RESUMO
Pluripotent stem cells (PSC) endocrine differentiation at a large scale allows sampling of transcriptome and proteome with phosphoproteome (proteoform) at specific time points. We describe the dynamic time course of changes in cells undergoing directed beta-cell differentiation and show target proteins or previously unknown phosphorylation of critical proteins in pancreas development, NKX6-1, and Chromogranin A (CHGA). We describe fluctuations in the correlation between gene expression, protein abundance, and phosphorylation, following differentiation protocol perturbations at all stages to identify proteoform profiles. Our modeling recognizes outliers on a phenomic landscape of endocrine differentiation, and we describe new biological pathways involved. We have validated our proteomic data by analyzing independent single-cell RNAseq datasets for in-vitro pancreatic islet production and corroborated our findings for several proteins suggestive as targets for future research. The single-cell analysis combined with proteoform data places new protein targets within the specific time point and at the specific pancreatic lineage of differentiating stem cells. We suggest that non-correlating proteins abundances or new phosphorylation motifs of NKX6.1 and CHGA point to new signaling pathways that may play an essential role in beta-cell development. We present our findings for the research community's use to improve endocrine differentiation protocols and developmental studies.
Assuntos
Células-Tronco Embrionárias Humanas , Células-Tronco Pluripotentes , Diferenciação Celular/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes/metabolismo , ProteômicaRESUMO
BACKGROUND: The dysfunction of pancreatic ß cells is related to the occurrence of gestational diabetes mellitus (GDM). This study aimed to investigate the mechanism underlying the effects of miR-190b on pancreatic ß cell proliferation and insulin secretion. METHODS: Quantitative real-time PCR was used to detect miR-190b expression in placenta tissues from GDM patients. The effects of miR-190b on islet cells activity, proliferation, and insulin secretion were measured using MTT assay, BrdU staining, and ELISA. The relationship between miR-190b and NK6 homeobox 1 (NKX6-1) was ensured by dual luciferase reporter assay. RESULTS: MiR-190b was overexpressed in placenta tissues from GDM patients compared to normal pregnant woman. MiR-190b inhibitor inhibited the cell activity, proliferation, and insulin secretion of islet ß cells, while miR-190b overexpression had an opposite effect. Additionally, miR-190b negatively regulated NKX6-1 expression. Overexpression of NKX6-1 reversed the inhibitory effect of miR-190b-mimics on islet ß cell activity, proliferation, and insulin secretion. In mouse islets, knockdown of miR-190b promoted insulin secretion by up-regulating NKX6-1 expression. CONCLUSION: Silence of miR-190b accelerated pancreatic ß cell proliferation and insulin secretion via targeting NKX6-1, which might be a mechanism underlying the effects of miR-190b on the progression of GDM.
Assuntos
Diabetes Gestacional/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , MicroRNAs/biossíntese , MicroRNAs/genética , Placenta/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Feminino , Inativação Gênica , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Camundongos , MicroRNAs/metabolismo , Gravidez , Reação em Cadeia da Polimerase em Tempo RealRESUMO
BACKGROUND: Leiomyosarcoma (LMS), the most common soft tissue sarcoma, exhibits heterogeneous and complex genetic karyotypes with severe chromosomal instability and rearrangement and poor prognosis. METHODS: Clinical variables associated with NKX6-1 were obtained from The Cancer Genome Atlas (TCGA). NKX6-1 mRNA expression was examined in 49 human uterine tissues. The in vitro effects of NXK6-1 in LMS cells were determined by reverse transcriptase PCR, western blotting, colony formation, spheroid formation, and cell viability assays. In vivo tumor growth was evaluated in nude mice. RESULTS: Using The Cancer Genome Atlas (TCGA) and human uterine tissue datasets, we observed that NKX6-1 expression was associated with poor prognosis and malignant potential in LMS. NKX6-1 enhanced in vitro tumor cell aggressiveness via upregulation of cell proliferation and anchorage-independent growth and promoted in vivo tumor growth. Moreover, overexpression and knockdown of NKX6-1 were associated with upregulation and downregulation, respectively, of stem cell transcription factors, including KLF8, MYC, and CD49F, and affected sphere formation, chemoresistance, NOTCH signaling and Sonic hedgehog (SHH) pathways in human sarcoma cells. Importantly, treatment with an SHH inhibitor (RU-SKI 43) but not a NOTCH inhibitor (DAPT) reduced cell survival in NKX6-1-expressing cancer cells, indicating that an SHH inhibitor could be useful in treating LMS. Finally, using the TCGA dataset, we demonstrated that LMS patients with high expression of NKX6-1 and HHAT, an SHH pathway acyltransferase, had poorer survival outcomes compared to those without. CONCLUSIONS: Our findings indicate that NKX6-1 and HHAT play critical roles in the pathogenesis of LMS and could be promising diagnostic and therapeutic targets for LMS patients.
