Iron is one of the essential mineral micronutrients for plants. Low concentrations of effective iron in soil can easily increase risk of plant iron deficiency. Several members of bHLH transcription factors family participate in the response to iron deficiency and play an important role in iron regulation of plants. In order to better understand the mechanism of iron deficiency response, an overview of the structure, classification, function and regulatory mechanism of bHLH transcription factors was given in this review as well as signaling pathway triggered by iron deficiency. It will provide theoretical basis and design strategies for cultivating iron deficiency tolerant or iron-rich crops using bHLH transcription factors.
Arabidopsis ; genetics ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Iron ; deficiency ; Signal Transduction ; physiology

Low density lipoprotein receptor (LDLR) plays an important role in the cholesterol homeostasis. We examined the possible circadian regulation of LDLR and mechanism(s) underlying it. In mice, blood glucose and plasma triglyceride, total and high density lipoprotein cholesterol varied distinctively throughout a day. In addition, LDLR mRNA oscillated in the liver in a functional clock-dependent manner. Accordingly, analysis of human LDLR promoter sequence revealed three putative E-boxes, raising the possible regulation of LDLR expression by E-box-binding transcription factors. To test this possibility, human LDLR promoter reporter constructs were transfected into HepG2 cells and the effects of CLOCK/BMAL1, Hes1, and Hes6 expression were analyzed. It was found that positive circadian transcription factor complex CLOCK/BMAL1 upregulated human LDLR promoter activity in a serum-independent manner, while Hes family members Hes1 and Hes6 downregulated it only under serum-depleted conditions. Both effects were mapped to proximal promoter region of human LDLR, where mutation or deletion of well-known sterol regulatory element (SRE) abolished only the repressive effect of Hes1. Interestingly, hes6 and hes1 mRNA oscillated in an anti-phasic manner in the wild-type but not in the per1-/-per2-/- mouse. Comparative analysis of mouse, rat and human hes6 genes revealed that three E-boxes are conserved among three species. Transfection and site-directed mutagenesis studies with hes6 reporter constructs confirmed that the third E-box in the exon IV is functionally induced by CLOCK/BMAL1. Taken together, these results suggest that LDLR expression is under circadian control involving CLOCK/BMAL1 and Hes family members Hes1 and Hes6.
ARNTL Transcription Factors/physiology ; Animals ; Base Sequence ; Basic Helix-Loop-Helix Transcription Factors/*genetics/metabolism/physiology ; CLOCK Proteins/physiology ; Cholesterol/blood ; *Circadian Rhythm ; E-Box Elements ; Exons ; *Gene Expression Regulation ; Hep G2 Cells ; Homeodomain Proteins/*genetics/metabolism/physiology ; Homeostasis ; Humans ; Liver/metabolism ; Male ; Mice ; Mice, Inbred C57BL ; *Promoter Regions, Genetic ; Receptors, LDL/*genetics/metabolism ; Repressor Proteins/*genetics/metabolism/physiology ; Transcription, Genetic

BACKGROUNDDelta-like 4 (DLL4) is an endothelium specific Notch ligand and has been shown to function as a regulating factor during physiological and pathological angiogenesis. It has been reported that the DLL4-Notch signaling pathway is regulated by hypoxia and may prevent excessive angiogenesis through the inhibition of angiogenic branching and by triggering vessel maturation. Choroidal neovascularization (CNV) is a pathological form of angiogenesis in which hypoxia is thought to play an important role. This study was aimed to evaluate the role of DLL4 in the development of CNV.

METHODSWe utilized chemical hypoxia induced by 200 µmol/L CoCl2 to observe the expression of DLL4 in choroid-retinal endothelial cells (RF/6A cells), which are the primary cells involved in CNV. After transfection of a DLL4 small interfering RNA (siRNA), mRNA and protein expression of DLL4 and key downstream genes, including HES1 and HEY1, in hypoxic RF/6A cells were investigated by RT-PCR, real-time PCR, and Western blotting analysis. Three controls were used: one without transfection, one with transfection reagent, and one with scrambled negative control siRNA. The effects of the DLL4 siRNA on the biological function of hypoxic RF/6A cells during angiogenesis, including cell proliferation, migration and tube formation, were investigated.

RESULTSThe results showed that hypoxic conditions led to upregulation of DLL4 expression in RF/6A cells in vitro. After transfection, siRNA-duplex1 targeting DLL4 depleted the DLL4 mRNA levels by as much as 91.4% compared with the scrambled siRNA control, and DLL4 protein expression was similarly effected. There was no significant difference in DLL4 expression among the blank control, transfection reagent control, and scrambled siRNA groups. In addition, after transfection of hypoxic RF/6A cells with the DLL4 siRNA-duplex1, the mRNA levels of HES1 and HEY1, which function downstream of DLL4-Notch signaling, were lowered by 75.1% and 86.3%, respectively, compared with the scrambled siRNA control. Furthermore, knockdown of DLL4 expression significantly promoted the proliferation of hypoxic RF/6A cells and led to their arrest in the S phase of the cell cycle. Migration and tube formation of hypoxic RF/6A cells were significantly induced by the DLL4 siRNA, with the number of migrated cells increased by 1.6-fold and total tube length increased by 82.3%, compared with the scrambled siRNA (P < 0.05).

