RESUMO
The cardiac outflow tract (OFT) transiently links the ventricles to the aortic sac and forms the arterial valves. Abnormalities in these valves, such as bicuspid aortic valve (BAV), are common congenital anomalies. GATA6-inactivating variants cause cardiac OFT defects and BAV, but their mechanisms are unclear. We generated Gata6STOP/+ mice using CRISPR-Cas9, which show highly penetrant BAV (70%) and membranous ventricular septal defects (43%). These mice exhibited decreased proliferation and increased ISL1-positive progenitor cells in the OFT, indicating abnormal cardiovascular differentiation. Gata6 deletion with the Mef2cCre driver line recapitulated Gata6STOP/+ phenotypes, indicating a cell-autonomous role for Gata6 in the second heart field. Gata6STOP/+ mice showed reduced OFT length and caliber, associated with deficient cardiac neural crest cell contribution, which may cause valvulo-septal defects. RNA-sequencing analysis showed depletion in pathways related to cell proliferation and migration, highlighting Cxcr7 (also known as Ackr3) as a candidate gene. Reduced mesenchymal cell migration and invasion were observed in Gata6STOP/+ OFT tissue. CXCR7 agonists reduced mesenchymal cell migration and increased invasion in wild-type but not in Gata6STOP/+ explants, indicating the GATA6-dependent role of CXCR7 in OFT development and its potential link to BAV.
Assuntos
Doença da Válvula Aórtica Bicúspide , Proliferação de Células , Fator de Transcrição GATA6 , Receptores CXCR , Transdução de Sinais , Animais , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Doença da Válvula Aórtica Bicúspide/patologia , Receptores CXCR/metabolismo , Receptores CXCR/genética , Crista Neural/metabolismo , Crista Neural/patologia , Camundongos , Movimento Celular , Valva Aórtica/anormalidades , Valva Aórtica/patologia , Valva Aórtica/metabolismo , Doenças das Valvas Cardíacas/patologia , Doenças das Valvas Cardíacas/metabolismo , Doenças das Valvas Cardíacas/genética , Fenótipo , Camundongos Endogâmicos C57BLRESUMO
Mutations in GATA6 are associated with congenital heart disease, most notably conotruncal structural defects. However, how GATA6 regulates cardiac morphology during embryogenesis is undefined. We used knockout and conditional mutant zebrafish alleles to investigate the spatiotemporal role of gata6 during cardiogenesis. Loss of gata6 specifically impacts atrioventricular valve formation and recruitment of epicardium, with a prominent loss of arterial pole cardiac cells, including those of the ventricle and outflow tract. However, there are no obvious defects in cardiac progenitor cell specification, proliferation or death. Conditional loss of gata6 starting at 24â h is sufficient to disrupt the addition of late differentiating cardiomyocytes at the arterial pole, with decreased expression levels of anterior secondary heart field (SHF) markers spry4 and mef2cb. Conditional loss of gata6 in the endoderm is sufficient to phenocopy the straight knockout, resulting in a significant loss of ventricular and outflow tract tissue. Exposure to a Dusp6 inhibitor largely rescues the loss of ventricular cells in gata6-/- larvae. Thus, gata6 functions in endoderm are mediated by FGF signaling to regulate the addition of anterior SHF progenitor derivatives during heart formation.
Assuntos
Diferenciação Celular , Endoderma , Fator de Transcrição GATA6 , Coração , Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Endoderma/metabolismo , Endoderma/embriologia , Endoderma/citologia , Diferenciação Celular/genética , Coração/embriologia , Organogênese/genética , Regulação da Expressão Gênica no Desenvolvimento , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/citologia , Transdução de Sinais , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fosfatase 6 de Especificidade Dupla/metabolismo , Fosfatase 6 de Especificidade Dupla/genética , Fatores de Transcrição GATARESUMO
Functional enhancer annotation is critical for understanding tissue-specific transcriptional regulation and prioritizing disease-associated non-coding variants. However, unbiased enhancer discovery in disease-relevant contexts remains challenging. To identify enhancers pertinent to diabetes, we conducted a CRISPR interference (CRISPRi) screen in the human pluripotent stem cell (hPSC) pancreatic differentiation system. Among the enhancers identified, we focused on an enhancer we named ONECUT1e-664kb, â¼664 kb from the ONECUT1 promoter. Previous studies have linked ONECUT1 coding mutations to pancreatic hypoplasia and neonatal diabetes. We found that homozygous deletion of ONECUT1e-664kb in hPSCs leads to a near-complete loss of ONECUT1 expression and impaired pancreatic differentiation. ONECUT1e-664kb contains a type 2 diabetes-associated variant (rs528350911) disrupting a GATA motif. Introducing the risk variant into hPSCs reduced binding of key pancreatic transcription factors (GATA4, GATA6, and FOXA2), supporting its causal role in diabetes. This work highlights the utility of unbiased enhancer discovery in disease-relevant settings for understanding monogenic and complex disease.