Assuntos
Proteínas Hedgehog/genética , Proteínas de Homeodomínio/genética , Leiomiossarcoma/metabolismo , Células-Tronco Neoplásicas/metabolismo , Transdução de Sinais , Animais , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Proteínas Hedgehog/metabolismo , Proteínas de Homeodomínio/metabolismo , Camundongos , Camundongos NusRESUMO
BACKGROUND: NKX6.1 is a transcription factor for insulin, as well as a marker for ß cell maturity. Abnormal NKX6.1 expression in ß cells, such as translocation from the nucleus to cytoplasm or lost expression, has been shown as a marker for ß cell dedifferentiation. METHODS: We obtained pancreatic sections from organ donors and immunofluorescence staining with NKX6.1 and insulin was performed to characterize NKX6.1 expression in subjects with or without type 2 diabetes mellitus (T2DM). RESULTS: Our results showed that cells with insulin expression but no nucleic NKX6.1 expression (NKX6.1Nuc-Ins+), and cells with cytoplasmic NKX6.1 expression but no insulin expression (NKX6.1cytIns-) were significantly increased in T2DM subjects and positively correlated with glycated hemoglobin (HbA1c), indicating the elevated ß cell dedifferentiation with NKX6.1 inactivation in T2DM. To investigate whether ß cell dedifferentiation has initiated in subjects with higher risks for T2DM, we next analyzed the association between ß-cell dedifferentiation level in ND subjects with different ages, body mass index, and HbA1c. The results showed the absolute number and percentage of dedifferentiated ß cells with NKX6.1 inactivation did not significantly change in subjects with advanced aging, obesity, or modest hyperglycemia, indicating that the ß cell dedifferentiation might mainly occur after T2DM was diagnosed. CONCLUSION: Our results suggested that NKX6.1 expression in ß cells was changed in type 2 diabetic subjects, evidenced by significantly increased NKX6.1Nuc-Ins+ and NKX6.1cytIns- cells. This abnormality did not occur more frequently in subjects with a higher risk for T2DM, suggesting that ß cell dedifferentiation might be secondary to the pathological changes in T2DM.
