Hypoxia-inducible Factor-1 Transcriptional Activity Research Articles

The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein.

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC (n = 73) and VHL-proficient pRCC human tumor tissue (n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = −0.38, p < 0.05 and r = −0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro, ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response.

β-TrCP upregulates HIF-1 in prostate cancer cells.

The substantial availability of hypoxia-inducible factor 1 (HIF-1) for pathophysiological states, such as malignancies and ischemia, is primarily regulated post-translationally through the ubiquitin proteolytic system. The balance between degradation and stabilization of HIF-1α protein is determined by specific E3 ligases. In our search for new E3 ligases that might affect HIF-1α protein expression, we studied the effects of beta-transducin repeat-containing protein (β-TrCP) on the hypoxic pathway in cancer cells. β-TrCP is overexpressed in many tumors and regulates various cellular processes through mediating the degradation of important targets. Unexpectedly, we found that β-TrCP overexpression increases HIF-1α protein expression level as well as HIF-1 transcriptional activity by stabilizing HIF-1α protein and preventing its ubiquitination and proteasomal degradation in prostate cancer cells. By using a proteomic approach, we succeeded in demonstrating that β-TrCP interferes with the association between HIF-1α and HSP70/CHIP, a HIF-1α established E3 ligase complex. Whereas the E3 ligase activity of β-TrCP is well known, antagonizing another E3 ligase is a new mechanism of action of this important E3. We suggest that destroying or suppressing β-TrCP and thereby interrupting the HIF-1 pathway, could be valuable antitumor therapy.

VHL-Mediated Regulation of CHCHD4 and Mitochondrial Function.

Dysregulated mitochondrial function is associated with the pathology of a wide range of diseases including renal disease and cancer. Thus, investigating regulators of mitochondrial function is of particular interest. Previous work has shown that the von Hippel-Lindau tumor suppressor protein (pVHL) regulates mitochondrial biogenesis and respiratory chain function. pVHL is best known as an E3-ubiquitin ligase for the α-subunit of the hypoxia inducible factor (HIF) family of dimeric transcription factors. In normoxia, pVHL recognizes and binds hydroxylated HIF-α (HIF-1α and HIF-2α), targeting it for ubiquitination and proteasomal degradation. In this way, HIF transcriptional activity is tightly controlled at the level of HIF-α protein stability. At least 80% of clear cell renal carcinomas exhibit inactivation of the VHL gene, which leads to HIF-α protein stabilization and constitutive HIF activation. Constitutive HIF activation in renal carcinoma drives tumor progression and metastasis. Reconstitution of wild-type VHL protein (pVHL) in pVHL-defective renal carcinoma cells not only suppresses HIF activation and tumor growth, but also enhances mitochondrial respiratory chain function via mechanisms that are not fully elucidated. Here, we show that pVHL regulates mitochondrial function when re-expressed in pVHL-defective 786O and RCC10 renal carcinoma cells distinct from its regulation of HIF-α. Expression of CHCHD4, a key component of the disulphide relay system (DRS) involved in mitochondrial protein import within the intermembrane space (IMS) was elevated by pVHL re-expression alongside enhanced expression of respiratory chain subunits of complex I (NDUFB10) and complex IV (mtCO-2 and COX IV). These changes correlated with increased oxygen consumption rate (OCR) and dynamic changes in glucose and glutamine metabolism. Knockdown of HIF-2α also led to increased OCR, and elevated expression of CHCHD4, NDUFB10, and COXIV in 786O cells. Expression of pVHL mutant proteins (R200W, N78S, D126N, and S183L) that constitutively stabilize HIF-α but differentially promote glycolytic metabolism, were also found to differentially promote the pVHL-mediated mitochondrial phenotype. Parallel changes in mitochondrial morphology and the mitochondrial network were observed. Our study reveals a new role for pVHL in regulating CHCHD4 and mitochondrial function in renal carcinoma cells.

