- Email: [email protected]
The AMP-activated protein kinase modifies gene expression in endothelial cells by interfering with protein lysine acetylation
320
Abstracts
Oxygen serves as primary electron acceptor in many cellular reactions and is central for generation of ATP in the mitochondria. Thus, an adequate supply with oxygen and a functional adaptive system to changes in oxygen homeostasis are essential for aerobic life. Thereby, the levels of molecular oxygen as well as its reactive derivates (reactive oxygen species, ROS), and redox equivalents can serve as signalling molecules to monitor disruption of oxygen homeostasis. Key players in oxygen homeostasis are a family of hypoxia-inducible transcription factors, (HIF) which control cellular adaptive processes in response to hypoxia including cellular metabolism and angiogenesis, but also proliferation or apoptosis. On the other hand, the family of NADPH oxidases has been recognised as important source of ROS in particular in the vascular system. Intriguingly, NADPH oxidases are involved in the control of the HIF pathway in the cardiovascular response to multiple stress factors independently of hypoxia. In fact, we could show a role of HIF transcription factors in flow-induced vascular remodelling as well as in the vascular prothrombotic or proinflammatory response. Their ability to be regulated by ROS and metabolic alterations under these conditions make them true redox-sensitive transcription factors. Intriguingly, some NADPH oxidase family members are even direct HIF target genes under these conditions. In addition, recent evidence suggests that NADPH oxidases are also involved in the cardiovascular adaptation to hypoxia. Dissecting the mechanisms controlling the ROS-HIF axis in cardiovascular physiology and pathophysiology appears to be a promising strategy to understand the importance of redox signalling in cardiovascular function and to delineate novel therapeutic options for a variety of cardiovascular disorders. doi:10.1016/j.vph.2011.08.041
L.6.2 Therapeutic manipulation of vascular signaling and cytoprotection Justin C. Mason Cardiovascular Sciences, Imperial College London, London, UK E-mail address: [email protected] Vascular endothelial injury predisposes to apoptosis, endothelial dysfunction and atherogenesis. This process may be accelerated in systemic inflammatory diseases such as systemic lupus erythematosus, in diabetes mellitus, chronic renal failure and post-transplantation. The endothelium has a variety of innate cytoprotective mechanisms aimed at minimizing injury and facilitating repair. These mechanisms may be regulated by specific signaling pathways, activated by both endogenous and exogenous mediators, and further influenced by patterns of shear stress exerted by the flowing blood. Cytoprotective genes induced by these pathways, including endothelial nitric oxide synthase, heme oxygenase-1, decay-accelerating factor and B cell lymphoma protein-2, play a central role in the maintenance of endothelial homeostasis and in the response to injury. We are currently exploring therapeutic options for their regulation, alongside the investigation of novel signaling mechanisms including those regulated by protein kinase C (PKC), and by specific nuclear receptors and their coregulators. For example, we have shown that PKCε activity in the vascular endothelium co-ordinates a diverse downstream cytoprotective response in EC. Likewise PPARδ, in conjunction with its co-regulator PGC1α, is able to regulate anti-oxidant responses. We propose that a detailed knowledge of cytoprotective mechanisms and their associated signaling pathways may ultimately reveal novel therapeutic targets. These may in turn provide the means by which vascular endothelial dysfunction can be reversed, so preventing or retarding accelerated atherogenesis in a variety of disease states. doi:10.1016/j.vph.2011.08.042
L.6.3 Blood viscosity limits maximal exercise performance in mice Max Gassmann Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zürich, Winterthurerstrasse 260, CH-8057 Zürich, Switzerland E-mail address: [email protected] Hypoxia induces erythropoietin (Epo) gene expression that in turn elevates erythropoiesis and hence the arterial O2 content thereby also improving exercise performance. It is well accepted that the highest attainable hematocrit cannot be associated with the maximal exercise capacity. To test as whether an optimal hematocrit in acute and chronic Epo-treated mice exists, i.e. a given hematocrit allowing the highest O2 transport during maximal exercise, hematocrit levels of wild type (wt) mice were acutely elevated by applying NESP (novel erythropoiesis stimulating protein, wtNESP). Furthermore, in the transgenic mouse line Tg6 that reaches hematocrit levels of up to 0.9 due to the constitutive over-expression of human Epo, the hematocrit was gradually reduced by application of the hemolysisinducing compound phenylhydrazine (PHZ, tg6PHZ). Maximal cardiovascular performance was measured by using telemetry in exercising mice. Highest maximal O2 uptake (˙VO2max) and maximal time to exhaustion at sub-maximal exercise intensities were reached at hematocrit values of 0.58 and 0.57 for wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. While maximal working capacity of the heart increased with elevated hematocrit values, blood viscosity correlated with ˙VO2max. Obviously, blood viscosity increases with rising hematocrit levels limiting the blood's O2 transport capacity. Of note, while performing this set of experiments, we observed that Epo present in the brain of both mouse models augmented exercise performance in a non-erythroid manner, too.
