RESUMO
The endothelial vascular permeability barrier has an important role throughout the body's extensive vasculature, and its disruption leads to vascular hyperpermeability (leakage), which is associated with numerous medical conditions. In the lung, vascular hyperpermeability can lead to pulmonary edema and acute respiratory distress syndrome (ARDS), the most severe and deadly complication of viral and bacterial infections, trauma and radiation exposure. There is currently no pharmacological treatment for ARDS with the only approved options being focused on supportive care. The development of effective treatments for ARDS has a potential to turn infectious diseases such as bacterial and viral pneumonia (including COVID-19) into manageable conditions, saving lives and providing a new tool to combat future epidemics. Strategies that aim to protect and augment the vascular endothelial barrier are important avenues to consider as potential treatments for ARDS and other conditions underlined by vascular hyperpermeability. We propose the activation of the MAPKAPK2 (MK2) kinase pathway as a new approach to augment the endothelial barrier and prevent or reverse ARDS and other conditions characterized by vascular barrier dysfunction.
Assuntos
Tratamento Farmacológico da COVID-19 , Síndrome do Desconforto Respiratório , Permeabilidade Capilar , Humanos , Pulmão/metabolismo , Síndrome do Desconforto Respiratório/tratamento farmacológico , Transdução de SinaisRESUMO
The ubiquitous α-catulin acts as a scaffold for distinct signalosomes including RhoA/ROCK; however, its function is not well understood. While α-catulin has homology to the cytoskeletal linkers α-catenin and vinculin, it appears to be functionally divergent. Here we further investigated α-catulin function in pulmonary vascular endothelial cells (VEC) on the premise that α-catulin has a unique cytoskeletal role. Examination of endogenous α-catulin intracellular localization by immunofluorescence revealed a highly organized cytosolic filamentous network suggestive of a cytoskeletal system in a variety of cultured VEC. Double-immunofluorescence analyses of VEC showed endogenous α-catulin co-localization with vimentin intermediate filaments. Similar to vimentin, α-catulin was found to distribute into detergent-soluble and -insoluble fractions. Treatment of VEC with withaferinA, an agent that targets vimentin filaments, disrupted the α-catulin network distribution and altered α-catulin solubility. Vimentin participates in cell migration, and withaferinA was found to inhibit VEC migration in vitro; similarly, α-catulin knock-down reduced VEC migration. Based on previous reports showing that ROCK modulates vimentin, we found that ROCK depletion attenuated VEC migration; furthermore, α-catulin depletion was shown to reduce ROCK-induced signaling. These findings indicate that α-catulin has a unique function in co-localization with vimentin filaments that contributes to VEC migration via a pathway that may involve ROCK signaling. J. Cell. Physiol. 231: 934-943, 2016. © 2015 Wiley Periodicals, Inc.
Assuntos
Movimento Celular , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Filamentos Intermediários/metabolismo , Pulmão/citologia , Vimentina/metabolismo , alfa Catenina/metabolismo , Quinases Associadas a rho/metabolismo , Animais , Bovinos , Movimento Celular/efeitos dos fármacos , Citosol/metabolismo , Células Endoteliais/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Filamentos Intermediários/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Solubilidade , Vitanolídeos/farmacologiaRESUMO
Anthrax is associated with severe vascular leak, which is caused by the bacterial lethal toxin (LeTx). Pleural effusions and pulmonary edema that occur in anthrax are believed to reflect endothelial injury caused by the anthrax toxin. Since vascular leak can also be observed consistently in rats injected intravenously with LeTx, the latter might present a simple physiologically relevant animal model of acute lung injury (ALI). Such a model could be utilized in evaluating and developing better treatment for ALI or acute respiratory distress syndrome (ARDS), as other available rodent models do not consistently produce the endothelial permeability that is a major component of ARDS. The biological activity of LeTx resides in the lethal factor metalloprotease that specifically degrades MAP kinase kinases (MKKs). Recently, we showed that LeTx inactivation of p38 MAP kinase signaling via degradation of MKK3 in pulmonary vascular endothelial cells can be linked to compromise of the endothelial permeability barrier. LeTx effects were linked specifically to blocking activation of p38 substrate and MAP kinase-activated protein kinase 2 (MAPKAPK2 or MK2) and phosphorylation of the latter's substrate, heat shock protein 27 (HSP27). We have now designed a peptide that directly and specifically activates MK2, causing HSP27 phosphorylation in cells and in vivo. The MK2-activating peptide (MK2-AP) also blocks the effects of LeTx on endothelial barriers in cultured cells and reduces LeTx-induced pulmonary vascular leak in rats. Hence, MK2-AP has the therapeutic potential to counteract anthrax or pulmonary edema and vascular leak due to other causes.
