cyclin-d1 and Hypoxia

Embryos of the annual killifish Austrofundulus limnaeus can enter into a state of metabolic dormancy, termed diapause, as a normal part of their development. In addition, these embryos can also survive for prolonged sojourns in the complete absence of oxygen. Dormant embryos support their metabolism using anaerobic metabolic pathways, regardless of oxygen availability. Dormancy in diapause is associated with high ATP and a positive cellular energy status, while anoxia causes a severe reduction in ATP content and large reductions in adenylate energy charge and ATP/ADP ratios. Most cells are arrested in the G 1/G 0 phase of the cell cycle during diapause and in response to oxygen deprivation. In this paper, we review what is known about the physiological and biochemical mechanisms that support metabolic dormancy in this species. We also highlight the great potential that this model holds for identifying novel therapies for human diseases such as heart attack, stroke and cancer.

Topics: Adenosine Triphosphate; Animals; Cell Cycle Checkpoints; Cell Division; Cyclin D1; Embryo, Nonmammalian; Embryonic Development; Enzyme Activation; Hypoxia; Killifishes; Life Cycle Stages; Oxygen; Stress, Physiological

Hypoxic pulmonary hypertension (HPH) is a common complication of chronic lung disease, which severely affects the survival and prognosis of patients. Several recent reports have shown that DNA damage and repair plays a crucial role in pathogenesis of pulmonary arterial hypertension. DNA-dependent protein kinase catalytic subunit (DNA-PKcs) as a part of DNA-PK is a molecular sensor for DNA damage that enhances DSB repair. This study aimed to demonstrate the expression and potential mechanism of DNA-PKcs on the pathogenesis of HPH.. Levels of DNA-PKcs and other proteins in explants of human and rats pulmonary artery from lung tissues and pulmonary artery smooth muscle cells (PASMC) were measured by immunohistochemistry and western blot analysis. The mRNA expression levels of DNA-PKcs and NOR1 in PASMCs were quantified with qRT-PCR. Meanwhile, the interaction among proteins were detected by Co-immunoprecipitation (Co-IP) assays. Cell proliferation and apoptosis was assessed by cell counting kit-8 assay(CCK-8), EdU incorporation and flow cytometry. Rat models of HPH were constructed to verify the role of DNA-PKcs in pulmonary vascular remodeling in vivo.. DNA-PKcs protein levels were both significantly up-regulated in explants of pulmonary artery from HPH models and lung tissues of patients with hypoxemia. In human PASMCs, hypoxia up-regulated DNA-PKcs in a time-dependent manner. Downregulation of DNA-PKcs by targeted siRNA or small-molecule inhibitor NU7026 both induced cell proliferation inhibition and cell cycle arrest. DNA-PKcs affected proliferation by regulating NOR1 protein synthesis followed by the expression of cyclin D1. Co-immunoprecipitation of NOR1 with DNA-PKcs was severely increased in hypoxia. Meanwhile, hypoxia promoted G. Our study indicated the potential mechanism of DNA-PKcs in the development of HPH. It might provide insights into new therapeutic targets for pulmonary vascular remodeling and pulmonary hypertension.

Topics: Animals; Cells, Cultured; Cyclin D1; DNA; DNA-Activated Protein Kinase; Humans; Hypertension, Pulmonary; Hypoxia; Rats; RNA, Messenger; RNA, Small Interfering; Vascular Remodeling

Hypoxia occurs naturally at high-altitudes and pathologically in hypoxic solid tumors. Here, we report that genes involved in various human cancers evolved rapidly in Tibetans and six Tibetan domestic mammals compared to reciprocal lowlanders. Furthermore, m

Topics: Adaptation, Physiological; Adenocarcinoma; Altitude; Animals; Animals, Domestic; Carcinoma, Non-Small-Cell Lung; Case-Control Studies; Cattle; Cyclin D1; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Dogs; Evolution, Molecular; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Genome; Goats; Horses; Humans; Hypoxia; Lung; Lung Neoplasms; Mice; Neoplasm Transplantation; RNA-Binding Proteins; Sheep; Sus scrofa; Swine; Tibet; Tumor Hypoxia

Objective- Hypoxic pulmonary hypertension (HPH) is characterized by proliferative vascular remodeling. Abnormal pulmonary artery smooth muscle cells proliferation and endothelial dysfunction are the primary cellular bases of vascular remodeling. AQP1 (aquaporin-1) is regulated by oxygen level and has been observed to play a role in the proliferation and migration of pulmonary artery smooth muscle cells. The role of AQP1 in HPH pathogenesis has not been directly determined to date. To determine the possible roles of AQP1 in the pathogenesis of HPH and explore its possible mechanisms. Approach and Results- Aqp1 knockout mice were used, and HPH model was established in this study. Primary pulmonary artery smooth muscle cells, primary mouse lung endothelial cells, and lung tissue sections from HPH model were used. Immunohistochemistry, immunofluorescence and Western blot, cell cycle, apoptosis, and migration analysis were performed in this study. AQP1 expression was upregulated by chronic hypoxia exposure, both in pulmonary artery endothelia and medial smooth muscle layer of mice. Aqp1 deficiency attenuated the elevation of right ventricular systolic pressures and mitigated pulmonary vascular structure remodeling. AQP1 deletion reduced abnormal cell proliferation in pulmonary artery and accompanied with accumulation of HIF (hypoxia-inducible factor). In vitro, Aqp1 deletion reduced hypoxia-induced proliferation, apoptosis resistance, and migration ability of primary cultured pulmonary artery smooth muscle cells and repressed HIF-1α protein stability. Furthermore, Aqp1 deficiency protected lung endothelial cells from apoptosis in response to hypoxic injury. Conclusions- Our data showed that Aqp1 deficiency could attenuate hypoxia-induced vascular remodeling in the development of HPH. AQP1 may be a potential target for pulmonary hypertension treatment.

