Journal of Leukocyte Biology eBioscience full spectrum cell analysis
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published online as doi:10.1189/jlb.0107060 on May 15, 2007

Published online before print May 15, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
jlb.0107060v1
82/2/392    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by De Ponti, C.
Right arrow Articles by Tacchini, L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by De Ponti, C.
Right arrow Articles by Tacchini, L.
(Journal of Leukocyte Biology. 2007;82:392-402.)
© 2007 by Society for Leukocyte Biology

Adenosine A2a receptor-mediated, normoxic induction of HIF-1 through PKC and PI-3K-dependent pathways in macrophages

Cristina De Ponti*, Rita Carini{dagger}, Elisa Alchera{dagger}, Maria Paola Nitti{ddagger}, Massimo Locati*,§, Emanuele Albano{dagger}, Gaetano Cairo*,1 and Lorenza Tacchini*

* Istituto di Patologia Generale, Università di Milano, Italy;
{dagger} Dipartimento di Scienze Mediche, Università "A. Avogadro," Novara, Italy;
{ddagger} Dipartimento di Medicina Sperimentale, Università di Genova, Italy; and
§ Istituto Clinico Humanitas, IRCCS, Rozzano, Italy

1 Correspondence: Istituto di Patologia Generale, University of Milan School of Medicine, Via Mangiagalli 31, 20133 Milano, Italy. E-mail: gaetano.cairo{at}unimi.it


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine released by cells in injurious or hypoxic environments has tissue-protecting and anti-inflammatory effects, which are also a result of modulation of macrophage functions, such as vascular endothelial growth factor (VEGF) production. As VEGF is a well-known target of hypoxia-inducible factor 1 (HIF-1), we hypothesized that adenosine may activate HIF-1 directly. Our studies using subtype-specific adenosine receptor agonists and antagonists showed that by activating the A2A receptor, adenosine treatment induced HIF-1 DNA-binding activity, nuclear accumulation, and transactivation capacity in J774A.1 mouse macrophages. Increased HIF-1 levels were also found in adenosine-treated mouse peritoneal macrophages. The HIF-1 activation induced by the A2A receptor-specific agonist CGS21680 required the PI-3K and protein kinase C pathways but was not mediated by changes in iron levels. Investigation of the molecular basis of HIF-1 activation revealed the involvement of transcriptional and to a larger extent, translational mechanisms. HIF-1 induction triggered the expression of HIF-1 target genes involved in cell survival (aldolase, phosphoglycerate kinase) and VEGF but did not induce inflammation-related genes regulated by HIF-1, such as TNF-{alpha} or CXCR4. Our results show that the formation of adenosine and induction of HIF-1, two events which occur in response to hypoxia, are linked directly and suggest that HIF-1 activation through A2A receptors may contribute to the anti-inflammatory and tissue-protecting activity of adenosine.

Key Words: hypoxia • inflammation • kinases


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The microenvironment of injured and inflamed tissues is characterized by low levels of O2, mainly caused by the disrupted microcirculation [1 ], and inflammation/injury-related hypoxia can be expected to activate hypoxia-inducible factor 1 (HIF-1), a ubiquitous transcription factor, which functions as a master regulator of oxygen homeostasis [2 3 4 ].

Active HIF-1 is a heterodimer consisting of an inducible HIF-1{alpha} subunit and a constitutively expressed HIF-1β subunit. HIF-1{alpha} is an extremely labile protein in normoxia, and hypoxia increases its intracellular levels by markedly inhibiting its rapid degradation. Under normoxic conditions, a family of prolyl-4-hydroxylase domain enzymes, which have an absolute requirement for oxygen, iron, and 2-oxoglutarate [5 ], hydroxylates HIF-1{alpha}, which is then bound by the von Hippel-Lindau tumor suppressor protein and targeted for proteasomal destruction [6 ]. In the absence of one of the three cofactors, hydroxylation is blocked, and the stabilized HIF-1{alpha} is free to bind HIF-1β to form an active heterodimer, which can bind to hypoxia response elements (HREs) located in the promoter regions of a variety of genes encoding proteins playing key roles in different cell functions, including angiogenesis, erythropoiesis, glucose transport, glycolysis, iron transport, and cell proliferation/survival [7 ]. Reactive oxygen species generated by mitochondria under hypoxic conditions also play an important role in inducing HIF-1 [8 ], which can be turned on as well by a number of nonhypoxic stimuli, such as growth factors, cytokines, hormones, NO, and LPS (see Dery et al. [9 ] for review). Unlike hypoxia, the stabilization of HIF-1{alpha} does not seem to play a role in the induction of HIF-1 exerted by these stimuli, the predominant mechanism being an increase in HIF-1{alpha} protein translation, which is in turn mediated by the PI-3K pathway and its downstream effectors [9 10 11 12 ]. In addition, increased HIF-1{alpha} mRNA levels have been found [10 12 13 ].

