CoCl2 induces apoptosis through the mitochondria- and death receptor-mediated pathway in the mouse embryonic stem cells

Embryonic hypoxia/ischemia is a major cause of a poor fetal outcome and future neonatal and adult handicaps. However, biochemical cellular events in mouse embryonic stem (mES) cells during hypoxia remains unclear. This study investigated the underlying mechanism of apoptosis in mES cells under CoCl₂-induced hypoxic/ischemic conditions. CoCl₂ enhanced the expression of hypoxia-inducible factor-1α (HIF-1α) and the accumulation of reactive oxygen species in mES cells. The CoCl₂-treated mES cells showed a decrease in cell viability as well as typical apoptotic changes, cell shrinkage, chromatin condensation, and nuclear fragmentation and an extended G₂/M phase of the cell cycle. CoCl₂ augmented the release of cytochrome c into the cytosol from the mitochondria with a concomitant loss of the mitochondrial transmembrane potential (ΔΨ_m) and upregulated the voltage-dependent anion channel. In addition, CoCl₂-induced caspase-3, -8, and -9 activation and upregulation of p53 level, whereas downregulated Bcl-2 and Bcl-xL, a member of the anti-apoptotic Bcl-2 family in mES cells. Furthermore, CoCl₂ led to the upregulation of Fas and Fas-ligand, which are the death receptor assemblies, as well as the cleavage of Bid in mES cells. These results suggest that CoCl₂ induces apoptosis through both mitochondria- and death receptor-mediated pathways that are regulated by the Bcl-2 family in mES cells.     

Hypoxia-inducible factor 1-alpha acts as a bridge factor for crosstalk between ERK1/2 and caspases in hypoxia-induced apoptosis of cementoblasts

Hypoxia-induced apoptosis of cementoblasts (OCCM-30) may be harmful to orthodontic treatment. Hypoxia-inducible factor 1-alpha (HIF-1α) mediates the biological effects during hypoxia. Little is known about the survival mechanism capable to counteract cementoblast apoptosis. We aimed to investigate the potential roles of HIF-1α, as well as the protein-protein interactions with ERK1/2, using an in-vitro model of chemical-mimicked hypoxia and adipokines. Here, OCCM-30 were co-stimulated with resistin, visfatin or ghrelin under CoCl₂-mimicked hypoxia. In-vitro investigations revealed that CoCl₂-induced hypoxia triggered activation of caspases, resulting in apoptosis dysfunction in cementoblasts. Resistin, visfatin and ghrelin promoted the phosphorylated ERK1/2 expression in OCCM-30 cells. Furthermore, these adipokines inhibited hypoxia-induced apoptosis at different degrees. These effects were reversed by pre-treatment with ERK inhibitor (FR180204). In cells treated with FR180204, HIF-1α expression was inhibited despite the presence of three adipokines. Using dominant-negative mutants of HIF-1α, we found that siHIF-1α negatively regulated the caspase-8, caspase-9 and caspase-3 gene expression. We concluded that HIF-1α acts as a bridge factor in lengthy hypoxia-induced apoptosis in an ERK1/2-dependent pathway. Gene expressions of the caspases-3, caspase-8 and caspase-9 were shown to be differentially regulated by adipokines (resistin, visfatin and ghrelin). Our study, therefore, provides evidence for the role of ERK1/2 and HIF-1α in the apoptotic response of OCCM-30 cells exposed to CoCl₂-mimicked hypoxia, providing potential new possibilities for molecular intervention in obese patients undergoing orthodontic treatment.     

Adolescence as a vulnerable period to alter rodent behavior

Induction of HIF-1α by oxygen limitation promotes increased phosphorylation and catalytic depression of mitochondrial pyruvate dehydrogenase (PDH) and an enhanced glycolytic poise in cells. Cobalt chloride and desferrioxamine are widely used as mimics for hypoxia because they increase the levels of HIF-1α. We evaluated the ability of these agents to elicit selected physiological responses to hypoxia as a means to metabolically precondition mammalian cells, but without the detrimental effects of hypoxia. We show that, while CoCl₂ does increase HIF-1α in a dose-dependent manner, it unexpectedly and strikingly decreases PDH phosphorylation at E1α sites 1, 2, and 3 (Ser²⁹³, Ser³⁰⁰, and Ser²³², respectively) in HepG2 cells. This same effect is also observed for site 1 in mouse NIH/3T3 fibroblasts and J774 macrophages. CoCl₂ unexpectedly decreases the mRNA expression for PDH kinase-2 in HepG2 cells, which likely explains the dephosphorylation of PDH observed. And nor does desferrioxamine promote the expected increase in PDH phosphorylation. Dimethyloxaloylglycine (a prolyl hydroxylase inhibitor) performs better in this regard, but failed to promote the stronger effects seen with hypoxia. Consequently, CoCl₂ and desferrioxamine are unreliable mimics of hypoxia for physiological events downstream of HIF-1α stabilization. Our study demonstrates that mimetic chemicals must be chosen with caution and evaluated thoroughly if bona fide cellular outcomes are to be promoted with fidelity.     

