Non-hypoxic Stimuli Research Articles

Protein kinase B/AKT phosphorylates hypoxia-inducible factor-3α1 in response to insulin, promoting cell growth and migration.

Hypoxia-inducible factors (HIFs) are best known for their roles in the adaptation to low oxygen environments. Besides hypoxia, HIF-1/2 α-subunits are also regulated by various non-hypoxic stimuli including insulin which can act via the PI3K/protein kinase B (PKB) signaling pathway. However, with respect to insulin little is known about HIF-3α. We aimed to investigate this relationship and found that insulin stimulates HIF-3α expression under both normal and low oxygen conditions. Blocking PKB activity reversed the effects of insulin, indicating that HIF-3α is a direct target of PKB. We identified serine 524, located in the oxygen-dependent degradation domain of HIF-3α, as a phosphorylation site of PKB. Mutating serine 524 impaired binding of PKB to HIF-3α and its ubiquitination, suggesting that PKB regulates HIF-3α stability through phosphorylation, thereby affecting important cellular processes such as cell viability and cell adhesion. Importantly, we discovered that this phosphorylation site also influenced insulin-dependent cell migration. These findings shed light on a novel mechanism by which insulin affects PKB-dependent HIF-3α expression and activity, with potential implications in metabolic diseases and cancer.

Carbon tetrachloride administration induces the expression of hypoxia inducible factor-1alpha in rat liver

Background: There is now increasing evidence that HIF-1 is also responsive to a variety of non-hypoxic stimuli. However, the mechanisms by which these non-hypoxic stimuli induce HIF-1α are not completely known, yet, although some evidence points to a role of ROS as messengers regulating HIF activity.
 Objective: To determine the expression of HIF-1α in liver rat tissue induced by carbon tetrachloride under normoxic conditions, with or without N-acetylcysteine protection.
 Methods: Twenty-five male Sprague-Dawley rats were divided into 5 groups: normal control rats, normal rats orally administered with coconut oil (1 mL/200 g body weight) for 1 day, rats orally administered with CCl4 (0.55 mg/g body weight) for 1 day, rats injected i.v. with NAC (0.15 mg/g body weight) for 8 days and then orally administered with CCl4 (0.55 mg/g body weight) for 1 day, rats orally administered with CCl4 (0.55 mg/g body weight) for 1 day and then injected i.v. with NAC (0.15 mg/g body weight) for 2 days. The expression of HIF-1α mRNA was measured by real-time RT-PCR using the Livak method. The expression of HIF-1α protein was measured by ELISA assay.
 Results: The highest HIF-1α mRNA and protein expression found in the group treated by CCl4 and then was gradually lowered in the pre-NAC group, post-NAC group, control group, and last, in the oil group.
 Conclusion: Our study shows the effect of CCl4-treated rats under normoxic conditions increased the mRNA and protein HIF-1α. NAC post-treatment provide a better protective effect compared with NAC pre-treatment

Selective HIF-1 Regulation under Nonhypoxic Conditions by the p42/p44 MAP Kinase Inhibitor PD184161

Hypoxia-inducible factor-1 (HIF-1) is a key gene regulator for cellular adaptation to low oxygen. In addition to hypoxia, several nonhypoxic stimuli, including hormones and growth factors, are essential for cell-specific HIF-1 regulation. Our studies have highlighted angiotensin II (AngII), a vasoactive hormone, as a potent HIF-1 activator in vascular smooth muscle cells (VSMC). AngII increases HIF-1 transcriptional activity by modulating specific signaling pathways. In VSMC, p42/p44 mitogen-activated protein kinase (MAPK) pathway activation is essential for HIF-1-mediated transcription during AngII treatment. The present study shows that PD184161, a potent MEK1/2 inhibitor, is an HIF-1 blocker in Ang II-treated VSMC. Unlike PD98059, a widely-used MEK1/2 inhibitor, we found that PD184161 blocked AngII-driven HIF-1α protein induction in a dose-dependent manner. Interestingly, the effect of PD184161 was specific to nonhypoxic activators, since HIF-1α induction by hypoxia (1% O2) was unaffected under similar conditions. VSMC treatment with MG132, a proteasome inhibitor, indicated that PD184161 influenced HIF-1α protein stability. PD184161 also increased HIF-1α binding to von Hippel-Lindau tumor suppressor protein, an E3 ligase component and an indication of HIF-1α hydroxylation. Finally, we show that PD184161 blocked mitochondrial ROS (mtROS) production and cellular ATP levels, at the same time enhancing ascorbate availability in AngII-treated VSMC. Taken together, our study indicates that, independently of p42/p44 MAPK activation, PD184161 blocks mtROS generation by AngII, leading to re-establishment of cellular ascorbate levels, increased VHL binding, and decreased HIF-1α stability. Therefore, this study reveals a previously unsuspected role for PD184161 as an HIF-1 inhibitor in VSMC under nonhypoxic conditions.

