Hypoxia-driven proliferation of embryonic neural stem/progenitor cells--role of hypoxia-inducible transcription factor-1alpha

We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1alpha, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O(2), 10% O(2), and 3% O(2)) for 3 days. The results showed that hypoxia, especially 10% O(2), promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1alpha mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O(2). To further elucidate the potential role of HIF-1alpha in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1alpha, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1alpha mRNA was used to knock down HIF-1alpha. We found that overexpression of HIF-1alpha caused the same proliferative effect on NPCs under 20% O(2) as under 10% O(2). In contrast, knockdown of HIF-1alpha inhibited NPC proliferation induced by 10% O(2). These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1alpha was critical in this process.     

Small ncRNA Expression and Regulation Under Hypoxia in Neural Progenitor Cells

Small non-coding RNA (ncRNA) plays critical roles in a large number of cellular processes, including neural development, cell survival and cell determination. Our previous work showed that low oxygen promoted the survival and proliferation of neural progenitor cells (NPCs) in vitro. In this study, we examine the expression and regulation of small ncRNAs in the hypoxia-driven proliferation of NPCs. The expression profiles of ncRNAs in NPCs under hypoxia were detected using microarray analysis. Results of significance analysis of microarrays (SAM) revealed that 15 small RNAs were up-regulated at least threefold and 11 were down-regulated under hypoxic conditions. The differentially expressed small ncRNAs were confirmed by quantitative RT-PCR, and miR-210 was observed to be highly expressed in NPCs under hypoxic conditions. Further study showed that hypoxia-inducible factor (HIF)-1α had a direct impact on the putative promoter regions of miR-210. From these results, we conclude that some small ncRNAs participate in the regulation of the proliferation of NPCs under hypoxia and that miR-210 is directly regulated by HIF-1α.     

Rapamycin decreases survivin expression to induce NSCLC cell apoptosis under hypoxia through inhibiting HIF-1α induction

Survivin is a member of the inhibitor of apoptosis protein family that is overexpressed in various tumors and is important in restricting apoptosis. Understanding the molecular events of apoptosis may provide information for developing novel therapeutic agents targeting non-small cell lung cancer (NSCLCs). This study used three human NSCLC cell lines, NCI-H1299, SK-MES-1, and NCI-H460. Changes in apoptosis, the mRNA and protein expression of survivin under normoxia and hypoxia, with or without rapamycin treatment were analyzed. In addition, siRNA and ChIP assay were further applied to demonstrate the role of hypoxia-inducible factor 1 (HIF-1)α in regulating survivin expression regulation under hypoxia during rapamycin induced NSCLC cell apoptosis. Treatment with rapamycin resulted in significantly increased NSCLC cells apoptosis under hypoxia. We demonstrated for the first time that rapamycin inhibited hypoxia-induced survivin expression in NSCLC cell lines. We further demonstrated that HIF-1α participated in hypoxia-induced survivin expression, and that rapamycin inhibited hypoxia-induced HIF-1α expression by enhancing its degradation. The results above collectively showed that rapamycin inhibits HIF-1α-induced survivin expression under hypoxia to induce NSCLC apoptosis.     

Hypoxia-induced compensatory effect as related to Shh and HIF-1α in ischemia embryo rat heart

Chronic cardiac ischemia/hypoxia induces coronary collateral formation and cardiomyocyte proliferation. Hypoxia can induce cellular adaptive responses, such as synthesis of VEGF for angiogenesis and IGF-2 for proliferation. Both reduce apoptotic effects to minimize injury or damage. To investigate the mechanism of neoangiogenesis and proliferation of fetal heart under umbilical cord compression situation, we used H9c2 cardiomyoblast cell culture, and in vivo embryonic hearts as our study models. Results showed hypoxia induced not only the increase of IGF-2 and VEGF expression but also the activation of their upstream regulatory genes, HIF-1α and Shh. The relationship between HIF-1α and Shh was further studied by using cyclopamine and 2-ME2, inhibitor of Shh and HIF-1α signaling, respectively, in the cardiomyoblast cell culture under hypoxia. We found that the two inhibitors not only blocked their own signal pathway, but also inhibited each other. The observations revealed when fetal heart under hypoxia that HIF-1α and Shh pathways maybe involve in cell proliferation and neoangiogenesis to minimize injury or damage, whereas the complex cross-talk between the two pathways remains unknown. Pei-Cheng Lin, Chih-Yang Huang, and Wei-Wen Kuo contributed equally to this work.     