Assuntos
Diabetes Mellitus Tipo 2/metabolismo , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Estado Pré-Diabético/metabolismo , Adulto , Idoso , Autopsia , Estudos de Casos e Controles , Contagem de Células , Diferenciação Celular , Diabetes Mellitus Tipo 2/patologia , Feminino , Hemoglobinas Glicadas/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Células Secretoras de Insulina/fisiologia , Masculino , Pessoa de Meia-Idade , Estado Pré-Diabético/patologia , Fatores de RiscoRESUMO
Nkx6-1 is a member of the Nkx family of homeodomain transcription factors (TFs) that regulates motor neuron development, neuron specification and pancreatic endocrine and ß-cell differentiation. To facilitate the isolation and tracking of Nkx6-1-expressing cells, we have generated a novel Nkx6-1 Venus fusion (Nkx6-1-VF) reporter allele. The Nkx6-1-VF knock-in reporter is regulated by endogenous cis-regulatory elements of Nkx6-1 and the fluorescent protein fusion does not interfere with the TF function, as homozygous mice are viable and fertile. The nuclear localization of Nkx6-1-VF protein reflects the endogenous Nkx6-1 protein distribution. During embryonic pancreas development, the reporter protein marks the pancreatic ductal progenitors and the endocrine lineage, but is absent in the exocrine compartment. As expected, the levels of Nkx6-1-VF reporter are upregulated upon ß-cell differentiation during the major wave of endocrinogenesis. In the adult islets of Langerhans, the reporter protein is exclusively found in insulin-secreting ß-cells. Importantly, the Venus reporter activities allow successful tracking of ß-cells in live-cell imaging and their specific isolation by flow sorting. In summary, the generation of the Nkx6-1-VF reporter line reflects the expression pattern and dynamics of the endogenous protein and thus provides a unique tool to study the spatio-temporal expression pattern of this TF during organ development and enables isolation and tracking of Nkx6-1-expressing cells such as pancreatic ß-cells, but also neurons and motor neurons in health and disease.
Assuntos
Técnicas Citológicas , Proteínas de Homeodomínio/genética , Células Secretoras de Insulina/citologia , Pâncreas/metabolismo , Alelos , Animais , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Perfilação da Expressão Gênica , Genes Reporter , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pâncreas/embriologia , Domínios Proteicos , Proteínas Recombinantes de Fusão/química , Fatores de Transcrição/metabolismoRESUMO
AIMS/HYPOTHESIS: Although obesity is a predisposing factor for pancreatic beta cell dysfunction, the mechanisms underlying its negative effect on insulin-secreting cells is still poorly understood. The aim of this study was to identify islet long non-coding RNAs (lncRNAs) involved in obesity-mediated beta cell dysfunction. METHODS: RNA sequencing was performed to analyse the islets of high-fat diet (HFD)-fed mice and those of normal chow-fed mice (NCD). The function in beta cells of the selected lncRNA 1810019D21Rik (referred to in this paper as ROIT [regulator of insulin transcription]) was assessed after its overexpression or knockdown in MIN6 cells and primary islet cells, as well as in siRNA-treated mice. Then, RNA pull-down, RNA immunoprecipitation, coimmunoprecipitation and bisulphite sequencing were performed to investigate the mechanism of ROIT regulation of islet function. RESULTS: ROIT was dramatically downregulated in the islets of the obese mice, as well as in the sera of obese donors with type 2 diabetes, and was suppressed by HNF1B. Overexpression of ROIT in MIN6 cells and islets led to improved glucose homeostasis and insulin transcription. Investigation of the mechanism involved showed that ROIT bound to DNA methyltransferase 3a and caused its degradation through the ubiquitin proteasome pathway, which blocked the methylation of the Nkx6.1 promoter. CONCLUSIONS/INTERPRETATION: These findings functionally suggest a novel link between obesity and beta cell dysfunction via ROIT. Elucidating a precise mechanism for the effect of obesity on lncRNA expression will broaden our understanding of the pathophysiological development of diabetes and facilitate the design of better tools for diabetes prevention and treatment. DATA AVAILABILITY: The raw RNA sequencing data are available from the NCBI Gene Expression Omnibus (GEO series accession number GSE139991).
Assuntos
Metilação de DNA , Proteínas de Homeodomínio/genética , Insulina/genética , Obesidade/genética , RNA Longo não Codificante/genética , Animais , Células Cultivadas , DNA Metiltransferase 3A , Dieta Hiperlipídica , Regulação para Baixo , Proteínas de Homeodomínio/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Obesidade/metabolismo , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic ß-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 µmol/l (226-416 µmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 µg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic ß-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic ß-cell functions that deserve further scrutiny.
