RESUMO
We addressed the heteromerization of the epidermal growth factor receptor (EGFR) with G-protein coupled receptors (GPCR) on the basis of angiotensin-II-receptor-subtype-1(AT1R)-EGFR interaction as proof-of-concept and show its functional relevance during synergistic nuclear information transfer, beyond ligand-dependent EGFR transactivation. Following in silico modelling, we generated EGFR-interaction deficient AT1R-mutants and compared them to AT1R-wildtype. Receptor interaction was assessed by co-immunoprecipitation (CoIP), Förster resonance energy transfer (FRET) and fluorescence-lifetime imaging microscopy (FLIM). Changes in cell morphology, ERK1/2-phosphorylation (ppERK1/2), serum response factor (SRF)-activation and cFOS protein expression were determined by digital high content microscopy at the single cell level. FRET, FLIM and CoIP confirmed the physical interaction of AT1R-wildtype with EGFR that was strongly reduced for the AT1R-mutants. Responsiveness of cells transfected with AT1R-WT or -mutants to angiotensin II or EGF was similar regarding changes in cell circularity, ppERK1/2 (direct and by ligand-dependent EGFR-transactivation), cFOS-expression and SRF-activity. By contrast, the EGFR-AT1R-synergism regarding these parameters was completely absent for in the interaction-deficient AT1R mutants. The results show that AT1R-EGFR heteromerisation enables AT1R-EGFR-synergism on downstream gene expression regulation, modulating the intensity and the temporal pattern of nuclear AT1R/EGFR-information transfer. Furthermore, remote EGFR transactivation, via ligand release or cytosolic tyrosine kinases, is not sufficient for the complete synergistic control of gene expression.
Assuntos
Núcleo Celular , Receptores ErbB , Receptor Tipo 1 de Angiotensina , Receptores ErbB/metabolismo , Humanos , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 1 de Angiotensina/genética , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Fosforilação , Receptores Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas G/genética , Membrana Celular/metabolismo , Angiotensina II/metabolismo , Angiotensina II/farmacologia , Fator de Crescimento Epidérmico/metabolismo , Células HEK293 , Ligação Proteica , Fator de Resposta Sérica/metabolismo , Fator de Resposta Sérica/genéticaRESUMO
The tyrosine kinase receptor EGFR and the G-protein-coupled receptor AT1R induce essential cellular responses, in part via receptor crosstalk with an unknown role in nuclear information transfer and transcription regulation. We investigated whether this crosstalk results in linear, EGFR-mediated nuclear signalling or in parallel, synergistic information transfer leading to qualitative and temporal variations, relevant for gene expression and environment interaction. AT1R and EGFR synergistically activate SRF via the ERK1/2-TCF and actin-MRTF pathways. Synergism, comprised of switch-like and graded single cell response, converges on the transcription factors AP1 and EGR, resulting in synergistic transcriptome alterations, in qualitative (over-additive number of genes), quantitative (over-additive expression changes of individual genes) and temporal (more late onset and prolonged expressed genes) terms. Gene ontology and IPA® pathway analysis indicate prolonged cell stress (e.g. hypoxia-like) and dysregulated vascular biology. Synergism occurs during separate but simultaneous activation of both receptors and during AT1R-induced EGFR transactivation. EGFR and AT1R synergistically regulate gene expression in qualitative, quantitative and temporal terms with (patho)physiological relevance, extending the importance of EGFR-AT1R crosstalk beyond cytoplasmic signalling.
Assuntos
Regulação da Expressão Gênica , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Receptor Cross-Talk , Transdução de Sinais , TranscriptomaRESUMO
MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among others cardiac hypertrophy and atrial fibrillation. The aim of our study was to evaluate the impact of miR-221/222 on cardiac electrical remodeling. Cardiac miR expression was analyzed in a mouse model with altered electrocardiography parameters and severe heart hypertrophy. Next generation sequencing revealed 14 differentially expressed miRs in hypertrophic hearts, with miR-221 and -222 being the strongest regulated miR-cluster. This increase was restricted to cardiomyocytes and not observed in cardiac fibroblasts. Additionally, we evaluated the change of miR-221/222 in vivo in two models of pharmacologically induced heart hypertrophy (angiotensin II, isoprenaline), thereby demonstrating a stimulus-induced increase in miR-221/222 in vivo by angiotensin II but not by isoprenaline. Whole transcriptome analysis by RNA-seq and qRT-PCR validation revealed an enriched number of downregulated mRNAs coding for proteins located in the T-tubule, which are also predicted targets for miR-221/222. Among those, mRNAs were the L-type Ca2+ channel subunits as well as potassium channel subunits. We confirmed that both miRs target the 3'-untranslated regions of Cacna1c and Kcnj5. Furthermore, enhanced expression of these miRs reduced L-type Ca2+ channel and Kcnj5 channel abundance and function, which was analyzed by whole-cell patch clamp recordings or Western blot and flux measurements, respectively. miR-221 and -222 contribute to the regulation of L-type Ca2+ channels as well as Kcnj5 channels and, therefore, potentially contribute to disturbed cardiac excitation generation and propagation. Future studies will have to evaluate the pathophysiological and clinical relevance of aberrant miR-221/222 expression for electrical remodeling.
Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , MicroRNAs/genética , Canais de Potássio/metabolismo , Animais , Canais de Cálcio Tipo L/genética , Cardiomegalia/genética , Cardiomegalia/patologia , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Canais de Potássio/genéticaRESUMO
AIMS/HYPOTHESIS: Obesity causes type 2 diabetes leading to vascular dysfunction and finally renal end-organ damage. Vascular smooth muscle (VSM) EGF receptor (EGFR) modulates vascular wall homeostasis in part via serum response factor (SRF), a major regulator of VSM differentiation and a sensor for glucose. We investigated the role of VSM-EGFR during obesity-induced renovascular dysfunction, as well as EGFR-hyperglycaemia crosstalk. METHODS: The role of VSM-EGFR during high-fat diet (HFD)-induced type 2 diabetes was investigated in a mouse model with inducible, VSM-specific EGFR-knockout (KO). Various structural and functional variables as well as transcriptome changes, in vivo and ex vivo, were assessed. The impact of hyperglycaemia on EGFR-induced signalling and SRF transcriptional activity and the underlying mechanisms were investigated at the cellular level. RESULTS: We show that VSM-EGFR mediates obesity/type 2 diabetes-induced vascular dysfunction, remodelling and transcriptome dysregulation preceding renal damage and identify an EGFR-glucose synergism in terms of SRF activation, matrix dysregulation and mitochondrial function. EGFR deletion protects the animals from HFD-induced endothelial dysfunction, creatininaemia and albuminuria. Furthermore, we show that HFD leads to marked changes of the aortic transcriptome in wild-type but not in KO animals, indicative of EGFR-dependent SRF activation, matrix dysregulation and mitochondrial dysfunction, the latter confirmed at the cellular level. Studies at the cellular level revealed that high glucose potentiated EGFR/EGF receptor 2 (ErbB2)-induced stimulation of SRF activity, enhancing the graded signalling responses to EGF, via the EGFR/ErbB2-ROCK-actin-MRTF pathway and promoted mitochondrial dysfunction. CONCLUSIONS/INTERPRETATION: VSM-EGFR contributes to HFD-induced vascular and subsequent renal alterations. We propose that a potentiated EGFR/ErbB2-ROCK-MRTF-SRF signalling axis and mitochondrial dysfunction underlie the role of EGFR. This advanced working hypothesis will be investigated in mechanistic depth in future studies. VSM-EGFR may be a therapeutic target in cases of type 2 diabetes-induced renovascular disease. DATA AVAILABILITY: The datasets generated during and/or analysed during the current study are available in: (1) share_it, the data repository of the academic libraries of Saxony-Anhalt ( https://doi.org/10.25673/32049.2 ); and (2) in the gene expression omnibus database with the study identity GSE144838 ( https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE144838 ). Graphical abstract.
Assuntos
Aorta/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Angiopatias Diabéticas/genética , Nefropatias Diabéticas/genética , Receptores ErbB/genética , Músculo Liso Vascular/metabolismo , Obesidade/metabolismo , Fator de Resposta Sérica/metabolismo , Actinas/metabolismo , Animais , Aorta/fisiopatologia , Linhagem Celular , Diabetes Mellitus Tipo 2/fisiopatologia , Angiopatias Diabéticas/metabolismo , Angiopatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/fisiopatologia , Dieta Hiperlipídica , Células HEK293 , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/fisiopatologia , Camundongos , Camundongos Knockout , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso , Obesidade/fisiopatologia , Transdução de Sinais , Remodelação Vascular , Quinases Associadas a rho/metabolismoRESUMO
Besides their importance for the vascular tone, vascular smooth muscle cells (VSMC) also contribute to pathophysiological vessel alterations. Various G-protein coupled receptor ligands involved in vascular dysfunction and remodeling can transactivate the epidermal growth factor receptor (EGFR) of VSMC, yet the importance of EGFR transactivation for the VSMC phenotype is incompletely understood. The aims of this study were (i) to characterize further the importance of the VSMC-EGFR for proliferation, migration and marker gene expression for inflammation, fibrosis and reactive oxygen species (ROS) homeostasis and (ii) to test the hypothesis that vasoactive substances (endothelin-1, phenylephrine, thrombin, vasopressin and ATP) rely differentially on the EGFR with respect to the abovementioned phenotypic alterations. In primary, aortic VSMC from mice without conditional deletion of the EGFR, proliferation, migration, marker gene expression (inflammation, fibrosis and ROS homeostasis) and cell signaling (ERK 1/2, intracellular calcium) were analyzed. VSMC-EGFR loss reduced collective cell migration and single cell migration probability, while no difference between the genotypes in single cell velocity, chemotaxis or marker gene expression could be observed under control conditions. EGF promoted proliferation, collective cell migration, chemokinesis and chemotaxis and leads to a proinflammatory gene expression profile in wildtype but not in knockout VSMC. Comparing the impact of five vasoactive substances (all reported to transactivate EGFR and all leading to an EGFR dependent increase in ERK1/2 phosphorylation), we demonstrate that the importance of EGFR for their action is substance-dependent and most apparent for crowd migration but plays a minor role for gene expression regulation.
