RESUMO
Myocardial fibrosis is a characteristic of various cardiomyopathies, and myocardial fibroblasts play a central role in this process. Gelsolin (GSN) is an actin severing and capping protein that regulates actin assembly and may be involved in fibroblast activation. While the role of GSN in mechanical stress-mediated cardiac fibrosis has been explored, its role in myocardial fibrosis in the absence of mechanical stress is not defined. In this study, we investigated the role of GSN in myocardial fibrosis induced by Angiotensin II (Ang II), a profibrotic hormone that is elevated in cardiovascular disease. We utilized mice lacking GSN (Gsn-/- ) and cultured primary adult cardiac fibroblasts (cFB). In vivo, Ang II infusion in mice resulted in significantly less severe myocardial fibrosis in Gsn-/- compared with Gsn+/+ mice, along with diminished activation of the TGFß1-Smad2/3 pathway, and reduced expression of cardiac extracellular matrix proteins (collagen, fibronectin, periostin). Moreover, Gsn-deficient hearts exhibited suppressed activity of the AMPK pathway and its downstream effectors, mTOR and P70S6Kinase, which could contribute to the suppressed TGFß1 activity. In vitro, the Ang II-induced activation of cFBs was reduced in Gsn-deficient fibroblasts evident from decreased expression of αSMA and periostin, diminished actin filament turnover; which also exhibited reduced activity of the AMPK-mTOR pathway, and P70S6K phosphorylation. AMPK inhibition compensated for the loss of GSN, restored the levels of G-actin in Gsn-/- cFBs and promoted activation to myofibroblasts by increasing αSMA and periostin levels. This study reveals a novel role for GSN in mediating myocardial fibrosis by regulating the AMPK-mTOR-P70S6K pathway in cFB activation independent from mechanical stress-induced factors.
Assuntos
Angiotensina II/farmacologia , Fibroblastos/efeitos dos fármacos , Fibrose/patologia , Gelsolina/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/química , Proteínas Quinases Ativadas por AMP/metabolismo , Actinas/metabolismo , Animais , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose/metabolismo , Gelsolina/deficiência , Gelsolina/genética , Homeostase , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/patologia , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Cross-presentation of antigens from dead tumor cells by type 1 conventional dendritic cells (cDC1s) is thought to underlie priming of anti-cancer CD8+ T cells. cDC1 express high levels of DNGR-1 (a.k.a. CLEC9A), a receptor that binds to F-actin exposed by dead cell debris and promotes cross-presentation of associated antigens. Here, we show that secreted gelsolin (sGSN), an extracellular protein, decreases DNGR-1 binding to F-actin and cross-presentation of dead cell-associated antigens by cDC1s. Mice deficient in sGsn display increased DNGR-1-dependent resistance to transplantable tumors, especially ones expressing neoantigens associated with the actin cytoskeleton, and exhibit greater responsiveness to cancer immunotherapy. In human cancers, lower levels of intratumoral sGSN transcripts, as well as presence of mutations in proteins associated with the actin cytoskeleton, are associated with signatures of anti-cancer immunity and increased patient survival. Our results reveal a natural barrier to cross-presentation of cancer antigens that dampens anti-tumor CD8+ T cell responses.
Assuntos
Apresentação Cruzada/imunologia , Gelsolina/metabolismo , Imunidade , Lectinas Tipo C/metabolismo , Neoplasias/imunologia , Receptores Imunológicos/metabolismo , Receptores Mitogênicos/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/metabolismo , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Apresentação Cruzada/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Gelsolina/química , Gelsolina/deficiência , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Imunidade/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Mutação/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Ligação Proteica/efeitos dos fármacos , Análise de SobrevidaRESUMO
Little is known about plasma proteins that can be used as biomarkers for early and late responses to radiation. The purpose of this study was to determine a link between depletion of plasma gelsolin (pGSN) and cell-death as well as inflammatory responses in the lung (one of the tissues known to be radiosensitive) of the same exposed CBA/CaJ mice after exposure to heavy silicon (28Si) ions. To prevent the development of multiple organ dysfunctions, pGSN (an important component of the extracellular actin-scavenging system) is responsible for the removal of actin that is released into the circulation during inflammation and from dying cells. We evaluated the levels of pGSN in plasma collected from groups of mice (5 mice in each) at 1 week (wk) and 1 month (1 mo) after exposure whole body to different doses of 28Si ions, i.e. 0, 0.1, 0.25, or 0.5â¯Gy (2 fractionated exposures, 15 days apart that totaled each selected dose). In the same mouse, the measurements of pGSN levels were coupled with the quantitation of injuries in the lung, determined by (a) the levels of cleaved poly (ADP-ribose) polymerase (cleaved-PARP), a marker of apoptotic cell-death, (b) the levels of activated nuclear factor-kappa B (NF-κB) and selected cytokines, i.e. tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1ß), and IL-6, from tissue-lysates of the lung. Further, the ratio of neutrophils and lymphocytes (N/L) was determined in the same mouse. Our data indicated: (i) the magnitude of pGSN depletion was dependent to radiation dose at both harvest times, (ii) a persistent depletion of pGSN up to 1 mo post-exposure to 0.25 or 0.5â¯Gy of 28Si ions, (iii) an inverse-correlation between pGSN depletion and increased levels of cleaved-PARP, including activated NF-κB/pro-inflammatory cytokines in the lung, and (iv) at both harvest times, statistically significant increases in the N/L ratio in groups of mice exposed to 0.5â¯Gy only. Our findings suggested that depletion in pGSN levels reflects not only the responses to 28Si-ion exposure at both harvest times but also early and late-occurring damage.