Abstract 51: Role of MUC13 as non-hypoxic stimuli inducing HIF-1α in pancreatic cancer under normoxia

Abstract Objective: Pancreatic cancer is the fourth most common cause of deaths occurring due to cancer, with an overall survival rate of just 5%. MUC13, a transmembrane mucin, is aberrantly expressed in pancreatic cancer, while an altered glucose metabolism is known to facilitate cancer cell survival and proliferation. Hypoxia-inducible factor 1 α (HIF-1 α) plays an important role in reprogramming of cancer cell metabolism by activating the transcription of genes which encode glucose transporters and enzymes involved in glycolysis. Recent reports suggest that several non-hypoxic stimuli such as lipopolysaccharides, thrombin, and angiotensin II can also increase HIF-1α under normoxia. Herein, we investigated the effects of MUC13 expression on glucose metabolism and elucidated underlying signaling mechanisms that might be involved in this process. Methods: MUC13 null pancreatic cancer Panc-1 cells were used for the study. Glucose and Lactate assay were performed in Panc-1 cells stably expressing MUC13 (Panc-1-M13) and vector (Panc-1-V). Cell culture media after 48 hrs was collected to measure the amount of unused glucose and L-lactate concentration using glucose and Lactate assay kits. Immunoblot and semi-quantitative PCR analyses were performed to check the expression of protein and mRNA levels, respectively. Cell proliferation and colony forming assays were performed to determine the effect of MUC13 on cellular growth and cell survival. Results: Our results demonstrate that MUC13 acts as a modulator of the glucose metabolism in pancreatic cancer cells by regulating the expression and activity of hypoxia-inducible factor-1α (HIF-1α) and its downstream targets. We observed increased amount of L-lactate production and glucose consumption in Panc-1-M13 cells as compared to Panc-1-V cells. This facilitates metabolic alterations and help tumor cells survive and proliferate under these conditions as indicated by increased cellular growth pattern in Panc-1-M13 cells. Our results demonstrate increased expression of the downstream targets of HIF-1α, such as cell regulatory (c-Myc), and cell survival (Bcl-2) proteins, while decreased the expression of tumor suppressor/cell cycle inhibitor (p-27) proteins in Panc-1M13 cells. Additionally, an increase in the expression of a critical downstream target of HIF-1α, Glut-1 was observed in Panc-1-M13 cells. Conclusion: Our studies indicate that MUC13 acts as a key regulator of the metabolic process and facilitates metabolic alterations in the non-hypoxic environment that help tumor cells survive and proliferate under these conditions. However, further studies are required to elucidate detailed molecular mechanisms that are involved in MUC13 mediated metabolic remodeling in pancreatic cancer cells. Citation Format: Sonam Kumari, Sheema Khan, Subhash Chauhan, Meena Jaggi. Role of MUC13 as non-hypoxic stimuli inducing HIF-1α in pancreatic cancer under normoxia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 51.

Hypoxia and Its Acid-Base Consequences: From Mountains to Malignancy.