Functional changes in murine mammary cancer cells elicited by CoCl2-induced hypoxia

Low O₂ levels in solid tumors are associated with increase in hypoxia-inducible factor 1α (HIF-1α). The present study examines functional changes involved in adaptation to hypoxia of the LMM3 mammary tumor cell line, using CoCl₂ as hypoxic mimetic. Our results showed that LMM3 cells were not only tolerant to 150 μM CoCl₂ but they can overgrowth in vitro respect to untreated cells. Hypoxia inhibited cell invasion, migration, MMP-9 activity and NO levels. Macrophage cytotoxicity augmented under hypoxia but was blunted by conditioned media from tumor cells. In vivo tumorigenicity of CoCl₂-treated cells was greater than controls. Our results show stabilization of HIF-1α in LMM3 cells under CoCl₂ and functional changes associated with enhanced cell survival and growth but not with tumor dissemination.     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型。研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化,基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征-DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14,0.49和1.43,与妊娠早期相比,妊娠中,晚期胎盘基因组DNA断裂值有显著性增加,TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层,免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax:Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势,实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax:Bcl-2的比例相关。     

妊娠兔胎盘细胞凋亡及凋亡相关基因Bcl-2和Bax表达的动态变化

以新西兰雌兔为动物模型,研究妊娠期间胎盘细胞凋亡及其凋亡调控蛋白Bcl-2和Bax表达的动态变化.基因组DNA凝胶电泳实验检测到妊娠中期和晚期胎盘基因组DNA中出现典型的凋亡特征——DNA梯带,而且DNA断裂值在妊娠早、中、晚期分别为：0.14、0.49和1.43,与妊娠早期相比,妊娠中、晚期胎盘基因组DNA断裂值有显著性增加.TUNEL实验和活化caspase-3的免疫定位实验表明,在妊娠早期胎盘中存在细胞凋亡,而且在各妊娠期中细胞凋亡主要发生于合体滋养层.免疫印迹法分析表明,Bcl-2和Bax随妊娠的进行其表达量明显增加,Bax∶Bcl-2比值在妊娠早、中、晚期分别为：0.89,0.91和1.25,呈增加趋势.实验结果说明,在兔正常妊娠中,胎盘合体滋养层细胞发生凋亡,且随妊娠的进行,凋亡细胞数量增多,胎盘细胞凋亡主要与细胞中Bax∶Bcl-2的比例相关.     

Hypoxia promotes Chlamydia trachomatis L2/434/Bu growth in immortal human epithelial cells via activation of the PI3K-AKT pathway and maintenance of a balanced NAD+/NADH ratio

Chlamydia trachomatis LGV (CtL2) causes systemic infection and proliferates in lymph nodes as well as genital tract or rectum producing a robust inflammatory response, presumably leading to a low oxygen environment. We therefore assessed how CtL2 growth in immortal human epithelial cells adapts to hypoxic conditions. Assessment of inclusion forming units, the quantity of chlamydial 16S rDNA, and inclusion size showed that hypoxia promotes CtL2 growth. Under hypoxia, HIF-1α was stabilized and p53 was degraded in infected cells. Moreover, AKT was strongly phosphorylated at S473 by CtL2 infection. This activation was significantly diminished by LY-294002, a PI3K-AKT inhibitor, which decreased the number of CtL2 progeny. HIF-1α stabilizers (CoCl₂, desferrioxamine) had no effect on increasing CtL2 growth, indicating no autocrine impact of growth factors produced by HIF-1α stabilization. Furthermore, in normoxia, CtL2 infection changed the NAD⁺/NADH ratio of cells with increased gapdh expression; in contrast, under hypoxia, the NAD⁺/NADH ratio was the same in infected and uninfected cells with high and stable expression of gapdh, suggesting that CtL2-infected cells adapted better to hypoxia. Together, these data indicate that hypoxia promotes CtL2 growth in immortal human epithelial cells by activating the PI3K-AKT pathway and maintaining the NAD⁺/NADH ratio with stably activated glycolysis.     