Topics: Animals; Aquaporin 1; Cells, Cultured; Cyclin D1; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Male; Mice; Mice, Knockout; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Vascular Remodeling

Endothelial injury is considered as a trigger of pulmonary vascular lesions in the pathogenesis of hypoxic pulmonary hypertension (HPH). Although endothelial colony-forming cells (ECFCs) have vascular regeneration potential to maintain endothelial integrity, hypoxia-induced precise alteration in ECFCs function remains controversial. This study investigated the impact of hypoxia on human ECFCs function in vitro and the underlying mechanism. We found that hypoxia inhibited ECFCs proliferation, migration and angiogenesis. Compared with no treatment, the expression of hypoxia inducible factor-1α (HIF-1α) in hypoxia-treated ECFCs was increased, with an up-regulation of p27 and a down-regulation of cyclin D1. The over-secreted vascular endothelial growth factor (VEGF) was detected, with the imbalanced expression of fetal liver kinase 1 (flk-1) and fms related tyrosine kinase 1 (flt-1). Hypoxia-induced changes in ECFCs could be reversed by HIF-1α inhibitor KC7F2. These data suggest that HIF-1α holds the key in regulating ECFCs function which may open a new perspective of ECFCs in HPH management.

Topics: Adult; Cardiovascular Agents; Cell Cycle; Cell Movement; Cell Proliferation; Cells, Cultured; Cyclin D1; Disulfides; Endothelial Cells; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Middle Aged; Receptors, Vascular Endothelial Growth Factor; Signal Transduction; Sulfonamides; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Young Adult

Rhodiola algida var. tangutica (Maxim.) S.H. Fu is a perennial plant of the Crassulaceae family that grows in the mountainous regions of Asia. The rhizome and roots of this plant have been long used as Tibetan folk medicine for preventing high latitude sickness.. The aim of this study was to determine the effect of bioactive fraction from R. algida (ACRT) on chronic hypoxia-induced pulmonary arterial hypertension (HPAH) and to understand the possible mechanism of its pharmacodynamic actions.. Male Sprague-Dawley rats were separated into five groups: control group, hypoxia group, and hypoxia+ACRT groups (62.5, 125, and 250mg/kg/day of ACRT). The chronic hypoxic environment was created in a hypobaric chamber by adjusting the inner pressure and oxygen content for 4 weeks. After 4 weeks, major physiological parameters of pulmonary arterial hypertension such as mPAP, right ventricle index (RV/LV+S, RVHI), hematocrit (Hct) levels and the medial vessel thickness (wt%) were measured. Protein and mRNA expression levels of proliferating cell nuclear antigen (PCNA), cyclin D1, p27Kip1 and cyclin-dependent kinase 4 (CDK4)) were detected by western blotting and real time PCR respectively. Chemical profile of ACRT was revealed by ultra performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UHPLC-Q-TOF-MS/MS).. The results showed that a successful HPAH rat model was established in a hypobaric chamber for 4 weeks, as indicated by the significant increase in mPAP, RV/LV+S, RV/BW and wt%. Compared with the normal group, administration of ACRT reduced mPAP, right ventricular hypertrophy, pulmonary small artery wall thickness, and damage in ultrastructure induced by hypoxia in rats. PCNA, cyclin D1, and CDK4 expression was reduced (p<0.05), and p27Kip1 expression increased (p<0.05) in hypoxia+ACRT groups compared to hypoxia. 38 constituents in bioactive fraction were identified by UHPLC-Q-TOF-MS/MS.. Our results suggest that ACRT could alleviate chronic hypoxia-induced pulmonary arterial hypertension. And its anti-proliferation mechanism in rats based on decreasing PCNA, cyclin D1, CDK4 expression level and inhibiting p27Kip1 degradation.