Hypoxia, depending upon its magnitude and circumstances, evokes a spectrum of mild to severe acid-base changes ranging from alkalosis to acidosis, which can alter many responses to hypoxia at both non-genomic and genomic levels, in part via altered hypoxia-inducible factor (HIF) metabolism. Healthy people at high altitude and persons hyperventilating to non-hypoxic stimuli can become alkalotic and alkalemic with arterial pH acutely rising as high as 7.7. Hypoxia-mediated respiratory alkalosis reduces sympathetic tone, blunts hypoxic pulmonary vasoconstriction and hypoxic cerebral vasodilation, and increases hemoglobin oxygen affinity. These effects and others can be salutary or counterproductive to tissue oxygen delivery and utilization, based upon magnitude of each effect and summation. With severe hypoxia either in the setting of profound arterial hemoglobin desaturation and reduced O2 content or poor perfusion (ischemia) at the global or local level, metabolic and hypercapnic acidosis develop along with considerable lactate formation and pH falling to below 6.8. Although conventionally considered to be injurious and deleterious to cell function and survival, both acidoses may be cytoprotective by various anti-inflammatory, antioxidant, and anti-apoptotic mechanisms which limit total hypoxic or ischemic-reperfusion injury. Attempts to correct acidosis by giving bicarbonate or other alkaline agents under these circumstances ahead of or concurrent with reoxygenation efforts may be ill advised. Better understanding of this so-called "pH paradox" or permissive acidosis may offer therapeutic possibilities. Rapidly growing cancers often outstrip their vascular supply compromising both oxygen and nutrient delivery and metabolic waste disposal, thus limiting their growth and metastatic potential. However, their excessive glycolysis and lactate formation may not necessarily represent oxygen insufficiency, but rather the Warburg effect-an attempt to provide a large amount of small carbon intermediates to supply the many synthetic pathways of proliferative cell growth. In either case, there is expression and upregulation of many genes involved in acid-base homeostasis, in part by HIF-1 signaling. These include a unique isoform of carbonic anhydrase (CA-IX) and numerous membrane acid-base transporters engaged to maintain an optimal intracellular and extracellular pH for maximal growth. Inhibition of these proteins or gene suppression may have important therapeutic application in cancer chemotherapy.

Expression of hypoxia-inducible factor-1 by trophectoderm cells in response to hypoxia and epidermal growth factor

The low oxygen environment in the uterine environment requires pre-implantation embryos to adapt to oxygen deficiency. Hypoxia-inducible factor (HIF)-1 is a master regulator whereby cells adapt to changes in oxygen concentrations. In addition to hypoxic conditions, non-hypoxic stimuli such as growth factors also activate expression of HIF-1. In this study, the mechanisms underlying low oxygen-dependent and epidermal growth factor (EGF)-dependent expression of HIF-1α were explored using porcine trophectoderm (pTr) cells. The results indicated that expression of HIF-1α and HIF-1β mRNAs was not affected by low concentrations of oxygen; however, hypoxic conditions markedly increased the abundance of HIF-1α protein, especially in nuclei of pTr cells. Even under normoxic conditions, the abundance of HIF-1α protein increased in response to EGF. This EGF-mediated increase in HIF-1α protein was blocked through inhibition of translation by cycloheximide. The inhibitors LY294002 (PI3K-AKT inhibitor), U0126 (inhibitor of ERK1/2) and rapamycin (mTOR inhibitor) also blocked the ability of EGF to increase HIF-1α protein and to phosphorylate AKT, ERK1/2 and mTOR proteins. Both hypoxia and EGF induced proliferation of pTr cells. This ability of EGF to stimulate proliferation of pTr cells was suppressed by EGFR siRNA, but not HIF-1α siRNA, but a significant decrease in EGF-induced HIF-1α protein occurred when pTr cells were transfected with HIF-1α siRNA. The results of the present study suggest that pTr cells adapt to oxygen deficiency and proliferate in response to an oxygen-dependent HIF-1 system, and that EGF at maternal–conceptus interface can increase the abundance of HIF-1α protein via translational regulation through AKT, ERK1/2 and mTOR signaling cascades.

Differential regulation of HIF-3α in LPS-induced BV-2 microglial cells: Comparison and characterization with HIF-1α

