RESUMO
During Freund's adjuvant induced inflammation rat mesenteric mesothelial cells transdifferentiate into mesenchymal cell. They express macrophage markers, inflammatory cytokines (TGF-ß, TNFα, IL-6), and specific receptors. When primary mesenteric cultures were treated with GM-CSF and/or TGF-ß (in vitro), similar phenotypic and biological changes were induced. It seemed likely that GM-CSF receptor-ligand complex should be internalized to initiate mesothelial-macrophage transition. To follow the intracellular route of GM-CSF receptor ß, we co-localized this receptor with various endocytic markers (Cav-1, EEA1, Rab7, and Rab11a), and carried out detailed immunocytochemical, statistical and biochemical analyses. Since STAT5 is one of the downstream element of GM-CSF signaling, we followed the expression and phosphorylation level of this transcription factor. Our results showed that in mesenteric mesothelial cells GM-CSF receptor ß is internalized by caveolae, delivered into early endosomes where the signaling events occur, STAT5A is phosphorylated by JAK2, and then translocated into the nucleus. When dynamin-dependent endocytosis of GM-CSFR ß is inhibited by dynasore, phosphorylation of STAT5A is not occurred, confirming, that the internalization of receptor ß is indispensable for signal transduction. At the early time of inflammation a significant receptor recycling can be found to the plasma membrane. Later (day 8) the receptor is delivered into late endosomes, indicating that its degradation has already started, and the regeneration of mesothelial cells can start. All of these data strongly support that the internalization of GM-CSF receptor ß is required and essential for signal transduction.
Assuntos
Transdiferenciação Celular/fisiologia , Subunidade beta Comum dos Receptores de Citocinas/metabolismo , Endocitose/fisiologia , Macrófagos/metabolismo , Transdução de Sinais , Animais , Cavéolas/efeitos dos fármacos , Cavéolas/metabolismo , Subunidade beta Comum dos Receptores de Citocinas/efeitos dos fármacos , Modelos Animais de Doenças , Hidrazonas/farmacologia , Inflamação/metabolismo , Janus Quinase 2/metabolismo , Macrófagos/citologia , Masculino , Fosforilação , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT5/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Inflammatory stimuli inducing epithelial-to-mesenchymal transition (EMT) can transdifferentiate mesenteric mesothelial cells into macrophages. METHODS: Sprague Dawley rat mesenteric mesothelial cells were used as a model. 1 ml Freund adjuvant was injected into the peritoneal cavity of rat and GM-CSF treatment was used to induce inflammation. IL-10 and IL-6 expression were studied by immunocytochemistry and Western blot analysis both in vivo and in vitro. RESULTS: Control mesothelial cell express anti-inflammatory IL-10, but no pro-inflammatory IL-6 expression could be detected in them. By the time of inflammation, IL-6 expression increased (reached the maximum level at the fifth day of inflammation), parallel to this the IL-10 entirely disappeared from these cells. In vitro GM-CSF treatment resulted in similar changes. As the mesothelial cells started to recover (at the eighth day of inflammation) IL-6 expression decreased and IL-10 level started to increase again. CONCLUSION: These data show that under inflammatory stimuli mesothelial cells-like macrophages-can produce pro-inflammatory cytokines.
Assuntos
Células Epiteliais/fisiologia , Interleucina-6/metabolismo , Macrófagos/metabolismo , Mesentério/citologia , Animais , Transdiferenciação Celular , Células Cultivadas , Transição Epitelial-Mesenquimal , Interleucina-10/metabolismo , Masculino , RatosRESUMO
In relation of immunobiology, the consequence of the crosstalk between TLR9-signaling and autophagy is poorly documented in HT29 cancer cells. To assess the TLR9-mediated biologic effects of modified self-DNA sequences on cell kinetics and autophagy response HT29 cells were incubated separately with intact genomic (g), hypermethylated (m), fragmented (f), and hypermethylated/fragmented (m/f) self-DNAs. Cell viability, apoptosis, cell proliferation, colonosphere-formation were determined. Moreover, the relation of TLR9-signaling to autophagy response was assayed by real-time RT-PCR, immunocytochemistry and transmission electron microscopy (TEM). After incubation with g-, m-, and m/f-DNAs cell viability and proliferation decreased, while apoptosis increased. F-DNA treatment resulted in an increase of cell survival. Methylation of self-DNA resulted in decrease of TLR9 expression, while it did not influence the positive effect of DNA fragmentation on MyD88 and TRAF6 overexpression, and TNFα downregulation. Fragmentation of DNA abrogated the positive effect of methylation on IRAK2, NFκB and IL-8 mRNA upregulations. In case of the autophagy genes and proteins, g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, and LC3B. According to TEM analyses, autophagy was present in each group of tumor cells, but to a varying degree. Incubation with m-DNA suppressed tumor cell survival by inducing features of apoptotic cell death, and activated mitophagy. F-DNA treatment enhanced cell survival, and activated macroautophagy and lipophagy. Colonospheres were only present after m-DNA incubation. Our data provided evidence for a close existing interplay between TLR9-signaling and the autophagy response with remarkable influences on cell survival in HT29 cells subjected to modified self-DNA treatments.