doi:10.1016/j.vph.2011.08.043
O.6.1 The AMP-activated protein kinase modifies gene expression in endothelial cells by interfering with protein lysine acetylation Beate Fisslthalera, Annemarieke Loota, Reinier Boonb, Eduard Hergenreiderb, Ingrid Fleminga a Institute for Vascular Signalling, Goethe University Frankfurt, Germany b Institute for Cardiovascular Regeneration, Goethe University Frankfurt, Germany E-mail addresses: [email protected](B. Fisslthaler), [email protected] (A. Loot), [email protected] (I. Fleming) The AMP-activated protein kinase (AMPK) is known to modulate the expression of mitochondrial and glucose utilising genes but the mechanisms are not well understood. In endothelial cells adenoviral overexpression of the AMPKα2 subunit resulted in the modulation of approximately 700 genes including the endothelial nitric oxide synthase (eNOS) and the transcription factor Krueppel-like factor-2 (KLF2) which is crucial for the shear stress dependent increase of eNOS expression. In murine aorta as well as in cultured cells deletion or inhibition of the expression of AMPKα2 resulted in a significantly increased eNOS protein expression. The NO-dependent vasodilation in response to bradykinin was increased in the perfused hindlimb from AMPKα2−/− animals compared to wild type littermates. As acetylation of histones is crucial for the cell type specific eNOS expression we analysed the effect of AMPK on the histone acetylase P300. In in vitro kinase assays P300 was phosphorylated by AMPK.
Abstracts
Also P300 protein expression was significantly decreased in aortic lysates from AMPKα2−/− mice. Inhibition of P300 by curcumin and downregulation of P300 with siRNA resulted in an increase in eNOS protein expression in HUVEC. To investigate if P300 attenuates eNOS expression by interference with KLF2 we used lentivirally infected HUVEC constitutively overexpressing KLF2. In these cells the effect of the P300 siRNA on the increase of eNOS expression was slightly enhanced. In summary these data suggest that AMPKα2 attenuates P300 activity and one consequence of this inhibition is the interference with KLF2 activity and a subsequent attenuation of eNOS expression.
