RESUMEN
Rationale: A mature tissue resident macrophage (TRM) population residing in the peritoneal cavity has been known for its unique ability to migrate to peritoneally located injured tissues and impart wound healing properties. Here, we sought to expand on this unique ability of large peritoneal macrophages (LPMs) by investigating whether these GATA6+ LPMs could also intravasate into systemic circulation and migrate to extra-peritoneally located lungs upon ablating lung-resident alveolar macrophages (AMs) by intranasally administered clodronate liposomes in mice. Methods: C12-200 cationic lipidoid-based nanoparticles were employed to selectively deliver a small interfering RNA (siRNA)-targeting CD-45 labeled with a cyanine 5.5 (Cy5.5) dye to LPMs in vivo via intraperitoneal injection. We utilized a non-invasive optical technique called Diffuse In Vivo Flow Cytometry (DiFC) to then systemically track these LPMs in real time and paired it with more conventional techniques like flow cytometry and immunocytochemistry to initially confirm uptake of C12-200 encapsulated siRNA-Cy5.5 (siRNA-Cy5.5 (C12-200)) into LPMs, and further track them from the peritoneal cavity to the lungs in a mouse model of AM depletion incited by intranasally administered clodronate liposomes. Also, we stained for LPM-specific marker zinc-finger transcription factor GATA6 in harvested cells from biofluids like broncho-alveolar lavage as well as whole blood to probe for Cy5.5-labeled LPMs in the lungs as well as in systemic circulation. Results: siRNA-Cy5.5 (C12-200) was robustly taken up by LPMs. Upon depletion of lung-resident AMs, these siRNA-Cy5.5 (C12-200) labeled LPMs rapidly migrated to the lungs via systemic circulation within 12-24 h. DiFC results showed that these LPMs intravasated from the peritoneal cavity and utilized a systemic route of migration. Moreover, immunocytochemical staining of zinc-finger transcription factor GATA6 further confirmed results from DiFC and flow cytometry, confirming the presence of siRNA-Cy5.5 (C12-200)-labeled LPMs in the peritoneum, whole blood and BALF only upon clodronate-administration. Conclusion: Our results indicate for the very first time that selective tropism, migration, and infiltration of LPMs into extra-peritoneally located lungs was dependent on clodronate-mediated AM depletion. These results further open the possibility of therapeutically utilizing LPMs as delivery vehicles to carry nanoparticle-encapsulated oligonucleotide modalities to potentially address inflammatory diseases, infectious diseases and even cancer.
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Ácido Clodrónico , Pulmón , Macrófagos Peritoneales , Nanopartículas , Animales , Ácido Clodrónico/farmacología , Ácido Clodrónico/administración & dosificación , Nanopartículas/química , Nanopartículas/administración & dosificación , Ratones , Pulmón/metabolismo , Macrófagos Peritoneales/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Alveolares/metabolismo , ARN Interferente Pequeño/administración & dosificación , Factor de Transcripción GATA6/metabolismo , Liposomas , Ratones Endogámicos C57BL , Carbocianinas/química , Movimiento Celular/efectos de los fármacos , Citometría de FlujoRESUMEN
Ovarian cancer is the common cause of cancer-related death in women and is considered the most deadly gynecological cancer. It has been established that GATA-binding protein 6 (GATA6) is abnormally expressed in several types of malignant tumors and acts as an oncogenic protein or a tumor suppressor. However, the underlying mechanism of GATA6 in ovarian cancer progression has not been elucidated. Data in the present study revealed that GATA6 expression was negatively correlated to microRNA-10a-5p (miR-10a-5p) in ovarian cancer tissue and cells and that GATA6 is directly targeted by miR-10a-5p. Notably, upregulated miR-10a-5p dramatically inhibited ovarian cancer cell proliferation, tumorigenic ability, migration, and invasion by targeting GATA6. In vitro and in vivo experiments confirmed that miR-10a-5p-mediated downregulation of GATA6 suppressed Akt pathway activation. Overall, our findings suggest that miR-10a-5p could be a novel therapeutic target for ovarian cancer, and targeting the miR-10a-5p/GATA6/Akt axis could improve outcomes in this patient population.