Assuntos
Autofagia , Neoplasias do Colo/patologia , Metilação de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Receptor Toll-Like 9/metabolismo , Apoptose , Proliferação de Células , Neoplasias do Colo/metabolismo , Genômica , Células HT29 , HumanosRESUMO
BACKGROUND: The outcome of cancer therapy is greatly defined by the ability of a tumor cell to evade treatment and re-establish its bulk mass after medical interventions. Consequently, there is an urgent need for the characterization of molecules affecting tumor reoccurrence. The phosphatase of regenerating liver 3 (PRL3) protein was recently emerged among the targets that could affect such a phenomenon. METHODS: The expression induction of PRL3 in melanoma cells treated with chemotherapeutic agents was assessed by western blotting. The effect of PRL3 expression on cancer growth was investigated both in vitro and in vivo. The association of PRL3 with the caveolae structures of the plasma membrane was analyzed by detergent free raft purification. The effect of PRL3 expression on the membrane organization was assayed by electron microscopy and by membrane biophysical measurements. Purification of the plasma membrane fraction and co-immunoprecipitation were used to evaluate the altered protein composition of the plasma membrane upon PRL3 expression. RESULTS: Here, we identified PRL3 as a genotoxic stress-induced oncogene whose expression is significantly increased by the presence of classical antitumor therapeutics. Furthermore, we successfully connected the presence of this oncogene with increased tumor growth, which implies that tumor cells can utilize PRL3 effects as a survival strategy. We further demonstrated the molecular mechanism that is connected with the pro-growth action of PRL3, which is closely associated with its localization to the caveolae-type lipid raft compartment of the plasma membrane. In our study, PRL3 was associated with distinct changes in the plasma membrane structure and in the caveolar proteome, such as the dephosphorylation of integrin ß1 at Thr788/Thr789 and the increased partitioning of Rac1 to the plasma membrane. These alterations at the plasma membrane were further associated with the elevation of cyclin D1 in the nucleus. CONCLUSIONS: This study identifies PRL3 as an oncogene upregulated in cancer cells upon exposure to anticancer therapeutics. Furthermore, this work contributes to the existing knowledge on PRL3 function by characterizing its association with the caveolae-like domains of the plasma membrane and their resident proteins.
Assuntos
Cavéolas/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Melanoma/patologia , Proteínas de Neoplasias/genética , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais/efeitos dos fármacos , Animais , Carcinogênese/efeitos dos fármacos , Cavéolas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
In our previous work, we showed that during inflammation-induced epithelial-to-mesenchymal transition (EMT), mesenteric mesothelial cells express ED1 (pan-macrophage marker), indicating that they are transformed into macrophage-like cells. In this paper, we provide additional evidences about this transition by following the phagocytic activity and the TNFα production of mesenteric mesothelial cells during inflammation. Upon injection of India ink particles or fluorescent-labeled bioparticles (pHrodo) into the peritoneal cavity of rats pretreated with Freund's adjuvant, we found that mesothelial cells efficiently engulfed these particles. A similar increase of internalization could be observed by mesothelial cells in GM-CSF pretreated primary mesenteric culture. Since macrophages are the major producers of tumor necrosis factor, TNFα, we investigated expression level of TNFα during inflammation-induced EMT and found that TNFα was indeed expressed in these cells, reaching the highest level at the 5th day of inflammation. Since TNFα is one of the target genes of early growth response (EGR1) transcription factor, playing important role in monocyte-macrophage differentiation, expression of EGR1 in mesothelial cells was also investigated by Western blot and immunocytochemistry. While mesothelial cells did not express EGR1, a marked increase was observed in mesothelial cells by the time of inflammation. Parallel to this, nuclear translocation of EGR1 was shown by immunocytochemistry at the day 5 of inflammation. Caveolin-1 level was high and ERK1/2 became phosphorylated as the inflammation proceeded showing a slight decrease when the regeneration started. Our present data support the idea that under special stimuli, mesenteric mesothelial cells are able to transdifferentiate into macrophages, and this transition is regulated by the caveolin-1/ERK1/2/EGR1 signaling pathway.