Topics: Animals; Arterial Pressure; Cell Proliferation; Chronic Disease; Cyclin D1; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase Inhibitor p27; Disease Models, Animal; Dose-Response Relationship, Drug; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Phytotherapy; Plant Extracts; Plants, Medicinal; Proliferating Cell Nuclear Antigen; Proteolysis; Pulmonary Artery; Rats, Sprague-Dawley; Rhodiola; Signal Transduction; Vascular Remodeling; Ventricular Function, Right; Ventricular Remodeling

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive and typically fatal lung disease with a very low survival rate. Excess accumulation of fibroblasts, myofibroblasts and extracellular matrix creates hypoxic conditions within the lungs, causing asphyxiation. Hypoxia is, therefore, one of the prominent features of IPF. However, there have been few studies concerning the effects of hypoxia on pulmonary fibroblasts. In this study, we investigated the molecular mechanisms of hypoxia-induced lung fibroblast proliferation. Hypoxia increased the proliferation of normal human pulmonary fibroblasts and IPF fibroblasts after exposure for 3-6 days. Cell cycle analysis demonstrated that hypoxia promoted the G1/S phase transition. Hypoxia downregulated cyclin D1 and A2 levels, while it upregulated cyclin E1 protein levels. However, hypoxia had no effect on the protein expression levels of cyclin-dependent kinase 2, 4, and 6. Chemical inhibition of hypoxia-inducible factor (HIF)-2 reduced hypoxia-induced fibroblast proliferation. Moreover, silencing of Nuclear Factor Activated T cell (NFAT) c2 attenuated the hypoxia-mediated fibroblasts proliferation. Hypoxia also induced the nuclear translocation of NFATc2, as determined by immunofluorescence staining. NFAT reporter assays showed that hypoxia-induced NFAT signaling activation is dependent on HIF-2, but not HIF-1. Furthermore, the inhibition or silencing of HIF-2, but not HIF-1, reduced the hypoxia-mediated NFATc2 nuclear translocation. Our studies suggest that hypoxia induces the proliferation of human pulmonary fibroblasts through NFAT signaling and HIF-2.

Topics: Adult; Aged; Basic Helix-Loop-Helix Transcription Factors; Cell Cycle; Cell Proliferation; Cells, Cultured; Cyclin A1; Cyclin D1; Cyclin E; Cyclin-Dependent Kinase 2; Cyclin-Dependent Kinase 4; Cyclin-Dependent Kinase 6; Female; Fibroblasts; Humans; Hypoxia; Idiopathic Pulmonary Fibrosis; Lung; Male; Middle Aged; NFATC Transcription Factors; Oncogene Proteins

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Animals; Animals, Newborn; bcl-2-Associated X Protein; Caspases; Cyclin D1; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Hypoxia; Membrane Potential, Mitochondrial; Mental Disorders; Mitochondrial Diseases; NAD; Oxidative Stress; Oxidoreductases; Pregnancy; Rats; Succinate Dehydrogenase

It has been shown that hypoxia stimulation promotes chondrocytes autophagy partly through HIF-1α, miR-146a and Bcl-2 progressively, and this mechanism represented the connection among hypoxia, miR-146a and autophagy, and provides a possible therapeutic strategy for osteoarthritis. However, the interaction between miR-146a and Bcl-2 is still unclear. Here in a hypoxic environment, we quantified the three reported miR-146a targets: two inflammation related targets Traf6, IRAK1; and the only reported target in chondrocytes Smad4. We confirmed the regulative function of miR-146a between hypoxia and these genes, and explored the Bcl-2 expression and autophagy level under extrinsic up-regulation of these three gene separately. All the three genes were down-regulated by hypoxia. Surprisingly, Traf6 and IRAK, but not the unique Smad4 in chondrocytes, were restored by antagomiR-146a. Both Ad-Traf6 and Ad-IRAK1 reinstated hypoxia or miR-146a repressed Bcl-2. However, Ad-Smad4 did not affect Bcl-2 in hypoxia or normoxia. The autophagy level showed a reverse variability compared to Bcl-2. Taken together, our results provided evidence that Smad4, the unique reported target for miR-146a in chondrocytes is unusually not involved in the chondrocytes autophagy, while the Traf6 and IRAK1 are the new targets for miR-146a in chondrocytes during autophagy.

Topics: Animals; Autophagy; Blotting, Western; Chondrocytes; Cyclin D1; Hypoxia; Interleukin-1 Receptor-Associated Kinases; Mice; MicroRNAs; Microscopy, Confocal; Real-Time Polymerase Chain Reaction; Smad4 Protein; TNF Receptor-Associated Factor 6

Hypoxic pulmonary hypertension (HPH), which is characterized by pulmonary arteriolar remodeling and right ventricular hypertrophy, is still a life-threatening disease with the current treatment strategies. The underlying molecular mechanisms of HPH remain unclear. Our previously published study showed that Wnt5a, one of the ligands in the Wnt family, was critically involved in the inhibition of hypoxia-induced pulmonary arterial smooth muscle cell proliferation by downregulation of β-catenin/cyclin D1 in vitro. In this study, we investigated the possible functions and mechanisms of Wnt5a in HPH in vivo. Recombinant mouse Wnt5a (rmWnt5a) or phosphate buffered saline (PBS) was administered to male C57/BL6 mice weekly from the first day to the end of the two or four weeks after exposed to hypoxia (10% O2). Hypoxia-induced pulmonary hypertension was associated with a marked increase in β-catenin/cyclin D1 expression in lungs. Right ventricular systolic pressure and right ventricular hypertrophy index were reduced in animals treated with rmWnt5a compared with PBS. Histology showed less pulmonary vascular remodeling and right ventricular hypertrophy in the group treated with rmWnt5a than with PBS. Treatment with rmWnt5a resulted in a concomitant reduction in β-catenin/cyclin D1 levels in lungs. These data demonstrate that Wnt5a exerts its beneficial effects on HPH by regulating pulmonary vascular remodeling and right ventricular hypertrophy in a manner that is associated with reduction in β-catenin/cyclin D1 signaling. A therapy targeting the β-catenin/cyclin D1 signaling pathway might be a potential strategy for HPH treatment.

