Regulation Of HIF-1alpha Research Articles

Key LncRNAs Associated with Distant Metastasis in Breast Cancer: A System Biology Analysis.

Breast cancer (BC) is the most prevalent cancer among women globally. Metastasis is the leading cause of mortality in most cancers. Early BC detection before metastasis can enhance survival rates. Understanding BC metastasis mechanisms could aid in developing metastasis-specific treatments. The role of long non-coding RNAs (lncRNA) in cancer progression is recognized, yet the importance of specific lncRNAs in BC, despite potential alterations, remains inadequately explored. We utilized bioinformatics tools to identify novel lncRNAs dysregulated in metastasis. To achieve this objective, the gene expression profile of GSE102484, encompassing metastatic and non-metastatic BC tissue samples, was analyzed using the limma package in R with cut-off criteria set at an adjusted p-value < 0.005 and |fold change (FC)| ≥ 0.5. We used WGCNA analysis to find co-expression genes for lncRNAs. Then, we identified hub genes and performed pathway enrichment to better understand the results. Considering the defined criteria, eight novels of dysregulated lncRNAs and top 10 miRNAs were identified. Dysregulated lncRNAs are found in yellow, green, brown, purple, and turquoise co-expression modules from WGCNA analysis. Enrichment analysis of these co-expressed modules revealed relevant pathways to metastasis, such as epithelial-to-mesenchymal transition and integrin cell-surface interactions, as well as regulation of HIF1-alpha. In addition, SDPR, TGFB1I1, ILF3, KIF4A, and COL5A1 were identified as hub genes. Based on DElncRNA-miRNADEmRNA connections and co-expression, we ultimately constructed lncRNA-associated ceRNA axes. The current study may identify novel lncRNAs implicated in BC metastasis; still, additional research is required to determine the potential functions of these lncRNAs in BC metastasis.

Feedback of hypoxia-inducible factor-1alpha (HIF-1alpha) transcriptional activity via redox factor-1 (Ref-1) induction by reactive oxygen species (ROS)

Hypoxia-inducible factor-1alpha (HIF-1alpha) is important for adaptation to hypoxia. Hypoxia is a common feature of cancer and inflammation, by which HIF-1alpha increases. However, prolonged hypoxia decreases HIF-1alpha, and the underlying mechanisms currently remain unclear. Cellular reactive oxygen species (ROS) increases in cancer and inflammation. In the present study, we demonstrated that prolonged hypoxia increased ROS, which induced prolyl hydroxylase domain-containing protein 2 (PHD2) and factor inhibiting HIF-1 (FIH-1), major regulators of HIF-1alpha. Cellular stress response (CSR) increased HIF-1alpha transcriptional activity by scavenging endogenous ROS. PHD2 and FIH-1 were induced by external hydrogen peroxide (H2O2) but were suppressed by ROS-scavenging catalase. We investigated the mechanisms by which PHD2 and FIH-1 are regulated by ROS. The knockdown of HIF-1alpha decreased PHD2 and FIH-1 mRNA levels, suggesting their regulation by HIF-1alpha. We then focused on redox factor-1 (Ref-1), which is a regulator of HIF-1alpha transcriptional activity. The knockdown of Ref-1 decreased PHD2 and FIH-1. Ref-1 was regulated by ROS. Prolonged hypoxia and the addition of H2O2 induced the expression of Ref-1. Furthermore, the knockdown of p65, a component of kappa-light-chain enhancer of activated B cells (NF-κB), efficiently inhibited the induction of Ref-1 by ROS. Collectively, the present results showed that prolonged hypoxia or increased ROS levels induced Ref-1, leading to the activation of HIF-1alpha transcriptional activity, while the activation of HIF-1alpha via Ref-1 induced PHD2 and FIH-1, causing the feedback of HIF-1alpha. To the best of our knowledge, this is the first study to demonstrate the regulation of HIF-1alpha via Ref-1 by ROS.

Endogenous reactive oxygen species and nitric oxide have opposite roles in regulating HIF-1alpha expression in hypoxic astrocytes.

