Nkx-2.5 Regulates MDR1 Expression via Its Upstream Promoter in Breast Cancer Cells

BACKGROUND: Increased expression of MDR1 gene is one of the major mechanisms responsible for multidrug resistance in cancer cells. Two alternative promoters, upstream and downstream, are responsible for transcription of MDR1 gene in the human. However, the molecular mechanism regarding the transactivation of MDR1 upstream promoter (USP) has not been determined. METHODS: Dual-luciferase reporter gene assays were used to assess the effect of Nkx-2.5 on MDR1 USP activity using reporter plasmids for human MDR1 USP and its mutants. MDR1 mRNA level was examined by quantitative real-time PCR. The direct binding of Nkx-2.5 to the USP of MDR1 was evaluated by promoter enzyme immunoassays and chromatin immunoprecipitation assays.

A family with NKX2.5 gene mutations presenting as familial atrial septal defect and atrioventricular block: A case report

Point mutations in the human cardiac homeobox gene NKX2.5 are associated with familial atrial septal defect (ASD), atrioventricular (AV) conduction disturbance, as well as sudden cardiac death. To date, more than 60 NKX2.5 mutations have been documented, but there are no reports in Korea. We are reporting the first Korean family with ASD and AV block associated with a novel mutation in the NKX2.5 coding region. A 9-year-old boy presented with a slow and irregular pulse, and was diagnosed with secundum ASD and first degree AV block. The boy's father, who had a history of ASD correction surgery, presented with second degree AV block and atrial fibrillation. The boy's brother was also found to have secundum ASD and first degree AV block. There were two sudden deaths in the family. Genetic testing revealed a novel mutation of NKX2.5 in all affected members of the family.

The c.63A>G polymorphism in the NKX2.5 gene is associated with thyroid hypoplasia in children with thyroid dysgenesis

Objective To search for genetic alteration in NKX2.5 gene in patients presenting both congenital heart disease (CHD) and TD. Subjects and methods Individual phenotypes were carefully analyzed in 86 children with thyroid dysgenesis (TD) using thyroid function tests, scintigraphy, ultrasound and echocardiography. DNA was extracted and NKX2.5 gene coding region was amplified by polymerase chain reaction (PCR) and sequenced. Results CHD were found in 8.1% of patients with TD. The mutation screening revealed two known polymorphisms in patients with isolated TD or TD associated with CHD. None of them are predicted to result in codon change in conserved domain. The c.63A>G polymorphism was detected in 54/86 patients (49 with isolated TD and 5 with TD combined with CHD). There was a significant association of c.63A>G polymorphism with hypoplasia (p < 0.036). The c.541G>A polymorphism was observed in only one patient with isolated thyroid hypoplasia. Conclusion NKX2.5 mutations were not found. The c.63A>G polymorphism might be associated with thyroid hypoplasia.

Two Novel Mutations in NKX 2.5 Gene Un‑translated Regions in Congenital Heart Diseases Patients from Pakistan.

Introduction: Congenital heart diseases (CHD) are one of the neglected and challenging areas in undeveloped countries. In Pakistan approximately 40-50,000 children are affected annually. A majority of these patients belongs to rural areas where properly medical facilities are out of reach. In the etiology of these diseases genetic profile, consanguinity and other factors should be examined carefully. The current study aims to check the genetic manipulations in patients for the NKX 2.5 gene specifically the untranslated gene of this gene. NKX 2.5, a transcription factor and first progenitor in cardiac formation, it encodes 324 amino acid and contains a homeodomain (142-200aa) which is highly conserved among vertebrates. To date, no data is available about the mutations in this gene responsible for CHD in Pakistani population. Material and Methods: A cohort of 225, CHD patients who were registered at National Institute of Cardiovascular Diseases (NICVD) from 2006-2009 were included in the study; these are non-syndromic and sporadic cases. Healthy 200 controls were also included with informed consents and detail family history was obtained. DNA was extracted and NKX 2.5 gene was amplified and sequenced to check mutations. Results: The mean age for patients TOF (2.97±1.21), PDA (2.95±2.55), D-TGA (1.84±2.26) and for controls (3.14±1.82) .In present study, two UTR alterations have been reported for NKX 2.5 one at 5' and other at 3'.In our study we look social and genetic aspects for these diseases. Conclusion: CHD is a major cause of child death in the first year of life. Our study concludes a few aspects and will broaden it to other genes as it is a need to find etiology of these diseases and to combat it with the modern genetic therapeutics.

Prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation

OBJECTIVE: The aim of this study was to evaluate the prevalence and spectrum of Nkx2.5 mutations associated with idiopathic atrial fibrillation (AF). METHODS: A cohort of 136 unrelated patients with idiopathic atrial fibrillation and 200 unrelated, ethnically matched healthy controls were enrolled. The coding exons and splice junctions of the Nkx2.5 gene were sequenced in 136 atrial fibrillation patients, and the available relatives of mutation carriers and 200 controls were subsequently genotyped for the identified mutations. The functional characteristics of the mutated Nkx2.5 gene were analyzed using a dual-luciferase reporter assay system. RESULTS: Two novel heterozygous Nkx2.5 mutations (p.N19D and p.F186S) were identified in 2 of the 136 unrelated atrial fibrillation cases, with a mutational prevalence of approximately 1.47%. These missense mutations co-segregated with atrial fibrillation in the families and were absent in the 400 control chromosomes. Notably, 2 mutation carriers also had congenital atrial septal defects and atrioventricular block. Multiple alignments of the Nkx2.5 protein sequences across various species revealed that the altered amino acids were completely conserved evolutionarily. Functional analysis demonstrated that the mutant Nkx2.5 proteins were associated with significantly reduced transcriptional activity compared to their wild-type counterpart. CONCLUSION: These findings associate the Nkx2.5 loss-of-function mutation with atrial fibrillation and atrioventricular block and provide novel insights into the molecular mechanism involved in the pathogenesis of atrial fibrillation. These results also have potential implications for early prophylaxis and allele-specific therapy of this common arrhythmia. .

Protective Effects of Folic Acid on Cardiac Development Related Genes GATA-4 and NKx2.5 Expression of Offspring Rats with Maternal Coxsackievirus B3 Infection / 天津医药

Objective To investigate the effects of oral folic acid on cardiac development related gene expression of offspring in an experimental model of coxsackievirus B3(CVB3) infection of pregnant rats. Methods SD female rats were randomized into control group, folic acid group, CVB3 group and CVB3+folic acid group. The female rats were given folic acid by gavage for 2 weeks before pregnancy in folic acid group and CVB3+folic acid group. After conception for 7 days, rat model was established by intraperitoneal injection of CVB3 for 5 days in CVB3 group and CVB3+folic acid group. After nat-ural childbirth neonatal heart was taken and stored in liquid nitrogen. The morphological changes of neonatal rat myocardial tissues were observed by HE staining. The expressions of GATA-4 and NKx2.5 mRNA were detected by RT-PCR and West-ern blot assay. Results There was significant myocardial injury, such as myocardial fiber disarray and myocardial fiber breakage, in neonatal rats in CVB3 group. These damages were improved in CVB3+folic acid group. The expression levels of GATA-4 and NKx2.5 genes in myocardial tissues were significantly lower in CVB3 group than those of control group (P<0.05). The expression levels of GATA-4 and NKx2.5 proteins in myocardial tissues were significantly higher in CVB3+folic acid group than those of CVB3 group (P<0.05). Conclusion CVB3 infection in the early pregnancy inhibited the expres-sion of neonatal rat cardiac development factor. Folic acid supplementation has obvious protective effects on the neonatal rat cardiac development.