It has been shown that adaptation to the low oxygen levels present in most areas of extensive inflammation is a key element of T cell activities [1 ]. However, the myeloid cells recruited to inflammatory sites also need to be able to survive and function under microenvironmental conditions characterized by low oxygen tension, and it has been demonstrated that targeted HIF-1 inactivation in macrophages and neutrophils affects several myeloid functions and leads to an impaired inflammatory response [14 15 ].

How is the low oxygen tension in tissues translated into the biochemical changes, which lead to the response of immune cells? One such mechanism could be direct hypoxia-generated HIF-1 activation, and another signal could be the accumulation of extracellular adenosine, which is produced by many different tissues and cell types and increases dramatically in response to hypoxia, ischemia, and inflammation to the point of reaching interstitial concentrations, which are several hundred-fold higher than those present under normal conditions [1 16 ]. Increased levels of extracellular adenosine trigger the G protein-coupled adenosine receptors on activated immune cells and modulate a variety of functions, including phagocytosis, antigen presentation, target cell killing, and the expression of cytokines and NO [1 16 17 ]. Furthermore, it has been demonstrated that adenosine plays a key role in promoting tissue protection by means of ischemic preconditioning (see Linden [16 ] for review).

Adenosine A2A receptors synergize with TLRs in up-regulating vascular endothelial growth factor (VEGF) expression in murine macrophages, thus acting as an angiogenic switch [18 ]. As VEGF induction is one of the best-characterized and most frequent responses to HIF-1 activation [19 20 ], this suggests that HIF-1 activity may be up-regulated by adenosine also. The aim of this study was to test this hypothesis, and we found that adenosine does in fact induce HIF-1 activity and the expression of HIF-1 target genes in macrophages. Furthermore, when exploring the signaling pathways downstream of the adenosine receptor, as well as the molecular mechanisms involved in HIF-1 induction, we found evidence indicating the involvement of protein kinase C (PKC) and PI-3K pathways and the importance of post-transcriptional mechanisms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell cultures and treatments
The J774A.1 murine macrophage cell line was obtained from the European Collection of Cell Cultures and cultured in E-MEM medium (Sigma, Milan, Italy) containing 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and 0.1 ng/ml streptomycin at 37°C in 5% CO2. Proteose peptone-elicited peritoneal macrophages were recovered from 8-week-old, pathogen-free, female CD1 mice (Charles River Italia, Calco, Italy), which were housed, fed, and handled in compliance with the prescriptions for the care and use of laboratory animals. Peritoneal macrophages were purified by adherence to plastic tissue-culture clusters (Corning-Costar Italia, Milano, Italy) for 2 h at 37°C in 5% CO2, as described previously [21 ]. Near-confluent J774A.1 cells were exposed to various concentrations of adenosine for 2 h or 100 µM adenosine for different times; cells were also treated with various concentrations of the A2A adenosine receptor agonist CGS21680 (Sigma) in serum-free medium for 4 h or 5 µM CGS21680 for different times. Control experiments demonstrated that incubation for up to 6 h in the absence of serum did not modify HIF-1 activity (not shown). When appropriate, 15 min before stimulation with adenosine or CGS21680, the cells were treated with the PI-3K inhibitor wortmannin (250 nM) and the PKC inhibitor chelerythrine (2 µM; both from Sigma) or with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 1 µM), 3,7-dimethyl-1-propargylxanthine (DMPX; 1 µM), alloxazine (1 µM), or VUF5574 (1 µM; all from Sigma), antagonists of, respectively, A1, A2, A2B, and A3 adenosine receptors. Different doses of the A2A receptor selective antagonist ZM241385 were also used. In some cases, the cells were exposed for 20 h to 100 µM desferrioxamine (DFO; Sigma) or 1 µg/ml LPS (Sigma) in complete medium.

