ester incubation stimulated the proliferation of normal and tumoral lactotroph cells, as determined by the measurement of the BrdU-labelling index. The translocation of PKCalpha to plasma and nuclear membranes induced by PMA was more marked than that observed for PKCepsilon in normal and tumoral lactotroph cells. Our results showed that PKCs translocation to the plasma and nuclear membranes varied from isozyme to isozyme emphasizing that PKCalpha could be related with the mitogenic stimulus exerted by phorbol ester. These data support the notion that specific PKC isozymes may exert spatially defined effects by virtue of their directed translocation to distinct intracellular sites.
Assuntos
Lactotrofos/enzimologia , Lactotrofos/patologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Feminino , Imunofluorescência , Lactotrofos/efeitos dos fármacos , Lactotrofos/ultraestrutura , Mitógenos/farmacologia , Membrana Nuclear/efeitos dos fármacos , Membrana Nuclear/enzimologia , Membrana Nuclear/ultraestrutura , Neoplasias Hipofisárias/enzimologia , Neoplasias Hipofisárias/patologia , Neoplasias Hipofisárias/ultraestrutura , Proteína Quinase C-alfa/ultraestrutura , Proteína Quinase C-épsilon/ultraestrutura , Transporte Proteico/efeitos dos fármacos , Ratos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
The aim of this investigation was to contribute to current knowledge about intracellular mechanisms that are involved in lactotroph cell proliferation, by evaluating the role of PKCalpha, PKCepsilon and extracellular-signal regulated kinase (ERK) 1/2 in response to phorbol 12-myristate13-acetate (PMA). In primary pituitary cultures, the activation of protein kinase C (PKC) by PMA for 15 min stimulated lactotroph proliferation; whereas a prolonged activation for 3-8h diminished this proliferative effect. The use of PMA for 15 min-activated PKCepsilon and ERK1/2, whereas incubation with PMA for 3 h induced PKCalpha activation and attenuated the PMA-triggered phosphorylation of ERK1/2. The following inhibitors: PKCs (bisindolylmaleimide I), PKCepsilon (epsilonV1 peptide) and ERK1/2 (PD98059) prevented the mitogenic activity induced by PMA for 15 min. Lactotroph cells stimulated with PMA for 15 min showed a translocation of PKCepsilon to membrane compartment and nucleus. These results thus establish that PKCepsilon plays an essential role in the lactotroph proliferation induced by PMA by triggering signals that involve ERK1/2 activation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Lactotrofos/citologia , Lactotrofos/fisiologia , Proteína Quinase C-épsilon/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Animais , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Feminino , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
The objectives of the present work were to assess whether epithelial cells from the different segments of epididymis express TR alpha 1-beta 1 isoforms, to depict its subcellular immunolocalization and to evaluate changes in their expression in rats experimentally submitted to a hypothyroid state by injection of 131I. In euthyroid and hypothyroid groups, TR protein was expressed in epididymal epithelial cells, mainly in the cytoplasmic compartment while only a few one showed a staining in the nucleus as well. A similar TR immunostaining pattern was detected in the different segments of the epididymis. In hypothyroid rats, the number of TR-immunoreactive epithelial cells as well as the intensity of the cytoplasmic staining significantly increased in all sections analyzed. In consonance to the immunocytochemical analysis, the expression of TR alpha 1-beta 1 isoforms, assessed by Western blot revealed significantly higher levels of TR in cytosol compared to the nuclear fractions. Furthermore, TR expression of both alpha 1 and beta 1 isoforms and their mRNA levels were increased by the hypothyroid state. The immuno-electron-microscopy showed specific reaction for TR in principal cells associated with eucromatin, cytosolic matrix and mitochondria. The differences in expression levels assessed in control and thyroidectomized rats ascertain a specific function of TH on this organ.
