SET1B Drives Sustained HIF activity and Disease Progression in Clear Cell Renal Cell Carcinoma.

Hypoxia-inducible factors (HIF) are heterodimeric (alpha/beta) transcription factors that play a fundamental role in cellular adaptation to low oxygen tension. In the presence of oxygen, the HIF-alpha subunit becomes hydroxylated at specific prolyl residues by prolyl hydroxylases. This post-translational modification is recognized by the von Hippel-Lindau (VHL) protein, which targets HIF-alpha for degradation. In the absence of oxygen, HIF-alpha hydroxylation is compromised and this subunit is stabilized. We have previously shown that the hypoxia-induced accumulation of HIF-alpha protein is strongly impaired by the inhibitor of diacylglycerol kinase, R59949. Here, we have investigated the mechanisms through which this inhibitor exerts its effect. We found that R59949 inhibits the accumulation of HIF-1/2alpha protein without affecting the expression of their mRNAs. We also determined that R59949 could only block the accumulation of HIF-alpha in the presence of VHL protein. In agreement with this, the binding of VHL to endogenous HIF-alpha was significantly enhanced after R59949 treatment, even under hypoxic conditions. In addition, we found that R59949 could stimulate prolyl hydroxylase both at 21% O2 as well as at 1% O2. Taken together, these results reveal that R59949 is an activator of HIF prolyl hydroxylases. This is of particular interest when we consider that, to date, mainly inhibitors of these enzymes have been described.

REDD1 (regulated in development and DNA damage responses) is essential for the inhibition of mTORC1 (mammalian target of rapamycin complex) signaling pathway in response to hypoxia. REDD1 expression is regulated by many stresses such as hypoxia, oxidative stress, and energy depletion. However, the regulation of REDD1 expression in response to insulin remains unknown. In the present study, we demonstrate that in murine and in human adipocytes, insulin stimulates REDD1 expression. Insulin-induced REDD1 expression occurs through phosphoinositide 3-kinase/mTOR-dependent pathways. Moreover, using echinomycin, a hypoxia-inducible factor 1 (HIF-1) inhibitor, and HIF-1alpha small interfering RNA, we demonstrate that insulin stimulates REDD1 expression only through the transcription factor HIF-1. In conclusion, our study shows that insulin stimulates REDD1 expression in adipocytes.

2] Detection of oxygen-sensing properties of mitochondria

BackgroundOsteosarcoma is the most common malignancy of bone. HIF-1 (hypoxia-inducible factor 1) activation is critical for the metabolic reprogramming and progression of solid tumors, and DEC2 (differentiated embryonic chondrocyte gene 2) has been recently reported to suppress HIF-1 in human breast and endometrial cancers. However, the roles of HIF-1 and DEC2 in human osteosarcomas remain unclear.MethodsWe evaluated the correlation of DEC2 and HIF-1 expression to the prognosis, and studied the roles of DEC2 and HIF-1 activation in the invasiveness of osteosarcoma. Multiple approaches including immunohistochemical staining of clinical osteosarcoma tissues, siRNA-based knockdown and other molecular biology techniques were used. Particularly, by using a repetitive trans-well culture-based in vitro evolution system, we selected a more invasive subpopulation (U2OS-M) of osteosarcoma cells from U2OS and used it as a model to study the roles of DEC2 and HIF-1 in the invasiveness of osteosarcoma.ResultsWe found that the expression of DEC2 was positively correlated with HIF-1α levels, and HIF-1α expression positively correlated with poor prognosis in osteosarcomas. DEC2 knockdown in osteosarcoma cell lines (U2OS, MNNG and 143B) attenuated HIF-1α accumulation and impaired the up-regulation of HIF-1 target genes in response to hypoxia. Compared with the low invasive parental U2OS, U2OS-M showed higher levels of DEC2 expression which were confirmed at both mRNA and protein levels. Importantly, we found that the increased DEC2 expression resulted in a more rapid accumulation of HIF-1α in U2OS-M cells in response to hypoxia. Finally, we found that HIF-1 activation is sufficient to upregulate DEC2 expression in osteosarcoma cells.ConclusionTaken together, whereas DEC2 was found to promote HIF-1α degradation in other types of tumors, our data indicate that DEC2 facilitates HIF-1α stabilization and promotes HIF-1 activation in osteosarcoma. This implies that DEC2 may contribute to the progression and metastasis of human osteosarcoma by sensitizing tumor cells to hypoxia. On the other hand, HIF-1 activation may contribute to the expression of DEC2 in osteosarcoma. This is the first demonstration of a novel DEC2-HIF-1 vicious cycle in osteosarcoma and a tumor-type specific role for DEC2.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-015-0135-8) contains supplementary material, which is available to authorized users.