doi:10.1016/j.vph.2011.08.044
O.6.2 Hypoxia and cytokines induce a natural antisense transcript (aHIF) to regulate HIF-1 mediated angiogenesis in rheumatoid arthritis Helene Larsen, Barbara Muz, Marc Feldmann, Ewa Paleolog Kennedy Institute of Rheumatology, Imperial College Faculty of Medicine, London, UK E-mail addresses: [email protected] (H. Larsen), [email protected] (E. Paleolog) Pathological angiogenesis is observed in solid tumours, retinopathies and in autoimmune diseases such as rheumatoid arthritis (RA) where it contributes to progression of disease and chronicity. These diseases are characterised by low oxygen tension ('hypoxia') and elevated levels of pro-inflammatory cytokines. It is becoming clear that key inflammatory and hypoxic signals cross-talk and are amplified through convergence upon powerful transcription factors. One such transcription factor is hypoxia-inducible factor (HIF), known to mediate hypoxic and cytokine-induced angiogenesis, an important early pathological feature of RA. The two best described isoforms HIF-1 and HIF-2 consist of two subunits; HIF-α which is targeted by the proteasome unless stabilised by environmental cues such as hypoxia or Th1 cytokines, and HIF-β which is constitutively expressed. A naturally occurring antisense complimentary to HIF-1α but not HIF-2α, termed aHIF, has been described to be a prognostic marker in diseases characterised by hypoxia and elevated inflammatory cytokines (Cayre, 2003; Zolk, 2008; Span, 2011), but has never been described in RA. There is speculation in the literature that the aHIF RNA transcript binds to and leads to degradation of HIF-1α mRNA, which would make it an important regulator of downstream HIF-1-controlled genes such as VEGF, known to be involved in synovial angiogenesis in RA. Our data has demonstrated that hypoxia and the Th1 cytokines TNFα and IL-1β, drive the expression of aHIF in RA synovial membrane cells, and that aHIF induction correlates with a decline of HIF-1α mRNA and protein in a temporal manner. When we performed actinomycin D chase experiments using quantitative-PCR to study mRNA stability, we found that the hypoxia-induced decline in HIF-1α mRNA was due to decreased mRNA stability. In contrast, the stability of HIF-2α remained unchanged in hypoxia. Importantly, using siRNA technology to knockdown HIF-1α and HIF-2α, we found that both hypoxia and cytokine-induced aHIF expression is driven by HIF-1. Thus, it appears that a negative feedback-loop exists between HIF-1 and aHIF, where HIF-1 induces aHIF which then destabilises HIF-1α mRNA and thereby reduces HIF-1α available for translation in RA synovial membrane cells. In comparison, knocking down HIF-2α in IL-1β-stimulated RA cells further induced aHIF, indicating that HIF2 represses HIF-1-induced aHIF. Based on our findings we suggest that aHIF plays an important regulatory role in diseases characterised by hypoxia and inflamma-
321
tion, such as RA, by influencing HIF-1 transcription factor levels through its ability to bind to and direct degradation of HIF-1α mRNA. Altered HIF-1 levels will in turn affect the magnitude and nature of well-known HIF-1-dependent pathological responses in RA, including synovial angiogenesis.
doi:10.1016/j.vph.2011.08.045
O.6.3 HIF-2α, but not HIF-1α, increases IL-8 expression in endothelial cells — The involvement of Sp-1 and c-Myc transcription factors Urszula Florczyka, Szymon Czaudernaa, Anna Stachurskaa, Magdalena Tertila, Magdalena Kozakowskaa, Lorenz Poellingerb, Alicja Jozkowicza, Agnieszka Lobodaa, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Karolinska Institute, Department of Cell and Molecular Biology, Stockholm, Sweden E-mail addresses: [email protected] (A. Loboda), [email protected] (J. Dulak) Hypoxia-inducible factors (HIF)-1α and HIF-2α are known to be the major mediators of hypoxic response and regulate the expression of genes involved in angiogenesis, cell proliferation and viability. Although highly related, owing to possess different transactivation domains and binding distinct cofactors, they may regulate not only shared but also unique target genes. Among HIF isoforms-interacting proteins c-Myc/ Max proteins and Sp-1 transcription factor were found. Interestingly, recently we have shown that interleukin-8 (IL-8), known mediator of angiogenesis, is downregulated by HIF-1α (Loboda et al., 2009), while other group has found that IL-8 is potently increased in Mxi1, c-Myc antagonist, knockdown