RESUMO
Pro-inflammatory cytokines are released in septic shock and impair cardiac function via the Jak-STAT pathway. It is well known that sympathetic stimulation leads to coupling of the ß-adrenergic receptor/Gs/adenylyl cyclase, a membrane-bound enzyme that catalyzes the conversion of ATP to cAMP, thereby stimulating protein kinase A (PKA) and ultimately compensating for cardiac dysfunction. The mechanism of such compensation by catecholamine has been traditionally understood as PKA-mediated enforcement of cardiac contractility. We hypothesized that exchange protein activated by cyclic AMP (Epac), a new target of cAMP signaling that functions independently of protein kinase A, also plays a key role in protection against acute stresses or changes in hemodynamic overload. Lipopolysaccharide injection induced cytokine release and severe cardiac dysfunction in mouse. In mouse overexpressing Epac1 in the heart, however, the magnitude of such dysfunction was significantly smaller. Epac1 overexpression inhibited the Jak-STAT pathway, as indicated by decreased phosphorylation of STAT3 and increased SOCS3 expression, with subsequent inhibition of iNOS expression. In cultured cardiomyocytes treated with isoproterenol or forskolin, the increase of SOCS3 expression was blunted when Epac1 or PKCα was silenced with siRNA. Activation of the cAMP/Epac/PKCα pathway protected the heart against cytokine-induced cardiac dysfunction, suggesting a new role of catecholamine signaling in compensating for cardiac dysfunction in heart failure. Epac1 and its downstream pathways may be novel targets for treating cardiac dysfunction in endotoxemia.
Assuntos
Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/genética , Janus Quinases/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais , Disfunção Ventricular/etiologia , Disfunção Ventricular/metabolismo , Animais , Biomarcadores , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Cardiomegalia/fisiopatologia , Catecolaminas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Testes de Função Cardíaca , Humanos , Lipopolissacarídeos/efeitos adversos , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Óxido Nítrico Sintase Tipo II/metabolismo , Proteína 3 Supressora da Sinalização de Citocinas/metabolismo , Disfunção Ventricular/tratamento farmacológico , Disfunção Ventricular/fisiopatologiaRESUMO
We describe the application of optical coherence tomography (OCT) for noninvasive analysis of follicular development in mouse ovaries. Ovaries contain many follicles and oocytes. Quantification of follicles at various developmental stages is an indication of the ability of an ovary to provide oocytes capable of fertilization. Three-dimensional structural OCT images identified each developmental stage, from a primary follicle (50 µm in diameter) to an antral follicle (350 µm in diameter), in the ovary of a 25.5-day-old mouse. We discovered time-varying OCT signals at the oocytes that differentiated them from surrounding tissues. These signals were clearly enhanced by interframe intensity-based Doppler OCT techniques. The OCT technology was effective in analyzing the development of follicles and oocytes in ovaries.
Assuntos
Oócitos/patologia , Folículo Ovariano/patologia , Ovário/patologia , Tomografia de Coerência Óptica/métodos , Animais , Desenho de Equipamento , Feminino , Humanos , Imageamento Tridimensional , Camundongos , Modelos Estatísticos , Oócitos/crescimento & desenvolvimento , Folículo Ovariano/crescimento & desenvolvimento , Ovário/crescimento & desenvolvimentoRESUMO
BACKGROUND: Secretoglobin (SCGB) 3A2, a cytokine-like secretory protein of small molecular weight, is predominantly expressed in airway epithelial cells. While SCGB3A2 is known to have anti-inflammatory, growth factor, and anti-fibrotic activities, whether SCGB3A2 has any other roles, particularly in lung homeostasis and disease has not been demonstrated in vivo. The aim of this study was to address these questions in mice. METHODS: A transgenic mouse line that expresses SCGB3A2 in the lung using the human surfactant protein-C promoter was established. Detailed histological, immunohistochemical, physiological, and molecular characterization of the Scgb3a2-transgenic mouse lungs were carried out. Scgb3a2-transgenic and wild-type mice were subjected to bleomycin-induced pulmonary fibrosis model, and their lungs and bronchoalveolar lavage fluids were collected at various time points during 9 weeks post-bleomycin treatment for further analysis. RESULTS: Adult Scgb3a2-transgenic mouse lungs expressed approximately five-fold higher levels of SCGB3A2 protein in comparison to wild-type mice as determined by western blotting of lung tissues. Immunohistochemistry showed that expression was