Absence of mutations in PAX8, NKX2.5, and TSH receptor genes in patients with thyroid dysgenesis / Ausência de mutações nos genes PAX-8, NKX2.5 e receptor de TSH em pacientes com disgenesia tireoidiana

OBJECTIVES: To precisely classify the various forms of TD, and then to screen for mutations in transcription factor genes active in thyroid development. SUBJECTS AND METHODS: Patients underwent ultrasound, thyroid scan, and serum thyroglobulin measurement to accurately diagnose the form of TD. DNA was extracted from peripheral leukocytes. The PAX8, and NKX2.5 genes were evaluated in all patients, and TSH receptor (TSHR) gene in those with hypoplasia. RESULTS: In 27 nonconsanguineous patients with TD, 13 were diagnosed with ectopia, 11 with hypoplasia, and 3 with athyreosis. No mutations were detected in any of the genes studied. CONCLUSION: Sporadic cases of TD are likely to be caused by epigenetic factors, rather than mutations in thyroid transcription factors or genes involved in thyroid development.

OBJETIVOS: Classificar corretamente as várias formas de DT e depois rastrear por mutações em genes que participam no desenvolvimento da tireoide. SUJEITOS E MÉTODOS: Os pacientes realizaram ultrassonografia, cintilografia e tireoglobulina sérica para o diagnóstico preciso de DT. DNA foi extraído de leucócitos periféricos. Os genes PAX8 e NKX2.5 foram estudados em todos os pacientes e o gene do receptor do TSH (TSHR) foi estudado na hipoplasia. RESULTADOS: Avaliaram-se 27 pacientes sem consanguinidade com DT, dos quais 13 foram diagnosticados com ectopia, 11 com hipoplasia e 3 com atireose. Nenhuma mutação foi detectada nos genes estudados. CONCLUSÃO: Casos esporádicos de DT são provavelmente causados mais por fatores epigenéticos do que por mutações em fatores de transcrição ou genes envolvidos no desenvolvimento tireoidiano.

The mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells into myocardial cells induced by 5-azacytidine.

Objective: To investigate the molecular mechanism underlying the differentiation of human umbilical cord-derived mesenchymal stem cells (hUCMSCs) into myocardial cells induced by 5-azacytidine (5-aza), and to explore the expression and significance of DLL4-Notch signaling in this process. Materials and Methods: hUCMSCs were isolated and purified from the umbilical cords of normal or cesarean term deliveries under sterile conditions. After treatment with 5-aza for 24 h, hUCMSCs was continued to culture, the expression of GATA4 and NKx2.5 at 4 weeks after induction, DLL4 and Notch1 mRNA at 1d, 3d, 5d, 7d after induction were detected. The expression of cardiac troponin I (cTnI) after 4 weeks was determined by immunocytochemistry. Results: hUCMSCs treated with 5-aza were stained positively for cTnI 4 weeks after induction. The expression of Notch1 and DLL4 mRNA in the 5-aza-induced group was stable and significantly higher than that in the control group (mean Ct value for the Notch1 gene: 0.51 ± 0.21 in the 5-aza-induced group vs. 7.85 ± 0.35 in the control group; mean Ct value for the DLL4 gene: 1.60 ± 0.49 in the 5-aza-induced group vs. 12.42 ± 0.73 in the control group). Similar results were observed for Nkx2.5 and GATA4 genes. The expressions of Nkx2.5 and GATA4 mRNA in the 5-aza group were 4.72 ± 0.58 and 3.76 ± 0.06 times higher than that in the control group, respectively, with statistical significance. Conclusions: hUCMSCs can be differentiated into myocardial cells by 5-aza induction in vitro. 5-Aza may affect this process by regulating the expression of GATA4 and Nkx2.5 genes. The DLL4-Notch signal pathway may be involved in this process.