Assuntos
Complicações do Diabetes/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Erros Inatos do Transporte Tubular Renal/genética , Cálculos Urinários/genética , Idoso , Complicações do Diabetes/complicações , Complicações do Diabetes/patologia , Glucose/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Homozigoto , Humanos , Insulina/biossíntese , Insulina/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Mutação/genética , Erros Inatos do Transporte Tubular Renal/complicações , Erros Inatos do Transporte Tubular Renal/patologia , Ácido Úrico/metabolismo , Cálculos Urinários/complicações , Cálculos Urinários/patologia , Sequenciamento do ExomaRESUMO
Accumulating evidence suggests that NKX6.1 (NK homeobox 1) plays a role in various types of cancer. In our previous studies, we identified NKX6.1 hypermethylation as a promising marker and demonstrated that the NKX6.1 gene functions as a metastasis suppressor through the epigenetic regulation of the epithelial-to-mesenchymal transition (EMT) in cervical cancer. More recently, we have demonstrated that NKX6.1 methylation is related to the chemotherapy response in colorectal cancer (CRC). Nevertheless, the biological function of NKX6.1 in the tumorigenesis of CRC remains unclear. In this study, we showed that NKX6.1 suppresses tumorigenic and metastatic ability both in vitro and in vivo. NKX6.1 represses cell invasion partly through the modulation of EMT. The overexpression of NKX6.1 enhances chemosensitivity in CRC cells. To further explore how NKX6.1 exerts its tumor-suppressive function, we used RNA sequencing technology for comprehensive analysis. The results showed that differentially expressed genes (DEGs) were mainly related to cell migration, response to drug, transcription factor activity, and growth factor activity, suggesting that these DEGs are involved in the function of NKX6.1 suppressing cancer invasion and metastasis. Our results demonstrated that NKX6.1 functions as a tumor suppressor partly by repressing EMT and enhancing chemosensitivity in CRC, making it a potential therapeutic target.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Proteínas de Homeodomínio/genética , Animais , Carcinogênese/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Transformação Celular Neoplásica/genética , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Ontologia Genética , Genes Supressores de Tumor , Células HCT116 , Células HT29 , Xenoenxertos , Proteínas de Homeodomínio/antagonistas & inibidores , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica/genética , Invasividade Neoplásica/prevenção & controle , Metástase Neoplásica/genética , Metástase Neoplásica/prevenção & controle , Oxaliplatina/farmacologia , Regulação para CimaRESUMO
The homeobox transcription factor Nkx6.1 is sufficient to increase functional ß-cell mass, where functional ß-cell mass refers to the combination of ß-cell proliferation, glucose-stimulated insulin secretion (GSIS) and ß-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional ß-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase ß-cell proliferation in primary rat islets and the INS-1 832/13 ß-cell line. The increase in HDAC1-mediated proliferation occurs while maintaining GSIS and increasing ß-cell survival in response to apoptotic stimuli. We demonstrate that HDAC1 overexpression results in decreased expression of the cell cycle inhibitor Cdkn1b/p27 which is essential for inhibiting the G1 to S phase transition of the cell cycle. This corresponds with increased expression of key cell cycle activators, such as Cyclin A2, Cyclin B1 and E2F1, which are activated by activation of the Cdk4/Cdk6/Cyclin D holoenzymes due to down-regulation of Cdkn1b/p27. Finally, we demonstrate that overexpression of Cdkn1b/p27 inhibits HDAC1-mediated ß-cell proliferation. Our data suggest that HDAC1 is critical for the Nkx6.1-mediated pathway that enhances functional ß-cell mass.