EMSA
The nuclear extracts were prepared as described by Tacchini et al. [10 ], and aliquots were incubated with {gamma}-32P-ATP-labeled oligonucleotides (Primm, Milan, Italy) encompassing the binding sites for HIF-1 (5'-AGCGTACGTGCCTCAGGA-3') and octamer-binding protein 1 (oct-1; 5'-TGCGAATGCAAATCACTAGAA-3') and then electrophoresed and autoradiographed [10 ]. The specificity of the assay was demonstrated by the disappearance of the signals after the addition of a 50-fold excess of specific but not nonspecific oligonucleotides (data not shown). The quantitative determinations were made by means of direct nuclear counting using an InstantImager (Packard Instruments, Milan, Italy), and the values were calculated after normalization to the activity of oct-1. For the supershift assays, the nuclear extracts were incubated first for 60 min with 1 µg anti-HIF-1{alpha} antibody (OZ15, LabVision-NeoMarkers, Fremont, CA, USA) or anti-heat shock factor 1 (anti-HSF1) antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on ice and then with the labeled oligonucleotide, followed by electrophoresis [22 ].

Transient transfection assay
Subconfluent J774A.1 cells were transiently transfected using the PlusTM and LipofectamineTM reagents (Invitrogen, Carlsbad, CA, USA) with a 50:1 mixture of the various constructs containing fragments of the HIF-1 promoter and the pRL-SV40 reporter vector containing renilla luciferase, which was used to normalize transfection efficiency. Ten minutes before transfection, the cells were exposed to CGS21680 in the absence of serum, and after 4 h, fresh medium was added, and the cells were reincubated. The transfected cells were treated with DFO, as described previously by Bianchi et al. [23 ]. The vectors (generous gifts of Darren E. Richard, Centre de Recherche, Quebec, Canada, originally prepared by Gregg L. Semenza’s laboratory, Johns Hopkins University, Baltimore, MD, USA) [12 ] contained a 604- or 856-bp fragment of the human HIF-1 promoter with (pHIF-1A) or without (pHIF-1B) the 5'-UTR region cloned in front of the firefly luciferase gene in the pGL2 vector (Promega, Milan, Italy). The cells were also transfected with the pGL3PGK6TKp vector containing a HRE multimer [24 ] (a kind gift of Peter J. Ratcliffe, Wellcome Trust Center for Human Genetics, Oxford, UK). When appropriate, the cells were cotransfected with 1 µg of the expression vector pcDNA3ARNT{Delta}_b ({Delta}ARNT), coding for the dominant-negative mutant form of the ARNT subunit (obtained from Michael Schwarz, University of Tübingen, Germany) [10 ]. After 24 h, the cells were collected, washed, and lysed using the reporter lysis buffer (Promega), and luciferase activities were measured in a Promega luminometer using the Dual-Luciferase reporter assay system (Promega), according to the manufacturer’s instructions [24 ]. The empty vectors showed practically undetectable luciferase activity. All of the transfection experiments were carried out on duplicate plates and were repeated at least three times (n=6).

Immunoblot analysis
HIF-1{alpha} was determined in nuclear extracts prepared according to Tacchini et al. [10 ]. Equal amounts of proteins were electrophoresed in acrylamide-SDS gels and electroblotted to Hybond membranes (Amersham Co., Milan, Italy). After assessing transfer by means of Ponceau S staining, the membranes were incubated with anti-human HIF-1{alpha} mAb (H1{alpha}67, Novus Biologicals, Littleton, CO, USA; 1:1000 dilution). The anti-TFIID antibody (Santa Cruz Biotechnology) was used to assess equal protein loading. After incubation with the appropriate secondary antibody and extensive washing, the antigens were detected using an immunodetection kit (ECL Plus, Amersham Co.). Antigens were quantitated by means of densitometry with the values being calculated after normalization to the amount of TFIID.

RNA-protein gel retardation assay
The cell lysates were prepared as described by Cairo et al. [25 ], and equal amounts of supernatant proteins were incubated with the iron-responsive element (IRE) probe, which was transcribed from the pSPT-fer plasmid containing the IRE of the human ferritin H chain [26 ] using T7 RNA polymerase in the presence of [{alpha}-32P]UTP and treated with RNase T1 and heparin [25 ]. After separation on 6% nondenaturing polyacrylamide gels, the RNA-protein complexes were visualized by autoradiography and quantitated by means of direct nuclear counting using an InstantImager (Packard Instruments).