Ischemic stroke results in cerebral tissue hypoxia and increased expression of hypoxia-inducible factor (HIF), which is critically implicated in ischemic brain injury. Understanding the mechanisms of HIF-1alpha regulation in the ischemic brain has been an important research focus. The generation of both nitric oxide (NO) and reactive oxygen species (ROS) is increased under hypoxic/ischemic conditions and each of them has been independently shown to regulate HIF-1alpha expression. In this study, we investigated the cross-effects of NO and ROS on the expression of HIF-1alpha in hypoxic astrocytes. Exposure of astrocytes to 2 h-hypoxia remarkably increased HIF-1alpha protein levels, which was accompanied by increased NO and ROS production. Decreasing ROS with NAC, NADPH oxidase inhibitor DPI, or SOD mimetic MnTMPyP decreased hypoxia-induced HIF-1alpha protein accumulation and increased NO level in hypoxic astrocytes. The NO synthase (NOS) inhibitor L-NAME inhibited ROS generation, which led to a reduction in hypoxia-induced HIF-1alpha protein expression. Although NOS inhibitor or ROS scavengers alone reduced HIF-1alpha protein levels, the reduction was reversed when NOS inhibitor and ROS scavenger present together. The NO scavenger PTIO increased hypoxia-induced HIF-1alpha protein expression and ROS production, while HIF-1alpha protein level was decreased in the presence of NO scavenger and ROS scavenger together. These results suggest that ROS, NO, and their interaction critically contribute to the regulation of hypoxia-induced HIF-1alpha protein accumulation under hypoxic condition. Furthermore, our results indicate that hypoxia-induced NO generation may represent an endogenous mechanism for balancing ROS-mediated hypoxic stress, as reflected by inhibiting hypoxia-induced HIF-1alpha protein accumulation.

Regulation of HIF1 alpha in hyperoxia and hyperglycaemia

Hypoxia inducible factor 1 alpha (HIF‐1α) is the master regulator of cell response to hypoxia. Increasing evidence demonstrates the existence of non‐hypoxic mediated HIF‐1α induction/activation1,2. Reactive oxygen species (ROS) production and increased levels of interleukin (IL)‐6 and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) are linked with hypoxia‐induced expression of HIF1a. Hyperoxia is known to stimulate ROS production and the concomitant effect of high glucose concentrations causes increases of ROS with known involvement of the nuclear factor (erythroid derived 2)‐like 2 (Nrf‐2), the principal regulator of Redox status in cells2. There is clearly a merit in understanding the effect of hyperoxia on HIF1α protein synthesis and stabilisation as it has been reported to result in both an upregulation and a downregulation of HIF1α from different groups2, 3.Here, primary human dermal microvascular endothelial cells (HDMECs) were maintained in growth media containing either 5.5 or 20mmol/L glucose and incubated at either 18–20% or 95% oxygen. RNA was extracted using Trizol and cDNAs prepared using a kit (Bioline UK), sub‐cellular fractions were prepared using lysis buffer and differential centrifugation and followed by Western blotting (WB) or the qRT‐PCR. Cells were fixed in 100% ice cold methanol and stained according to standard immunocytochemistry (ICC) protocol. HIF1α and Nrf2 expression were examined with ICC using HIF1a (1:20) (Thermo Fischer Scientific) and Nrf2 (1:20) (Abcam) and visualized with immunofluorescence (DayLight 488)(Thermo Fischer Scientific). WB was used to confirm the presence of the respective proteins; HIF1α (1:100) (Thermo Fischer Scientific), Nrf2 (1:100)(Santa Cruz), NFκB (1:100)(Santa Cruz) and gene expression was examined by qRT‐PCR to detect and quantify HIF1α using β2‐microglobulin as the internal control.Results showed an absence of HIF1a protein in the nuclear fraction and no change in the level of cytosolic HIF1α protein in basal or treated conditions. However, an increase in HIF1α mRNA level in hyperoxia/hyperglycemia relative to control was evident We also found an acute increase IL‐6 mRNA level and NFκB protein in normoglycemia/hyperoxia, which was further enhanced in hyperglycemia/hyperoxia with an increased expression of Nrf2 protein.This study demonstrates a likely transcriptional link between HIF1α expression and hyperoxia in HDMECs. The possibility of the existence of a transcriptional effect was further strengthened when we examined the gene and protein expression levels of IL‐6 and NFκB respectively as both IL‐6 and NFκB are transcriptional activators of HIF1α mRNA. The hyperoxia‐mediated expression of HIF1α protein via accumulation of ROS2 is supported with the corresponding increased level of nrf‐2. The relevance of this data points to the role of hyperglycemia/hyperoxia, highlighting associated cell response to redox imbalance and inflammation and the convergence on HIF1α regulation at a transcriptional level. HIF1α signalling is involved in several functions and the understanding of its regulation is warranted.Support or Funding InformationThe first author, Benedicta U. Iwuagwu received an academic scholarship for her PhD (3 years) and is at the last stage of PhD (4th year) finalising data collection and writing which is nonfunded.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 5026: Association to drug-induced leukopenia using whole-exome sequencing of non-small cell lung cancer patients on gemcitabine/carboplatin regimen

