RESUMO
Adult neural stem cells (NSCs) reside in specialized niches, which hold a balanced number of NSCs, their progeny, and other cells. How niche capacity is regulated to contain a specific number of NSCs remains unclear. Here, we show that ependyma-derived matricellular protein CCN1 (cellular communication network factor 1) negatively regulates niche capacity and NSC number in the adult ventricular-subventricular zone (V-SVZ). Adult ependyma-specific deletion of Ccn1 transiently enhanced NSC proliferation and reduced neuronal differentiation in mice, increasing the numbers of NSCs and NSC units. Although proliferation of NSCs and neurogenesis seen in Ccn1 knockout mice eventually returned to normal, the expanded NSC pool was maintained in the V-SVZ until old age. Inhibition of EGFR signaling prevented expansion of the NSC population observed in CCN1 deficient mice. Thus, ependyma-derived CCN1 restricts NSC expansion in the adult brain to maintain the proper niche capacity of the V-SVZ.
Assuntos
Proteína Rica em Cisteína 61/metabolismo , Neurogênese/fisiologia , Transdução de Sinais , Células-Tronco Adultas/fisiologia , Animais , Encéfalo , Proteína Rica em Cisteína 61/genética , Epêndima/citologia , Epêndima/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismoRESUMO
BackgroundCystein-rich protein 61 (Cyr61/CCN1) is a member of the CCN family of matricellular proteins that has an important role in tissue development and remodeling. However, the role of CCN1 in the pathogenesis of bronchopulmonary dysplasia (BPD) is unknown. Accordingly, we have investigated the effects of CCN1 on a hyperoxia-induced lung injury model in neonatal rats.MethodsIn experiment 1, newborn rats were randomized to room air (RA) or 85% oxygen (O2) for 7 or 14 days, and we assessed the expression of CCN1. In experiment 2, rat pups were exposed to RA or O2 and received placebo or recombinant CCN1 by daily intraperitoneal injection for 10 days. The effects of CCN1 on hyperoxia-induced lung inflammation, alveolar and vascular development, vascular remodeling, and right ventricular hypertrophy (RVH) were observed.ResultsIn experiment 1, hyperoxia downregulated CCN1 expression. In experiment 2, treatment with recombinant CCN1 significantly decreased macrophage and neutrophil infiltration, reduced inflammasome activation, increased alveolar and vascular development, and reduced vascular remodeling and RVH in the hyperoxic animals.ConclusionThese results demonstrate that hyperoxia-induced lung injury is associated with downregulated basal CCN1 expression, and treatment with CCN1 can largely reverse hyperoxic injury.
Assuntos
Anti-Inflamatórios/farmacologia , Displasia Broncopulmonar/prevenção & controle , Proteína Rica em Cisteína 61/farmacologia , Hiperóxia/complicações , Lesão Pulmonar/prevenção & controle , Pulmão/efeitos dos fármacos , Artéria Pulmonar/efeitos dos fármacos , Animais , Animais Recém-Nascidos , Displasia Broncopulmonar/etiologia , Displasia Broncopulmonar/metabolismo , Displasia Broncopulmonar/patologia , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Modelos Animais de Doenças , Hipertrofia Ventricular Direita/etiologia , Hipertrofia Ventricular Direita/prevenção & controle , Inflamassomos/efeitos dos fármacos , Inflamassomos/metabolismo , Pulmão/metabolismo , Pulmão/patologia , Lesão Pulmonar/etiologia , Lesão Pulmonar/metabolismo , Lesão Pulmonar/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Macrófagos/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Infiltração de Neutrófilos/efeitos dos fármacos , Pneumonia/etiologia , Pneumonia/prevenção & controle , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Remodelação Vascular/efeitos dos fármacosAssuntos
Proliferação de Células , Senescência Celular , Proteína Rica em Cisteína 61/metabolismo , Fibroblastos/metabolismo , Traumatismos Cardíacos/metabolismo , Miócitos Cardíacos/metabolismo , Regeneração , Compostos de Anilina/farmacologia , Animais , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Proteína Rica em Cisteína 61/genética , Modelos Animais de Doenças , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Fibrose , Traumatismos Cardíacos/genética , Traumatismos Cardíacos/patologia , Traumatismos Cardíacos/fisiopatologia , Camundongos Knockout , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Comunicação Parácrina , Regeneração/efeitos dos fármacos , Transdução de Sinais , Sulfonamidas/farmacologia , Fatores de Tempo , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