Assuntos
Proliferação de Células/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Regulação para Baixo/fisiologia , Regulação Enzimológica da Expressão Gênica , Histona Desacetilase 1/biossíntese , Células Secretoras de Insulina/metabolismo , Animais , Células Cultivadas , Inibidor de Quinase Dependente de Ciclina p27/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p27/genética , Histona Desacetilase 1/genética , Humanos , Masculino , Ratos , Ratos WistarRESUMO
Colorectal cancer (CRC) is one of the most common cancers and the second leading cause of cancer-related deaths. Discrepancies in clinical outcomes are observed even among patients with same-stage CRC due to molecular heterogeneity. Thus, biomarkers for predicting prognosis in CRC patients are urgently needed. We previously demonstrated that stage II CRC patients with NKX6.1 methylation had poor 5-year overall survival. However, the methylation frequency of NKX6.1 was only 23% in 151 pairs of CRC tissues. Thus, we aimed to develop a more robust prognostic panel for CRC using NKX6.1 in combination with three genes: LIM homeobox transcription factor 1α (LMX1A), sex-determining region Y-box 1 (SOX1), and zinc finger protein 177 (ZNF177). Through quantitative methylation analysis, we found that LMX1A, SOX1, and ZNF177 were hypermethylated in CRC tissues. LMX1A methylation was significantly associated with poor 5-year overall, and disease-free survivals in stage I and II CRC patients. Sensitivity and specificity analyses of the four-gene combination revealed the best sensitivity and optimal specificity. Moreover, patients with the four-gene methylation profile exhibited poorer disease-free survival than those without methylation. A significant effect of the four-gene methylation status on overall survival and disease-free survival was observed in early stage I and II CRC patients (p = 0.0016 and p = 0.0230, respectively). Taken together, these results demonstrate that the combination of the methylation statuses of NKX6.1, LMX1A, SOX1, and ZNF177 creates a novel prognostic panel that could be considered a molecular marker for outcomes in CRC patients.
Assuntos
Neoplasias Colorretais , Metilação de DNA , DNA de Neoplasias , Proteínas de Neoplasias , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Intervalo Livre de Doença , Feminino , Células HCT116 , Células HT29 , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estadiamento de Neoplasias , Taxa de SobrevidaRESUMO
BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença/epidemiologia , Adolescente , Adulto , Estudos de Coortes , Exoma , Feminino , Biblioteca Gênica , Genômica , Hemoglobinas Glicadas/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Índia/epidemiologia , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo , Análise de Sequência de DNA , Receptores de Sulfonilureias/genética , Receptores de Sulfonilureias/metabolismo , Adulto JovemRESUMO
Among breast cancer subtypes, basal-like breast cancer is particularly aggressive, and research on the molecules involved in its pathology might contribute to therapy. In this study, we found that expression of NKX6.1, a homeobox transcription factor, is higher in basal-like breast cancer than in other subtypes. In loss-of-function experiments on basal-like breast cancer cell lines, NKX6.1-depleted cells exhibited reduced cell growth. Because cytokine interleukin-6 (IL-6) is expressed in basal-like breast cancer, and increases cell growth, we analyzed expression levels of IL6, an IL-6 gene, and observed reduced IL6 expression in NKX6.1-depleted cells. In a reporter assay, IL6 promoter activity was reduced by loss of NKX6.1 function. A pull-down assay showed that NKX6.1 binds to the proximal region in IL6 promoter. These results indicate that NKX6.1 directly up-regulates IL6 expression. To investigate further, we established cells with forced expression of IL-6. We observed that exogenous IL-6 expression restored the reduced cell growth of NKX6.1-depleted cells. Furthermore, orthotopic xenografts showed that NKX6.1-depleted cells lost the capacity for tumor formation. We therefore conclude that NKX6.1 is a factor for IL-6-regulated growth and tumor formation in basal-like breast cancer. Our findings facilitate profound understanding of basal-like breast cancer, and the development of suitable therapy.
Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Regulação Neoplásica da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Interleucina-6/genética , Regulação para Cima/genética , Animais , Sequência de Bases , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células , Retroalimentação Fisiológica , Feminino , Humanos , Interleucina-6/metabolismo , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Induced pluripotent stem cells (iPSC) possess an enormous potential as both, scientific and therapeutic tools. Their application in the regenerative medicine provides new treatment opportunities for numerous diseases, including type 1 diabetes. In this work we aimed to derive insulin producing cells (IPC) from iPS cells established in defined conditions. METHODS: We optimized iPSC generation protocol and created pluripotent cell lines with stably integrated PDX1 and NKX6.1 transgenes under the transcriptional control of doxycycline-inducible promoter. These cells were differentiated using small chemical molecules and recombinant Activin A in the sequential process through the definitive endoderm, pancreatic progenitor cells and insulin producing cells. Efficiency of the procedure was assessed by quantitative gene expression measurements, immunocytochemical stainings and functional assays for insulin secretion. RESULTS: Generated cells displayed molecular markers characteristic for respective steps of the differentiation. The obtained IPC secreted insulin and produced C-peptide with significantly higher hormone release level in case of the combined expression of PDX1 and NKX6.1 induced at the last stage of the differentiation. CONCLUSIONS: Efficiency of differentiation of iPSC to IPC can be increased by concurrent expression of PDX1 and NKX6.1 during progenitor cells maturation. Protocols established in our study allow for iPSC generation and derivation of IPC in chemically defined conditions free from animal-derived components, which is of the utmost importance in the light of their prospective applications in the field of regenerative medicine.
Assuntos
Diferenciação Celular , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/citologia , Transativadores/metabolismo , Animais , Peptídeo C/biossíntese , Células Cultivadas , Reprogramação Celular , Endoderma/citologia , Células Epiteliais/citologia , Fibroblastos/citologia , Técnicas de Transferência de Genes , Humanos , Insulina/biossíntese , Células Secretoras de Insulina/metabolismo , TransgenesRESUMO
A commonality between type 1 and type 2 diabetes is the decline in functional ß-cell mass. The transcription factor Nkx6.1 regulates ß-cell development and is integral for proper ß-cell function. We have previously demonstrated that Nkx6.1 depends on c-Fos mediated upregulation and the nuclear hormone receptors Nr4a1 and Nr4a3 to increase ß-cell insulin secretion, survival, and replication. Here, we demonstrate that Nkx6.1 overexpression results in upregulation of the bZip transcription factor CEBPA and that CEBPA expression is independent of c-Fos regulation. In turn, CEBPA overexpression is sufficient to enhance INS-1 832/13 ß-cell and primary rat islet proliferation. CEBPA overexpression also increases the survival of ß-cells treated with thapsigargin. We demonstrate that increased survival in response to ER stress corresponds with changes in expression of various genes involved in the unfolded protein response, including decreased Ire1a expression. These data show that CEBPA is sufficient to enhance functional ß-cell mass by increasing ß-cell proliferation and modulating the unfolded protein response.
RESUMO
The multipotent pancreatic progenitor cells (MPCs) co-expressing the transcription factors, PDX1 and NKX6.1, are the source of functional pancreatic ß-cells. The aim of this study was to examine the effect of p53 inhibition in MPCs on the generation of PDX1+/NKX6.1+ MPCs and pancreatic ß-cell generation. Human embryonic stem cells (hESCs) were differentiated into MPCs and ß-cells. hESC-MPCs (stage 4) were treated with different concentrations of p53 inhibitors, and their effect was evaluated using different approaches. NKX6.1 was overexpressed during MPCs specification. Inhibition of p53 using pifithrin-µ (PFT-µ) at the MPC stage resulted in a significant increase in the number of PDX1+/NKX6.1+ cells and a reduction in the number of CHGA+/NKX6.1- cells. Further differentiation of MPCs treated with PFT-µ into pancreatic ß-cells showed that PFT-µ treatment did not significantly change the number of C-Peptide+ cells; however, the number of C-PEP+ cells co-expressing glucagon (polyhormonal) was significantly reduced in the PFT-µ treated cells. Interestingly, overexpression of NKX6.1 in hESC-MPCs enhanced the expression of key MPC genes and dramatically suppressed p53 expression. Our findings demonstrated that the p53 inhibition during stage 4 of differentiation enhanced MPC generation, prevented premature endocrine induction and favored the differentiation into monohormonal ß-cells. These findings suggest that adding a p53 inhibitor to the differentiation media can significantly enhance the generation of monohormonal ß-cells.