Northern blot analysis
Total cell RNA was isolated, and equal amounts were electrophoresed under denaturing conditions [10 ]. To confirm that each lane contained equal amounts of RNA, the rRNA content in each lane was estimated in the ethidium bromide-stained gels by laser densitometry. The RNA was transferred to Hybond-N filters (Amersham Co.), which were hybridized with 32P-labeled human cDNAs of HIF-1{alpha} (kindly provided by Ruth D. Thornton, Philadelphia College of Osteopathic Medicine, Philadelphia, PA, USA), aldolase [Clone HFBCC77, obtained from American Type Culture Collection (ATCC), Manassas, VA, USA], and phosphoglycerate kinase 1 (PGK-1; plasmid pHPGK-7e, obtained from ATTC). The quantitative determinations were made by means of direct nuclear counting using an InstantImager (Packard Instruments), with the values being calculated after normalization to the amount of rRNA.

Evaluation of c- and n-PKCs in cytosol and membrane fractions
The cells were lysed by sonication in 10 mM HEPES, pH 7.5, 0.25 M sucrose, 5 mM EDTA, 10 mM 2-ME, 2 mM PMSF, and 1 mM leupeptin. The unbroken cells and nuclei were separated by 10 min centrifugation at 13,000 g. The soluble fraction was separated by centrifugation at 100,000 g for 30 min, and the cell debris were treated for 20 min with the lysis buffer containing 0.2% Triton X-100 on ice and further centrifuged for 30 min at 100,000 g to separate the membrane fraction. The cytosolic and membrane proteins were separated by electrophoresis on 8% SDS-polyacrylamide gel and blotted onto nitrocellulose membranes. The PKC-{delta} isoenzyme was revealed using rabbit polyclonal antibodies against the PKC-{delta} isoform (Santa Cruz Biotechnology) and ECL (Amersham Co.) [27 ]. Antigens were quantitated by means of densitometry.

In vitro PKC activity assay
The different PKC isoforms were immunoprecipitated with a mouse anti-c-PKC mAb or with rabbit polyclonal anti-{delta} and anti-{epsilon} antibodies (Santa Cruz Biotechnology) and protein A sepharose (Sigma). The sepharose beads were then washed three times in the buffer described previously [27 ]. The histone kinase activity of c-PKC ({alpha}, β1, β2 isoforms) was evaluated by incubating 30 µl of the buffer for 10 min with 0.1 mM ATP, 2 µCi/sample [32P]ATP (Amersham Co.), 1 µg phosphatidylserine, 0.4 µg diacylglycerol, 0.5 mM CaCl2, and 10 µg histone H1 at 37°C. To assay n-PKC-{delta} and n-PKC-{epsilon} activity, CaCl2 was omitted from the incubation buffer. The reaction was stopped by the addition of Laemmli’s buffer, and the histone was separated by electrophoresis on a 12.5% SDS-polyacrylamide gel. After blotting on a nitrocellulose Hybond C membrane (Amersham Co.), [32P] incorporation was revealed by autoradiography, and the relative intensity of phosphorylation was evaluated densitometrically [27 ].

Analysis of Akt phosphorylation state
Cell lysates prepared as described previously [28 ] were centrifuged at 13,000 g for 10 min. For immunoblot analysis, 30 µg protein aliquots of the supernatants were electrophoresed on a 10% SDS-polyacrylamide gel, blotted onto nitrocellulose membranes, and probed sequentially with rabbit polyclonal antibodies against (Ser473) phospho-Akt and Akt (Cell Signaling Technology, Beverly, MA, USA). The antibody binding was revealed by HRP-conjugated anti-rabbit Igs (Bio-Rad, Hercules, CA, USA) using Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer, Boston, MA, USA). The relative intensity of the bands was measured by densitometry [28 ].

Real-time PCR
RT reaction from 1 µg total RNA template was performed using TaqMan reagents (Applied Biosystems, Foster City, CA, USA) as per the manufacturer’s instructions. Real-time PCR was done using SyBr Green PCR Master Mix (Applied Biosystems) and detected by an ABI-Prism 5700 sequence detector (Applied Biosystems). Schioppa et al. [29 ] described the primers used. Data were processed using the GeneAmp software (Applied Biosystems), normalized to the expression of the housekeeping gene β-actin, and expressed as fold changes in mRNA levels with respect to the control, untreated cells. Data represent mean ± SE from three independent experiments done in triplicate.