Activation of RhoA, a low molecular-weight G-protein, plays an important role in protecting the heart against ischemic stress. Studies using non-cardiac cells demonstrate that the expression and subsequent secretion of the matricellular protein CCN1 is induced by GPCR agonists that activate RhoA. In this study we determined whether and how CCN1 is induced by GPCR agonists in cardiomyocytes and examined the role of CCN1 in ischemic cardioprotection in cardiomyocytes and the isolated perfused heart. In neonatal rat ventricular myocytes (NRVMs), sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA) and endothelin-1 induced robust increases in CCN1 expression while phenylephrine, isoproterenol and carbachol had little or no effect. The ability of agonists to activate the small G-protein RhoA correlated with their ability to induce CCN1. CCN1 induction by S1P was blocked when RhoA function was inhibited with C3 exoenzyme or a pharmacological RhoA inhibitor. Conversely overexpression of RhoA was sufficient to induce CCN1 expression. To delineate the signals downstream of RhoA we tested the role of MRTF-A (MKL1), a co-activator of SRF, in S1P-mediated CCN1 expression. S1P increased the nuclear accumulation of MRTF-A and this was inhibited by the functional inactivation of RhoA. In addition, pharmacological inhibitors of MRTF-A or knockdown of MRTF-A significantly diminished S1P-mediated CCN1 expression, indicating a requirement for RhoA/MRTF-A signaling. We also present data indicating that CCN1 is secreted following agonist treatment and RhoA activation, and binds to cells where it can serve an autocrine function. To determine the functional significance of CCN1 expression and signaling, simulated ischemia/reperfusion (sI/R)-induced apoptosis was assessed in NRVMs. The ability of S1P to protect against sI/R was significantly reduced by the inhibition of RhoA, ROCK or MRTF-A or by CCN1 knockdown. We also demonstrate that ischemia/reperfusion induces CCN1 expression in the isolated perfused heart and that this functions as a cardioprotective mechanism, evidenced by the significant increase in infarct development in response to I/R in the cardiac specific CCN1 KO relative to control mice. Our findings implicate CCN1 as a mediator of cardioprotection induced by GPCR agonists that activate RhoA/MRTF-A signaling.
Assuntos
Cardiotônicos/metabolismo , Proteína Rica em Cisteína 61/metabolismo , Isquemia Miocárdica/metabolismo , Fatores de Transcrição/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Animais Recém-Nascidos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ventrículos do Coração/citologia , Técnicas In Vitro , Lisofosfolipídeos/farmacologia , Camundongos Knockout , Modelos Biológicos , Isquemia Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Esfingosina/análogos & derivados , Esfingosina/farmacologiaRESUMO
CCN1 is a matricellular protein involved in normal vascular development and tissue repair. CCN1 exhibits cell- and context-dependent activities that are reflective of its tetramodular structure phylogenetically linked to four domains found in various matrix proteins. Here, we show that vitreal fluids from patients with proliferative diabetic retinopathy (PDR) were enriched with a two-module form of CCN1 comprising completely or partially the insulin-like growth factor-binding protein (IGFBP) and von Willebrand factor type C (vWC) domains. The two- and three-module forms comprising, in addition to IGFBP and vWC, the thrombospondin type 1 (TSP1) repeats are CCN1 degradome products by matrix metalloproteinase-2 and -14. The functional significance of CCN1 and its truncated variants was determined in the mouse model of oxygen-induced retinopathy, which simulates neovascular growth associated with PDR and assesses treatment outcomes. In this model, lentivirus-mediated expression of either CCN1 or the IGFBP-vWC-TSP1 form reduced ischemia-induced neovascularization, whereas ectopic expression of the IGFBP-vWC variant exacerbated pathological angiogenesis. The IGFBP-vWC form has potent proangiogenic properties promoting retinal endothelial cell growth, migration, and three-dimensional tubular structure formation, whereas the IGFBP-vWC-TSP1 variant suppressed cell growth and angiogenic gene expression. Both IGFBP-vWC and IGFBP-vWC-TSP1 forms exhibited predictable variations of their domain folding that enhanced their functional potential. These data provide new insights into the formation and activities of CCN1-truncated variants and raise the predictive value of the form containing completely or partially the IGFBP and vWC domains as a surrogate marker of CCN1 activity in PDR distinguishing pathological from physiological angiogenesis.