Assuntos
Células-Tronco Pluripotentes , Proteína Supressora de Tumor p53 , Humanos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Diferenciação Celular/genéticaRESUMO
Aims: Differentiating human embryonic stem cells into pancreatic ß cells has been proposed as a practical approach to managing diabetes. There have been several protocols attempting to generate ß-like cells or insulin-producing cells (IPCs), but their low efficiency is a common issue. The expression level of Nkx6.1 is crucial for maintaining pancreatic ß cell identity, while the proportion of PDX1 and Nkx6.1 double positive cells were not satisfied in the present protocols, leading to relative low efficiency in the differentiation into IPCs. This study aims to identify the mechanism underlying the regulation of Nkx6.1 during IPC differentiation and provide new insights for diabetes therapy. Methods: In the current study, human embryonic stem cell (hESC) line H1 was used to perform IPC specifications. Immunofluorescence, flow cytometry, and qPCR were conducted to analyze gene expression. In addition, insulin and C-peptide were measured through glucose-stimulated insulin secretion (GSIS) assays and ELISA. Results: We found that the transcription factor NKX6.1, a crucial inducer of early pancreatic development and IPC generation, was downregulated by micro-RNA-124-5p (miR-124-5p) in hESCs during IPC differentiation. Also, we observed that miR-124-5p was upregulated and bound to the 3' untranslated region (3' UTR) of NKX6.1 in pancreatic progenitor (PP), which subsequently suppressed PP differentiation. Moreover, inhibiting miR-124-5p induced the generation of IPCs. Conclusion: The current study results demonstrated an important role for miR-124-5p in regulating NKX6.1 expression, which appears to be a practical strategy for producing IPCs.
RESUMO
Nkx6.1 plays an essential role during the embryonic development of the spinal cord. However, its role in the adult and injured spinal cord is not well understood. Here we show that lentivirus-mediated Nkx6.1 expression in the adult injured mouse spinal cord promotes cell proliferation and activation of endogenous neural stem/progenitor cells (NSPCs) at the acute phase of injury. In the chronic phase, Nkx6.1 increases the number of interneurons, reduces the number of reactive astrocytes, minimizes glial scar formation, and represses neuroinflammation. Transcriptomic analysis reveals that Nkx6.1 upregulates the sequential expression of genes involved in cell proliferation, neural differentiation, and Notch signaling pathway, downregulates genes and pathways involved in neuroinflammation, reactive astrocyte activation, and glial scar formation. Together, our findings support the potential role of Nkx6.1 in neural regeneration in the adult injured spinal cord.
Assuntos
Gliose/metabolismo , Proteínas de Homeodomínio/biossíntese , Células-Tronco Neurais/metabolismo , Doenças Neuroinflamatórias/metabolismo , Traumatismos da Medula Espinal/metabolismo , Fatores Etários , Animais , Feminino , Gliose/patologia , Gliose/prevenção & controle , Células HEK293 , Humanos , Locomoção/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neuroinflamatórias/prevenção & controle , Traumatismos da Medula Espinal/patologiaRESUMO
GATA6 is a critical regulator of pancreatic development, with heterozygous mutations in this transcription factor being the most common cause of pancreatic agenesis. To study the variability in disease phenotype among individuals harboring these mutations, a patient-induced pluripotent stem cell model was used. Interestingly, GATA6 protein expression remained depressed in pancreatic progenitor cells even after correction of the coding mutation. Screening the regulatory regions of the GATA6 gene in these patient cells and 32 additional agenesis patients revealed a higher minor allele frequency of a SNP 3' of the GATA6 coding sequence. Introduction of this minor allele SNP by genome editing confirmed its functionality in depressing GATA6 expression and the efficiency of pancreas differentiation. This work highlights a possible genetic modifier contributing to pancreatic agenesis and demonstrates the usefulness of using patient-induced pluripotent stem cells for targeted discovery and validation of non-coding gene variants affecting gene expression and disease penetrance.
