Transcription Factor HIF-1alpha Research Articles

Transcriptome analysis of FOXO-dependent hypoxia gene expression identifies Hipk as a regulator of low oxygen tolerance in Drosophila.

When exposed to low oxygen or hypoxia, animals must alter their metabolism and physiology to ensure proper cell-, tissue-, and whole-body level adaptations to their hypoxic environment. These alterations often involve changes in gene expression. While extensive work has emphasized the importance of the HIF-1 alpha transcription factor on controlling hypoxia gene expression, less is known about other transcriptional mechanisms. We previously identified the transcription factor FOXO as a regulator of hypoxia tolerance in Drosophila larvae and adults. Here, we use an RNA-sequencing approach to identify FOXO-dependent changes in gene expression that are associated with these tolerance effects. We found that hypoxia altered the expression of over 2,000 genes and that ∼40% of these gene expression changes required FOXO. We discovered that hypoxia exposure led to a FOXO-dependent increase in genes involved in cell signaling, such as kinases, GTPase regulators, and regulators of the Hippo/Yorkie pathway. Among these, we identified homeodomain-interacting protein kinase as being required for hypoxia survival. We also found that hypoxia suppresses the expression of genes involved in ribosome synthesis and egg production, and we showed that hypoxia suppresses tRNA synthesis and mRNA translation and reduces female fecundity. Among the downregulated genes, we discovered that FOXO was required for the suppression of many ribosomal protein genes and genes involved in oxidative phosphorylation, pointing to a role for FOXO in limiting energetically costly processes such as protein synthesis and mitochondrial activity upon hypoxic stress. This work uncovers a widespread role for FOXO in mediating hypoxia changes in gene expression.

What (H)IF isoform matters? Adeubiquitinase can tune the hypoxic response.

Context-specific control mechanisms of hypoxia-inducible transcription factors HIF-1alpha and HIF-2alpha in tumors exposed to oxygen shortage remain incompletely understood. In this issue, Zhang et al (2022) identify a deubiquitinase that differentially stabilizes HIF-2alpha in stem-like glioblastoma cells, suggesting potential implications for regulation of the hypoxic response in a wide array of tissues and cancers.

The Vhl E3 ubiquitin ligase complex regulates melanisation via sima, cnc and the copper import protein Ctr1A

VHL encodes a tumour suppressor, which possesses E3 ubiquitin ligase activity in complex with EloC and Cul2. In tumour cells or in response to hypoxia, VHL activity is lost, causing accumulation of the transcription factor HIF-1alpha. In this study, we demonstrated that in Drosophila, Rpn9, a regulatory component of the 26 s proteasome, participates in the Vhl-induced proteasomal degradation of sima, the Drosophila orthologue of HIF-1alpha. Knockdown of Vhl induces increased melanisation in the adult fly thorax and concurrent decrease in pigmentation in the abdomen. Both these defects are rescued by knockdown of sima and partially by knockdown of cnc, which encodes the fly orthologue of the transcription factor Nrf2, the master regulator of oxidative stress response. We further show that sima overexpression and Rpn9 knockdown both result in post-translational down-regulation of the copper uptake transporter Ctr1A in the fly eye and that Ctr1A expression exacerbates Vhl knockdown defects in the thorax and rescues these defects in the abdomen. We conclude that Vhl negatively regulates both sima and cnc and that in the absence of Vhl, these transcription factors interact to regulate Ctr1A, copper uptake and consequently melanin formation. We propose a model whereby the co-regulatory relationship between sima and cnc flips between thorax and abdomen: in the thorax, sima is favoured leading to upregulation of Ctr1A; in the abdomen, cnc dominates, resulting in the post-translational downregulation of Ctr1A.

