ILKAP Binding to and Dephosphorylating HIF-1α is Essential for Apoptosis Induced by Severe Hypoxia

Background/Aims: Integrin-linked kinase-associated phosphatase (ILKAP), a serine/threonine phosphatase that belongs to the protein phosphatase 2C family, has a role in cell survival and apoptosis. Hypoxia-inducible factor 1α (HIF-1α) is the key transcription factor in the response to oxygen deficiency in mammals. Direct phosphorylation and dephosphorylation of HIF-1α affect its function. The present study investigated the role of ILKAP on HIF-1α dephosphorylation and cell behavior. Methods: HIF-1α was induced by hypoxia. Physical binding between ILKAP and HIF-1α was demonstrated by a co-immunoprecipitation assay. HIF-1α transcriptional activity was investigated using a hypoxia-response element-containing luciferase reporter plasmid. Cell viability was evaluated by a trypan blue dye exclusion assay. ILKAP function was explored by a gain and loss assay with an overexpression plasmid and shRNA infection. Results: ILKAP physically interacted with HIF-1α and induced its dephosphorylation. Both the HIF-1α-p53 interaction and apoptosis relied on ILKAP. Conclusion: The results indicated that the ILKAP directly binds and dephosphorylates HIF-1α and responsible for severe hypoxia-induced cell apoptosis.

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Hypoxia-inducible factor (HIF): fuel for cancer progression

Current Molecular Pharmacology ◽

10.2174/1874467214666210120154929 ◽

2021 ◽

Vol 14 ◽

Author(s):

Saurabh Satija ◽

Harpreet Kaur ◽

Murtaza M. Tambuwala ◽

Prabal Sharma ◽

Manish Vyas ◽

...

Keyword(s):

Biological Sciences ◽

Tumor Microenvironment ◽

Tumor Cells ◽

Cancer Progression ◽

Oxygen Deficiency ◽

Hypoxia Inducible Factor ◽

Transcription Factor Activation ◽

Underlying Mechanisms ◽

Adaptive Reactions

Hypoxia is an integral part of tumor microenvironment, caused primarily due to rapidly multiplying tumor cells and a lack of proper blood supply. Among the major hypoxic pathways, HIF-1 transcription factor activation is one of the widely investigated pathways in the hypoxic tumor microenvironment (TME). HIF-1 is known to activate several adaptive reactions in response to oxygen deficiency in tumor cells. HIF-1 has two subunits, HIF-1β (constitutive) and HIF-1α (inducible). The HIF-1α expression is largely regulated via various cytokines (through PI3K-ACT-mTOR signals), which involves the cascading of several growth factors and oncogenic cascades. These events lead to the loss of cellular tumor suppressant activity through changes in the level of oxygen via oxygen-dependent and oxygen-independent pathways. The significant and crucial role of HIF in cancer progression and its underlying mechanisms have gained much attention lately among the translational researchers in the fields of cancer and biological sciences, which have enabled them to correlate these mchanisms with various other disease modalities. In the present review, we have summarized the key findings related to the role of HIF in the progression of tumors.

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miR-182 facilitates invasion and EMT by targeting Numb in Malignant Pleural Mesothelioma

10.21203/rs.3.rs-27018/v1 ◽

2020 ◽

Author(s):

Yanmeng Kang ◽

Degan Lu ◽

Lingxia Meng ◽

Ruiping Ma ◽

Chuanjun Huang ◽

...

Keyword(s):

Malignant Pleural Mesothelioma ◽

Luciferase Reporter ◽

Pleural Mesothelioma ◽

Reporter Plasmid ◽

Luciferase Reporter Plasmid ◽

Aggressive Tumor ◽

High Level ◽

The Impact ◽

Test Correlation

Abstract Background Malignant Pleural Mesothelioma (MPM) is a highly aggressive tumor which need effective therapeutic methods to improve the prognosis. We carried out this study to explore the role of miR-182 in MPM development, its correlation with Numb expression and EMT. Methods First, we investigated the level of miR-182 and Numb-mRNA by qRT-PCR. Furthermore, we introduced the putative miR-182 binding site into a luciferase reporter plasmid to illustrate the impact of miR-182 on Numb. Then, we down-regulated the expression of miR-182 with/without Numb knocked down in NCI-H2452 cells to investigated their effect. Data were presented as mean ± SD of three independent experiments. Student’s test, correlation analysis and analysis of variance (ANOVA) were used. Results Our results revealed that miR-182 has a high level of expression in MPM, it has a negative correlation with Numb and targeted Numb in MPM cells. miR-182 facilitated the invasion of MPM cells while down-regulation of miR-182 restrained the progression of EMT and made MPM cells more susceptible to pemetrexed. Conclusions miR-182 and Numb can serve as potential therapeutic targets for MPM.

