Vitronectina e integrina αvβ3 en la interfase placentaria porcina

La cerda posee una placenta de tipo epiteliocorial y no invasiva, por lo que las moléculas de adhesión y sus ligandos cumplen un rol fundamental en la adhesión de ambos epitelios y el correcto desarrollo de la interfase feto-materna. El objetivo de este trabajo fue determinar la expresión de la vitronectina (VN) y su receptor, la integrina αvβ3 en placenta porcina. Se recolectó úteros de cerdas no gestantes (n=5) y placentas de 17 (n=5), 30 (n=5) (n=5), 60 (n=5), 70 (n=5) y 114 (n=5) días de gestación (dg). La detección de VN y de αvβ3 se realizó por inmunoperoxidasa indirecta sobre cortes histológicos de útero no gestante y de placentas porcinas maternas y fetales en los periodos de gestación seleccionados. Los resultados se expresaron de un modo semicualitativo en función de la coloración detectada, determinando que: (-)= negativo, (+)= positividad leve, (++)= positividad moderada y (+++)= positividad fuerte. En útero no gestante la expresión de VN fue moderada y la integrina estuvo ausente en el epitelio. A los 17 dg, la VN se halló elevada en el trofoblasto (+++), y se expresó moderadamente (++) en el epitelio endometrial. La integrina αvβ3 tuvo una expresión moderada en ambos epitelios (++). A los 30 y 60 dg la expresión de ambas moléculas se halló elevada (+++) o moderada (++) en los epitelios que conforman la interfase placentaria. Desde los 70 dg VN y αvβ3 disminuyeron su expresión en los epitelios. La expresión de la vitronectina y de la integrina αvβ3 desde los 17 dg en epitelio materno y trofoblástico hasta los 70 dg permitiría la adhesión entre los epitelios placentarios materno y fetal durante la gestación porcina. Su ausencia al finalizar la gestación sería necesaria para permitir el desprendimiento de la placenta fetal en el momento del parto

NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) modulation by intracellular Cl– concentration

The impairment of the cystic fibrosis transmembrane conductance regulator (CFTR) activity induces intracellular chloride (Cl–) accumulation. The anion Cl–, acting as a second messenger, stimulates the secretion of interleukin-1β (IL-1β), which starts an autocrine positive feedback loop. Here, we show that NLR family pyrin domain containing 3 (NLRP3) and caspase 1 (CASP1) are indirectly modulated by the intracellular Cl– concentration, showing maximal expression and activity at 75 mM Cl–, in the presence of the ionophores nigericin and tributyltin. The expression of PYD and CARD domain containing (PYCARD/ASC) remained constant from 0 to 125 mM Cl–. The CASP1 inhibitor VX-765 and the NLRP3 inflammasome inhibitor MCC950 completely blocked the Cl–-stimulated IL-1β mRNA expression and partially the IL-1β secretion. DCF fluorescence (cellular reactive oxygen species, cROS) and MitoSOX fluorescence (mitochondrial ROS, mtROS) also showed maximal ROS levels at 75 mM Cl–, a response strongly inhibited by the ROS scavenger N-acetyl-L-cysteine (NAC) or the NADPH oxidase (NOX) inhibitor GKT137831. These inhibitors also affected CASP1 and NLRP3 mRNA and protein expression. More importantly, the serum/glucocorticoid regulated kinase 1 (SGK1) inhibitor GSK650394, or its shRNAs, completely abrogated the IL-1β mRNA response to Cl– and the IL-1β secretion, interrupting the autocrine IL-1β loop. The results suggest that Cl– effects are mediated by SGK1, in which under Cl– modulation stimulates the secretion of mature IL-1β, in turn, responsible for the upregulation of ROS, CASP1, NLRP3 and IL-1β itself, through autocrine signalling.Fil: Clauzure, Mariangeles. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentina. Universidad Nacional de La Pampa; ArgentinaFil: Valdivieso, Ángel Gabriel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Dugour, Andrea Vanesa. Fundación Pablo Cassará; ArgentinaFil: Mori, Consuelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Massip Copiz, María Macarena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Aguilar, María Á.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Sotomayor, Veronica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Asensio, Cristian Jorge Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; ArgentinaFil: Figueroa, Juan M.. Fundación Pablo Cassará; ArgentinaFil: Santa Coloma, Tomás Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Ciencias Biológicas y Biomédicas del Sur. Universidad Nacional del Sur. Departamento de Biología, Bioquímica y Farmacia. Instituto de Ciencias Biológicas y Biomédicas del Sur; Argentin