Assuntos
Transdiferenciação Celular/efeitos dos fármacos , Transição Epitelial-Mesenquimal , Inflamação/complicações , Macrófagos/citologia , Mesentério/citologia , Animais , Caveolina 1/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos , Sistema de Sinalização das MAP Quinases , Ratos , Transdução de Sinais , Fator de Necrose Tumoral alfa/análiseRESUMO
BACKGROUND: Although membrane-associated estrogen receptors (mERs) have been known to play important role in steroid-induced signal transmission, we still know little about their function in the estrogen-induced proliferation of breast cancer cells. METHODS: In our current work we tried to separate membrane-initiated estrogen receptor signaling from the overall estrogenic effect in MCF-7 breast carcinoma cells. Re-analyzing expression data from multiple microarray experiments, we selected a set of key regulatory genes involved in proliferation regulation and estrogen signaling to monitor estrogen-induced transcription changes. We then compared these expression changes after 17ß-estradiol and a membrane receptor selective estrogen-BSA treatment using quantitative real-time PCR. In order to follow receptor trafficking we used light and electron microscopy. RESULTS: Our quantitative real-time PCR results confirmed that the selective membrane receptor agonist, estrogen-BSA induces similarly pronounced expression changes regarding these genes as 17ß-estradiol. Morphological study revealed that the membrane-bound form of classical estrogen receptor alpha is internalized after ligand binding via dynamin-dependent, caveola-mediated endocytosis. Inhibition of this internalization with dynamin inhibitor, dynasore practically abolished the regulatory effect of E2-BSA, suggesting that interaction and internalization with the scaffold protein is necessary for effective signaling. CONCLUSIONS: The physiological role of plasma membrane estrogen receptor alpha is intensively studied, yet there are still several aspects of it to be resolved. The dynamin-dependent, ligand-mediated internalization of mERs seems to play an important role in estrogen signaling. Our results may serve as another example of how membrane initiated estrogen signaling and nuclear receptor initiated signaling overlap and form an intertwined system.
Assuntos
Neoplasias da Mama/metabolismo , Membrana Celular/metabolismo , Dinaminas/metabolismo , Receptor alfa de Estrogênio/metabolismo , Transdução de Sinais/fisiologia , Feminino , Humanos , Células MCF-7RESUMO
In previous studies we showed that during Freund's adjuvant induced inflammation rat mesenteric mesothelial cells undergo epithelial-mesenchymal transition type II (EMT). This process was characterized by a dramatic increase of the number of cell organelles and volume of mesothelial cells. After the inflammation reached its maximum, the mesenchymal-like cells gradually regained their epithelial phenotype (mesenchymal-epithelial transition, MET). During the recovery process, the decrease of the number of cell organelles was accompanied by an increasing number of autophagic structures in the cytoplasm, indicating that autophagy might play crucial role in MET. Morphometric data of this study showed that the number of the autophagic organelles increased by the time of inflammation and was the highest at day 7-8, when regeneration started. These morphological observations were supported by immunocytochemistry and Western blot analyses with various markers, directly or indirectly involved in this process. Endocytic markers were expressed at high level during both EMT and MET, while the expression of factors regulating autophagy simultaneously changed with the morphology: p-Akt and p-mTOR level was high at day 3-5 and significantly decreased when autophagy speeded up. The Beclin-1, which is the key factor of initiating autophagy, was expressed at the early time of inflammation. These results strongly suggest that autophagy plays important role in regeneration (MET), and it is regulated and synchronized by various signalling events during inflammation.