Assuntos
Movimento Celular , Receptores ErbB/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Trifosfato de Adenosina/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células , Células Cultivadas , Endotelina-1/farmacologia , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/agonistas , Receptores ErbB/deficiência , Receptores ErbB/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibrose , Regulação da Expressão Gênica , Genótipo , Inflamação/genética , Inflamação/metabolismo , Ligantes , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Estresse Oxidativo , Fenótipo , Fenilefrina/farmacologia , Cultura Primária de Células , Transdução de Sinais , Trombina/farmacologia , Fatores de Tempo , Vasopressinas/farmacologiaRESUMO
Inappropriately activated mineralocorticoid receptor (MR) is a risk factor for vascular remodeling with unclear molecular mechanism. Recent findings suggest that post-transcriptional regulation by micro-RNAs (miRs) may be involved. Our aim was to search for MR-dependent miRs in vascular smooth muscle cells (VSMCs) and to explore the underlying molecular mechanism and the pathologic relevance. We detected that aldosteroneviathe MR reduces miR-29bin vivoin murine aorta and in human primary and cultured VSMCs (ED50= 0.07 nM) but not in endothelial cells [quantitative PCR (qPCR), luciferase assays]. This effect was mediated by an increased decay of miR-29b in the cytoplasm with unchanged miR-29 family member or primary-miR levels. Decreased miR-29b led to an increase in extracellular matrix measured by ELISA and qPCR and enhanced VSMC migration in single cell-tracking experiments. Additionally, cell proliferation and the apoptosis/necrosis ratio (caspase/lactate dehydrogenase assay) was modulated by miR-29b. Enhanced VSMC migration by aldosterone required miR-29b regulation. Control experiments were performed with scrambled RNA and empty plasmids, by comparing aldosterone-stimulated with vehicle-incubated cells. Overall, our findings provide novel insights into the molecular mechanism of aldosterone-mediated vascular pathogenesis by identifying miR-29b as a pathophysiologic relevant target of activated MR in VSMCs and by highlighting the importance of miR processing for miR regulation.-Bretschneider, M., Busch, B., Mueller, D., Nolze, A., Schreier, B., Gekle, M., Grossmann, C. Activated mineralocorticoid receptor regulates micro-RNA-29b in vascular smooth muscle cells.
Assuntos
MicroRNAs/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Receptores de Mineralocorticoides/genética , Aldosterona/farmacologia , Animais , Aorta/efeitos dos fármacos , Aorta/metabolismo , Apoptose/genética , Linhagem Celular , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Proliferação de Células/genética , Células Cultivadas , Colágeno/metabolismo , Fibronectinas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Miócitos de Músculo Liso/efeitos dos fármacos , Análise de Sequência com Séries de Oligonucleotídeos , Receptores de Mineralocorticoides/agonistas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacos , Transcriptoma/genéticaRESUMO
Epi dermal growth factor (EGF) receptor (EGFR) is activated by its canonical ligands and transactivated by various vasoactive substances, e.g. angiotensin II (Ang II). Vascular EGFR has been proposed to be involved in vascular tissue homoeostasis and remodelling. Thus, most studies have focused on its role during long-term vascular changes whereas the relevance for acute regulation of vascular function in vivo and ex vivo is insufficiently understood. To investigate the postnatal role of VSMCs (vascular smooth muscle cells) EGFR in vivo and ex vivo, we generated a mouse model with cell-specific and inducible deletion of VSMC EGFR and studied the effect on basal blood pressure, acute pressure response to, among others, Ang II in vivo as well as ex vivo, cardiovascular tissue homoeostasis and vessel morphometry in male mice. In knockout (KO) animals, systolic, diastolic and mean blood pressures were reduced compared with wild-type (WT). Furthermore, Ang II-induced pressure load was lower in KO animals, as was Ang II-induced force development and extracellular-signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation in aortic rings from KO animals. By contrast, we observed no difference in force development during application of serotonin, KCl, endothelin-1 or endothelin-1-induced pressure load in KO animals. In addition, nitric oxide (NO)-mediated vasodilation was not affected. Heart weight (HW) increase and up-regulation of aortic and cardiac expression of Ccl2 (chemoattractant protein-2) and serpinE1 (plasminogen activator inhibitor 1) during the transition from 4- to 10-months of age were prevented by VSMC EGFR KO. We conclude that VSMC EGFR is involved in basal blood pressure homoeostasis and acute pressure response to Ang II, and thereby contributes to maturation-related remodelling.
