hes1-protein--human and Hypoxia

Ischemic preconditioning (IPC), and ischemic postconditioning (IPost) have a significant protective effect on myocardial ischemia/reperfusion (MI/R) injury by alleviating oxidative stress and mitochondrial disturbances, although the underlying molecular mechanisms are unclear. The study was to demonstrate that cardioprotection against anoxia/reoxygenation (A/R) injury is transduced via the Notch1/Hes1/VDAC1 signaling pathway. Using mass spectrometry and tandem affinity purification (TAP), to screen for differentially expressed proteins associated with Hes1, followed by standard bioinformatics analysis. The co-immunoprecipitation (Co-IP) assay confirmed an interaction between Hes1 and VDAC1 proteins. H9c2 cells were transfected with Hes1 adenoviral N-terminal TAP vector (AD-NTAP/Hes1) and Hes1-short hairpin RNA adenoviral vector (AD-Hes1-shRNA) to establish A/R injury, IPC, and IPost models, respectively. The expression of Hes1 and VDAC1 proteins were measured by western blot analysis, while the levels of reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and apoptosis were evaluated by flow cytometry. AD-NTAP/Hes1 can activate the exogenous protein expression of Hes1, thus decreasing creatine phosphokinase (CPK) and lactate dehydrogenase (LDH) activity and promoting cell viability. The study found that VDAC1 was a potential target protein for Hes1 and the overexpression of Hes1 protein expression downregulated protein expression levels of VDAC1, reduced ROS production, stabilized ΔΨm, and inhibited apoptosis in H9c2 cells. Additionally, downregulation of Hes1 protein expression also upregulated VDAC1 protein expression, increased ROS production, imbalanced ΔΨm, promoted cell apoptosis, and attenuated the cardioprotection afforded by IPC and IPost. The Notch1/Hes1 signaling pathway activated by IPC/IPost can directly downregulate the protein expression of VDAC1 and consequently relieve A/R injury.

Topics: Apoptosis; Humans; Hypoxia; Ischemic Postconditioning; Ischemic Preconditioning; Myocardial Reperfusion Injury; Myocytes, Cardiac; Reactive Oxygen Species; Receptor, Notch1; Transcription Factor HES-1; Voltage-Dependent Anion Channel 1

Tetralogy of Fallot (TF) is a severe congenital defect of heart development. Fine-tuned sequential activation of Notch signaling genes is responsible for proper heart chamber development. Mutations in Notch genes have been associated with TF. The aim of this study was to analyze the activity of the Notch pathway in cardiac mesenchymal cells derived from ventricular tissue of TF patients.. Cardiac mesenchymal cells were isolated from 42 TF patients and from 14 patients with ventricular septal defects (VSDs), used as a comparison group. The Notch pathway was analyzed by estimating the expression of Notch-related genes by qPCR. Differentiation and proliferation capacity of the cells was estimated.. The TF-derived cells demonstrated a dysregulated pattern of Notch-related gene expression comparing to VSD-derived cells. Correlation of Notch signaling activation level by HEY1/HES1 expression level with proliferation and cardiogenic-like differentiation of cardiac mesenchymal cells was observed but not with clinical parameters nor with the age of the patients.. The data suggest a contribution of dysregulated Notch signaling to the pathogenesis of tetralogy of Fallot and importance of Notch signaling level for the functional state of cardiac mesenchymal cells, which could be critical considering these cells for potential cell therapy approaches.

Topics: Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Cell Differentiation; Cell Proliferation; Female; Gene Expression Profiling; Gene Expression Regulation; Heart; Heart Septal Defects, Ventricular; Heart Ventricles; Humans; Hypoxia; Immunophenotyping; Infant; Infant, Newborn; Male; Mesenchymal Stem Cells; Mutation; Myocardium; Receptors, Notch; Signal Transduction; Tetralogy of Fallot; Transcription Factor HES-1

