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1.
Histochem Cell Biol ; 139(4): 605-13, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23076260

RESUMEN

The family of resistin-like molecules (RELM), also known as found in inflammatory zone (FIZZ), consists of four members in mouse (RELMα/FIZZ1/HIMF, RELMß/FIZZ2, Resistin/FIZZ3, and RELMγ/FIZZ4) and two members in human (resistin and RELMß). The importance of these proteins in many aspects of physiology and pathophysiology, especially inflammatory processes, is rapidly evolving in the literature, and many investigators are beginning to work in this field. Most published studies focus on only one isoform, do not evaluate other isoforms that might be present, and have not tested for the specificity of the antibody used. Because RELM isoforms have high sequence and structural similarity and both distinct and overlapping functions, it is important to use a specific antibody to distinguish each isoform in the study. We constructed and established HEK 293 cell lines that constitutively express each isoform. Using these cell lines, we determined the specificity of antibodies (both commercially available and laboratory-made) to each isoform by Western blot and immunofluorescence. Some of the antibodies showed specificity in Western blotting but were not applicable in immunofluorescence. Others showed cross reactivity in Western blot assays. Our results indicate that RELM antibody specificity should be taken into account when using them in research and interpreting data obtained with them.


Asunto(s)
Anticuerpos/inmunología , Hormonas Ectópicas/inmunología , Péptidos y Proteínas de Señalización Intercelular/inmunología , Resistina/inmunología , Animales , Especificidad de Anticuerpos , Línea Celular , Células HEK293 , Humanos , Ratones , Isoformas de Proteínas/inmunología , Proteínas Recombinantes de Fusión/inmunología
2.
Microvasc Res ; 88: 19-24, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23583905

RESUMEN

We have previously demonstrated that PKC-potentiated inhibitory protein of protein phosphatase-1 (CPI-17) is expressed in lung endothelium. CPI-17, a specific inhibitor of myosin light chain phosphatase (MLCP), is involved in the endothelial cytoskeletal and barrier regulation. In this paper, we report the identification of fourteen putative CPI-17 interacting proteins in the lung using BacterioMatch Two-Hybrid System. Five of them: plectin 1 isoform 1, alpha II spectrin, OK/SW-CL.16, gelsolin isoform a, and junction plakoglobin are involved in actin cytoskeleton organization and cell adhesion, suggesting possible significance of these binding partners in CPI-17-mediated cytoskeletal reorganization of endothelial cells. Furthermore, we confirmed the specific interaction between plakoglobin and CPI-17, which is affected by the phosphorylation status of CPI-17 in human lung microvascular endothelial cells.


Asunto(s)
Fosfoproteínas Fosfatasas/metabolismo , Técnicas del Sistema de Dos Híbridos , Actinas/metabolismo , Citoesqueleto/metabolismo , Endotelio/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Inmunoprecipitación , Péptidos y Proteínas de Señalización Intracelular , Pulmón/irrigación sanguínea , Microcirculación , Microscopía Fluorescente , Proteínas Musculares , Fosfatasa de Miosina de Cadena Ligera/metabolismo , Fosforilación , Unión Proteica , Mapeo de Interacción de Proteínas , Transducción de Señal , gamma Catenina/metabolismo
3.
Circ Res ; 97(2): 115-24, 2005 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-15994434

RESUMEN

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca2+ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically Galphaq and Galphai2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of Galphaq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.


Asunto(s)
Adenosina Trifosfato/farmacología , Células Endoteliales/efectos de los fármacos , Transducción de Señal , Animales , Calcio/metabolismo , Bovinos , Moléculas de Adhesión Celular/fisiología , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Impedancia Eléctrica , Células Endoteliales/metabolismo , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Humanos , Uniones Intercelulares/efectos de los fármacos , Proteínas de Microfilamentos , Cadenas Ligeras de Miosina/metabolismo , Fosfatasa de Miosina de Cadena Ligera/fisiología , Fosfoproteínas/fisiología , Fosforilación
4.
Stem Cells Dev ; 22(2): 239-47, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-22891677