Assuntos
Angiotensina II , Pressão Sanguínea , Receptores ErbB/deficiência , Hipertensão/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Fatores Etários , Animais , Pressão Sanguínea/efeitos dos fármacos , Cardiomegalia/induzido quimicamente , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Quimiocina CCL2/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Receptores ErbB/genética , Deleção de Genes , Humanos , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/fisiopatologia , Hipertensão/prevenção & controle , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso/efeitos dos fármacos , Fosforilação , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Remodelação Vascular , Vasoconstritores/farmacologiaRESUMO
The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1-MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.
Assuntos
Receptores ErbB/genética , Receptores de Mineralocorticoides/metabolismo , Elementos de Resposta , Fator de Transcrição Sp1/metabolismo , Transcrição Gênica , Aldosterona/farmacologia , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Ratos , Receptores de Mineralocorticoides/química , Transdução de Sinais , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp3/metabolismo , Regulação para CimaRESUMO
PURPOSE OF THE REVIEW: The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase with a wide implication in tumor biology, wound healing and development. Besides acting as a growth factor receptor activated by ligands such as EGF, the EGFR can also be transactivated and thereby mediate cross-talk with different signaling pathways. The aim of this review is to illustrate the Janus-faced function of the EGFR in the vasculature with its relevance for vascular biology and disease. RECENT FINDINGS: Over recent years, the number of identified signaling partners of the EGFR has steadily increased, as have the biological processes in which the EGFR is thought to be involved. Recently, new models have allowed investigation of EGFR effects in vivo, shedding some light on the overall function of the EGFR in the vasculature. At the same time, EGFR inhibitors and antibodies have become increasingly established in cancer therapy, providing potential therapeutic tools for decreasing EGFR signaling. SUMMARY: The EGFR is a versatile signaling pathway integrator associated with vascular homeostasis and disease. In addition to modulating basal vascular tone and tissue homeostasis, the EGFR also seems to be involved in proinflammatory, proliferative, migratory and remodeling processes, with enhanced deposition of extracellular matrix components, thereby promoting vascular diseases such as hypertension or atherosclerosis.
Assuntos
Vasos Sanguíneos/enzimologia , Receptores ErbB/metabolismo , Transdução de Sinais , Animais , Anticorpos/uso terapêutico , Antineoplásicos/uso terapêutico , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Vasos Sanguíneos/fisiopatologia , Células Endoteliais/enzimologia , Receptores ErbB/antagonistas & inibidores , Humanos , Ligantes , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptor Cross-Talk , Transdução de Sinais/efeitos dos fármacos , Doenças Vasculares/tratamento farmacológico , Doenças Vasculares/enzimologiaRESUMO
Alternative splicing is one of the major cellular processes that determine the tissue-specific expression of protein variants. However, it remains challenging to identify physiologically relevant and tissue-selective proteins that are generated by alternative splicing. Hence, we investigated the target spectrum of the splicing factor Rbfox1 in the cardiac muscle context in more detail. By using a combination of in silico target prediction and in-cell validation, we identified several focal adhesion proteins as alternative splicing targets of Rbfox1. We focused on the alternative splicing patterns of vinculin (metavinculin isoform) and paxillin (extended paxillin isoform) and identified both as potential Rbfox1 targets. Minigene analyses suggested that both isoforms are promoted by Rbfox1 due to binding in the introns. Focal adhesions play an important role in the cardiac muscle context, since they mainly influence cell shape, cytoskeletal organization, and cell-matrix association. Our data confirmed that depletion of Rbfox1 changed cardiomyoblast morphology, cytoskeletal organization, and multinuclearity after differentiation, which might be due to changes in alternative splicing of focal adhesion proteins. Hence, our results indicate that Rbfox1 promotes alternative splicing of focal adhesion genes in cardiac muscle cells, which might contribute to heart disease progression, where downregulation of Rbfox1 is frequently observed.
