scholarly journals Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

2012 ◽  
Vol 198 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Yanmei Qi ◽  
Xiaoxiang Tian ◽  
Jie Liu ◽  
Yaling Han ◽  
Alan M. Graham ◽  
...  
Keyword(s):  
Protein A ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Epithelial Morphogenesis ◽  
Dependent Manner ◽  
Interacting Protein ◽  
The Core ◽  
Adenovirus E1b ◽  
Induced Apoptosis

Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell–differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)–2. Ablation of HIF-2α or HIF-1β, the common β subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2α stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.

scholarly journals MicroRNA-30e targets BNIP3L to protect against aldosterone-induced podocyte apoptosis and mitochondrial dysfunction

2017 ◽  
Vol 312 (4) ◽  
pp. F589-F598 ◽  
Author(s):  
Yan Guo ◽  
Xu Deng ◽  
Shuang Chen ◽  
Lingyun Yang ◽  
Jiajia Ni ◽  
...  
Keyword(s):  
Mitochondrial Dysfunction ◽  
Underlying Mechanism ◽  
Protective Role ◽  
Early Event ◽  
Dependent Manner ◽  
Podocyte Injury ◽  
Interacting Protein ◽  
Adenovirus E1b ◽  
Induced Apoptosis

MicroRNAs are essential for the maintenance of podocyte homeostasis. Emerging evidence has demonstrated a protective role of microRNA-30a (miR-30a), a member of the miR-30 family, in podocyte injury. However, the roles of other miR-30 family members in podocyte injury are unclear. The present study was undertaken to investigate the contribution of miR-30e to the pathogenesis of podocyte injury induced by aldosterone (Aldo), as well as the underlying mechanism. After Aldo treatment, miR-30e was reduced in a dose-and time-dependent manner. Notably, overexpression of miR-30e markedly attenuated Aldo-induced apoptosis in podocytes. In agreement with this finding, miR-30e silencing led to significant podocyte apoptosis. Mitochondrial dysfunction (MtD) has been shown to be an early event in Aldo-induced podocyte injury. Here we found that overexpression of miR-30e improved Aldo-induced MtD while miR-30e silencing resulted in MtD. Next, we found that miR-30e could directly target the BCL2/adenovirus E1B-interacting protein 3-like (BNIP3L) gene. Aldo markedly enhanced BNIP3L expression in podocytes, and silencing of BNIP3L largely abolished Aldo-induced MtD and cell apoptosis. On the contrary, overexpression of BNIP3L induced MtD and apoptosis in podocytes. Together, these findings demonstrate that miR-30e protects mitochondria and podocytes from Aldo challenge by targeting BNIP3L.

scholarly journals Embryonal Carcinoma P19 Cells Produce Erythropoietin Constitutively But Express Lactate Dehydrogenase in an Oxygen-Dependent Manner

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1185-1195 ◽  
Author(s):  
Taiho Kambe ◽  
Junko Tada ◽  
Mariko Chikuma ◽  
Seiji Masuda ◽  
Masaya Nagao ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Binding Site ◽  
Embryonal Carcinoma ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Gene Promoter ◽  
Dependent Manner ◽  
P19 Cells ◽  
Independent Manner ◽  
Hep3b Cells

Abstract Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3′ enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

278 EFFECT OF XENOESTROGENS ON THE DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS

2009 ◽  
Vol 21 (1) ◽  
pp. 236
Author(s):  
E.-M. Jeung ◽  
K.-C. Choi ◽  
E.-B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
Time Dependent ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Markers ◽  
Oct4 Expression