Assuntos
Autofagia , Transição Epitelial-Mesenquimal , Fenótipo , Animais , Proteína Beclina-1/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Masculino , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE AND DESIGN: During peritonitis, mesothelial cells assume macrophage characteristics, expressing macrophage markers, indicating that they might differentiate into macrophage-like cells. MATERIALS AND SUBJECTS: Twenty-five male rats were used for in vivo experiments. For in vitro experiments, a primary mesentery culture model was developed. The mesothelial cell to macrophage-like cell transition was followed by studying ED1 expression. TREATMENTS: In vitro primary mesenteric culture was treated with granulocyte-macrophage colony-stimulating factor (GM-CSF, 1 ng/ml). Blocking internalization of receptor-ligand complex, Dynasore (80 µM) was used. Acute peritonitis was induced by Freund's adjuvant's (1 ml) intraperitoneal injection. RESULTS: Immunohistochemistry: GM-CSF in vitro treatment resulted in a prominent ED1 expression in transformed mesothelial cells. Blocking the internalization, ED1 expression could not be detected. GM-CSF receptor (both α and ß) was expressed in mesothelial cells in vitro (even if the GM-CSF was not present) and in vivo. Inflammation resulted in an increasing GM-CSF and GM-CSF-receptor level in the lysate of mesothelial cells. CONCLUSIONS: Mesothelial cells can differentiate into macrophage-like cells, and GM-CSF, produced by the mesothelial cells, has probably an autocrine regulatory role in this transition. Our results provide new data about the plasticity of mesothelial cell and support the idea that during inflammation macrophages can derive from non-hematopoietic sources as well.
Assuntos
Células Epiteliais/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos Peritoneais/citologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Animais , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Epitélio/efeitos dos fármacos , Epitélio/metabolismo , Epitélio/ultraestrutura , Adjuvante de Freund , Masculino , Peritonite/induzido quimicamente , Peritonite/metabolismo , Ratos Sprague-DawleyRESUMO
Transformation of epithelial cells into connective tissue cells (epithelial-mesenchymal transition, EMT) is a complex mechanism involved in tumor metastasis, and in normal embryogenesis, while type II EMT is mainly associated with inflammatory events and tissue regenaration. In this study we examined type II EMT at the ultrastructural and molecular level during the inflammatory process induced by Freund's adjuvant treatment in rat mesenteric mesothelial cells. We found that upon the inflammatory stimulus mesothelial cells lost contact with the basal lamina and with each other, and were transformed into spindle-shaped cells. These morphological changes were accompanied by release of interleukins IL-1alpha, -1beta and IL-6 and by secretion of transforming growth factor beta (TGF-ß) into the peritoneal cavity. Mesothelial cells also expressed estrogen receptor alpha (ER-α) as shown by immunolabeling at the light and electron microscopical levels, as well as by quantitative RT-PCR. The mRNA level of ER-α showed an inverse correlation with the secretion of TGF-ß. At the cellular and subcellular levels ER-α was colocalized with the coat protein caveolin-1 and was found in the plasma membrane of mesothelial cells, in caveolae close to multivesicular bodies (MVBs) or in the membrane of these organelles, suggesting that ER-α is internalized via caveola-mediated endocytosis during inflammation. We found asymmetric, thickened, electron dense areas on the limiting membrane of MVBs (MVB plaques) indicating that these sites may serve as platforms for collecting and organizing regulatory proteins. Our morphological observations and biochemical data can contribute to form a potential model whereby ER-α and its caveola-mediated endocytosis might play role in TGF-ß induced type II EMT in vivo.
Assuntos
Cavéolas/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Receptor alfa de Estrogênio/metabolismo , Adjuvante de Freund/farmacologia , Mesentério/efeitos dos fármacos , Mesentério/metabolismo , Animais , Caveolina 1/genética , Caveolina 1/metabolismo , Células Epiteliais/ultraestrutura , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Receptor alfa de Estrogênio/genética , Feminino , Imuno-Histoquímica , Masculino , Ligação Proteica , RatosRESUMO
Although clathrin-mediated endocytosis constitutes the main pathway for internalization of extracellular ligands and plasma membrane components it has generally been accepted that other uptake mechanisms-caveolae-mediated and noncaveolar raft-dependent endocytosis-also exist. During the last 20 years many papers have been published about caveolar endocytosis. These studies have fundamentally changed our view about the endocytotic role of caveolae. Views that caveolae are permanently static structures1 have been extensively considered and rejected. Although the initial steps leading to the pinching off of caveolae from the plasma membrane have been studied in details, there are still contradictory data about the intracellular trafficking of caveolae. It is still not entirely clear whether caveolar endocytosis represents an uptake pathway with distinct cellular compartments to avoid lysosomal degradation or ligands taken up by caveolae can also be targeted to late endosomes/lysosomes.In this chapter, we summarize the data available about caveolar endocytosis focusing on the intracellular route of caveolae and we provide data supporting that caveolar endocytosis can join the classical endocytotic pathway.