Inmunolocalización de receptores de estrógenos acoplados a proteína g en placentas porcinas.

En la gestación porcina, las hormonas esteroideas Estrógenos y Progesterona interaccionan con receptores nucleares y de membrana. El propósito de esta investigación fue determinar la distribución y localización del receptor de estrógenos acoplado a proteína G (GPER) en placentas porcinas de diversos periodos de gestación. Se utilizaron cerdas mestizas gestantes (G) de 15-17 días de gestación (dg) (n= 4), de 30-35 dg (n= 4) y de 60-70 dg (n= 4), provenientes de frigoríficos de la zona de General Pico, La Pampa. Y cerdas no gestantes (NG) (n=4). De cada muestra se realizó una tinción de Hematoxilina/Eosina para determinar la integridad de los tejidos, y posteriormente se realizó la determinación de los receptores de estrógenos por la técnica de inmunoperoxidasa indirecta LSAB (Labelled Streptavidin Biotin). Los resultados de la técnica de inmunomarcación para la determinación de receptores de hormonas esteroideas, se expresaron en forma cualitativa. Se evaluó la expresión según la coloración detectada (diferentes intensidades de marrón). En todos los períodos estudiados se observó inmunomarcación de GPER en células trofoblásticas. En el componente materno se expresó en epitelio luminal y glandular además de músculo liso. Los resultados de este estudio confirman la localización de GPER en endometrio de placentas porcinas de 5, 17, 30 y 70 dg y en células trofoblásticas de 17, 30 y 70 dg. La expresión de los receptores de estrógenos de membrana acuerda con estudios previos en los cuales se cuantificó los niveles de estrógenos en sueros y en el componente materno y fetal de similares etapas de la gestación. Sugerimos que en la interfase materno fetal, los estrógenos podrían ligarse a GPER y establecer vías de señalización específicamente reguladas. Esa comunicación entre la hembra y sus conceptus permitiría inducir y sostener la expresión de moléculas que modulan la implantación, el desarrollo y el mantenimiento de la gestació

Inmunolocalización de la integrina a5B1, la laminina y el colágeno tipo V en placenta porcina en diferentes etapas gestacionales