Endocrine disruptors (ED) may have adverse impacts on reproductive and immune systems in human and wild animals. It has been shown that octyl-phenol (OP) and nonyl-phenol (NP) have estrogenicity in estrogen-responding cells or tissues. In this study, we further investigated the effect(s) of OP and NP on the expression of undifferentiation and differentiation markers in mouse embryonic stem cells (ESC), which function as an important factor in the differentiation of ESC into cardiomyocytes. Mouse ESC were cultured in hanging drops to form embryoid bodies (EB). The medium was replaced with phenol red-free DMEM/F-12 supplemented with 5% charcoal-dextran-stripped FBS. The ESC were treated with OP, NP (1Ã-10-6 and 1Ã-10-7 M) or 17β-estradiol (E2; 1Ã-10-8 and 1Ã-10-9 M) in a time-dependent manner (1, 2 and 3 days), and EB were treated with identical concentrations for 4 and 8 days, respectively. High increasing doses of OP and NP were employed in this study because a binding affinity of ED to estrogen receptors (ER) is about 1000 less than that of E2. We determined the mRNA expression of undifferentiation markers (Oct4, Sox2 and Zfp206) and cardiomyocyte differentiation markers (cardiac alpha-MHC, beta-MHC and myosin light chain isoform-2V) using real-time PCR. In ESC, undifferentiation markers were identified. It is of interest that treatment with OP, NP or E2 induced a significant increase (1.4 5.5-fold) in Oct4 expression at the transcription levels according to a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2 and Zfp206 genes in ESC, suggesting that OP and NP may play a role as an Oct4 enhancer in ESC. In addition, both undifferentiation and cardiomyocyte differentiation markers were identified in EB. Treatment with OP and NP induced a significant increase in the expression of Oct4, Sox2 and Zfp206 genes at the transcription levels in a dose-dependent manner for 4 days, whereas Oct4 expression was only induced at these doses for 8 days. In contrast, cardiomyocyte differentiation markers were reduced by these ED in EB. Taken together, these results suggest that OP and NP play a role as a positive regulator in the undifferentiation process of ESC and EB, and maintenance and differentiation of mouse ESC.

scholarly journals miR-103 Regulates Oxidative Stress by Targeting the BCL2/Adenovirus E1B 19 kDa Interacting Protein 3 in HUVECs

2015 ◽  
Vol 2015 ◽  
pp. 1-11 ◽  
Author(s):  
Mao-Chun Xu ◽  
Xiu-Fang Gao ◽  
Changwu Ruan ◽  
Zhi-Ru Ge ◽  
Ji-De Lu ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Critical Role ◽  
Cell Activity ◽  
Antioxidant Effect ◽  
Dependent Manner ◽  
Ros Production ◽  
Interacting Protein ◽  
Adenovirus E1b ◽  
Antioxidative Therapy ◽  
First Time

Oxidative stress plays a critical role in cardiovascular diseases. Salidroside, a glycoside fromRhodiola rosea, has been used as an antioxidative therapy for oxidative injury in cardiac diseases. However, the mechanism underlying its antioxidant effect needs to be elucidated. Treatment of HUVECs with H2O2significantly decreased the expression of miR-103 in a dose- and time-dependent manner, whereas pretreatment with salidroside significantly inhibited this decrease. Subsequent analysis showed that overexpression of miR-103 abrogated cell activity and ROS production induced by H2O2. Bcl2/adenovirus E1B 19 kDa interacting protein 3 (BNIP3) was determined to be a novel miR-103 target in HUVECs. Interestingly, H2O2treatment upregulated BNIP3 expression; in turn, this effect was inhibited by pretreatment with salidroside. Further studies confirmed that the knockdown of BNIP3 enhanced cell activity and suppressed the ROS production induced by H2O2. These results demonstrated for the first time that salidroside protects HUVECs in part by upregulating the expression of miR-103, which mediates BNIP3 downregulation and plays an important role in the cytoprotective actions.

scholarly journals Protective effects of ginsenoside Rb1 on H2O2-induced oxidative injury in human endothelial cell line (EA.hy926) via miR-210

2019 ◽  
Vol 33 ◽  
pp. 205873841986602 ◽  
Author(s):  
Fubao Jia ◽  
Lei Mou ◽  
Hanming Ge
Keyword(s):  
Oxidative Injury ◽  
Nuclear Factor Κb ◽  
Migration And Invasion ◽  
Protective Effects ◽  
Ginsenoside Rb1 ◽  
Interacting Protein ◽  
Positive Effects ◽  
Adenovirus E1b ◽  
And Migration ◽  
Induced Apoptosis

Ginsenoside Rb1 (Rb1) possesses a cardioprotective effect via mediating microRNAs (miRs), while it is unexplored whether miR-210 is regulated by Rb1 in response to oxidative stress. Human endothelial EA.hy926 cells were stimulated with H2O2 before Rb1 treatment. After transfection, cell viability, apoptosis, migration, and invasion assays were conducted. Western blot was applied to quantify protein. BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and miR-210 were analyzed with quantitative reverse transcription polymerase chain reaction. Dual luciferase activity assay was performed. Rb1 elevated viability, migration, and invasion of H2O2-treated cells. H2O2-induced apoptosis was moderated by Rb1. miR-210 was augmented in H2O2-treated cells after Rb1 stimulation. miR-210 inhibitor abolished the positive effects of Rb1. BNIP3 was negatively modulated by miR-210 and implicated in modulating viability, apoptosis, and migration and invasion. In addition, BNIP3 modulated phosphorylation of regulators. Rb1 repressed oxidative injury via elevating miR-210. miR-210 negatively mediated BNIP3, which participated in oxidative damage via regulating mammalian targets of rapamycin (mTOR) and nuclear factor-κB (NF-κB).