Assuntos
Cavéolas/metabolismo , Endocitose , Animais , Células Hep G2 , Humanos , Espaço Intracelular/metabolismoRESUMO
Intraperitoneal injection of Freund's adjuvant induces acute peritonitis. By the time of the Freund's adjuvant treatment the flat, simple squamous epithelial cells became rounded, cuboidal shaped, many of them have lost their connection with the neighbouring cells and detached from the basement membrane. The macrophage markers' (ED1, OX43 and CD68) expression also increased in the mesothelial cells and more mesothelin and anti-ED1 double-labelled cells were found freely present close to the surface. The cytokeratin expression of the mesothelial cells has gradually decreased. At the 5th day of the inflammation practically there was no cytokeratin labelling present in the mesothelial cells and the mesothelin expression has significantly decreased. Parallel to this mesothelial cells started to express vimentin, a characteristic mesenchymal intermediate filament protein indicating that they gradually lost their epithelial character and gained mesenchymal phenotype. These results strongly suggest that under the effect of Freund's adjuvant treatment (inflammation) mesothelial cells can undergo epithelial-to-mesenchymal transition and differentiate into phagocytotic (macrophage-like) cells. Studying the caveolae/caveolin-1 on the plasma membrane of mesothelial cells we found that the Freund's adjuvant treatment has changed the cellular distribution of caveolin-1: as the inflammation progressed strong caveolin-1 labelling was found inside of the cytoplasm (in perinuclear localization) indicating that inflammation induced the caveolae internalization. These results indicate that caveolae/caveolin-1 might play important regulatory role in signal transduction leading to trasdifferentiation.
Assuntos
Adjuvantes Imunológicos/toxicidade , Biomarcadores/metabolismo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Adjuvante de Freund/toxicidade , Inflamação/metabolismo , Inflamação/patologia , Animais , Caveolina 1/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Técnicas Imunoenzimáticas , Inflamação/induzido quimicamente , Queratinas/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Vimentina/metabolismoRESUMO
Peritoneal cell suspension is composed of heterogeneous cell population. Macrophages are the most numerous cells among them. They can originate from different sources and can be resident, exudate and elicited. When we used Freund's adjuvant to elicit peritoneal macrophages, cells having large amount of caveolae on their plasma membrane appeared in the peritoneal wash. The number of these caveolae-rich cells increased by the time of the Freund's adjuvant treatment. Although their morphology was different form from the common macrophages, they were labelled with pan-macrophage antibodies. As the origin of these cells is unknown in this work, we tried to find out where they can originate from. Our interest turned towards the mesothelial cells. We found that the adjuvant treatment resulted in significant morphological changes in these cells and stimulate them to leave the surface of the mesentery. By the time of the adjuvant treatment, the macrophage markers expression increased in the mesothelial cells and more cells were found to detach from the mesentery. These results strongly suggest that under special stimuli mesothelial cells can leave the mesentery and differentiate into phagocytotic (macrophage-like) cells. These data raises the idea that mesothelial cells might not entirely differentiated and represent a multipotential cell lineage. To study whether this is the case we used anti-nestin antibody, which is a specific marker for multifunctional, multi-lineage progenitor cells. Mesothelial cells showed strong labelling with this antibody indicating that these cells really represent a 'young', not entirely differentiated cell population.