Assuntos
Lesão Pulmonar Aguda/prevenção & controle , Antígenos de Bactérias , Toxinas Bacterianas , Células Endoteliais/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Pulmão/efeitos dos fármacos , Peptídeos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Edema Pulmonar/prevenção & controle , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/enzimologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Citoproteção , Modelos Animais de Doenças , Células Endoteliais/enzimologia , Ativação Enzimática , Proteínas de Choque Térmico HSP27/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Pulmão/irrigação sanguínea , Pulmão/enzimologia , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Edema Pulmonar/induzido quimicamente , Edema Pulmonar/enzimologia , Interferência de RNA , Ratos , Ratos Endogâmicos F344 , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
Despite high expression levels, the role of Tsc1 in cardiovascular tissue is ill defined. We launched this study to examine the role of Tsc1 in cardiac physiology and pathology. Mice in which Tsc1 was deleted in cardiac tissue and vascular smooth muscle (Tsc1c/cSM22cre(+/-)), developed progressive cardiomegaly and hypertension and died early. Hearts of Tsc1c/cSM22cre(+/-) mice displayed a progressive increase in cardiomyocyte number, and to a lesser extent, size between the ages of 1 and 6 weeks. In addition, compared to control hearts, proliferation markers (phospho-histone 3 and PCNA) were elevated in Tsc1c/cSM22cre(+/-) cardiomyocytes at 0-4 weeks, suggesting that cardiomyocyte proliferation was the predominant mechanism underlying cardiomegaly in Tsc1c/cSM22cre(+/-) mice. To examine the contribution of Tsc1 deletion in peripheral vascular smooth muscle to the cardiac phenotype, Tsc1c/cSM22cre(+/-) mice were treated with the antihypertensive, hydralazine. Prevention of hypertension had no effect on survival, cardiac size, or cardiomyocyte number in these mice. We furthermore generated mice in which Tsc1 was deleted only in vascular smooth muscle but not in cardiac tissue (Tsc1c/cSMAcre-ER(T2+/-)). The Tsc1c/cSMAcre-ER(T2+/-) mice also developed hypertension. However, their survival was normal and no cardiac abnormalities were observed. Our results suggest that loss of Tsc1 in the heart causes cardiomegaly, which is driven by increased cardiomyocyte proliferation that also appears to confer relative resistance to afterload reduction. These findings support a critical role for the Tsc1 gene as gatekeeper in the protection against uncontrolled cardiac growth.
Assuntos
Cardiomegalia/metabolismo , Proliferação de Células/genética , Miócitos Cardíacos/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Cardiomegalia/genética , Cardiomegalia/patologia , Modelos Animais de Doenças , Hemodinâmica/fisiologia , Hiperplasia/genética , Hiperplasia/metabolismo , Immunoblotting , Imuno-Histoquímica , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Multiplex , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Reação em Cadeia da Polimerase , Proteína 1 do Complexo Esclerose Tuberosa , Proteínas Supressoras de Tumor/genéticaRESUMO
Vimentin is a major intermediate filament protein in vascular endothelial cells which might be involved in their function as a barrier tissue. It is proposed to dynamically maintain integrity of the endothelium as a tightly regulated permeability barrier that is subjected to a variety of shear and contractile forces. The results described in this report demonstrate that vimentin plays that role through mechanisms that are dependent on its phosphorylation state. Withaferin A (WFA), a vimentin targeting drug is shown to disrupt endothelial barrier function through its effects on vimentin filament distribution and physical properties. These effects are related to WFA's ability to increase vimentin phosphorylation. Through overexpressing a non-phosphorylatable vimentin mutant we can block the effects of WFA on vimentin distribution and barrier permeability. The barrier augmentation effect appears to extend to endothelial cells that do not express detectable mutant vimentin which might suggest transmissible effects across cells. Blocking vimentin phosphorylation also protects the endothelial barrier against LPS endotoxin, implicating it as a target for drug development against pulmonary edema and acute respiratory distress syndrome (ARDS).