Assuntos
Processamento Alternativo , Adesões Focais , Miócitos Cardíacos , Paxilina , Fatores de Processamento de RNA , Processamento Alternativo/genética , Fatores de Processamento de RNA/metabolismo , Fatores de Processamento de RNA/genética , Adesões Focais/metabolismo , Adesões Focais/genética , Animais , Paxilina/metabolismo , Paxilina/genética , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Camundongos , Vinculina/metabolismo , Vinculina/genética , Humanos , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genéticaRESUMO
The thromboxane A2 receptor (TP) has been shown to play a role in angiotensin II (Ang II)-mediated hypertension and pathological vascular remodeling. To assess the impact of vascular TP on Ang II-induced hypertension, atherogenesis, and pathological aortic alterations, i.e. aneurysms, we analysed Western-type diet-fed and Ang II-infused TPVSMC KO/Ldlr KO, TPEC KO/Ldlr KO mice and their respective wild-type littermates (TPWT/Ldlr KO). These analyses showed that neither EC- nor VSMC-specific deletion of the TP significantly affected basal or Ang II-induced blood pressure or aortic atherosclerotic lesion area. In contrast, VSMC-specific TP deletion abolished and EC-specific TP deletion surprisingly reduced the ex vivo reactivity of aortic rings to the TP agonist U-46619, whereas VSMC-specific TP knockout also diminished the ex vivo response of aortic rings to Ang II. Furthermore, despite similar systemic blood pressure, there was a trend towards less atherogenesis in the aortic arch and a trend towards fewer pathological aortic alterations in Ang II-treated female TPVSMC KO/Ldlr KO mice. Survival was impaired in male mice after Ang II infusion and tended to be higher in TPVSMC KO/Ldlr KO mice than in TPWT/Ldlr KO littermates. Thus, our data may suggest a deleterious role of the TP expressed in VSMC in the pathogenesis of Ang II-induced aortic atherosclerosis in female mice, and a surprising role of the endothelial TP in TP-mediated aortic contraction. However, future studies are needed to substantiate and further elucidate the role of the vascular TP in the pathogenesis of Ang II-induced hypertension, aortic atherosclerosis and aneurysm formation.
Assuntos
Aterosclerose , Hipertensão , Receptores de Tromboxanos , Animais , Feminino , Masculino , Camundongos , Angiotensina II/toxicidade , Aorta , Aterosclerose/induzido quimicamente , Aterosclerose/genética , Aterosclerose/patologia , Hipertensão/induzido quimicamente , Hipertensão/genética , Hipertensão/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Tromboxanos/genéticaRESUMO
The epidermal growth factor receptor (EGFR) belongs to the ErbB-family of receptor tyrosine kinases that are of importance in oncology. During the last years, substantial evidence accumulated for a crucial role of EGFR concerning the action of the angiotensin II type 1 receptor (AT1R) in blood vessels, resulting form AT1R-induced EGFR transactivation. This transactivation occurs through the release of membrane-anchored EGFR-ligands, cytosolic tyrosine kinases, heterocomplex formation or enhanced ligand expression. AT1R-EGFR crosstalk amplifies the signaling response and enhances the biological effects of angiotensin II. Downstream signaling cascades include ERK1/2 and p38 MAPK, PLCγ and STAT. AT1R-induced EGFR activation contributes to vascular remodeling and hypertrophy via e.g. smooth muscle cell proliferation, migration and extracellular matrix production. EGFR transactivation results in increased vessel wall thickness and reduced vascular compliance. AT1R and EGFR signaling pathways are also implicated the induction of vascular inflammation. Again, EGFR transactivation exacerbates the effects, leading to endothelial dysfunction that contributes to vascular inflammation, dysfunction and remodeling. Dysregulation of the AT1R-EGFR axis has been implicated in the pathogenesis of various cardiovascular diseases and inhibition or prevention of EGFR signaling can attenuate part of the detrimental impact of enhanced renin-angiotensin-system (RAAS) activity, highlighting the importance of EGFR for the adverse consequences of AT1R activation. In summary, EGFR plays a critical role in vascular AT1R action, enhancing signaling, promoting remodeling, contributing to inflammation, and participating in the pathogenesis of cardiovascular diseases. Understanding the interplay between AT1R and EGFR will foster the development of effective therapeutic strategies of RAAS-induced disorders.