Hypoxia inducible factor(s) (HIF) are transcription factors that respond to a low level of oxygen or hypoxic conditions. The HIF pathway has been poorly studied under neuroinflammatory conditions, and no reports are available on the regulation of HIF-3α. Several studies have established that non-hypoxic stimuli can modulate the HIF pathway in a cell-specific manner. Recent reports suggest that hypoxia elicits inflammation or that inflammation during hypoxia is involved in a wide array of human diseases. In the present study, we used lipopolysaccharide (LPS), a well know inflammatory agent, to characterize the HIF-3α expression pattern and compare it with that of HIF-1α under inflammatory conditions in BV-2 microglial cells. Moreover, we used reactive oxygen species inhibitors (rotenone, diphenyleneiodonium, and N-acetyl-l-cysteine) under inflammatory conditions to determine the role of the functional electron transport chain in the regulation of HIF-3α in BV-2 microglial cells. Additionally, we utilized YC-1, a specific inhibitor of HIF-1α, to determine the role of HIF-3α in inflammatory conditions after inhibiting the HIF-1α pathway. YC-1 inhibited nuclear localization of HIF-1α following treatment with LPS in BV-2 microglia cells. Immunoblot and immunocytochemistry revealed a transient effect on HIF-3α after pre-treating the cells with YC-1. Furthermore, we determined the role of nuclear factor kappa B (NF-κB) in the regulation of HIF-3α using the NF-κB inhibitor PDTC in LPS-stimulated BV-2 microglia cells. PDTC altogether abolished LPS-induced nuclear translocation of HIF-3α with a partial effect on HIF-1α, suggesting that HIF-3α expression under inflammatory conditions may be directly under the control of the NF-κB pathway in BV-2 microglial cells. Interestingly, HIF-3α and HIF-1α exhibited almost similar responses to a variety of activating or inhibiting pharmacological agents. These results provide the first evidence for regulation of HIF-3α under inflammatory conditions in BV-2 microglial cells.

Transcriptional repression of hypoxia-inducible factor-1 (HIF-1) by the protein arginine methyltransferase PRMT1

Hypoxia-inducible factors (HIF-1 and HIF-2) are essential mediators for the adaptive transcriptional response of cells and tissues to low-oxygen conditions. Under hypoxia or when cells are treated with various nonhypoxic stimuli, the active HIF-α subunits are mainly regulated through increased protein stabilization. For HIF-1α, it is clear that further transcriptional, translational, and posttranslational regulations are important for complete HIF-1 activity. Novel evidence links hypoxia and HIF-1 to arginine methylation, an important protein modification. These studies suggest that arginine methyltransferases may be important for hypoxic responses. Protein arginine methyltransferase 1 (PRMT1), the predominant arginine methyltransferase, can act as a transcriptional activator or repressor by modifying a diverse set of substrates. In this work, we show that PRMT1 is a repressor of both HIF-1 and HIF-2. The cellular depletion of PRMT1 by small interference RNA targeting leads to increased HIF transcriptional activity. This activation is the result of enhanced HIF-α subunit transcription, which allows increased HIF-α subunit availability. We provide evidence that PRMT1-dependent HIF-1α regulation is mediated through the activities of both specificity protein 1 (Sp1) and Sp3, two transcription factors known to control HIF-1α expression. This study therefore identifies PRMT1 as a novel regulator of HIF-1- and HIF-2-mediated responses.

In vivoevidence suggesting reciprocal renal hypoxia-inducible factor-1 upregulation and signal transducer and activator of transcription 3 activation in response to hypoxic and non-hypoxic stimuli

In vitro studies suggest that combined activation of hypoxia-inducible factor (HIF) and signal transducer and activator of transcription 3 (STAT3) promotes the hypoxia response. However, their interrelationship in vivo remains poorly defined. The present study investigated the possible relationship between HIF-1 upregulation and STAT3 activation in the rodent kidney in vivo. Activation of HIF-1 and STAT3 was analysed by immunohistochemical staining and western blot analysis in: (i) models of hypoxia-associated kidney injury induced by radiocontrast media or rhabdomyolysis; (ii) following activation of STAT3 by the interleukin (IL)-6-soluble IL-6 receptor complex; or (iii) following HIF-1α stabilization using hypoxic and non-hypoxic stimuli (mimosine, FG-4497, CO, CoCl(2)) and in targeted von Hippel-Lindau-knockout mice. Western blot analysis and immunostaining revealed marked induction of both transcription factors under all conditions tested, suggesting that in vivo STAT3 can trigger HIF and vice versa. Colocalization of HIF-1α and phosphorylated STAT3 was detected in some, but not all, renal cell types, suggesting that in some cells a paracrine mechanism may be responsible for the reciprocal activation of the two transcription factors. Nevertheless, in several cell types spatial concordance was observed under the majority of conditions tested, suggesting that HIF-1 and STAT3 may act as cotranscription factors. These in vivo studies suggest that, in response to renal hypoxic-stress, upregulation of HIF-1 and activation of STAT3 may be both reciprocal and cell type dependent.