Assuntos
Permeabilidade Capilar , Células Endoteliais/metabolismo , Vimentina/metabolismo , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Resistência a Medicamentos , Células Endoteliais/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Mutação , Fosforilação , Ratos , Serina , Solubilidade , Fatores de Tempo , Transfecção , Vimentina/química , Vimentina/genética , Vitanolídeos/farmacologia , Quinases Ativadas por p21/metabolismoRESUMO
Exposure to anthrax causes life-threatening disease through the action of the toxin produced by the Bacillus anthracis bacteria. Lethal factor (LF), an anthrax toxin component which causes severe vascular leak and edema, is a protease which specifically degrades MAP kinase kinases (MKK). We have recently shown that p38 MAP kinase activation leading to HSP27 phosphorylation augments the endothelial permeability barrier. We now show that treatment of rat pulmonary microvascular endothelial cells with anthrax lethal toxin (LeTx), which is composed of LF and the protective antigen, increases endothelial barrier permeability and gap formation between endothelial cells through disrupting p38 signaling. LeTx treatment increases MKK3b degradation and in turn decreases p38 activity at baseline as well as after activation of p38 signaling. Consequently, LeTx treatment decreases activation of the p38 substrate kinase, MK2, and the phosphorylation of the latter's substrate, HSP27. LeTx treatment disrupts other signaling pathways leading to suppression of Erk-mediated signaling, but these effects do not correlate with LeTx-induced barrier compromise. Overexpressing phosphomimicking (pm)HSP27, which protects the endothelial permeability barrier against LeTx, blocks LeTx inactivation of p38 and MK2, but it does not block MKK3b degradation or Erk inactivation. Our results suggest that LeTx might cause vascular leak through inactivating p38-MK2-HSP27 signaling and that activating HSP27 phosphorylation specifically restores p38 signaling and blocks anthrax LeTx toxicity. The fact that barrier integrity could be restored by pmHSP27 overexpression without affecting degradation of MKK3b, or inactivation of Erk, suggests a specific and central role for p38-MK2-HSP27 in endothelial barrier permeability regulation.
Assuntos
Antígenos de Bactérias/toxicidade , Toxinas Bacterianas/toxicidade , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/fisiologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Animais , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Células Endoteliais/ultraestrutura , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/ultraestrutura , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico HSP27/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresRESUMO
Hypoxia triggers responses in endothelial cells that play roles in many conditions including high-altitude pulmonary edema and tumor angiogenesis. Signaling pathways activated by hypoxia modify cytoskeletal and contractile proteins and alter the biomechanical properties of endothelial cells. Intermediate filaments are major components of the cytoskeleton whose contribution to endothelial physiology is not well understood. We have previously shown that hypoxia-activated signaling in endothelial cells alters their contractility and adhesiveness. We have also linked p38-MAP kinase signaling pathway leading to HSP27 phosphorylation and increased actin stress fiber formation to endothelial barrier augmentation. We now show that vimentin, a major intermediate filament protein in endothelial cells, is regulated by hypoxia. Our results indicate that exposure of endothelial cells to hypoxia causes vimentin filament networks to initially redistribute perinuclearly. However, by 1 hour hypoxia these networks reform and appear more continuous across cells than under normoxia. Hypoxia also causes transient changes in vimentin phosphorylation, and activation of PAK1, a kinase that regulates vimentin filament assembly. In addition, exposure to 1 hour hypoxia increases the ratio of insoluble/soluble vimentin. Overexpression of phosphomimicking mutant HSP27 (pmHSP27) causes changes in vimentin distribution that are similar to those observed in hypoxic cells. Knocking-down HSP27 destroys the vimentin filamentous network, and disrupting vimentin filaments with acrylamide increases endothelial permeability. Both hypoxia- and pmHSP27 overexpression-induced changes are reversed by inhibition of phosphatase activity. In conclusion hypoxia causes redistribution of vimentin to a more insoluble and extensive filamentous network that could play a role in endothelial barrier stabilization. Vimentin redistribution appears to be mediated through altering the phosphorylation of the protein and its interaction with HSP27.
Assuntos
Células Endoteliais/fisiologia , Filamentos Intermediários/fisiologia , Vimentina/fisiologia , Animais , Hipóxia Celular/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP27/deficiência , Proteínas de Choque Térmico HSP27/genética , Fosforilação/fisiologia , Ratos , Vimentina/metabolismoRESUMO
This manuscript describes how the permeability of pulmonary artery microvascular endothelial cell (RPMEC) monolayer is elevated by hypoxia and the role played by HSP27 phosphorylation. p38 MAP kinase activation leading to HSP27 phosphorylation was previously shown by our laboratory to alter the actin cytoskeleton and tethering properties of RPMEC. This effect was independent of hypoxia-induced contractility which was ROCK-dependent rather than HSP27-dependent. Results described here show that increased HSP27 phosphorylation not only does not underlie hypoxia-induced permeability, but may actually augment the endothelial barrier. Hypoxia causes gap formation between RPMEC and increases MLC2 phosphorylation. The phosphorylation of MYPT1, which inhibits MLC2 phosphatase, is also increased in hypoxia. In addition, FAK phosphorylation, which alters focal adhesion signaling, is increased in hypoxia. Overexpressing phosphomimicking HSP27 (pmHSP27), which induces significant actin stress fiber formation, surprisingly renders RPMEC resistant to hypoxia- or TGFbeta-induced permeability. siRNA against pmHSP27 reverses the increased actin stress fiber formation in pmHSP27-overexpressing cells, and disrupting actin stress fibers in pmHSP27-overexpressing RPMEC renders them more susceptible to hypoxia. Finally, hypoxia-induced gap formation, as well as phosphorylation of MLC2, MYPT1 and FAK are almost abolished by overexpressing pmHSP27 in RPMEC. These effects of pmHSP27 overexpression might represent decreased cytoskeletal plasticity and increased tethering which counteracts permeability-inducing contractility. Thus hypoxia activates two pathways one leading to contractility and increased permeability, the other leading to actin stress fibers, stronger adhesion, and reduced permeability. Altering HSP27 phosphorylation, which tips the balance towards decreased permeability, might be targeted in managing endothelial barrier dysfunction.
