RESUMO
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
BACKGROUND: PTP4A3 is a subclass of a protein tyrosine phosphatase super family and is expressed in a range of epithelial neoplasms. We evaluated PTP4A3 expression and its association with clinicopathological parameters in different types of functioning pituitary adenomas. METHODS: A total of 34 functioning pituitary adenomas samples were evaluated in this observational study. PTP4A3 expression was examined by immunohistochemical staining, and, possible correlations between PTP4A3 and some clinicopathological variables were investigated. RESULTS: PTP4A3 was expressed in 19 out of 34 tumours (55%), at the cytoplasmic level of tumorous cells. Moreover, there was significant association (p=0.042) between PTP4A3 expression and tumorous size. CONCLUSIONS: PTP4A3 was expressed in more than half of the tumours analysed, with there being a significant association with the tumorous size of functioning adenomas. This allows to speculate that PTP4A3 may regulate tumour growth, although further investigations are necessary to determine if this phosphatase can serve as a biomarker or used as a therapeutic target in pituitary macroadenomas.
Assuntos
Adenoma/diagnóstico , Regulação Neoplásica da Expressão Gênica , Proteínas de Neoplasias/metabolismo , Neoplasias Hipofisárias/diagnóstico por imagem , Proteínas Tirosina Fosfatases/metabolismo , Adenoma/metabolismo , Adenoma/patologia , Adulto , Citoplasma/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , Hipófise/metabolismo , Neoplasias Hipofisárias/metabolismo , Neoplasias Hipofisárias/patologia , Proteínas Tirosina Fosfatases/genética , Estudos Retrospectivos , Adulto JovemRESUMO
Androgens play a central role in homeostatic and pathological processes of the prostate gland. At the cellular level, testosterone activates both the genomic signaling pathway, through the intracellular androgen receptor (AR), and membrane-initiated androgen signaling (MIAS), by plasma membrane receptors. We have previously shown that the activation of MIAS induces uncontrolled proliferation and fails to stimulate the beneficial immunomodulatory effects of testosterone in prostatic cells, becoming necessary to investigate if genomic signaling mediates homeostatic effects of testosterone. However, the lack of specific modulators for genomic androgen signaling has delayed the understanding of this mechanism. In this article, we demonstrate that monosialoganglioside (GM1) micelles are capable of delivering testosterone into the cytoplasm to specifically activate genomic signaling. Stimulation with testosterone-loaded GM1 micelles led to the activation of androgen response element (ARE)-regulated genes in vitro as well as to the recovery of normal prostate size and histology after castration in mice. In addition, these micelles avoided MIAS, as demonstrated by the absence of rapid signaling pathway activation and the inability to induce uncontrolled cell proliferation. In conclusion, our results validate a novel tool for the specific activation of genomic androgen signaling and demonstrate the importance of selective pathway activation in androgen-mediated proliferation.
Assuntos
Neoplasias da Próstata , Receptores Androgênicos , Androgênios , Animais , Gangliosídeo G(M1) , Genômica , Humanos , Masculino , Camundongos , Micelas , Receptores Androgênicos/genética , Transdução de Sinais , TestosteronaRESUMO
Humans are exposed to numerous endocrine disruptors on a daily basis, which may interfere with endogenous estrogens, with Di-(2-ethylhexyl) phthalate (DEHP) being one of the most employed. The anterior pituitary gland is a target of 17ß-estradiol (E2) through the specific estrogen receptors (ERs) α and ß, whose expression levels fluctuate in the gland under different contexts, and the ERα/ß index is responsible for the final E2 effect. The aim of the present study was to evaluate in vivo and in vitro the DEHP effects on ERα and ß expression in the pituitary cell population, and also its impact on lactotroph and somatotroph cell growth. Our results revealed that perinatal exposure to DEHP altered the ERα and ß expression pattern in pituitary glands from prepubertal and adult female rats and increased the percentage of lactotroph cells in adulthood. In the in vitro system, DEHP down-regulated ERα and ß expression, and as a result increased the ERα/ß ratio and decreased the percentages of lactotrophs and somatotrophs expressing ERα and ß. In addition, DEHP increased the S + G2M phases, Ki67 index and cyclin D1 in vitro, leading to a rise in the lactotroph and somatotroph cell populations. These results showed that DEHP modified the pituitary ERα and ß expression in lactotrophs and somatotrophs from female rats and had an impact on the pituitary cell growth. These changes in ER expression may be a mechanism underlying DEHP exposure in the pituitary gland, leading to cell growth deregulation.
Assuntos
Dietilexilftalato/toxicidade , Ácidos Ftálicos/toxicidade , Receptores de Estrogênio/metabolismo , Animais , Proliferação de Células/efeitos dos fármacos , Dietilexilftalato/metabolismo , Disruptores Endócrinos/metabolismo , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Estrogênios/farmacologia , Feminino , Lactotrofos/efeitos dos fármacos , Lactotrofos/metabolismo , Hipófise/efeitos dos fármacos , RatosRESUMO
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
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
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
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
Chlamydomonas reinhardtii centrin is a member of the EF-hand calcium-binding superfamily. It is found in the basal body complex and is important for flagellar motility. Like other members of the EF-hand family, centrin interacts with and modulates the function of other proteins in a calcium-dependent manner. To understand how C. reinhardtii centrin interacts with its protein targets, it has been crystallized in the presence of the model peptide melittin and X-ray diffraction data have been collected to 2.2 A resolution. The crystals are orthorhombic, with unit-cell parameters a = 52.1, b = 114.4, c = 34.8 A, and are likely to belong to space group P2(1)2(1)2.