Assuntos
Células Epiteliais/citologia , Macrófagos/citologia , Mesentério/citologia , Animais , Diferenciação Celular , Células Epiteliais/química , Adjuvante de Freund/farmacologia , Proteínas de Filamentos Intermediários/análise , Queratinas/análise , Masculino , Proteínas do Tecido Nervoso/análise , Nestina , Ratos , Ratos Sprague-DawleyRESUMO
Caveola-mediated endocytosis exists parallel to other forms of endocytosis. Being ligand-triggered, caveolar endocytosis provides a more selective and highly regulated way for uptake of specified substances. Internalized caveolae accumulate in intermediate organelles called caveosomes. It is still debated whether caveosomes are independent organelles or the downstream caveosomes interact with the classical endocytotic compartments. In our work caveola internalization was stimulated with a serine/threonine phosphatase (PP1 and PP2A) inhibitor (ocadaic acid-OA). To find out whether caveolar clusters are really independent organelles or they are still connected to the cell surface we used an electron dense surface marker, ruthenium red (Ru red). Since we were especially interested in the fate of caveolar clusters, the cells were treated with OA for longer time. Stimulating caveola-mediated endocytosis, OA treatment resulted in a significant increase in the number of caveolar cluster. Most of these clusters were found Ru red positive indicating that they were still conneted to the cell surface. Our double labeling experiments on ultrathin frozen sections clearly showed that in OA-treated cells caveolae are not transported to late endosomes instead they are accumulted in large multicaveolar clusters. We think that PP2A can be one of the key components to regulate the fusion of various endocytotic compartments and /or the trafficking along the microtubules.
Assuntos
Cavéolas/metabolismo , Cavéolas/ultraestrutura , Endocitose/fisiologia , Inibidores Enzimáticos/farmacologia , Ácido Okadáico/farmacologia , Proteína Fosfatase 2/metabolismo , Cavéolas/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Células Hep G2 , Humanos , Microscopia Eletrônica de Transmissão , Microscopia ImunoeletrônicaRESUMO
The effects of intraperitoneally administered plant lectins were examined in rats and mice. Intraperitoneally injected ConA transiently decreased the leukocyte count in the peritoneal cavity, due to the agglutination and attachment of cells to the peritoneal lining. Subsequently the total cell count was increased for hours, exceeding initial values. Peritoneal fluid aspartate transaminase (AST) concentration showed little change during the accumulation of ascitic fluid. The most marked histological alterations were found when wheat germ lectin was injected ip. (WGA, 10 mg/kg, 6 h). Neutrophil granulocytes migrated across the wall of both arterioles and venules, but the response was highly variable among adjacent vessels. The wall of the arterioles may have impeded the migration of neutrophil granulocytes, resulting in their accumulation in the muscular layer. Granulocyte accumulation was also observed in patches under the mesothelium and in other sites of the interstitium. Marked dilatation and thrombosis of a few venules were also observed. Kidney bean lectin (PHA) induced similar but less pronounced changes. The neutrophil diapedesis suggests the release of mediator(s) from mesothelial cells and/or peritoneal white cells. The cytokine-induced neutrophil chemoattractant CINC-1, injected as control, resulted in the diapedesis of predominantly mononuclear cells in the omentum within 40 minutes. In rats ip. injected ConA increased the wet weight of spleen and liver within 6 and 10 h, respectively, but kidney weight did not change. Intravascular clumping of red blood cells, thrombosis and organ weight changes also suggest the absorption of ConA into the circulation. The experiments show that plant lectins, used as models of bacterial lectins, can reproduce some aspects of peritonitis.
Assuntos
Movimento Celular/efeitos dos fármacos , Leucócitos/citologia , Lectinas de Plantas/farmacologia , Vísceras/anatomia & histologia , Animais , Concanavalina A/administração & dosagem , Concanavalina A/farmacologia , Feminino , Injeções Intraperitoneais , Rim/anatomia & histologia , Rim/efeitos dos fármacos , Leucócitos/efeitos dos fármacos , Fígado/anatomia & histologia , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Mitógenos/farmacologia , Tamanho do Órgão/efeitos dos fármacos , Fito-Hemaglutininas/administração & dosagem , Fito-Hemaglutininas/farmacologia , Lectinas de Plantas/administração & dosagem , Ratos , Ratos Wistar , Proteínas de Soja/administração & dosagem , Proteínas de Soja/farmacologia , Baço/anatomia & histologia , Baço/efeitos dos fármacos , Vísceras/efeitos dos fármacos , Aglutininas do Germe de Trigo/administração & dosagem , Aglutininas do Germe de Trigo/farmacologiaRESUMO
PURPOSE: Caveolin-1 has been identified in Müller and pigment cells of rodents, but the distribution of caveolin isoforms has not been studied in the human retina. Since there are no relevant data in humans, we aimed to study the distribution of caveolins in the human retina. METHODS: Our samples were derived from eyes affected by melanoma malignum choroideae that were enucleated. The distribution of the caveolins was examined by immunocytochemistry using isoform-specific antibodies. RESULTS: In this study we report on the presence of different caveolin isoforms in many cell types of the human retina. These isoforms were present in several regions and layers in the human retina. Centro-peripheral changes have been detected: the distribution altered following the radier direction. CONCLUSIONS: This is the first demonstration of caveolin expression in the human retina. Our data suggest that caveolins play an important role in the function of retinal cells. Our observations refute previous assumptions that there is a shortage of caveolins in the retina. Since the retina contains a number of different neuronal and glial cell types, the caveolin expression of these cells can no longer be a matter of dispute.