Assuntos
Proteína Rica em Cisteína 61/metabolismo , Retinopatia Diabética/metabolismo , Células Endoteliais/metabolismo , Neovascularização Patológica/metabolismo , Proteólise , Animais , Biomarcadores/metabolismo , Linhagem Celular , Proteína Rica em Cisteína 61/genética , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Células Endoteliais/patologia , Regulação da Expressão Gênica/genética , Humanos , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/genética , Proteínas de Ligação a Fator de Crescimento Semelhante a Insulina/metabolismo , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Dobramento de Proteína , Estrutura Terciária de Proteína , Ratos , Trombospondina 1/genética , Trombospondina 1/metabolismo , Fator de von Willebrand/genética , Fator de von Willebrand/metabolismoRESUMO
Repair of the lung epithelium after injury is integral to the pathogenesis and outcomes of diverse inflammatory lung diseases. We previously reported that ß-catenin signaling promotes epithelial repair after inflammatory injury, but the ß-catenin target genes that mediate this effect are unknown. Herein, we examined which ß-catenin transcriptional coactivators and target genes promote epithelial repair after inflammatory injury. Transmigration of human neutrophils across cultured monolayers of human lung epithelial cells resulted in a fall in transepithelial resistance and the formation of discrete areas of epithelial denudation ("microinjury"), which repaired via cell spreading by 96 h. In mice treated with intratracheal (i.t.) LPS or keratinocyte chemokine, neutrophil emigration was associated with increased permeability of the lung epithelium, as determined by increased bronchoalveolar lavage (BAL) fluid albumin concentration, which decreased over 3-6 days. Activation of ß-catenin/p300-dependent gene expression using the compound ICG-001 accelerated epithelial repair in vitro and in murine models. Neutrophil transmigration induced epithelial expression of the ß-catenin/p300 target genes Wnt-induced secreted protein (WISP) 1 and cysteine-rich (Cyr) 61, as determined by real-time PCR (qPCR) and immunostaining. Purified neutrophil elastase induced WISP1 upregulation in lung epithelial cells, as determined by qPCR. WISP1 expression increased in murine lungs after i.t. LPS, as determined by ELISA of the BAL fluid and qPCR of whole lung extracts. Finally, recombinant WISP1 and Cyr61 accelerated repair, and Cyr61-neutralizing antibodies delayed repair of the injured epithelium in vitro. We conclude that ß-catenin/p300-dependent expression of WISP1 and Cyr61 is critical for epithelial repair and represents a potential therapeutic target to promote epithelial repair after inflammatory injury.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Proteínas de Sinalização Intercelular CCN/fisiologia , Proteína Rica em Cisteína 61/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Mucosa Respiratória/metabolismo , Migração Transendotelial e Transepitelial , beta Catenina/fisiologia , Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Animais , Proteínas de Sinalização Intercelular CCN/genética , Proteínas de Sinalização Intercelular CCN/metabolismo , Células Cultivadas , Técnicas de Cocultura , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Proteína p300 Associada a E1A/metabolismo , Proteína p300 Associada a E1A/fisiologia , Feminino , Expressão Gênica , Regulação da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Mucosa Respiratória/imunologia , Transdução de Sinais , beta Catenina/metabolismoRESUMO
The transcription factor FoxM1 is over-expressed in most human malignancies. Although it is evident that FoxM1 has critical functions in tumour development and progression, the mechanisms by which FoxM1 participates in those processes are not understood. Here, we describe an essential role of FoxM1 in the regulation of oxidative stress that contributes to malignant transformation and tumour cell survival. We identify a negative feedback loop involving FoxM1 that regulates reactive oxygen species (ROS) in proliferating cells. We show that induction of FoxM1 by oncogenic Ras requires ROS. Elevated FoxM1, in turn, downregulates ROS levels by stimulating expression of ROS scavenger genes, such as MnSOD, catalase and PRDX3. FoxM1 depletion sensitizes cells to oxidative stress and increases oncogene-induced premature senescence. Moreover, tumour cells expressing activated AKT1 are 'addicted' to FoxM1, as they require continuous presence of FoxM1 for survival. Together, our results identify FoxM1 as a key regulator of ROS in dividing cells, and provide insights into the mechanism how tumour cells use FoxM1 to control oxidative stress to escape premature senescence and apoptosis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Estresse Oxidativo , Animais , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Proteína Forkhead Box M1 , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Genes ras , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The remarkable regenerative capability of the liver has long been appreciated. Upon significant loss of liver tissue, the remnant liver can grow rapidly to restore the original liver mass through a combination of hepatocyte proliferation and hypertrophy to maintain homeostasis. Experimentally, 2/3 partial hepatectomy in mice has been used extensively as a model to dissect the molecular mechanism of liver regeneration and the genetic networks involved. Herein, we describe the protocols for partial hepatectomy and analyses of pertinent CCN protein functions.