Abstract A classical non-small cell lung cancer (NSCLC) chemotherapy treatment is gemcitabine in combination with carboplatin. The treatment is known to cause severe hematological toxicity such as leukopenia, which can lead to chemotherapy cessation or even death. It would therefore a priori be of advantage to identify patients at risk of severe leukopenia to allow for a personalized treatment approach. In this study we aim to identify genetic markers for chemotherapy induced leukopenia in non-small cell lung cancer. In total, 212 non-small cell lung cancer patients treated with gemcitabine and carboplatin regimen were included in the study. Whole blood extracted DNA was prepared with Nextera Rapid Capture kit and whole exome sequenced using Illumina HiSeq 2500. Leukopenia was assessed from leukocyte particle count at baseline and the first cycle nadir values. The statistical approach was to study association of single common variants (MAF &amp;gt; 0.01) to leukopenia using linear regression in PLINK and association of genes (common and rare variants) to leukopenia using SKATO in the R-package SKAT. An enrichment analysis, with input from the association results (p &amp;lt; 0.001), was performed using the online tool ConsensusPathDB-human to identify overrepresented pathways. A prediction model was created from the single variant analysis results (n=10 causal and n=10 protective) using weighted genetic risk score from the R-package PredictABEL. The single variant analysis of common variants identified 133 variants (p &amp;lt; 0.001) and the gene based analysis identified 54 genes (p &amp;lt; 0.001). The pathway analysis identified 20 enriched pathways (p &amp;lt; 0.05). A prediction model was created to assess the risk of leukopenia. The top pathway, HIF-1-alpha transcription factor network, overlaps with four genes (HDAC7, NDRG1, HK2 and CP). It can be of interest to leukopenia as regulation of HIF-1 alpha is essential for maintenance of hematopoietic stem cells in the bone marrow hypoxic niche. It has also been shown that HIF-1 alpha knocked mice are more sensitive to myelosuppressive treatment compared to unknocked mice, supporting the involvement of the pathway in chemotherapy induced myelosuppression. We have identified variants located in genes and pathways likely to be involved in leukocyte sensitivity after exposure of gemcitabine and carboplatin. A prediction model has been created to estimate the risk that patients will suffer from severe leukopenia. The biological connection of the pathway HIF-1-alpha transcription factor network to leukopenia supports the relevance of these results. Further studies on this are of interest to improve identification of patients at high risk of severe leukopenia after gemcitabine and carboplatin treatment. Citation Format: Anna Svedberg, Benjamín Sigurgeirsson, Niclas Björn, Sailendra Pradhananga, Eva Brandén, Hirsh Koyi, Rolf Lewensohn, Luigi De Petris, Cristina Rodríguez-Antona, María Apellániz-Ruiz, Joakim Lundeberg, Henrik Gréen. Association to drug-induced leukopenia using whole-exome sequencing of non-small cell lung cancer patients on gemcitabine/carboplatin regimen [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 5026. doi:10.1158/1538-7445.AM2017-5026

Quantification of VEGF and Screening of Transcription Factor HIF-1α in Synovial Fluid of Polyarthritic Patients: Targets for Potential Anti-angiogenic Molecules

Rheumatoid arthritis (RA) is a polyarticular inflammatory autoimmune disease and osteoarthritis (OA) is an inflammatory degenerative disease of the articular cartilage. The current study efforts have been made to understand the relationship between the angiogenic genes VEGF, its regulator HIF-1α and MVD in RA and OA. The level of VEGF in serum and synovial fluid was increased in both RA and OA, but the levels were slightly more in cases of RA compared to that of OA. A corresponding increase in expression of HIF-1α and MVD counts was also noted in both conditions. Similar to VEGF levels, the expression of HIF-1α , MVD and CD-31 counts was higher among RA patients. These results thus indicate that there is a significant association between the angiogenic peptide VEGF in serum and synovial fluids, expression levels of transcription factor HIF-1α and microvessel density in the synovium of polyarthritic patients. The increased expression and regulation of VEGF, HIF-1alpha and MVD in these chronic inflammatory conditions are not only early indicators but also potential targets for antiangiogenic factors in the therapeutics of novel drugs for these smoldering conditions.