Assuntos
Proteínas Sanguíneas/deficiência , Gelsolina/deficiência , Pneumonia/sangue , Silício/toxicidade , Oligoelementos/toxicidade , Animais , Proteínas Sanguíneas/efeitos da radiação , Morte Celular , Gelsolina/sangue , Gelsolina/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos CBA , Pneumonia/induzido quimicamente , Pneumonia/patologiaRESUMO
Evidence for a critical pathophysiological role of aberrant cytoskeletal dynamics is being uncovered in a growing number of neuropsychiatric syndromes. A sedentary lifestyle as well as overt psychopathology is prevalent in patients with the metabolic syndrome. Using mice deficient in gelsolin (Gsn-/-), a crucial actin-severing protein, we here investigated reduced actin turnover as a potential common driver of metabolic disturbances, sedentary behavior, and an anxious/depressive phenotype. Gelsolin deficiency resulted in reduced lifespan. As compared to wildtype controls, Gsn-/- mice (~ 9 weeks) fed a high-fat diet (HFD) over a span of 12 weeks showed increased body weight gain, fat mass, hepatic steatosis, and adipocyte hypertrophy as well as a significantly reduced respiratory quotient. Moreover, increased rigidity of the actin cytoskeleton in mice on HFD induced mRNA expression of Acc1, Acc2, Fasn, and Lipe, key genes involved in fatty acid metabolism in the liver. Glucose tolerance and insulin sensitivity were worsened in Gsn-/- HFD relative to Gsn+/+ HFD mice. Hypertension in Gsn-/- mice was associated with reduced endothelial NO synthase (eNOS) mRNA expression and reduced eNOS protein trafficking to the plasma membrane. Furthermore, acetylcholine-induced cGMP production and relaxation of aortic rings were impaired by actin filament stabilization. Gsn-/- mice on HFD displayed reduced corticosterone concentrations and reduced energy expenditure as compared to Gsn+/+ HFD mice. Moreover, Gsn-/- HFD mice displayed an overall pattern of hypoactive and anxious/depressive-like behavior. In aggregate, our results demonstrate that impaired actin filament dynamics promote the development of key behavioral and physiological aspects of the metabolic syndrome.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto/patologia , Comportamento Sedentário , Adipócitos/patologia , Animais , Comportamento Animal , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Gelsolina/deficiência , Gelsolina/genética , Regulação da Expressão Gênica , Hipertensão/etiologia , Hipertensão/fisiopatologia , Fígado/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/efeitos dos fármacos , Obesidade/complicações , Obesidade/genética , Obesidade/patologia , Aumento de PesoRESUMO
BACKGROUND & AIMS: Cell stress signaling pathways result in phosphorylation of the eukaryotic translation initiation factor 2 subunit alpha (EIF2S1 or EIF2A), which affects regulation of protein translation. Translation reprogramming mitigates stress by activating pathways that result in autophagy and cell death, to eliminate damaged cells. Actin is modified during stress and EIF2A is dephosphorylated to restore homeostasis. It is not clear how actin affects EIF2A signaling. We studied the actin-binding proteins villin 1 (VIL1) and gelsolin (GSN) in intestinal epithelial cells (IECs) to determine whether they respond to cell stress response and affect signaling pathways. METHODS: We performed studies with mice with disruptions in Vil1 and Gsn (double-knockout mice). Wild-type (WT) mice either were or were not (controls) exposed to cell stressors such as tumor necrosis factor and adherent-invasive Escherichia coli. Distal ileum tissues were collected from mice; IECs and enteroids were cultured and analyzed by histology, immunoblots, phalloidin staining, immunohistochemistry, electron microscopy, and flow cytometry. HT-29 cells were incubated with cell stressors such as DTT, IFN, and adherent-invasive E coli or control agents; cells were analyzed by immunoblots and quantitative polymerase chain reaction. Green fluorescent protein and green fluorescent protein tagged mutant EIF2A were expressed from a lentiviral vector. The mouse immunity-related GTPase (IRGM1) was overexpressed in embryonic fibroblasts