Assuntos
Permeabilidade Capilar , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Transdução de Sinais , Animais , Miosinas Cardíacas/metabolismo , Hipóxia Celular , Células Cultivadas , Células Endoteliais/enzimologia , Quinase 1 de Adesão Focal/metabolismo , Junções Comunicantes/enzimologia , Proteínas de Choque Térmico HSP27/genética , Proteínas de Choque Térmico , Chaperonas Moleculares , Mutação , Cadeias Leves de Miosina/metabolismo , Fosforilação , Proteína Fosfatase 1/metabolismo , Interferência de RNA , Ratos , Fibras de Estresse/metabolismo , Fatores de Tempo , Transfecção , Fator de Crescimento Transformador beta/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Mechanical ventilation, a fundamental therapy for acute lung injury, worsens pulmonary vascular permeability by exacting mechanical stress on various components of the respiratory system causing ventilator associated lung injury. We postulated that MK2 activation via p38 MAP kinase induced HSP25 phosphorylation, in response to mechanical stress, leading to actin stress fiber formation and endothelial barrier dysfunction. We sought to determine the role of p38 MAP kinase and its downstream effector MK2 on HSP25 phosphorylation and actin stress fiber formation in ventilator associated lung injury. Wild type and MK2(-/-) mice received mechanical ventilation with high (20 ml/kg) or low (7 ml/kg) tidal volumes up to 4 hrs, after which lungs were harvested for immunohistochemistry, immunoblotting and lung permeability assays. High tidal volume mechanical ventilation resulted in significant phosphorylation of p38 MAP kinase, MK2, HSP25, actin polymerization, and an increase in pulmonary vascular permeability in wild type mice as compared to spontaneous breathing or low tidal volume mechanical ventilation. However, pretreatment of wild type mice with specific p38 MAP kinase or MK2 inhibitors abrogated HSP25 phosphorylation and actin polymerization, and protected against increased lung permeability. Finally, MK2(-/-) mice were unable to phosphorylate HSP25 or increase actin polymerization from baseline, and were resistant to increases in lung permeability in response to HV(T) MV. Our results suggest that p38 MAP kinase and its downstream effector MK2 mediate lung permeability in ventilator associated lung injury by regulating HSP25 phosphorylation and actin cytoskeletal remodeling.
Assuntos
Actinas/metabolismo , Permeabilidade Capilar , Proteínas de Choque Térmico/fisiologia , Lesão Pulmonar/etiologia , MAP Quinase Quinase 2/fisiologia , Proteínas de Neoplasias/fisiologia , Respiração Artificial/efeitos adversos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Pulmão , Lesão Pulmonar/metabolismo , Camundongos , Camundongos Knockout , Chaperonas Moleculares , Fosforilação , Estresse Mecânico , Ventiladores Mecânicos/efeitos adversosRESUMO
The ubiquitous alpha(E)-catenin is an essential actin cytoskeletal linker. The transcription factor, serum response factor (SRF), induces transcription via binding to the serum response element (SRE) in gene promoters, and in many cases responds to actin dynamics. Here, we report that alpha(E)-catenin expression in HEK293 cells activates the SRE.L transcriptional reporter, a reporter containing the isolated SRF-binding site, and a stably integrated SRE.L reporter in fibroblasts. alpha-Catenin-induced reporter activity appears only partly dependent on RhoA GTPase and Rho kinase function. alpha-Catenin expression has no effect on RhoA activation or localization, and alpha-catenin-induced SRE.L reporter activation is insensitive to the actin-modulating agent latrunculin B. Ectopic alpha-catenin expression was not sufficient to induce actin filament assembly as measured by stress fiber formation. SRE.L reporter is activated by the C-terminal approximately 300 residue region of alpha(E)-catenin. These results suggest induction of SRF-mediated transcription by alpha(E)-catenin either downstream of RhoA or via a parallel pathway.