CONCLUSIONSDLL4 functions as a negative regulator of angiogenic branching and sprouting. Based on our results, DLL4 signaling appears to play an essential role in the biological behavior of choroid vascular endothelial cells under hypoxia. Therefore, DLL4 may represent a novel target for CNV therapy in the future.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Blotting, Western ; Cell Cycle ; genetics ; physiology ; Cell Cycle Proteins ; genetics ; metabolism ; Cell Hypoxia ; genetics ; physiology ; Cell Line ; Cell Movement ; genetics ; physiology ; Cell Proliferation ; Choroidal Neovascularization ; Endothelial Cells ; cytology ; metabolism ; Homeodomain Proteins ; genetics ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; RNA, Small Interfering ; Reverse Transcriptase Polymerase Chain Reaction ; Transcription Factor HES-1

BACKGROUND AND OBJECTIVEHuman achaete-scute homolog 1 (hASH1) gene plays a critical role in development of the central nervous system, automatic nervous system, adrenal medullary chromaffin cells, thyroid C cells and pulmonary neuroendocrine cells. The aim of this study is to determine hASH1 gene expression in the normal lung tissue and various types of lung tumors, to analyze whether its expression correlated with pulmonary neuroendocrine markers, and to explore the possibility of hASH1 as clinical pathological markers in the neuroendocrine tumors compared with previous neuroendocrine tumor markers.

METHODShASH1, Chromogranin A, Synaptophysin and CD56 expression were examined in lung tumor specimens (lung inflammatory pseudotumor, squamous cell carcinoma, adenocarcinomas, large cell carcinoma, typical carcinoids, atypical carcinoids, large cell neuroendocrine carcinomas and small cell lung carcinoma and corresponding normal lung specimens) using immunohistochemistry (S-P method). Western blot and reverse transcription polymerase chain reaction (RT-PCR) assay were applied to detect the expressions of hASH1 protein and mRNA in lung cancer tissues.

RESULTShASH1 expression was positive in 2/16 (12.5%) typical carcinoids, 15/20 (75%) atypical carcinoids, 6/10 (60%) large cell neuroendocrine carcinomas and 31/40 (77.5%) small cell lung carcinoma, respectively, but not in any normal lung tissue (0/10), lung inflammatory pseudotumor (0/49), squamous cell carcinoma (0/30), adenocarcinomas (0/30) or large cell carcinoma (0/20). There was a significant difference in hASH1 expression between typical carcinoids and atypical carcinoids (P < 0.01), but not in large cell neuroendocrine carcinomas and small cell lung carcinoma (P > 0.05). hASH1 expression highly closely correlated with Chromogranin A, Synaptophysin and CD56 expression (P < 0.05).

CONCLUSIONhASH1 is a new kind of highly specific markers of pulmonary neuroendocrine tumours, and may be applied to clinical pathology diagnosis of the pulmonary neuroendocrine tumors.

Adenocarcinoma ; genetics ; metabolism ; pathology ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Carcinoma, Large Cell ; genetics ; metabolism ; pathology ; Carcinoma, Neuroendocrine ; genetics ; metabolism ; pathology ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Gene Expression Regulation, Neoplastic ; genetics ; physiology ; Humans ; Immunohistochemistry ; Lung Neoplasms ; genetics ; metabolism ; pathology ; Neuroendocrine Tumors ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Small Cell Lung Carcinoma ; genetics ; metabolism ; pathology

BACKGROUNDThe tendency of tumor cells to disperse throughout the liver is a distinct feature of hepatocellular carcinoma (HCC). Nck family adaptor proteins function to regulate actin cytoskeletal reorganization that leads to cell motility. We previously found that Max binding protein (MNT) was differentially expressed in HCC, and interacted with Nck1 by 2-DE. MNT is a protein member of the Myc/Max/Mad network which plays roles in cell proliferation, differentiation, and death. We investigated the effects of MNT on migration of human liver cancer SK-HEP-1 cells to study the migration regulatory role of MNT in HCC cells.

METHODSInteraction between MNT and Nck1 was further validated in hepatoma cells by GST-pull down assay and immunoprecipitation. siRNAs specific to MNT (MNT siRNA) were used to knockdown MNT expression. Western blotting, transwell assay were used to determine the migration potential of cells.