Novel mutations in the TBX5 gene in patients with Holt-Oram Syndrome

The Holt-Oram syndrome (HOS) is an autosomal dominant condition characterized by upper limb and cardiac malformations. Mutations in the TBX5 gene cause HOS and have also been associated with isolated heart and arm defects. Interactions between the TBX5, GATA4 and NKX2.5 proteins have been reported in humans. We screened the TBX5, GATA4, and NKX2.5 genes for mutations, by direct sequencing, in 32 unrelated patients presenting classical (8) or atypical HOS (1), isolated congenital heart defects (16) or isolated upper-limb malformations (7). Pathogenic mutations in the TBX5 gene were found in four HOS patients, including two new mutations (c.374delG; c.678G > T) in typical patients, and the hotspot mutation c.835C > T in two patients, one of them with an atypical HOS phenotype involving lower-limb malformations. Two new mutations in the GATA4 gene were found in association with isolated upper-limb malformations, but their clinical significance remains to be established. A previously described possibly pathogenic mutation in the NKX2.5 gene (c.73C > 7) was detected in a patient with isolated heart malformations and also in his clinically normal father.

Nkx2-5 gene promotes expression of cardiac markers in 5-azacytidine-induced monolayer P19 cells / 第二军医大学学报

Objective: To study the role of Nkx2-5 gene in 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells. Methods: The experiment was divided into two groups: an experimental group and a control group. The cells in the experimental group were P19 cells stably expressing Nkx2-5 gene, and cells in the control group were P19 cells. Under the monolayer-culture condition, the cells of two groups were induced by 5-azacytidine (1 μmol/L). The growth of cells were observed by inverted microscope. On the 4th day, 8th day, 12th day and 16th day after induction, RT-PCR was used to detect the expression of GATA-4, α-MHC and ANP gene. Results: In control group, there was no ANP expression after induction; GATA-4 expression was seen on the 8 th day, 12th day, and 16th day after induction; and α-MHC expression was found on the 12th day and 16 th day. In experimental group, the expression of GATA-4 was detected on the 4th day, 8th day, 12th day and 16 th day after induction; Alpha-MHC and ANP expression was noticed on the 8th day, 12th day and 16th day after induction. RT-PCR results showed that the expression of GATA-4 and α-MHC in the experimental group was earlier than that in the control group. And at all time points of observation, the expression of GATA-4 and α-MHC in the experimental group was significantly increased compared with that in the control group (P<0. 05,P<0. 01), except for α-MHC expression on the 12 th day. Conclusion: Nkx2-5 gene can promote 5-azacytidine-induced differentiation of monolayer P19 cells into myocardial cells.

Cardiomyocyte Differentiation from P19 Cells Induced by NKX2-5 / 中国医科大学学报

Objective To identify the rale of NKX2-5 gene in cardiomyocyte differentiation and its mechanism.Methods P19 cells were divided into transfected and non-transfected groups.In the transfected group,P19 cells were with stable expression of NKX2-5 gene.The P19 cells were cultured in suspension for 4 days,and the formed aggregates were transferred to Petri dish for adherent culture.On days 4,8,12,and 16 of the adherent culture,the expressions of ct-saicomeric actin(α-SA)and cardiac troponin T(cTnT)were detected with double-labeling immunofluorescence and Western blot.The ultrastruetural changes were observed on day 16.Results In the transfected group,no expression of α-SA and cTnT was found on day 4,and the expression of these 2 proteins or co-expression existed on days 8,12,and 16.There were early cell junction and myofilament-like structure in the cytoplasm of some cells in the transfected group.In the non-transfected group,these 2 proteins were negative,and no differentiated cell was found.Conclusion Stable expression of NKX2-5 gene can induce cardiomyocyte differentiation from P19 cells,but the P19 cells with stable expression of JVKX2-5 gene is not suitable to be an in vitro model of cardiac development.