Assuntos
Hepatectomia , Regeneração Hepática , Camundongos , Animais , Hepatectomia/métodos , Hepatócitos/metabolismo , Fígado/cirurgia , Hiperplasia , Proliferação de CélulasRESUMO
Vascular stiffness is an independent predictor of human vascular diseases and is linked to ischemia, diabetes, high blood pressure, hyperlipidemia, and/or aging. Blood vessel stiffening increases owing to changes in the microscale architecture and/or content of extracellular, cytoskeletal, and nuclear matrix proteins. These alterations, while best appreciated in large blood vessels, also gradually occur in the microvasculature and play an important role in the initiation and progression of numerous microangiopathies including diabetic retinopathy. Although macroscopic measurements of arterial stiffness by pulse wave velocity are often used for clinical diagnosis, stiffness changes of intact microvessels and their causative factors have not been characterized. Herein, we describe the use of atomic force microscopy (AFM) to determine stiffness of mouse retinal capillaries and assess its regulation by the cellular communication network (CCN) 1, a stiffness-sensitive gene-encoded matricellular protein. AFM yields reproducible measurements of retinal capillary stiffness in lightly fixed freshly isolated retinal flat mounts. AFM measurements also show significant changes in compliance properties of the retinal microvasculature of mice with endothelial-specific deletion of CCN1, indicating that CCN1 expression, or lack thereof, affects the mechanical properties of microvascular cells in vivo. Thus, AFM has the force sensitivity and the spatial resolution necessary to measure the local modulus of retinal capillaries in situ and eventually to investigate microvascular compliance heterogeneities as key components of disease pathogenesis.
Assuntos
Análise de Onda de Pulso , Doenças Vasculares , Camundongos , Humanos , Animais , Microscopia de Força Atômica , Retina/metabolismo , Endotélio , Microvasos , Doenças Vasculares/metabolismoRESUMO
CCN1 (CYR61) is a matricellular protein that is highly expressed at sites of inflammation and wound repair. In these contexts, CCN1 can modify the activities of specific cytokines, enabling TNF-alpha to be cytotoxic without blocking NF-kappaB activity and enhancing the apoptotic activity of Fas ligand and TRAIL. In this paper, we show that CCN1 supports the adhesion of macrophages through integrin alpha(M)beta(2) and syndecan-4, activates NFkappaB-mediated transcription, and induces a proinflammatory genetic program characteristic of classically activated M1 macrophages that participates in Th1 responses. The effects of CCN1 include upregulation of cytokines (TNF-alpha, IL-1alpha, IL-1beta, IL-6, and IL-12b), chemokines (MIP-1alpha; MCP-3; growth-related oncogenes 1 and 2; and inflammatory protein 10), and regulators of oxidative stress and complement (inducible NO synthase and C3) and downregulation of specific receptors (TLR4 and IL-10Rbeta) and anti-inflammatory factors (TGF-beta1). CCN1 regulates this genetic program through at least two distinct mechanisms: an immediate-early response resulting from direct activation of NF-kappaB by CCN1, leading to the synthesis of cytokines including TNF-alpha and inflammatory protein 10; and a delayed response resulting from CCN1-induced TNF-alpha, which acts as an autocrine/paracrine mediator to activate the expression of other cytokines including IL-1beta and IL-6. These results identify CCN1 as a novel component of the extracellular matrix that activates proinflammatory genes in macrophages, implicating its role in regulating macrophage function during inflammation.