Abstract 5494: Hypoxia via HIF1alpha can regulate Wnt signaling in human colon cancer cells

Abstract Standard-of-care treatment for metastatic colorectal cancer combines chemotherapy with bevacizumab, an angiogenesis inhibitor that depletes nutrients and triggers hypoxia. Unfortunately, this strategy extends patient survival only a few months because tumors acquire adaptive resistance, enabling resumption of angiogenesis. Adaptive resistance arises from reprogrammed metabolism, but the regulatory networks that govern reprogramming are not defined. Our studies have shown that overactive Wnt signaling, which is the most common cause of colon cancer, programs cancer metabolism by promoting glycolysis and angiogenesis. Interestingly, metabolic genes targeted by Wnt are also regulated by the hypoxia transcription factor HIF1alpha suggesting there may be crosstalk between the two pathways. Several studies suggest that HIF1alpha can influence Wnt signaling, but the reported effects are variable ranging from positive to negative. We find that HIF1alpha and LEF/TCF/beta-catenin complexes co-regulate metabolic targets including LEF1 and TCF1 (TCF7) expression. Using luciferase reporters driven by LEF/TCF promoters, we observe significantly increased LEF1/TCF1 but not TCF3(TCF7L1)/TCF4(TCF7L2) promoter activity in hypoxic colon cancer cells. We identified putative hypoxia responsive elements (HREs) in the human LEF1 promoter and deletion of these HREs reduced responsiveness to hypoxia. Furthermore, when colon cancer cells were treated with inhibitors that either inactivate or reduce HIF1alpha protein, there was a significant decrease in Wnt reporter activity. These results suggest that hypoxia and the Wnt pathway crosstalk wherein hypoxia co-regulates metabolism genes and increases Wnt signaling capacity via LEF/TCF expression. Current studies are underway to investigate the mechanisms of crosstalk and joint regulation of metabolic gene programs during adaptive resistance. Citation Format: Yung Lyou, Amber Habowski, Stephanie Sprowl-Tanio, Kira Pate, George Chen, Marian L. Waterman. Hypoxia via HIF1alpha can regulate Wnt signaling in human colon cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5494. doi:10.1158/1538-7445.AM2017-5494

Abstract A23: AMPK's role in glioblastoma survival: An insight into its regulation on transcription factors and mTOR

Abstract AMP kinase (AMPK) is an evolutionarily conserved enzyme required for adaptive responses to various physiological and pathological conditions. The multisubunit structure and sensitivity to various stresses confer complexity to AMPK function which scores beyond energy sensing. Here we show that inhibition of AMPK reduces viability of patient-derived primary Glioblastoma Multiforme (GBM) lines and tumors. We demonstrate that primary GBM cells coopt the AMPK-CREB1 pathway to coordinate global bioenergetics program through control of the transcription factors HIF1alpha and GABPA. But a growing number of AMPK activators are being developed to treat human diseases such as cancer and diabetes. This may be because AMPK activity can inhibit biosynthetic kinases like mammalian target of rapamycin (mTOR) and acetyl Co-A carboxylase (ACC). We found that AMPK silencing in glioma cells did not affect mTOR or downstream targets. AMPK is not absolutely necessary for mTOR inhibition, suggesting that in certain contexts, activated AMPK and mTOR may coexist. Indeed, this is what we observed in glioblastoma suggesting that basally active AMPK may not be sufficient to put a brake on mTOR. Further analysis of our preliminary data from primary tumor cells on Cap-dependent and well reported procancer IRES dependent translation under tumor microenvironment context will demonstrate significance of AMPK in translational machinery in GBM and will benefit in finding new therapeutic targets. Citation Format: Rishi Raj Chhipa, Biplab Dasgupta. AMPK's role in glioblastoma survival: An insight into its regulation on transcription factors and mTOR. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr A23.

Poor prognosis in human papillomavirus-positive oropharyngeal squamous cell carcinomas that overexpress hypoxia inducible factor-1α.

Hypoxia induces stabilization of the transcription factor HIF-1alpha (HIF-1α), associated with (chemo-)radiotherapy resistance in oropharyngeal squamous cell carcinoma (SCC). We investigated the effect of HIF-1α expression on survival in relation to human papillomavirus (HPV) status in oropharyngeal SCC. We conducted an immunohistochemical analysis of HIF-1α protein expression and downstream targets carbonic anhydrase-IX (CA-IX) and glucose transporter-1 (GLUT-1) in 274 patients with oropharyngeal SCC. Overall survival (OS) was analyzed in total and stratified for HPV status and treatment. In HPV-positive tumors (n = 44), HIF-1α overexpression predicted worse OS (hazard ratio [HR] = 6.23; p = .012), whereas TNM classification or treatment modality did not. In HPV-negative tumors (n = 218), advanced T and N classification and HIF-1α overexpression all independently predicted worse OS. However, the effect of HIF-1α overexpression on OS was lower in HPV-negative (HR = 1.50; p = .024) than in HPV-positive tumors. HIF-1α overexpression is associated with worse OS and characterized a subgroup of patients with HPV-positive oropharyngeal SCC with poor prognosis. Possibly, patients with HIF-1α overexpressing HPV-positive tumors should not be eligible for treatment dose deescalation. © 2016 Wiley Periodicals, Inc. Head Neck 38: 1338-1346, 2016.