cells. Thus, we aimed to examine the effect of HIF-2α on IL-8 expression and the involvement of c-Myc/Mxi1/Sp-1 proteins in such regulation. Human microvascular endothelial cells (HMEC-1) were transduced with adenoviral vectors to overexpress HIF-1α or HIF-2α. In contrast to HIF-1α, overexpression of HIF-2α resulted in significantly increased expression of IL-8. Accordingly, HIF-2α was found to augment the activity of Sp-1. Importantly, inhibition of the latter with mithramycin A decreased, while its overexpression with pCMV-Sp-1 vector increased IL-8 expression. Mutation of putative Sp-1-binding site within IL-8 promoter revealed however no direct interaction of Sp-1 and IL-8. On the other hand, we confirmed that in contrast to HIF-1α, overexpression of HIF-2α isoform results in increased expression of c-Myc. We checked the effect of c-Myc silencing and revealed diminishment of HIF-2αdependent induction of IL-8 in such conditions. Conversely, inhibition of Mxi1 by siRNA reversed the effect of HIF-1α on IL-8. Moreover, we showed that severe hypoxia (0.5% oxygen), simultaneously with decrease in IL-8 expression, augments Mxi1, but reduces c-Myc mRNA level. In accordance with the latter, transduction of cells with AdHIF-1α caused decrease in c-Myc level suggesting that effect of hypoxia is primarily mediated by HIF-1α. Current study revealed for the first time opposite role of HIF-1α and HIF-2α in regulation of IL-8 expression in endothelial cells and the involvement of Sp-1 and c-Myc in the enhancement of IL-8 by HIF-2α. Supported by grant nos. NN301 314837 and NN301 009639 (Ministry of Science and Higher Education). The Faculty of Biochemistry, Biophysics and Biotechnology is a beneficiary of grant nos.: POIG.02.01.00-12 064/08, POIG 01.01.02-00-109/09, POIG.02.02.00014/08 and 01.01.02-00-069/09.
doi:10.1016/j.vph.2011.08.046
Abstracts
Oxygen serves as primary electron acceptor in many cellular reactions and is central for generation of ATP in the mitochondria. Thus, an adequate supply with oxygen and a functional adaptive system to changes in oxygen homeostasis are essential for aerobic life. Thereby, the levels of molecular oxygen as well as its reactive derivates (reactive oxygen species, ROS), and redox equivalents can serve as signalling molecules to monitor disruption of oxygen homeostasis. Key players in oxygen homeostasis are a family of hypoxia-inducible transcription factors, (HIF) which control cellular adaptive processes in response to hypoxia including cellular metabolism and angiogenesis, but also proliferation or apoptosis. On the other hand, the family of NADPH oxidases has been recognised as important source of ROS in particular in the vascular system. Intriguingly, NADPH oxidases are involved in the control of the HIF pathway in the cardiovascular response to multiple stress factors independently of hypoxia. In fact, we could show a role of HIF transcription factors in flow-induced vascular remodelling as well as in the vascular prothrombotic or proinflammatory response. Their ability to be regulated by ROS and metabolic alterations under these conditions make them true redox-sensitive transcription factors. Intriguingly, some NADPH oxidase family members are even direct HIF target genes under these conditions. In addition, recent evidence suggests that NADPH oxidases are also involved in the cardiovascular adaptation to hypoxia. Dissecting the mechanisms controlling the ROS-HIF axis in cardiovascular physiology and pathophysiology appears to be a promising strategy to understand the importance of redox signalling in cardiovascular function and to delineate novel therapeutic options for a variety of cardiovascular disorders. doi:10.1016/j.vph.2011.08.041
L.6.2 Therapeutic manipulation of vascular signaling and cytoprotection Justin C. Mason Cardiovascular Sciences, Imperial College London, London, UK E-mail address: [email protected] Vascular endothelial injury predisposes to apoptosis, endothelial dysfunction and atherogenesis. This process may be accelerated in systemic inflammatory diseases such as systemic lupus erythematosus, in diabetes mellitus, chronic renal failure and post-transplantation. The endothelium has a variety of innate cytoprotective mechanisms aimed at minimizing injury and facilitating repair. These mechanisms may be regulated by specific signaling pathways, activated by both endogenous and exogenous mediators, and further influenced by patterns of shear stress