RM-01 * RADIATION INDUCED PRMT5 EXPRESSION IS REGULATED BY miRNA-96 IN GLIOBLASTOMA

BACKGROUND: Glioblastoma (GB) represent the most common and aggressive histological subtype among malignant gliomas and are associated with poor outcomes. It is composed of multiple types of tumor cells displaying multiple genetic and epigenetic defects affecting tumor suppressor gene expression, regulation of growth and apoptosis. Recurrence is the major concern with the current standard therapy. Protein arginine methyltransferase enzyme 5 (PRMT5), that regulates many cellular processes through its methylation-dependent and independent activity is overexpressed in this disease. We hypothesize that PRMT5 contributes to radioresistance in GB. Using patient derived GB neurosphere models we are probing the role of PRMT5 in inducing radioresistance in GB. METHODS: GB neurospheres dispersed into single cell, were treated with radiation or transfected with specified siRNAs. At appropriate time, cells were collected and probed for mRNA expression by qPCR and protein level by immunblotting. RESULTS: We found that PRMT5 associates with Hsp90 in GB neurospheres and that this binding is essential for PRMT5 stability. In addition to higher levels of baseline expression, radiation treatment increases PRMT5 mRNA level by 2 fold and protein expression by 60%. This increase is independent of its post-translational stability conferred by its association with Hsp90. However radiation treatment decreases miRNA-96 level significantly (p ≤ 0.01). Overxpression of miRNA-96 down regulates the expression of PRMT5 protein. Additionally we found that PRMT5 is important for regulation of HIF1alpha, a transcription factor induced post radiation and known to be involved in radiation resistance. We are currently investigating the implications of PRMT5 inhibition in conjunction with radiation in vivo. CONCLUSIONS: Based on our findings we conclude that radiation treatment induced suppression of miRNA-96 causes increased expression of PRMT5. This suggests that increased expression of PRMT5 may contribute for HIF-mediated radioresistance in GB.

Regulation of HIF-1 alpha by the proprotein convertases furin and PC7 in human squamous carcinoma cells

Proprotein convertases (PC), a family of serine proteases, process cancer-related substrates such as growth factors, growth factor receptors, cell adhesion molecules, metalloproteinases, etc. HIF-1α is a major transcription factor involved in tumorigenesis by sensing intratumoral hypoxia. Furin (PCSK3) is one of the numerous target genes regulated by HIF-1α transactivation and its distribution into endosomal compartments and onto the cell surface can be triggered by hypoxia via HIF-1α. siRNAs to knockdown PCs were transfected into cells alone or in combination with different drug treatments. Protein and RNA expression levels were analyzed by Western blotting or RT-PCR, respectively. PC7 (PCSK7) and furin siRNAs upregulated HIF-1α protein under normoxic condition to a level similar to that obtained by cobalt chloride treatment, eventually leading to activation of VEGF-A synthesis in two human head and neck squamous cell carcinoma cell lines. The unchanged levels of HIF-1α mRNA expression under siRNA treatment and the additive HIF-1α induction of PC siRNAs and either cobalt chloride or the 26S ribosome inhibitor, MG-132, suggested a post-transcriptional PC-mediated regulation. Furthermore, cycloheximide chase showed that PC7/furin siRNA regulation occurred at the level of HIF-1α translation. A specific IGF-1R signaling inhibitor was able to attenuate the PC siRNA induction of HIF-1α, suggesting the involvement of the IGF-1R pathway. Thus, the data show that PCs regulate HIF-1α. Furin and PC7 siRNAs induced HIF-1α protein by increasing its translation, resulting in upregulation of VEGF-A. This finding may provide insight into intricate PC functions that seem to be independent from their substrate-processing activity.

Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

BackgroundOur previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α.Methodology/Principal FindingsIn primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule–depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells.Conclusions/SignificanceThis study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes.