Quantification of the Expression of HIF-1alpha by Real-Time PCR in Rat Hepatocytes Cultures Invaded by <i>Shigella flexneri</i> under Normoxic and Hypoxic Conditions

Aim: Shigella flexneri (S. flexneri) is a gram-negative enterobacterium responsible for severe intestinal end systemic infection in humans. The bacteria can reach the liver due to degeneration of the colonic epithelium. Hypoxia is present in many human diseases and can induce the expression of the transcription factor HIF-1alpha that may have a cell protective role. The influence of hypoxia and HIF-1alpha on bacterial infection, studied in this work, is unclear. Hypoxia inducible factor-1alpha (HIF-1alpha) is a transcription factor that acts as a master regulator of gene expression induced by hypoxia. Methods: We compared the ability of S. flexneri to invade rat hepatocytes in primary culture both in normoxic and hypoxic conditions. We evaluated TNF-alpha released by hepatocytes, apoptosis rate and HIF-1alpha expression by confocal microscopy as well as real time PCR technique. Results: We showed that S. flexneri invaded less hepatocytes previously submitted to 24 h hypoxia (6.5% O2) than those cultivated in normoxia (21% O2). S. flexneri also induced HIF-1α expression in hepatocytes, TNF-α secretion and apoptosis. Conclusion: a) Hypoxia alone was not a stimulus to TNF-α secretion, but induced cell apoptosis and HIF-1α expression; b) S. flexneri was able to invade rat hepatocytes and hypoxia apparently influenced significantly bacterial cell invasiveness; c) HIF-1α was expressed in hypoxic conditions, and it was also stimulated by S. flexneri.

Tumor Suppressor Role of HIF-1alpha in Leukemia-Initiating Cells in Flt3-ITD-Induced Myeloproliferative Neoplasm

Hypoxia-induced signalling is a major regulator in normal and malignant hematopoiesis. The transcription factor HIF-1alpha plays a crucial role in the quiescence and self-renewal of hematopoietic stem cells as well as leukemia-initiating cells (LICs) of acute myeloid leukemia and chronic myeloid leukemia. Better understanding of the requirement of the molecular response to hypoxia in LICs could lead to new therapies targeting this pathway.We have therefore investigated the effect of HIF-1alpha loss on the phenotype and biology of FLT3-ITD induced myeloproliferative neoplasm (MPN).Using a combined transgenic mouse model (Mx1-Cre; Hif-1alphafl/fl; Flt3ITD/+) we showed that deletion of HIF-1alpha leads to a more severe MPN phenotype reflected by higher numbers of white blood cells and myeloid cells in peripheral blood, as well as a more severe splenomegaly. Loss of long-term hematopoietic stem cells (LT-HSCs: Lin- Sca1+ cKit+ CD48- CD150+) and increased number of lineage-restricted progenitors (Lin- Sca1+ cKit+ CD48+ CD150-) were the most pronounced effects on a cellular level upon the loss of HIF-1alpha. The proliferative effect of the HIF-1alpha loss was cell intrinsic and not at the expense of the ability of the LICs to self-renew because the disease was transplantable into secondary recipients recapitulating the same phenotype. Mice transplanted with FLT3-ITD induced MPN lacking HIF-1alpha succumbed to their disease (average survival of 35 weeks after transplant), while animals transplanted with MPN with wild-type HIF-1alpha suffered from MPN but did not die in the observation period of 60 weeks.These findings are in contrary to what has been previously described for the role of HIF-1alpha in leukemia initiating cells and lead us to propose that HIF-1alpha could act as a tumor suppressor gene, inhibiting proliferation in myeloid malignancies. Our results provide evidence that targeting HIF-1alpha can lead to disease progression of MPN while not affecting self-renewal of LICs. DisclosuresNo relevant conflicts of interest to declare.