exerted by the flowing blood. Cytoprotective genes induced by these pathways, including endothelial nitric oxide synthase, heme oxygenase-1, decay-accelerating factor and B cell lymphoma protein-2, play a central role in the maintenance of endothelial homeostasis and in the response to injury. We are currently exploring therapeutic options for their regulation, alongside the investigation of novel signaling mechanisms including those regulated by protein kinase C (PKC), and by specific nuclear receptors and their coregulators. For example, we have shown that PKCε activity in the vascular endothelium co-ordinates a diverse downstream cytoprotective response in EC. Likewise PPARδ, in conjunction with its co-regulator PGC1α, is able to regulate anti-oxidant responses. We propose that a detailed knowledge of cytoprotective mechanisms and their associated signaling pathways may ultimately reveal novel therapeutic targets. These may in turn provide the means by which vascular endothelial dysfunction can be reversed, so preventing or retarding accelerated atherogenesis in a variety of disease states. doi:10.1016/j.vph.2011.08.042
L.6.3 Blood viscosity limits maximal exercise performance in mice Max Gassmann Institute of Veterinary Physiology, Vetsuisse Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zürich, Winterthurerstrasse 260, CH-8057 Zürich, Switzerland E-mail address: [email protected] Hypoxia induces erythropoietin (Epo) gene expression that in turn elevates erythropoiesis and hence the arterial O2 content thereby also improving exercise performance. It is well accepted that the highest attainable hematocrit cannot be associated with the maximal exercise capacity. To test as whether an optimal hematocrit in acute and chronic Epo-treated mice exists, i.e. a given hematocrit allowing the highest O2 transport during maximal exercise, hematocrit levels of wild type (wt) mice were acutely elevated by applying NESP (novel erythropoiesis stimulating protein, wtNESP). Furthermore, in the transgenic mouse line Tg6 that reaches hematocrit levels of up to 0.9 due to the constitutive over-expression of human Epo, the hematocrit was gradually reduced by application of the hemolysisinducing compound phenylhydrazine (PHZ, tg6PHZ). Maximal cardiovascular performance was measured by using telemetry in exercising mice. Highest maximal O2 uptake (˙VO2max) and maximal time to exhaustion at sub-maximal exercise intensities were reached at hematocrit values of 0.58 and 0.57 for wtNESP, and 0.68 and 0.66 for tg6PHZ, respectively. While maximal working capacity of the heart increased with elevated hematocrit values, blood viscosity correlated with ˙VO2max. Obviously, blood viscosity increases with rising hematocrit levels limiting the blood's O2 transport capacity. Of note, while performing this set of experiments, we observed that Epo present in the brain of both mouse models augmented exercise performance in a non-erythroid manner, too.
doi:10.1016/j.vph.2011.08.043
O.6.1 The AMP-activated protein kinase modifies gene expression in endothelial cells by interfering with protein lysine acetylation Beate Fisslthalera, Annemarieke Loota, Reinier Boonb, Eduard Hergenreiderb, Ingrid Fleminga a Institute for Vascular Signalling, Goethe University Frankfurt, Germany b Institute for Cardiovascular Regeneration, Goethe University Frankfurt, Germany E-mail addresses: [email protected](B. Fisslthaler), [email protected] (A. Loot), [email protected] (I. Fleming) The AMP-activated protein kinase (AMPK) is known to modulate the expression of mitochondrial and glucose utilising genes but the mechanisms are not well understood. In endothelial cells adenoviral overexpression of the AMPKα2 subunit resulted in the modulation of approximately 700 genes including the endothelial nitric oxide synthase (eNOS) and the transcription factor Krueppel-like factor-2 (KLF2) which is crucial for the shear stress dependent increase of eNOS expression. In murine aorta as well as in cultured cells deletion or inhibition of the expression of AMPKα2 resulted in a significantly increased eNOS protein expression. The NO-dependent vasodilation in response to bradykinin was increased in the perfused hindlimb from AMPKα2−/− animals compared to wild type littermates. As acetylation of histones is crucial for the cell type specific eNOS expression we analysed the effect of AMPK on the histone acetylase P300. In in vitro kinase assays P300 was phosphorylated by AMPK.