from dynamin1 like (DNM1L) protein-knockout mice or their WT littermates. IRGM1 was overexpressed in embryonic fibroblasts from receptor interacting serine/threonine kinase 1-knockout mice or their WT littermates. Human IRGM was overexpressed in human epithelial cell lines incubated with the DNM1L-specific inhibitor Mdivi-1. Mitochondria were analyzed by semi-quantitative confocal imaging. We performed immunohistochemical analyses of distal ileum tissues from 6-8 patients with Crohn's disease (CD) and 6-8 individuals without CD (controls). RESULTS: In IECs exposed to cell stressors, EIF2A signaling reduced expression of VIL1 and GSN. However, VIL1 and GSN were required for dephosphorylation of EIF2A and recovery from cell stress. In mouse and human IECs, prolonged, unresolved stress was accompanied by continued down-regulation of VIL1 and GSN, resulting in constitutive phosphorylation of EIF2A and overexpression of IRGM1 (or IRGM), which regulates autophagy. Overexpression of IRGM1 (or IRGM) induced cell death by necroptosis, accompanied by release of damage-associated molecular patterns (DAMPs). In double-knockout mice, constitutive phosphorylation of EIF2A and over-expression of IRGM1 resulted in spontaneous ileitis that resembled human CD in symptoms and histology. Distal ileum tissues from patients with CD had lower levels of VIL1 and GSN, increased phosphorylation of EIF2A, increased levels of IRGM and necroptosis, and increased release of nuclear DAMPs compared with controls. CONCLUSIONS: In studies of intestinal epithelial tissues from patients with CD and embryonic fibroblasts from mice, along with enteroids and human IEC lines, we found that induction of cell stress alters the cytoskeleton in IECs via changes in the actin-binding proteins VIL1 and GSN. Acute changes in actin dynamics increase IEC survival, whereas long-term changes in actin dynamics lead to IEC death and intestinal inflammation. IRGM regulates necroptosis and release of DAMPs to induce gastrointestinal inflammation, linking IRGM activity with CD.
Assuntos
Citoesqueleto de Actina/metabolismo , Doença de Crohn/metabolismo , Células Epiteliais/metabolismo , Gelsolina/metabolismo , Íleo/metabolismo , Mucosa Intestinal/metabolismo , Proteínas dos Microfilamentos/metabolismo , Transdução de Sinais , Estresse Fisiológico , Citoesqueleto de Actina/patologia , Alarminas/metabolismo , Animais , Morte Celular , Sobrevivência Celular , Doença de Crohn/genética , Doença de Crohn/patologia , Modelos Animais de Doenças , Células Epiteliais/patologia , Fator de Iniciação 2 em Eucariotos/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Gelsolina/deficiência , Gelsolina/genética , Células HT29 , Células HeLa , Humanos , Íleo/patologia , Mucosa Intestinal/patologia , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação , Interferência de RNA , Fatores de Tempo , TransfecçãoRESUMO
Adseverin is an actin-severing/capping protein that may contribute to osteoclast differentiation in vitro but its role in bone remodeling of healthy animals is not defined. We analyzed bone and osteoclast structure in adseverin conditional null mice at alveolar and long bone sites. In wild-type and adseverin null mice, as measured by dual-energy X-ray absorptiometry, there were no differences of bone mineral content or bone mineral density, indicating no change of bone metabolism. In tibiae, TRAcP+ osteoclasts were formed in comparable numbers in adseverin null and wild-type mice. Ultrastructural analysis showed normal and similar abundance of ruffled borders, sealing zones, and mitochondria, and with no difference of osteoclast nuclear numbers. In contrast, analyses of long bone showed that in the absence of adseverin osteoclasts were smaller (120 ± 13 vs. 274 ± 19 µm2; p < 0.05), as were nuclear size and the surface area of cytoplasm. The nuclei of adseverin null osteoclasts exhibited more heterochromatin (31 ± 3%) than wild-type cells (8 ± 1%), suggesting that adseverin affects cell differentiation. The data indicate that in healthy, developing tissues, adseverin contributes to the regulation of osteoclast structure but not to bone metabolism in vivo.