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Construction and Functional Analysis of Luciferase Reporter Plasmid Containing PCNA Gene Promoter

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.556-562.257 ◽

2014 ◽

Vol 556-562 ◽

pp. 257-260

Author(s):

Tong Cun Zhang ◽

Yue Wang ◽

Xing Hua Liao ◽

Nan Wang ◽

Hao Zhou

Keyword(s):

Breast Cancer ◽

Cell Proliferation ◽

Transcription Factors ◽

Transcriptional Activity ◽

Nuclear Antigen ◽

Luciferase Reporter ◽

Reporter Plasmid ◽

Luciferase Reporter Construct ◽

Luciferase Reporter Plasmid ◽

Mcf 7

PCNA (proliferating cell nuclear antigen) is a protein related to tumor development, which has been used extensively in breast cancer diagnosis and prognosis. PCNA has proven to be a useful marker to evaluate cell proliferation and prognosis when combined with other breast cancer markers. Construction of PCNA promoter luciferase reporter plasmid will provide the theory basis for researching the effect of other transcription factors on regulating PCNA transcription. In this study, a human PCNA promoter luciferase reporter construct was generated by PCR amplification of PCNA promoter. The PCR fragment was digested and cloned into pGL3 vector. The promoter sequence was verified by sequencing. The results showed that luciferase reporter plasmids of PCNA promoter were successfully constructed. Then the effects of some key transcription factors, which play important roles in breast cancer cell proliferation, were investigated by luciferase reporter assays in MCF-7 cells. The results showed that ERα can enhance transcriptional activity of PCNA. Furthermore, 17-β-estradiol (E2) also shows an obvious impact in activating PCNA transcription. Our data illuminated that E2 enhances ERα-induced proliferation potential of MCF-7 cells by stimulating the transcriptional activity of PCNA. Our research will provide a model to screen some novel factors in regulating proliferation marker transcription.

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Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis in vitro

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00167.2014 ◽

2014 ◽

Vol 307 (10) ◽

pp. L791-L799 ◽

Cited By ~ 15

Author(s):

Shashi Chillappagari ◽

Shalini Venkatesan ◽

Virajith Garapati ◽

Poornima Mahavadi ◽

Antje Munder ◽

...

Keyword(s):

Cystic Fibrosis ◽

Iron Homeostasis ◽

Hypoxia Inducible Factor ◽

Immunohistochemical Analysis ◽

Surface Expression ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Intracellular Iron

Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1α (HIF-1α), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1α were performed on lung sections of CFTR−/− and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1α, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1α stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1α, downregulate HO-1 expression in CFBE41o- cells.

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Compounds from Cynomorium songaricum with Estrogenic and Androgenic Activities Suppress the Oestrogen/Androgen-Induced BPH Process

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/6438013 ◽

2017 ◽

Vol 2017 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Xueni Wang ◽

Rui Tao ◽

Jing Yang ◽

Lin Miao ◽

Yu Wang ◽

...

Keyword(s):