Assuntos
Rim/metabolismo , Fator de Resposta Sérica/metabolismo , Ativação Transcricional/fisiologia , alfa Catenina/metabolismo , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Linhagem Celular , Humanos , Estrutura Terciária de Proteína , Transdução de Sinais/fisiologiaRESUMO
Transforming growth factor (TGF)-beta1 activity has been shown to increase vascular endothelial barrier permeability, which is believed to precede several pathologic conditions, including pulmonary edema and vessel inflammation. In endothelial monolayers, TGF-beta1 increases permeability, and a number of studies have demonstrated the alteration of cell-cell contacts by TGF-beta1. We hypothesized that focal adhesion complexes also likely contribute to alterations in endothelial permeability. We examined early signal transduction events associated with rapid changes in monolayer permeability and the focal adhesion complex of bovine pulmonary artery endothelial cells. Western blotting revealed rapid tyrosine phosphorylation of focal adhesion kinase (FAK) and Src kinase in response to TGF-beta1; inhibition of both of these kinases using pp2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), ameliorates TGF-beta1-induced monolayer permeability. Activation of FAK/Src requires activation of the epidermal growth factor receptor downstream of the TGF-beta receptors, and is blocked by the epidermal growth factor receptor inhibitor AG1478. Immunohistochemistry showed that actin and the focal adhesion proteins paxillin, vinculin, and hydrogen peroxide-inducible clone-5 (Hic-5) are rearranged in response to TGF-beta1; these proteins are released from focal adhesion complexes. Rearrangement of paxillin and vinculin by TGF-beta1 is not blocked by the FAK/Src inhibitor, pp2, or by SB431542 inhibition of the TGF-beta type I receptor, anaplastic lymphoma kinase 5; however, pp1 (4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), which inhibits both type I and type II TGF-beta receptors, does block paxillin and vinculin rearrangement. Hic-5 protein rearrangement requires FAK/Src activity. Together, these results suggest that TGF-beta1-induced monolayer permeability involves focal adhesion and cytoskeletal rearrangement through both FAK/Src-dependent and -independent pathways.
Assuntos
Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/enzimologia , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Quinase do Linfoma Anaplásico , Animais , Bovinos , Células Cultivadas , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Receptores ErbB/genética , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Adesões Focais/efeitos dos fármacos , Adesões Focais/metabolismo , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Paxilina/metabolismo , Fosfotirosina/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Receptores Proteína Tirosina Quinases , Ativação Transcricional/efeitos dos fármacos , Vinculina/metabolismoRESUMO
Fibroblasts play a major role in tissue repair and remodeling. Their differentiation into myofibroblasts, marked by increased expression of smooth muscle-specific alpha-actin (alpha-SMA), is believed to be important in wound healing and fibrosis. We have recently described a role for MK2 in this phenotypic differentiation in culture. In this article, we demonstrate that MK2 also regulates myofibroblasts in vivo. Disruption of MK2 in mice prevented myofibroblast formation in a model of pulmonary fibrosis. However, MK2 disruption and consequent lack of myofibroblast formation exacerbated fibrosis rather than ameliorated it as previously postulated. When mice lacking MK2 (MK2-/-) were exposed to bleomycin, more collagen accumulated and more fibroblasts populated fibrotic regions in their lungs than in similarly treated wild-type mice. While there were many vimentin-positive cells in the bleomycin-treated MK2-/- mouse lungs, few alpha-SMA-positive cells were observed in these lungs compared with wild-type mouse lungs. siRNA against MK2 reduced alpha-SMA expression in wild-type mouse embryonic fibroblasts (MEF), consistent with its suppression in MK2-/- MEF. On the other hand expressing constitutively active MK2 in MK2-/- MEF significantly increased alpha-SMA expression. MK2-/-MEF proliferated at a faster rate and produced more collagen; however, they migrated at a slower rate than wild-type MEF. Overexpressing phosphomimicking HSP27, a target of MK2, did not reverse the effect of MK2 disruption on fibroblast migration. MK2 disruption did not affect Smad2 activation by transforming growth factor-beta. Thus, MK2 appears to mediate myofibroblast differentiation, and inhibiting that differentiation might contribute to fibrosis rather than protect against it.