Early atherosclerotic plaques show evidence of infection by Chlamydia pneumoniae

Chlamydia pneumoniae (Cpn) could play an important role in the development of atherosclerosis. Cpn interferes with HIF-1alpha regulation in infected host cells during intracellular replication in hypoxia. We obtained carotid artery specimens with low (n=38), high (n=25) levels of stenosis and 10 middle cerebral arteries. Fifty eight percent of the carotids with low levels of stenosis showed evidence of the viable organism. Ninety one percent of the positive results were derived from pre-atheromatous lesions. Only 12 percent of plaques removed at endarterectomy showed the presence of Cpn DNA. All middle cerebral arteries failed to show evidence of live Chlamydia. Ninety one percent of sera from 22 endarterectomy patients failed to show the presence of Cpn antibodies. Immunohistology of carotid arteries with low levels of stenosis was used to confirm the presence of HIF-1alpha in infected specimens and showed a correlation between the over-expression of HIF-1alpha and Cpn in the plaque (p less than 0.05). Cpn might play an important role in activation and development of the initial stages of atherosclerotic lesions.

Abstract 2047: Betulinic acid exerts anti-angiogenic activity via regulation of HIF-1 alpha and STAT3 in PC-3 prostate cancer cells under hypoxia

Abstract Signal transducer and activator of transcription 3 (STAT3) is a transcription factor constitutively overexpressed in several human cancer cells and related to various cellular processes such as cell survival, angiogenesis and proliferation. In the present study, we examined whether betulinic acid (BA), a triterpene from the bark of white birch, could inhibit hypoxia-mediated activation of STAT3 and HIF-1 alpha in androgen independent human prostate cancer PC-3 cells. Our results show that BA inhibited the transcriptional activities and nuclear accumulation of both HIF-1 alpha and STAT3 under hypoxia. In addition, BA significantly reduced cellular and secreted levels of hypoxia-induced vascular endothelial growth factor (VEGF), a critical angiogenic factor, in PC-3 cells. BA prevented in vitro capillary tube formation in human umbilical vein endothelial cells (HUVECs) maintained in conditioned medium of hypoxia-induced PC-3 cells. Moreover, silencing HIF 1 alpha and STAT3 by siRNA transfection augmented the suppression of VEGF expression by BA under hypoxia. Mechanistically, BA inhibited the recruitment of HIF-1 alpha and STAT3 on the VEGF promoter. Taken together, these findings suggest that BA may exert angio-prevention via inactivation of hypoxia-induced STAT3 and HIF-1 alpha in PC-3 cells. The in vivo anti-angiogenic and anti-PCa efficacy are being evaluated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2047. doi:10.1158/1538-7445.AM2011-2047

Abstract 2082: ERK activation mediated by Duox2 expression and reactive oxygen production is responsible for HIF-1α accumulation and VEGF induction in IFN-γ-treated BxPC-3 pancreatic cancer cells

Abstract Reactive oxygen species (ROS) play an important role in tumor angiogenesis by up-regulating pro-angiogenic effectors such as HIF-1alpha and vascular endothelial growth factor (VEGF). However, the sources of ROS and the pro-angiogenic signaling pathways they target in tumor cells remain incompletely understood. We recently found that several members of the NADPH Oxidase (Nox) gene family are expressed in many human tumors; of these, Duox (Dual oxidase) 2 is highly expressed in tumors of the gastrointestinal tract. Recently, we demonstrated that IFN-gamma treatment of Bx-PC-3 cells upregulates the expression of Duox2 (and its maturation factor DuoxA2; but no other Nox isoform) leading to long-lived production of H2O2. Using this system, we investigated the role of Duox2-mediated ROS in the regulation of HIF-1alpha and VEGF expression. In BxPC-3 cells, IFN-gamma induction of Duox2/A2 expression was accompanied by both intra- and extracellular ROS accumulation, and by the simultaneous upregulation of HIF-1alpha and VEGF. However, in the MIA-PaCa-2 and PANC-1 lines that do not upregulate Duox2 following IFN-gamma exposure, no HIF-1alpha or VEGF induction was observed. In BxPC-3 cells, both the Nox inhibitor diphenylene iodonium and the ROS scavenger N-acetyl-L-cysteine suppressed IFN-gamma-induced HIF-1alpha accumulation and VEGF upregulation, suggesting that Duox2-derived ROS are involved in VEGF induction. Further experiments revealed that IFN-gamma induced a sustained activation of ERK1/2, which subsequently activated p90 ribosomal S6 kinases (RSKs) and the regulator of protein translation, ribosomal protein S6, resulting in enhanced synthesis of HIF-1alpha. ERK activation was required for IFN-gamma-induced HIF-1alpha and VEGF expression, since treatment with the MEK inhibitor U0126 completely abrogated IFN-gamma-induced HIF-1alpha and VEGF expression. Duox2-specific siRNA experiments showed that IFN-gamma-induced ROS, ERK activation, and HIF-1alpha accumulation were significantly attenuated following silencing of Duox2 expression. However, IFN-gamma-induced expression of VEGF was unchanged following Duox2 knockdown. Ongoing studies are evaluating the role of residual Duox2 or Duox2-independent pathways for VEGF activation following IFN-gamma exposure as well as other downstream, pro-angiogenic targets of Duox2-mediated HIF-1alpha expression under normoxic conditions. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2082. doi:10.1158/1538-7445.AM2011-2082