Assuntos
Epididimo/metabolismo , Células Epiteliais/metabolismo , Receptores alfa dos Hormônios Tireóideos/genética , Receptores beta dos Hormônios Tireóideos/genética , Animais , Western Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Epididimo/patologia , Epididimo/ultraestrutura , Células Epiteliais/patologia , Células Epiteliais/ultraestrutura , Expressão Gênica , Hipotireoidismo/genética , Hipotireoidismo/metabolismo , Hipotireoidismo/fisiopatologia , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores dos Hormônios Tireóideos/análise , Receptores dos Hormônios Tireóideos/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Glândula Tireoide/metabolismo , Glândula Tireoide/fisiopatologia , Receptores alfa dos Hormônios Tireóideos/análise , Receptores beta dos Hormônios Tireóideos/análise , Tiroxina/sangue , Tri-Iodotironina/sangueRESUMO
We have investigated the expression of receptors for insulin and insulin-like growth factor 1 (IGF-1) in rat pituitary cells in vitro and examined the morphological and proliferative changes induced in adenohypophyseal cells by insulin and IGF-1. The proliferation of lactotrophs was determined by double-immunostaining for bromodeoxyuridine and prolactin. Incubation with insulin (10, 100 or 1000 ng/ml) or IGF-1 (5, 30 or 100 ng/ml) for 48 or 72 h significantly increased the number of lactotrophs undergoing mitosis. Co-incubation of insulin or IGF-1 with genistein (25 microM), an inhibitor of the tyrosine kinase receptor, reduced the proliferation of lactotrophs elicited by the hormone and the growth factor. The receptors for insulin and IGF-1 were localized in intact pituitary cells by ultrastructural immunocytochemistry with the colloidal gold-protein A technique. Gonadotrophs expressed both receptors, specific labelling being restricted to this cell type. Electron-microscopical observations of pituitary cell cultures incubated with insulin or IGF-1 revealed gonadotroph cells exhibiting the fine-structural features of enhanced protein synthetic activity. These findings suggest that both insulin and IGF-1 are able to induce the proliferation of lactotrophs through an indirect mechanism mediated by a factor synthesized by gonadotroph cells, in addition to stimulating the biosynthetic activity of the gonadotroph in a direct manner.
Assuntos
Gonadotrofos/citologia , Lactotrofos/citologia , Microscopia Eletrônica de Transmissão/métodos , Adeno-Hipófise/metabolismo , Receptor IGF Tipo 1/metabolismo , Receptor de Insulina/metabolismo , Animais , Contagem de Células , Proliferação de Células , Células Cultivadas , DNA/biossíntese , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Feminino , Técnica Direta de Fluorescência para Anticorpo , Gonadotrofos/metabolismo , Gonadotrofos/ultraestrutura , Técnicas Imunoenzimáticas , Insulina/farmacologia , Fator de Crescimento Insulin-Like I/farmacologia , Lactotrofos/metabolismo , Lactotrofos/ultraestrutura , Adeno-Hipófise/ultraestrutura , Ratos , Ratos Wistar , Receptor IGF Tipo 1/ultraestrutura , Receptor de Insulina/ultraestruturaRESUMO
Clara cells are nonciliated secretory cells implicated in lung homeostasis by the synthesis of immunomodulatory and host defense products, being one of the most important the CC16 protein. In this study, we compared the effects of budesonide (BUD), an inhaled corticoid, on Clara cell biology and its ability to reverse morphofunctional changes induced in an allergic airway hyper-responsiveness mouse model. In normal mice, exposure to BUD induced morphological changes compatible with a state of maximal differentiation on CC16 positive cells which developed a prominent cupola filled up with numerous mitochondria rich in CYP2E1, a member of the cytochrome P450 family. Consequently, CYP2E1 expression raised significantly. Exposure to OVA provoked hypertrophy of Clara cells and an increment in their number per millimeter of basal membrane. These cells acquired a mucous cell phenotype characterized by a notorious expansion of the secretory granular content. Synthesis of CC16 was greatly up-regulated concurrent to the finding of MUC5AC expression and the increment of epidermal growth factor receptor (EGFR). Mitochondrial content decreased significantly with a consequent reduction in CYP2E1 expression. After BUD treatment of OVA-challenged animals, the majority of Clara cells regained their normal morphology and functional characteristics; CYP2E1 levels raised when compared to the OVA exposed group. The BUD potential to differentiate Clara cells appeared to be important for the regression of the profound changes generated by the allergic injury. These results demonstrated the wide range of stimuli that can modify different aspects of Clara cell biology, and highlighted the effects of budesonide as a modulator of P450 enzymes, which probably contributes to a complementary antiinflamatory activity.