localized to alveolar type II cells in addition to airway epithelial cells, thus accurately reflecting the site of surfactant protein-C expression. Scgb3a2-transgenic mice showed normal lung development and histology, and no overt gross phenotypes. However, when subjected to a bleomycin-induced pulmonary fibrosis model, they initially exhibited exacerbated fibrosis at 3 weeks post-bleomycin administration that was more rapidly resolved by 6 weeks as compared with wild-type mice, as determined by lung histology, Masson Trichrome staining and hydroxyproline content, inflammatory cell numbers, expression of collagen genes, and proinflammatory cytokine levels. The decrease of fibrosis coincided with the increased expression of SCGB3A2 in Scgb3a2-transgenic lungs. CONCLUSIONS: These results demonstrate that SCGB3A2 is an anti-fibrotic agent, and suggest a possible therapeutic use of recombinant SCGB3A2 in the treatment of pulmonary fibrosis.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pulmão/metabolismo , Fibrose Pulmonar/genética , RNA/genética , Secretoglobinas/genética , Animais , Bleomicina/toxicidade , Northern Blotting , Western Blotting , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Pulmão/patologia , Camundongos , Camundongos Transgênicos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Secretoglobinas/biossínteseRESUMO
Ghrelin, an endogenous ligand for the growth hormone secretagogue receptor, is produced predominantly in the stomach. It has been reported that endogenous ghrelin levels are increased by fasting and decreased immediately after feeding and that fasting-induced ghrelin release is controlled by the sympathetic nervous system. However, the mechanisms of plasma ghrelin decrement after feeding are poorly understood. Here, we studied the control of ghrelin secretion using ghrelin-producing cell lines and found that these cells express high levels of mRNA encoding G-protein coupled receptor 120 (GPR120). Addition of GW-9508 (a GPR120 chemical agonist) and α-linolenic acid (a natural ligand for GPR120) inhibited the secretion of ghrelin by â¼50 and 70%, respectively. However, the expression levels of preproghrelin and ghrelin O-acyltransferase (GOAT) mRNAs were not influenced by GW-9508. In contrast, the expression levels of prohormone convertase 1 were decreased significantly by GW-9508 incubation. Moreover, we observed that the inhibitory effect of GW-9508 on ghrelin secretion was blocked by a small interfering RNA (siRNA) targeting the sequence of GPR120. Furthermore, pretreatment with GW-9508 blocked the effect of the norepinephrine (NE)-induced ghrelin elevation in ghrelin cell lines. In addition, we showed that GW-9508 inhibited ghrelin secretion via extracellular signal-regulated kinase activity in ghrelin cell lines. Finally, we found that GW-9508 decreased plasma ghrelin levels in mice. These results suggest that the decrease of ghrelin secretion after feeding is induced partially by long-chain fatty acids that act directly on gastric GPR120-expressing ghrelin cells.
Assuntos
Grelina/metabolismo , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Linhagem Celular Tumoral , Ácidos Graxos/farmacologia , Alimentos , Mucosa Gástrica/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Norepinefrina/farmacologia , Pró-Proteína Convertase 1/genética , RNA Mensageiro/análise , RNA Interferente Pequeno/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Estômago/química , Neoplasias Gástricas/metabolismo , Ácido alfa-Linolênico/farmacologiaRESUMO
Secretoglobin (SCGB) 3A2 was originally identified as a downstream target for the homeodomain transcription factor NKX2-1 in the lung. NKX2-1 plays a role in the genesis and expression of genes in the thyroid, lung and ventral forebrain; Nkx2-1-null mice have no thyroid and pituitary and severely hypoplastic lungs and hypothalamus. To demonstrate whether SCGB3A2 plays any role in pituitary hormone production, NKX2-1 and SCGB3A2 expression in the mouse pituitary gland was examined by immunohistochemical analysis and RT-PCR. NKX2-1 was localized in the posterior pituitary lobe, whereas SCGB3A2 was observed in both anterior and posterior lobes as shown by immunohistochemistry and RT-PCR. Expression of CCAAT-enhancer binding proteins (C/EBPs), which regulate mouse Scgb3a2 transcription, was also examined by RT-PCR. C/EBPß, γ, δ and ζ were expressed in the adult mouse pituitary gland. SCGB3A2 was expressed in the anterior and posterior lobes from postnatal days 1 and 5, respectively and the areas where SCGB3A2 expression was found coincided with the area where FSH-secreting cells were found. Double-staining for SCGB3A2 and pituitary hormones revealed that SCGB3A2 was mainly localized in gonadotrophs in 49 % of FSH-secreting cells and 47 % of LH-secreting cells. In addition, SCGB3A2 dramatically inhibited LH and FSH mRNA expression in rat pituitary primary cell cultures. These results suggest that SCGB3A2 regulates FSH/LH production in the anterior pituitary lobe and that transcription factors other than NKX2-1 may regulate SCGB3A2 expression.