Topics: Animals; beta Catenin; Cyclin D1; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Male; Mice; Mice, Inbred C57BL; Recombinant Proteins; Signal Transduction; Vascular Remodeling; Wnt Proteins; Wnt-5a Protein

Chronic hypoxia-induced pulmonary arterial hypertension (HPH) is closely associated with profound vascular remodeling, especially pulmonary arterial medial hypertrophy and muscularization due to hyperplasia of pulmonary artery smooth muscle cells (PASMCs). Aberrant Wnt signaling has been associated with lung diseases, but its role in pulmonary hypertension is unclear. This study evaluated the effect of Wnt5a on hypoxia-induced proliferation of human PASMCs and its possible mechanism. The results show that hypoxia (3% O(2), 48 h) induced proliferation of human PASMCs, accompanied with a significant decrease in Wnt5a gene expression, increase in β-catenin and Cyclin D1 expression, as well as β-catenin nuclear translocation. Treatment with recombinant mouse Wnt5a significantly inhibited hypoxia-induced proliferation of human PASMCs, upregulation of Cyclin D1 and β-catenin expression, as well as the nuclear translocation of β-catenin. These effects were inhibited by Wnt5a antibody. Knocking down β-catenin or Cyclin D1 gene expression inhibited hypoxia-induced human PASMC proliferation, whereas overexpression of β-catenin increased hypoxia-induced human PASMC proliferation and counteracted the inhibitory effect of Wnt5a. These results suggest that Wnt5a has an antiproliferative effect on hypoxia-induced human PASMC proliferation by downregulation of β-catenin and its target gene Cyclin D1. Hypoxia-induced downregulation of Wnt5a may be a way to facilitate hypoxia-induced human PASMC proliferation. The results of this study will help to understand the novel strategies for PH treatment involving Wnt signaling.

Topics: Animals; beta Catenin; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Cyclin D1; Down-Regulation; Humans; Hypertension, Pulmonary; Hypoxia; Mice; Myocytes, Smooth Muscle; Proto-Oncogene Proteins; Pulmonary Artery; Wnt Proteins; Wnt-5a Protein

Pulmonary arterial hypertension (PAH) is a devastating disease, and no effective treatments are available. Hypoxia-induced pulmonary artery remodeling, including smooth muscle cell proliferation, contributes to PAH, but the exact mechanisms underlying this abnormal process are largely undefined. The forkhead box M1 (FoxM1) transcription factor regulates cancer cell growth by modulating gene expression critical for cell cycle progression. Here, we report for the first time, to the best of our knowledge, a novel function of FoxM1 in the hypoxia-stimulated proliferation of human pulmonary artery smooth muscle cells (HPASMCs). Exposure to hypoxia caused a marked up-regulation of FoxM1 gene expression, mainly at the transcription level, and this induction correlated with HPASMC cell proliferation. The knockdown of FoxM1 inhibited the hypoxia-stimulated proliferation of HPASMCs. We found that the knockdown of HIF-2α, but not HIF-1α, diminished FoxM1 induction in response to hypoxia. However, the knockdown of FoxM1 did not alter expression levels of HIF-2α or HIF-1α, suggesting that HIF-2α is an upstream regulator of FoxM1. Furthermore, the knockdown of FoxM1 prevented the hypoxia-induced expression of aurora A kinase and cyclin D1. Collectively, our results suggest that hypoxia induces FoxM1 gene expression in an HIF-2α-dependent pathway, thereby promoting HPASMC proliferation.

Topics: Aurora Kinases; Basic Helix-Loop-Helix Transcription Factors; Cell Hypoxia; Cell Proliferation; Cells, Cultured; Cyclin B; Cyclin D1; Familial Primary Pulmonary Hypertension; Forkhead Box Protein M1; Forkhead Transcription Factors; Gene Expression; Humans; Hypertension, Pulmonary; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Myocytes, Smooth Muscle; Protein Serine-Threonine Kinases; Pulmonary Artery