Statistical analysis
Densitometric values were analyzed by ANOVA, and P values of <0.05 were considered significant. The differences from control were evaluated using the original experimental data.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adenosine induces HIF-1 activation in J774A.1 cells and mouse macrophages
To study the effect of adenosine on HIF-1, mouse J774A.1 macrophages were exposed to increasing adenosine concentrations for 2 h, and the DNA-binding activity of HIF-1 was evaluated by EMSA. Adenosine increased HIF-1 activity in a dose-dependent manner, and activation was already detectable at 50 µM, and maximal binding levels were reached at 100 µM (Fig. 1A ) and maintained at higher doses (data not shown). A similar binding activity was found in extracts of cells exposed to the iron chelator DFO, a well-known inducer of HIF-1 [30 ]. Competition experiments using unlabeled oligonucleotides demonstrated the specificity of the interaction between the DNA probe and the nuclear factors induced by adenosine. The complex that migrates faster represents a so-called constitutive factor, which is closely related or identical to the transcription factors, activating transcription factor 1 and CREB-1, and has been shown to be induced by hypoxia, iron chelation, and adenosine [31 32 33 ]. The addition of an antibody against HIF-1{alpha} (but not of an irrelevant antibody) in supershift assays immunodepleted the slower-migrating bands induced by adenosine or DFO, thus indicating that HIF-1 was the nuclear protein activated by the treatments (Fig. 1B) . Immunoblot analysis of the nuclear extracts showed that HIF-1 protein levels were also increased greatly in the cells exposed to adenosine, and a dose response reflected that shown by the DNA-binding activity (Fig. 1C) . Time-course experiments showed that HIF-1 levels increased after 1 h, remained high until the 4th h, and decreased thereafter (Fig. 1D) . To investigate whether the HIF-1 induction observed in the J774A.1 cell line was also detectable in primary mononuclear cells, immunoblotting assays were performed with nuclear extracts from mouse peritoneal macrophages incubated in the presence of 100 µM adenosine for 2 h. As shown in Figure 1E , exposure to adenosine was able to increase HIF-1 content to an extent almost similar to that obtained with DFO.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		Figure 1. Adenosine induces the HIF-1 transcription complex. (A) EMSA analysis of the HIF-1 DNA-binding activity of nuclear extracts from J774A.1 cells treated for 2 h with different concentrations of adenosine (Ado) or 100 µM DFO for 20 h. s.c., Specific competition of the 100 µM sample; arrow indicates the inducible HIF-1 complex, whereas the arrowhead indicates the constitutive (const) complex. The binding activity of the constitutively expressed transcription factor oct-1 was used to assess equal loading. The values indicate the fold difference ± SE of the HIF-1 band in relation to the untreated sample. (B) Supershift assay of the nuclear extracts of cells treated with adenosine, 100 µM for 2 h, and DFO, 100 µM for 20 h, using antibodies against the HIF-1{alpha} subunit or HSF. (C) Immunoblot analysis of nuclear extracts from J774A.1 cells treated for 2 h with different concentrations of adenosine. TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the control. (D) Immunoblot analysis of nuclear extracts from cells untreated (Lane C) or treated with 100 µM adenosine for different times or DFO for 20 h. TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the control. (E) Immunoblot analysis with an antibody against HIF-1 of nuclear extracts from untreated mouse peritoneal macrophages or treated with 100 µM adenosine for 2 h or DFO for 20 h. TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the control. All of the results shown are representative of at least three independent experiments.

 
Activation of HIF-1-binding activity and transactivation capacity is mediated by the A2A receptor
To assess which member of the adenosine receptor family was involved in HIF-1 activation, we first used a panel of adenosine receptor antagonists. The accumulation of HIF-1 in J774A.1 cells treated for 4 h with 100 µM adenosine was prevented almost completely by the simultaneous addition of the A2 receptor inhibitor DMPX but not by the antagonists of the A1, A2B, and A3 receptors (Fig. 2A ). Treatment with the antagonists alone did not influence HIF-1 activity (results not shown). The involvement of the A2A receptor was indicated by the progressive down-regulation of HIF-1 protein levels obtained when the cells were incubated in the presence of increasing concentrations of the selective inhibitor of the A2A receptor (ZM241385; Fig. 2B ).


Figure 2
View larger version (31K):
[in this window]
[in a new window]

  		Figure 2. Adenosine stimulates HIF-1 transcriptional activity through the A2A receptor. (A) Immunoblot analysis with an antibody against HIF-1 of the nuclear extracts from J774A.1 cells treated with 100 µM adenosine for 4 h in the presence or absence of specific receptor antagonists, as described in Materials and Methods. (B) Immunoblot analysis of nuclear extracts from J774A.1 macrophages treated for 4 h with 100 µM adeonsine in the absence or presence of increasing concentrations of the A2A receptor antagonist ZM241385. TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the sample treated with adenosine alone. All of the results shown are representative of at least three independent experiments.