Assuntos
Brônquios/patologia , Hiper-Reatividade Brônquica/tratamento farmacológico , Células Epiteliais/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Brônquios/efeitos dos fármacos , Hiper-Reatividade Brônquica/patologia , Broncodilatadores/farmacologia , Budesonida , Citocromo P-450 CYP2E1/biossíntese , Glucocorticoides/farmacologia , Hipersensibilidade/tratamento farmacológico , Inflamação/tratamento farmacológico , Camundongos , Regulação para Cima/efeitos dos fármacos , Uteroglobina/biossínteseRESUMO
The effects of IGF-1, 17 beta oestradiol and its functional interaction on lactotrophs cell proliferation were evaluated. In addition we investigated the involvement of PKC alpha, epsilon and phosphorilated ERK, in the mitogenic process. Primary cell cultures of adenohypophysis from female Wistar rats were studied in serum free conditions. The proliferation of lactotrophs was determined by double immunostaining for BrdU and PRL. The incubation with IGF-1 5, 30 or 100 ng/ml during 48 or 72 h increased lactotrophs proliferation two-threefold depending on IGF-1 concentration. Co-incubation of IGF-1 (30 ng/ml) with genistein (25 microM) or BIM (0.5 or 2 microM), lowered of tyrosine kinase receptor or of PKC respectively, inhibited the induced IGF-1 lactotrophs proliferation. 17 beta oestradiol (1, 10 or 100 nM) had not mitogenic effect, whereas in the presence of serum PRL cells proliferation was stimulated. Co-incubation with 1 nM oestradiol and IGF-1 significantly decreased the lactotroph BrdU-labelling achieved with IGF-1. PKC alpha, epsilon and ERK1/2 levels measured by western blot augmented in the presence of IGF-1 and were inhibited with the addition of genistein, supporting a participation of these enzymes in the proliferate process. Co-incubation of IGF-1 with 1 nM oestradiol decreased both PKC isoforms and activated ERK1/2 levels, suggesting that oestradiol would exert its antiproliferative effect by acting on the signalling pathway of IGF-1. The results revealed antagonic effects of oestradiol on lactotroph proliferation depending on its concentration and the presence of IGF-1.
Assuntos
Estradiol/fisiologia , Fator de Crescimento Insulin-Like I/fisiologia , Adeno-Hipófise/citologia , Animais , Proliferação de Células , Células Cultivadas , Estradiol/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Fator de Crescimento Insulin-Like I/farmacologia , Fosforilação , Adeno-Hipófise/metabolismo , Adeno-Hipófise/ultraestrutura , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-épsilon/metabolismo , Ratos , Ratos WistarRESUMO
Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the pituitary gland. The objective of this study was to unveil the VEGF subcellular localisation in different pituitary cell types and to evaluate changes in its expression at different time intervals after oestrogen stimulation. A relevant feature demonstrated was the identification of this cytokine in the nucleus and cytoplasm of lactotrophs, somatotrophs and gonadotrophs, as well as in follicle-stellate cells of male rats. Oestrogen treatment increased the number of VEGF immunopositive cells and its expression detected differentially by western blot in both nucleus and cytoplasm of pituitary cells when compared to the control. At ultrastructural level VEGF appeared associated with nucleolus and euchromatin involving a possible internal autocrine loop. In lactotrophs, the predominant cell of the tumour, VEGF was immunodetected in RER, Golgi complex, and vesicular organelles, supporting further the association with an auto-paracrine effect exerted by VEGF. The nucleus/cytoplasm ratio of VEGF revealed a prevalent accumulation of VEGF in the cytoplasm. The presence of VEGF in the nucleus may probably be associated with a translocation to this cell compartment. This study demonstrated a cytoplasmic and nuclear immunolocalisation of VEGF in normal and tumoural adenohypophyseal cells. In the course of prolactinoma development, the oestrogen stimulated VEGF expression in tumoural cells, promoting a vascular adaptation which contributes to growth and progression of the tumour.