Assuntos
Proteína Rica em Cisteína 61/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Regulação da Expressão Gênica/imunologia , Mediadores da Inflamação/fisiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/patologia , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Linhagem Celular , Linhagem Celular Transformada , Linhagem Celular Tumoral , Quimiocinas/biossíntese , Quimiocinas/genética , Citocinas/biossíntese , Citocinas/genética , Mediadores da Inflamação/metabolismo , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Antígeno de Macrófago 1/fisiologia , Macrófagos Peritoneais/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , NF-kappa B/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Sindecana-4/fisiologiaRESUMO
CCN1 (CYR61) is a dynamically expressed, multifunctional matricellular protein that plays essential roles in cardiovascular development during embryogenesis, and regulates inflammation, wound healing and fibrogenesis in the adult. Aberrant CCN1 expression is associated with myriad pathologies, including various cancers and diseases associated with chronic inflammation. CCN1 promotes diverse and sometimes opposing cellular responses, which can be ascribed, as least in part, to disparate activities mediated through its direct binding to distinct integrins in different cell types and contexts. Accordingly, CCN1 promotes cell proliferation, survival and angiogenesis by binding to integrin α(v)ß(3), and induces apoptosis and senescence through integrin α(6)ß(1) and heparan sulfate proteoglycans. The ability of CCN1 to trigger the accumulation of a robust and sustained level of reactive oxygen species underlies some of its unique activities as a matrix cell-adhesion molecule. Emerging studies suggest that CCN1 might be useful as a biomarker or therapeutic target in certain diseases.
Assuntos
Proteína Rica em Cisteína 61/fisiologia , Modelos Biológicos , Animais , Adesão Celular/fisiologia , Movimento Celular/fisiologia , Proliferação de Células , Senescência Celular , Proteína Rica em Cisteína 61/química , Proteína Rica em Cisteína 61/genética , DNA/biossíntese , Desenvolvimento Embrionário/genética , Feminino , Regulação da Expressão Gênica , Integrinas/metabolismo , Camundongos , Neoplasias/genética , Neoplasias/patologia , Gravidez , Espécies Reativas de Oxigênio/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
Following inflammatory injury in the liver, neutrophils quickly infiltrate the injured tissue to defend against microbes and initiate the repair process; these neutrophils are short lived and rapidly undergo apoptosis. Hepatic stellate cells (HSCs) are the principal precursor cells that transdifferentiate into myofibroblast-like cells, which produce a large amount of extracellular matrix that promotes repair but can also lead to fibrosis if the injury becomes chronic. The matricellular protein cellular communication network factor 1 (CCN1) acts as a bridging molecule by binding phosphatidylserine in apoptotic cells and integrin αv ß3 in phagocytes, thereby triggering efferocytosis or phagocytic clearance of the apoptotic cells. Here, we show that CCN1 induces liver macrophage efferocytosis of apoptotic neutrophils in carbon tetrachloride (CCl4 )-induced liver injury, leading to the production of activated transforming growth factor (TGF)-ß1, which in turn induces HSC transdifferentiation into myofibroblast-like cells that promote fibrosis development. Consequently, knock-in mice expressing a single amino acid substitution in CCN1 rendering it unable to bind αv ß3 or induce efferocytosis are impaired in neutrophil clearance, production of activated TGF-ß1, and HSC transdifferentiation, resulting in greatly diminished liver fibrosis following exposure to CCl4 . Conclusion: These results reveal the crucial role of CCN1 in stimulating liver macrophage clearance of apoptotic neutrophils, a process that drives HSC transdifferentiation into myofibroblastic cells and underlies fibrogenesis in chronic liver injury.