Assuntos
Diferenciação Celular/genética , Fibroblastos/enzimologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mioblastos/enzimologia , Proteínas Serina-Treonina Quinases/genética , Fibrose Pulmonar/genética , Animais , Fibroblastos/citologia , Fibroblastos/patologia , Predisposição Genética para Doença , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mioblastos/citologia , Mioblastos/patologia , Proteínas Serina-Treonina Quinases/deficiência , Fibrose Pulmonar/enzimologia , Fibrose Pulmonar/patologiaRESUMO
Fibroblasts play a major role in processes such as wound repair, scarring, and fibrosis. Differentiation into myofibroblasts, characterized by upregulation of smooth muscle alpha-actin (smalpha) in response to profibrotic agents such as TGFbeta is believed to be an important step in fibrosis. Therefore, elucidating mechanisms of myofibroblast differentiation might reveal novel targets in treating diseases such as idiopathic pulmonary fibrosis (IPF). MK2 is a kinase substrate of p38 MAP kinase that mediates some effects of p38 activation on the actin cytoskeleton. Using mouse embryonic fibroblasts (MEF) from MK2 knockout (MK2(-/-)) mice, we demonstrate that disrupting expression of MK2 expression reduces filamentous actin and stress fibers. It also causes MK2(-/-) MEF to express less smalpha than their corresponding wild-type (WT) MEF at baseline and in response to TGFbeta. Furthermore, TGFbeta causes downregulation of smalpha in MK2(-/-) MEF, instead of upregulation observed in WT MEF. Expression of other fibroblast markers, such as collagen, is not altered in MK2(-/-) MEF. Our results further suggest that downregulation of smalpha in MK2(-/-) MEF is not due to lack of activation of serum responsive promoter elements, but probably due to reduced smalpha message stability in these cells. These results indicate that MK2 plays a key role in regulation of smalpha expression, and that targeting MK2 might present a therapeutic approach in managing conditions such as pulmonary fibrosis.
Assuntos
Actinas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Proteínas Quinases/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Actinas/genética , Animais , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fibroblastos/citologia , Fibroblastos/metabolismo , Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Immunoblotting , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Luciferases/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso/citologia , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Piridinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Elemento de Resposta Sérica/genética , Transfecção , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Xanthine oxidoreductase (XOR) plays a prominent role in acute lung injury because of its ability to generate reactive oxygen species. We investigated the role of XOR in ventilator-induced lung injury (VILI). Male C57BL/6J mice were assigned to spontaneous ventilation (sham) or mechanical ventilation (MV) with low (7 ml/kg) and high tidal volume (20 ml/kg) for 2 h after which lung XOR activity and expression were measured and the effect of the specific XOR inhibitor allopurinol on pulmonary vascular leakage was examined. In separate experiments, rat pulmonary microvascular endothelial cells (RPMECs) were exposed to cyclic stretch (5% and 18% elongation, 20 cycles/min) for 2 h before intracellular XOR activity measurement. Lung XOR activity was significantly increased at 2 h of MV without changes in XOR expression. There was evidence of p38 MAP kinase, ERK1/2, and ERK5 phosphorylation, but no change in JNK phosphorylation. Evans blue dye extravasation and bronchoalveolar lavage protein concentration were significantly increased in response to MV, changes that were significantly attenuated by pretreatment with allopurinol. Cyclic stretch of RPMECs also caused MAP kinase phosphorylation and a 1.7-fold increase in XOR activity, which was completely abrogated by pretreatment of the cells with specific MAP kinase inhibitors. We conclude that XOR enzymatic activity is significantly increased by mechanical stress via activation of p38 MAP kinase and ERK and plays a critical role in the pathogenesis of pulmonary edema associated with VILI.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Pneumopatias/enzimologia , Sistema de Sinalização das MAP Quinases , Síndrome do Desconforto Respiratório/enzimologia , Estresse Mecânico , Xantina Oxidase/metabolismo , Animais , Permeabilidade Capilar , Endotélio Vascular/metabolismo , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Pulmão/metabolismo , Pneumopatias/etiologia , Pneumopatias/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Circulação Pulmonar/fisiologia , Síndrome do Desconforto Respiratório/etiologia , Síndrome do Desconforto Respiratório/metabolismo , Transcrição Gênica , Ventiladores Mecânicos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Hypoxia alters the barrier function of the endothelial cells that line the pulmonary vasculature, but underlying biophysical mechanisms remain unclear. Using rat pulmonary microvascular endothelial cells (RPMEC) in culture, we report herein changes in biophysical properties, both in space and in time, that occur in response to hypoxia. We address also the molecular basis of these changes. At the level of the single cell, we measured cell stiffness, the distribution of traction forces exerted by the cell on its substrate, and spontaneous nanoscale motions of microbeads tightly bound to the cytoskeleton (CSK). Hypoxia increased cell stiffness and traction forces by a mechanism that was dependent on the activation of Rho kinase. These changes were followed by p38-mediated decreases in spontaneous bead motions, indicating stabilization of local cellular-extracellular matrix (ECM) tethering interactions. Cells overexpressing phospho-mimicking small heat shock protein (HSP27-PM), a downstream effector of p38, exhibited decreases in spontaneous bead motions that correlated with increases in actin polymerization in these cells. Together, these findings suggest that hypoxia differentially regulates endothelial cell contraction and cellular-ECM adhesion.