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Hormônio Luteinizante/metabolismo , Hipófise/metabolismo , Secretoglobinas/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Gonadotrofos/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Hipófise/citologia , Hormônios Hipofisários/metabolismo , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Coloração e Rotulagem , Fator Nuclear 1 de Tireoide , Fatores de Transcrição/metabolismoRESUMO
We investigated the role of macrophage colony-stimulating factor (M-CSF) in the pituitary gland to understand the effect of M-CSF on pituitary hormones and the relationship between the endocrine and immune systems. When we attempted to establish pituitary cell lines from a thyrotropic pituitary tumor (TtT), a macrophage cell line, TtT/M-87, was established. We evaluated M-CSF-like activity in conditioned media (CM) from seven pituitary cell lines using TtT/M-87 cells. TtT/M-87 proliferation significantly increased in the presence of CM from TtT/GF cells, a pituitary folliculostellate (FS) cell line. M-CSF mRNA was detected in TtT/GF and MtT/E cells by reverse transcriptase-polymerase chain reaction (RT-PCR), and its expression in TtT/GF cells was increased in a lipopolysaccharide (LPS) dose-dependent manner. M-CSF mRNA expression was also increased in rat anterior pituitary glands by LPS. M-CSF receptor (M-CSFR) mRNA was only detected in TtT/ M-87 cells and increased in the LPS-stimulated rat pituitary glands. In rat pituitary glands, M-CSF and M-CSFR were found to be localized in FS cells and prolactin (PRL)-secreting cells, respectively, by immunohistochemistry. The PRL concentration in rat sera was significantly increased at 24 h after M-CSF administration, and mRNA levels significantly increased in primary culture cells of rat anterior pituitary glands. In addition, TNF-α mRNA was increased in the primary culture cells by M-CSF. These results revealed that M-CSF was secreted from FS cells and M-CSF regulated PRL expression in rat pituitary glands.
Assuntos
Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Hipófise/citologia , Hipófise/metabolismo , Prolactina/metabolismo , Animais , Linhagem Celular , Meios de Cultivo Condicionados , Lipopolissacarídeos/toxicidade , Fator Estimulador de Colônias de Macrófagos/genética , Macrófagos/metabolismo , Masculino , Camundongos , Hipófise/efeitos dos fármacos , Prolactina/genética , Ratos , Ratos Wistar , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.
Assuntos
Fumar Cigarros , Doença Pulmonar Obstrutiva Crônica , Enfisema Pulmonar , Fibrose Pulmonar , Camundongos , Animais , Secretoglobinas/genética , Secretoglobinas/metabolismo , Enfisema Pulmonar/genética , Enfisema Pulmonar/prevenção & controle , Fumar Cigarros/efeitos adversos , Pulmão/patologia , Fibrose Pulmonar/metabolismo , Inflamação/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismoRESUMO
Receptor-independent G-protein regulators provide diverse mechanisms for signal input to G-protein-based signaling systems, revealing unexpected functional roles for G-proteins. As part of a broader effort to identify disease-specific regulators for heterotrimeric G-proteins, we screened for such proteins in cardiac hypertrophy using a yeast-based functional screen of mammalian cDNAs as a discovery platform. We report the identification of three transcription factors belonging to the same family, transcription factor E3 (TFE3), microphthalmia-associated transcription factor, and transcription factor EB, as novel receptor-independent activators of G-protein signaling selective for Gα(16). TFE3 and Gα(16) were both up-regulated in cardiac hypertrophy initiated by transverse aortic constriction. In protein interaction studies in vitro, TFE3 formed a complex with Gα(16) but not with Gα(i3) or Gα(s). Although increased expression of TFE3 in heterologous systems had no influence on receptor-mediated Gα(16) signaling at the plasma membrane, TFE3 actually translocated Gα(16) to the nucleus, leading to the induction of claudin 14 expression, a key component of membrane structure in cardiomyocytes. The induction of claudin 14 was dependent on both the accumulation and activation of Gα(16) by TFE3 in the nucleus. These findings indicate that TFE3 and Gα(16) are up-regulated under pathologic conditions and are involved in a novel mechanism of transcriptional regulation via the relocalization and activation of Gα(16).
Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/biossíntese , Cardiomegalia/metabolismo , Membrana Celular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/biossíntese , Regulação da Expressão Gênica , Transdução de Sinais , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células COS , Cardiomegalia/genética , Chlorocebus aethiops , Claudinas , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Proteínas de Membrana/biossíntese , CamundongosRESUMO
With increasing worldwide rates of morbidity and mortality of pulmonary fibrosis, the development of effective therapeutics for this disease is of great interest. Secretoglobin (SCGB) 3A2, a novel cytokine-like molecule predominantly expressed in pulmonary airways epithelium, exhibits anti-inflammatory and growth factor activities. In the current study SCGB3A2 was found to inhibit TGFß-induced differentiation of fibroblasts to myofibroblasts, a hallmark of the fibrogenic process, using pulmonary fibroblasts isolated from adult mice. This induction was through increased phosphorylation of STAT1 and expression of SMAD7 and decreased phosphorylation of SMAD2 and SMAD3. To demonstrate the effect of SCGB3A2 on the TGFß signaling in vivo, a bleomycin-induced pulmonary fibrosis mouse model was used. Mice were administered bleomycin intratracheally followed by intravenous injection of recombinant SCGB3A2. Histological examination in conjunction with inflammatory cell counts in bronchoalveolar lavage fluids demonstrated that SCGB3A2 suppressed bleomycin-induced pulmonary fibrosis. Microarray analysis was carried out using RNAs from lungs of bleomycin-treated mice with or without SCGB3A2 and normal mice treated with SCGB3A2. The results demonstrated that SCGB3A2 affects TGFß signaling and reduces the expression of genes involved in fibrosis. This study suggests the potential utility of SCGB3A2 for targeting TGFß signaling in the treatment of pulmonary fibrosis.
Assuntos
Antibióticos Antineoplásicos/efeitos adversos , Bleomicina/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Proteínas/metabolismo , Fibrose Pulmonar/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Bleomicina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Regulação para Baixo/genética , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Perfilação da Expressão Gênica , Humanos , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Proteínas/genética , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Secretoglobinas , Transdução de Sinais/genética , Proteínas Smad/genética , Proteínas Smad/metabolismo , Fator de Crescimento Transformador beta/genéticaRESUMO
RATIONALE: Prostaglandin (PG)E(2), which increases intracellular cAMP via activation of adenylyl cyclases (ACs), induces vasodilation and hyaluronan-mediated intimal thickening (IT) in the ductus arteriosus (DA) during late gestation. After birth, however, differential regulation of vasodilation and IT is preferable for treatment of patients with patent DA and DA-dependent congenital cardiac malformations. OBJECTIVE: Our objectives were to examine whether AC isoforms play differential roles in DA vasodilation and IT. METHODS AND RESULTS: AC2 and AC6 were more highly expressed in rat DA than in the aorta during the perinatal period. AC6-targeted siRNA counteracted PGE(1)-induced hyaluronan production in rat DA smooth muscle cells. Overexpression of AC6 enhanced PGE(1)-induced hyaluronan production and induced IT in DA explants. Furthermore, IT of the DA was less marked in mice lacking AC6 than in wild-type and AC5-deficient mice. Stimulation of AC2 attenuated AC6-induced hyaluronan production via inhibition of the p38 mitogen-activated protein kinase pathway and AC6-induced IT of the DA. An AC2/6 activator, 6-[N-(2-isothiocyanatoethyl) aminocarbonyl] forskolin (FD1), did not induce hyaluronan-mediated IT in DA explants, although an AC5/6 activator, 6-[3-(dimethylamino)propionyl]-14,15-dihydroforskolin (FD6) did. Moreover, FD1 induced longer vasodilation of the DA than did PGE(1) without significant adverse effects in vivo. CONCLUSIONS: AC6 is responsible for hyaluronan-mediated IT of the DA and AC2 inhibited AC6-induced hyaluronan production. Stimulation of both AC2 and AC6 by FD1 induced longer vasodilation without hyaluronan-mediated IT in the DA in vivo. FD1 may be a novel alternative therapy to currently available PGE therapy for patients with DA-dependent congenital heart disease.