Hypoxic stabilization of mRNA is HIF-independent but requires mtROS

BackgroundTissue ischemia can arise in response to numerous physiologic and pathologic conditions. The cellular response to decreased perfusion, most notably a decrease in glucose and oxygen, is important for cellular survival. In response to oxygen deprivation or hypoxia, one of the key response elements is hypoxia inducible factor (HIF) and a key protein induced by hypoxia is vascular endothelial growth factor (VEGF). Under hypoxia, we and others have reported an increase in the half-life of VEGF and other hypoxia related mRNAs including MYC and CYR61; however, the mediator of this response has yet to be identified. For this study, we sought to determine if HIF-mediated transcriptional activity is involved in the mRNA stabilization induced by hypoxia.MethodsHEK293T or C6 cells were cultured in either normoxic or hypoxic (1% oxygen) conditions in the presence of 1 g/L glucose for all experiments. Pharmacological treatments were used to mimic hypoxia (desferroxamine, dimethyloxaloglutamate, CoCl2), inhibit mitochondrial respiration (rotenone, myxothiazol), scavenge reactive oxygen species (ROS; ebselen), or generate mitochondrial ROS (antimycin A). siRNAs were used to knock down components of the HIF transcriptional apparatus. mRNA half-life was determined via actinomycin D decay and real time PCR and western blotting was used to determine mRNA and protein levels respectively.ResultsTreatment of HEK293T or C6 cells with hypoxic mimetics, desferroxamine, dimethyloxaloglutamate, or CoCl2 showed similar induction of HIF compared to hypoxia treatment, however, in contrast to hypoxia, the mimetics caused no significant increase in VEGF, MYC or CYR61 mRNA half-life. Knockdown of HIF-alpha or ARNT via siRNA also had no effect on hypoxic mRNA stabilization. Interestingly, treatment of HEK293T cells with the mitochondrial inhibitors rotenone and myxothiazol, or the glutathione peroxidase mimetic ebselen did prevent the hypoxic stabilization of VEGF, MYC, and CYR61, suggesting a role for mtROS in the process. Additionally, treatment with antimycin A, which has been shown to generate mtROS, was able to drive the normoxic stabilization of these mRNAs.ConclusionOverall these data suggest that hypoxic mRNA stabilization is independent of HIF transcriptional activity but requires mtROS.

Hypoxia-inducible factor 1-dependent expression of adenosine receptor 2B promotes breast cancer stem cell enrichment

Breast cancer stem cells (BCSCs), which are characterized by a capacity for unlimited self-renewal and for generation of the bulk cancer cell population, play a critical role in cancer relapse and metastasis. Hypoxia is a common feature of the cancer microenvironment that stimulates the specification and maintenance of BCSCs. In this study, we found that hypoxia increased expression of adenosine receptor 2B (A2BR) in human breast cancer cells through the transcriptional activity of hypoxia-inducible factor 1. The binding of adenosine to A2BR promoted BCSC enrichment by activating protein kinase C-δ, which phosphorylated and activated the transcription factor STAT3, leading to increased expression of interleukin 6 and NANOG, two key mediators of the BCSC phenotype. Genetic or pharmacological inhibition of A2BR expression or activity decreased hypoxia- or adenosine-induced BCSC enrichment in vitro, and dramatically impaired tumor initiation and lung metastasis after implantation of MDA-MB-231 human breast cancer cells into the mammary fat pad of immunodeficient mice. These data provide evidence that targeting A2BR might be an effective strategy to eradicate BCSCs.

Abstract B44: Regulation of macrophage activation by vascular niche in glioblastoma