Molecular Evolution of the Metazoan PHD–HIF Oxygen-Sensing System

Metazoans rely on aerobic energy production, which requires an adequate oxygen supply. During reduced oxygen supply (hypoxia), the most profound changes in gene expression are mediated by transcription factors known as hypoxia-inducible factors (HIFs). HIF alpha proteins are commonly posttranslationally regulated by prolyl-4-hydroxylase (PHD) enzymes, which are direct "sensors" of cellular oxygen levels. We examined the molecular evolution of the metazoan PHD-HIF oxygen-sensing system by constructing complete phylogenies for PHD and HIF alpha genes and used computational tools to characterize the molecular changes underlying the functional divergence of PHD and HIF alpha duplicates. The presence of PHDs in metazoan genomes predates the emergence of HIF alphas. Our analysis revealed an unexpected diversity of PHD genes and HIF alpha sequence characteristics in invertebrates, suggesting that the simple oxygen-sensing systems of Caenorhabditis and Drosophila may not be typical of other invertebrate bilaterians. We studied the early vertebrate evolution of the system by sequencing these genes in early-diverging cartilaginous fishes, elasmobranchs. Cartilaginous fishes appear to have three paralogs of both PHD and HIF alpha. The novel sequences were used as outgroups for a detailed molecular analysis of PHD and HIF alpha duplicates in a major air-breathing vertebrate lineage, the mammals, and a major water-breathing vertebrate lineage, the teleosts. In PHDs, functionally divergent amino acid sites were detected near the HIF alpha-binding channel and beta2beta3 loop that defines its substrate specificity. In HIF alphas, more functional divergence was found in teleosts than in mammals, especially in the HIF-1 alpha PAS domain and HIF-2 alpha oxygen-dependent degradation (ODD) domains, which interact with PHDs. Overall, in the vertebrates, elevated substitution rates in the HIF-2 alpha N-terminal ODD domain, together with a functional divergence associated with the known differences in PHD2 versus PHD1/3 substrate specificity, have contributed to the tighter oxygen-sensitive regulation of HIF-1 alpha than that of HIF-2 alpha.

Placenta Growth Factor-induced Early Growth Response 1 (Egr-1) Regulates Hypoxia-inducible Factor-1α (HIF-1α) in Endothelial Cells

Leukotrienes, the lipid inflammatory products derived from arachidonic acid, are involved in the pathogenesis of respiratory and cardiovascular diseases and reactive airway disease in sickle cell disease. Placenta growth factor (PlGF), elaborated from erythroid cells, increased the mRNA expression of 5-lipoxygenase and 5-lipoxygenase-activating protein (FLAP) in human pulmonary microvascular endothelial cells. PlGF-induced both promoter activity and mRNA expression of hypoxia-inducible factor-1alpha (HIF-1alpha), which was abrogated by early growth response-1 (EGR-1) small interfering RNA. PlGF showed a temporal reciprocal relationship in the mRNA levels of EGR-1 and NAB2, the latter a repressor of Egr-1. Moreover, Nab2, but not mutant Nab2, significantly reduced promoter activity and mRNA expression of HIF-1alpha and also reduced expression of the HIF-1alpha target gene FLAP. Furthermore, overexpression of Egr-1 led to increased promoter activities for both HIF-1alpha and FLAP in the absence of PlGF. Additionally, the Egr-1-mediated induction of HIF-1alpha and FLAP promoters was reduced to basal levels by EGR-1 small interfering RNA. The binding of Egr-1 to HIF-1alpha promoter was corroborated by electrophoretic mobility shift assay and chromatin immunoprecipitation assay, which showed increased Egr-1 binding to the HIF-1alpha promoter in response to PlGF stimulation. These studies provide a novel mechanism for PlGF-mediated regulation of HIF-1alpha via Egr-1, which results in increased FLAP expression. This study provides a new therapeutic target, namely Egr-1, for attenuation of elevated leukotriene levels in patients with sickle cell disease and other inflammatory diseases.