Adhesion molecules are essential for anchoring and adhesion during swine placentation in both the maternal and the fetal epithelia. The aim of this work was to determine the expression of ?5?1 integrin and its receptors, laminin and collagen type V, in swine placentas at different gestation intervals: 30, 60, 70 and 114 days of gestation (dg; n = 23). The expression of these molecules was determined by immunohistochemistry. Here wefound that the ?5?1 integrin was expressed at high levels in endometrial connective tissue and around blood vessels of the maternal and fetal placenta. Laminin was expressed in the swine fetal-maternal interface, at 30, 60 and 114 dg in a mild manner. On the other hand, the expression was strong in endometrial stroma, endometrial glands, blood vessels and basal membrane of the villi throughout pregnancy. Collagen type V was strongly expressed in the mesenchyme villi of the maternal placenta and around the fetal and maternal placenta vessels.\nBased on our results, we postulate that ?5?1 integrin, and its receptors, could play a crucial role in the development and maintenance of placental architecture and angiogenesis.Fil: Vélez, C.L. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias. La Pampa, ArgentinaFil: Vélez, C.L. CONICET. La Pampa, ArgentinaFil: Williamson, D.M. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias. La Pampa, ArgentinaFil: Clauzure, M. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias. La Pampa, ArgentinaFil: Clauzure, M. CONICET. La Pampa, ArgentinaFil: Koncurat, M.A. Universidad Nacional de La Pampa. Facultad de Ciencias Veterinarias. La Pampa, ArgentinaFil: Barbeito, C.G. CONICET. La Pampa, ArgentinaFil: Barbeito, C.G. Universidad Nacional de La Plata. Facultad de Ciencias Veterinarias. Laboratorio de Histología y Embriología Descriptiva, Experimental y Comparada. La Plata, ArgentinaLas moléculas de adhesión son indispensables para el anclaje y la adhesión de los epitelios materno y fetal en la placentación porcina. El objetivo fue determinar la inmunolocalización de la integrina ?5?1, la laminina y el colágeno V, en placentas porcinas de diferentes períodos de gestación: 30, 60, 70 y 114 días de gestación (dg; n=23). Se determinó la inmunolocalización de las moléculas por inmunohistoquímica. La integrina ?5?1 fue fuerte en el tejido conectivo del endometrio y alrededor de vasos sanguíneos en placenta materna y fetal. La laminina se identificó en la interfase feto-materna porcina, a los 30, 60 y 114 dg de forma leve; en cambio, la intensidad de marcación fue fuerte en estroma endometrial, glándulas endometriales, en los vasos sanguíneos y en la membrana basal de las vellosidades a lo largo de toda la gestación. El colágeno V se evidenció con una fuerte intensidad en el mesénquima de las vellosidades placentarias maternas, y alrededor de losvasos sanguíneos en placenta fetal y materna. En base a nuestros resultados, postulamos que la integrina ?5?1, la laminina y el colágeno tipo V, podrían cumplir un rol crucial en el desarrollo y el mantenimiento de la arquitectura placentaria y angiogénesis. \

N-acetyl cysteine reverts the proinflammatory state induced by cigarette smoke extract in lung Calu-3 cells

Chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF) are lethal pulmonary diseases. Cigarette consumption is the main cause for development of COPD, while CF is produced by mutations in the CFTR gene. Although these diseases have a different etiology, both share a CFTR activity impairment and proinflammatory state even under sterile conditions. The aim of this work was to study the extent of the protective effect of the antioxidant N-acetylcysteine (NAC) over the proinflammatory state (IL-6 and IL-8), oxidative stress (reactive oxygen species, ROS), and CFTR levels, caused by Cigarette Smoke Extract (CSE) in Calu-3 airway epithelial cells. CSE treatment (100 µg/ml during 24 h) decreased CFTR mRNA expression and activity, and increased the release of IL-6 and IL-8. The effect on these cytokines was inhibited by N-acetyl cysteine (NAC, 5 mM) or the NF-kB inhibitor, IKK-2 (10 µM). CSE treatment also increased cellular and mitochondrial ROS levels. The cellular ROS levels were normalized to control values by NAC treatment, although significant effects on mitochondrial ROS levels were observed only at short times (5´) and effects on CFTR levels were not observed. In addition, CSE reduced the mitochondrial NADH-cytochrome c oxidoreductase (mCx I-III) activity, an effect that was not reverted by NAC. The reduced CFTR expression and the mitochondrial damage induced by CSE could not be normalized by NAC treatment, evidencing the need for a more specific reagent. In conclusion, CSE causes a sterile proinflammatory state and mitochondrial damage in Calu-3 cells that was partially recovered by NAC treatment. Keywords: Cigarette smoke extract, Mitochondria, CFTR, ROS, COPD, Cystic fibrosi