Abstracts
Also P300 protein expression was significantly decreased in aortic lysates from AMPKα2−/− mice. Inhibition of P300 by curcumin and downregulation of P300 with siRNA resulted in an increase in eNOS protein expression in HUVEC. To investigate if P300 attenuates eNOS expression by interference with KLF2 we used lentivirally infected HUVEC constitutively overexpressing KLF2. In these cells the effect of the P300 siRNA on the increase of eNOS expression was slightly enhanced. In summary these data suggest that AMPKα2 attenuates P300 activity and one consequence of this inhibition is the interference with KLF2 activity and a subsequent attenuation of eNOS expression.
doi:10.1016/j.vph.2011.08.044
O.6.2 Hypoxia and cytokines induce a natural antisense transcript (aHIF) to regulate HIF-1 mediated angiogenesis in rheumatoid arthritis Helene Larsen, Barbara Muz, Marc Feldmann, Ewa Paleolog Kennedy Institute of Rheumatology, Imperial College Faculty of Medicine, London, UK E-mail addresses: [email protected] (H. Larsen), [email protected] (E. Paleolog) Pathological angiogenesis is observed in solid tumours, retinopathies and in autoimmune diseases such as rheumatoid arthritis (RA) where it contributes to progression of disease and chronicity. These diseases are characterised by low oxygen tension ('hypoxia') and elevated levels of pro-inflammatory cytokines. It is becoming clear that key inflammatory and hypoxic signals cross-talk and are amplified through convergence upon powerful transcription factors. One such transcription factor is hypoxia-inducible factor (HIF), known to mediate hypoxic and cytokine-induced angiogenesis, an important early pathological feature of RA. The two best described isoforms HIF-1 and HIF-2 consist of two subunits; HIF-α which is targeted by the proteasome unless stabilised by environmental cues such as hypoxia or Th1 cytokines, and HIF-β which is constitutively expressed. A naturally occurring antisense complimentary to HIF-1α but not HIF-2α, termed aHIF, has been described to be a prognostic marker in diseases characterised by hypoxia and elevated inflammatory cytokines (Cayre, 2003; Zolk, 2008; Span, 2011), but has never been described in RA. There is speculation in the literature that the aHIF RNA transcript binds to and leads to degradation of HIF-1α mRNA, which would make it an important regulator of downstream HIF-1-controlled genes such as VEGF, known to be involved in synovial angiogenesis in RA. Our data has demonstrated that hypoxia and the Th1 cytokines TNFα and IL-1β, drive the expression of aHIF in RA synovial membrane cells, and that aHIF induction correlates with a decline of HIF-1α mRNA and protein in a temporal manner. When we performed actinomycin D chase experiments using quantitative-PCR to study mRNA stability, we found that the hypoxia-induced decline in HIF-1α mRNA was due to decreased mRNA stability. In contrast, the stability of HIF-2α remained unchanged in hypoxia. Importantly, using siRNA technology to knockdown HIF-1α and HIF-2α, we found that both hypoxia and cytokine-induced aHIF expression is driven by HIF-1. Thus, it appears that a negative feedback-loop exists between HIF-1 and aHIF, where HIF-1 induces aHIF which then destabilises HIF-1α mRNA and thereby reduces HIF-1α available for translation in RA synovial membrane cells. In comparison, knocking down HIF-2α in IL-1β-stimulated RA cells further induced aHIF, indicating that HIF2 represses HIF-1-induced aHIF. Based on our findings we suggest that aHIF plays an important regulatory role in diseases characterised by hypoxia and inflamma-
321
tion, such as RA, by influencing HIF-1 transcription factor levels through its ability to bind to and direct degradation of HIF-1α mRNA. Altered HIF-1 levels will in turn affect the magnitude and nature of well-known HIF-1-dependent pathological responses in RA, including synovial angiogenesis.