RESUMEN

Resistin-like molecule α (RELMα) is highly upregulated in the lungs of mice subjected to hypoxia. It is secreted from pulmonary epithelium and causes potent mitogenic, angiogenic, and vasoconstrictive effects in the lung vasculature. By using bone marrow transplantation in mice, we previously showed that RELMα is able to increase the number of bone marrow-derived cells in lung tissue, especially in the remodeling pulmonary vasculature. The current study investigated the effect of RELMα on progenitor stem cell content in mouse lung. Hypoxia, while stimulating RELMα expression, caused an increase in the number of Sca1(+)/CD45(-) progenitor cells in lungs of wild-type mice, but not in lungs of RELMα knockout mice. An in vitro study with cultured mesenchymal stem cells (MSCs) showed that RELMα induced a robust proliferative response that was dependent on Phosphatidylinositol 3-kinase/Akt and Erk activation. RELMα treatment of MSCs caused upregulation of a large number of genes involved in cell cycle, mitosis, organelle, and cytoskeleton biogenesis, and DNA metabolism. MSCs cultured in RELMα-supplemented media were able to maintain their differentiation potential into adipogenic, osteogenic, or mesenchymal phenotypes, although adipogenic differentiation was partially inhibited. These results demonstrate that RELMα may be involved in stem cell proliferation in the lung, without affecting differentiation potential.


Asunto(s)
Proliferación Celular/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/efectos de los fármacos , Animales , Apoptosis , Médula Ósea/metabolismo , Recuento de Células , Diferenciación Celular/efectos de los fármacos , División Celular , Medios de Cultivo/metabolismo , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Humanos , Hipoxia/metabolismo , Hipoxia/patología , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/farmacología , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Pulmón/metabolismo , Pulmón/patología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Osteogénesis/efectos de los fármacos , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
5.
Curr Signal Transduct Ther ; 6(3): 428-440, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-28373830

RESUMEN

This review summarizes the key role of Toll-Like Receptor (TLRs) molecules for igniting the immune system. Activated by a broad spectrum of pathogens, cytokines or other specific molecules, TLRs trigger innate immune responses. Published data demonstrate that the targeting and suppression of TLRs and TLR-related proteins with particular inhibitors may provide pivotal treatments for patients with cancer, asthma, sepsis, Crohn's disease and thrombosis. Many drugs that target cytokines act in the late phases of the activated pathways, after the final peptides, proteins or glycoproteins are formed in the cell environment. TLR activity occurs in the early activation of cellular pathways; consequently inhibiting them might be most beneficial in the treatment of human diseases.

6.
PLoS One ; 5(6): e11251, 2010 Jun 22.
Artículo en Inglés | MEDLINE | ID: mdl-20582166

RESUMEN

BACKGROUND: Pulmonary hypertension (PH) is a disease of multiple etiologies with several common pathological features, including inflammation and pulmonary vascular remodeling. Recent evidence has suggested a potential role for the recruitment of bone marrow-derived (BMD) progenitor cells to this remodeling process. We recently demonstrated that hypoxia-induced mitogenic factor (HIMF/FIZZ1/RELM alpha) is chemotactic to murine bone marrow cells in vitro and involved in pulmonary vascular remodeling in vivo. METHODOLOGY/PRINCIPAL FINDINGS: We used a mouse bone marrow transplant model in which lethally irradiated mice were rescued with bone marrow transplanted from green fluorescent protein (GFP)(+) transgenic mice to determine the role of HIMF in recruiting BMD cells to the lung vasculature during PH development. Exposure to chronic hypoxia and pulmonary gene transfer of HIMF were used to induce PH. Both models resulted in markedly increased numbers of BMD cells in and around the pulmonary vasculature; in several neomuscularized small (approximately 20 microm) capillary-like vessels, an entirely new medial wall was made up of these cells. We found these GFP(+) BMD cells to be positive for stem cell antigen-1 and c-kit, but negative for CD31 and CD34. Several of the GFP(+) cells that localized to the pulmonary vasculature were alpha-smooth muscle actin(+) and localized to the media layer of the vessels. This finding suggests that these cells are of mesenchymal origin and differentiate toward myofibroblast and vascular smooth muscle. Structural location in the media of small vessels suggests a functional role in the lung vasculature. To examine a potential mechanism for HIMF-dependent recruitment of mesenchymal stem cells to the pulmonary vasculature, we performed a cell migration assay using cultured human mesenchymal stem cells (HMSCs). The addition of recombinant HIMF induced migration of HMSCs in a phosphoinosotide-3-kinase-dependent manner. CONCLUSIONS/SIGNIFICANCE: These results demonstrate HIMF-dependent recruitment of BMD mesenchymal-like cells to the remodeling pulmonary vasculature.