The increased expression of DEC1 gene is related to HIF-1α protein in gastric cancer cell lines

Overexpression of differentiated embryo chondrocyte 1 (DEC1) has been reported to contribute to the cellular differentiation, proliferation, and apoptosis of various cancers. Our previous studies have shown that DEC1 was highly expressed in gastric cancer (GCa) tissues. However, there is no report about the expression of DEC1 in GCa cell lines until now. In this study, We evaluated the mRNA and protein expression of DEC1 and hypoxia-inducible factor 1α (HIF-1α) under normoxic and hypoxic conditions in six GCa cell lines: BGC-823, MGC80-3, MKN1, AGS, FU97 and SGC-7901. An HIF-1α protein inhibitor was used to analyze the association of DEC1 and HIF-1α expression. Under normoxia, the mRNA expression of both HIF-1α and DEC1 was moderate, whereas the protein expression of DEC1 was higher than that of HIF-1α. Hypoxia induced the mRNA expression of DEC1 and the protein expression of HIF-1α and DEC1 in a time-dependent manner but had no effect on the mRNA expression of HIF-1α. Furthermore, inhibition of HIF-1α protein expression resulted in a significant decrease in both the mRNA and protein expression of DEC1. Taken together, DEC1 expression is correlated with HIF-1α protein in GCa cell line, blockage of HIF-1α protein led to reduced DEC1 expression. The efficacy of inhibiting HIF-1α and DEC1 expression should be tested in clinical trials as possible treatment for GCa.     

Glucose up-regulates HIF-1 alpha expression in primary cortical neurons in response to hypoxia through maintaining cellular redox status

It has been suggested that hypoxia-inducible factor 1 (HIF-1), a key regulator in cell's adaptation to hypoxia, plays an important role in the fate of neurons during ischemia. However, the mechanism of HIF-1 regulation is still not fully understood in neurons subjected to ischemia. In this study, we demonstrated that glucose up-regulated the expression of HIF-1α, the oxygen-dependent subunit of HIF-1, in rat primary cortical neurons exposed to hypoxia. To understand the mechanism of glucose-regulated HIF-1α expression, we investigated the relationships between HIF-1α expression, reactive oxygen species (ROS), and redox status. Low levels of HIF-1α protein expression were observed in the neurons exposed to in vitro ischemic conditions that had high levels of ROS (oxidizing environments), and vice versa . The glutathione (GSH) precursor, N -acetyl cysteine, induced HIF-1α protein expression in hypoxic neurons while the GSH synthesis inhibitor, l -buthionine sulfoximine, inhibited the expression. Moreover, (−)-epicatechin gallate, a ROS scavenger, elevated HIF-1α expression in the neurons subjected to in vitro ischemia. Furthermore, results from a systemic hypoxia model showed that a reducing environment increased HIF-1α expression in rat brains. Taken together, these data presented the first evidence that glucose promoted HIF-1α stabilization through regulating redox status in primary neurons exposed to hypoxia. The results imply that hypoxia only may not be sufficient to stabilize HIF-1α and that a reducing environment is required to stabilize HIF-1α in neurons exposed to hypoxia.     

Interaction of the PAS B domain with HSP90 accelerates hypoxia-inducible factor-1alpha stabilization.

Hypoxia-inducible factor (HIF) alpha subunits are induced under hypoxic conditions, when limited oxygen supply prevents prolyl hydroxylation-dependent binding of the ubiquitin ligase pVHL and subsequent proteasomal degradation. A short normoxic half-life of HIF-alpha and a very rapid hypoxic protein stabilization are crucial to the cellular adaptation to changing oxygen supply. However, the molecular requirements for the unusually rapid mechanisms of protein synthesis, folding and nuclear translocation are not well understood. We and others previously found that the chaperone heat-shock protein 90 (HSP90) can interact with HIF-1alpha in vitro. Here we show that HSP90 also interacts with HIF-2alpha and HIF-3alpha, suggesting a general involvement of HSP90 in HIF-alpha stabilization. The PAS B domain, common to all three alpha subunits, was required for HSP90 interaction. ARNT competed with HSP90 for binding to the PAS B domain since an excess of either component inhibited the activity of the other. HSP90 as well as the heterocomplex members HSP70 and p23, but not HSP40, were detected in immunoprecipitations of endogenous cellular HIF-1alpha. While HSP90 and HSP70 bound to HIF-1alpha predominantly under normoxic conditions, ARNT bound to HIF-1alpha primarily under hypoxic conditions, suggesting that ARNT displaced HSP90 from HIF-1alpha following nuclear translocation. Hypoxic accumulation of HIF-1alpha was delayed in a novel cell model deficient for HSP90beta as well as after treatment of wild-type cells with the HSP90 inhibitor geldanamycin, suggesting that HSP90 activity is involved in the rapid HIF-1alpha protein induction.     