Assuntos
Hipóxia/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Mucosa Respiratória/metabolismo , Resistência à Tração/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Transporte Biológico/fisiologia , Fenômenos Biofísicos , Biofísica , Adesão Celular/fisiologia , Células Cultivadas , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Proteínas de Choque Térmico HSP27 , Proteínas de Choque Térmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Microesferas , Proteínas de Neoplasias/metabolismo , Fosforilação , Ratos , Mucosa Respiratória/citologia , Quinases Associadas a rhoRESUMO
In the present work, we tested the hypothesis that serotonin (5-hydroxytryptamine = 5-HT) might activate the extracellular signal-regulated kinase (ERK) pathway in human peripheral blood mononuclear cells (PBMC). PBMC were maintained in culture for 72 hrs at 37 degrees C prior to the addition of 5-HT. Our results showed an increase in ERK activation by 5-HT with a peak effect at 30 min and maximal stimulation with 5-HT at 1microM. This activation of ERK did not occur in adherent monocytes suggesting that the effect was on lymphocytes. In addition, p38 MAP kinase was not activated under these conditions. The effect of 5-HT on ERK activation appeared to be mediated through the activation of 5-HT1A receptors since similar results were obtained with R-+-8-hydroxy-DPAT, a selective 5-HT1A receptor agonist and WAY100635, a selective 5-HT1A receptor antagonist, reversed the 5-HT and the R-+-8-hydroxy-DPAT effects. Results from Western blot analysis confirmed the presence of 5-HT1A receptors on the PBMC. A 5-HT2A antagonist, ketanserin, and a 5-HT transport inhibitor, fluoxetine, both failed to block the activation of ERK by 5-HT. Our results indicate that 5-HT activates ERK, but not p38, MAP kinase of human PBMC via a 5-HT1A receptor.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Leucócitos Mononucleares/efeitos dos fármacos , Agonistas do Receptor 5-HT1 de Serotonina , Serotonina/farmacologia , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Fluoxetina/farmacologia , Humanos , Fatores Imunológicos/farmacologia , Ketanserina/farmacologia , Leucócitos Mononucleares/enzimologia , Piperazinas/farmacologia , Piridinas/farmacologia , Receptor 5-HT1A de Serotonina/metabolismoRESUMO
Nitric oxide (NO) has an important role in modulating the pulmonary vascular tone. NO acts, in part, by stimulating soluble guanylate cyclase (sGC) to synthesize the intracellular second messenger cyclic GMP. In vascular smooth muscle cells, sGC is a heterodimer composed of alpha1 and beta1 subunits. The objective of this study was to test whether oxygen concentration regulates sGC expression in cultured rat pulmonary artery smooth muscle cells (rPaSMC). rPaSMC were exposed to 0, 3, and 20% oxygen for 1-48 h, and sGC subunit mRNA levels were measured. Compared with rPaSMC exposed to 20% oxygen, sGC alpha1 and beta1 subunit mRNA levels were markedly decreased in rPaSMC exposed to 0% and 3% oxygen. The decrease in sGC subunit mRNA levels in hypoxic rPaSMC was detected as early as 6 h of exposure. Compared with rPaSMC exposed to 20% oxygen, exposure of rPaSMC to 3% oxygen progressively decreased sGC subunit protein levels at 24 and 48 h. There was also a 30% and 50% decrease in sGC enzyme activity in cells exposed to hypoxia for 24 and 48 h (P < 0.05 and P < 0.001, respectively, as compared with cells maintained in normoxia). These results demonstrate that hypoxia decreases sGC expression in cultured pulmonary artery smooth muscle cells and suggest that, in hypoxic vascular smooth muscle, decreased cyclic GMP synthesis may limit the vasodilator response to NO.
Assuntos
Guanilato Ciclase/metabolismo , Hipóxia/metabolismo , Miócitos de Músculo Liso/metabolismo , Subunidades Proteicas/metabolismo , Artéria Pulmonar/anatomia & histologia , Animais , Células Cultivadas , Regulação Enzimológica da Expressão Gênica , Guanilato Ciclase/genética , Concentração de Íons de Hidrogênio , Miócitos de Músculo Liso/citologia , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
Remodeling of the airway smooth muscle (ASM) cell has been proposed to play an important role in airway hyperresponsiveness. Using a functional assay, we have assessed remodeling of the cultured rat ASM cell and the role of heat shock protein (HSP) 27 in that process. To probe remodeling dynamics, we measured spontaneous motions of an individual Arg-Gly-Asp-coated microbead that was anchored to the cytoskeleton. We reasoned that the bead could not move unless the microstructure to which it is attached rearranged; if so, then its mean square displacement (MSD) would report ongoing internal reorganizations over time. Each bead displayed a random, superdiffusive motion; MSD increased with time as approximately t(1.7), whereas an exponent of unity would be expected for a simple passive diffusion. Increasing concentrations of cytochalasin-D or latrunculin-A caused marked increases in the MSD, whereas colchicine did not. Treatments with PDGF or IL-1beta, but not transforming growth factor-beta, caused decreases in the MSD, the extent of which rank-ordered with the relative potency of these agents in eliciting the phosphorylation of HSP27. The chemical stressors anisomycin and arsenite each increased the levels of HSP27 phosphorylation and, at the same time, decreased bead motions. In particular, arsenite prevented and even reversed the effects of cytochalasin-D on bead motions. Finally, ASM cells overexpressing phospho-mimicking human HSP27, but not wild-type or phosphorylation-deficient HSP27, exhibited decreases in bead motions that were comparable to the arsenite response. Taken together, these results show that phosphorylated HSP27 favors reduced bead motions that are probably due to stabilization of the actin cytoskeleton.