Hypoxia and the hypoxia-regulated transcription factor HIF-1alpha regulate innate immune responses in respiratory epithelial cells

Introduction: Acute and chronic diseases of the respiratory tract, such as pneumonia, cystic fibrosis, and COPD, are associated with mucosal and systemic hypoxia. Bacterial infections contribute to exacerbations in these diseases. Innate immune functions of respiratory epithelial cells are required to control and prevent colonization of the lung with bacterial pathogens. Our aim was to examine whether hypoxia and HIF-1alpha regulate innate immune mechanisms of respiratory epithelial cells.

Cellular Test Systems for the Search for Transcription Factor Activity Modulators

Test systems for monitoring activities and the search for substances activating or inhibiting transcription factors as biotargets have been designed on the basis of luciferase constructs containing binding sites for transcription factors CREB, NFAT, NF-kB, p53, STAT1, GAS, VDR, HSF1, and HIF1alpha. An assessment of the functional activity of reporter constructs has been carried out using their transient transfection into HEK293 cells followed by treatment with specific inducers. The functional activity of all reporter constructs was observed based on the increased luciferase expression. In order to evaluate the efficiency of the suggested test systems, aspirin was used. Incubation of cells transfected with the above-mentioned constructs treated with aspirin was accompanied by the suppression of NF-kB, HIF1alpha, GAS, VDR, and HSF binding activity. The findings revealed for NF-kB, NFAT, and STAT1 confirm the published data concerning the mechanisms of aspirin action. The detected effects of this drug on the HIF1alpha, GAS, VDR, and CREB activity have been demonstrated for the first time.

Heat shock protein 90 (hsp90) expression and breast cancer.

Hsp90 is an abundant protein in mammalian cells. It forms several discrete complexes, each containing distinct groups of co-chaperones that assist protein folding and refolding during stress, protein transport and degradation. It interacts with a variety of proteins that play key roles in breast neoplasia including estrogen receptors, tumor suppressor p53 protein, angiogenesis transcription factor HIF-1alpha, antiapoptotic kinase Akt, Raf-1 MAP kinase and a variety of receptor tyrosine kinases of the erbB family. Elevated Hsp90 expression has been documented in breast ductal carcinomas contributing to the proliferative activity of breast cancer cells; whilst a significantly decreased Hsp90 expression has been shown in infiltrative lobular carcinomas and lobular neoplasia. Hsp90 overexpression has been proposed as a component of a mechanism through which breast cancer cells become resistant to various stress stimuli. Therefore, pharmacological inhibition of HSPs can provide therapeutic opportunities in the field of cancer treatment. 17-allylamino,17-demethoxygeldanamycin is the first Hsp90 inhibitor that has clinically been investigated in phase II trial, yielding promising results in patients with HER2-overexpressing metastatic breast cancer, whilst other Hsp90 inhibitors (retaspimycin HCL, NVP-AUY922, NVP-BEP800, CNF2024/BIIB021, SNX-5422, STA-9090, etc.) are currently under evaluation.

I14 Metabolic regulation of IL-1beta transcription

In the field of inflammation research, the most important advances in the past 10 years has been in the uncovering of multiple pathways involved in innate immunity. The best characterised involve the Toll-like receptors (TLRs) and NOD-like receptors (NLRs). From work on Nlrp3 there has also been a resurgence of interest in the IL1 system as a key driver of inflammation in diseases such as gout and diabetes (both Type I and Type II). Glucose has also been shown to drive IL-1beta production in beta cells in the pancreas. We have uncovered a process whereby glucose metabolism is required for induction of IL-1beta mRNA. We have evidence that this could involve the transcription factor HIF-1alpha which is stabilised by LPS in a process involving induction of succinate. The role of metabolic processes including glycolysis in the regulation of signalling in inflammation is also likely to be important here. The so-called Warburg effect of aerobic glycolysis is a feature of cells activated during inflammation (including Th17 cells) providing an added level of complexity to signalling pathways activated by TLRs during inflammation.