The Role of Nkx2.5 in Keratinocyte Differentiation

BACKGROUND: Nkx2.5 is a homeodomain-containing nuclear transcription protein that has been associated with acute T-lymphoblastic leukemia. In addition, Nkx2.5 has an essential role in cardiomyogenesis. However, the expression of Nkx2.5 in the skin has not been investigated. OBJECTIVE: In an attempt to screen the differentially regulated genes involved in keratinocyte differentiation, using a cDNA microarray, we identified Nkx2.5 as one of the transcription factors controlling the expression of proteins associated with keratinocyte differentiation. METHODS: To investigate the expression of Nkx2.5 during keratinocyte differentiation, we used a calcium-induced keratinocyte differentiation model. RESULTS: RT-PCR and Western blot analysis revealed that the expression of Nkx2.5, in cultured human epidermal keratinocytes, increased with calcium treatment in a time-dependent manner. In normal skin tissue, the expression of Nkx2.5 was detected in the nuclei of the keratinocytes in all layers of the epidermis except the basal layer by immunohistochemistry. In addition, the expression of Nkx2.5 was significantly increased in psoriasis and squamous cell carcinoma, but was barely detected in atopic dermatitis and basal cell carcinoma. CONCLUSION: These results suggest that Nkx2.5 may play a role in the change from proliferation to differentiation of keratinocytes and in the pathogenesis of skin disease with aberrant keratinocyte differentiation.

An understanding the genetic basis of congenital heart disease.

The recent exponential increase in the knowledge of genetics has revolutionized the understanding of congenital heart diseases (CHDs) during the past few decades. Prior studies have reported the influence of Mendelian disorders on CHDs to be very small, when compared to the polygenic inheritance, which constituted a higher percentage. The recent findings of candidate genes responsible for CHDs have provided new insights into the genetic basis of heart malformation. Here we reviewed the understandings of different types of heart lesions associated with syndromes for which genetic etiologies are apparent, as well as the recent developments involving the molecular pathways involved in CHDs in case of human beings. The similar mutations, which are the devastating events of molecular mechanism, may be the cause of different types of CHDs indicating single gene defects as the cause of different apparent phenotypes. An integrated simple model will explain the causes of presently well known CHDs. This review provides updated information on the genetic basis for cardiac defects which helps to understand, identify, prevent and treat individuals who might be at risk at an early stage. There is a need to find heart defects as early as possible so that they can be treated while the heart is still forming.

Genetic Reveal Study of NKX2.5,TBX5 and GATA4 Genes in Patients With Tetralogy of Fallot / 中国循环杂志

Objective:To investigate mRNA expression of cardiac related genes of NKX2.5,TBX5 and GATA4 in patients with tetralogy of fallot(TOF). Methods:A total of 10 TOF patients(TOF group)from 4 months to 8 years with the mean age of 3.5 years were recruited in our hospital from June to December 2005.The patients were diagnosed by typical clinical manifestation and cardiac color echocardiogram, the diagnosis was confirmed by cardiac surgery.6 non-congenital heart disease children were selected as Control group, and they were from 4 months to 9 years with the mean age of 3.8 years.The myocardial total RNA was extracted,the related cDNA was obtained by RT-PCR.The product cDNA was amplified with fluorescent quantitative PCR in order to compare the differences of NKX2.5,TBX5,GATA4 and GAPDH mRNA expression between TOF group and Control group. Results:NKX2.5 mRNA expression in TOF group was statistically decreased than that in Control group,while there were no statistical changes found in TBX5 and GATA4 mRNA expression between TOF group and Control group. Conclusion:The mRNA expression of NKX2.5,TBX5 and GATA4 were found in myocardium development.TOF was possibly related to decreased NKX2.5 mRNA expression.

Studies on Differentiation of Cardiac Myoblast Induced by Co-culture with Isolated Neonatal Rat Cardiac Myocytes / 대한해부학회지