Assuntos
Diferenciação Celular , Elementos Facilitadores Genéticos , Pâncreas , Humanos , Elementos Facilitadores Genéticos/genética , Diferenciação Celular/genética , Pâncreas/metabolismo , Pâncreas/patologia , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Células-Tronco Pluripotentes/metabolismo , Sistemas CRISPR-Cas/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genéticaRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at metastatic stage and typically treated with fluorouracil, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX). Few patients benefit from this treatment. Molecular subtypes are prognostic in particularly resectable PDAC and might predict treatment response. This study aims to correlate molecular subtypes in metastatic PDAC with FOLFIRINOX responses using real-world data, providing assistance in counselling patients. We collected 131 RNA-sequenced metastatic biopsies and applied a network-based meta-analysis using published PDAC classifiers. Subsequent survival analysis was performed using the most suitable classifier. For validation, we developed an immunohistochemistry (IHC) classifier using GATA6 and keratin-17 (KRT17), and applied it to 86 formalin-fixed paraffin-embedded samples of advanced PDAC. Lastly, GATA6 knockdown models were generated in PDAC organoids and cell lines. We showed that the PurIST classifier was the most suitable classifier. With this classifier, classical tumors had longer PFS and OS than basal-like tumors (PFS: 216 vs. 78 days, p = 0.0002; OS: 251 vs. 195 days, p = 0.049). The validation cohort showed a similar trend. Importantly, IHC GATA6low patients had significantly shorter survival with FOLFIRINOX (323 vs. 746 days, p = 0.006), but no difference in non-treated patients (61 vs. 54 days, p = 0.925). This suggests that GATA6 H-score predicts therapy response. GATA6 knockdown models did not lead to increased FOLFIRINOX responsiveness. These data suggest a predictive role for subtyping (transcriptomic and GATA6 IHC), though no direct causal relationship was found between GATA6 expression and chemoresistance. GATA6 immunohistochemistry should be seamlessly added to current diagnostics and integrated into upcoming clinical trials.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Fluoruracila , Fator de Transcrição GATA6 , Irinotecano , Leucovorina , Oxaliplatina , Neoplasias Pancreáticas , Transcriptoma , Humanos , Leucovorina/uso terapêutico , Oxaliplatina/uso terapêutico , Irinotecano/uso terapêutico , Fluoruracila/uso terapêutico , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Estudos de Coortes , Idoso , Linhagem Celular Tumoral , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/mortalidade , Metástase Neoplásica , Resultado do TratamentoRESUMO
There is a significant difference in prognosis and response to chemotherapy between basal and classical subtypes of pancreatic ductal adenocarcinoma (PDAC). Further biomarkers are required to identify subtypes of PDAC. We selected candidate biomarkers via review articles. Correlations between these candidate markers and the PDAC molecular subtype gene sets were analyzed using bioinformatics, confirming the biomarkers for identifying classical and basal subtypes. Subsequently, 298 PDAC patients were included, and their tumor tissues were immunohistochemically stratified using these biomarkers. Survival data underwent analysis, including Cox proportional hazards modeling. Our results indicate that the pairwise and triple combinations of KRT5/KRT17/S100A2 exhibit a higher correlation coefficient with the basal-like subtype gene set, whereas the corresponding combinations of GATA6/HNF4A/TFF1 show a higher correlation with the classical subtype gene set. Whether analyzing unmatched or propensity-matched data, the overall survival time was significantly shorter for the basal subtype compared with the classical subtype (p < .001), with basal subtype patients also facing a higher risk of mortality (HR = 4.017, 95% CI 2.675-6.032, p < .001). In conclusion, the combined expression of KRT5, KRT17, and S100A2, in both pairwise and triple combinations, independently predicts shorter overall survival in PDAC patients and likely identifies the basal subtype. Similarly, the combined expression of GATA6, HNF4A, and TFF1, in the same manner, may indicate the classical subtype. In our study, the combined application of established biomarkers offers valuable insights for the prognostic evaluation of PDAC patients.