Assuntos
Adenilil Ciclases/metabolismo , Canal Arterial/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatação/fisiologia , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/genética , Animais , Células Cultivadas , Colforsina/farmacologia , AMP Cíclico/metabolismo , Canal Arterial/citologia , Ácido Hialurônico/metabolismo , Isoenzimas/efeitos dos fármacos , Isoenzimas/genética , Isoenzimas/metabolismo , MAP Quinase Quinase 3/metabolismo , Camundongos , Camundongos Knockout , Modelos Animais , Músculo Liso Vascular/citologia , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Túnica Íntima/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We previously demonstrated that type 5 adenylyl cyclase (AC5) functions in autonomic regulation in the heart. Based on that work, we hypothesized that pharmacological modulation of AC5 activity could regulate the autonomic control of the heart rate under micro- and hypergravity. To test this hypothesis, we selected the approach of activating AC5 activity in mice with a selective AC5 activator (NKH477) or inhibitor (vidarabine) and examining heart rate variability during parabolic flight. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater under micro- and hypergravity in the vidarabine group, while there were no significant changes in the NKH477 group, suggesting that autonomic regulation was unstable in the vidarabine group. The ratio of low frequency and high frequency (HF) in heart rate variability analysis, a marker of sympathetic activity, became significantly decreased under micro- and hypergravity in the NKH477 group, while there was no such decrease in the vidarabine group. Normalized HF, a marker of parasympathetic activity, became significantly greater under micro- and hypergravity in the NKH477 group. In contrast, there was no such increase in the vidarabine group. This study is the first to indicate that pharmacological modulation of AC5 activity under micro- and hypergravity could be useful to regulate the autonomic control of the heart rate.
Assuntos
Adenilil Ciclases/fisiologia , Frequência Cardíaca/fisiologia , Hipergravidade , Ausência de Peso , Inibidores de Adenilil Ciclases , Animais , Colforsina/análogos & derivados , Colforsina/farmacologia , Frequência Cardíaca/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vidarabina/farmacologiaRESUMO
Secretoglobin (SCGB) 3A2 was first identified in 2001 as a protein exhibiting similarities in amino acid sequence and gene structure to SCGB1A1, a multi-functional cytokine-like molecule highly expressed in airway epithelial Club cells that was the first identified and extensively studied member of the SCGB gene superfamily. SCGB3A2 is a small secretory protein of ~10 kDa that forms a dimer and a tetramer. SCGB3A2 is predominantly expressed in airway epithelial Club cells, and has anti-inflammatory, growth factor, anti-fibrotic, and anti-cancer activities that influence various lung diseases. This review summarizes the current understanding of SCGB3A2 biological functions and its role in human diseases with emphasis on its mechanisms of actions and signaling pathway.
Assuntos
Citocinas , Sistema Respiratório , Secretoglobinas , Citocinas/metabolismo , Humanos , Sistema Respiratório/metabolismo , Secretoglobinas/genética , Secretoglobinas/metabolismoRESUMO
Background: Secretoglobin (SCGB) 3A2 is a novel bioactive molecule with anti-inflammatory and anti-fibrotic activities. SCGB3A2 also promotes the maturation of bronchial divergence and the lungs during embryonic development. However, much remains unknown concerning the roles of SCGB3A2 in diseases associated with aging. Methods: The lungs of Scgb3a2-knockout (KO) mice and their wild-type (WT) littermates were subjected to histological analysis, Victoria blue staining to evaluate of elastic fibers, and lung morphometric analysis during the postnatal period (birth to 8 weeks) and during aging (8 weeks to 2 years). Their spleens were also histologically evaluated. The expression of lung surfactant protein (SP) mRNAs was examined by quantitative reverse transcriptase-polymerase chain reaction. RNA sequencing (RNAseq) analysis was performed on 3-month-old KO and WT mouse lungs. Results: The alveolar spaces of KO mice continuously expanded between 0.5 and 2 years of age, accompanied by increases of the mean linear intercept and destructive index. KO mouse lungs displayed inflammation associated with lymphocyte aggregate starting at 1 year of age, and the inflammation was worse than that of WT mouse lungs. A high number of lymphoma-like cells were presented in 2-year-old KO mouse lungs. White pulp fusion was detected in the spleens of both WT and KO mice older than 0.5 years; however, the fusion was more severe in KO mice than in WT mice. The expression of surfactant protein (SP)-A, SP-B, SP-C, and SP-D mRNAs in KO mouse lungs decreased with age, and after 1 year of age, the expression of most SPs was significantly lower in KO mice than in WT mice. RNAseq demonstrated that the expression of immune system-related genes was highly altered in KO mouse lungs. Conclusion: SCGB3A2 may be required for maintaining homeostasis and immune activity in the lungs during aging. SCGB3A2 deficiency might increase the risk of emphysema of the lung.