doi:10.1016/j.vph.2011.08.045
O.6.3 HIF-2α, but not HIF-1α, increases IL-8 expression in endothelial cells — The involvement of Sp-1 and c-Myc transcription factors Urszula Florczyka, Szymon Czaudernaa, Anna Stachurskaa, Magdalena Tertila, Magdalena Kozakowskaa, Lorenz Poellingerb, Alicja Jozkowicza, Agnieszka Lobodaa, Jozef Dulaka a Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland b Karolinska Institute, Department of Cell and Molecular Biology, Stockholm, Sweden E-mail addresses: [email protected] (A. Loboda), [email protected] (J. Dulak) Hypoxia-inducible factors (HIF)-1α and HIF-2α are known to be the major mediators of hypoxic response and regulate the expression of genes involved in angiogenesis, cell proliferation and viability. Although highly related, owing to possess different transactivation domains and binding distinct cofactors, they may regulate not only shared but also unique target genes. Among HIF isoforms-interacting proteins c-Myc/ Max proteins and Sp-1 transcription factor were found. Interestingly, recently we have shown that interleukin-8 (IL-8), known mediator of angiogenesis, is downregulated by HIF-1α (Loboda et al., 2009), while other group has found that IL-8 is potently increased in Mxi1, c-Myc antagonist, knockdown cells. Thus, we aimed to examine the effect of HIF-2α on IL-8 expression and the involvement of c-Myc/Mxi1/Sp-1 proteins in such regulation. Human microvascular endothelial cells (HMEC-1) were transduced with adenoviral vectors to overexpress HIF-1α or HIF-2α. In contrast to HIF-1α, overexpression of HIF-2α resulted in significantly increased expression of IL-8. Accordingly, HIF-2α was found to augment the activity of Sp-1. Importantly, inhibition of the latter with mithramycin A decreased, while its overexpression with pCMV-Sp-1 vector increased IL-8 expression. Mutation of putative Sp-1-binding site within IL-8 promoter revealed however no direct interaction of Sp-1 and IL-8. On the other hand, we confirmed that in contrast to HIF-1α, overexpression of HIF-2α isoform results in increased expression of c-Myc. We checked the effect of c-Myc silencing and revealed diminishment of HIF-2αdependent induction of IL-8 in such conditions. Conversely, inhibition of Mxi1 by siRNA reversed the effect of HIF-1α on IL-8. Moreover, we showed that severe hypoxia (0.5% oxygen), simultaneously with decrease in IL-8 expression, augments Mxi1, but reduces c-Myc mRNA level. In accordance with the latter, transduction of cells with AdHIF-1α caused decrease in c-Myc level suggesting that effect of hypoxia is primarily mediated by HIF-1α. Current study revealed for the first time opposite role of HIF-1α and HIF-2α in regulation of IL-8 expression in endothelial cells and the involvement of Sp-1 and c-Myc in the enhancement of IL-8 by HIF-2α. Supported by grant nos. NN301 314837 and NN301 009639 (Ministry of Science and Higher Education). The Faculty of Biochemistry, Biophysics and Biotechnology is a beneficiary of grant nos.: POIG.02.01.00-12 064/08, POIG 01.01.02-00-109/09, POIG.02.02.00014/08 and 01.01.02-00-069/09.
doi:10.1016/j.vph.2011.08.046