Assuntos
Reabsorção Óssea/metabolismo , Osso e Ossos/metabolismo , Gelsolina/deficiência , Osteoclastos/metabolismo , Absorciometria de Fóton/métodos , Animais , Densidade Óssea/fisiologia , Reabsorção Óssea/genética , Diferenciação Celular/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismoRESUMO
Cytoskeleton filaments play an important role in cellular functions such as maintaining cell shape, cell motility, intracellular transport, and cell division. Actin-binding proteins (ABPs) have numerous functions including regulation of actin filament nucleation, elongation, severing, capping, cross linking, and actin monomer sequestration. Gelsolin (GSN) is one of the actin-binding proteins. Gelsolin (GSN) is one of the actin-binding proteins that regulate cell morphology, differentiation, movement, and apoptosis. GSN also regulates cell morphology, differentiation, movement, and apoptosis. In this study, we have used H9c2 cardiomyoblast cell and H9c2-GSN stable clones to understand the roles and mechanisms of GSN overexpression in hypoxia-induced cardiomyoblast cell death. The data show that hypoxia or GSN overexpression induces HIF-1α expression and reduces the expression of survival markers p-Akt and Bcl-2 in H9c2 cardiomyoblast cells. Under hypoxic conditions, GSN overexpression further reduces p-Akt expression and elevates total as well as cleaved GSN levels and HIF-1α levels. In addition, GSN overexpression enhances apoptosis in cardiomyoblasts under hypoxia. Hypoxic challenge further induced activated caspase-3 and cell death that was attenuated after GSN knock down, which implies that GSN is a critical therapeutic target against hypoxia-induced cardiomyoblast cell death.
Assuntos
Apoptose , Gelsolina/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mioblastos/citologia , Miócitos Cardíacos/citologia , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Biomarcadores/metabolismo , Caspase 3/metabolismo , Hipóxia Celular , Linhagem Celular , Gelsolina/deficiência , Gelsolina/genética , Técnicas de Silenciamento de Genes , Humanos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RatosRESUMO
Ion channels involved in cardiac excitation-contraction coupling are linked to the cytoskeleton. Therefore changes in the cytoskeletal actin filaments may influence cardiac membrane currents and electro-mechanical coupling. Depolymerization of actin filaments by gelsolin (gsn) is involved in the organisation of the cytoskeleton by leading to a lower polymerization state. Gsn is activated by Ca(2+) and inhibited by phosphoinositol-bisphosphate (PIP2). Furthermore, gsn has been linked to pathological conditions with reduced contractility like heart failure, amyloidosis and apoptosis. Thus, we hypothesize, that gsn deficiency may change electromechanical properties of freshly isolated ventricular cardiomyocytes. We recorded L-type Ca(2+) current (ICa,L) in whole-cell patch clamp mode in freshly isolated ventricular cardiomyocytes from gsn deficient ((-/-)) and control (gsn(+/+)) mice. Sarcomere shortening was monitored in field-stimulated myocytes from 0.5 Hz to 10 Hz by video microscopy. Shortening-frequency relation, post-rest potentiation and ß-adrenergic stimulation were investigated. ICa,L was increased in gsn(-/-) vs. gsn(+/+) myocytes. Sarcomere shortening amplitude and velocity were enhanced in gsn(-/-) vs. gsn(+/+) at all frequencies. Shortening-frequency relationship showed a biphasic pattern with decay in shortening amplitude between 0.5 and 2 Hz and an increase at higher frequencies in both genotypes. Post-rest characteristics revealed a frequency-dependent decay of post-rest potentiation in gsn(+/+) while it remained stable in gsn(-/-). In gsn(-/-) a reduced response to ß-adrenergic stimulation was observed. Resting sarcomere length was shorter in gsn(-/-) but neither increasing frequency nor ß-adrenergic stimulation induced further decay in any of the genotypes. In summary, gsn deficiency had a profound effect on excitiation-contraction properties and improved systolic function while not affecting diastolic function in unloaded isolated cardiomyocytes. Therefore, gsn mediated effects on contractility may play a role in patients with heart failure and cancer, where gsn levels are known to be elevated.
Assuntos
Gelsolina/fisiologia , Miócitos Cardíacos/fisiologia , Animais , Canais de Cálcio Tipo L/fisiologia , Acoplamento Excitação-Contração , Feminino , Gelsolina/deficiência , Gelsolina/genética , Coração/anatomia & histologia , Masculino , Camundongos Knockout , Sarcômeros/fisiologiaRESUMO
Osteoclast differentiation and function are highly dependent on the assembly and turnover of actin filaments, but little is known about the roles of actin binding proteins in these processes. Adseverin (Ads), a member of the gelsolin superfamily of actin capping and severing proteins, regulates actin filament turnover and can regulate the turnover of cortical actin filaments of chromaffin cells during exocytosis. Using a conditional Ads knockout mouse model, we confirmed our previous finding in cultured cells that Ads plays a role in osteoclastogenesis (OCG) and actin cytoskeletal organization in osteoclasts. Here we show that Ads is required for osteoclast formation and that when alveolar bone resorption is experimentally induced in mice, genetic deletion of Ads prevents osteoclast-mediated bone loss. Further, when Ads-null osteoclasts are cultured, they exhibit defective OCG, disorganized podosome-based actin filament superstructures, and decreased bone resorption. Reintroduction of Ads into Ads-null osteoclast precursor cells restored these osteoclast defects. Collectively, these data demonstrate a unique and osteoclast-specific role for Ads in OCG and osteoclast function.