Assuntos
Enfisema , Linfoma , Doença Pulmonar Obstrutiva Crônica , Envelhecimento , Animais , Feminino , Humanos , Inflamação/metabolismo , Pulmão/metabolismo , Linfoma/metabolismo , Linfoma/patologia , Camundongos , Camundongos Knockout , Gravidez , Doença Pulmonar Obstrutiva Crônica/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Secretoglobinas/genética , Secretoglobinas/metabolismo , Tensoativos/metabolismoRESUMO
Cell survival and death play critical roles in tissues composed of post-mitotic cells. Cyclic AMP (cAMP) has been known to exert a distinct effect on cell susceptibility to apoptosis, protecting neuronal cells and deteriorating myocardial cells. These effects are primarily studied using protein kinase A activation. In this study we show the differential roles of Epac, an exchange protein activated by cAMP and a new effector molecule of cAMP signaling, in regulating apoptosis in these cell types. Both stimulation of Epac by 8-p-methoxyphenylthon-2'-O-methyl-cAMP and overexpression of Epac significantly increased DNA fragmentation and TUNEL (terminal deoxynucleotidyltransferase-mediated biotin nick end-labeling)-positive cell counts in mouse cortical neurons but not in cardiac myocytes. In contrast, stimulation of protein kinase A increased apoptosis in cardiac myocytes but not in neuronal cells. In cortical neurons the expression of the Bcl-2 interacting member protein (Bim) was increased by stimulation of Epac at the transcriptional level and was decreased in mice with genetic disruption of Epac1. Epac-induced neuronal apoptosis was attenuated by the silencing of Bim. Furthermore, Epac1 disruption in vivo abolished the 3-nitropropionic acid-induced neuronal apoptosis that occurs in wild-type mice. These results suggest that Epac induces neuron-specific apoptosis through increasing Bim expression. Because the disruption of Epac exerted a protective effect on neuronal apoptosis in vivo, the inhibition of Epac may be a consideration in designing a therapeutic strategy for the treatment of neurodegenerative diseases.
Assuntos
Regulação da Expressão Gênica , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miocárdio/metabolismo , Neurônios/metabolismo , Animais , Apoptose , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Musculares/metabolismo , Miocárdio/citologia , Doenças Neurodegenerativas/metabolismo , Neurônios/citologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de SinaisRESUMO
Caveolin, a member of the membrane-anchoring protein family, accumulates various growth receptors in caveolae and inhibits their function. Upregulation of caveolin attenuates cellular proliferation and growth. However, the role of caveolin in regulating insulin signals remains controversial. Here, we demonstrate that caveolin potently enhances insulin receptor (IR) signaling when overexpressed in the liver in vivo. Adenovirus-mediated gene transfer was used to overexpress caveolin specifically in the liver of diabetic obese mice, which were generated with a high-fat diet. Expression of molecules involved in IR signaling, such as IR or Akt, remained unchanged after gene transfer. However, hepatic glycogen synthesis was markedly increased with a decrease in phosphoenolpyruvate carboxykinase protein expression. Insulin sensitivity was increased after caveolin gene transfer as determined by decreased blood glucose levels in response to insulin injection and fasting blood glucose levels. Glucose tolerant test performance was also improved. Similar improvements were obtained in KKA(y) genetically diabetic mice. Adenovirus-mediated overexpression of caveolin-3 in hepatic cells also enhanced IR signaling, as shown by increased phosphorylation of IR in response to insulin stimulation and higher glycogen synthesis at baseline. These effects were attributed mostly to increased insulin receptor activity and caveolin-mediated, direct inhibition of protein tyrosine phosphatase 1B, which was increased in obese mouse livers. In conclusion, our results suggest that caveolin is an important regulator of glucose metabolism that can enhance insulin signals.