Cell Proliferation ◽

Transcriptional Activity ◽

Nuclear Translocation ◽

Immunofluorescence Assay ◽

Luciferase Reporter ◽

Reporter Assay ◽

Lncap Cells ◽

Mcf 7

Objective. To investigate the phytoestrogenic and phytoandrogenic activities of compounds isolated from CS and uncover the role of CS in prevention of oestrogen/androgen-induced BPH. Methods. Cells were treated with CS compounds, and immunofluorescence assay was performed to detect the nuclear translocation of ERα or AR in MCF-7 or LNCaP cells; luciferase reporter assay was performed to detect ERs or AR transcriptional activity in HeLa or AD293 cells; MTT assay was performed to detect the cell proliferation of MCF-7 or LNCaP cells. Oestrogen/androgen-induced BPH model was established in rat and the anti-BPH, anti-estrogenic, and anti-androgenic activities of CS in vivo were further investigated. Results. The nuclear translocation of ERα was stimulated by nine CS compounds, three of which also stimulated AR translocation. The transcriptional activities of ERα and ERβ were induced by five compounds, within which only ECG induced AR transcriptional activity as well. Besides, ECG stimulated the proliferation of both MCF-7 cells and LNCaP cells. CS extract suppressed oestrogen/androgen-induced BPH progress in vivo by downregulation of E2 and T level in serum and alteration of the expressions of ERα, ERβ, and AR in the prostate. Conclusion. Our data demonstrates that compounds from CS exhibit phytoestrogenic and phytoandrogenic activities, which may contribute to inhibiting the oestrogen/androgen-induced BPH development.

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Sirt-1 Regulates Physiological Process and Exerts Protective Effects against Oxidative Stress

BioMed Research International ◽

10.1155/2021/5542545 ◽

2021 ◽

Vol 2021 ◽

pp. 1-12

Author(s):

Lei Liu ◽

Guangyuan Xia ◽

Peifan Li ◽

Yiming Wang ◽

Qian Zhao

Keyword(s):

Oxidative Stress ◽

Transcriptional Activity ◽

Cell Counting ◽

Luciferase Reporter ◽

Therapeutic Drug ◽

Mrna Levels ◽

P53 Expression ◽

Protein Levels ◽

Gene Assay

Background. Recent studies suggest a correlation between the reduced Sirt-1 expression with Alzheimer’s diseases (AD) and depression, respectively, suggesting a possible pathogenic role of the altered Sirt-1 expression in neuronal degenerative diseases, such as AD and depression. However, the molecular mechanisms underlying how Sirt-1 reduction impairs neuronal functions remain unknown. Methods. We used the SK-N-SH neuroblastoma cells to study the role of Sirt-1 expression on physiological roles in neuronal cells. Gain of Sirt-1 was achieved by transiently transfecting Sirt-1 expression plasmid. Sirt-1-specific shRNA was used to elucidate the role of Sirt-1 loss of function. CCK-8 (Cell Counting Kit-8) assay and flow cytometry were used to evaluate cell proliferation. Semiquantitative western blotting was used to detect relative protein levels. A further luciferase reporter gene assay was employed to examine the effect of Sirt-1 expression on the transcriptional activity of p53. RT-qPCR was used to determine the mRNA levels of p21, Bax, and Bcl-2, which were the downstream target genes of p53. Results. Sirt-1 suppressed the p53 downstream gene p21 transcription, while shRNA-mediated Sirt-1 knockdown resulted in a significant increase in p21 expression, implying a possibility that Sirt-1 promotes neuron proliferation through suppressing p53 transcriptional activity. The mRNA and protein levels of p53 were not affected by the altered Sirt-1 expression, suggesting that Sirt-1 regulates the transcriptional regulatory activity of p53 rather than p53 expression. Indeed, we further confirmed that Sirt-1 appeared to inhibit p53 transcriptional activity by attenuating its acetylation and resulted in a decrease of p53’s binding to the p21 promoter. Overexpressed Sirt-1 scavenged reactive oxygen species (ROS) production in SK-N-SH with H2O2. Knockdown of Sirt-1 presented opposite effect; the addition of EX527 (Sirt-1 inhibitor) increased ROS accumulation. Conclusions. Oxidative stress induces Sirt-1 in neuron cells, and Sirt-1 promotes proliferation in SK-N-SH cells, which protects them from oxidative stress-induced cell death, potentially via suppressing the transcriptional activity of p53. These results provide a molecular explanation underlying how the reduced Sirt-1 potentially causes the AD and depression-related diseases, supporting the idea that Sirt-1 can possibly be used as a diagnostic biomarker and/or therapeutic drug target for the AD and depression-related diseases.

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Regulation of tyrosinase gene expression by cAMP in B16 melanoma cells involves two CATGTG motifs surrounding the TATA box: implication of the microphthalmia gene product.