Assuntos
Células Estreladas do Fígado , Fator de Crescimento Transformador beta1 , Animais , Tetracloreto de Carbono/toxicidade , Células Estreladas do Fígado/metabolismo , Integrinas/metabolismo , Células de Kupffer/metabolismo , Cirrose Hepática/induzido quimicamente , Camundongos , Fosfatidilserinas , Fatores de Crescimento TransformadoresRESUMO
Senescent cells have long been associated with deleterious effects in aging-related pathologies, although recent studies have uncovered their beneficial roles in certain contexts, such as wound healing. We have found that hepatic stellate cells (HSCs) underwent senescence within 2 days after 2/3 partial hepatectomy (PHx) in young (2-3 months old) mice, and the elimination of these senescent cells by using the senolytic drug ABT263 or by using a genetic mouse model impaired liver regeneration. Senescent HSCs secrete IL-6 and CXCR2 ligands as part of the senescence-associated secretory phenotype, which induces multiple signaling pathways to stimulate liver regeneration. IL-6 activates STAT3, induces Yes-associated protein (YAP) activation through SRC family kinases, and synergizes with CXCL2 to activate ERK1/2 to stimulate hepatocyte proliferation. The administration of either IL-6 or CXCL2 partially restored liver regeneration in mice with senescent cell elimination, and the combination of both fully restored liver weight recovery. Furthermore, the matricellular protein central communication network factor 1 (CCN1, previously called CYR61) was rapidly elevated in response to PHx and induced HSC senescence. Knockin mice expressing a mutant CCN1 unable to bind integrin α6ß1 were deficient in senescent cells and liver regeneration after PHx. Thus, HSC senescence, largely induced by CCN1, is a programmed response to PHx and plays a critical role in liver regeneration through signaling pathways activated by IL-6 and ligands of CXCR2.
Assuntos
Células Estreladas do Fígado , Regeneração Hepática , Animais , Hepatectomia , Células Estreladas do Fígado/metabolismo , Interleucina-6/metabolismo , Ligantes , Regeneração Hepática/fisiologia , Camundongos , Receptores de Interleucina-8BRESUMO
Smooth muscle-rich tissues respond to mechanical overload by an adaptive hypertrophic growth combined with activation of angiogenesis, which potentiates their mechanical overload-bearing capabilities. Neovascularization is associated with mechanical strain-dependent induction of angiogenic factors such as CCN1, an immediate-early gene-encoded matricellular molecule critical for vascular development and repair. Here we have demonstrated that mechanical strain-dependent induction of the CCN1 gene involves signaling cascades through RhoA-mediated actin remodeling and the p38 stress-activated protein kinase (SAPK). Actin signaling controls serum response factor (SRF) activity via SRF interaction with the myocardin-related transcriptional activator (MRTF)-A and tethering to a single CArG box sequence within the CCN1 promoter. Such activity was abolished in mechanically stimulated mouse MRTF-A(-/-) cells or upon inhibition of CREB-binding protein (CBP) histone acetyltransferase (HAT) either pharmacologically or by siRNAs. Mechanical strain induced CBP-mediated acetylation of histones 3 and 4 at the SRF-binding site and within the CCN1 gene coding region. Inhibition of p38 SAPK reduced CBP HAT activity and its recruitment to the SRF.MRTF-A complex, whereas enforced induction of p38 by upstream activators (e.g. MKK3 and MKK6) enhanced both CBP HAT and CCN1 promoter activities. Similarly, mechanical overload-induced CCN1 gene expression in vivo was associated with nuclear localization of MRTF-A and enrichment of the CCN1 promoter with both MRTF-A and acetylated histone H3. Taken together, these data suggest that signal-controlled activation of SRF, MRTF-A, and CBP provides a novel connection between mechanical stimuli and angiogenic gene expression.
Assuntos
Proteína de Ligação a CREB/fisiologia , Proteína Rica em Cisteína 61/genética , Proteínas de Ligação a DNA/fisiologia , Histona Acetiltransferases/metabolismo , Proteínas de Fusão Oncogênica/fisiologia , Estresse Mecânico , Transativadores/fisiologia , Acetilação/efeitos dos fármacos , Animais , Sequência de Bases , Western Blotting , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Camundongos , Dados de Sequência Molecular , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Elemento de Resposta Sérica/genética , Transativadores/genética , Transativadores/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The Forkhead box M1 (FoxM1) transcription factor is critical for expression of the genes essential for G(1)/S transition and mitotic progression. To explore the cell cycle regulation of FoxM1, we examined the phosphorylation profile of FoxM1. Here, we show that the phosphorylated status and the activity of FoxM1 increase as cells progress from S to G(2)/M phases. Moreover, dephosphorylation of FoxM1 coincides with exit from mitosis. Using mass spectrometry, we have identified a new conserved phosphorylation site (Ser-251) within the forkhead domain of FoxM1. Disruption of Ser-251 inhibits phosphorylation of FoxM1 and dramatically decreases its transcriptional activity. We demonstrate that the Ser-251 residue is required for CDK1-dependent phosphorylation of FoxM1 as well as its interaction with the coactivator CREB-binding protein (CBP). Interestingly, the transcriptional activity of the S251A mutant protein remains responsive to activation by overexpressed Polo-like kinase 1 (PLK1). Cells expressing the S251A mutant exhibit reduced expression of the G(2)/M phase genes and impaired mitotic progression. Our results demonstrate that the transcriptional activity of FoxM1 is controlled in a cell cycle-dependent fashion by temporally regulated phosphorylation and dephosphorylation events, and that the phosphorylation at Ser-251 is critical for the activation of FoxM1.