Assuntos
Glicemia/metabolismo , Caveolina 3/metabolismo , Diabetes Mellitus Tipo 2/sangue , Técnicas de Transferência de Genes , Insulina/sangue , Fígado/metabolismo , Obesidade/complicações , Adenoviridae/genética , Fatores Etários , Envelhecimento/sangue , Animais , Glicemia/efeitos dos fármacos , Caveolina 3/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/fisiopatologia , Diabetes Mellitus Tipo 2/terapia , Gorduras na Dieta , Modelos Animais de Doenças , Vetores Genéticos , Teste de Tolerância a Glucose , Glicogênio/biossíntese , Células Hep G2 , Humanos , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Resistência à Insulina , Fígado/efeitos dos fármacos , Camundongos , Obesidade/sangue , Obesidade/genética , Obesidade/fisiopatologia , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 1/metabolismo , Ratos , Receptor de Insulina/metabolismo , Transdução de SinaisRESUMO
Ischemic injury of the heart is associated with activation of multiple signal transduction systems including the heterotrimeric G-protein system. Here, we report a role of the ischemia-inducible regulator of G betagamma subunit, AGS8, in survival of cardiomyocytes under hypoxia. Cultured rat neonatal cardiomyocytes (NCM) were exposed to hypoxia or hypoxia/reoxygenation following transfection of AGS8siRNA or pcDNA::AGS8. Hypoxia-induced apoptosis of NCM was completely blocked by AGS8siRNA, whereas overexpression of AGS8 increased apoptosis. AGS8 formed complexes with G-proteins and channel protein connexin 43 (CX43), which regulates the permeability of small molecules under hypoxic stress. AGS8 initiated CX43 phosphorylation in a G betagamma-dependent manner by providing a scaffold composed of G betagamma and CX43. AGS8siRNA blocked internalization of CX43 following exposure of NCM to repetitive hypoxia; however it did not influence epidermal growth factor-mediated internalization of CX43. The decreased dye flux through CX43 that occurred with hypoxic stress was also prevented by AGS8siRNA. Interestingly, the G betagamma inhibitor Gallein mimicked the effect of AGS8 knockdown on both the CX43 internalization and the changes in cell permeability elicited by hypoxic stress. These data indicate that AGS8 is required for hypoxia-induced apoptosis of NCM, and that AGS8-G betagamma signal input increased the sensitivity of cells to hypoxic stress by influencing CX43 regulation and associated cell permeability. Under hypoxic stress, this unrecognized response program plays a critical role in the fate of NCM.
Assuntos
Apoptose , Conexina 43/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Hipóxia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Miócitos Cardíacos/metabolismo , Transdução de Sinais , Animais , Hipóxia Celular , Células Cultivadas , Conexina 43/genética , Subunidades beta da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Hipóxia/genética , Hipóxia/fisiopatologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Miócitos Cardíacos/citologia , Ligação Proteica , RatosRESUMO
Melanoma, the most malignant form of human skin cancer, has a poor prognosis due to its strong metastatic ability. It was recently demonstrated that Epac, an effector molecule of cAMP, is involved in regulating cell migration; however, the role of Epac in melanoma cell migration remains unclear. We thus examined whether Epac regulates cell migration and metastasis of melanoma. Epac activation, by either specific agonist or overexpression of Epac, increased melanoma cell migration. Deletion of endogenous Epac with small interfering RNA decreased basal melanoma cell migration. These data suggested a major role of Epac in melanoma cell migration. Epac-induced cell migration was mediated by translocation of syndecan-2, a cell-surface heparan sulfate proteoglycan, to lipid rafts. This syndecan-2 translocation was regulated by tubulin polymerization via the Epac/phosphoinositol-3 kinase pathway. Epac-induced cell migration was also regulated by the production of heparan sulfate, a major extracellular matrix. Epac-induced heparan sulfate production was attributable to the increased expression of N-deacetylase/N-sulfotransferase-1 (NDST-1) accompanied by an increased NDST-1 translation rate. Finally, Epac overexpression enhanced lung colonization of melanoma cells in mice. Taken together, these data indicate that Epac regulates melanoma cell migration/metastasis mostly via syndecan-2 translocation and heparan sulfate production.