scholarly journals DIPA-family coiled-coils bind conserved isoform-specific head domain of p120-catenin family: potential roles in hydrocephalus and heterotopia

2014 ◽  
Vol 25 (17) ◽  
pp. 2592-2603 ◽  
Author(s):  
Nicholas O. Markham ◽  
Caleb A. Doll ◽  
Michael R. Dohn ◽  
Rachel K. Miller ◽  
Huapeng Yu ◽  
...  
Keyword(s):  
Epithelial To Mesenchymal Transition ◽  
Specific Binding ◽  
Protein A ◽  
Family Members ◽  
Adherens Junction ◽  
Dependent Manner ◽  
Interacting Protein ◽  
P120 Catenin ◽  
Mesenchymal Transition ◽  
Head Domain

p120-catenin (p120) modulates adherens junction (AJ) dynamics by controlling the stability of classical cadherins. Among all p120 isoforms, p120-3A and p120-1A are the most prevalent. Both stabilize cadherins, but p120-3A is preferred in epithelia, whereas p120-1A takes precedence in neurons, fibroblasts, and macrophages. During epithelial-to-mesenchymal transition, E- to N-cadherin switching coincides with p120-3A to -1A alternative splicing. These isoforms differ by a 101–amino acid “head domain” comprising the p120-1A N-terminus. Although its exact role is unknown, the head domain likely mediates developmental and cancer-associated events linked to p120-1A expression (e.g., motility, invasion, metastasis). Here we identified delta-interacting protein A (DIPA) as the first head domain–specific binding partner and candidate mediator of isoform 1A activity. DIPA colocalizes with AJs in a p120-1A- but not 3A-dependent manner. Moreover, all DIPA family members (Ccdc85a, Ccdc85b/DIPA, and Ccdc85c) interact reciprocally with p120 family members (p120, δ-catenin, p0071, and ARVCF), suggesting significant functional overlap. During zebrafish neural tube development, both knockdown and overexpression of DIPA phenocopy N-cadherin mutations, an effect bearing functional ties to a reported mouse hydrocephalus phenotype associated with Ccdc85c. These studies identify a novel, highly conserved interaction between two protein families that may participate either individually or collectively in N-cadherin–mediated development.

scholarly journals Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-16 ◽  
Author(s):  
Ade Kallas ◽  
Martin Pook ◽  
Annika Trei ◽  
Toivo Maimets
Keyword(s):  
Cell Cycle Progression ◽  
Single Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Flow Cytometric Method ◽  
Histone 3 ◽  
Flow Cytometric ◽  
M Phase ◽  
Induced Apoptosis ◽  
Hes Cells

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

scholarly journals FoxO1 induces apoptosis in skeletal myotubes in a DNA-binding-dependent manner

2009 ◽  
Vol 297 (3) ◽  
pp. C548-C555 ◽  
Author(s):  
Thomas J. McLoughlin ◽  
Sierra M. Smith ◽  
Alissa D. DeLong ◽  
Hengbing Wang ◽  
Terry G. Unterman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Muscle Atrophy ◽  
Ring Finger ◽  
Skeletal Muscle Atrophy ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dependent Manner ◽  
Interacting Protein ◽  
Skeletal Myotubes ◽  
Adenovirus E1b

Recent studies indicate that FoxO transcription factors play an important role in promoting muscle atrophy. To study mechanisms mediating effects of FoxO proteins on muscle wasting, FoxO1-estrogen receptor fusion proteins that are activated by treatment with 4-hydroxytamoxifen (4-OH-T) were stably transfected in C2C12 skeletal myoblasts using the pBABE retroviral system and grown into multinucleated skeletal myotubes. Activation of FoxO1 resulted in significant muscle atrophy, which was accompanied by DNA fragmentation, evidenced by terminal deoxynucleotidyl transferase dUTP-mediated nick end labeling. Cells expressing a DNA-binding-deficient form of FoxO1 also exhibited significant atrophy on FoxO1 activation but no hallmark signs of apoptosis. FoxO1 activation resulted in a significant increase in muscle atrophy F-box (MAFbx)/atrogin-1, muscle-specific RING finger protein 1 (MuRF-1), and Bcl-2-interacting mediator of cell death (Bim) gene expression, with no significant increase in Bcl-2/adenovirus E1B 19-kDa-interacting protein 3 (BNip3) gene expression. Although the ability of FoxO1 to induce MuRF-1 gene expression appeared to be independent of DNA binding, expression of MAFbx/atrogin-1 and Bim was significantly blunted in cells expressing DNA-binding-deficient FoxO1. BNip3 gene expression was significantly elevated in DNA-binding-deficient mutant cells. These findings indicate that FoxO1 promotes skeletal muscle atrophy through induction of proteolytic and apoptotic machinery via DNA-binding-dependent and -independent mechanisms.