Assuntos
Perda do Osso Alveolar/fisiopatologia , Diferenciação Celular/fisiologia , Gelsolina/fisiologia , Osteoclastos/metabolismo , Doenças Periodontais/fisiopatologia , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/metabolismo , Perda do Osso Alveolar/genética , Animais , Western Blotting , Diferenciação Celular/genética , Células Cultivadas , Recuperação de Fluorescência Após Fotodegradação , Gelsolina/deficiência , Gelsolina/genética , Camundongos Knockout , Microscopia Confocal , Osteoclastos/citologia , Doenças Periodontais/genética , TransfecçãoRESUMO
Integrins are functionally regulated by "inside-out" signaling, in that stimulus-induced signaling pathways act on the intracellular integrin tail to regulate the activity of the receptor on the outside. Both a change in conformation (affinity) and clustering (avidity/valency) of the receptors occurs, but the mechanisms that regulate inside out signaling are not completely understood. Previously, we identified gelsolin in a proteomics screen to identify proteins involved in inside-out control of integrins using the lymphocytic leukemia cell line L1210. Furthermore, we showed that gelsolin was involved in affinity regulation of ß1 -integrins in the leukemic cell line U937. Here, we examined how gelsolin regulates ß1 -integrin affinity in the leukemia cell line L1210. We show that gelsolin is mainly expressed at the cell membrane and is present near ß1 -integrins. The role for actin polymerization in integrin affinity regulation was examined using the actin modulating agent jasplakinolide, which decreased ß1 -integrin affinity. Similarly, knock-down of gelsolin in L1210 cells also decreased ß1 -integrin affinity and cell adhesion to collagen. These data suggest that increased actin polymerization through gelsolin regulates ß1 -integrin affinity and cell adhesion.
Assuntos
Adesão Celular/fisiologia , Gelsolina/biossíntese , Integrina beta1/metabolismo , Actinas/metabolismo , Animais , Membrana Celular/metabolismo , Gelsolina/antagonistas & inibidores , Gelsolina/deficiência , Gelsolina/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Integrina beta1/biossíntese , Leucemia L1210 , Camundongos , Transdução de SinaisRESUMO
Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.
Assuntos
Neoplasias Colorretais/patologia , Gelsolina/metabolismo , Transdução de Sinais , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Linhagem Celular Tumoral , Fibrinolisina/metabolismo , Gelsolina/deficiência , Gelsolina/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Invasividade Neoplásica , RNA Interferente Pequeno/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
BACKGROUND: During late differentiation, erythroid cells undergo profound changes involving actin filament remodeling. One of the proteins controlling actin dynamics is gelsolin, a calcium-activated actin filament severing and capping protein. Gelsolin-null (Gsn(-/-)) mice generated in a C57BL/6 background are viable and fertile.1 DESIGN AND METHODS: We analyzed the functional roles of gelsolin in erythropoiesis by: (i) evaluating gelsolin expression in murine fetal liver cells at different stages of erythroid differentiation (using reverse transcription polymerase chain reaction analysis and immunohistochemistry), and (ii) characterizing embryonic and adult erythropoiesis in Gsn(-/-) BALB/c mice (morphology and erythroid cultures). RESULTS: In the context of a BALB/c background, the Gsn(-/-) mutation causes embryonic death. Gsn(-/-) embryos show defective erythroid maturation with persistence of circulating nucleated cells. The few Gsn(-/-) mice reaching adulthood fail to recover from phenylhydrazine-induced acute anemia, revealing an impaired response to stress erythropoiesis. In in vitro differentiation assays, E13.5 fetal liver Gsn(-/-) cells failed to undergo terminal maturation, a defect partially rescued by Cytochalasin D, and mimicked by administration of Jasplakinolide to the wild-type control samples. CONCLUSIONS: In BALB/c mice, gelsolin deficiency alters the equilibrium between erythrocyte actin polymerization and depolymerization, causing impaired terminal maturation. We suggest a non-redundant role for gelsolin in terminal erythroid differentiation, possibly contributing to the Gsn(-/-) mice lethality observed in mid-gestation.