Assuntos
Biomarcadores Tumorais , Carcinoma Ductal Pancreático , Queratina-17 , Queratina-5 , Neoplasias Pancreáticas , Proteínas S100 , Humanos , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Masculino , Feminino , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Proteínas S100/genética , Proteínas S100/metabolismo , Queratina-5/genética , Queratina-5/metabolismo , Idoso , Queratina-17/genética , Queratina-17/metabolismo , Prognóstico , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Regulação Neoplásica da Expressão Gênica , Adulto , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Fatores QuimiotáticosRESUMO
Endometriosis is a chronic inflammatory disease that causes debilitating pelvic pain in women. Macrophages are considered to be key players in promoting disease progression, as abundant macrophages are present in ectopic lesions and elevated in the peritoneum. In the present study, we examined the role of GATA6+ peritoneal macrophages on endometriosis-associated hyperalgesia using mice with a specific myeloid deficiency of GATA6. Lesion induction induced the disappearance of TIM4hi MHCIIlo residential macrophages and the influx of increased Ly6C+ monocytes and TIM4lo MHCIIhi macrophages. The recruitment of MHCIIhi inflammatory macrophages was extensive in Mac Gata6 KO mice due to the severe disappearance of TIM4hi MHCIIlo residential macrophages. Ki67 expression confirmed GATA6-dependent proliferative ability, showing different proliferative phenotypes of TIM4+ residential macrophages in Gata6f/f and Mac Gata6 KO mice. Peritoneal proinflammatory cytokines were elevated after lesion induction. When cytokine levels were compared between Gata6f/f and Mac Gata6 KO mice, TNFα at day 21 in Gata6f/f mice was higher than in Mac Gata6 KO mice. Lesion induction increased both abdominal and hind paw sensitivities. Gata6f/f mice tended to show higher sensitivity in the abdomen after day 21. Elevated expression of TRPV1 and CGRP was observed in the dorsal root ganglia after ELL induction in Gata6f/f mice until days 21 and 42, respectively. These results support that peritoneal GATA6+ macrophages are involved in the recruitment and reprogramming of monocyte-derived macrophages. The extensive recruitment of monocyte-derived macrophages in Mac Gata6 KO mice might protect against inflammatory stimuli during the resolution phase, whereas GATA6 deficiency did not affect lesion initiation and establishment at the acute phase of inflammation. GATA6+ residential macrophages act to sustain local inflammation in the peritoneum and sensitivities in the neurons, reflecting endometriosis-associated hyperalgesia.
Assuntos
Endometriose , Fator de Transcrição GATA6 , Macrófagos Peritoneais , Animais , Feminino , Camundongos , Citocinas/metabolismo , Modelos Animais de Doenças , Endometriose/imunologia , Endometriose/patologia , Endometriose/metabolismo , Gânglios Espinais/metabolismo , Gânglios Espinais/imunologia , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Hiperalgesia/etiologia , Hiperalgesia/metabolismo , Hiperalgesia/imunologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peritônio/patologia , Peritônio/imunologia , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/genéticaRESUMO
Although sequence-based studies show that basal-like features lead to worse prognosis and chemotherapy-resistance compared to the classical subtype in advanced pancreatic ductal adenocarcinoma (PDAC), a surrogate biomarker distinguishing between these subtypes in routine diagnostic practice remains to be identified. We aimed to evaluate the utility of immunohistochemistry (IHC) expression subtypes generated by unsupervised hierarchical clustering based on staining scores of four markers (CK5/6, p63, GATA6, HNF4a) applied to endoscopic ultrasound-guided fine needle aspiration biopsy (EUS-FNAB) materials. EUS-FNAB materials taken from 190 treatment-naïve advanced PDAC patients were analyzed, and three IHC patterns were established (Classical, Transitional, and Basal-like pattern). Basal-like pattern (high co-expression of CK5/6 and p63 with low expression of GATA6 and HNF4a) was significantly associated with squamous differentiation histology (p < 0.001) and demonstrated the worst overall survival among our cohort (p = 0.004). IHC expression subtype (Transitional, Basal vs Classical) was an independent poor prognosticator in multivariate analysis [HR 1.58 (95% CI 1.01-2.38), p = 0.047]. Furthermore, CK5/6 expression was an independent poor prognostic factor in histological glandular type PDAC [HR 2.82 (95% CI 1.31-6.08), p = 0.008]. Our results suggest that IHC expression patterns successfully predict molecular features indicative of the Basal-like subgroup in advanced PDAC. These results provide the basis for appropriate stratification for therapeutic selection and prognostic estimation of advanced PDAC in a simplified manner.