Tumor angiogenesis plays important roles in the pathogenesis and prognosis of lung cancer. Both vascular endothelial growth factor (VEGF) and Dll4/Notch pathways are critical for angiogenesis, whereas their relationship under hypoxia in lung cancer remains unknown. Thus, in the present study, we evaluated the expression of VEGF and Dll4/Notch signaling molecules, and assessed their association with the microvessel density (CD31) and hypoxia (HIF1a) in lung cancer and normal lung tissues using immunohistochemical and Real-time RT-PCR techniques. Then, we investigated the biological function of Dll4 by transfecting Dll4 into HUVECs. In lung cancer tissues, Notch pathway molecules (HES1) and VEGF pathway molecules (VEGFR1 and VEGFR2) were significantly up-regulated, while the ratio of VEGFR1/VEGFR2 was decreased. CD31 and HIF1a were also found to be elevated in lung cancer. VEGFR1 was negatively correlated with Notch1 while positively correlated with Dll4. CD31 was positively correlated with HIF1a but negatively correlated with VEGFR1. Moreover, HIF1a was nearly positively correlated with HES1 in lung cancer tissues. After transfection, Dll4, Notch1 and VEGFR1 were up-regulated while VEGF and VEGFR2 were down-regulated in Dll4-transfected HUVECs compared with controls. Also, our findings suggest that the expression of VEGF and VEGFR2 increased gradually with the disease progression of lung cancer. In summary, VEGF and Notch signaling pathway molecules were overexpressed in lung cancer, which positively correlates with hypoxia (HIF1a) and angiogenesis (CD31). There might be a negative feedback loop between VEGF and Dll4/Notch signaling pathway in lung tumor angiogenesis.

Topics: Adaptor Proteins, Signal Transducing; Adenocarcinoma; Adenocarcinoma of Lung; Adult; Aged; Aged, 80 and over; Basic Helix-Loop-Helix Transcription Factors; Calcium-Binding Proteins; Carcinoma, Squamous Cell; Cells, Cultured; Disease Progression; Female; Follow-Up Studies; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Intercellular Signaling Peptides and Proteins; Lung; Lung Neoplasms; Male; Middle Aged; Neovascularization, Pathologic; Platelet Endothelial Cell Adhesion Molecule-1; Receptors, Notch; Retrospective Studies; Signal Transduction; Transcription Factor HES-1; Umbilical Veins; Up-Regulation; Vascular Endothelial Growth Factor A

Prostate carcinoma is among the most common causes of cancer-related death in men, representing 15% of all male malignancies in developed countries. Neuroendocrine differentiation (NED) has been associated with tumor progression, poor prognosis, and with the androgen-independent status. Currently, no successful therapy exists for advanced, castration-resistant disease. Because hypoxia has been linked to prostate cancer progression and unfavorable outcome, we sought to determine whether hypoxia would impact the degree of neuroendocrine differentiation of prostate cancer cells in vitro.. Exposure of LNCaP cells to low oxygen tension induced a neuroendocrine phenotype, associated with an increased expression of the transcription factor neurogenin3 and neuroendocrine markers, such as neuron-specific enolase, chromogranin A, and β3-tubulin. Moreover, hypoxia triggered a significant decrease of Notch 1 and Notch 2 mRNA and protein expression, with subsequent downregulation of Notch-mediated signaling, as shown by reduced levels of the Notch target genes, Hes1 and Hey1. NED was promoted by attenuation of Hes1 transcription, as cells expressing a dominant-negative form of Hes1 displayed increased levels of neuroendocrine markers under normoxic conditions. Although hypoxia downregulated Notch 1 and Notch 2 mRNA transcription and receptor activation also in the androgen-independent cell lines, PC-3 and Du145, it did not change the extent of NED in these cultures, suggesting that androgen sensitivity may be required for transdifferentiation to occur.. Hypoxia induces NED of LNCaP cells in vitro, which seems to be driven by the inhibition of Notch signaling with subsequent downregulation of Hes1 transcription.