Hypoxia-Inducible Factor-lα Increase is an Early and Sensitive Marker of Lung Cells Responding to Benzo[a]Pyrene

Hypoxia inducible factor-1α (HIF-lα) is a central regulator of tumor survival and metastasis, responsible for metabolic adaptation to hypoxic conditions and promotion of angiogenesis. It has been also shown to respond to non-hypoxic stimuli, such as growth factors and moderate oxidative stress. We examined the protein levels of HIF-lα in A549 human lung cells exposed to the typical carcinogen benzo[a]pyrene (B[a]P). Our results revealed that B[a]P, at low, non-cytotoxic concentrations, induced a transient increase of nuclear HIF-lα and its target, GLUT1. HIF-lα upregulation was partly mediated by Akt kinase and coincided with increased nuclear levels of the redox-sensitive marker, nuclear factor erythroid 2-related factor-2 (NrF-2). B[a]P-induced HIF-lα was also detected during serum depletion or treatment with the hypoxia-mimicking agent, CoCl2. In addition, exposure of A549 cells to B[a]P containing diesel exhaust particles enhanced HIF-lα accumulation, probably due to the presence of additional carcinogenic compounds. B[a]P-induced increase of HIF-lα was further confirmed in normal rat and human lung fibroblasts. Our findings indicate that HIF-lα stimulation may act as an early and sensitive marker of exposure to low, non-cytotoxic concentrations of B[a]P and/or other carcinogens.

Functional regulation of HIF‐1α under normoxia—is there more than post‐translational regulation?

The hypoxia-inducible factor-1 (HIF-1) is an oxygen-regulated transcriptional activator playing a pivotal role in mammalian physiology and disease pathogenesis, e.g., HIF-1 is indispensable in a broad range of developmental stages in different tumors. Its post-translational regulation via PHDs under the influence of hypoxia is widely investigated and accepted. Different non-hypoxic stimuli such as lipopolysaccharides (LPS), thrombin, and angiotensin II (Ang II), have been proven to enhance HIF-1 levels through activation of regulative mechanisms distinct from protein stabilization. Some of these stimuli specifically regulate HIF-1α at the transcriptional, post-transcriptional, or translational level, whereas others additionally influence post-translational modifications. Thus, it is difficult for the investigators to discern the impact of the different mechanisms leading to functional HIF-1 protein. Nevertheless, profound knowledge of additional regulatory networks appears to depict new therapeutic opportunities and thus is an interesting and important field for further investigations.

Immunohistochemical assessment of intrinsic and extrinsic markers of hypoxia in reproductive tissue: differential expression of HIF1α and HIF2α in rat oviduct and endometrium

Hypoxia is thought to be critical in regulating physiological processes within the female reproductive system, including ovulation, composition of the fluid in the oviductal/uterine lumens and ovarian follicle development. This study examined the localisation of exogenous (pimonidazole) and endogenous [hypoxia inducible factor 1α and 2α (HIF1α, -2α), glucose transporter type 1 (GLUT1) and carbonic anhydrase 9 (CAIX)] hypoxia-related antigens within the oviduct and uterus of the rat reproductive tract. The extent to which each endogenous antigen co-compartmentalised with pimonidazole was also assessed. Female Wistar Furth rats (n = 10) were injected intraperitoneally with pimonidazole (60 mg/kg) 1 h prior to death. Reproductive tissues were removed immediately following death and fixed in 4% paraformaldehyde before being embedded in paraffin. Serial sections were cut (6–7 μm thick) and antigens of interest identified using standard immunohistochemical procedures. The mucosal epithelia of the ampulla, isthmus and uterus were immunopositive for pimonidazole in most sections. Co-compartmentalisation of pimonidazole with HIF1α was only expressed in the mucosa of the uterus whilst co-compartmentalisation with HIF2α was observed in the mucosa of the ampulla, isthmus and uterus. Both GLUT1 and CAIX were co-compartmentalised with pimonidazole in mucosa of the isthmus and uterus. This study confirms that mucosal regions of the rat oviduct and uterus frequently experience severe hypoxia and there are compartment specific variations in expression of endogenous hypoxia-related antigens, including the HIF isoforms. The latter observation may relate to target gene specificity of HIF isoforms or perhaps HIF2α’s responsiveness to non-hypoxic stimuli such as hypoglycaemia independently of HIF1α.