Transitions towards either slow‐oxidative or fast‐glycolytic phenotype can be induced in the murine WTt myogenic cell line

Contraction and energy metabolism are functions of skeletal muscles co-regulated by still largely unknown signals. To help elucidating these interconnecting pathways, we are developing new cellular models that will allow to control the switch from a neonatal to an adult slow-oxidative or fast-glycolytic phenotype of myofibers, during in vitro differentiation. Thus, our purpose was to direct the differentiation of the newly characterized WTt clone, from a mixed towards either fast or slow phenotype, by modifying amounts of two transcription factors respectively involved in control of glycolytic and oxidative energy metabolism, namely HIF-1alpha and PPARdelta. Our data support the idea that HIF-1alpha protein stabilization would favor expression of fast phenotypic markers, accompanied or not by a decreased expression of slow markers, depending on treatment conditions. Conversely, PPARdelta over-expression appears to enhance the slow-oxidative phenotype of WTt myotubes. Furthermore, we have observed that expression of PGC-1alpha, a coregulator of PPAR, is also modified in this cell line upon conditions that stabilize HIF-1alpha protein. This observation points to the existence of a regulatory link between pathways controlled by the two transcription factors HIF-1alpha and PPARdelta. Therefore, these cells should be useful to analyze the balance between oxidative and glycolytic energy production as a function of phenotypic transitions occurring during myogenic maturation. The newly characterized murine WTt clone will be a good tool to investigate molecular mechanisms implicating HIF-1alpha and PPARdelta in the coordinated metabolic and contractile regulations involved in myogenesis.

Sirtuin 1 Modulates Cellular Responses to Hypoxia by Deacetylating Hypoxia-Inducible Factor 1α

To survive in hypoxic environments, organisms must be able to cope with redox imbalance and oxygen deficiency. The SIRT1 deacetylase and the HIF-1alpha transcription factor act as redox and oxygen sensors, respectively. Here, we found that SIRT1 binds to HIF-1alpha and deacetylates it at Lys674, which is acetylated by PCAF. By doing so, SIRT1 inactivated HIF-1alpha by blocking p300 recruitment and consequently repressed HIF-1 target genes. During hypoxia, SIRT1 was downregulated due to decreased NAD(+) levels, which allowed the acetylation and activation of HIF-1alpha. Conversely, when the redox change was attenuated by blocking glycolysis, SIRT1 was upregulated, leading to the deacetylation and inactivation of HIF-1alpha even in hypoxia. In addition, we confirmed the SIRT1-HIF-1alpha interaction in hypoxic mouse tissues and observed in vivo that SIRT1 has negative effects on tumor growth and angiogenesis. Our results suggest that crosstalk between oxygen- and redox-responsive signal transducers occurs through the SIRT1-HIF-1alpha interaction.

Abstract 1699: The effects of hypoxia and oxidative stress on transcriptional regulation of carcinogen metabolizing enzymes

Abstract Enzymes are essential to every biochemical process; it is through the action of enzymes that the many metabolic pathways of the body can synchronize with one another to coordinate the processes needed to maintain life. Enzymes are also involved in the metabolism of xenobiotics including dietary chemicals, drugs and environmental chemicals. In this capacity they are very involved in chemical carcinogenesis. Cytochrome P450 1A1 (CYP1A1) and NAD(P)H: quinone oxidoreductase (NQO1) are two enzymes that play a vital role in the process of chemical carcinogenesis. The aryl hydrocarbon receptor (AhR), located in the cytoplasm, mediates various biological responses to environmental pollutants via the adaptive pathway. The binding of a xenobiotic to the AhR triggers transcriptional activation of xenobiotic metabolizing enzymes such as CYP1A1 and NQO1. Our current research investigates the contrast between oxidative stress and hypoxia on the transcriptional activation of CYP1A1 and NQO1 enzyme expression. Hypoxia is a frequent characteristic of tumors as a result of low vascualrization. The transcription factor HIF1alpha can serve as a marker of hypoxia, and aids as a key regulator of the hypoxic response. Previous research in our lab has shown that oxidative stress results in an increase in CYP1A1 and NQO1. We hypothesize that under hypoxic conditions the expression of NQO1 and CYP1A1 are altered which could indicate that hypoxia may possibly play a role in carcinogen metabolism. Two human cancerous cell lines, HEPG2 and HT29, were used to test this hypothesis. Both cells lines were treated with 500 µM of the hypoxic mimetic agent cobalt chloride and harvested at one, two and four hour increments. Hypoxic conditions were then confirmed via HIF1 alpha staining in immunocytochemistry. We then observed mRNA, protein and enzyme expression via RTPCR, Western Blots and enzyme assays, respectively. Altered levels of NQO1 and CYP1A1 suggest that these enzymes respond to hypoxia in a similar manner as they do to oxidative stress. This may provide preliminary evidence into one of the potential mechanisms by which hypoxia is chemopreventive. (Support: NIH MBRS/GM028248; RCMI RR033032). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1699.