Assuntos
Células-Tronco Pluripotentes Induzidas , Diferenciação Celular/genética , Fator de Transcrição GATA6/genética , Humanos , Organogênese , PâncreasRESUMO
Recent studies have reported significant advances in the differentiation of human pluripotent stem cells to clinically relevant cell types such as the insulin producing beta-like cells and motor neurons. However, many of the current differentiation protocols lead to heterogeneous cell cultures containing cell types other than the targeted cell fate. Genetically modified human pluripotent stem cells reporting the expression of specific genes are of great value for differentiation protocol optimization and for the purification of relevant cell populations from heterogeneous cell cultures. Here we present the generation of human induced pluripotent stem cell (iPSC) lines with a GFP reporter inserted in the endogenous NKX6.1 locus. Characterization of the reporter lines demonstrated faithful GFP labelling of NKX6.1 expression during pancreas and motor neuron differentiation. Cell sorting and gene expression profiling by RNA sequencing revealed that NKX6.1-positive cells from pancreatic differentiations closely resemble human beta cells. Furthermore, functional characterization of the isolated cells demonstrated that glucose-stimulated insulin secretion is mainly confined to the NKX6.1-positive cells. We expect that the NKX6.1-GFP iPSC lines and the results presented here will contribute to the further refinement of differentiation protocols and characterization of hPSC-derived beta cells and motor neurons for disease modelling and cell replacement therapies.
Assuntos
Diferenciação Celular , Genes Reporter , Loci Gênicos , Proteínas de Fluorescência Verde , Proteínas de Homeodomínio/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células Secretoras de Insulina/metabolismo , Neurônios Motores/metabolismo , Linhagem Celular , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Neurônios Motores/citologiaRESUMO
BACKGROUND: Homeobox transcription factors have demonstrated utility in diagnosing neuroendocrine tumors. Orthopedia homeobox protein (OTP) has a well-defined role in embryonic neurodevelopment and has also been described as a prognostic marker in lung neuroendocrine tumors (NET). Additionally, NK6 homeobox-1 (NKX6.1) has been described to be necessary for the development of neuroendocrine cells in the pancreas. We evaluated immunohistochemical (IHC) expression of OTP and NKX6.1 to determine their utility in the diagnosis of NETs from lung and pancreas fine-needle aspirations (FNA). METHODS: Our study examined 50 FNA specimens, including 30 primary pulmonary NETs (8 carcinoid tumors (CT), 6 atypical carcinoids (AC), 11 small-cell neuroendocrine carcinomas (SCNEC), 5 large-cell neuroendocrine carcinomas (LCNEC)) and 20 primary pancreatic NETs (17 well-differentiated pancreatic neuroendocrine tumors (PanNET) and 3 poorly differentiated pancreatic neuroendocrine carcinomas (PanNEC)). IHC expression of OTP, NKX6.1, and Ki-67 was evaluated on FNA cell blocks. RESULTS: Half of the pulmonary TC tumors expressed OTP, while only 17% of AC and 20% of LCNEC expressed OTP. Neither SCNECs nor any pancreatic NET expressed OTP. In contrast, intermediate and high-grade tumors expressed NKX6.1 (LCNEC-80%, SCNEC-82%, and AC-83%) more often than low-grade tumors (TC-63%, PanNET-71%). All three PanNECs expressed NKX6.1. CONCLUSIONS: OTP may be useful in diagnosing well-differentiated NETs of pulmonary origin. NKX6.1 may have utility in segregating high from low-grade NETs of both pulmonary and pancreatic origin, although other methods will be required to determine site of origin.