BRAFV600E mutation influences hypoxia-inducible factor-1α expression levels in papillary thyroid cancer

Hypoxia-inducible factor-1α is found frequently overexpressed in solid tumors cells, exerting an important role in angiogenesis, glucose metabolism, cell proliferation, survival and invasion. In thyroid carcinomas, hypoxia-inducible factor-1α expression was found increased in differentiated, poorly differentiated, medullary and anaplastic variants. Hypoxia represents the principal stimulus responsible for hypoxia-inducible factor-1α induction. Other nonhypoxic stimuli increase hypoxia-inducible factor-1α synthesis through the activation of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in a cell-type-specific manner. We have previously shown the role of BRAFV600E mutation in papillary thyroid cancer cells as a factor that facilitates tumor cell growth and progression. In this study, we tested the hypothesis that BRAFV600E mutation influences hypoxia-inducible factor-1α expression in papillary thyroid carcinoma cells. We analyzed 27 papillary thyroid carcinomas, 13 of which presented BRAFV600E mutation. In tumor tissues, immunoreactivity for hypoxia-inducible factor-1α was detected in the majority of analyzed BRAFV600E mutated cases. Transcriptional analyses revealed elevated hypoxia-inducible factor-1α levels with significant differences between wild-type and mutated group. A BRAF wild-type papillary thyroid carcinoma cell line and a BRAFV600E mutated papillary thyroid carcinoma cell line were selected to study the effects of BRAF mutation on hypoxia-inducible factor-1α expression in vitro. Knockdown of mutant BRAFV600E or both the wild type and the BRAFV600E by RNA interference induced a significant reduction of hypoxia-inducible factor-1α expression at mRNA and protein levels. Pharmacological inhibition of BRAF significantly reduces hypoxia-inducible factor-1α expression levels in papillary thyroid carcinoma cell line harboring BRAFV600E mutation. Our results suggest that hypoxia-inducible factor-1α is expressed in papillary thyroid carcinomas and is regulated not only by hypoxia but also by BRAFV600E-mediated signaling pathway.

Hypoxia-inducible Factor-1 Activation in Nonhypoxic Conditions: The Essential Role of Mitochondrial-derived Reactive Oxygen Species

Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor for responses to low oxygen. Different nonhypoxic stimuli, including hormones and growth factors, are also important HIF-1 activators in the vasculature. Angiotensin II (Ang II), the main effecter hormone in the renin-angiotensin system, is a potent HIF-1 activator in vascular smooth muscle cells (VSMCs). HIF-1 activation by Ang II involves intricate mechanisms of HIF-1α transcription, translation, and protein stabilization. Additionally, the generation of reactive oxygen species (ROS) is essential for HIF-1 activation during Ang II treatment. However, the role of the different VSMC ROS generators in HIF-1 activation by Ang II remains unclear. This work aims at elucidating this question. Surprisingly, repression of NADPH oxidase-generated ROS, using Vas2870, a specific inhibitor or a p22(phox) siRNA had no significant effect on HIF-1 accumulation by Ang II. In contrast, repression of mitochondrial-generated ROS, by complex III inhibition, by Rieske Fe-S protein siRNA, or by the mitochondrial-targeted antioxidant SkQ1, strikingly blocked HIF-1 accumulation. Furthermore, inhibition of mitochondrial-generated ROS abolished HIF-1α protein stability, HIF-1-dependent transcription and VSMC migration by Ang II. A large number of studies implicate NADPH oxidase-generated ROS in Ang II-mediated signaling pathways in VSMCs. However, our work points to mitochondrial-generated ROS as essential intermediates for HIF-1 activation in nonhypoxic conditions.