 
To assess the involvement of the A2A receptor further in HIF-1 activation, J774A.1 cells were incubated in the presence of CGS21680, a highly selective receptor agonist of the A2A receptor. Concentration response experiments showed that HIF-1 nuclear accumulation (Fig. 3A ) was induced at 0.5 µM, increased further at 1 µM, and remained high at 5 µM. HIF-1 was also activated in a time-dependent manner, full activation being reached at 2 h and maintained for 4–6 h (Fig. 3B) . The inducing effect of CGS21680 was prevented by the addition of the A2A receptor antagonist ZM241385 (Fig. 3B) .


Figure 3
View larger version (25K):
[in this window]
[in a new window]

  		Figure 3. The A2A receptor agonist CGS21680 stimulates HIF-1 transcriptional activity. (A) Immunoblot analysis with an antibody against HIF-1 of nuclear extracts from J774A.1 cells treated with different concentrations of CGS21680 (CGS) for 4 h. (B) Immunoblot analysis of nuclear extracts from untreated J774A.1 cells or cells treated for different times with 1 µM CGS21680 in the presence or in the absence of the A2A receptor antagonist ZM241385 (1 µM). TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the control. All of the results shown are representative of three independent experiments. (C) The J774A.1 cells were transiently transfected with the empty pGL2 basic vector or a construct in which luciferase is controlled by a HRE multimer and were left untreated (C) or exposed to 5 µM CGS21680 for 4 h or 100 µM DFO for 20 h. When appropriate, they were also cotransfected with an expression vector coding for a dominant-negative mutant of the constitutive HIF-1 β subunit. The cells were cotransfected with a control vector containing the Renilla luciferase gene. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity, and normalized to the activity recorded in untreated cells, arbitrarily defined as 1. Mean values ± SE of three independent experiments. *, P < 0.001, versus untreated control; **, P < 0.001, versus cells treated with CGS21680; +, P < 0.001, versus cells treated with DFO.

 
We then used transactivation capacity experiments to verify whether the HIF-1 induced by CGS21680 was transcriptionally active. In J774A.1 cells transiently transfected with a luciferase reporter gene controlled by a DNA fragment containing multiple consensus HREs, which have been shown previously to drive HIF-1-dependent transcription in response to hypoxia and hypoxia-mimics [24 ], the expression of the reporter gene increased more than fivefold in response to CGS21680 or DFO (Fig. 3C) . Further indications of the involvement of HIF-1 in the CGS21680- and DFO-dependent activation of luciferase activity were obtained by means of experiments in which the HIF-1-transactivating capacity was abolished almost completely by the cotransfection of a plasmid expressing a dominant-negative of the β subunit of the HIF-1 heterodimer (HRE/{Delta}ARNT), which maintains the capacity of forming a heterodimer but cannot bind DNA [10 ] (Fig. 3C) .

CGS21680 was used to represent adenosine-mediated effects in all of the subsequent experiments because of its higher stability in solution.

HIF-1 activation is iron-independent and occurs through the PKC and PI-3K pathways
Iron is required for HIF-1 degradation in normoxic cells, as it is a necessary cofactor for prolyl and asparginyl hydroxylases [5 ], and so decreased iron availability activates HIF-1 [30 ]. To investigate the possibility that the adenosine/A2A receptor pathway leads to HIF-1 activation by reducing the intracellular iron pool, we used a RNA bandshift assay to measure the activity of the iron regulatory proteins 1 and 2 (IRP1 and IRP2), key regulators of iron homeostasis, which are activated by decreased intracellular iron availability [34 35 ]. Figure 4A shows that the RNA-binding activity of IRP1 and IRP2 remained unaltered in J774A.1 cells treated with CGS21680 for 2 h but as expected, was remarkably activated in cells deprived of iron by exposure to the iron chelator DFO [34 35 ]. Further evidence that CGS21680 activates HIF-1 in an iron-independent manner was provided by transfection experiments showing that DFO and CGS21680 had an additive effect on luciferase activity driven by the HRE multimer (Fig. 4B) , indicating that different mechanisms are involved in the activation of HIF-1 induced by iron deficiency or CGS21680 in J774A.1 cells.