scholarly journals Neural Differentiation of Mouse Embryonic Stem Cells—An in vitro Approach to Profile DNA Methylation of Reprogramming Factor Sox2-SRR2

2021 ◽  
Vol 12 ◽  
Author(s):  
Sajida Batool ◽  
Mahmood Akhtar Kayani ◽  
Martin Valis ◽  
Kamil Kuca
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Neuronal Morphology ◽  
Embryoid Bodies ◽  
The Core ◽  
Pluripotency Genes

Sox2 is one of the core transcription factors maintaining the embryonic stem cells (ES) pluripotency and, also indispensable for cellular reprogramming. However, limited data is available about the DNA methylation of pluripotency genes during lineage-specific differentiations. This study investigated the DNA methylation of Sox2 regulatory region 2 (SRR2) during directed differentiation of mouse ES into neural lineage. ES cells were first grown to form embryoid bodies in suspension which were then dissociated, and cultured in defined medium to promote neural differentiation. Typical neuronal morphology together with the up-regulation of Pax6, neuroepithelial stem cell intermediate filament and β-tubulin III and, down-regulation of pluripotency genes Oct4, Nanog and Sox2 showed the existence of neural phenotype in cells undergoing differentiation. Three CpGs in the core enhancer region of neural-specific SRR2 were individually investigated by direct DNA sequencing post-bisulfite treatment and, found to be unmethylated in differentiated cells at time-points chosen for analysis. This analysis does not limit the possibility of methylation at other CpG sites than those profiled here and/or transient methylation. Hence, similar analyses exploring the DNA methylation at other regions of the Sox2 gene could unravel the onset and transitions of epigenetic signatures influencing the outcome of differentiation pathways and neural development. The data presented here shows thatin vitroneural differentiation of embryonic stem cells can be employed to study and characterize molecular regulatory mechanisms governing neurogenesis by applying diverse pharmacological and toxicological agents.

385 ESTROGENIC EVALUATION OF ALKYPHENOLS IN MOUSE EMBRYONIC STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 349
Author(s):  
E. M. Jung ◽  
E. B. Jeung
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Methylation Pattern ◽  
Embryoid Bodies ◽  
Time Dependent ◽  
Target Cells ◽  
Transcriptional Level ◽  
Dependent Manner ◽  
Cardiomyocyte Differentiation ◽  
Differentiation Markers

Xenoestrogens can have adverse effects on the reproductive and immune systems; for example, 4-tert-octylphenol (OP) and 4-nonylphenol (NP) can have estrogenic effects in target cells. In this study, we investigated the effects of xenoestrogens on the expression of undifferentiation and differentiation markers in mouse embryonic stem (ES) cells, which are important mediators of the differentiation of ES cells into cardiomyocytes. The ES cells were treated with 17β-estradiol or OP and NP in a time-dependent manner (for 1, 2, or 3 days), and embryoid bodies (EB) cells were given the same treatment for 5, 8, 12, or 16 days. The mRNA expressions of undifferentiation markers (Oct-4, Sox2, Zfp206, and Rex-1) and cardiomyocyte differentiation markers (α-MHC, β-MHC, ANF, and MLC-2V) were determined by semi- and quantitative real-time PCR. Treatment with E2 induced an increase (1.3- to 4.6-fold) in Oct-4 expression at the transcriptional level in a dose- and time-dependent manner. However, no difference was observed in the expression of Sox2, Zfp206, or Rex-1 genes in ES cells, suggesting that E2 might be an Oct-4 enhancer in ES cells. However, induction of Oct-4 expression by E2 might result from changes in the Oct-4 promoter methylation pattern rather than from other regulatory mechanisms. We also found that cardiomyocyte differentiation markers were differentially expressed in response to xenoestrogens in EB cells. Taken together, these results suggest that xenoestrogens might play a role as a positive regulator of the undifferentiation process in mouse ES and EB cells and might be involved in the maintenance and differentiation of mouse ES cells.

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