Assuntos
Biomarcadores Tumorais , Carcinoma Ductal Pancreático , Fator de Transcrição GATA6 , Fator 4 Nuclear de Hepatócito , Imuno-Histoquímica , Neoplasias Pancreáticas , Humanos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/mortalidade , Masculino , Feminino , Fator 4 Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Idoso , Biomarcadores Tumorais/metabolismo , Pessoa de Meia-Idade , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/genética , Prognóstico , Queratina-5/metabolismo , Queratina-6/metabolismo , Idoso de 80 Anos ou mais , Adulto , Aspiração por Agulha Fina Guiada por Ultrassom Endoscópico , Fatores de Transcrição , Proteínas Supressoras de TumorRESUMO
Vascular calcification in diabetes patients is a major independent risk factor for developing diabetic cardiovascular complications. However, the mechanisms by which diabetes leads to vascular calcification are complex and not yet fully understood. Our previous study revealed that miR-32-5p is a potential new diagnostic marker for coronary artery calcification. In this study, we found that miR-32-5p levels were significantly greater in the plasma of type 2 diabetes patients with coronary artery calcification and were positively correlated with the coronary artery calcification score. In type 2 diabetic mice, miR-32-5p levels were also elevated in the aorta, and knockout of miR-32-5p inhibited the osteogenic differentiation of vascular smooth muscle cells in vivo. Furthermore, overexpression of miR-32-5p promoted vascular smooth muscle cell calcification, while antagonism of miR-32-5p inhibited vascular smooth muscle cell calcification under high-glucose conditions. GATA binding protein 6 (GATA6) was identified as the key target gene through which miR-32-5p promotes vascular smooth muscle cell calcification. Overexpression of GATA6 antagonized the effects of miR-32-5p on vascular calcification. Additionally, high glucose levels were shown to induce the upregulation of miR-32-5p by activating CCAAT/enhancer binding protein beta (CEBPB). These results suggest that miR-32-5p is an important procalcification factor in vascular calcification associated with type 2 diabetes and identify the CEBPB/miR-32-5p/GATA6 axis as a potential biomarker and therapeutic target for preventing and treating vascular calcification in type 2 diabetes.
Assuntos
Proteína beta Intensificadora de Ligação a CCAAT , Diabetes Mellitus Tipo 2 , Fator de Transcrição GATA6 , MicroRNAs , Calcificação Vascular , Animais , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/genética , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diabetes Mellitus Tipo 2/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/genética , MicroRNAs/metabolismo , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/genéticaRESUMO
BACKGROUND: Lung squamous cell carcinoma (LUSC) is associated with high mortality and has limited therapeutic treatment options. Plasminogen activator urokinase (PLAU) plays important roles in tumor cell malignancy. However, the oncogenic role of PLAU in the progression of LUSC remains unknown. GATA-binding factor 6 (GATA6), a key regulator of lung development, inhibits LUSC cell proliferation and migration, but the underlying regulatory mechanism remains to be further explored. Moreover, the regulatory effect of GATA6 on PLAU expression has not been reported. The aim of this study was to identify the role of PLAU and the transcriptional inhibition mechanism of GATA6 on PLAU expression in LUSC. METHODS: To identify the potential target genes regulated by GATA6, differentially expressed genes (DEGs) obtained from GEO datasets analysis and RNA-seq experiment were subjected to Venn analysis and correlation heatmap analysis. The transcriptional regulatory effects of GATA6 on PLAU expression were detected by real-time PCR, immunoblotting, and dual-luciferase reporter assays. The oncogenic effects of PLAU on LUSC cell proliferation and migration were evaluated by EdU incorporation, Matrigel 3D culture and Transwell assays. PLAU expression was detected in tissue microarray of LUSC via immunohistochemistry (IHC) assay. To determine prognostic factors for prognosis of LUSC patients, the clinicopathological characteristics and PLAU expression were subjected to univariate Cox regression analysis. RESULTS: PLAU overexpression promoted LUSC cell proliferation and migration. PLAU is overexpressed in LUSC tissues compared with normal tissues. Consistently, high PLAU expression, which acts as an independent risk factor, is associated with poor prognosis of LUSC patients. Furthermore, the expression of PLAU is transcriptionally regulated by GATA6. CONCLUSION: In this work, it was revealed that PLAU is a novel oncogene for LUSC and a new molecular regulatory mechanism of GATA6 in LUSC was unveiled. Targeting the GATA6/PLAU pathway might help in the development of novel therapeutic treatment strategies for LUSC.