Assuntos
Células-Tronco Embrionárias/patologia , Eritrócitos/patologia , Eritropoese/genética , Gelsolina/genética , Fígado/patologia , Actinas/antagonistas & inibidores , Actinas/metabolismo , Anemia/induzido quimicamente , Animais , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Citocalasina D/farmacologia , Depsipeptídeos/farmacologia , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Eritrócitos/efeitos dos fármacos , Eritrócitos/metabolismo , Feto , Gelsolina/deficiência , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fenil-Hidrazinas/toxicidadeRESUMO
Protein-energy wasting (PEW) is increasingly recognized to be a prevalent and significant contributor to the clinical deterioration of patients with chronic kidney disease (CKD). While factors reflecting various aspects of PEW correlate with outcomes in CKD, the mechanism linking PEW and CKD outcomes is not completely understood. Plasma gelsolin (pGSN) is a highly abundant circulating protein that is depleted by inflammatory mediators and mainly produced by muscles. Recent data documenting the prognostic ability of low pGSN levels in hemodialysis patients suggests that circulating pGSN levels incorporate the degree of systemic inflammation and muscle wasting. Therefore, pGSN deficiency appears to be a powerful biomarker and a potential therapeutic target in CKD patients.
Assuntos
Gelsolina/sangue , Falência Renal Crônica/sangue , Atrofia Muscular/metabolismo , Desnutrição Proteico-Calórica/etiologia , Diálise Renal , Actinas/sangue , Biomarcadores , Gelsolina/deficiência , Gelsolina/fisiologia , Humanos , Inflamação/sangue , Mediadores da Inflamação/sangue , Falência Renal Crônica/complicações , Falência Renal Crônica/mortalidade , Falência Renal Crônica/patologia , Falência Renal Crônica/terapia , Lipídeos/sangue , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Atrofia Muscular/etiologia , Diálise Renal/efeitos adversos , Resultado do TratamentoRESUMO
Reorganization of the actin cytoskeleton is necessary for Schwann cell proliferation, migration and for the morphological changes associated with sorting, ensheathing and myelination of axons. Such reorganization requires regulated severing and depolymerization of actin filaments. Gelsolin is an actin filament severing protein expressed in many cell types including Schwann cells. Using Gelsolin knockout mice, we investigated the role of this protein in the myelination and remyelination of the peripheral nervous system. Our results show that although gelsolin is not necessary for developmental myelination, it is required for timely remyelination of the sciatic nerve following crush injury. Gelsolin is necessary for macrophage motility in culture, and its absence is likely to impair the recruitment of macrophages to the injury site.
Assuntos
Gelsolina/metabolismo , Macrófagos/fisiologia , Células de Schwann/fisiologia , Neuropatia Ciática/patologia , Neuropatia Ciática/fisiopatologia , Fatores Etários , Animais , Antígenos de Diferenciação/metabolismo , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citoesqueleto/metabolismo , Gelsolina/deficiência , Marcação In Situ das Extremidades Cortadas/métodos , Macrófagos/ultraestrutura , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Proteínas do Tecido Nervoso/metabolismo , Netrinas , Células de Schwann/ultraestrutura , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
BACKGROUND: Despite intense research efforts, the aetiology and pathogenesis of idiopathic pulmonary fibrosis remain poorly understood. Gelsolin, an actin-binding protein that modulates cytoskeletal dynamics, was recently highlighted as a likely disease modifier through comparative expression profiling and target prioritisation. METHODS: To decipher the possible role of gelsolin in pulmonary inflammation and fibrosis, immunocytochemistry on tissue microarrays of human patient samples was performed followed by computerised image analysis. The results were validated in the bleomycin-induced animal model of pulmonary inflammation and fibrosis using genetically-modified mice lacking gelsolin expression. Moreover, to gain mechanistic insights into the mode of gelsolin activity, a series of biochemical analyses was performed ex vivo in mouse embryonic fibroblasts. RESULTS: Increased gelsolin expression was detected in lung samples of patients with idiopathic interstitial pneumonia as well as in modelled pulmonary inflammation and fibrosis. Genetic ablation of gelsolin protected mice from the development of modelled pulmonary inflammation and fibrosis attributed to attenuated epithelial apoptosis. CONCLUSIONS: Gelsolin expression is necessary for the development of modelled pulmonary inflammation and fibrosis, while the caspase-3-mediated gelsolin fragmentation was shown to be an apoptotic effector mechanism in disease pathogenesis and a marker of lung injury.