L-Mimosine, an iron chelator and a prolyl 4-hydroxylase inhibitor, blocks many cancer cells at the late G1 phase. B-cell translocation gene 2 (Btg2) regulates the G1/S transition phases of the cell cycle. N-myc downstream regulated gene 1 (Ndrg1) is a differentiation-inducing gene upregulated by hypoxia. We evaluated the molecular mechanisms of L-mimosine on cell cycle modulation in PC-3 and LNCaP prostate carcinoma cells. The effect of L-mimosine on cell proliferation of prostate carcinoma cells was determined by the [3H]thymidine incorporation and flow cytometry assays. L-Mimosine arrested the cell cycle at the G1 phase in PC-3 cells and at the S phase in LNCaP cells, thus attenuating cell proliferation. Immunoblot assays indicated that hypoxia and L-mimosine stabilized hypoxia-inducible factor-1α (HIF-1α) and induced Btg2 and Ndrg1 protein expression, but downregulated protein levels of cyclin A in both PC-3 and LNCaP cells. L-Mimosine treatment decreased cyclin D1 protein in PC-3 cells, but not in LNCaP cells. Dimethyloxalylglycine, a pan-prolyl hydroxylase inhibitor, also induced Btg2 and Ndrg1 protein expression in LNCaP cells. The transient gene expression assay revealed that L-mimosine treatment or cotransfection with HIF-1α expression vector enhanced the promoter activities of Btg2 and Ndrg1 genes. Knockdown of HIF-1α attenuated the increasing protein levels of both Btg2 and Ndrg1 by hypoxia or L-mimosine in LNCaP cells. Our results indicated that hypoxia and L-mimosine modulated Btg2 and Ndrg1 at the transcriptional level, which is dependent on HIF-1α. L-Mimosine enhanced expression of Btg2 and Ndrg1, which attenuated cell proliferation of the PC-3 and LNCaP prostate carcinoma cells.

Topics: B-Lymphocytes; Carcinoma; Cell Cycle; Cell Cycle Proteins; Cell Line, Tumor; Cell Proliferation; Cyclin D1; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immediate-Early Proteins; Intracellular Signaling Peptides and Proteins; Male; Mimosine; Organ Specificity; Procollagen-Proline Dioxygenase; Prostate; Prostatic Neoplasms; Signal Transduction; Transcriptional Activation; Transfection; Tumor Suppressor Proteins

The clinical side-effects of increased cyclooxygenase (COX) activity induced by pathologic conditions have raised concerns recently. However, a better understanding of the mechanisms underlying the subsequent neurotoxicity requires knowledge of pathways downstream of COX, especially prostaglandin E2 (PGE2) and its receptors. Therefore, this study was performed to investigate the effects of PGE2 receptor 1 (EP1) activity on neuronal cell death resulting from hypoxia/reoxygenation (Hyp). As cyclinD1 activity has been shown to regulate neuronal apoptosis as well, the role of cyclinD1 was investigated, as well. Cortical neural cells isolated from fetal Wistar rats were cultured for 12 d and exposed to Hyp conditions to establish an in vitro Hyp model. To determine the effects of EP1 activity on Hyp-induced neurotoxicity, cells were treated with 17-phenyl trinor-PGE2 (17-pt), a synthetic EP1 agonist, or sc-51089, an EP1 antagonist, then exposed to hypoxic conditions for 3h and reoxygenated for 21 h. Following Hyp, cell viability was quantified by MTT assays, and apoptosis was assessed by flow cytometry. Protein expression levels of caspase-3 and cyclinD1 were examined by Western blot analysis. Treatment of cultured cortical neurons with 17-pt significantly decreased the survival rate of Hyp-treated neurons (p<0.05), while treatment with sc-51089 increased the survival rate. Treatment with 17-pt also led to increased expression levels of caspase-3, further supporting a role for EP1 in the observed neurotoxicity. However, cyclinD1 expression levels were unchanged following treatment with either 17-pt or sc-51089. Therefore, EP1 may play an important role in Hyp-induced neuronal apoptosis, but this neurotoxic activity is unlikely to involve cyclinD1.

Topics: Analysis of Variance; Animals; Annexin A5; Caspase 3; Cell Survival; Cells, Cultured; Cerebral Cortex; Cyclin D1; Dinoprostone; Dose-Response Relationship, Drug; Embryo, Mammalian; Hydrazines; Hypoxia; Neurons; Oxazepines; Oxygen; Rats; Rats, Wistar; Receptors, Prostaglandin E, EP1 Subtype; Tetrazolium Salts; Thiazoles; Time Factors; Up-Regulation

Chronic hypoxia causes pulmonary vascular remodeling leading to pulmonary hypertension (PH) and right ventricle (RV) hypertrophy. Aberrant expression of microRNA (miRNA) is closely associated with a number of pathophysiologic processes. However, the role of miRNAs in chronic hypoxia-induced pulmonary vascular remodeling and PH has not been well characterized. In this study, we found increased expression of miR-21 in distal small arteries in the lungs of hypoxia-exposed mice. Putative miR-21 targets, including bone morphogenetic protein receptor (BMPR2), WWP1, SATB1, and YOD1, were downregulated in the lungs of hypoxia-exposed mice and in human pulmonary artery smooth muscle cells (PASMCs) overexpressing miR-21. We found that sequestration of miR-21, either before or after hypoxia exposure, diminished chronic hypoxia-induced PH and attenuated hypoxia-induced pulmonary vascular remodeling, likely through relieving the suppressed expression of miR-21 targets in the lungs of hypoxia-exposed mice. Overexpression of miR-21 enhanced, whereas downregulation of miR-21 diminished, the proliferation of human PASMCs in vitro and the expression of cell proliferation associated proteins, such as proliferating cell nuclear antigen, cyclin D1, and Bcl-xL. Our data suggest that miR-21 plays an important role in the pathogenesis of chronic hypoxia-induced pulmonary vascular remodeling and also suggest that miR-21 is a potential target for novel therapeutics to treat chronic hypoxia associated pulmonary diseases.

