RESUMO
Serratia marcescens is an opportunistic human pathogen involved in antibiotic-resistant hospital acquired infections. Upon contact with the host epithelial cell and prior to internalization, Serratia induces an early autophagic response that is entirely dependent on the ShlA toxin. Once Serratia invades the eukaryotic cell and multiples inside an intracellular vacuole, ShlA expression also promotes an exocytic event that allows bacterial egress from the host cell without compromising its integrity. Several toxins, including ShlA, were shown to induce ATP efflux from eukaryotic cells. Here, we demonstrate that ShlA triggered a nonlytic release of ATP from Chinese hamster ovary (CHO) cells. Enzymatic removal of accumulated extracellular ATP (eATP) or pharmacological blockage of the eATP-P2Y2 purinergic receptor inhibited the ShlA-promoted autophagic response in CHO cells. Despite the intrinsic ecto-ATPase activity of CHO cells, the effective concentration and kinetic profile of eATP was consistent with the established affinity of the P2Y2 receptor and the known kinetics of autophagy induction. Moreover, eATP removal or P2Y2 receptor inhibition also suppressed the ShlA-induced exocytic expulsion of the bacteria from the host cell. Blocking α5ß1 integrin highly inhibited ShlA-dependent autophagy, a result consistent with α5ß1 transactivation by the P2Y2 receptor. In sum, eATP operates as the key signaling molecule that allows the eukaryotic cell to detect the challenge imposed by the contact with the ShlA toxin. Stimulation of P2Y2-dependent pathways evokes the activation of a defensive response to counteract cell damage and promotes the nonlytic clearance of the pathogen from the infected cell.
Assuntos
Autofagia , Interações Hospedeiro-Patógeno , Integrina alfa5beta1 , Receptores Purinérgicos P2Y2 , Serratia , Toxinas Biológicas , Animais , Cricetinae , Trifosfato de Adenosina/metabolismo , Autofagia/efeitos dos fármacos , Células CHO , Cricetulus , Exocitose/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Serratia/química , Serratia/efeitos dos fármacos , Serratia/fisiologia , Toxinas Biológicas/farmacologia , HumanosRESUMO
Mammalian ejaculated spermatozoa must undergo a series of changes in the female reproductive tract, collectively called capacitation, in order to fertilize the oocyte. We reported that fibronectin (Fn), a glycoprotein from the extracellular matrix, and anandamide (AEA), one of the major members of the endocannabinoid family, are present in the bovine oviductal fluid and regulate bull sperm function. Also, AEA induces bovine sperm capacitation, through CB1 and TRPV1 receptors. In this work, we investigated if Fn induces bovine sperm capacitation thought the activation of the endocannabinoid system in this process. We incubated sperm with Fn (100 µg/ml) and/or capsazepine, a TRPV1 antagonist (0.1 µM) and some events related to sperm capacitation such as LPC-induced acrosome reaction, sperm-release from the oviduct, induction of PKA phosphorylated substrates (pPKAs) and protein tyrosine phosphorylation (pY) and nitric oxide (NO) production were assessed. Also, we studied the activity of fatty acid amide hydrolase (FAAH), the enzyme that degrades AEA. We found that Fn, via α5ß1 integrin, induced capacitation-associated events. Also, Fn stimulated signaling pathways associated to capacitation as cAMP/PKA and NO/NO synthase. Moreover, Fn decreased the FAAH activity and this correlated with sperm capacitation. Capsazepine reversed fibronectin-induced capacitation, and pPKAs and NO levels. The incubation of spermatozoa with R-methanandamide (1.4 nM), a stable analogue of AEA, increased cAMP and pPKAs levels. The presence of H89 (50 µM) or KT5720 (100 nM) (PKA inhibitors) prevented AEA-induced capacitation. In addition, R-methanandamide and capsaicin (0.01 µM), a TRPV1 agonist, increased NO production via the PKA pathway. These results indicate that Fn, through α5ß1, supports capacitation in bovine spermatozoa. This effect is dependent on the activation of TRPV1 through cAMP/PKA and NO signaling pathways. We propose that Fn could be considered as a new agent that promotes sperm capacitation in bull sperm. Our findings contribute to better understand the significance of Fn signaling in the capacitating events that lead to successful fertilization and embryo development in mammals including humans.