Assuntos
Carcinoma de Células Escamosas , Movimento Celular , Proliferação de Células , Fator de Transcrição GATA6 , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Humanos , Proliferação de Células/genética , Movimento Celular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Linhagem Celular Tumoral , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Proteínas de MembranaRESUMO
This study underscores GATA6's role in distinguishing classical and basal-like pancreatic ductal adenocarcinoma (PDAC) phenotypes. Retrospective studies associate GATA6 immunohistochemistry (IHC) expression with survival outcomes, warranting prospective validation. In a prospective treatment-naive cohort of patients with resected PDAC, GATA6 IHC proves a prognostic discriminator, associating high GATA6 expression with extended survival and the classical PDAC phenotype. However, GATA6's prognostic significance is numerically lower after gemcitabine-based neoadjuvant chemoradiotherapy compared to its significance in patients treated with upfront surgery. Furthermore, GATA6 is implicated in immunomodulation, although a comprehensive investigation of its immunological role is lacking. Treatment-naive PDAC tumors with varying GATA6 expression yield distinct immunological landscapes. Tumors highly expressing GATA6 show reduced infiltration of immunosuppressive regulatory T cells and M2 macrophages but increased infiltration of immune-stimulating, antigen-presenting, and activated T cells. Our findings caution against solely relying on GATA6 for molecular subtyping in clinical trials and open avenues for exploring immune-based combination therapies.
Assuntos
Carcinoma Ductal Pancreático , Fator de Transcrição GATA6 , Neoplasias Pancreáticas , Fenótipo , Humanos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Masculino , Feminino , Prognóstico , Idoso , Pessoa de Meia-Idade , Macrófagos/imunologia , Macrófagos/metabolismo , Resultado do Tratamento , Terapia Neoadjuvante/métodos , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/genéticaRESUMO
GATA6 is expressed during early embryogenesis and localizes to endoderm- and mesoderm-derived tissues during later embryogenesis. Here, we established a human induced pluripotent stem cell (hiPSC) line expressing EGFP under GATA6 gene. EGFP coding sequence was introduced into the C-terminus of GATA6 in KSCBi017-A hiPSCs through homologous recombination using CRISPR/Cas9 system. The successfully edited line, KSCBi017-A-1, was selected and confirmed by sequencing. The line had a normal karyotype and exhibited potential to differentiate into three germ layers while it expressed EGFP upon endoderm induction. KSCBi017-A-1 cells can be used to monitor the expression of GATA6 during differentiation. This cell line is available from Korea National Stem Cell Bank.
Assuntos
Sistemas CRISPR-Cas , Fator de Transcrição GATA6 , Proteínas de Fluorescência Verde , Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Humanos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Linhagem Celular , Diferenciação CelularRESUMO
The traditional view of hematopoiesis is that myeloid cells derive from a common myeloid progenitor (CMP), whereas all lymphoid cell populations, including B, T, and natural killer (NK) cells and possibly plasmacytoid dendritic cells (pDCs), arise from a common lymphoid progenitor (CLP). In Max41 transgenic mice, nearly all B cells seem to be diverted into the granulocyte lineage. Here, we show that these mice have an excess of myeloid progenitors, but their CLP compartment is ablated, and they have few pDCs. Nevertheless, T cell and NK cell development proceeds relatively normally. These hematopoietic abnormalities result from aberrant expression of Gata6 due to serendipitous insertion of the transgene enhancer (Eµ) in its proximity. Gata6 mis-expression in Max41 transgenic progenitors promoted the gene-regulatory networks that drive myelopoiesis through increasing expression of key transcription factors, including PU.1 and C/EBPa. Thus, mis-expression of a single key regulator like GATA6 can dramatically re-program multiple aspects of hematopoiesis.
Assuntos
Fator de Transcrição GATA6 , Hematopoese , Camundongos Transgênicos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Animais , Camundongos , Linhagem da Célula , Células Matadoras Naturais/metabolismo , Células Matadoras Naturais/imunologia , Camundongos Endogâmicos C57BL , Células Dendríticas/metabolismo , Diferenciação Celular , Linfócitos T/metabolismo , Linfócitos T/citologia , Proteínas Proto-Oncogênicas , TransativadoresRESUMO
Recent interest in the biology and function of peritoneal tissue resident macrophages (pMΦ) has led to a better understanding of their cellular origin, programming, and renewal. The programming of pMΦ is dependent on microenvironmental cues and tissue-specific transcription factors, including GATA6. However, the contribution of microRNAs remains poorly defined. We conducted a detailed analysis of the impact of GATA6 deficiency on microRNA expression in mouse pMΦ. Our data suggest that for many of the pMΦ, microRNA composition may be established during tissue specialization and that the effect of GATA6 knockout is largely unable to be rescued in the adult by exogenous GATA6. The data are consistent with GATA6 modulating the expression pattern of specific microRNAs, directly or indirectly, and including miR-146a, miR-223, and miR-203 established by the lineage-determining transcription factor PU.1, to achieve a differentiated pMΦ phenotype. Lastly, we showed a significant dysregulation of miR-708 in pMΦ in the absence of GATA6 during homeostasis and in response to LPS/IFN-γ stimulation. Overexpression of miR-708 in mouse pMΦ in vivo altered 167 mRNA species demonstrating functional downregulation of predicted targets, including cell immune responses and cell cycle regulation. In conclusion, we demonstrate dependence of the microRNA transcriptome on tissue-specific programming of tissue macrophages as exemplified by the role of GATA6 in pMΦ specialization.