Physiologically, angiogenesis is tightly regulated, or otherwise it leads to pathological processes, such as tumors, inflammatory diseases, gynecological diseases and diabetic retinopathy. The vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis. The VEGF gene expression is regulated by a variety of stimuli. Hypoxia is one of the most potent inducers of the VEGF expression. The hypoxia inducible factor 1 (HIF-1) plays as a key transcription factor in hypoxia-mediated VEGF gene upregulation. Nitric oxide (NO) as well as hypoxia is reported to upregulate the VEGF gene by enhancing HIF-1 activity. The Akt/protein kinase B (PKB) pathway may be involved in NO-mediated HIF-1 activation in limited cell lines. There are some reports of negative effects of NO on HIF-1 and VEGF activity. These conflicting data of NO effects may be attributed mainly to the amount of released NO. Indeed, NO can be a positive or negative modulator of the VEGF gene under the same conditions simply by changing its amounts. The VEGF-mediated angiogenesis requires NO production from activated endothelial NO synthase (eNOS). Activation of eNOS by VEGF involves several pathways including Akt/PKB, Ca(2+)/calmodulin, and protein kinase C. The NO-mediated VEGF expression can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity, and the VEGF-mediated NO production by eNOS can be also modulated by HIF-1 and HO-1 activity, depending upon the amount of produced NO. These reciprocal relations between NO and VEGF may contribute to regulated angiogenesis in normal tissues.

Physiologically, angiogenesis is tightly regulated, or otherwise it leads to pathological processes, such as tumors, inflammatory diseases, gynecological diseases and diabetic retinopathy. The vascular endothelial growth factor (VEGF) is a potent and critical inducer of angiogenesis. The VEGF gene expression is regulated by a variety of stimuli. Hypoxia is one of the most potent inducers of the VEGF expression. The hypoxia inducible factor 1 (HIF-1) plays as a key transcription factor in hypoxia-mediated VEGF gene upregulation. Nitric oxide (NO) as well as hypoxia is reported to upregulate the VEGF gene by enhancing HIF-1 activity. The Akt/protein kinase B (PKB) pathway may be involved in NO-mediated HIF-1 activation in limited cell lines. There are some reports of negative effects of NO on HIF-1 and VEGF activity. These conflicting data of NO effects may be attributed mainly to the amount of released NO. Indeed, NO can be a positive or negative modulator of the VEGF gene under the same conditions simply by changing its amounts. The VEGF-mediated angiogenesis requires NO production from activated endothelial NO synthase (eNOS). Activation of eNOS by VEGF involves several pathways including Akt/PKB, Ca(2+)/calmodulin, and protein kinase C. The NO-mediated VEGF expression can be regulated by HIF-1 and heme oxygenase 1 (HO-1) activity, and the VEGF-mediated NO production by eNOS can be also modulated by HIF-1 and HO-1 activity, depending upon the amount of produced NO. These reciprocal relations between NO and VEGF may contribute to regulated angiogenesis in normal tissues.