Assuntos
Bovinos , Endocanabinoides/metabolismo , Fibronectinas/farmacologia , Preservação do Sêmen/veterinária , Capacitação Espermática/efeitos dos fármacos , Animais , Criopreservação/veterinária , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Endocanabinoides/genética , Integrina alfa5beta1/genética , Integrina alfa5beta1/metabolismo , Masculino , Óxido Nítrico , Motilidade dos EspermatozoidesRESUMO
NOD (non-obese diabetic) mice spontaneously develop type 1 diabetes following T cell-dependent destruction of pancreatic β cells. Several alterations are observed in the NOD thymus, including the presence of giant perivascular spaces (PVS) filled with single-positive (SP) CD4⺠and CD8⺠T cells that accumulate in the organ. These cells have a decreased expression of membrane CD49e (the α5 integrin chain of the fibronectin receptor VLA-5 (very late antigen-5). Herein, we observed lower sphingosine-1-phosphate receptor 1 (S1P1) expression in NOD mouse thymocytes when compared with controls, mainly in the mature SP CD4âºCD62Lhi and CD8âºCD62Lhi subpopulations bearing the CD49e− phenotype. In contrast, differences in S1P1 expression were not observed in mature CD49e⺠thymocytes. Functionally, NOD CD49e− thymocytes had reduced S1P-driven migratory response, whereas CD49e⺠cells were more responsive to S1P. We further noticed a decreased expression of the sphingosine-1-phosphate lyase (SGPL1) in NOD SP thymocytes, which can lead to a higher sphingosine-1-phosphate (S1P) expression around PVS and S1P1 internalization. In summary, our results indicate that the modulation of S1P1 expression and S1P/S1P1 interactions in NOD mouse thymocytes are part of the T-cell migratory disorder observed during the pathogenesis of type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Receptores de Lisoesfingolipídeo/genética , Timócitos/metabolismo , Animais , Movimento Celular , Diabetes Mellitus Tipo 1/imunologia , Modelos Animais de Doenças , Regulação da Expressão Gênica , Integrina alfa5/genética , Integrina alfa5/metabolismo , Integrina alfa5beta1/metabolismo , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Receptores de Lisoesfingolipídeo/metabolismo , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. METHODS: We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. RESULTS: We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. CONCLUSIONS: We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.
Assuntos
Movimento Celular , Matriz Extracelular/metabolismo , Fator de Crescimento de Hepatócito/farmacologia , Sistema de Sinalização das MAP Quinases , Metaloproteinases da Matriz/metabolismo , Mioblastos/metabolismo , Células Cultivadas , Humanos , Integrina alfa5beta1/metabolismo , Metaloproteinases da Matriz/genética , Mioblastos/efeitos dos fármacos , Mioblastos/fisiologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores de Laminina/metabolismoRESUMO
The formation of new types of sensitive conductive surfaces for the detection and transduction of cell-extracellular matrix recognition events in a real time, label-free manner is of great interest in the field of biomedical research. To study molecularly defined cell functions, biologically inspired materials that mimic the nanoscale order of extracellular matrix protein fibers and yield suitable electrical charge transfer characteristics are highly desired. Our strategy to achieve this goal is based on the spatial self-organization of patches of cell-adhesive molecules onto a gold-nanoparticle-patterned indium tin oxide electrode. Fibroblast adhesion response to selective ligands for integrins α5ß1 and αvß3, which are both relevant in cancer progression, is investigated by simultaneous electrochemical impedance spectroscopy and optical microscopy. Adhesive cells on α5ß1-selective nanopatterns showed enhanced membrane dynamics and tighter binding, compared with cells on αvß3-selective nanopatterns. The surface of the electrode exhibits high sensitivity to small changes in surface properties, because of the constitution of specific cell-surface interactions. Moreover, such sensitivity enables differentiation between cell types. This is exemplified by analyzing distinct features in the electrochemical readout of MCF-7 breast cancer cells versus MCF-10A mammary epithelial cells, when subjected to individual adhesive nanopatches.