Asunto(s)
Vasos Sanguíneos/citología , Células de la Médula Ósea/citología , Hipoxia/fisiopatología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Pulmón/irrigación sanguínea , Animales , Western Blotting , Trasplante de Médula Ósea , Quimiotaxis , Dependovirus/genética , Femenino , Vectores Genéticos , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Fluorescente
7.
Am J Physiol Lung Cell Mol Physiol ; 294(2): L319-24, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17993588

RESUMEN

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are major causes of acute respiratory failure associated with high morbidity and mortality. Although ALI/ARDS pathogenesis is only partly understood, pulmonary endothelium plays a major role by regulating lung fluid balance and pulmonary edema formation. Consequently, endothelium-targeted therapies may have beneficial effects in ALI/ARDS. Recently, attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. Extracellular purines (adenosine, ADP, and ATP) and pyrimidines (UDP and UTP) are important signaling molecules that mediate diverse biological effects via cell-surface P2Y receptors. We previously described ATP-induced endothelial cell (EC) barrier enhancement via a complex cell signaling and hypothesized endothelial purinoreceptors activation to exert anti-inflammatory barrier-protective effects. To test this hypothesis, we used a murine model of ALI induced by intratracheal administration of endotoxin/lipopolysaccharide (LPS) and cultured pulmonary EC. The nonhydrolyzed ATP analog ATPgammaS (50-100 muM final blood concentration) attenuated inflammatory response with decreased accumulation of cells (48%, P < 0.01) and proteins (57%, P < 0.01) in bronchoalveolar lavage and reduced neutrophil infiltration and extravasation of Evans blue albumin dye into lung tissue. In cell culture model, ATPgammaS inhibited junctional permeability induced by LPS. These findings suggest that purinergic receptor stimulation exerts a protective role against ALI by preserving integrity of endothelial cell-cell junctions.


Asunto(s)
Adenosina Trifosfato/análogos & derivados , Purinas/agonistas , Síndrome de Dificultad Respiratoria/prevención & control , Adenosina Trifosfato/farmacología , Animales , Barrera Alveolocapilar/efectos de los fármacos , Barrera Alveolocapilar/patología , Peso Corporal/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Células Endoteliales/patología , Humanos , Leucocitos/citología , Leucocitos/efectos de los fármacos , Lipopolisacáridos , Pulmón/irrigación sanguínea , Pulmón/efectos de los fármacos , Pulmón/enzimología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Peroxidasa/metabolismo , Neumonía/prevención & control , Pérdida de Peso/efectos de los fármacos
8.
Mol Biol Rep ; 33(2): 83-9, 2006 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-16817016

RESUMEN

BacterioMatch Two-Hybrid System (Stratagene) was applied in order to identify potential human TIMAP interaction proteins in the lung. TIMAP highly expressed in endothelial cells and may be involved in endothelial cytoskeletal and barrier regulation. Seven TIMAP interacting partner proteins were identified. Four of identified proteins: cystein and glycine-rich protein 1, eukaryotic translation elongation factor 2, U5 snRNP-specific protein 116 kD, and solute carrier family 3 member 2 are involved in actin cytoskeleton organization, cell adhesion or translation and transcriptional regulation.


Asunto(s)
Endotelio/metabolismo , Pulmón/metabolismo , Proteínas de la Membrana/metabolismo , Técnicas del Sistema de Dos Híbridos , Humanos , Proteínas Recombinantes/metabolismo
9.
Am J Physiol Lung Cell Mol Physiol ; 291(2): L289-95, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16825658

RESUMEN

ATP is a physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of P(2) purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier enhancement evoked by ATP has been described. ATP effects on EC barrier function and vascular permeability, however, remain poorly characterized. Although the mechanisms involved are unclear, we previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier augmentation induced by simvastatin and sphingosine 1-phosphate and therefore examined the role of these molecules in ATP-induced EC barrier enhancement. ATP induced rapid, dose-dependent barrier enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25 min (39% increase, 10 microM) and persisting at 2 h. These effects were associated with rearrangement of the EC actin cytoskeleton, early myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (10 microM)-treated EC demonstrated a significant increase in Rac activation relative to controls, with a maximal effect (approximately 4-fold increase) at 10 min. Finally, ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to controls. Our results suggest for the first time that ATP-mediated barrier protection is associated with cytoskeletal activation and is dependent on both Rac activation and cortactin.