Hormonally regulated follicle differentiation and luteinization in the mouse is associated with hypoxia inducible factor activity

Hypoxia inducible factors (HIFs) are transcription factors that mediate physiological responses to hypoxia. Hypoxia is established as the major inducer of HIFs, but stimuli such as transition metals and hormones also induce HIF target genes. Whilst the ovarian granulosa cell layer is known to be avascular and the follicle is vascularised via the thecal cell layer, little is known about the role of hypoxia or HIFs in regulating ovarian function. In this study, we hypothesized that hypoxia as well as non-hypoxic stimuli cooperate in promoting follicle differentiation and luteinization via HIF activity and resultant gene regulation. We quantitatively measured the HIF1α protein response to hCG in ovarian granulosa cell cultures and in vivo and developed a transgenic (HRE(4)-SV40-EGFP) HIF reporter mouse line. We observed a time-dependent increase of HIF1α protein levels in granulosa cells post-hCG in vivo, maximal around time of ovulation. hCG alone was unable to promote HIF1α protein accumulation in cultured granulosa cells, but increased protein abundance was observed when combined with a hypoxic stimulus. HRE-EGFP ovaries showed no follicular EGFP in stages prior to antrum formation. However, HIF regulated EGFP was maximally induced in granulosa cells around the time of ovulation and readily observed in corpora lutea. There was also an increase in HIF regulated EGFP activity in the corpora lutea from functional to regressing stages. Taken together, these observations establish the notion that HIFs play a role during follicular differentiation and luteinization.

Inflammatory stimulation and hypoxia cooperatively activate HIF-1α in bronchial epithelial cells: involvement of PI3K and NF-κB

The transcription factor hypoxia-inducible factor (HIF)-1 plays a central physiological role in oxygen and energy homeostasis, and is activated during hypoxia by stabilization of the subunit HIF-1α. Recent studies have demonstrated that non-hypoxic stimuli can also activate HIF-1α in a cell-specific manner. Here, we demonstrate that stimulation of BEAS-2B cells and primary human bronchial epithelial cells by proinflammatory cytokines TNFα/IL-4 strongly induced expression and transcriptional activity of HIF-1α under normoxic conditions and amplified hypoxic HIF-1α activation. TNFα/IL-4 stimulated de novo HIF-1α gene transcription and translation rather than affected HIF-1α protein degradation and mRNA decay process. The activation of HIF-1α by TNFα/IL-4 was countered by the phosphoinositol 3-kinase (PI3K) inhibitor LY-294002 and rapamycin, an antagonist of mammalian target of rapamycin (mTOR), but not by inhibition of the MAPK pathway. In line, TNFα/IL-4 also activated NF-κB, whereas blocking of NF-κB by an inhibitor or silencing NF-κB subunit p65 attenuated HIF-1α activation by TNFα/IL-4. We also found the collaborative induction of VEGF, a potent angiogenic factor required for airway remodeling, by TNFα/IL-4 and hypoxia partially via HIF-1α pathway in BEAS-2B cells. This study reports the previously unsuspected collaborative regulation of HIF-1α by TNFα/IL-4 and hypoxia in bronchial epithelial cells partially via PI3K-mTOR and NF-κB pathway, and thereby will lead to the elucidation of the importance of HIF-1 in integrating inflammatory and hypoxic response in the pathogenesis of airway diseases.

HIF-1 is induced via EGFR activation and mediates resistance to anoikis-like cell death under lipid rafts/caveolae-disrupting stress

The plasma membrane microdomains, lipid rafts, are involved in regulation of cellular functions such as cell survival and adhesion. Cholesterol is a critical component of lipid rafts in terms of their integrity and functions and rafts disruption by cholesterol depletion can induce detachment-induced cell death. Hypoxia inducible factor-1 (HIF-1) alpha is stabilized in hypoxia and transactivates numerous genes required for cellular adaptation to hypoxia. It is also induced by non-hypoxic stimuli and contributes to cell survival. Because hypoxia inhibits cholesterol synthesis and HIF-1alpha plays a role in this process, we here explored a possible connection between lipid rafts and HIF-1alpha. We investigated whether HIF-1alpha is regulated during cholesterol depletion/rafts disruption in A431 cells in normoxic conditions. Methyl-beta cyclodextrin (MbetaCD), which induces cholesterol depletion, upregulated HIF-1alpha even under normoxic conditions and this upregulation required epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase 1 and 2 activation, but not Akt activation. MbetaCD treatment induced HIF-1alpha upregulation at both the transcriptional and translational levels but not at the posttranslational levels. In addition, MbetaCD robustly induced vascular endothelial growth factor production and stimulated an hypoxia response element-driven luciferase reporter activity under normoxic conditions, indicating that MbetaCD-induced HIF-1alpha is functionally activated. Both EGFR activity and HIF-1alpha expression were higher in the attached cells than in the detached cells after MbetaCD treatment. Furthermore, inhibition of HIF-1alpha by RNA interference accelerated cell detachment, thus increasing cell death, indicating that HIF-1alpha expression attenuates MbetaCD-induced anoikis-like cell death. These data suggest that, depending on cholesterol levels, lipid rafts or membrane fluidity are probably to regulate HIF-1alpha expression in normoxia by modulating rafts protein activities such as EGFR, and this connection between lipid rafts and HIF-1alpha regulation may provide cell survival under membrane-disturbing stress.