Cellular cardiomyoplasty has recently emerged as a potential new treatment of ischemic heart disease. Combining cellular cardiomyoplasty with gene therapy using myogenic transcription factor might facilitate myocardial regeneration. In this study, we engineered H9c2, L6 using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. Each cell overexpressing MEF2 and Nkx2.5 was generated by gene transfection and clonal selection. CO-culture was performed that each cell line added over cultured cardiomyocyte. H9c2-MEF2c and H9c2-Nkx2.5 became long, spindle shape like cardiomyocyte. Troponin T, cardiac specific marker, was found spot-like pattern in H9c2-Nkx2.5. However, co-culture with cardiomyocyte did not induce differentiation all kinds of cells into cardiomyocyte. Connexin43, which is gap junction marker was increased in H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c and L6-Nkx2.5. Especially, co-culture with cardiomyocyte resulted in elevation of connexin43 levels more than monoculture. Ultrastructurally, formations of gap junction and desmosome were found apparently in L6-Nkx2.5. Long-standing, strong, regular and more frequent contraction were observed in cardiomyocyte co-cultured with H9c2-MEF2c, H9c2-Nkx2.5, L6-MEF2c, L6-Nkx2.5, respectively. Neverthless, any cell did not have active contraction itself, but passive movement except cardiomyocyte. H9c2-MEF2c, L6-MEF2c and L6-Nkx2.5 had resistance to hypoxia compared with other groups. These results suggested that co-culture and overexpressions of MEF2c and Nkx2.5 induced differentiation into cardiomyocyte and played an important role on intercellular junction formation and hypoxic resistance. This would be a promising source of cellular cardiomyoplasty. Therefore, much more research would be essential for clinical application of cellular cardiomyoplasty and this study would be a basic source for further study of MEF2c and Nkx2.5 in cellular cardiomyoplasty.

Role of Cardiac Transcription Factor Nkx2.5 on Cardiomyoplasty Model in vitro / 대한해부학회지

Despite therapeutic advance, the prevalence of ischemic heart disease continues to increase. Recently, cell transplantation of stem cell has been proposed as a strategy for cardiac repair following myocardial damage. However, low differentiation efficiency into cardiomyocyte and poor cell viability associated with transplantation have limited the reparative capacity of these cell. In this study, we engineered P19 embryonal carcinoma cells using plasmid vector to overexpress the transcription factor MEF2c, Nkx2.5 involved in cardiomyogenesis. We investigated 1) formation of intercellular junction of P19 in mono-culture and co-culture with cardiomyocyte for functional and structural synchronous contraction after transplantation, 2) differentiation into cardiomyocyte, 3) resistance to hypoxic condition. An P19 embryonal carcinoma cell line expressing GFP, MEF2c, Nkx2.5 was generated by gene transfection and clonal selection. Nkx2.5 overexpression induced connexin43 expression level decrease. Electron microscopy revealed myofibril organization and immunostaining with cTnT showed positive staining in P19-Nkx2.5, consistent with early stage cardiomyocyte. Connexin43 and N-cadherin was expressed between P19-MEF2c and cardiomyocyte, P19- Nkx2.5 and cardiomyocyte in co-culture. And beating rate of cardiomyocyte co-cultured with P19-Nkx2.5 increased much more than other group, even if P19-Nkx2.5 did not have synchronous contraction with cardiomyocyte. Additionally, P19-Nkx2.5 had a resistance against hypoxia. These result suggest that overexpression of Nkx2.5 induced differentiation of P19 into cardiomyocyte and would be electro-mechanical coupling with cardiomyocyte after transplantation. Futhermore, Nkx2.5 overexpression had protection potential to hypoxic injury. Therefore, P19 cell overexpressed Nkx2.5 would be promising cell source for further study of new therapy of myocardial disease and building up in vitro model.

Effects of Folate Deficiency during Pregnancy on Expression of NKx2.5 Gene in Heart of Offspring / 实用儿科临床杂志

Objective To study the expression of NKx2.5 on the heart of offspring during the development of embryo,whose mother is deficient of folic acid.Methods 1.Control group involving 18 rats and study group involving 18 rats were chosen from the total 36 adult female SD rats randomly copulate with the male normal rats after feeding different fodder for 2 weeks.The heart of the 13.5 days,17.5 days embryos and the newborns were obtained;2.the expression of NKx2.5mRNA by RT-PCR was observed;3.the expression of NKx2.5 protein by Western-blotting was investigated.Results 1.The expression of NKx2.5 mRNA of study group was weaker than control group in heart of the 13.5 days,17.5 days embryos and the newborns(P