The Role of Hypoxia in the Differentiation of P19 Embryonal Carcinoma Cells into Dopaminergic Neurons

Nervous system development at early stage is in hypoxic environment. Very little is known about the role of hypoxia in neuronal development. P19 embryonal carcinoma (EC) cells are a widely used model for studying early neuronal development. In this study we investigated the roles of hypoxia in differentiation of dopaminergic neurons derived from P19 EC cells. Results demonstrate that hypoxia increases the percentage of differentiated neurons, especially neurons of dopaminergic phenotype. To investigate the potential mechanism involved in hypoxia promoted differentiation of dopaminergic neurons, we measured the expression of hypoxia-inducible factor 1α (HIF-1α), based on its characteristic response to hypoxia. The result shows that HIF-1α mRNA level in P19 EC cells increases after hypoxia treatment. It is known that HIF-1α regulates the expression of tyrosine hydroxylase (TH) gene through binding to its promoter. Therefore, we propose that the underlying mechanism for hypoxia promoted differentiation of dopaminergic neurons was mediated by HIF-1α up-regulation under hypoxia. Yue Wang—Co-first author. Special Issue in honor of Dr. Ji-Sheng Han.     

Effect of hypoxia-inducible factor-1alpha silencing on the sensitivity of human brain glioma cells to doxorubicin and etoposide

Multidrug resistance (MDR) is a significant problem underlying the poor prognosis associated with gliomas. Hypoxia-inducible factor-1α (HIF-1α) is thought to induce the genes expression involved in MDR. To evaluate the effect of silencing HIF-1α in human glioma T98G cells, cells were transfected with HIF-1α-small interference RNA (HIF-1α-siRNA) and cultured under hypoxic conditions. The effect of HIF-1α-siRNA on HIF-1α and multidrug resistance-associated protein 1 gene (MRP1) and protein levels was determined. Silencing rates of HIF-1α were 90%, 85%, and 88% at 24, 48, 72 h post-transfection, respectively. Corresponding rates of HIF-1α protein were 74.5%, 61.1% and 59.1%. MRP1 protein levels decreased by 7.6%, 36.8% and 45.2%. HIF-1α-siRNA transfected cells were significantly more sensitive to doxorubicin and etoposide compared to non-transfected cells. These findings suggest that the HIF-1α plays a role in mediating chemotherapeutic drug resistance in glioma cells. HIF-1α silencing may prove to be an effective therapeutic means of treating gliomas.     

HIF-1α and iNOS levels in crucian carp gills during hypoxia-induced transformation

Hypoxia inducible factor 1 alpha (HIF-1α) initiates expression of a wide variety of genes, some of which are involved in apoptosis and cell cycle arrest. We have previously shown that crucian carp increases its respiratory surface area 7.5-fold in response to hypoxia. This change is due to apoptosis and cell cycle arrest in specific parts of its gills. Here we have characterized crucian carp HIF-1α, and measured mRNA, protein and DNA binding levels during hypoxia exposure in crucian carp gills. We have also measured an HIF-1α-induced gene, the inducible nitric oxide synthase (iNOS), which has the ability to initiate apoptosis and cell cycle arrest. Crucian carp HIF-1α was found to have all critical domains known to be important for function. Comparison of the peptide sequence with other species indicated high similarity with other cyprinid fish, but a pronounced variation compared to the salmonid, rainbow trout. Further, we found HIF-1α protein to be stabilized during hypoxia. Further, HIF-1α was often present in normoxia, and showed marked individual weight-dependent variation. We found no alteration of iNOS mRNA levels during hypoxia exposure. These findings suggest HIF-1α involvement in hypoxia-induced change of respiratory surface area in crucian carp gills. However, its activity does not seem to be mediated through iNOS.