Assuntos
Fator de Transcrição GATA6 , Macrófagos Peritoneais , MicroRNAs , Transcriptoma , Animais , Camundongos , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/genética , Regulação da Expressão Gênica , Lipopolissacarídeos/farmacologia , Macrófagos Peritoneais/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , MicroRNAs/metabolismo , Especificidade de Órgãos , Proteínas Proto-Oncogênicas , Transativadores/genética , Transativadores/metabolismoRESUMO
PURPOSE: This study aims to explore the role of GATA6 in lung cancer, with a focus on its impact on metabolic processes. METHODS: We assessed GATA6 expression in lung cancer tissues and its association with patient prognosis. In vitro cell function experiments were conducted to investigate the effects of altered GATA6 levels on lung cancer cell proliferation and migration. Mechanistic insights were gained by examining GATA6's influence on glucose metabolism-related genes, particularly its effect on c-Myc mRNA expression. RESULTS: Our study revealed significant down-regulation of GATA6 in lung cancer tissues, and this down-regulation was strongly correlated with unfavorable patient prognosis. Elevating GATA6 levels effectively inhibited the proliferation and migration of lung cancer cells in our cell function experiments. Mechanistically, we found that GATA6 suppressed the expression of c-Myc mRNA, impacting genes related to glucose metabolism. As a result, glucose uptake and metabolism in lung cancer cells were disrupted, ultimately impeding their malignant behaviors. CONCLUSION: Our study provides crucial insights into the metabolic regulation of GATA6 in lung cancer cells. These findings have the potential to offer a solid theoretical foundation for the development of novel clinical treatments for lung cancer.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Linhagem Celular Tumoral , RNA Mensageiro/genética , Glucose , Proliferação de Células , Regulação Neoplásica da Expressão Gênica , Fator de Transcrição GATA6/genéticaRESUMO
ABSTRACT: Epithelioid hemangioma (EH) is a benign vascular tumor displaying diverse histomorphologies. Among these, one EH subtype comprises cellular sheets of atypical epithelioid cells, posing potential challenges in distinguishing it from malignant vascular lesions. In this case report, we present a cutaneous cellular EH that carries the rare GATA6::FOXO1 gene fusion, a recent discovery. Our aim is to provide an updated insight into the evolving knowledge of EHs while delving into the histologic and molecular characteristics of the primary differential diagnoses.
Assuntos
Hiperplasia Angiolinfoide com Eosinofilia , Hemangioendotelioma Epitelioide , Hemangioma , Neoplasias Vasculares , Humanos , Hiperplasia Angiolinfoide com Eosinofilia/patologia , Hemangioma/patologia , Fusão Gênica , Diagnóstico Diferencial , Hemangioendotelioma Epitelioide/genética , Proteína Forkhead Box O1/genética , Fator de Transcrição GATA6/genéticaRESUMO
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.
Assuntos
Fator de Transcrição GATA6 , MicroRNAs , Neoplasias Ovarianas , Fator de Transcrição GATA6/antagonistas & inibidores , Fator de Transcrição GATA6/genética , MicroRNAs/genética , Neoplasias Ovarianas/terapia , Progressão da Doença , Humanos , Linhagem Celular Tumoral , Células HEK293 , Ensaios Antitumorais Modelo de Xenoenxerto , Camundongos Nus , Animais , Camundongos , Lentivirus , Vetores Genéticos , Feminino , Movimento Celular , Proliferação de CélulasRESUMO
BACKGROUND: Pathogenic GATA6 variants have been associated with congenital heart disease (CHD) and a spectrum of extracardiac abnormalities, including pancreatic agenesis, congenital diaphragmatic hernia, and developmental delay. However, the comprehensive genotype-phenotype correlation of pathogenic GATA6 variation in humans remains to be fully understood. METHODS: Exome sequencing was performed in a family where four members had CHD. In vitro functional analysis of the GATA6 variant was performed using immunofluorescence, western blot, and dual-luciferase reporter assay. RESULTS: A novel, heterozygous missense variant in GATA6 (c.1403 G > A; p.Cys468Tyr) segregated with affected members in a family with CHD, including three with persistent truncus arteriosus. In addition, one member had childhood onset diabetes mellitus (DM), and another had necrotizing enterocolitis (NEC) with intestinal perforation. The p.Cys468Tyr variant was located in the c-terminal zinc finger domain encoded by exon 4. The mutant protein demonstrated an abnormal nuclear localization pattern with protein aggregation and decreased transcriptional activity. CONCLUSIONS: We report a novel, familial GATA6 likely pathogenic variant associated with CHD, DM, and NEC with intestinal perforation. These findings expand the phenotypic spectrum of pathologic GATA6 variation to include intestinal abnormalities. IMPACT: Exome sequencing identified a novel heterozygous GATA6 variant (p.Cys468Tyr) that segregated in a family with CHD including persistent truncus arteriosus, atrial septal defects and bicuspid aortic valve. Additionally, affected members displayed extracardiac findings including childhood-onset diabetes mellitus, and uniquely, necrotizing enterocolitis with intestinal perforation in the first four days of life. In vitro functional assays demonstrated that GATA6 p.Cys468Tyr variant leads to cellular localization defects and decreased transactivation activity. This work supports the importance of GATA6 as a causative gene for CHD and expands the phenotypic spectrum of pathogenic GATA6 variation, highlighting neonatal intestinal perforation as a novel extracardiac phenotype.