Asunto(s)
Adenosina Trifosfato/metabolismo , Cortactina/metabolismo , Células Endoteliales/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Cortactina/genética , Citoesqueleto/metabolismo , Electrofisiología , Células Endoteliales/citología , Activación Enzimática , Humanos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteína de Unión al GTP rac1/genética
10.
J Cell Physiol ; 203(3): 520-8, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15521070

RESUMEN

The actin- and myosin-binding protein, caldesmon (CaD) is an essential component of the cytoskeleton in smooth muscle and non-muscle cells and is involved in the regulation of cell contractility, division, and assembly of actin filaments. CaD is abundantly present in endothelial cells (EC); however, the contribution of CaD in endothelial cytoskeletal arrangement is unclear. To examine this contribution, we generated expression constructs of l-CaD cloned from bovine endothelium. Wild-type CaD (WT-CaD) and truncated mutants lacking either the N-terminal myosin-binding site or the C-terminal domain 4b (containing actin- and calmodulin-binding sites) were transfected into human pulmonary artery EC. Cell fractionation experiments and an actin overlay assay demonstrated that deleting domain 4b, but not the N-terminal myosin-binding site, resulted in decreased affinity to both the detergent-insoluble cytoskeleton and soluble actin. Recombinant WT-CaD co-localized with acto-myosin filaments in vivo, but neither of CaD mutants did. Thus both domain 4b and the myosin-binding site are essential for proper localization of CaD in EC. Overexpression of WT-CaD led to cell rounding and formation of a thick peripheral subcortical actin rim in quiescent EC, which correlated with decreased cellular migration. Pharmacological inhibition of p38 MAPK, but not ERK MAPK, caused disassembly of this peripheral actin rim in CaD-transfected cells and decreased CaD phosphorylation at Ser531 (Ser789 in human h-CaD). These results suggest that CaD is critically involved in the regulation of the actin cytoskeleton and migration in EC, and that p38 MAPK-mediated CaD phosphorylation may be involved in endothelial cytoskeletal remodeling.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Unión a Calmodulina/metabolismo , Movimiento Celular/fisiología , Citoesqueleto/metabolismo , Células Endoteliales/metabolismo , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Sitios de Unión/fisiología , Proteínas de Unión a Calmodulina/genética , Bovinos , Línea Celular , Movimiento Celular/efectos de los fármacos , Forma de la Célula/fisiología , Citoesqueleto/ultraestructura , Células Endoteliales/ultraestructura , Inhibidores Enzimáticos/farmacología , Humanos , Mutación/fisiología , Miosinas/metabolismo , Fosforilación/efectos de los fármacos , Unión Proteica/fisiología , Estructura Terciaria de Proteína/fisiología , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo
11.
Am J Physiol Lung Cell Mol Physiol ; 287(5): L970-80, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15234908

RESUMEN

We have previously shown that myosin light chain (MLC) phosphatase (MLCP) is critically involved in the regulation of agonist-mediated endothelial permeability and cytoskeletal organization (Verin AD, Patterson CE, Day MA, and Garcia JG. Am J Physiol Lung Cell Mol Physiol 269: L99-L108, 1995). The molecular mechanisms of endothelial MLCP regulation, however, are not completely understood. In this study we found that, similar to smooth muscle, lung microvascular endothelial cells expressed specific endogenous inhibitor of MLCP, CPI-17. To elucidate the role of CPI-17 in the regulation of endothelial cytoskeleton, full-length CPI-17 plasmid was transiently transfected into pulmonary artery endothelial cells, where the background of endogenous protein is low. CPI-17 had no effect on cytoskeleton under nonstimulating conditions. However, stimulation of transfected cells with direct PKC activator PMA caused a dramatic increase in F-actin stress fibers, focal adhesions, and MLC phosphorylation compared with untransfected cells. Inflammatory agonist histamine and, to a much lesser extent, thrombin were capable of activating CPI-17. Histamine caused stronger CPI-17 phosphorylation than thrombin. Inhibitory analysis revealed that PKC more significantly contributes to agonist-induced CPI-17 phosphorylation than Rho-kinase. Dominant-negative PKC-alpha abolished the effect of CPI-17 on actin cytoskeleton, suggesting that the PKC-alpha isoform is most likely responsible for CPI-17 activation in the endothelium. Depletion of endogenous CPI-17 in lung microvascular endothelial cell significantly attenuated histamine-induced increase in endothelial permeability. Together these data suggest the potential importance of PKC/CPI-17-mediated pathway in histamine-triggered cytoskeletal rearrangements leading to lung microvascular barrier compromise.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Endotelio Vascular/metabolismo , Pulmón/irrigación sanguínea , Proteínas Musculares/metabolismo , Fosfoproteínas/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Adhesiones Focales/metabolismo , Histamina/farmacología , Humanos , Péptidos y Proteínas de Señalización Intracelular , Microcirculación/fisiología , Proteínas Musculares/genética , Cadenas Ligeras de Miosina/metabolismo , Fosfoproteínas Fosfatasas , Fosfoproteínas/genética , Fosforilación , Proteína Quinasa C/metabolismo , Arteria Pulmonar/citología , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venas Umbilicales/citología
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