Assuntos
Doenças Cardiovasculares , Receptor Tipo 1 de Angiotensina , Humanos , Angiotensina II/metabolismo , Receptores ErbB/metabolismo , Inflamação , Receptor Tipo 1 de Angiotensina/metabolismo , TirosinaRESUMO
(1) Background: Obesity is associated with hypertension because of endocrine dysregulation of the adrenergic and the renin-angiotensin-aldosterone systems. The epidermal growth factor receptor (EGFR) is an important signaling hub in the cardiovascular system. In this study, we investigate the role of smooth muscle cell (VSMC) and endothelial cell (EC) EGFRs for blood pressure homeostasis and acute vascular reactivity in vivo. (2) Methods: Mice with deletion of the EGFR in the respective cell type received either a high-fat (HFD) or standard-fat diet (SFD) for 18 weeks. Intravascular blood pressure was measured via a Millar catheter in anesthetized animals upon vehicle load, angiotensin II (AII) and phenylephrine (PE) stimulation. (3) Results: We confirmed that deletion of the EGFR in VSMCs leads to reduced blood pressure and a most probably compensatory heart rate increase. EC-EGFR and VSMC-EGFR had only a minor impact on volume-load-induced blood pressure changes in lean as well as in obese wild-type animals. Regarding vasoactive substances, EC-EGFR seems to have no importance for angiotensin II action and counteracting HFD-induced prolonged blood pressure increase upon PE stimulation. VSMC-EGFR supports the blood pressure response to adrenergic and angiotensin II stimulation in lean animals. The responsiveness to AII and alpha-adrenergic stimulation was similar in lean and obese animals despite the known enhanced activity of the RAAS and the sympathetic nervous system under a high-fat diet. (4) Conclusions: We demonstrate that EGFRs in VSMCs and to a lesser extent in ECs modulate short-term vascular reactivity to AII, catecholamines and volume load in lean and obese animals.
RESUMO
Vascular smooth muscle cells (VSMC) are critical for the vascular tone, but they can also drive the development of vascular diseases when they lose their contractile phenotype and de-differentiate. Previous studies showed that the epidermal growth factor receptor (EGFR) of VSMC is critical for vascular health, but most of the underlying mechanisms by which VSMC-EGFR controls vascular fate have remained unknown. We combined RNA-sequencing and bioinformatics analysis to characterize the effect of EGFR-activation on the transcriptome of human primary VSMC (from different female donors) and to identify potentially affected cellular processes. Our results indicate that the activation of human VSMC-EGFR is sufficient to trigger a phenotypical switch toward a proliferative and inflammatory phenotype. The extent of this effect is nonetheless partly donor-dependent. Our hypothesis-generating study thus provides a first insight into mechanisms that could partly explain variable susceptibilities to vascular diseases in between individuals.
RESUMO
Endothelial cells (EC) are key players in vascular function, homeostasis and inflammation. EC show substantial heterogeneity due to inter-individual variability (e.g. sex-differences) and intra-individual differences as they originate from different organs or vessels. This variability may lead to different responsiveness to external stimuli. Here we compared the responsiveness of female human primary EC from the aorta (HAoEC) and coronary arteries (HCAEC) to Epidermal Growth Factor Receptor (EGFR) activation. EGFR is an important signal integration hub for vascular active substances with physiological and pathophysiological relevance. Our transcriptomic analysis suggested that EGFR activation differentially affects the inflammatory profiles of HAoEC and HCAEC, particularly by inducing a HCAEC-driven leukocyte attraction but a downregulation of adhesion molecule and chemoattractant expression in HAoEC. Experimental assessments of selected inflammation markers were performed to validate these predictions and the results confirmed a dual role of EGFR in these cells: its activation initiated an anti-inflammatory response in HAoEC but a pro-inflammatory one in HCAEC. Our study highlights that, although they are both arterial EC, female HAoEC and HCAEC are distinguishable with regard to the role of EGFR and its involvement in inflammation regulation, what may be relevant for vascular maintenance but also the pathogenesis of endothelial dysfunction.