Histamine H4 receptor agonism induces antitumor effects in human T-cell lymphoma

Abstract: The discovery of the human histamine H4 receptor (H4R) has contributed to our understanding of the role of histamine in numerous physiological and pathological conditions, including tumor development and progression. The lymph nodes of patients with malignant lymphomas have shown to contain high levels of histamine, however, less is known regarding the expression and function of the H4R in T-cell lymphoma (TCL). In this work we demonstrate the expression of H4R isoforms (mRNA and protein) in three human aggressive TCL (OCI-Ly12, Karpas 299, and HuT78). Histamine and specific H4R agonists (VUF8430 and JNJ28610244) significantly reduced cell viability in a dose-dependent manner (p < 0.05). The combined treatment with the H4R antagonist (JNJ7777120, 10 µM) reversed the effects of the H4R ligands. Importantly, we screened a drug repurposing library of 433 FDA-approved compounds (1 µM) in combination with histamine (10 µM) in Hut78 cells. Histamine produced a favorable antitumor effect with 18 of these compounds, including the histone deacetylase inhibitor panobinostat. Apoptosis, proliferation, and oxidative stress studies confirmed the antitumoral effects of the combination. We conclude that the H4R is expressed in TCL, and it is involved in histamine-mediated responses

Disruption of interleukin-1β autocrine signaling rescues complex I activity and improves ROS levels in immortalized epithelial cells with impaired cystic fibrosis transmembrane conductance regulator (CFTR) function.

Patients with cystic fibrosis (CF) have elevated concentration of cytokines in sputum and a general inflammatory condition. In addition, CF cells in culture produce diverse cytokines in excess, including IL-1β. We have previously shown that IL-1β, at low doses (∼30 pM), can stimulate the expression of CFTR in T84 colon carcinoma cells, through NF-κB signaling. However, at higher doses (>2.5 ng/ml, ∼150 pM), IL-1β inhibit CFTR mRNA expression. On the other hand, by using differential display, we found two genes with reduced expression in CF cells, corresponding to the mitochondrial proteins CISD1 and MTND4. The last is a key subunit for the activity of mitochondrial Complex I (mCx-I); accordingly, we later found a reduced mCx-I activity in CF cells. Here we found that IB3-1 cells (CF cells), cultured in serum-free media, secrete 323±5 pg/ml of IL-1β in 24 h vs 127±3 pg/ml for S9 cells (CFTR-corrected IB3-1 cells). Externally added IL-1β (5 ng/ml) reduces the mCx-I activity and increases the mitochondrial (MitoSOX probe) and cellular (DCFH-DA probe) ROS levels of S9 (CFTR-corrected IB3-1 CF cells) or Caco-2/pRSctrl cells (shRNA control cells) to values comparable to those of IB3-1 or Caco-2/pRS26 cells (shRNA specific for CFTR). Treatments of IB3-1 or Caco-2/pRS26 cells with either IL-1β blocking antibody, IL-1 receptor antagonist, IKK inhibitor III (NF-κB pathway) or SB203580 (p38 MAPK pathway), restored the mCx-I activity. In addition, in IB3-1 or Caco-2/pRS26 cells, IL-1β blocking antibody, IKK inhibitor III or SB203580 reduced the mitochondrial ROS levels by ∼50% and the cellular ROS levels near to basal values. The AP-1 inhibitors U0126 (MEK1/2) or SP600125 (JNK1/2/3 inhibitor) had no effects. The results suggest that in these cells IL-1β, through an autocrine effect, acts as a bridge connecting the CFTR with the mCx-I activity and the ROS levels

Identification and characterization of human PEIG-1/GPRC5A as a 12-Otetradecanoyl phorbol-13-acetate (TPA) and PKC-induced gene