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Factor de Transcripción GATA6 , MicroARNs , Neoplasias Ováricas , Factor de Transcripción GATA6/antagonistas & inhibidores , Factor de Transcripción GATA6/genética , MicroARNs/genética , Neoplasias Ováricas/terapia , Progresión de la Enfermedad , Humanos , Línea Celular Tumoral , Células HEK293 , Ensayos Antitumor por Modelo de Xenoinjerto , Ratones Desnudos , Animales , Ratones , Lentivirus , Vectores Genéticos , Femenino , Movimiento Celular , Proliferación CelularRESUMEN
A 2-year-old boy was admitted to Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine in Nov 30th, 2018, due to polydipsia, polyphagia, polyuria accompanied with increased glucose levels for more than 2 weeks. He presented with symmetrical short stature [height 81 cm (ï¼2.2 SD), weight 9.8 kg (ï¼2.1 SD), body mass index 14.94 kg/m2 (P10-P15)], and with no special facial or physical features. Laboratory results showed that the glycated hemoglobin A1c was 14%, the fasting C-peptide was 0.3 ng/mL, and the islet autoantibodies were all negative. Oral glucose tolerance test showed significant increases in both fasting and postprandial glucose, but partial islet functions remained (post-load C-peptide increased 1.43 times compared to baseline). A heterozygous variant c.1366C>T (p.R456C) was detected in GATA6 gene, thereby the boy was diagnosed with a specific type of diabetes mellitus. The boy had congenital heart disease and suffered from transient hyperosmolar hyperglycemia after a patent ductus arteriosus surgery at 11 months of age. Insulin replacement therapy was prescribed, but without regular follow-up thereafter. The latest follow-up was about 3.5 years after the diagnosis of diabetes when the child was 5 years and 11 months old, with the fasting blood glucose of 6.0-10.0 mmol/L, and the 2 h postprandial glucose of 17.0-20.0 mmol/L.
Asunto(s)
Diabetes Mellitus Tipo 2 , Masculino , Niño , Humanos , Preescolar , Lactante , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/complicaciones , Mutación Missense , Péptido C/genética , China , Insulina/genética , Glucosa , Glucemia , Factor de Transcripción GATA6/genéticaRESUMEN
Damaging GATA6 variants can cause moderate congenital heart defects. With the application of next-generation sequencing approaches, various novel GATA6 variants with unknown significance have been identified from a broad spectrum of congenital heart defects. However, functional assessment for distinct GATA6 variants from different severity of congenital heart defects, especially from mild defects, is lacking, which hinders our understanding of the genotype-phenotype correlations and underlying mechanisms. Here, we assessed the functional consequences of nine rare GATA6 variants, which had been implicated as the most significant variants associated with mild congenital heart defects using the largest case and control cohort. We examined the effects of these variants on subcellular localization, transcriptional activity, and protein interactions in 293T or AC16 cells and their ability to rescue heart malformation in gata6 zebrafish mutant. We found that two of these nine variants, Q120X and S424I, significantly decreased transcriptional activity. Additionally, Q120X altered subcellular localization. Consistent with the in vitro results, the in vivo results showed that Q120X and S424I lost their potency to rescue ventricular malformation in gata6 -/- embryos. The results indicated that Q120X and S424I are pathogenic in mild congenital heart defects. Further, the inconsistence of severely impaired Q120X function and mild CHDs phenotype suggested the complexity of the genotype-phenotype correlation between the GATA6 variant and heart phenotype, which may help to inform prenatal genetic counseling and pre-implantation genotyping for congenital heart defects.
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Cardiopatías Congénitas , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Cardiopatías Congénitas/genética , Cardiopatías Congénitas/metabolismo , Corazón , Fenotipo , Estudios de Asociación Genética , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismoRESUMEN
A 2-year-old boy was admitted to Shanghai Children's Medical Center, Shanghai Jiao Tong University School of Medicine in Nov 30th, 2018, due to polydipsia, polyphagia, polyuria accompanied with increased glucose levels for more than 2 weeks. He presented with symmetrical short stature [height 81 cm (-2.2 SD), weight 9.8 kg (-2.1 SD), body mass index 14.94 kg/m2 (P10-P15)], and with no special facial or physical features. Laboratory results showed that the glycated hemoglobin A1c was 14%, the fasting C-peptide was 0.3 ng/mL, and the islet autoantibodies were all negative. Oral glucose tolerance test showed significant increases in both fasting and postprandial glucose, but partial islet functions remained (post-load C-peptide increased 1.43 times compared to baseline). A heterozygous variant c.1366C>T (p.R456C) was detected in GATA6 gene, thereby the boy was diagnosed with a specific type of diabetes mellitus. The boy had congenital heart disease and suffered from transient hyperosmolar hyperglycemia after a patent ductus arteriosus surgery at 11 months of age. Insulin replacement therapy was prescribed, but without regular follow-up thereafter. The latest follow-up was about 3.5 years after the diagnosis of diabetes when the child was 5 years and 11 months old, with the fasting blood glucose of 6.0-10.0 mmol/L, and the 2 h postprandial glucose of 17.0-20.0 mmol/L.