Topics: Airway Remodeling; Animals; Apoptosis; bcl-X Protein; Bone Morphogenetic Protein Receptors, Type II; Cell Cycle Proteins; Cell Line; Cell Proliferation; Cyclin D1; Down-Regulation; Humans; Hypertension, Pulmonary; Hypertrophy, Right Ventricular; Hypoxia; Lung; Matrix Attachment Region Binding Proteins; Mice; MicroRNAs; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Pulmonary Artery; Ubiquitin-Protein Ligases; Up-Regulation

The red-eared slider turtle (Trachemys scripta elegans) has a well-developed natural tolerance for oxygen deprivation that derives from biochemical adaptations, including anoxia-induced suppression of metabolic rate. We hypothesized that mechanisms that suppress ATP-expensive cell cycle activity would contribute significantly to establishing the hypometabolic state during anaerobiosis. Cyclin D1 is a critical regulator of the G 1 phase of the cell cycle and is regarded as key to initiating cell proliferation. The relative protein expression of cyclin D1 was analyzed in both whole-cell and nuclear fractions of liver, kidney and skeletal muscle from turtles exposed to 5 or 20 h of submergence anoxia. Expression of cyclin D1 in both total and nuclear fractions decreased significantly under anoxia in liver and kidney as compared with aerobic controls, but no significant change occurred in muscle. The relative phosphorylation state of cyclin D1 (threonine 286) was also unchanged during anoxia in all tissues. Since phosphorylation of threonine 286 is necessary for proteasomal degradation of cyclin D1, this implies that an alternative mechanism is responsible for cyclin D1 suppression in anoxia. Levels of cyclin D1 mRNA transcripts did not change under anoxia in any tissue, so a post-transcriptional method of regulation was implicated. Analysis of the 3'UTR of cyclin D1 showed the presence of both an AU-rich region and a conserved binding site for microRNA-16-1 and microRNA-15a. Levels of both microRNAs increased in liver and kidney (but not in muscle) under anoxic conditions, implicating microRNA inhibition of mRNA translation as the mechanism underlying the suppression of cyclin D1 protein levels in the anoxic turtle.

Topics: 3' Untranslated Regions; Anaerobiosis; Animals; Cell Cycle Checkpoints; Cell Proliferation; Cyclin D1; Hypoxia; Kidney; Liver; MicroRNAs; Oxygen; Phosphorylation; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Proteolysis; Regulatory Elements, Transcriptional; RNA, Messenger; Threonine; Turtles

Embryos of the annual killifish Austrofundulus limnaeus can enter into dormancy associated with diapause and anoxia-induced quiescence. Dormant embryos are composed primarily of cells arrested in the G(1)/G(0) phase of the cell cycle based on flow cytometry analysis of DNA content. In fact, most cells in developing embryos contain only a diploid complement of DNA, with very few cells found in the S, G(2), or M phases of the cell cycle. Diapause II embryos appear to be in a G(0)-like state with low levels of cyclin D1 and p53. However, the active form of pAKT is high during diapause II. Exposure to anoxia causes an increase in cyclin D1 and p53 expression in diapause II embryos, suggesting a possible re-entry into the cell cycle. Post-diapause II embryos exposed to anoxia or anoxic preconditioning have stable levels of cyclin D1 and stable or reduced levels of p53. The amount of pAKT is severely reduced in 12 dpd embryos exposed to anoxia or anoxic preconditioning. This study is the first to evaluate cell cycle control in embryos of A. limnaeus during embryonic diapause and in response to anoxia and builds a foundation for future research on the role of cell cycle arrest in supporting vertebrate dormancy.

Topics: Adaptation, Physiological; Analysis of Variance; Animals; Blotting, Western; Cell Cycle Checkpoints; Cyclin D1; Embryo, Nonmammalian; Flow Cytometry; Hypoxia; Killifishes; Oncogene Protein v-akt; Tumor Suppressor Protein p53