Figure 4
View larger version (29K):
[in this window]
[in a new window]

  		Figure 4. The induction of HIF-1 transcriptional activity is iron-independent. (A) RNA bandshift analysis of cytoplasmic IRP activity from J774A.1 cells. Cytosolic extracts from untreated J774A.1 cells or cells treated with 5 µM CGS21680 for 2 h or 100 µM DFO for 20 h were incubated with an excess of a 32P-labeled iron responsive element probe, and the RNA protein complexes were separated on nondenaturing polyacrylamide gels. The autoradiogram is representative of three independent experiments. (B) J774A.1 cells were transiently transfected with a construct in which luciferase is controlled by a HRE multimer and were left untreated or exposed to 5 µM CGS21680 for 4 h and/or 100 µM DFO for 20 h. The cells were cotransfected with a control vector containing the Renilla luciferase gene, and luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity, and normalized to the activity recorded in untreated cells, arbitrarily defined as 1. Mean values ± SE of three independent experiments. *, P < 0.001, versus untreated control; **, P = 0.001, versus cells treated with CGS21680; +, P = 0.002, versus cells treated with DFO.

 
Hypoxia and iron deficiency activate HIF-1 by stabilizing the HIF-1 protein, but it has been shown that nonhypoxic stimuli increase HIF-1 levels by stimulating HIF-1 mRNA transcription and translation through kinase signaling pathways [9 ]. We analyzed the involvement of the PKC and PI-3K pathways, as they are stimulated by adenosine [27 ] and required for HIF-1 activation [12 ]. Treatment with CGS21680 did not modify the activity of PKC-{alpha}, β1, or β2 isoenzymes (data not shown) but induced membrane translocation of PKC-{delta} (Fig. 5A ) and increased its activity in the membrane fraction by ~70% (Table 1 ). Akt phosphorylation, which was used as a reporter for PI-3K activity, was increased promptly by exposure to CGS21680, whereas total Akt content remained unchanged (Fig. 5B) . To investigate the role of these pathways in HIF-1 activation, we performed luciferase expression experiments using the HRE multimer construct. The presence of wortmannin and chelerythrine, specific inhibitors of PI-3K and PKC, respectively, prevented the CGS21680-induced stimulation of the transactivation capacity of HIF-1, and addition of the inhibitors alone had no effect (Fig. 5C) . Immunoblot experiments indicated that the modulation of HIF-1 transactivation capacity was paralleled by changes in HIF-1 protein levels (Fig. 5D) . Altogether, these results indicated that the induction of HIF-1 activity by CGS21680 requires PI-3K and PKC activity.


Figure 5
View larger version (28K):
[in this window]
[in a new window]

  		Figure 5. Involvement of the PKC and PI-3K pathways in HIF-1 induction by CGS21680. (A) The levels of PKC-{delta} were estimated by means of Western blotting in cytosolic and membrane extracts prepared from untreated cells or cells treated with 5 µM CGS21680 for 30 min. The figure shows one blot representative of three independent experiments. The values indicate the fold difference ± SE in relation to the untreated control. (B) Equal amounts of cytosolic and membrane extracts prepared from untreated cells or cells exposed to CGS21680 for 15 or 30 min were separated and probed with antibodies against the phosphorylated or nonphosphorylated form of Akt. The figure shows one blot representative of four independent experiments. The values indicate the fold difference ± SE in relation to the untreated control. (C) J774A.1 cells were transiently transfected with a construct in which luciferase is controlled by a HRE multimer and were left untreated or exposed to 5 µM CGS21680 for 4 h in the presence or absence of wortmannin (wortm) and chelerythrine (cheler), as described in Materials and Methods. The cells were cotransfected with a control vector containing the Renilla luciferase gene. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity, and normalized to the activity recorded in untreated cells, arbitrarily defined as 1. Mean values ± SE of three independent experiments. *, P < 0.001, versus untreated control; **, P < 0.001, versus cells treated with CGS21680. (D) Immunoblot analysis of nuclear extracts from J774A.1 cells left untreated or treated for 4 h with 5 µM CGS21680 in the absence or presence of wortmannin and chelerythrine. TFIID was used as a loading control. The values indicate the fold difference ± SE in relation to the control. All of the results shown are representative of three independent experiments.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Effect of CGS21680 on PKC Activity

 
HIF-1 up-regulation involves transcriptional and translational events
To examine the molecular mechanisms involved in the induction of HIF-1, we determined HIF-1 mRNA expression. Northern blot analysis (Fig. 6A ) showed that the exposure of J774A.1 cells to CGS21680 led to a time-dependent increase in steady-state HIF-1 mRNA levels after 4–6 h of treatment. To investigate HIF-1 gene transcription further and verify whether CGS21680 also favored HIF-1 mRNA translation, we performed transient transfection experiments using two reporter constructs (Fig. 6B) : one in which the luciferase was controlled by the 5' flanking and 5'-UTR regions of the HIF-1 gene (pHIF-1A) and a shorter one in which the 5'-UTR region, important for HIF-1 mRNA translation [10 12 ], was removed (pHIF-1B). Exposure to CGS21680 doubled the luciferase activity of pHIF-1B (Fig. 6C) , thus demonstrating the role of transcriptional activation and confirming the result of the Northern blot analysis. However, the response to CGS21680 was much greater in the cells transfected with the plasmid pHIF-1A encompassing the entire HIF-1 regulatory region (Fig. 6C) , thus indicating that the 5'-UTR of HIF-1 mRNA is involved in the CGS21680-induced expression of a heterologous protein at the translational level.