Assuntos
Caveolinas/metabolismo , Neoplasias Oculares/metabolismo , Neoplasias Oculares/patologia , Melanoma/metabolismo , Melanoma/patologia , Retina/metabolismo , Retina/patologia , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Caveolina 3/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Isoformas de Proteínas/metabolismoRESUMO
The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.
Assuntos
Cavéolas/metabolismo , Monoéster Fosfórico Hidrolases/fisiologia , Fosfotransferases/fisiologia , Western Blotting , Caveolinas/análise , Linhagem Celular Tumoral , Membrana Celular/química , Citoplasma/química , Inibidores Enzimáticos/farmacologia , Humanos , Imuno-Histoquímica , Imunoprecipitação , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Ácido Okadáico/farmacologia , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Vanadatos/farmacologiaRESUMO
Recently, it has been shown that 17beta estradiol (E2) induces a rapid and transient activation of the Src ERK phosphorylation cascade: a clear indication that the alpha oestrogen receptor (ERalpha) is able to associate with the plasma membrane. Increasing evidence suggests that caveolae, which are caveolin-1 containing, highly hydrophobic membrane domains, play an important role in E2 induced signal transduction. Caveolae can accumulate signalling molecules preferentially; thus, they may have a regulatory role in signalling processes. Results from previous experiments have shown that E2 treatment decreased the number of surface connected caveolae significantly in uterine smooth muscle cells and also downregulated the expression of caveolin-1. In addition to providing further evidence that ERalpha interacts with caveolin/caveolae in uterine smooth muscle cells, this study also shows that the interaction between caveolin-1 and ERalpha is actually facilitated by E2. One of the signal transduction components found to accumulate in caveolae is Src kinase in an amount that increases simultaneously with increases in the amount of ERalpha. Upon E2 treatment, Src kinase is tyrosine phosphorylated, which, in turn, stimulates Src kinase to phosphorylate caveolin-1. Phosphorylation of caveolin-1 can drive caveolae to pinch off from the plasma membrane, thereby decreasing the amount of plasma membrane-associated caveolin-1. This loss of caveolin/caveolae activates the signal cascade that triggers cell proliferation.
Assuntos
Cavéolas/química , Caveolina 1/fisiologia , Estradiol/farmacologia , Receptor alfa de Estrogênio/análise , Tirosina/fisiologia , Animais , Cavéolas/enzimologia , Cavéolas/fisiologia , Caveolina 1/análise , Caveolina 1/genética , Membrana Celular/química , Membrana Celular/fisiologia , Proliferação de Células , Regulação para Baixo/fisiologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/fisiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Microscopia de Fluorescência , Músculo Liso/citologia , Músculo Liso/enzimologia , Músculo Liso/fisiologia , Fosforilação , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Útero/química , Útero/enzimologia , Útero/fisiologia , Quinases da Família src/metabolismoRESUMO
Pathological circumstances like inflammation or ischemic insult facilitate the release of adenine nucleotides from several types of cells. These extracellular nucleotides are rapidly converted to adenosine by ectonucleotidases, mainly ectonucleoside triphosphate diphosphohydrolase1 (NTPDase1/CD39) and CD73. NTPDase1/CD39 can interact with caveolins, structural proteins of signal-transducing microdomains termed caveolae. Caveolins are thought to have physiological roles in heart ageing and cardiac diseases. The aim of this study was to investigate the expression of NTPDase1 together with caveolins in chronic human cardiovascular diseases and elucidate their role in human heart. The HPLC analysis showed significant increase in ATPase activity in pathological samples from patients with ischemic heart disease. Immunostaining also showed alterations in the expression and distribution of NTPDase1. Caveolin-1 and caveolin-2 expression was much alike in control and pathological cases, while expression of caveolin-3 was lower in pathological samples. Changes in the expression of NTPDase1 and caveolins seem to be independent of human cardiovascular disease.