Cyclin D1 and insulin-like growth factor 1 receptor (IGF-1R) are key regulators of cell proliferation that are overexpressed in most breast cancers. The purpose of the present study was to investigate the molecular mechanism by which hemin exerts its inhibitory effects on aggressive breast cancer cells. We found that hemin regulates cyclin D1 and IGF-1R proteins and insulin-like growth factor-1 gene expression through STAT5b in breast cancer cells. We confirmed that STAT5b, cyclin D1, and IGF-1R is up-regulated by hypoxia, and the increased STAT5b binds strongly to the STAT5-binding sites contained within the distal 5'-flanking region of IGF-1 gene in breast cancer cells. EMSA studies showed that STAT5 binding activity to the IGF-1 and cyclin D1 promoter was distinctly decreased by hemin in STAT5b-transfected COS-7 or MDA-MB 231 cells. IGF-1 gene expression was also decreased by hemin in mammary epithelial cells. STAT5b expression was inhibited in siRNA experiments and by hemin, leading to decreased levels of IGF-1. These results provide a basis for molecular targets in cancer treatment via the STAT5b/IGF-1 or /cyclin D1 pathway in solid tumor cells. These data indicate that hemin inhibits the cyclin D1 and IGF-1 expression via STAT5b under hypoxia in ERalpha-negative breast cancer cells. These findings are valuable toward understanding the role of hemin-induced inhibition of cyclin D1 and IGF-1 expression under hypoxia in invasive and metastatic breast cancer.

Topics: Animals; Cell Line, Tumor; Chlorocebus aethiops; CHO Cells; COS Cells; Cricetinae; Cricetulus; Cyclin D1; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Hemin; Humans; Hypoxia; Insulin-Like Growth Factor I; Neoplasm Invasiveness; Neoplasm Metastasis; STAT5 Transcription Factor

A few highly aggressive and malignant tumor cells could acquire identities by turning on genes expressed by endothelial cells and recruit blood vessels to sustain tumor growth. Hypoxia was reported recently to play an essential role in these events. These 'plastic' tumor-cell phenotypes and the exact mechanism driving transendothelial differentiation by hypoxia-inducible factor (HIF)-1alpha is unclear. In this study, epithelial ovarian carcinoma cells were exposed to hypoxia and the tumor cells were transformed into endothelial cells-like (ECs-like). Typical endothelial features such as cell markers and uptaking of acetylated low density lipoprotein were identified constantly. Small interference RNA was used to block the expression of HIF-1alpha. Analysis revealed that hypoxia promotes transendothelial differentiation through stimulating HIF-1-dependent transcriptional expression of vascular endothelial growth factor (VEGF), VEGF receptor-2 (Flk-1) and P53, and through decreasing HIF-1-independent transcriptional expression of Cyclin D1. These results demonstrate that ECs-like derived from epithelial ovarian cancer cells are similar to endothelial progenitor cells rather than endothelial cells. HIF-1alpha is crucial but not unique in alternation of tumor cells towards ECs-like.

Topics: Cell Differentiation; Cyclin D1; Endothelial Cells; Endothelium, Vascular; Epithelial Cells; Female; Humans; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Microscopy, Fluorescence; Ovarian Neoplasms; RNA, Small Interfering; Tumor Cells, Cultured; Tumor Suppressor Protein p53; Vascular Endothelial Growth Factor Receptor-2

The Ets family of transcription factors is defined by a conserved DNA-binding Ets domain that forms a winged helix-turn-helix structure motif. The Ets family is involved in a diverse array of biologic functions, including cellular growth, migration, and differentiation. The hypothesis in this study was that Ets-1 is re-expressed during regeneration after acute renal failure (ARF) and plays a key role in the transcriptional regulation of cyclin D1 and the cell cycle progression in renal tubular cells. For clarifying the significance of Ets-1 in ARF, a rat ARF model in vivo and LLC-PK1 cells as an in vitro model were used. After the left rat renal artery was clamped for 1 h, the whole kidney homogenate was examined and total RNA was extracted at 6, 12, 24, 48, and 72 h after reperfusion by Western blot analysis and real-time reverse transcription-PCR. Ets-1 mRNA and protein expression were strongly increased at 6 to 24 h after the ischemia, respectively. The expression of hypoxia-inducible factor-1alpha was increased dramatically as early as 6 h after ischemia-reperfusion and decreased at 48 and 72 h after ischemia-reperfusion. In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with proliferating cell nuclear antigen (PCNA). Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells. Ets-1 promoter activity increased between 3 and 6 h in hypoxia, and hypoxia also induced changes in the Ets-1 protein level in LLC-PK1 cells. The Ets-1 induction by hypoxia was abolished by the transfection of dominant-negative hypoxia-inducible factor-1alpha. A gel shift assay demonstrated that Ets-1 binds to the ets-1 binding site of the cyclin D1 promoter in the ischemia-reperfusion condition. Overexpression of Ets-1 did not significantly change the caspase 3 activity or the value of cell death ELISA in LLC-PK1 cells. Taken together, these data suggest that Ets-1 plays a key role in the cell-cycle progression of renal tubules in ARF. The Ets-1 pathway may regulate the transcription of cyclin D1 and control the regeneration of renal tubules in ARF.