Topics: Androgens; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle Proteins; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Gene Expression Regulation, Neoplastic; Homeodomain Proteins; Humans; Hypoxia; Male; Nerve Tissue Proteins; Neuroendocrine Tumors; Oxygen; Prostatic Neoplasms; Receptor, Notch1; Receptor, Notch2; Transcription Factor HES-1

Epithelial-to-mesenchymal transition (EMT) is associated with decreased adhesion and acquisition of metastatic potential of breast cancer cells. Epithelial-to-mesenchymal transition is mediated, in part, by two transcription repressors, Snail and Slug, that are known to be targets of the Notch signaling pathway, and JAGGED1-induced Notch activation increases EMT. However, the events that lead to increased Notch activity during EMT of breast cancer cells are unknown.. The accumulation of hypoxia inducible factors (HIFs) under hypoxia was detected by western blot analysis, and their effects on Notch signaling were measured by an in vitro Notch reporter assay. The expression of Notch target genes under hypoxia was tested by real-time PCR. The knockdown of HIF-1alpha was mediated by retroviral delivery of shRNA. The expression of Slug and Snail under hypoxia was measured by real-time PCR. Breast cancer cell migration and invasion under hypoxia were tested with cell migration and invasion kits.. Hypoxia increased the expression of Notch target genes such as HES1 and HEY1 in breast cancer cells, as was expression of Notch receptors and ligands. The mechanism is likely to involve the accumulation of HIF-1alpha and HIF-2alpha in these cells by hypoxia, which synergised with the Notch co-activator MAML1 in potentiating Notch activity. Hypoxia inducible factor-1alpha was found to bind to HES1 promoter under hypoxia. Knockdown of HIF-1alpha with shRNA inhibited both HES1 and HEY1 expression under hypoxia. Hypoxia increased the expression of Slug and Snail, and decreased the expression of E-cadherin, hallmarks of EMT. Notch pathway inhibition abrogated the hypoxia-mediated increase in Slug and Snail expression, as well as decreased breast cancer cell migration and invasion.. Hypoxia-mediated Notch signaling may have an important role in the initiation of EMT and subsequent potential for breast cancer metastasis.

Topics: Basic Helix-Loop-Helix Transcription Factors; Breast Neoplasms; Cadherins; Cell Cycle Proteins; Cell Movement; Female; Homeodomain Proteins; Humans; Hypoxia; Neoplasm Invasiveness; Receptors, Notch; Signal Transduction; Transcription Factor HES-1

The centrality of the transcriptional regulator Snail in epithelial-to-mesenchymal transformation (EMT), known to occur in models of diabetic nephropathy, has not been established. Transforming growth factor beta-1 (TGFbeta1) is induced in diabetic nephropathy and induces both Snail and EMT. Hypoxia inducible factors (HIFs) are known to induce Snail, independent of TGFbeta1. Notch induction is integral to Snail induction and EMT in tumour cells, but its role in the kidney is unknown. The present study was undertaken to determine the upstream regulators of Snail in the kidney in high glucose and hypoxic conditions. HK-2 cells were cultured in normoxic, hypoxic, high glucose and combined hypoxic/high glucose conditions. The expression of HIF1alpha, NotchIC, Snail, Lysyl oxidase-like 2 (Loxl2), and Hairy and Enhancer Split-1 (Hes1) were measured. We found that hypoxia increased HIF1alpha expression; however, concurrent exposure to high glucose blunted this effect. A similar pattern was observed in Lox12 expression, suggesting that Loxl2 was downstream of HIF1alpha, which was confirmed using siRNA techniques. Snail was upregulated by hypoxia and high glucose and in combination the effect was additive, suggesting independent upstream activation pathways by the two stimuli. Hes1 was upregulated by high glucose and to a lesser extent by hypoxia, but the effect of the combined stimuli was no greater than that observed with high glucose alone. NotchIC was downregulated by both hypoxia and high glucose, and in combination the effect was additive. Therefore, this study suggests that hypoxia and high glucose induce Snail expression through distinct pathways, independent of Notch signalling.

Topics: Amino Acid Oxidoreductases; Basic Helix-Loop-Helix Transcription Factors; Cell Line; Diabetic Nephropathies; Epithelial-Mesenchymal Transition; Homeodomain Proteins; Humans; Hyperglycemia; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Kidney Tubules, Proximal; Receptor, Notch1; RNA, Small Interfering; Signal Transduction; Snail Family Transcription Factors; Transcription Factor HES-1; Transcription Factors; Up-Regulation