Role of hypoxia-inducible transcription factor 1α for progression and chemosensitivity of murine hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a hypervascularized tumor entity with association of arterial vessel density with poor prognosis. The hypoxia-inducible transcription factor HIF-1alpha represents a pivotal regulator of angiogenesis and is thought to determine the angiogenic nature of HCC. However, the precise role of HIF-1alpha during the pathogenesis of HCC remains elusive. We established a functional inactivation of HIF-1alpha in vitro and in vivo via RNAi and Cre/loxP-mediated recombination, respectively, to determine HIF-1alpha's role for tumor growth and chemosensitivity in transgenic and orthotopic murine HCC models. HIF-1alpha-deficient HCC cells displayed significantly reduced anchorage-independent growth and enhanced sensitivity toward etoposide, while basic cellular proliferation was unaffected. Analysis of gross tumor growth failed to detect reduced growth of HIF-1alpha-deficient tumors in the orthotopic and the transgenic HCC model, respectively. In line with the in vitro data, treatment of HIF-1alpha-deficient tumors with etoposide resulted in greater antiproliferative efficacy when compared to wild-type mice. Taken together, our study does not support a pivotal role of HIF-1alpha for tumor growth and angiogenesis in two murine HCC models. However, our data point toward a significant function of HIF-1alpha in determining chemosensitivity of HCC and therefore warrant validation of HIF-1alpha-inhibitors as adjuvant therapeutic agents in clinical studies of human HCC.

Astrocyte hypoxic response is essential for pathological but not developmental angiogenesis of the retina

Vascular/parenchymal crosstalk is increasingly recognized as important in the development and maintenance of healthy vascularized tissues. The retina is an excellent model in which to study the role of cell type-specific contributions to the process of blood vessel and neuronal growth. During retinal vascular development, glial cells such as astrocytes provide the template over which endothelial cells migrate to form the retinal vascular network, and hypoxia-regulated vascular endothelial growth factor (VEGF) has been demonstrated to play a critical role in this process as well as pathological neovascularization. To investigate the nature of cell-specific contributions to this process, we deleted VEGF and its upstream regulators, the hypoxia-inducible transcription factors HIF-1 alpha and HIF-2 alpha, and the negative regulator of HIF alpha, von Hippel-Lindau protein (VHL), in astrocytes. We found that loss of hypoxic response and VEGF production in astrocytes does not impair normal development of retinal vasculature, indicating that astrocyte-derived VEGF is not essential for this process. In contrast, using a model of oxygen-induced ischemic retinopathy, we show that astrocyte-derived VEGF is essential for hypoxia-induced neovascularization. Thus, we demonstrate that astrocytes in the retina have highly divergent roles during developmental, physiological angiogenesis, and ischemia-driven, pathological neovascularization.

Altered expression of mRNA for HIF‐1α and its target genes RTP801and VEGF in patients with oral lichen planus

To explore a potential causal contribution of the transcription factor HIF-1alpha and its target gene, RTP801 and VEGF, to the development of oral lichen planus (OLP). Design relevant: Twenty-two adult OLP patients were enrolled in this study. All OLP diagnoses were verified by histopathological characteristics. Normal mucous specimens were collected from 12 controls after various oral surgeries. RNA was isolated from OLP and control specimens. Microarray was performed using BiostarH-40s gene chip. Expression of HIF-1alpha, VEGF and RTP801 was evaluated using quantitative real-time polymerase chain reaction (qPCR). Unpaired t-test and one-way ANOVA was used for statistical analysis. Microarray results showed that RTP801 expression was lower in OLP than in controls (779 vs 3090). qPCR further confirmed that expression of RTP801 was similarly lower in OLP than in controls (0.363 vs 1.473, P < 0.001); expression of VEGF was also lower in OLP (0.448 vs 1.74, P = 0.012). In contrast, expression of HIF-1alpha was higher in OLP than in controls (11.12 vs 1.628, P < 0.001). The oral mucosa of OLP is hypoxic. Genes that are activated by hypoxia, such as RTP801 and VEGF, and their signal cascades may be novel potential therapeutic targets for OLP.