Assuntos
Diabetes Mellitus , Enterocolite Necrosante , Doenças Fetais , Cardiopatias Congênitas , Perfuração Intestinal , Persistência do Tronco Arterial , Feminino , Recém-Nascido , Humanos , Criança , Cardiopatias Congênitas/genética , Fator de Transcrição GATA6/genéticaRESUMO
Arterial calcification is a hallmark of vascular pathology in the elderly and in individuals with chronic kidney disease (CKD). Vascular smooth muscle cells (VSMCs), after attaining a senescent phenotype, are implicated in the calcifying process. However, the underlying mechanism remains to be elucidated. Here, we reveal an aberrant upregulation of transcriptional factor GATA6 in the calcified aortas of humans, mice with CKD and mice subjected to vitamin D3 injection. Knockdown of GATA6, via recombinant adeno-associated virus carrying GATA6 shRNA, inhibited the development of arterial calcification in mice with CKD. Further gain- and loss-of function experiments in vitro verified the contribution of GATA6 in osteogenic differentiation of VSMCs. Samples of human aorta exhibited a positive relationship between age and GATA6 expression and GATA6 was also elevated in the aortas of old as compared to young mice. Calcified aortas displayed senescent features with VSMCs undergoing premature senescence, blunted by GATA6 downregulation. Notably, abnormal induction of GATA6 in senescent and calcified aortas was rescued in Sirtuin 6 (SIRT6)-transgenic mice, a well-established longevity mouse model. Suppression of GATA6 accounted for the favorable effect of SIRT6 on VSMCs senescence prevention. Mechanistically, SIRT6 inhibited the transcription of GATA6 by deacetylation and increased degradation of transcription factor Nkx2.5. Moreover, GATA6 was induced by DNA damage stress during arterial calcification and subsequently impeded the Ataxia-telangiectasia mutated (ATM)-mediated DNA damage repair process, leading to accelerated VSMCs senescence and osteogenic differentiation. Thus, GATA6 is a novel regulator in VSMCs senescence. Our findings provide novel insight in arterial calcification and a potential new target for intervention.
Assuntos
Insuficiência Renal Crônica , Sirtuínas , Calcificação Vascular , Humanos , Camundongos , Animais , Idoso , Músculo Liso Vascular , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/farmacologia , Osteogênese , Células Cultivadas , Insuficiência Renal Crônica/patologia , Dano ao DNA , Senescência Celular/genética , Envelhecimento/genética , Sirtuínas/genética , Sirtuínas/metabolismo , Calcificação Vascular/genética , Calcificação Vascular/metabolismo , Miócitos de Músculo Liso/metabolismoRESUMO
OBJECTIVE: To study the effect of adenomyosis on the localized expression of the GATA binding proteins 2 and 6 (GATA2 and GATA6) zinc-finger transcription factors that are involved in proliferation of hematopoietic and endocrine cell lineages, cell differentiation, and organogenesis, potentially leading to impaired endometrial implantation. DESIGN: Laboratory based experimental study. SETTING: Academic hospital and laboratory. PATIENTS: Human endometrial stromal cells (HESCs) of reproductive age patients, 18-45 years of age, with adenomyosis were compared with patients with no pathology and leiomyomatous uteri as controls (n = 4 in each group, respectively). Additionally, midsecretory phase endometrial sections were obtained from patients with adenomyosis and control patients with leiomyoma (n = 8 in each group, respectively). INTERVENTIONS: GATA2 and GATA6 immunohistochemistry and H-SCORE were performed on the midsecretory phase endometrial sections from adenomyosis and leiomyoma control patients (n = 8 each, respectively). Control and adenomyosis patient HESC cultures were treated with placebo or 10-8 M estradiol (E2), or decidualization media (EMC) containing 10-8 M E2, 10-7 M medroxyprogesterone acetate, and 5 × 10-5 M cAMP for 6 and 10 days. Additionally, control HESC cultures (n = 4) were transfected with scrambled small interfering RNA (siRNA) (control) or GATA2-specific siRNAs for 6 days while adenomyosis HESC cultures (n = 4) were transfected with human GATA2 expression vectors to silence or induce GATA2 overexpression. MAIN OUTCOME MEASURES: Immunohistochemistry was performed to obtain GATA2 and GATA6 H-SCORES in