Assuntos
Proteína Quinase CDC2/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Sítios de Ligação , Proteína de Ligação a CREB/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Sequência Conservada , Proteína Forkhead Box M1 , Humanos , Fosforilação , Serina/metabolismo , Transcrição GênicaRESUMO
Integrin-mediated cell adhesion to extracellular matrix proteins is known to promote cell survival, whereas detachment from the matrix can cause rapid apoptotic death in some cell types. Contrary to this paradigm, we show that fibroblast adhesion to the angiogenic matrix protein CCN1 (CYR61) induces apoptosis, whereas endothelial cell adhesion to CCN1 promotes cell survival. CCN1 induces fibroblast apoptosis through its adhesion receptors, integrin alpha6beta1 and the heparan sulfate proteoglycan (HSPG) syndecan-4, triggering the transcription-independent p53 activation of Bax to render cytochrome c release and activation of caspase-9 and -3. Neither caspase-8 activity nor de novo transcription or translation is required for this process. These results show that cellular interaction with a specific matrix protein can either induce or suppress apoptosis in a cell type-specific manner and that integrin alpha6beta1-HSPGs can function as receptors to induce p53-dependent apoptosis.
Assuntos
Apoptose/fisiologia , Células Endoteliais/fisiologia , Fibroblastos/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Animais , Caspases/metabolismo , Sobrevivência Celular/fisiologia , Células Cultivadas , Proteína Rica em Cisteína 61 , Humanos , Integrina alfa6beta1/metabolismo , Glicoproteínas de Membrana/metabolismo , Biossíntese de Proteínas , Proteoglicanas/metabolismo , Ratos , Sindecana-4 , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismoRESUMO
Nog1 is a conserved eukaryotic GTPase of the Obg family involved in the biogenesis of 60S ribosomal subunits. Here we report the unique dominant-inhibitory properties of a point mutation in the switch II region of mouse Nog1; this mutation is predicted to restrict conformational mobility of the GTP-binding domain. We show that although the mutation does not significantly affect GTP binding, ectopic expression of the mutant in mouse cells disrupts productive assembly of pre-60S subunits and arrests cell proliferation. The mutant impairs processing of multiple pre-rRNA intermediates, resulting in the degradation of the newly synthesized 5.8S/28S rRNA precursors. Sedimentation analysis of nucleolar preribosomes indicates that defective Nog1 function inhibits the conversion of 32S pre-rRNA-containing complexes to a smaller form, resulting in a drastic accumulation of enlarged pre-60S particles in the nucleolus. These results suggest that conformational changes in the switch II element of Nog1 have a critical importance for the dissociation of preribosome-bound factors during intranucleolar maturation and thereby strongly influence the overall efficiency of the assembly process.
Assuntos
GTP Fosfo-Hidrolases/química , GTP Fosfo-Hidrolases/metabolismo , Sequência de Aminoácidos , Animais , Nucléolo Celular/metabolismo , Centrifugação com Gradiente de Concentração , Guanosina Trifosfato/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Fenótipo , Mutação Puntual/genética , Estrutura Terciária de Proteína , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Ribossomos/metabolismo , Relação Estrutura-AtividadeRESUMO
Expression of the matricellular protein CCN1 (CYR61) is associated with inflammation and is required for successful wound repair. Here, we show that CCN1 binds bacterial pathogen-associated molecular patterns including peptidoglycans of Gram-positive bacteria and lipopolysaccharides of Gram-negative bacteria. CCN1 opsonizes methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa and accelerates their removal by phagocytosis and increased production of bactericidal reactive oxygen species in macrophages through the engagement of integrin αvß3. Mice with myeloid-specific Ccn1 deletion and knock-in mice expressing CCN1 unable to bind αvß3 are more susceptible to infection by S. aureus or P. aeruginosa, resulting in increased mortality and organ colonization. Furthermore, CCN1 binds directly to TLR2 and TLR4 to activate MyD88-dependent signaling, cytokine expression and neutrophil mobilization. CCN1 is therefore a pattern recognition receptor that opsonizes bacteria for clearance and functions as a damage-associated molecular pattern to activate inflammatory responses, activities that contribute to wound healing and tissue repair.