Src induces expression of carbonic anhydrase IX via hypoxia-inducible factor 1.

Carbonic anhydrase IX (CA IX) belongs to the physiologically important enzymes which contribute to tumor physiology. Tumor-associated expression of CA IX is induced mainly due to its strong transcriptional activation via hypoxia-inducible factor 1 (HIF-1). Therefore, CA IX can serve as a surrogate marker of hypoxia and a prognostic indicator. HIF-1 is a master transcription factor that mediates essential homeostatic responses to cellular and systemic hypoxia by activating transcription of multiple genes including those encoding glycolytic enzymes and vascular endothelial growth factor. In addition to hypoxia, HIF-1alpha expression can be up-regulated by growth factors and oncogenic signals (e.g. Src oncogene). Consequently, induction of the HIF-1alpha transcription factor up-regulates target gene expression. The results from the present study suggest that Src oncogene induces CA IX expression under normoxic, as well as hypoxic conditions. Moreover, we demonstrate that Src-mediated induction of CA IX expression is critically dependent on HIF-1alpha activity. Transcriptional activity of the CA9 promoter was significantly increased by expression of v-Src or c-Src. The effect was more prominent in normoxia, most likely because of already high level of HIF-1alpha expression in hypoxia. By co-transfection with dominant-negative HIF-1alpha we confirmed that Src-induced stimulation of CA9 transcription is mediated via HIF-1alpha. Consistent with this, Src-expressing HeLa cells displayed higher levels of HIF-1alpha protein. Finally, these results indicate a novel regulatory pathway responsible for increased CA IX expression in tumor cells and define CA IX as a new down-stream target for Src oncogene.

Hyperglycemia and hypoxia are interrelated in their teratogenic mechanism: studies on cultured rat embryos

Hyperglycemia and hypoxia are well-known teratogens that may affect many animal species, including man. One of the main mechanisms of teratogenic action of both seems to be increased oxidative stress. The purpose of this study was to evaluate the hypothesis that in the developing embryo hyperglycemia also leads to hypoxia, both resulting in oxidative damage. The study was performed on 10.5-day-old rat embryos of the regular Sabra strain cultured for 28 hours in hyperglycemic or hypoxic conditions. Embryonic growth and rate of anomalies was assessed at the end of the culture period. The embryonic oxidative damage was investigated by studying the levels of Malondialdehyde (MDA) to determine the lipid peroxidation. The redox status was studied by measuring the activity of Catalase-like (CAT) and Super Oxide Dismutase (SOD) enzymes and the amount of Low Molecular Weight Antioxidants (LMWA). In addition, we studied by immunohistochemistry in the embryos and yolk sacs the amount of nitrotyrosine as an additional marker for the extent of oxidative stress. The amounts of the redox and hypoxia sensitive transcription factors HIF1alpha, NFkB, and IkB were also studied by immunohistochemistry. Both hyperglycemia and hypoxia increased the rate of congenital anomalies mainly of the heart, neural tube, and brain. Embryonic growth and scoring were decreased only by hypoxia. Both hyperglycemia and hypoxia increased embryonic oxidative stress as evidenced by increased lipid peroxidation, increased nitrotyrosine and LMWA, but only minimal changes in CAT and SOD activity. Severe hyperglycemia also caused hypoxia, as evidenced by increased HIF1alpha. Thus, there seems to be an interrelation between hyperglycemia and hypoxia, both resulting in embryonic damage apparently by enhanced oxidative stress. Both hyperglycemia and hypoxia seem to exert their embryotoxicity through a similar mechanism of increased oxidative stress in the embryo in a stage when its antioxidant capacity is still weak. Moreover, hyperglycemia also seems to induce hypoxia, intensifying diabetes-induced embryopathy.