adenomyosis vs. control patient endometrial tissue. Expression of GATA2, GATA6, insulin-like growth factor-binding protein 1 (IGFBP1), prolactin (PRL), progesterone receptor (PGR), estrogen receptor 1 (ESR1), leukemia inhibitory factor (LIF), and Interleukin receptor 11 (IL11R) messenger RNA (mRNA) levels were analyzed using by qPCR with normalization to ACTB. Silencing and overexpression experiments also had the corresponding mRNA levels of the above factors analyzed. Western blot analysis was performed on isolated proteins from transfection experiments. RESULTS: Immunohistochemistry revealed an overall fourfold lower GATA2 and fourfold higher GATA6 H-SCORE level in the endometrial stromal cells of patients with adenomyosis vs. controls. Decidual induction with EMC resulted in significantly lower GATA2, PGR, PRL and IGFBP1 mRNA levels in HESC cultures from patients with adenomyosis patient vs. controls. Leukemia inhibitory factor and IL11R mRNA levels were also significantly dysregulated in adenomyosis HESCs compared with controls. . Silencing of GATA2 expression in control HESCs induced an adenomyosis-like state with significant reductions in GATA2, increases in GATA6 and accompanying aberrations in PGR, PRL, ESR1 and LIF levels. Conversely, GATA2 overexpression via vector in adenomyosis HESCs caused partial restoration of the defective decidual response with significant increases in GATA2, PGR, PRL and LIF expression. CONCLUSION: In-vivo and in-vitro experiment results demonstrate that there is an overall inverse relationship between endometrial GATA2 and GATA6 levels in patients with adenomyosis who have diminished GATA2 levels and concurrently elevated GATA6 levels. Additionally, lower GATA2 and higher GATA6 levels, together with aberrant levels of important receptors and implantation factors, such as ESR1, PGR, IGFBP1, PRL, LIF, and IL11R mRNA in HESCs from patients with adenomyosis or GATA2-silenced control HESCs, support impaired decidualization. These effects were partially restored with GATA2 overexpression in adenomyosis HESCs, demonstrating a potential therapeutic target.
Assuntos
Adenomiose , Fator de Transcrição GATA2 , Fator de Transcrição GATA6 , Adolescente , Adulto , Feminino , Humanos , Pessoa de Meia-Idade , Adulto Jovem , Adenomiose/genética , Adenomiose/metabolismo , Adenomiose/patologia , Decídua/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismo , Fator de Transcrição GATA2/farmacologia , Fator de Transcrição GATA6/genética , Fator de Transcrição GATA6/metabolismo , Fator de Transcrição GATA6/farmacologia , Leiomioma , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Prolactina/metabolismo , Prolactina/farmacologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/farmacologia , Fatores de TranscriçãoRESUMO
Monogenic diabetes caused by GATA6 mutations were almost described as neonatal diabetes, and the phenotypic spectrum has expanded since then. Our study underscores the broad phenotypic spectrum by reporting a de novo GATA6 mutation in a family. Furthermore, we reviewed related literature to summarize the clinical and genetic characteristics of monogenic diabetes with GATA6 mutations (n = 39) in order to improve clinicians' understanding of the disease. We conclude that the GATA6 missense mutation (c. 749G > T, p. Gly250Val) is not reported presently, characterized by adult-onset diabetes with pancreatic dysplasia and located in transcriptional activation region. Carries with GATA6 mutations (n = 55) have a variable spectrum of diabetes, ranging from neonatal (72.7%), childhood-onset (20%) to adults-onset (7.5%). 83.5% of patients with abnormal pancreatic development. Heart and hepatobillary defects are the most common abnormalities of extrapancreatic features. Most mutations with GATA6 are loss of function (LOF, 71.8%) and located in functional region. Functional studies mostly support loss-of-function as the pathophysiological mechanism. In conclusion, there are various types of diabetes with GATA6 mutations, which can also occur in adult diabetes. Phenotypic defects with GATA6 mutations are most frequently malformations of pancreas and heart. This highlights the importance of comprehensive clinical evaluation of identified carriers to evaluate their full phenotypic spectrum.