Assuntos
Proteína Rica em Cisteína 61/metabolismo , Proteínas Opsonizantes/metabolismo , Infecções por Pseudomonas/imunologia , Infecções Estafilocócicas/imunologia , Receptores Toll-Like/metabolismo , Animais , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/imunologia , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Técnicas de Introdução de Genes , Técnicas de Silenciamento de Genes , Humanos , Integrina alfaVbeta3/imunologia , Integrina alfaVbeta3/metabolismo , Masculino , Staphylococcus aureus Resistente à Meticilina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Opsonizantes/genética , Moléculas com Motivos Associados a Patógenos/metabolismo , Fagocitose/imunologia , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Células Sf9 , Transdução de Sinais/imunologia , Infecções Estafilocócicas/microbiologia , Receptores Toll-Like/imunologiaRESUMO
Benign hepatosteatosis, affected by lipid uptake, de novo lipogenesis and fatty acid (FA) oxidation, progresses to non-alcoholic steatohepatitis (NASH) on stress and inflammation. A key macronutrient proposed to increase hepatosteatosis and NASH risk is fructose. Excessive intake of fructose causes intestinal-barrier deterioration and endotoxaemia. However, how fructose triggers these alterations and their roles in hepatosteatosis and NASH pathogenesis remain unknown. Here we show, using mice, that microbiota-derived Toll-like receptor (TLR) agonists promote hepatosteatosis without affecting fructose-1-phosphate (F1P) and cytosolic acetyl-CoA. Activation of mucosal-regenerative gp130 signalling, administration of the YAP-induced matricellular protein CCN1 or expression of the antimicrobial peptide Reg3b (beta) peptide counteract fructose-induced barrier deterioration, which depends on endoplasmic-reticulum stress and subsequent endotoxaemia. Endotoxin engages TLR4 to trigger TNF production by liver macrophages, thereby inducing lipogenic enzymes that convert F1P and acetyl-CoA to FA in both mouse and human hepatocytes.
Assuntos
Frutose/farmacologia , Inflamação/metabolismo , Lipogênese/efeitos dos fármacos , Acetilcoenzima A/farmacologia , Animais , Endotoxemia/sangue , Feminino , Frutosefosfatos/farmacologia , Microbioma Gastrointestinal , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Intestinos/efeitos dos fármacos , Lipidômica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/metabolismo , Regeneração/efeitos dos fármacos , Receptores Toll-Like/agonistasRESUMO
The matricellular protein CCN1 (formerly named CYR61) regulates cell adhesion, migration, proliferation, survival, and differentiation through binding to integrin receptors and heparan sulfate proteoglycans. Here we show that Ccn1-null mice are impaired in cardiac valvuloseptal morphogenesis, resulting in severe atrioventricular septal defects (AVSD). Remarkably, haploinsufficiency for Ccn1 also results in delayed formation of the ventricular septum in the embryo and persistent ostium primum atrial septal defects (ASD) in approximately 20% of adults. Mechanistically, Ccn1 is not required for epithelial-to-mesenchymal transformation or cell proliferation and differentiation in the endocardial cushion tissue. However, Ccn1 deficiency leads to precocious apoptosis in the atrial junction of the cushion tissue and impaired gelatinase activities in the muscular component of the interventricular septum at embryonic day 12.5, when fusion between the endocardial cushion tissue and the atrial and ventricular septa occurs, indicating that these defects may underlie the observed AVSD. Moreover, human CCN1 maps to 1p21-p31, the chromosomal location of an AVSD susceptibility gene. Together, these results provide evidence that deficiency in matrix signaling can lead to autosomal dominant AVSD, identify Ccn1(+/-) mice as a genetic model for ostium primum ASD, and implicate CCN1 as a candidate gene for AVSD in humans.