Hypoxia-inducible factor-1 (HIF-1) plays a central role in tumor progression by regulating genes involved in proliferation, glycolysis, angiogenesis, and metastasis. To improve our understanding of HIF-1 regulation by kinome, we screened a kinase-specific small interference RNA library using a hypoxia-response element (HRE) luciferase reporter assay under hypoxic conditions. This screen determined that depletion of cellular SMG-1 kinase most significantly modified cellular HIF-1 activity in hypoxia. SMG-1 is the newest and least studied member of the phosphoinositide 3-kinase-related kinase family, which consists of ATM, ATR, DNA-PKcs, mTOR, and SMG-1. We individually depleted members of the phosphoinositide 3-kinase-related kinase family, and only SMG-1 deficiency significantly augmented HIF-1 activity in hypoxia. We subsequently discovered that SMG-1 kinase activity was activated by hypoxia, and depletion of SMG-1 up-regulated MAPK activity under low oxygen. Suppressing cellular MAPK by silencing ERK1/2 or by treatment with U0126, a MAPK inhibitor, partially blocked the escalation of HIF-1 activity resulting from SMG-1 deficiency in hypoxic cells. Increased expression of SMG-1 but not kinase-dead SMG-1 effectively inhibited the activity of HIF-1α. In addition, cellular SMG-1 deficiency increased secretion of the HIF-1α-regulated angiogenic factor, vascular epidermal growth factor, and survival factor, carbonic anhydrase IX (CA9), as well as promoted the hypoxic cell motility. Taken together, we discovered that SMG-1 negatively regulated HIF-1α activity in hypoxia, in part through blocking MAPK activation.

The transcription factor hypoxia inducible factor-1 (HIF-1) is a master regulator of oxygen homeostasis in cells. HIF-1 consists of a hypoxia-dependent alpha (α) subunit and an oxygen-independent beta (β) subunit. HIF-1 activates transcription of over 100 genes involved in a wide range of cellular and molecular responses to ischemia and hypoxia including angiogenesis, glucose transport, glycolysis, antiapopotic and proapoptotic pathways, and cellular proliferation. In most tissues, HIF-1α is ubiquitinated and rapidly degraded under normoxic conditions, but stabilized from degradation and activated by hypoxia. Previously we have demonstrated that HIF-1 is abundant in Leydig cells and that, unlike HIF-1 in other tissues, testicular HIF-1 exists in a non-ubiquitinated form that is constitutively expressed in both the normoxic and hypoxic rat testis. We hypothesize that HIF-1 plays key roles in protecting Leydig cells from apoptosis and that HIF-1 is important for regulating oxygen microenvironments in the testis. Studies in our laboratory have focused on the role of HIF-1 in the normoxic testis and in conditions of testis ischemia such as testicular torsion. Prolyl hydroxylases 1 and 2 (PHD1 and PHD2) and Factor Inhibiting HIF (FIH1) are oxygen-dependent enzymes that hydroxylate the HIF-1α subunit under normoxic conditions. Hydroxylated HIF-1α binds to the von Hippel-Lindau (pVHL) tumor suppressor protein, a ubiquitin ligase that targets HIF-1α for ubiquitination and subsequent degradation by the proteasome. The purpose of this study was to examine expression of PHDs, FIH1, and pVHL in the rat testis as an initial step towards understanding the regulatory mechanisms responsible for constitutive expression of testicular HIF-1. Experimental unilateral testicular ischemia (I) was created by 720° of testicular torsion for 1h followed by 4h or 24h of reperfusion (I/R). Cytoplasmic and nuclear proteins were isolated from sham-operated and ischemic testes and steady-state levels of PHD1, PHD2, FIH-1, and pVHL proteins detected by immunoblot analysis. PHD1 and 2 were constitutively expressed in normoxic and ischemic testes (P<0.05), and PHD2 was the predominant isoform detected. FIH1 and pVHL were also abundant in the testis and the levels of these proteins showed no significant changes following I or I/R (P<0.05). In conclusion, constitutive expression of HIF-1 and HIF-1 regulatory proteins in the normoxic and ischemic testis suggest that stabilization and activation of HIF-1 in the testis may occur through pathways that are fundamentally different from HIF-1 activation mechanisms in other tissues. Further studies on the functions of testicular HIF-1 and HIF-1 regulatory pathways will advance an understanding of the role of Leydig cells in oxygen homeostasis of the testis and cellular and molecular responses to testis ischemia and hypoxia. Supported by NIH grant R15HD046451 to M.A.P.