Assuntos
Técnicas Eletroquímicas , Ouro/química , Nanopartículas Metálicas/química , Imagem Óptica , Compostos de Estanho/química , Animais , Adesão Celular , Células Cultivadas , Humanos , Integrina alfa5beta1/antagonistas & inibidores , Integrina alfa5beta1/metabolismo , Integrina alfaVbeta3/antagonistas & inibidores , Integrina alfaVbeta3/metabolismo , Ligantes , Células MCF-7 , Microeletrodos , Tamanho da Partícula , Ratos , Propriedades de SuperfícieRESUMO
Staphylococcus aureus has the ability to invade mammary epithelial cells (bMECs) causing mastitis. This event depends primarily on the α5ß1 integrin in the host cell. In addition, bMECs are a target for the hormone prolactin (PRL), which can regulate ß1 integrin-dependent actions related to differentiation and lactation. Previously, we demonstrated that bovine PRL (bPRL, 5 ng/ml) stimulates S. aureus internalization into bMECs. TLR2 is important during S. aureus infections, but its activation by PRL has not yet been established. The objective of this study was to determine the role of α5ß1 integrin and TLR2 during S. aureus internalization into bMECs stimulated with bPRL. We demonstrated that the prolactin-stimulated internalization of S. aureus decreases in response to the blockage of α5ß1 integrin (â¼ 80%) and TLR2 (â¼ 80%). bPRL increases the membrane abundance (MA) of α5ß1 integrin (â¼ 20%) and induces TLR2 MA (â¼ 2-fold). S. aureus reduces the α5ß1 integrin MA in bMECs treated with bPRL (â¼ 75%) but induces TLR2 MA in bMECs (â¼ 3-fold). Bacteria and bPRL did not modify TLR2 MA compared with the hormone alone. S. aureus induces the activation of the transcription factor AP-1, which was inhibited in bMECs treated with bPRL and infected. In general, bPRL induces both pro- and anti-inflammatory responses in bMECs, which are abated in response to bacterial challenge. Interestingly, the canonical Stat-5 transcription factor was not activated in the challenged bMECs and/or treated with bPRL. Taken together, these results support novel functions of prolactin as a modulator of the innate immune response that do not involve the classical prolactin pathway.
Assuntos
Endocitose , Células Epiteliais/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Fatores Imunológicos/metabolismo , Prolactina/metabolismo , Staphylococcus aureus/fisiologia , Receptor 2 Toll-Like/metabolismo , Animais , Bovinos , Células Cultivadas , Células Epiteliais/imunologia , Integrina alfa5beta1/metabolismoRESUMO
Adherence to the intestinal epithelia is a key feature in enteroaggregative Escherichia coli (EAEC) infection. The aggregative adherence fimbriae (AAFs) are involved in EAEC interaction with receptors at the surface of intestinal cells. We and others have demonstrated that fibronectin is a receptor for AAF/II fimbriae. Considering that the major cellular receptor of fibronectin is integrin α5ß1, in this study we evaluated the participation of this receptor in the fibronectin-mediated adherence of EAEC strain 042 to intestinal cells. We found that EAEC strain 042 has the ability to bind directly and indirectly to integrin α5ß1; direct binding was not mediated by AAF/II fimbriae and indirect binding was mediated by AAF/II and fibronectin. Coimmunoprecipitation assays confirmed the formation of the complex AafA/fibronectin/integrin α5ß1. To evaluate EAEC adherence to intestinal cells, T84 cells were incubated with fibronectin and an antibody that blocks the interaction region of integrin α5ß1 to fibronectin, the RGD site. Under these conditions, we found the number of adherent bacteria to epithelial cells significantly reduced. Additionally, fibronectin-mediated adherence of EAEC strain 042 was abolished in HEp-2 cells transfected with integrin α5 shRNA. Altogether, our data support the involvement of integrin α5ß1 in the fibronectin-mediated EAEC binding to intestinal cells.