Topics: Acute Kidney Injury; Animals; Apoptosis; Blotting, Western; Cell Division; Cyclin D1; Electrophoretic Mobility Shift Assay; Gene Expression; Hypoxia; Hypoxia-Inducible Factor 1, alpha Subunit; Immunohistochemistry; LLC-PK1 Cells; Male; Polymerase Chain Reaction; Promoter Regions, Genetic; Proto-Oncogene Protein c-ets-1; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-ets; Rats; Rats, Sprague-Dawley; Regeneration; Swine; Transcription Factors; Transcription, Genetic

Apoptosis may contribute to the myocardial dysfunction associated with heart failure (HF). Activation of the p38 MAPK cascade can induce apoptosis in non-cardiac cells through increased expression of Fas-L, or through decreased expression of cyclin D(1).. We tested the hypothesis that hypoxia (HX), angiotensin-II (A-II) and norepinephrine (NEPI) can mediate apoptosis by activating p38 MAPK, and thus initiating stimulus specific changes in Fas-L and cyclin D(1) expression in failing cardiomyocytes.. Cardiomyocytes isolated from ten dogs with HF induced by coronary microembolizations were subjected to HX or A-II or NEPI with and without a p38 MAPK inhibitor (SB 203580). TUNEL staining for DNA fragmentation and Western blots for p38 MAPK, Fas-L and cyclin D(1) detection were performed. HX-induced apoptosis was associated with increased Fas-L expression, A-II-induced apoptosis was associated with increased Fas-L and decreased cyclin D(1) expression, and NEPI-induced apoptosis was associated with decreased cyclin D(1) expression. Inhibition of p38 MAPK activity attenuated stress-induced apoptosis in all experiments and reversed changes in Fas-L and cyclin D(1) expression.. HX, A-II and NEPI mediate apoptosis in failing cardiomyocytes via different effects on Fas-L and cyclin D(1) expression. Inhibition of p38 MAPK reversed these effects, suggesting that apoptosis induced by HX, A-II and NEPI involves activation of p38 MAPK upstream from Fas-L and cyclin D(1).

Topics: Angiotensin II; Animals; Apoptosis; Cyclin D1; Disease Models, Animal; Dogs; Fas Ligand Protein; Follow-Up Studies; Heart Failure; Hypoxia; Incidence; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; Models, Cardiovascular; Myocytes, Cardiac; Norepinephrine; p38 Mitogen-Activated Protein Kinases

Ischemic heart disease is more prevalent in men than in women. The remodeling of extracellular matrix, is a structural correlate of heart failure of ischemic origin and proliferation of cardiac fibroblasts is a key factor in this remodeling. We asked if proliferative response of male and female cardiac fibroblasts is differentially susceptible to hypoxia. DNA synthesis, using 3H-thymidine incorporation was compared under hypoxia (2% O2) in cardiac fibroblasts obtained from adult, age-matched male and female rat heart. In female cells DNA synthesis remained unchanged under hypoxia and this resistance was dependent on tyrosine kinase activation, as it was abolished in the presence of genistein, a tyrosine kinase inhibitor. Male cells, on the other hand, were susceptible to hypoxia and their DNA synthesis was reduced significantly (70%, (p < 0.0001). This effect was partially reversed by inhibition of tyrosine kinase. Western analysis showed a higher abundance of tyrosine phosphorylated proteins in male cells compared to female cells as well as differences in molecular weight of basal and hypoxia-induced tyrosine-phosphorylated proteins between male and female cells. The presence of estrogen (17-beta estradiol, 10 nM) altered the response of both cells to hypoxia. In female cells the combined effect of hypoxia and estrogen led to inhibition of DNA synthesis, whereas in male cells estrogen partially reversed the hypoxia-induced inhibition of DNA synthesis (37% (p < 0.01) inhibition in the presence of estrogen vs. 70% (p < 0.0001) inhibition in the absence of estrogen). The effects of estrogen in male and female cells were mediated via estrogen receptors as they were reversed by the pure anti-estrogen, ICI 182,780. Western analysis of cell lysate showed hypoxia-induced increase in the level of estrogen receptor beta in both male and female cells. Gel shift analysis showed hypoxia-induced increase in cytoplasmic ERE (estrogen response element)-binding activity and decrease in nuclear ERE-binding in male cells. In female cells cytoplasmic and nuclear ERE-binding activities remained unchanged under hypoxia. Together, these data demonstrate that while female cells are resistant to hypoxia-induced inhibition in DNA synthesis, male cells are susceptible; intracellular pathways involving tyrosine phosphorylation are involved in the response of both cells; and estrogen, via estrogen-receptor-dependent mechanisms, differentially alters the response of male and female c

Topics: Age Factors; Animals; Blotting, Western; Cell Division; Cell Nucleus; Cells, Cultured; Cyclin B; Cyclin B1; Cyclin D1; Cytoplasm; Cytosol; DNA; Enzyme Activation; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor beta; Female; Fibroblasts; Fulvestrant; Genistein; Hypoxia; Male; Myocardium; Phosphorylation; Precipitin Tests; Proliferating Cell Nuclear Antigen; Protein Binding; Protein-Tyrosine Kinases; Rats; Rats, Inbred F344; Receptors, Estrogen; Response Elements; Sex Factors; Tyrosine