Abstract Spatiotemporal regulation of tumor immunity remains largely unexplored. Here we identify a vascular niche that controls alternative macrophage activation in glioblastoma (GBM). We show that tumor-promoting macrophages are spatially proximate to GBM-associated endothelial cells (ECs), permissive for angiocrine-induced macrophage polarization. We identify ECs as a major source for interleukin (IL)-6 expression in GBM microenvironment. Furthermore, we reveal that colony stimulating factor-1 (CSF-1) and angiocrine IL-6 induce robust arginase-1 expression and macrophage alternative activation, mediated through peroxisome proliferator-activated receptor (PPAR)-γ-dependent transcriptional activation of hypoxia-inducible factor (HIF)-2α. Finally, utilizing a genetic murine GBM model, we show that EC-specific knockout of IL-6 inhibits macrophage alternative activation and improves survival in the GBM-bearing mice. These findings illustrate a vascular niche-dependent mechanism for alternative macrophage activation and cancer progression, and suggest that targeting endothelial IL-6 may offer a selective and efficient therapeutic strategy for GBM, and possibly other solid malignant tumors. Note: This abstract was not presented at the conference. Citation Format: Qirui Wang, Yanqing Anna Gong, Yi Fan. Regulation of macrophage activation by vascular niche in glioblastoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B44.

The role of hypoxic signalling in metastasis: towards translating knowledge of basic biology into novel anti-tumour strategies

Hypoxia is a characteristic feature of many cancer types, which ensues when the growth of a tumour outpaces its oxygen supply. The cellular response to reduced oxygen tension is centred around the hypoxia-inducible transcription factors (HIFs), which become stabilized under hypoxic conditions. In addition, a number of oxygen-independent mechanisms of HIF regulation have been described, which also play a role at distinct stages of tumour progression. Hypoxia and HIF activity have been linked to the control of all hallmarks of cancer, and increased levels of hypoxia or HIFs in human tumours are typically associated with poor prognosis. In this review, we describe the current knowledge about the role of hypoxic signalling in tumour metastasis, which is the main cause of cancer-related mortality. The members of the HIF family, HIF1α, HIF2α and HIF3α, play important functions at all key stages of metastatic dissemination. This includes local migration within the tumour and invasion of the surrounding stromal tissue through induction of an epithelial-mesenchymal transition (EMT)-like process, remodelling of the extracellular matrix, intravasation and extravasation, survival and dissemination through the circulation, generation of premetastatic niches to support secondary tumour growth, colonisation of distant sites, and tumour cell dormancy. The central role of hypoxic signalling in tumour growth and metastasis, as well as its involvement in therapy resistance, have motivated efforts to monitor tumour hypoxia through various invasive and non-invasive techniques, and to identify inhibitors of HIFs, their regulators or their targets. Recent progress in these areas has provided indications that such approaches may represent a viable strategy for translating basic knowledge about tumour hypoxia and HIF biology into novel therapeutic strategies for metastatic cancer.

Abstract 5474: Activity of the S1P pathway promotes ovarian cancer and serves as a novel metabolic target of metformin

Abstract Sphingosine-1-phosphate (S1P), a metabolite of the phospholipid sphingosine, is a bioactive lipid that directly interacts with specific G-protein-coupled receptors to transduce signals that regulate growth, proliferation and motility. Sphingosine kinases (SPHK1 and 2) catalyze the formation of S1P from sphingosine, thereby regulating S1P homeostasis and the sphingolipid rheostat. Recently, using TCGA data, we noted SPHK1 amplification in several gynecologic cancers and that high expression of SPHK1 was associated with increased mortality in high-grade serous ovarian cancer (HGSOC). The findings reported here show that exposure of HGSOC tumor cells to exogenous S1P, or overexpression of SPHK1, induced migration, proliferation and clonogenecity in multiple ovarian cancer cell lines in vitro. Likewise, in a xenograft mouse model of ovarian cancer, overexpression of SPHK1 markedly enhanced tumor growth. In prior pre-clinical studies, we have demonstrated a strong protective effect of the diabetes medication metformin in ovarian cancer. Interestingly, we found that patients with HGSOC that use metformin for diabetes have reduced serum S1P levels compared to controls, suggesting that the sphingolipid rheostat may be a novel metabolic target of metformin in ovarian cancer. Supporting this, we identified that SPHK1 expression in ovarian cancer cell lines is reduced by treatment with metformin, and further that the reduction of SPHK1 by metformin was mediated through inhibition of transcriptional activity of hypoxia-inducible factors (HIF1α and HIF2α). Finally, we show that overexpression of SPHK1 in HGSOC cell lines enhanced the cytotoxic effects of metformin. Taken together, our data indicates that hypoxia-induced SPHK1 expression and downstream S1P signaling promote tumor progression in HGSOC, and that tumors utilizing this pathway may be particularly vulnerable to the anti-cancer effects of metformin. Citation Format: Peter C. Hart, Tatsuyuki Chiyoda, Marion Curtis, Xiaojing Liu, Chun-Yi Chiang, Stephanie McGregor, Ricardo Lastra, Jason Locasale, Ernst Lengyel, Iris L. Romero. Activity of the S1P pathway promotes ovarian cancer and serves as a novel metabolic target of metformin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5474.