Assuntos
Citoesqueleto/fisiologia , Proteínas de Choque Térmico/fisiologia , Miócitos de Músculo Liso/fisiologia , Traqueia/fisiologia , Animais , Anisomicina/farmacologia , Arsenitos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Células Cultivadas , Materiais Revestidos Biocompatíveis , Citocalasina D/farmacologia , Citoesqueleto/efeitos dos fármacos , Difusão , Feminino , Substâncias de Crescimento/farmacologia , Proteínas de Choque Térmico/metabolismo , Interleucina-1/farmacologia , Toxinas Marinhas/farmacologia , Microesferas , Movimento (Física) , Miócitos de Músculo Liso/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Oligopeptídeos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tiazóis/farmacologia , Tiazolidinas , Traqueia/citologia , Traqueia/efeitos dos fármacosRESUMO
Hypoxia causes up-regulation and activation of xanthine dehydrogenase/xanthine oxidase (XDH/XO) in vitro and in the lungs in vivo. This up-regulation, and the likely corresponding production of reactive oxygen species, may underlie the pathogenesis of an array of disorders. Thus, compounds that prevent hypoxia-induced increase in XDH/XO activity may provide a therapeutic strategy in such disorders. The antioxidant properties of estrogens have been demonstrated in several studies. However, the effect of these compounds on XDH/XO has not been explored previously. The aim of this study was to investigate the effects of estrogen on hypoxia-induced increase in XDH/XO activity. Rat pulmonary artery microvascular endothelial cells were exposed to normoxia or hypoxia in the presence or absence of 17beta- or 17alpha-estradiol. The XDH/XO enzyme and gene promoter activities were measured in different groups of cells. Hypoxia caused a twofold increase in XDH/XO enzymatic and promoter activity. Either of the estradiol stereoisomers prevented the hypoxia-induced increase in XDH/XO enzymatic activity, but not the promoter activity. ICI 182,780, an antagonist of the estrogen receptor, failed to block the inhibitory effect of estradiol on XDH/XO. In conclusion, 17alpha- and 17beta-estradiol modulate the hypoxia-induced regulation of XDH/XO activity at a posttranscriptional level by a receptor-independent mechanism.
Assuntos
Estradiol/análogos & derivados , Estrogênios/metabolismo , Regulação Enzimológica da Expressão Gênica , Xantina Desidrogenase/metabolismo , Xantina Oxidase/metabolismo , Animais , Antioxidantes/metabolismo , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Endotélio Vascular/patologia , Estradiol/metabolismo , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Fulvestranto , Hipóxia , Microcirculação/citologia , Testes de Precipitina , Regiões Promotoras Genéticas , Artéria Pulmonar/patologia , Processamento Pós-Transcricional do RNA , Ratos , Espécies Reativas de Oxigênio , Regulação para CimaRESUMO
Tobacco smoking has been causally linked to the development of chronic obstructive pulmonary disease. It has been reported that the reactive oxygen species (ROS)- generating enzyme xanthine dehydrogenase/oxidase (XO) is increased in smoking-related stomach ulcers and that gastric mucosal damage caused by tobacco smoke can be blocked by the XO inhibitor allopurinol. In order to test the hypothesis that tobacco may cause the upregulation of XO in the lung, cultured rat pulmonary microvascular endothelial cells were exposed to tobacco smoke condensate (TSC). TSC at a concentration of 20 microg/mL significantly upregulated XO activity after 24 h of exposure. Longer exposure (1 week) to a lower concentration of TSC (2 microg/mL) also caused an increase in XO activity. Unlike hypoxia, TSC treatment did not alter the phosphorylation of XO. However, TSC treatment increased XO mRNA expression and the XO gene promoter activity. Furthermore, actinomycin D blocked the activation of XO by TSC. In conclusion, our results indicate that tobacco smoke condensate causes upregulation of XO transcription and activity.