The Journal of Cell Biology ◽

10.1083/jcb.134.3.747 ◽

1996 ◽

Vol 134 (3) ◽

pp. 747-755 ◽

Cited By ~ 108

Author(s):

C Bertolotto ◽

K Bille ◽

J P Ortonne ◽

R Ballotti

Keyword(s):

Gene Expression ◽

Transcriptional Activity ◽

Transcriptional Start Site ◽

Regulatory Elements ◽

Melanoma Cells ◽

Tata Box ◽

Luciferase Reporter ◽

Tyrosinase Gene ◽

Luciferase Reporter Plasmid ◽

E Box

In melanocytes and in melanoma cells, upregulation of melanogenesis, by cAMP elevating agents, results from a stimulation of tyrosinase activity that has been ascribed to an increase in tyrosinase protein and messenger amount. However, the mechanism by which cAMP elevating agents increase tyrosinase mRNA remains to be elucidated. In this study, using a luciferase reporter plasmid containing the 2.2-kb fragment 5' of the transcriptional start site of the mouse tyrosinase gene, we showed that cAMP elevating agents lead to a strong stimulation (20-fold) of transcriptional activity of the tyrosinase promoter. Deletions and mutations in the mouse tyrosinase promoter showed that the M-box 70-bp upstream from the TATA-box and the E-box located downstream the TATA-box, near to the initiator site, are involved in the regulation of the tyrosinase promoter activity by cAMP. Additionally, we showed that microphthalmia, a b-HLH transcription factor associated with pigmentation disorders in mouse, binds to these regulatory elements and modulates the transcriptional activity of the tyrosinase promoter. Since cAMP stimulates the binding of microphthalmia to the M-box and to the E-box; it is tempting to propose that microphthalmia, through its interaction with cis-acting elements surrounding the TATA-box, plays a key role in the regulation of the mouse tyrosinase gene expression by cAMP.

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The Anti-Atherogenic Properties of Sesamin are Mediated via Improved Macrophage Cholesterol Efflux Through PPARγ1-LXRα and MAPK Signaling

International Journal for Vitamin and Nutrition Research ◽

10.1024/0300-9831/a000195 ◽

2014 ◽

Vol 84 (1-2) ◽

pp. 79-91 ◽

Cited By ~ 7

Author(s):

Amin F. Majdalawieh ◽

Hyo-Sung Ro

Keyword(s):

Cholesterol Efflux ◽

Transcriptional Activity ◽

Molecular Mechanisms ◽

Foam Cell ◽

Mapk Signaling ◽

Luciferase Reporter ◽

Foam Cell Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Macrophage Cholesterol Efflux

Background: Foam cell formation resulting from disrupted macrophage cholesterol efflux, which is triggered by PPARγ1 and LXRα, is a hallmark of atherosclerosis. Sesamin and sesame oil exert anti-atherogenic effects in vivo. However, the exact molecular mechanisms underlying such effects are not fully understood. Aim: This study examines the potential effects of sesamin (0, 25, 50, 75, 100 μM) on PPARγ1 and LXRα expression and transcriptional activity as well as macrophage cholesterol efflux. Methods: PPARγ1 and LXRα expression and transcriptional activity are assessed by luciferase reporter assays. Macrophage cholesterol efflux is evaluated by ApoAI-specific cholesterol efflux assays. Results: The 50 μM, 75 μM, and 100 μM concentrations of sesamin up-regulated the expression of PPARγ1 (p< 0.001, p < 0.001, p < 0.001, respectively) and LXRα (p = 0.002, p < 0.001, p < 0.001, respectively) in a concentration-dependent manner. Moreover, 75 μM and 100 μM concentrations of sesamin led to 5.2-fold (p < 0.001) and 6.0-fold (p<0.001) increases in PPAR transcriptional activity and 3.9-fold (p< 0.001) and 4.2-fold (p < 0.001) increases in LXR transcriptional activity, respectively, in a concentration- and time-dependent manner via MAPK signaling. Consistently, 50 μM, 75 μM, and 100 μM concentrations of sesamin improved macrophage cholesterol efflux by 2.7-fold (p < 0.001), 4.2-fold (p < 0.001), and 4.2-fold (p < 0.001), respectively, via MAPK signaling. Conclusion: Our findings shed light on the molecular mechanism(s) underlying sesamins anti-atherogenic effects, which seem to be due, at least in part, to its ability to up-regulate PPARγ1 and LXRα expression and transcriptional activity, improving macrophage cholesterol efflux. We anticipate that sesamin may be used as a therapeutic agent for treating atherosclerosis.

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