Experimental study of expression of hypoxia-inducible factor-1α and its role in traumatic brain injury

Decades of traumatic brain injury (TBI) research have not led to the development of novel drugs that successfully improve the outcome of injured patients. Thus, new research approaches or medicine are required. Vascular injury is the primary key event that can cause a range of secondary injury, and become a key part of TBI repairmen. Reduced blood flow is one pathophysiological symptom of TBI. Regenerative mechanisms are then initiated to reduce cellular damage, including the activation of hypoxia-inducible transcription factor 1 (HIF-1). Successful revascularization can provide critical neurovascular microenvironment, which produces effective perfusion to promote nerve repair and growth by clearing Toxic factors, providing nutrition, and making it a critical treatment for TBI. Ninety-six male SD rats were divided into four groups: sham group ( n = 8), control (sham operate, n = 8), traumatic brain injury group (TBI group, n = 40) and TBI treated with 2ME2 group ( n = 40). Feeney's method was applied to establish TBI model. At the time point 6 h, 24 h, 3d, 7d, 14d after modeling, the expression of PHD2/HIF-1α/VEGF/Ang-1 were observed by RT-PCR\Western blot analysis. Compared with sham-operated controls, the expression of HIF-1α in TBI group have been significantly ( P < 0.05) enhanced, which related to the degradation of PHD2 following TBI induction. HIF-1α expression began to increase after 6 h, within 24 h to 3d reached peak and fall to common levels before 7d. Nevertheless, VEGF/Ang-1 expression was remarkably ( P < 0.01) decreased after inhibition of HIF-1α by 2-ME-2. The expression of HIF-1α increased after experimental TBI, which can induce its downstream target gene expression to promote the repairment of injured vascular and angiogenesis.

Abstract 5427: Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α

Abstract Hypoxia-inducible factor 1 (HIF-1) is a main regulator of cellular response to hypoxia and its transcriptional activity is crucial for cancer cell migration and invasion. Post-translational modifications of the α subunit of HIF-1 (HIF-1α) play dominant roles in regulating its function. In this study, we demonstrated that the lysine methyltransferases G9a and GLP catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. Functionally, K674 methylation suppressed HIF-1 transcriptional activity as well as expression of the HIF-1 target genes including PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Moreover, mutation of K674 of HIF-1α significantly enhanced U251MG cell migration under hypoxia. Importantly, we found that G9a level was downregulated by hypoxia in glioblastoma, which was negatively correlated with PTGS1 expression and survival rate in glioblastoma patients. These findings reveal a novel naturally existing negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma. Citation Format: Lei Bao, Yan Chen, Hsien-Tsung Lai, Cheng-Ming Chiang, Gregg L. Semenza, Yingfei Wang, Weibo Luo. Inhibition of hypoxia-inducible factor 1 (HIF-1) transcriptional activity and cell mobility via G9a/GLP-mediated methylation of HIF-1α [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5427.