Assuntos
Aderência Bacteriana/fisiologia , Enterócitos/fisiologia , Escherichia coli/fisiologia , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Linhagem Celular , Enterócitos/citologia , Escherichia coli/citologia , HumanosRESUMO
The objective of this study was to compare the immunoexpression of integrin α5ß1, fibronectin, and the Bcl-2 protein in normal oral mucosa (NOM), inflammatory fibroepithelial hyperplasia (IFH), oral epithelial dysplasia (OED), and oral squamous cell carcinoma (OSCC). Eleven cases of NOM, 16 IFH, 20 OED, and 27 OSCC were selected for analysis of the immunoexpression of integrin α5ß1, fibronectin, and bcl-2 protein. There was an association between the intensity and location of the integrin α5ß1 expression, especially in the OSCC, that 48.1% of cases showed weak immunoreactivity and 40.7% in the suprabasal layer (P < 0.05). There was an association between the pattern and distribution of fibronectin expression in basement membrane, where 90% of NOM showed a pattern of linear continuous and 80% of OED exhibited focal distribution (P < 0.05). The fibronectin expression in connective tissue was predominantly intense with an association of staining pattern among the different specimens, where 37% of OSCC showed a reticular pattern (P < 0.05). There was an association of bcl-2 protein among the types of specimens, especially in IFH and OSCC, where 100% of the cases exhibited scores 1 of staining (P < 0.05). Within this context, the interaction of integrin α5ß1 with its main ligand in the extracellular matrix, fibronectin, is suggested to influence the survival of tumor cells and to favor their proliferation by modulating apoptosis through the upregulation of antiapoptotic proteins or the suppression of apoptotic mediators.
Assuntos
Carcinoma de Células Escamosas/patologia , Fibronectinas/metabolismo , Hiperplasia/patologia , Integrina alfa5beta1/metabolismo , Mucosa Bucal/metabolismo , Neoplasias Fibroepiteliais/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Adulto , Idoso , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-IdadeRESUMO
Brown spider (Loxosceles sp.) venom affects the endothelium of vessels and triggers disruptive activity in the subendothelial matrix. The vascular disorders observed after venom exposure include leukocyte and platelet activation, disseminated intravascular coagulation, an increase in vessel permeability and hemorrhage into the dermis. In this study, we report additional evidence regarding the mechanism of endothelial cell cytotoxicity induced by Loxosceles intermedia venom. Exposure to venom led to endothelial cell detachment in a time-dependent manner. Loss of cell anchorage and cell-cell adhesion following venom exposure was accompanied by changes in the distribution of the α5ß1 integrin and VE-cadherin. An ultrastructural analysis of cells treated with venom revealed morphological alterations characteristic of apoptosis. Moreover, after venom exposure, the ratio between Bax and Bcl-2 proteins was disturbed in favor of Bax. In addition, late apoptosis was only observed in cells detached by the action of venom. Accordingly, there was no increase in apoptosis when cells were exposed to L. intermedia venom in suspension, suggesting that the loss of cell anchorage provides the signal to initiate apoptosis. Thus, L. intermedia venom likely triggers endothelial cell death indirectly through an apoptotic mechanism known as anoikis.
Assuntos
Anoikis/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Venenos de Aranha/farmacologia , Aranhas/metabolismo , Animais , Antígenos CD/metabolismo , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Aorta Torácica/ultraestrutura , Proteínas Reguladoras de Apoptose/metabolismo , Brasil , Caderinas/metabolismo , Adesão Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Integrina alfa5beta1/metabolismo , Cinética , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , CoelhosRESUMO
Little is known about the immunologic consequences from endocrine changes observed in diabetes. Since a preserved thymic microenvironment is of critical importance for the T cell development and maturation, we have examined the thymus from alloxan-diabetic mice. An intense thymic atrophy accompanied by changes in histological pattern and in thymocyte subpopulations were observed in diabetic mice. Laminin and fibronectin, which are closely associated with thymocytes maturation, were evaluated, but, only laminin presented an altered distribution and density in thymuses from diabetes group. the expression of fibronectin and laminin receptors was found to be decreased in diabetic mice. There was also intense decrease in the expression of two important chemokines for thymus, CCL25 and CXCL12, and in the CCR9 (CCL25 receptor), but the expression of CXCR4 (CXCL12 receptor) did not drop on cells. However, no significant difference was observed in the in vitro thymocytes migratory capacity from diabetic mice. The results show significant alterations in thymus microenvironment in diabetes and offer insights for studies involving endocrine influences on lymphatic organs and T cell maturation.