RESULTSInteraction between MNT and Nck1 was validated in hepatoma cells. MNT knockdown promoted the migration of human liver cancer SK-HEP-1 cells (P < 0.01).

CONCLUSIONThe results suggest that MNT, via interaction with Nck1, inhibits hepatoma cell migration.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors ; genetics ; metabolism ; Blotting, Western ; Cell Differentiation ; genetics ; physiology ; Cell Line, Tumor ; Cell Movement ; genetics ; physiology ; Humans ; Immunoprecipitation ; Liver Neoplasms ; Oncogene Proteins ; genetics ; metabolism ; Protein Binding ; genetics ; physiology ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

BACKGROUNDHepatocyte growth factor (HGF) treats ischemic disease by promoting arteriogenesis, however, its mechanism of action is not known. The notch signaling pathway plays an important role in neovascularization. The relationship between the proliferation and migration ability of artery endothelial cells and the Dll4-Notch-Hey2 signaling pathway in the process of arteriogenesis was investigated as a mechanism of action of HGF.

METHODSBased on the prophase study cells and supernatant were harvested at the indicated time after human femoral artery endothelial cells (HFAECs) were infected with adenovirus-HGF (Ad-HGF) at 200 pfu/cell. Cells were analyzed for HGF expression and Notch1, Dll4 and Hey2 expression by ELISA and reverse transcription-PCR (RT-PCR). The changes in the proliferation and migration ability of HFAECs were observed by MTT and Transwell migration experiments. Ad-GFP-infected HFAECs were used as control.

RESULTSCompared with the control group the Ad-HGF group's HGF expression was not increased with time, and the induction by HGF of Notch1, Dll4 and Hey2 gene transcription was not enhanced with an increase of HGF. The proliferation ability of Ad-HGF-transduced HFAECs was enhanced and their migration ability was also enhanced in the presence of HGF.

CONCLUSIONSThrough activating the Dll4-Notch-Hey2 signaling pathway, HGF indirectly promotes the proliferation and migration ability of cells, so that offspring artery branches are formed.

Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; Hepatocyte Growth Factor ; genetics ; metabolism ; Humans ; Intercellular Signaling Peptides and Proteins ; genetics ; metabolism ; Receptors, Notch ; genetics ; metabolism ; Repressor Proteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; genetics ; physiology

BACKGROUNDTranscription factors hypoxia inducible factor 1alpha (HIF 1alpha) and endothelial PAS domain protein 1 (EPAS1) promote the transcription of vascular endothelial growth factor (VEGF). VEGF enhances angiogenesis and vascular permeability of tumours, which promotes tumour growth and facilitates entry of cancer cells into blood circulation and metastasizing. This study examined whether HIF 1alpha and EPAS1 stimulated angiogenesis through activation of VEGF in human pancreatic carcinoma.

METHODSSpecimens from pancreatic carcinoma and healthy parts of same pancreas were taken from 60 patients. Real time quantitative reverse transcription polymerase chain reaction estimated expression of HIF 1alpha, EPAS1, and VEGF mRNAs. Western blotting and immunohistochemical, streptavidin peroxidase method assessed expression of HIF 1alpha, EPAS1, and VEGF proteins. Microvessel density (MVD) was assessed.

RESULTSHighly significant increases in expression of EPAS1, VEGF, and MVD were found in pancreatic carcinoma tissue but not in normal pancreatic tissue: VEGF at mRNA and protein levels (t = 17.32, P = 0.0001; t = 98.41, P = 0.0001); EPAS1 protein level (t = 22.51, P = 0.0001). Expression of HIF 1alpha was similar in pancreatic carcinoma and normal pancreatic tissues at both mRNA and protein levels. Significant correlations were observed between EPAS1 and VEGF (r = 0.736, P = 0.0041), between VEGF and MVD (r = 0.858, P = 0.0001), and between EPAS1 and MVD (r = 0.641, P = 0.0003). No significant correlations were observed between HIF 1alpha and VEGF, or between HIF 1alpha and MVD. MVD and expression of EPAS1 and VEGF were significantly related with TNM staging, so was EPASI and VEGF with size of tumour.

CONCLUSIONSEPAS1 and VEGF, but not HIF1alpha, are overexpressed in pancreatic carcinoma. The expression of EPAS1 is correlated with that of VEGF and MVD. EPAS1 may be involved in the angiogenesis of pancreatic carcinoma by upregulating the expression of VEGF. Targeting EPAS1 may be a new method of antiangiogenic tumour therapy for pancreatic carcinoma.