Pilocarpine Protects Cobalt Chloride-induced Apoptosis of RGC-5 Cells: Involvement of Muscarinic Receptors and HIF-1α Pathway

The retina is the most metabolically active tissue in the human body and hypoxia-induced retinal ganglion cell (RGC) death has been implicated in glaucomatous optic neuropathy. The aim of this study is to determine whether muscarinic receptor agonist pilocarpine, a classic antiglaucoma drug, possesses neuroprotection against cobalt chloride (CoCl₂)-mimetic hypoxia-induced apoptosis of rat retinal ganglion cells (RGC-5 cells) and its underlying mechanisms. Cell viability was determined by Cell Counting Kit-8 assay and apoptosis was examined by annexin V and mitochondrial membrane potential (MMP) assays. Expressions of hypoxia-induced factor-1α (HIF-1α), p53, and BNIP3 were investigated by quantitative real-time PCR and western blot analysis. After treatment of 200 μM CoCl₂ for 24 h, RGC-5 cells showed a marked decrease of cell viability by approximately 30%, increased apoptosis rate and obvious decline in MMP, which could largely be reversed by the pretreatment of 1 μM pilocarpine mainly via the activation of muscarinic receptors. Meanwhile, pretreatment of 1 μM pilocarpine could significantly prevent CoCl₂-induced HIF-1α translocation from cytoplasm to nucleus and down-regulate the expression of HIF-1α, p53, and BNIP3. These studies demonstrated that pilocarpine had effective protection against hypoxia-induced apoptosis in RGCs via muscarinic receptors and HIF-1α pathway. The findings suggest that HIF-1α pathway as a “master switch” may be used as a therapeutic target in the cholinergic treatment of glaucoma.     

Effect of IFNgamma on caspase-3, Bcl-2 and Bax expression, and apoptosis in rabbit placenta

The purpose of this study was to determine whether apoptosis in placenta was affected by IFNgamma, which can induce abortion, and whether the effect of IFNgamma on apoptosis resulted from an intrinsic program of apoptosis, which was regulated by Bcl-2 and Bax. DNA fragmentation analysis indicated that cleavage of DNA into 180 bp and its polymers were recognized in placenta in control and IFNgamma treated groups. Quantitative analysis of low molecular weight fragments of DNA revealed a significant increase in cases of 100,000 IU IFNgamma treatment compared with those in normal pregnancy (P<0.05). An analysis in situ revealed that apoptosis occurred predominantly in syncytiotrophoblast. Expression of Bcl-2 and Bax in placenta was evaluated by immunoblot analysis and immunohistochemistry study. Bcl-2 was expressed predominantly in syncytiotrophoblast, and was not expressed in cytotrophoblast of all cases. Whereas Bax was expressed in cytotrophoblast, syncytiotrophoblasts were found to be negative for Bax protein expression in all cases. Both Bcl-2 and Bax expression was decreased 0.44 fold and 0.46 fold by 50,000 IU IFNgamma and 0.41 fold and 0.03 fold by 100,000 IU IFNgamma. This resulted in change of a 0.07 fold increase in the Bax:Bcl-2 ratio in 50,000 IU IFNgamma treated groups and 0.41 fold increase in 100,000 IU IFNgamma treated groups as compared with those in control groups. The difference in Bax to Bcl-2 ratio between control and 100,000 IU IFNgamma treated groups was significant (P<0.05). The localization of caspase-3, the executioner of apoptosis, was detected in some cytotrophoblast and syncytiotrophoblast and increased 0.03 fold and 0.68 fold in 50,000 IU IFNgamma and 100,000 IU IFNgamma treated groups, respectively. There was significant difference between control and 100,000 IU IFNgamma treated groups (P<0.05). The results showed that high dose of IFNgamma administration increased the extent of apoptosis in placenta, the Bax to Bcl-2 ratio, and the activated caspase-3.     

Hypoxia-mimetic agents desferrioxamine and cobalt chloride induce leukemic cell apoptosis through different hypoxia-inducible factor-1α independent mechanisms