Selective virtual screening of hypoxia inducible factor agonists and antagonists using artificial intelligence and linear response theory.

Hepatocyte growth factor signalling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells.

The hypoxia inducible factor (HIF) plays an important role in the progression of a number of pathophysiological processes including tumorigenesis. In addition to several well characterized oxygen-dependent modes of regulation, the function of the HIF transcription factor can also be influenced through the action of other regulatory pathways. Misregulation of these factors resulting in inappropriate HIF expression or activity can contribute to the progression of human cancers through the induction of genes promoting angiogenesis, glycolysis, cell survival, and metastasis, among other processes. The candidate tumor suppressor protein inhibitor of growth family member 4 (ING4) has recently been implicated as a repressor of angiogenesis and tumor growth through association with NF-kappaB. Here we demonstrate that suppression of ING4 further induces HIF transcriptional activity as well. ING4 directly associates with the HIF prolyl hydroxylase, an Fe(II)-dependent oxygenase previously shown to mediate HIF stability as a function of oxygen availability. However, rather than affecting HIF's stability, ING4 mediates HIF's activity. These data support a model in which, in addition to regulating HIF stability, HIF prolyl hydroxylases can modulate HIF function through the recruitment of ING4, a likely component of a chromatin-remodeling complex.

Hypoxia-inducible factor-1 (HIF-1) is a transcriptional activator involved in adaptation to hypoxic stress. Previous studies from our laboratory demonstrated that pharmacological activators of HIF-1 (e.g. deferoxamine, cobalt chloride) could also protect cultured primary neurons or an immortalized hippocampal neuroblast line (HT22) from oxidative stress-induced death. However, whether HIF-1 activation is sufficient to abrogate neuronal death resulting from oxidative stress or other hypoxia-independent death inducers remains unclear. To address this question we utilized a HIF-1alpha fusion protein that partially lacks the domain required for oxygen-dependent degradation of HIF-1alpha and that has a VP16 transcriptional activation domain from herpes simplex virus. HT22 cells were infected with a retrovirus encoding either the HIF-1alpha-VP16 fusion protein or the activation domain of the VP16 protein alone as a control. Expression of HIF-1alpha-VP16, but not VP16 alone, increased luciferase activity driven by a canonical hypoxia response element, increased mRNA of established HIF-1 target genes, and increased activity of one of these HIF-1 target genes. Unexpectedly, enhanced HIF-1 activity in HT22 cells enhanced sensitivity to oxidative death induced by glutathione depletion. Accordingly, suppression of HIF-1alpha expression using RNA interference prevented oxidative death. By contrast, HIF-1alpha-VP16-expressing HT22 cells were more resistant to DNA damage (induced by camptothecin) or endoplasmic reticulum stress (induced by thapsigargin and tunicamycin) than were VP16-expressing cells, and suppression of HIF-1alpha expression using RNA interference rendered HT22 cells more sensitive to death induced by DNA damage or endoplasmic reticulum stress. Together, these data demonstrate that HIF-1 can mediate prodeath or prosurvival responses in the same cell type depending on the injury stimulus.