Extended Ischemia Prevents HIF1α Degradation at Reoxygenation by Impairing Prolyl-hydroxylation: ROLE OF KREBS CYCLE METABOLITES

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor that activates the cellular response to hypoxia. The HIF1alpha subunit is constantly synthesized and degraded under normoxia, but degradation is rapidly inhibited when oxygen levels drop. Oxygen-dependent hydroxylation by prolyl-4-hydroxylases (PHD) mediates HIF1alpha proteasome degradation. Brain ischemia limits the availability not only of oxygen but also of glucose. We hypothesized that this circumstance could have a modulating effect on HIF. We assessed the separate involvement of oxygen and glucose in HIF1alpha regulation in differentiated neuroblastoma cells subjected to ischemia. We report higher transcriptional activity and HIF1alpha expression under oxygen deprivation in the presence of glucose (OD), than in its absence (oxygen and glucose deprivation, OGD). Unexpectedly, HIF1alpha was not degraded at reoxygenation after an episode of OGD. This was not due to impairment of proteasome function, but was associated with lower HIF1alpha hydroxylation. Krebs cycle metabolites fumarate and succinate are known inhibitors of PHD, while alpha-ketoglutarate is a co-substrate of the reaction. Lack of HIF1alpha degradation in the presence of oxygen was accompanied by a very low alpha-ketoglutarate/fumarate ratio. Furthermore, treatment with a fumarate analogue prevented HIF1alpha degradation under normoxia. In all, our data suggest that postischemic metabolic alterations in Krebs cycle metabolites impair HIF1alpha degradation in the presence of oxygen by decreasing its hydroxylation, and highlight the involvement of metabolic pathways in HIF1alpha regulation besides the well known effects of oxygen.

Expression and regulation of HIF-1alpha in macrophages under inflammatory conditions; significant reduction of VEGF by CaMKII inhibitor

BackgroundMacrophages expressing the pro-angiogenic transcription factor hypoxia-inducible factor (HIF)-1alpha have been demonstrated in rheumatoid arthritis (RA) in the synovial tissue. Aim of the present study was to investigate intracellular signal transduction regulation of pro-inflammatory HIF-1 alpha expression in macrophages to identify possible new intervention strategies. We investigated the effects of CaMKII-inhibitors amongst other kinase inhibitors, on HIF-1 alpha expression and downstream production of pro-angiogenic factors in macrophages.MethodsDifferentiated THP-1 cells and synovial fluid (SF) macrophages were stimulated with 1 μg/ml LPS with or without pretreatment with specific inhibitors of the ERK pathway (PD98059), the PI3K pathway (LY294002), and the CaMKII pathway (KN93 and SMP-114). mRNA and protein expression of HIF-1 alpha, VEGF, MMP-9, and IL-8 was measured in cell lysates and cell supernatants.ResultsHIF-1 alpha protein expression in LPS-stimulated THP-1 macrophages could be blocked by ERK- and PI3K-inhibitors, but also by the CaMKII inhibitor KN93. THP-1 and SF macrophages produced high levels of VEGF, IL-8, and MMP-9, and VEGF protein production was significantly inhibited by PI3K-inhibitor, and by both CaMKII inhibitors. LPS stimulation in an hypoxic environment did not change VEGF levels, suggesting that LPS induced VEGF production in macrophages is more important than the hypoxic induction.ConclusionsExpression of HIF-1 alpha and downstream effects in macrophages are regulated by ERK-, PI3K, but also by CaMKII pathways. Inhibition of HIF-1α protein expression and significant inhibition of VEGF production in macrophages was found using CaMKII inhibitors. This is an unknown but very interesting effect of the CaMKII inhibitor SMP-114, which has been in clinical trial as DMARD for the treatment of RA. This effect may contribute to the anti-arthritic effects of SMP-114.