Hypoxia presents pro-apoptotic and anti-apoptotic biphasic effects that appear to be dependent upon cell types and conditions around cells. The substantial reports demonstrated that commonly used hypoxia-mimetic agents cobalt chloride (CoCl₂) and desferrioxamine (DFO) could also induce apoptosis in many different kinds of cells, but the mechanism was poorly understood. In this work, we compare the apoptosis-inducing effects of these two hypoxia-mimetic agents with acute myeloid leukemic cell lines NB4 and U937 as in vitro models. The results show that both of them induce these leukemic cells to undergo apoptosis with a loss of mitochondrial transmembrane potentials (ΔΨ m), the activation of caspase-3/8 and the cleavage of anti-apoptotic protein Mcl-1, together with the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) protein, a critical regulator for the cellular response to hypoxia. Metavanadate and sodium nitroprusside significantly abrogate DFO rather than CoCl₂-induced mitochondrial Δ Ψ m collapse, caspase-3/8 activation, Mcl-1 cleavage and apoptosis, but they fail to influence DFO and CoCl₂-induced HIF-1α protein accumulation. Moreover, inducible expression of HIF-1α gene dose not alter DFO and CoCl₂-induced apoptosis in U937 cells. In conclusion, these results propose that although both DFO and CoCl₂-induced leukemic cell apoptosis by mitochondrial pathway-dependent and HIF-1α-independent mechanisms, DFO and CoCl₂-induced apoptosis involves different initiating signal pathways that remain to be investigated.     

Deferoxamine Preconditioning of Neural-Like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells as a Strategy to Promote Their Tolerance and Therapeutic Potential: An In Vitro Study

Transplantation of neural-like cells is considered as a promising therapeutic strategy developed for neurodegenerative disease in particular for ischemic stroke. Since cell survival is a major concern following cell implantation, a number of studies have underlined the protective effects of preconditioning with hypoxia or hypoxia mimetic pharmacological agents such as deferoxamine (DFO), induced by activation of hypoxia inducible factor-1 (HIF-1) and its target genes. The present study has investigated the effects of DFO preconditioning on some factors involved in cell survival, angiogenesis, and neurogenesis of neural-like cells derived from human Wharton’s jelly mesenchymal stem cells (HWJ-MSCs) in presence of hydrogen peroxide (H₂O₂). HWJ-MSCs were differentiated toward neural-like cells for 14 days and neural cell markers were identified using immunocytochemistry. HWJ-MSC-derived neural-like cells were then treated with 100 µM DFO, as a known hypoxia mimetic agent for 48 h. mRNA and protein expression of HIF-1 target genes including brain-derived neurotrophic factors (BDNF) and vascular endothelial growth factor (VEGF) significantly increased using RT-PCR and Western blotting which were reversed by HIF-1α inhibitor, while, gene expression of Akt-1, Bcl-2, and Bax did not change significantly but pAkt-1 was up-regulated as compared to poor DFO group. However, addition of H₂O₂ to DFO-treated cells resulted in higher resistance to H₂O₂-induced cell death. Western blotting analysis also showed significant up-regulation of HIF-1α, BDNF, VEGF, and pAkt-1, and decrease of Bax/Bcl-2 ratio as compared to poor DFO. These results may suggest that DFO preconditioning of HWJ-MSC-derived neural-like cells improves their tolerance and therapeutic potential and might be considered as a valuable strategy to improve cell therapy.     

Podocytes protect glomerular endothelial cells from hypoxic injury via deSUMOylation of HIF-1α signaling

Hypoxia can cause severe tubulointerstitial injury and peritubular capillary loss. However, hypoxia-induced injury in glomerular capillaries is far milder than tubulointerstitium, but the reason for this difference is unclear. We hypothesized that the phenomenon is due to the protective crosstalk among intrinsic glomerular cells. To mimic the microenvironment and investigate the crosstalk process temporally, we established co-culture models of glomerular endothelial cells (GEnCs) with podocytes or with mesangial cells. We found that podocytes rather than mesangial cells prevented GEnCs from injury and hypoxia-induced apoptosis and promoted migration and angiogenesis of GEnCs under hypoxic conditions. We then identified that increased activation of the hypoxia inducible factor 1α (HIF-1α) pathway as the major mechanism enabling podocytes to protect GEnCs against hypoxia. HIF-1α stabilization during hypoxia is known to be dependent on SUMO-specific protease 1 (SENP1)-mediated deSUMOylate modifications. Therefore, we further targeted deSUMOylation, regulated by SENP1, by short hairpin RNA (shRNA) knockdown of SENP1 mRNA in vitro and measured expression of HIF-1α and its downstream gene VEGF in hypoxic podocytes. Our results showed that SENP1 was essential for HIF-1α deSUMOylation in podocytes. The blockade of deSUMOylation by SENP1 shRNA successfully abolished the activation of HIF-1α signaling and consequently suppressed the protective effects of podocytes on GEnCs. In conclusion, we demonstrate for the first time that hypoxia may promote HIF-1α stabilization and activation by increasing SENP1 expression in podocytes, which induce GEnCs survival and angiogenesis to resist hypoxia. Thus, deSUMOylation of HIF-1α signaling is a potentially novel therapeutic target for treating hypoxic renal disorders.