Abstract: Homo sapiens orphan G protein-coupling receptor PEIG-1 was first cloned and characterized by applying differential display to T84 colonic carcinoma cells incubated in the presence of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) (GenBank AF506289.1). Later, Lotan's laboratory found the same gene product in response to retinoic acid analogues, naming it with the symbol RAIG1. Now the official HGNC symbol is GPRC5A. Here, we report the extension of its original cDNA fragment towards the 5' and 3' end. In addition, we show that TPA (100 ng/ml, 162 nM) strongly stimulated GPRC5A mRNA in T84 colonic carcinoma cells, with maximal expression at 4 h and 100 ng/ml (162 nM). Western blots showed several bands between 35 and 50 kDa, responding to TPA stimulation. Confocal microscopy confirmed its TPA upregulation and the location in the plasma membrane. The PKC inhibitor Gö 6983 (10 μM), and the Ca2+ chelator BAPTA-AM (150 μM), strongly inhibited its TPA induced upregulation. The PKA inhibitor H-89 (10 μM), and the MEK1/2 inhibitor U0126 (10 μM), also produced a significant reduction in the TPA response (~50%). The SGK1 inhibitor GSK650394 stimulated GPRC5A basal levels at low doses and inhibit its TPA-induced expression at concentrations ≥10 μM. The IL-1β autocrine loop and downstream signalling did not affect its expression. In conclusion, RAIG1/RAI3/GPRC5A corresponds to the originally reported PEIG-1/TIG1; the inhibition observed in the presence of Gö 6983, BAPTA and U0126, suggests that its TPA-induced upregulation is mediated through a PKC/Ca2+ →MEK1/2 signalling axis. PKA and SGK1 kinases are also involved in its TPA-induced upregulation

CFTR chloride channel activity modulates the mitochondrial morphology in cultured epithelial cells

Abstract: The impairment of the CFTR channel activity, a cAMP-activated chloride (Cl− ) channel responsible for cystic fibrosis (CF), has been associated with a variety of mitochondrial alterations such as modified gene expression, impairment in oxidative phosphorylation, increased reactive oxygen species (ROS), and a disbalance in calcium homeostasis. The mechanisms by which these processes occur in CF are not fully understood. Previously, we demonstrated a reduced MTND4 expression and a failure in the mitochondrial complex I (mCx-I) activity in CF cells. Here we hypothesized that the activity of CFTR might modulate the mitochondrial fission/fusion balance, explaining the decreased mCx-I. The mitochondrial morphology and the levels of mitochondrial dynamic proteins MFN1 and DRP1 were analysed in IB3− 1 CF cells, and S9 (IB3− 1 expressing wt-CFTR), and C38 (IB3− 1 expressing a truncated functional CFTR) cells. The mitochondrial morphology of IB3− 1 cells compared to S9 and C38 cells showed that the impaired CFTR activity induced a fragmented mitochondrial network with increased rounded mitochondria and shorter branches. Similar results were obtained by using the CFTR pharmacological inhibitors CFTR(inh)-172 and GlyH101 on C38 cells. These morphological changes were accompanied by modifications in the levels of the mitochondrial dynamic proteins MFN1, DRP1, and p(616)-DRP1. IB3− 1 CF cells treated with Mdivi-1, an inhibitor of mitochondrial fission, restored the mCx-I activity to values similar to those seen in S9 and C38 cells. These results suggest that the mitochondrial fission/fusion balance is regulated by the CFTR activity and might be a potential target to treat the impaired mCx-I activity in CF

Effect of IL-1β on mitochondrial Complex I activity (mCx-I).

<p>IB3-1 and S9 cells pre-incubated in serum free media for 24 h were incubated for additional 24 h (plus CFTR-stimulating cocktail) in the presence or absence of IL-1β (5 ng/ml) and the mCx-I activity was determined. A: mCx-I in-gel activity (IGA) of treated and untreated S9 and IB3-1 cells; mCx-III correspond to the WB for the UQCRC1 subunit of the mitochondrial complex III used as internal standard. B: Densitometric quantification and statistical analysis of the results shown in panel A (for n = 3); IGA of mCx-I was calculated as the ratio mCx-I IGA/mCx-III WB. C: Spectrophotometric measurements of mitochondrial NADH-cytochrome c reductase activity for the same experiments shown in panel A and B. Results were expressed as percentage (%) relative to S9 control values. Measurements were performed in triplicate and data were expressed as mean ± SE of three independent experiments (n = 3). *indicates p<0.05 compared with basal S9 cells.</p