HIF involvement in the regulation of rat Sertoli cell proliferation by FSH

The final number of Sertoli cells reached during the proliferative periods determines sperm production capacity in adulthood. It is well known that FSH increases the rate of proliferation of Sertoli cells; however, little is known about the transcription factors that are activated by the hormone in order to regulate Sertoli cell proliferation. On the other hand, Hypoxia Inducible Factors (HIFs) are master regulators of cell growth. HIFs are dimers of HIF-β and HIF-α subunits. Considering that HIF-β is constitutively expressed, HIF transcriptional activity is regulated through the abundance of HIF-α subunits. To date, three HIF-α isoforms have been described. The association of the different HIF-α subunits with HIF-β subunit constitutes three active transcription factors —HIF-1, HIF-2 and HIF-3— which interact with consensus hypoxia-response elements in the promoter region of target genes. Hypoxia has been classically considered the main stimulus that increases HIF transcriptional activity, however, regulation by hormones under normoxic conditions was also demonstrated. The aim of this work has been to investigate whether HIFs participate in the regulation of rat Sertoli cell proliferation by FSH. Sertoli cells obtained from 8-day old rats were cultured in the absence or presence of FSH. It has been observed that FSH increases HIF transcriptional activity and HIF-2α mRNA levels without modifying either HIF-1α or HIF-3α expression. Incubations with FSH have been also performed in the absence or presence of a pharmacological agent that promotes HIF-α subunit degradation, LW6. It has been observed that LW6 inhibits the FSH effect on proliferation, CCND1 expression and c-Myc transcriptional activity. Altogether, these results suggest that HIFs might be involved in the regulation of Sertoli cell proliferation by FSH.

Aryl Hydrocarbon Receptor Nuclear Translocator in Vascular Smooth Muscle Cells Is Required for Optimal Peripheral Perfusion Recovery.

BackgroundLimb ischemia resulting from peripheral vascular disease is a common cause of morbidity. Vessel occlusion limits blood flow, creating a hypoxic environment that damages distal tissue, requiring therapeutic revascularization. Hypoxia‐inducible factors (HIFs) are key transcriptional regulators of hypoxic vascular responses, including angiogenesis and arteriogenesis. Despite vascular smooth muscle cells’ (VSMCs’) importance in vessel integrity, little is known about their functional responses to hypoxia in peripheral vascular disease. This study investigated the role of VSMC HIF in mediating peripheral ischemic responses.Methods and ResultsWe used Arnt SMKO mice with smooth muscle–specific deletion of aryl hydrocarbon receptor nuclear translocator (ARNT, HIF‐1β), required for HIF transcriptional activity, in a femoral artery ligation model of peripheral vascular disease. Arnt SMKO mice exhibit impaired perfusion recovery despite normal collateral vessel dilation and angiogenic capillary responses. Decreased blood flow manifests in extensive tissue damage and hypoxia in ligated limbs of Arnt SMKO mice. Furthermore, loss of aryl hydrocarbon receptor nuclear translocator changes the proliferation, migration, and transcriptional profile of cultured VSMCs. Arnt SMKO mice display disrupted VSMC morphologic features and wrapping around arterioles and increased vascular permeability linked to decreased local blood flow.ConclusionsOur data demonstrate that traditional vascular remodeling responses are insufficient to provide robust peripheral tissue reperfusion in Arnt SMKO mice. In all, this study highlights HIF responses to hypoxia in arteriole VSMCs critical for the phenotypic and functional stability of vessels that aid in the recovery of blood flow in ischemic peripheral tissues.

Methylation of hypoxia-inducible factor (HIF)-1α by G9a/GLP inhibits HIF-1 transcriptional activity and cell migration.

Hypoxia-inducible factor 1 (HIF-1) is a master transcriptional regulator in response to hypoxia and its transcriptional activity is crucial for cancer cell mobility. Here we present evidence for a novel epigenetic mechanism that regulates HIF-1 transcriptional activity and HIF-1-dependent migration of glioblastoma cells. The lysine methyltransferases G9a and GLP directly bound to the α subunit of HIF-1 (HIF-1α) and catalyzed mono- and di-methylation of HIF-1α at lysine (K) 674 in vitro and in vivo. K674 methylation suppressed HIF-1 transcriptional activity and expression of its downstream target genes PTGS1, NDNF, SLC6A3, and Linc01132 in human glioblastoma U251MG cells. Inhibition of HIF-1 by K674 methylation is due to reduced HIF-1α transactivation domain function but not increased HIF-1α protein degradation or impaired binding of HIF-1 to hypoxia response elements. K674 methylation significantly decreased HIF-1-dependent migration of U251MG cells under hypoxia. Importantly, we found that G9a was downregulated by hypoxia in glioblastoma, which was inversely correlated with PTGS1 expression and survival of patients with glioblastoma. Therefore, our findings uncover a hypoxia-induced negative feedback mechanism that maintains high activity of HIF-1 and cell mobility in human glioblastoma.