Assuntos
Vasos Coronários , Células Endoteliais , Humanos , Feminino , Vasos Coronários/metabolismo , Células Endoteliais/metabolismo , Células Cultivadas , Aorta , Receptores ErbB/metabolismo , Inflamação/metabolismo , Endotélio Vascular/metabolismoRESUMO
OBJECTIVE: Pathophysiological effects of the epidermal growth factor receptor (EGFR or ErbB1) include vascular remodeling. EGFR transactivation is proposed to contribute significantly to heterologous signaling and remodeling in vascular smooth muscle cells (VSMC). METHODS AND RESULTS: We investigated the importance of EGFR in primary VSMC from aorta of mice with targeted deletion of the EGFR (EGFR(Δ/Δ VSMC)âVSMC(EGFR-/-) and EGFR(Δ/+ VSMC)âVSMC(EGFR+/-)) and the respective littermate controls (EGFR(+/+ VSMC)âVSMC(EGFR+/+)) with respect to survival, pentose phosphate pathway activity, matrix homeostasis, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and Ca(2+) homeostasis. In VSMC(EGFR-/-), epidermal growth factor-induced signaling was abolished; VSMC(EGFR+/-) showed an intermediate phenotype. EGFR deletion enhanced spontaneous cell death, reduced pentose phosphate pathway activity, disturbed cellular matrix homeostasis (collagen III and fibronectin), and abolished epidermal growth factor sensitivity. In VSMC(EGFR-/-) endothelin-1- or α(1)-adrenoceptor-induced ERK1/2 phosphorylation and the fraction of Ca(2+) responders were significantly reduced, whereas responsive cells showed a significantly stronger Ca(2+) signal. Oxidative stress (H(2)O(2)) induced ERK1/2 activation in VSMC(EGFR+/+) and VSMC(EGFR+/-) but not in VSMC(EGFR-/-). The Ca(2+) signal was enhanced in VSMC(EGFR-/-), similar to purinergic stimulation by ATP. CONCLUSIONS: In conclusion, EGFR was found to be important for basal VSMC homeostasis and ERK1/2 activation by the tested G-protein-coupled receptors or radical stress. Ca(2+) signaling was modulated by EGFR differentially with respect to the fraction of responders and magnitude of the signal. Thus, EGFR seems to be Janus-faced for VSMC biology.
Assuntos
Receptores ErbB/deficiência , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Transdução de Sinais , Trifosfato de Adenosina/metabolismo , Agonistas de Receptores Adrenérgicos alfa 1/farmacologia , Animais , Sinalização do Cálcio , Sobrevivência Celular , Células Cultivadas , Endotelina-1/metabolismo , Receptores ErbB/efeitos dos fármacos , Receptores ErbB/genética , Matriz Extracelular/metabolismo , Genótipo , Homeostase , Peróxido de Hidrogênio/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Oxidantes/farmacologia , Estresse Oxidativo , Via de Pentose Fosfato , Fenótipo , Fenilefrina/farmacologia , Fosforilação , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de TempoRESUMO
Liver diseases are the fourth common death in Europe responsible for about 2 million death per year worldwide. Among the known detrimental causes for liver dysfunction are virus infections, intoxications and obesity. The mineralocorticoid receptor (MR) is a ligand-dependent transcription factor activated by aldosterone or glucocorticoids but also by pathological milieu factors. Canonical actions of the MR take place in epithelial cells of kidney, colon and sweat glands and contribute to sodium reabsorption, potassium secretion and extracellular volume homeostasis. The non-canonical functions can be initiated by inflammation or an altered micro-milieu leading to fibrosis, hypertrophy and remodelling in various tissues. This narrative review summarizes the evidence regarding the role of MR in portal hypertension, non-alcoholic fatty liver disease, liver fibrosis and cirrhosis, demonstrating that inhibition of the MR in vivo seems to be beneficial for liver function and not just for volume regulation. Unfortunately, the underlying molecular mechanisms are still not completely understood. LINKED ARTICLES: This article is part of a themed issue on Emerging Fields for Therapeutic Targeting of the Aldosterone-Mineralocorticoid Receptor Signaling Pathway. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.13/issuetoc.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Receptores de Mineralocorticoides , Aldosterona , Fibrose , Homeostase , Humanos , Cirrose Hepática , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Receptores de Mineralocorticoides/metabolismoRESUMO
In vivo, cells are simultaneously exposed to multiple stimuli whose effects are difficult to distinguish. Therefore, they are often investigated in experimental cell culture conditions where stimuli are applied separately. However, it cannot be presumed that their individual effects simply add up. As a proof-of-principle to address the relevance of transcriptional signaling synergy, we investigated the interplay of the Epidermal Growth Factor Receptor (EGFR) with the Angiotensin-II (AT1R) or the Thromboxane-A2 (TP) receptors in murine primary aortic vascular smooth muscle cells. Transcriptome analysis revealed that EGFR-AT1R or EGFR-TP simultaneous activations led to different patterns of regulated genes compared to individual receptor activations (qualitative synergy). Combined EGFR-TP activation also caused a variation of amplitude regulation for a defined set of genes (quantitative synergy), including vascular injury-relevant ones (Klf15 and Spp1). Moreover, Gene Ontology enrichment suggested that EGFR and TP-induced gene expression changes altered processes critical for vascular integrity, such as cell cycle and senescence. These bioinformatics predictions regarding the functional relevance of signaling synergy were experimentally confirmed. Therefore, by showing that the activation of more than one receptor can trigger a synergistic regulation of gene expression, our results epitomize the necessity to perform comprehensive network investigations, as the study of individual receptors may not be sufficient to understand their physiological or pathological impact.