Hypoxia is a common feature of solid tumors, and the extent of tumor hypoxia correlates with advanced disease stages and treatment resistance. The transcription factor hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective molecular target for anticancer drug discovery directed at tumor hypoxia. A natural product chemistry-based approach was employed to discover small molecule inhibitors of HIF-1. Bioassay-guided isolation of an active lipid extract of the tropical legumaceous plant Lonchocarpus glabrescens and structure elucidation afforded two new HIF-1 inhibitors: alpinumisoflavone (compound 1) and 4'-O-methylalpinumisoflavone (compound 2). In human breast tumor T47D cells, compounds 1 and 2 inhibited hypoxia-induced HIF-1 activation with IC(50) values of 5 and 0.6 mum, respectively. At the concentrations that in hibited HIF-1 activation, compound 2 inhibited hypoxic induction of HIF-1 target genes (CDKN1A, GLUT-1, and VEGF), tumor angiogenesis in vitro, cell migration, and chemotaxis. Compound 2 inhibits HIF-1 activation by blocking the induction of nuclear HIF-1alpha protein, the oxygen-regulated subunit that controls HIF-1 activity. Mechanistic studies indicate that, unlike rotenone and other mitochondrial inhibitors, compound 2 represents the first small molecule that inhibits HIF-1 activation by simultaneously suppressing mitochondrial respiration and disrupting protein translation in vitro. This unique mechanism distinguishes compound 2 from other small molecule HIF-1 inhibitors that are simple mitochondrial inhibitors or flavanoid-based protein kinase inhibitors.

Bone marrow-derived cells are recruited to sites of ischemia, where they promote tissue vascularization. This response is dependent upon the expression of vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1), which mediates cell migration in response to VEGF or placental growth factor (PLGF). In this study, we found that exposure of cultured mouse bone marrow-derived mesenchymal stromal cells (MSC) to hypoxia or an adenovirus encoding a constitutively active form of hypoxia-inducible factor 1 (HIF-1) induced VEGFR1 mRNA and protein expression and promoted ex vivo migration in response to VEGF or PLGF. MSC in which HIF-1 activity was inhibited by a dominant negative or RNA interference approach expressed markedly reduced levels of VEGFR1 and failed to migrate or activate AKT in response to VEGF or PLGF. Thus, loss-of-function and gain-of-function approaches demonstrated that HIF-1 activity is necessary and sufficient for basal and hypoxia-induced VEGFR1 expression in bone marrow-derived MSC.

Hypoxia-inducible factor-1 (HIF-1) is an essential transcription factor involved in the oxygen-dependent regulation of gene expression. Thiol groups in HIF-1 or in proteins that modify HIF-1 are conventional targets for regulation by nitric oxide (NO). Moreover, NO delivery to tissue by hemoglobin appears to be oxygen dependent. Therefore, the role NO plays in regulating HIF-1 activity and expression was examined. The 1-substituted diazen-1-ium-1, 2-diolate NOC-18 induced HIF-1 DNA-binding activity in normoxic bovine pulmonary artery endothelial cells and rat aortic smooth muscle cells in a time- and dose-dependent manner. Induction of HIF-1-binding activity was consistent with an increased expression of HIF-1 subunit proteins HIF-1alpha and HIF-1beta. The effect of NOC-18 on HIF-1 activity was blocked by cycloheximide, consistent with a post-transcriptional effect. NOC-18 induction of HIF-1 DNA-binding activity was not blocked with oxyhemoglobin, nor was it related to the rate of NO evolution, arguing against NO-mediation of the effect. Additionally, the effect of NOC-18 could not be mimicked by Angeli's salt, arguing against nitroxyl mediation. However, the NOC-18 effect could be reproduced by S-nitrosoglutathione (GSNO), an endogenous nitrosonium donor formed in the presence of deoxyhemoglobin. Furthermore, the GSNO effect could be reversed by dithiothreitol as well as acivicin, an inhibitor of GSNO bioactivation. Taken together, these results suggest that an S-nitrosylation reaction stabilizes HIF-1 protein expression and activity. We speculate that one signaling mechanism by which deoxyhemoglobin may activate HIF-1 involves NO.