Notch Signaling Enhances Stemness by Regulating Metabolic Pathways Through Modifying p53, NF-κB, and HIF-1α.

Human adipose-derived mesenchymal stromal cells (hASCs) are attractive for regenerative medicine, but their limited in vitro life span limits their therapeutic applicability. Recent data demonstrate that hypoxia may benefit the ex vivo culture of stem cells. Such cells exhibit a high level of glycolytic metabolism under hypoxic conditions. However, the physiological role of glycolytic activation and its underlying regulatory mechanism are incompletely understood. We have shown that when activated under conditions of 5% O2, Notch signaling dramatically increases the rate of glycolysis, improves proliferation efficiency, prevents senescence, and maintains the multipotency of hASCs. In the present study, we found that activated Notch1 enhanced nuclear p65 levels, resulting in an increase in glucose metabolism through the upregulation of glycolytic factors, including GLUT3. Notch signaling was also involved in glucose metabolism through p53 inactivation. We also found that NF-κB signaling was regulated by p53. These data suggest that Notch-HES1 signaling enhances the glycolytic pathway through p53 and NF-κB. Our data also revealed that activated Notch1 markedly increased the transcriptional activity of hypoxia-inducible factor 1 (HIF-1). Knockdown of HIF-1α significantly attenuated glycolysis induced by activated Notch1, indicating that the glycolysis pathway is regulated by the coordination of Notch signaling and HIF. Overall, our observations provide new regulatory mechanisms for the glycolysis by Notch signaling to maintain the stemness of hASCs.

Hif1a inactivation rescues photoreceptor degeneration induced by a chronic hypoxia-like stress

Reduced choroidal blood flow and tissue changes in the ageing human eye impair oxygen delivery to photoreceptors and the retinal pigment epithelium. As a consequence, mild but chronic hypoxia may develop and disturb cell metabolism, function and ultimately survival, potentially contributing to retinal pathologies such as age-related macular degeneration (AMD). Here, we show that several hypoxia-inducible genes were expressed at higher levels in the aged human retina suggesting increased activity of hypoxia-inducible transcription factors (HIFs) during the physiological ageing process. To model chronically elevated HIF activity and investigate ensuing consequences for photoreceptors, we generated mice lacking von Hippel Lindau (VHL) protein in rods. This activated HIF transcription factors and led to a slowly progressing retinal degeneration in the ageing mouse retina. Importantly, this process depended mainly on HIF1 with only a minor contribution of HIF2. A gene therapy approach using AAV-mediated RNA interference through an anti-Hif1a shRNA significantly mitigated the degeneration suggesting a potential intervention strategy that may be applicable to human patients.

Hypoxia-inducible factor prolyl hydroxylase inhibitor roxadustat (FG-4592) protects against cisplatin-induced acute kidney injury.