Assuntos
Receptores ErbB/metabolismo , Músculo Liso Vascular , Miócitos de Músculo Liso , Receptor Tipo 1 de Angiotensina/metabolismo , Tromboxano A2/metabolismo , Angiotensina II/metabolismo , Animais , Receptores ErbB/genética , Regulação da Expressão Gênica , Camundongos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismoRESUMO
The F2-isoprostane 8-iso-PGF2α (also known as 15-F2t-isoprostane, iPF2α-III, 8-epi PGF2α, 15(S)-8-iso-PGF2α, or 8-Isoprostane), a thromboxane A2 receptor (TP) agonist, stable biomarker of oxidative stress, and risk marker of cardiovascular disease, has been proposed to aggravate atherogenesis in genetic mouse models of atherosclerotic vascular disease. Moreover, the TP plays an eminent role in the pathophysiology of endothelial dysfunction, atherogenesis, and cardiovascular disease. Yet it is unknown, how the TP expressed by vascular cells affects atherogenesis or 8-iso-PGF2α-related effects in mouse models of atherosclerosis. We studied Ldlr-deficient vascular endothelial-specific (EC) and vascular smooth muscle cell (VSMC)-specific TP knockout mice (TPECKO/Ldlr KO; TPVSMCKO/Ldlr KO) and corresponding wild-type littermates (TPWT/Ldlr KO). The mice were fed a Western-type diet for eight weeks and received either 8-iso-PGF2α or vehicle infusions via osmotic pumps. Subsequently, arterial blood pressure, atherosclerotic lesion formation, and lipid profiles were analyzed. We found that VSMC-, but not EC-specific TP deletion, attenuated atherogenesis without affecting blood pressure or plasma lipid profiles of the mice. In contrast to a previous report, 8-iso-PGF2α tended to reduce atherogenesis in TPWT/Ldlr KO and TPEC KO/Ldlr KO mice, again without significantly affecting blood pressure or lipid profiles of these mice. However, no further reduction in atherogenesis was observed in 8-iso-PGF2α-treated TPVSMC KO/Ldlr KO mice. Our work suggests that the TP expressed in VSMC but not the TP expressed in EC is involved in atherosclerotic lesion formation in Ldlr-deficient mice. Furthermore, we report an inhibitory effect of 8-iso-PGF2α on atherogenesis in this experimental atherosclerosis model, which paradoxically appears to be related to the presence of the TP in VSMC.
Assuntos
Aterosclerose , Doenças Cardiovasculares , Animais , Aterosclerose/genética , Dinoprosta/análogos & derivados , F2-Isoprostanos , Camundongos , Camundongos Knockout , Fator de Crescimento Placentário , Receptores de Tromboxanos/genética , Tromboxano A2 , TromboxanosRESUMO
The mineralocorticoid receptor (MR), a ligand-activated transcription factor expressed in various cell types (e.g. epithelial cells, neurons, smooth muscle cells, immune cells), plays important roles in neurohumoral, neuronal, cardiovascular, renal and intestinal function. Pathophysiological relevant signaling mechanisms include nongenomic pathways involving the EGF receptor (EGFR). We investigated whether a MR-EGFR colocalization may underlie the functional MR-EGFR interaction by coimmunoprecipitation, fluorescence resonance energy transfer (FRET) and confocal microscopy in a heterologous expression system. EGFR and a small fraction of MR colocalize at the cell membrane, independently of short time exposure (=60min) to receptor ligands. Twenty-four-hour-exposure to saturating concentrations of aldosterone (10nmol/l) resulted in an almost complete nuclear translocation of MR and disappearance of MR-EGFR colocalization. EGFR transactivation is enhanced only after MR stimulation. Inhibition of HSP90 by geldanamycin did not reduce the fraction of MR interacting with EGFR. Disruption of cholesterol-rich membrane domains by cyclodextrin reduced MR-EGFR interaction. In conclusion, a subfraction of MR interacts with EGFR at the plasma membrane in our heterologous expression system, possibly at cholesterol-rich domains, to form a steroid receptor/growth factor receptor signaling module.