PI4KIIα is a novel regulator of tumor growth by its action on angiogenesis and HIF-1α regulation

Tumor growth is the orchestration of various oncogenes and tumor suppressors, and the regulation of these genes offers a rational therapeutic approach to cancer treatment. In this study, we found a new regulator of tumor growth, phosphatidylinositol 4-kinase type IIalpha (PI4KIIalpha), the mechanism of which is involved in angiogenesis and hypoxia-inducible factor HIF-1alpha regulation. Results obtained from a human cancer tissue microarray showed that PI4KIIalpha protein expression increases markedly in seven types of cancers compared with normal tissues. Suppression of PI4KIIalpha leads to retarded tumor growth in nude mice. Downregulation of PI4KIIalpha in cancer cells eliminates tumor cell-induced endothelial cell tubulogenesis and migration, and results in impaired angiogenesis. Further investigation showed that PI4KIIalpha can directly regulate HIF-1alpha expression and that the expression of these two proteins is correlated in vivo. At the same time, downregulation of PI4KIIalpha markedly reduces HER-2 autophosphorylation, and PI4KIIalpha specifically triggers HIF-1alpha accumulation through a phosphatidylinositol 3-kinase (PI3K)- and extracellular signal-regulated protein kinase (ERK)-dependent pathway, suggesting that PI4KIIalpha may regulate HIF-1alpha through the HER-2/PI3K, ERK cascade. In summary, we discovered a pivotal role for PI4KIIalpha in the regulation of tumor growth. Our results shed new light on understanding the novel functions of PI4KIIalpha in cancer and suggest that PI4KIIalpha may be a promising specific target for tumor therapy.

Regulation of HIF-1α and VEGF by miR-20b Tunes Tumor Cells to Adapt to the Alteration of Oxygen Concentration

The regulation of HIF-1α is considered to be realized by pVHL-mediated ubiquitin-26S proteasome pathway at a post-transcriptional level. The discovery of a class of small noncoding RNAs, called microRNAs, implies alternative mechanism of regulation of HIF-1α. Here, we show that miR-20b plays an important role in fine-tuning the adaptation of tumor cells to oxygen concentration. The inhibition of miR-20b increased the protein levels of HIF-1α and VEGF in normoxic tumor cells; the increase of miR-20b in hypoxic tumor cells, nevertheless, decreased the protein levels of HIF-1α and VEGF. By using luciferase reporter vector system, we confirmed that miR-20b directly targeted the 3′UTR of Hif1a and Vegfa. On the other hand, the forced overexpression of HIF-1α in normoxic tumor cells downregulated miR-20b expression. However, HIF-1α knockdown in hypoxic tumor cells caused the increase of miR-20b. The differential expression of miR-20b has important biological significance in tumor cells, either enhancing the growth or favoring the survival of tumor cells upon the oxygen supply. Thus, we identify a novel molecular regulation mechanism through which miR-20b regulates HIF-1α and VEGF and is regulated by HIF-1α so to keep tumor cells adapting to different oxygen concentrations.

COMMD1 Promotes pVHL and O2-Independent Proteolysis of HIF-1α via HSP90/70

BackgroundThe Copper Metabolism MURR1 Domain containing 1 protein COMMD1 has been associated with copper homeostasis, NF-κB signaling, and sodium transport. Recently, we identified COMMD1 as a novel protein in HIF-1 signaling. Mouse embryos deficient for Commd1 have increased expression of hypoxia/HIF-regulated genes i.e. VEGF, PGK and Bnip3. Hypoxia-inducible factors (HIFs) are master regulators of oxygen homeostasis, which control angiogenesis, erythropoiesis, glycolysis and cell survival/proliferation under normal and pathologic conditions. Although HIF activity is mainly controlled by ubiquitination and protein degradation by the von Hippel Lindau (pVHL) tumor suppressor gene other mechanisms have recently been identified that regulate HIF signaling independently of pVHL.Principal FindingsHere we characterized the mechanism by which COMMD1 regulates HIF-1α protein degradation. We show that COMMD1 competes with the chaperone heat shock protein HSP90β for binding to the NH2-terminal DNA-binding and heterodimerization domain of HIF-1α to regulate HIF-1α stability together with HSP70. Inhibition of HSP90 activity with 17-Allylamino-17-demethoxygeldanamycin (17-AAG) increased COMMD1-mediated HIF-1α degradation independent of ubiquitin and pVHL.Conclusion/SignificanceThese data reveal a novel role for COMMD1 in conjunction with HSP90β/HSP70 in the ubiquitin and O2-independent regulation of HIF-1α.

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.