Assuntos
Gelsolina/metabolismo , Pneumonia/metabolismo , Fibrose Pulmonar/metabolismo , Adulto , Idoso , Animais , Apoptose , Bleomicina , Modelos Animais de Doenças , Células Epiteliais/patologia , Feminino , Gelsolina/deficiência , Gelsolina/fisiologia , Humanos , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Infiltração de Neutrófilos , Pneumonia/induzido quimicamente , Pneumonia/patologia , Pneumonia/prevenção & controle , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fibrose Pulmonar/prevenção & controle , Mucosa Respiratória/patologiaRESUMO
Gelsolin, a calcium-regulated actin severing and capping protein, is highly expressed in murine and human hearts after myocardial infarction and is associated with progression of heart failure in humans. The biological role of gelsolin in cardiac remodeling and heart failure progression after injury is not defined. To elucidate the contribution of gelsolin in these processes, we randomly allocated gelsolin knockout mice (GSN(-/-)) and wild-type littermates (GSN(+/+)) to left anterior descending coronary artery ligation or sham surgery. We found that GSN(-/-) mice have a surprisingly lower mortality, markedly reduced hypertrophy, smaller late infarct size, less interstitial fibrosis, and improved cardiac function when compared with GSN(+/+) mice. Gene expression and protein analysis identified significantly lower levels of deoxyribonuclease (DNase) I and reduced nuclear translocation and biological activity of DNase I in GSN(-/-) mice. Absence of gelsolin markedly reduced DNase I-induced apoptosis. The association of hypoxia-inducible factor (HIF)-1alpha with gelsolin and actin filaments cleaved by gelsolin may contribute to the higher activation of DNase. The expression pattern of HIF-1alpha was similar to that of gelsolin, and HIF-1alpha was detected in the gelsolin complex by coprecipitation and HIF-1alpha bound to the promoter of DNase I in both gel-shift and promoter activity assays. Furthermore, the phosphorylation of Akt at Ser473 and expression of Bcl-2 were significantly increased in GSN(-/-) mice, suggesting that gelsolin downregulates prosurvival factors. Our investigation concludes that gelsolin is an important contributor to heart failure progression through novel mechanisms of HIF-1alpha and DNase I activation and downregulation of antiapoptotic survival factors. Gelsolin inhibition may form a novel target for heart failure therapy.
Assuntos
Apoptose , Desoxirribonuclease I/metabolismo , Gelsolina/metabolismo , Insuficiência Cardíaca/enzimologia , Infarto do Miocárdio/enzimologia , Miocárdio/enzimologia , Remodelação Ventricular , Citoesqueleto de Actina/metabolismo , Animais , Caspases/metabolismo , Desoxirribonuclease I/genética , Modelos Animais de Doenças , Progressão da Doença , Ativação Enzimática , Fibrose , Gelsolina/deficiência , Gelsolina/genética , Regulação da Expressão Gênica , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Miocárdio/patologia , Regiões Promotoras Genéticas , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , Função Ventricular EsquerdaRESUMO
Mechanical ventilation, an essential life-support modality of patients with acute lung injury (ALI) or the acute respiratory distress syndrome (ARDS), exerts its detrimental effects through largely unknown mechanisms. Gelsolin (GSN), an actin-binding protein and a substrate of caspase-3, was recently shown to play a major role in bleomycin- or lipopolysaccharide-induced lung injury. To dissect a possible role of GSN in the pathogenesis of ventilator-induced lung injury (VILI), genetically modified mice lacking GSN expression and wild-type controls underwent mechanical ventilation with high tidal volumes. GSN was found up-regulated in the airways upon VILI, and its genetic ablation led to almost complete disease protection as manifested by reduced edema formation, reduced lung injury, attenuated epithelial apoptosis, diminished cytokine expression, and impaired neutrophil infiltration. GSN fragmentation was shown to be an effector mechanism in VILI-induced apoptosis, while GSN expression was shown to be necessary for efficient neutrophil infiltration, which was found to be a prerequisite for VILI induction in this model. Therefore, intracellular GSN and GSN-mediated responses were shown to be an important player in the pathogenesis of VILI.