Sphingosine-1-Phosphate

Sphingosine-1-phosphate (S1P) is a potent bioactive phospholipid responsible for a variety of vascular cell responses. Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator of genes essential for adaptation to low oxygen. S1P and HIF-1 are both important mediators of vascular cell responses such as migation, proliferation, and survival. Studies have shown that nonhypoxic stimuli can activate HIF-1 in oxygenated conditions. Here, we attempt to determine whether S1P can modulate the vascular activation of HIF-1. We show that in vascular endothelial and smooth muscle cells, activation of the S1P type-2 receptor by S1P strongly increases HIF-1 alpha protein levels, the active subunit of HIF-1. This is achieved through pVHL-independent stabilization of HIF-1 alpha. We demonstrate that the HIF-1 nuclear complex, formed on S1P stimulation, is transcriptionally active and specifically binds to a hypoxia-responsive elements. Moreover, S1P activates the expression of genes known to be closely regulated by HIF-1. Our results identify S1P as a novel and potent nonhypoxic activator of HIF-1. We believe that understanding the role played by HIF-1 in S1P gene regulation will have a strong impact on different aspects of vascular biology.

Non-hypoxic Stabilization of Hypoxia-Inducible Factor Alpha (HIF-α): Relevance in Neural Progenitor/Stem Cells

Hypoxia-inducible factor-1 (HIF-1) plays an important role in neural progenitor cell (NPC) propagation and dopaminergic differentiation. In the presence of oxygen and iron, hypoxia-inducible factor 1 alpha (HIF-1alpha) is rapidly degraded via the prolyl hydroxylase (PHD)/VHL pathway. In addition to hypoxia, various non-hypoxic stimuli can stabilize HIF-1alpha in NPCs and influence the transcription of HIF-regulated genes. Here, we investigate various hypoxia mimetics: deferoxamine (DFO), ciclopirox olamine (CPX), dimethyloxallyl glycine (DMOG), a novel HIF-PHD inhibitor (FG-4497) and cobalt chloride (CoCl(2)) with respect to their ability to enhance in vitro proliferation, neurogenesis and dopaminergic differentiation of human fetal mesencephalic NPCs (hmNPCs) in ambient oxygen (21%). Although able to stabilize HIF-1alpha, iron chelators (DFO and CPX) and DMOG were toxic to hmNPCs. CoCl(2) was beneficial only towards neuronal and dopaminergic differentiation, while FG-4497 enhanced proliferation, neurogenesis and dopaminergic differentiation of hmNPCs. Both CoCl(2) and FG-4497 were protective to human dopaminergic neurons. Finally, exposure to hyperbaric oxygen (HBO) also stabilized HIF-1alpha in hmNPCs and induced neurogenesis in vitro. These findings suggest that several HIF stabilizing agents or conditions can rescue impaired neurons and promote neurogenesis in vitro.

Induction of hypoxia inducible factor 1α by Toll-like receptors in human dendritic cells

Nonhypoxic stimuli can induce the expression of hypoxia-inducible factor 1α (HIF1α). Only recently has it been demonstrated that lipopolysaccharide (LPS) induces the expression of HIF1α in macrophages and that the induction of hypoxic genes in macrophages is also Toll-like receptor 4 (TLR4)-dependent. We hypothesized that HIF1α expression is induced in dendritic cells in a TLR-dependent manner, plays a crucial role in linking the innate with the adaptive immune system and may also influence mitochondrial respiration.