Adult ; Aged ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; physiology ; Blotting, Western ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Immunohistochemistry ; Male ; Middle Aged ; Pancreatic Neoplasms ; genetics ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; physiology ; Young Adult

BACKGROUNDNeural stem cells (NSCs) are a self-renewing and multipotent population of the central nervous system (CNS), which are active during development and maintain homeostasis and tissue integrity throughout life. Microglias are an immune cell population resident in the CNS, which have crucial physiological functions in the developing and adult CNS. This study aimed to investigate that whether microglia co-cultured with NSCs could promote astrogliogenesis from NSCs.

METHODSMicroglia and NSCs were co-cultured in 24-well insert plates. NSCs were plated in the bottom of the well and microglia in the insert. Fluorescent staining, Western blotting and RT-PCR were used to determine the effect of microglia on NSCs differentiation.

RESULTSCo-culture of microglia and NSCs promoted astrogliogenesis from NSCs. Several key genes, such as Notch 1, Notch 2, Notch 3, Hes 5, and NRSF were downregulated, while the critical genes Id1 and Id2 were upregulated. BMP2 and FGF2 were upregulated.

CONCLUSIONMicroglias act as a regulator of NSCs astrogliogenesis.

Animals ; Astrocytes ; cytology ; metabolism ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; Blotting, Western ; Bone Morphogenetic Protein 2 ; genetics ; Cell Differentiation ; genetics ; physiology ; Cells, Cultured ; Coculture Techniques ; methods ; Fibroblast Growth Factor 2 ; genetics ; Inhibitor of Differentiation Protein 1 ; genetics ; Inhibitor of Differentiation Protein 2 ; genetics ; Microglia ; cytology ; metabolism ; Microscopy, Fluorescence ; Neural Stem Cells ; cytology ; metabolism ; Rats ; Repressor Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVE: To investigate the role of Notch signaling in differentiation of Sprague-Dawley (SD) rat retinal progenitor cells (RPCs). METHODS: RPCs were isolated from 16-day embryonic SD rats and cultured in suspension. RPCs were cultured respectively in media with (treatment group) or without (control group) gamma-secretase inhibitor X which was used to block Notch signaling. Morphological observation and immunocytochemistry were applied at day 14 to determine the cell types and analyze the expression of Notch pathway genes in both groups. RESULTS: Most RPCs expressed Notch1 intracellular domains or its downstream transcriptional factor Hes1. A few expressed bHLH transcriptional factors NeuroD and Mash1. Most auto-differentiated RPCs expressed NeuroD or Mash1, while a few of them expressed Notch1 intracellular domains or Hes1. In the group treated with gamma-secretase inhibitor X, the positive rate of Nestin or GFAP was much lower than that in the control group while the positive rate of beta-tubulin was much higher than that in the control group. The difference in the positive rate of recovering between the two groups was not significant. CONCLUSION In vitro Notch signaling may inhibit retinal stem cells differentiation. Inhibiting Notch signaling in vitro may promote differentiation to neurons and partially inhibit glial differentiation.
Animals ; Basic Helix-Loop-Helix Transcription Factors ; metabolism ; Cell Differentiation ; physiology ; Cells, Cultured ; Female ; Fetus ; Homeodomain Proteins ; metabolism ; Neurons ; cytology ; Rats ; Rats, Sprague-Dawley ; Receptor, Notch1 ; genetics ; metabolism ; Retina ; cytology ; Signal Transduction ; drug effects ; physiology ; Stem Cells ; cytology ; Transcription Factor HES-1

OBJECTIVETo study the role of Ezh2 in the all-trans retinoic acid RA induced P19 neural differentiation.

METHODSThe expression of Ngn1 in the RA induced P19 cells was detected at the mRNA and protein levels using real time RT-PCR and Western blot assays. The binding of Ezh2 and H3K27me3 on the Ngn 1 promoter was analyzed using chromatin immunoprecipitation assay.

RESULTIn the RA induced P19 cells, the recruitment of Ezh2 and its methylated substrate H3K27me3 on the promoter of Ngn 1 gene elevated in the first 2 days, and then declined rapidly, followed by the initiation of neuronal differentiation.

CONCLUSIONSEzh2 produces a repressive histone mark H3K27me3 in the early stage of RA induced P12 cells. By avoiding the premature expression of Ngn1 gene, Ezh2 can ensure the normal differentiation of P19 cells.

Animals ; Basic Helix-Loop-Helix Transcription Factors ; genetics ; metabolism ; Cell Differentiation ; drug effects ; physiology ; Enhancer of Zeste Homolog 2 Protein ; Histone-Lysine N-Methyltransferase ; genetics ; metabolism ; Histones ; metabolism ; Mice ; Nerve Tissue Proteins ; genetics ; metabolism ; Neurons ; cytology ; drug effects ; metabolism ; Polycomb Repressive Complex 2 ; Tretinoin ; pharmacology ; Tumor Cells, Cultured