Assuntos
Gelsolina/fisiologia , Ventilação de Alta Frequência/efeitos adversos , Lesão Pulmonar Induzida por Ventilação Mecânica/fisiopatologia , Resistência das Vias Respiratórias , Animais , Apoptose , Líquido da Lavagem Broncoalveolar/citologia , Permeabilidade Capilar , Citocinas/metabolismo , Gelsolina/deficiência , Gelsolina/genética , Pulmão/patologia , Complacência Pulmonar , Camundongos , Camundongos Knockout , Neutrófilos/patologia , Edema Pulmonar/etiologia , Edema Pulmonar/fisiopatologia , Edema Pulmonar/prevenção & controle , Estresse Mecânico , Lesão Pulmonar Induzida por Ventilação Mecânica/etiologia , Lesão Pulmonar Induzida por Ventilação Mecânica/patologia , Lesão Pulmonar Induzida por Ventilação Mecânica/prevenção & controleRESUMO
Acetylation/deactylation of histones is an important mechanism to regulate gene expression and chromatin remodeling. We have previously demonstrated that the HDAC inhibitor trichostatin A (TSA) protects cortical neurons from oxygen/glucose deprivation in vitro which is mediated--at least in part--via the up regulation of gelsolin expression. Here, we demonstrate that TSA treatment dose-dependently enhances histone acetylation in brains of wildtype mice as evidenced by immunoblots of total brain lysates and immunocytochemical staining. Along with increased histone acetylation dose-dependent up regulation of gelsolin protein was observed. Levels of filamentous actin were largely decreased by TSA pre-treatment in brain of wildtype but not gelsolin-deficient mice. When exposed to 1 h filamentous occlusion of the middle cerebral artery followed by reperfusion TSA pre-treated wildtype mice developed significantly smaller cerebral lesion volumes and tended to have improved neurological deficit scores compared to vehicle-treated mice. These protective effects could not be explained by apparent changes in physiological parameters. In contrast to wildtype mice, TSA pre-treatment did not protect gelsolin-deficient mice against MCAo/reperfusion suggesting that enhanced gelsolin expression is an important mechanism by which TSA protects against ischemic brain injury. Our results suggest that HDAC inhibitors such as TSA are a promising therapeutic strategy for reducing brain injury following cerebral ischemia.
Assuntos
Lesões Encefálicas/etiologia , Lesões Encefálicas/metabolismo , Isquemia Encefálica/complicações , Gelsolina/deficiência , Histonas/metabolismo , Acetilação/efeitos dos fármacos , Animais , Lesões Encefálicas/patologia , Lesões Encefálicas/prevenção & controle , Isquemia Encefálica/tratamento farmacológico , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Embrião de Mamíferos , Inibidores Enzimáticos/uso terapêutico , Glucose/deficiência , Ácidos Hidroxâmicos/uso terapêutico , Hipóxia , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfopiruvato Hidratase/metabolismo , RatosRESUMO
Histone acetylation and deacetylation participate in the epigenetic regulation of gene expression. In this paper, we demonstrate that pre-treatment with the histone deacetylation inhibitor trichostatin A (TSA) enhances histone acetylation in primary cortical neurons and protects against oxygen/glucose deprivation, a model for ischaemic cell death in vitro. The actin-binding protein gelsolin was identified as a mediator of neuroprotection by TSA. TSA enhanced histone acetylation of the gelsolin promoter region, and up-regulated gelsolin messenger RNA and protein expression in a dose- and time-dependent manner. Double-label confocal immunocytochemistry visualized the up-regulation of gelsolin and histone acetylation within the same neuron. Together with gelsolin up-regulation, TSA pre-treatment decreased levels of filamentous actin. The neuroprotective effect of TSA was completely abolished in neurons lacking gelsolin gene expression. In conclusion, we demonstrate that the enhancement of gelsolin gene expression correlates with neuroprotection induced by the inhibition of histone deacetylation.
Assuntos
Inibidores Enzimáticos/farmacologia , Gelsolina/deficiência , Glucose/fisiologia , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Hipóxia/patologia , Neurônios/fisiologia , Fármacos Neuroprotetores , Acetilação/efeitos dos fármacos , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Animais , Morte Celular/efeitos dos fármacos , Cromatina/metabolismo , Remoção de Radical Alquila , Feminino , Gelsolina/biossíntese , Gelsolina/genética , Glucose/deficiência , Imunoensaio , Immunoblotting , Imuno-Histoquímica , Microscopia Confocal , Gravidez , RNA Mensageiro/biossíntese , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Regulação para Cima/fisiologiaRESUMO
The Drosophila melanogaster flightless I protein and its homologues in higher eukaryotes (FliI) are conserved members of the gelsolin family of actin-binding proteins. Members of the gelsolin family generally contain three or six copies of a 125-amino-acid residue gelsolin-related repeating unit, and may contain additional domains including the C-terminal villin-related 'headpiece' or N-terminal extensions such as the leucine-rich repeat of the FliI protein. Numerous studies including work done with mouse knockouts for gelsolin, villin and CapG support a role for the family in cytoskeletal actin dynamics. In both fruitfly and mouse, the FliI protein is also essential for early development. Recent studies indicate that supervillin, gelsolin and FliI are involved in intracellular signalling via nuclear hormone receptors including the androgen, oestrogen and thyroid hormone receptors. This unexpected role in signalling has opened a new area in research on the gelsolin family and is providing important new insights into the mechanisms of gene regulation via nuclear receptors.