Assuntos
Movimento Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Heparitina Sulfato/biossíntese , Melanoma/metabolismo , Sulfotransferases/metabolismo , Sindecana-2/metabolismo , Animais , Linhagem Celular Tumoral , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Melanoma/patologia , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Fosfatidilinositol 3-Quinases/fisiologia , Transporte Proteico , Transdução de Sinais , Tubulina (Proteína)/metabolismoRESUMO
Inhibitor of growth 2 (ING2) is associated with chromatin remodeling and regulation of gene expression by binding to a methylated histone H3K4 residue and recruiting HDAC complexes to the region. The aim of our study is to investigate the regulation of ING2 expression and the clinical significance of upregulated ING2 in colon cancer. Here, we show that the ING2 mRNA level in colon cancer tissue increased to more than twice than that in normal mucosa in the 45% of colorectal cancer cases that we examined. A putative NF-kappaB binding site was found in the ING2 promoter region. We confirmed that NF-kappaB could bind to the ING2 promoter by EMSA and luciferase assays. Subsequent microarray analyses revealed that ING2 upregulates expression of matrix metalloproteinase 13 (MMP13), which enhances cancer invasion and metastasis. ING2 regulation of MMP13 expression was confirmed in both ING2 overexpression and knock down experiments. MMP13 expression was further induced by coexpression of ING2 with HDAC1 or with mSin3A, suggesting that the ING2-HDAC1-mSin3A complex members regulates expression of MMP13. In vitro invasion assay was performed to determine functional significance of ING2 upregulation. ING2 overexpressed cells exhibited greater invasive potential. Taken together, upregulation of ING2 was associated with colon cancer and MMP13-dependent cellular invasion, indicating that ING2 expression might be involved with cancer invasion and metastasis.
Assuntos
Neoplasias do Colo/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/genética , Metaloproteinase 13 da Matriz/genética , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Supressoras de Tumor/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Western Blotting , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Perfilação da Expressão Gênica , Histona Desacetilase 1 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Luciferases/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Pessoa de Meia-Idade , NF-kappa B/genética , NF-kappa B/metabolismo , Invasividade Neoplásica , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Complexo Correpressor Histona Desacetilase e Sin3 , Transfecção , Regulação para CimaRESUMO
RATIONALE: Secretoglobin 3A2 (SCGB3A2) was originally identified as a downstream target in lung for the homeodomain transcription factor NKX2-1, whose null mutation resulted in severely hypoplastic lungs. A very low level of SCGB3A2 is expressed in lungs at Embryonic Day (E) 11.5 during mouse development, which markedly increases by E16.5, the time when lung undergoes dramatic morphologic changes, suggesting that SCGB3A2 may be involved in lung development in addition to a known role in lung inflammation. OBJECTIVES: To determine whether SCGB3A2 plays a role in lung development. METHODS: To assess a potential role for SCGB3A2 during early lung development, wild-type and Nkx2-1-null fetal lungs of early developmental stages were subjected to ex vivo organ culture in the presence of SCGB3A2. Nkx2-1-null fetuses were exposed to SCGB3A2 during early organogenesis period through intravenous administration of this protein to Nkx2-1-heterozygous pregnant females carrying these null fetuses. Cultured lungs and fetal lungs were subjected to histologic and immunohistochemical analyses. To assess a role for SCGB3A2 in late lung development, SCGB3A2 was administered to pregnant wild-type females during mid- to late organogenesis stages, and the preterm pups and/or their lungs were evaluated for extent of maturity using breathing motion, gross morphology and histology of lungs, expression of gestational stage-specific genes, and phospholipid profiles. MEASUREMENTS AND MAIN RESULTS: SCGB3A2 significantly promoted both early and late stages of lung development. CONCLUSIONS: SCGB3A2 is a novel growth factor in lung.
Assuntos
Maturidade dos Órgãos Fetais/genética , Pulmão/embriologia , Proteínas Nucleares/genética , Organogênese/genética , Proteínas/genética , Fatores de Transcrição/genética , Animais , Diferenciação Celular/genética , Divisão Celular/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Idade Gestacional , Pulmão/patologia , Camundongos , Análise de Sequência com Séries de Oligonucleotídeos , Técnicas de Cultura de Órgãos , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Secretoglobinas , Fator Nuclear 1 de TireoideRESUMO
The article Epac activation inhibits IL-6-induced cardiac myocyte dysfunction.