Renal hypoxia occurs in acute kidney injury (AKI) of various etiologies. Activation of hypoxia-inducible transcription factor (HIF) has been identified as an important mechanism of cellular adaptation to low oxygen. Preconditional HIF activation protects against AKI, suggesting a new approach in AKI treatment. HIF is degraded under normoxic conditions mediated by oxygen-dependent hydroxylation of specific prolyl residues of the regulative α-subunits by HIF prolyl hydroxylases (PHD). FG-4592 is a novel, orally active, small-molecule HIF PHD inhibitor for the treatment of anemia in patients with chronic kidney disease (CKD). The current study aimed to evaluate the effect of FG-4592 (Roxadustat) on cis-diamminedichloroplatinum (cisplatin)-induced kidney injury. In mice, pretreatment with FG-4592 markedly ameliorated cisplatin-induced kidney injury as shown by the improved renal function (blood urea nitrogen (BUN), serum creatinine (Scr), and cystatin C) and kidney morphology (periodic acid-Schiff (PAS) staining) in line with a robust blockade of renal tubular injury markers of kidney injury molecule 1 (KIM-1) and neutrophil gelatinase-associated lipocalin (NGAL). Meanwhile, the renal apoptosis and inflammation induced by cisplatin were also strikingly attenuated in FG-4592-treated mice. Along with the protective effects shown above, FG-4592 pretreatment strongly enhanced HIF-1α in tubular cells, as well as the expressions of HIF target genes. FG-4592 alone did not affect the renal function and morphology in mice. In vitro, FG-4592 treatment significantly up-regulated HIF-1α and protected the tubular cells against cisplatin-induced apoptosis. In summary, FG-4592 treatment remarkably ameliorated the cisplatin-induced kidney injury possibly through the stabilization of HIF. Thus, besides the role in treating CKD anemia, the clinical use of FG-4592 also could be extended to AKI.

Acquisition of Temporal HIF Transcriptional Activity Using a Secreted Luciferase Assay.

Here we describe a simple method based on secreted luciferase driven by a hypoxia-inducible factor (HIF) response element (HRE) that allows the acquisition of dynamic and high-throughput data on HIF transcriptional activity during hypoxia and pharmacological activation of HIF. The sensitivity of the assay allows for the secreted luciferase to be consecutively sampled (as little as 1% of the total supernatant) over an extended time period, thus allowing the acquisition of time-resolved HIF transcriptional activity.

2017 SfRBM Discovery Award Lecture Maintenance Of Oxygen And Redox Homeostasis By Hypoxia-Inducible Factor 1

In virtually every metazoan species, cells express the transcriptional activator hypoxia-inducible factor 1 (HIF-1) in response to decreased O2 availability. HIF-1 activates the transcription of a large battery of target genes, many of which encode proteins that serve either to increase O2 delivery or to decrease O2 consumption. The latter is likely to represent a primordial function of HIF-1, which is expressed even in animals with bodies that are too simple to require specialized systems for O2 delivery. Studies performed with mouse embryo fibroblasts revealed coordinate, HIF-1-dependent activation of genes that inhibit respiration and stimulate glycolysis. This metabolic reprogramming is required to maintain redox homeostasis: HIF-1-deficient fibroblasts exposed to chronic hypoxia do not switch from respiration to glycolysis and die due to ROS toxicity. More recently, analysis of human breast cancer cells has revealed that hypoxia induces coordinate, HIF-1-dependent transcriptional activation of genes encoding enzymes of the serine synthesis pathway and one-carbon metabolism that increase mitochondrial NADPH production, which is required to detoxify ROS produced under hypoxic conditions. Failure to do so leads to increased cell death and impairs the enrichment of breast cancer stem cells that otherwise occurs in response to hypoxia. Thus, HIF-1 controls both the production and detoxification of ROS under conditions of chronic hypoxia. Different cell types use different strategies to modulate metabolism in order to maintain redox homeostasis under hypoxic conditions. These studies have led to major paradigm shifts in our understanding of oxygen homeostasis: (i) ROS levels do not increase monotonically with increasing pO2; rather, mitochondrial function is optimized for physiological pO2 and any increase or decrease in O2 availability leads to increased ROS levels; and (ii) the switch from oxidative to glycolytic metabolism does not occur in order to maintain energy homeostasis, but rather to maintain redox homeostasis under hypoxic conditions.

Epigenetic regulators: multifunctional proteins modulating hypoxia-inducible factor-α protein stability and activity.

The hypoxia-inducible factor (HIF) is a heterodimeric transcription factor governing a transcriptional program in response to reduced O2 availability in metazoans. It contributes to physiology and pathogenesis of many human diseases through its downstream target genes. Emerging studies have shown that the transcriptional activity of HIF is highly regulated at multiple levels and the epigenetic regulators are essential for HIF-mediated transactivation. In this review, we will discuss the comprehensive regulation of HIF transcriptional activity by different types of epigenetic regulators.

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.