Asunto(s)
Masculino , Niño , Humanos , Preescolar , Lactante , Diabetes Mellitus Tipo 2/complicaciones , Mutación Missense , Péptido C/genética , China , Insulina/genética , Glucosa , Glucemia , Factor de Transcripción GATA6/genéticaRESUMEN
BACKGROUND: Several transcription factors regulate cardiac conduction system (CCS) development and function but the role of each in specifying distinct CCS components remains unclear. GATA-binding factor 6 (GATA6) is a zinc-finger transcription factor that is critical for patterning the cardiovascular system. However, the role of GATA6 in the embryonic heart and CCS has never been shown. METHODS AND RESULTS: We report that Gata6 is expressed abundantly in the proximal CCS during midgestation in mice. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 induces loss of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the compact atrioventricular node with some retention of hyperpolarizing cyclic nucleotide-gated channel, subtype 4 staining in the atrioventricular bundle, but has no significant effect on the connexin-40-positive bundle branches. Furthermore, myocardial-specific deletion of the carboxyl zinc-finger of Gata6 alters atrioventricular conduction in postnatal life as assessed by surface and invasive electrophysiological evaluation, as well as decreasing the number of ventricular myocytes and inducing compensatory myocyte hypertrophy. Myocardial-specific deletion of the carboxyl zinc-finger of Gata6 is also associated with downregulation of the transcriptional repressor ID2 and the cardiac sodium-calcium exchanger NCX1 in the proximal CCS, where GATA6 transactivates both of these factors. Finally, carboxyl zinc-finger deletion of Gata6 reduces cell-cycle exit of TBX3+ myocytes in the developing atrioventricular bundle during the period of atrioventricular node specification, which results in fewer TBX3+ cells in the proximal CCS of mature mutant mice. CONCLUSIONS: GATA6 contributes to the development and postnatal function of the murine atrioventricular node by promoting cell-cycle exit of specified cardiomyocytes toward a conduction system lineage.
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Nodo Atrioventricular/embriología , Factor de Transcripción GATA6/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Animales , Factor de Transcripción GATA6/genética , Ratones , Ratones MutantesRESUMEN
RATIONALE: Small heterodimer partner (SHP; NR0B2) is an atypical orphan nuclear receptor that lacks a conventional DNA-binding domain. Through interactions with other transcription factors, SHP regulates diverse biological events, including glucose metabolism in liver. However, the role of SHP in adult heart diseases has not yet been demonstrated. OBJECTIVE: We aimed to investigate the role of SHP in adult heart in association with cardiac hypertrophy. METHODS AND RESULTS: The roles of SHP in cardiac hypertrophy were tested in primary cultured cardiomyocytes and in animal models. SHP-null mice showed a hypertrophic phenotype. Hypertrophic stresses repressed the expression of SHP, whereas forced expression of SHP blocked the development of hypertrophy in cardiomyocytes. SHP reduced the protein amount of Gata6 and, by direct physical interaction with Gata6, interfered with the binding of Gata6 to GATA-binding elements in the promoter regions of natriuretic peptide precursor type A. Metformin, an antidiabetic agent, induced SHP and suppressed cardiac hypertrophy. The metformin-induced antihypertrophic effect was attenuated either by SHP small interfering RNA in cardiomyocytes or in SHP-null mice. CONCLUSIONS: These results establish SHP as a novel antihypertrophic regulator that acts by interfering with GATA6 signaling. SHP may participate in the metformin-induced antihypertrophic response.
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Cardiomegalia/prevención & control , Factor de Transcripción GATA6/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Animales , Factor Natriurético Atrial/genética , Factor Natriurético Atrial/metabolismo , Sitios de Unión , Cardiomegalia/genética , Cardiomegalia/metabolismo , Cardiomegalia/patología , Modelos Animales de Enfermedad , Factor de Transcripción GATA6/genética , Regulación de la Expresión Génica , Genotipo , Células HEK293 , Humanos , Masculino , Metformina/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/patología , Fenotipo , Regiones Promotoras Genéticas , Interferencia de ARN , Ratas , Ratas Sprague-Dawley , Receptores Citoplasmáticos y Nucleares/deficiencia , Receptores Citoplasmáticos y Nucleares/genética , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
Objective To identify novel mutations in the GATA6 gene associated with congenital atrial septal defects (ASD).Methods This was a case-control study.A cohort of 220 unrelated Han-race patients with congenital ASD and 200 unrelated ethnically matched healthy individuals used as controls,who were admitted to Tongji University Affiliated Tongji Hospital from January,2007 to October,2011,were recruited.The peripheral venous blood samples from the participants were prepared.All the coding exons and their flanking sequences of the GATA6 gene were amplified by polymerase chain reaction and sequenced using the di-deoxynucleotide chain termination technique.The acquired sequences were aligned with the sequences derived from GenBank by BLAST to identify the sequence variations.The software ClustalW was used to analyze the conservation of the altered amino acids.Results Three novel heterozygous missense GATA6 mutations,c.250G >A (p.A84T),c.649G >C (p.G217R) and c.1270A >C (p.S424R),were identified in 3 of 220 ASD patients,respectively.None of the three mutations was detected in 200 healthy control individuals.A cross-species alignment of GATA6 encoded protein sequences showed that the mutated amino acids were relatively conserved evolutionarily.Conclusion The identification of novel GATA6 mutations associated with ASD contributes to the reveal of the mechanism involved in the pathogenesis of ASD.