Figure 6
View larger version (11K):
[in this window]
[in a new window]

  		Figure 6. CGS21680 increases HIF-1 mRNA transcription and translation. (A) Northern blot analysis: a filter with equal amounts of total RNA (as revealed by the ethidium bromide fluorescence of rRNA) extracted from untreated cells or cells treated with 5 µM CGS21680 for different times was hybridized with HIF-1 cDNA. The result shown is representative of three separate experiments. The values indicate the fold difference ± SE in relation to the untreated control. (B) Structure of the HIF-1 constructs used in the transfection experiments. The reporter plasmids contained a 856- or 604-bp fragment of the human HIF-1 promoter with (pHIF-1A) or without (pHIF-1B) the 5'-UTR region cloned in front of the luciferase gene (Luc). (C) J774A.1 cells were transiently transfected with the reporter plasmids shown in B and were left untreated or exposed to 5 µM CGS21680 for 4 h. The cells were cotransfected with a control vector containing the Renilla luciferase gene. Luciferase activity was determined after 24 h, corrected for transfection efficiency on the basis of Renilla luciferase activity, and normalized to the activity recorded in untreated cells, arbitrarily defined as 1. Mean values ± SE of three independent experiments. *, P < 0.001, versus the corresponding untreated control.

 
Effect of A2A receptor activation on the expression of HIF-1 target genes in J774A.1 macrophages
To investigate further the transcriptional function of activated HIF-1 in CGS21680-treated cells, we examined the expression of endogenous genes known to be under the transcriptional control of HIF-1. The Northern blot in Figure 7A shows that aldolase and PGK-1 mRNA levels increased after 4 and 6 h of CGS21680 treatment. Real-time PCR analysis of VEGF mRNA showed a strong increase after 4 h exposure to CGS1680 (Fig. 7B) . These results indicate that adenosine triggers the expression of genes, which may have a positive effect on macrophage survival and function. To verify whether the adenosine-dependent stimulation of HIF-1 activity was involved in modulating the synthesis of specific macrophage products, we analyzed the expression of the CXCR4 chemokine receptor, which has been shown to be regulated by HIF-1{alpha} [29 36 ]. Moreover, we examined TNF-{alpha} mRNA, as its induction in response to LPS is greater under hypoxic conditions [37 38 ], and its activation by bacterial infection is impaired in HIF-1{alpha}–/– macrophages [15 ]. In J774A.1 cells exposed to CGS21680 for 6 h, CXCR4 and TNF-{alpha} transcript levels (Fig. 7B) were not modulated, but as expected, a significant increase of CXCR4 and TNF-{alpha} mRNA was observed in cells stimulated with LPS, used as a positive control.


Figure 7
View larger version (23K):
[in this window]
[in a new window]

  		Figure 7. Effects of treatment with CGS21680 on the levels of hypoxia-responsive genes. (A) Northern blot analysis: a filter with equal amounts of total RNA (as revealed by the ethidium bromide fluorescence of rRNA), extracted from untreated cells or cells treated with 5 µM CGS21680 for different times, was hybridized with aldolase and PGK-1 cDNA probes. The result shown is representative of three separate experiments. The values indicate the fold difference ± SE in relation to the untreated control. (B) Real-time PCR analysis of VEGF, CXCR4, and TNF-{alpha} mRNA levels. Total RNA extracted from untreated cells or cells treated with 5 µM CGS21680 for 6 h or LPS for 20 h were quantified by real-time PCR as described in Materials and Methods. The levels in untreated cells were arbitrarily defined as 1. Mean values ± SE of three independent experiments. *, P = 0.002, versus the corresponding, untreated control; **, P = 0.005, versus the corresponding untreated control.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
One of the consequences of the local tissue hypoxia caused by inflammation or tissue damage is the extracellular accumulation of adenosine released by the damaged cells, which is involved physiologically in protecting tissues from acute inflammatory injury [17 ]. Adenosine exerts this cytoprotective effect by increasing oxygen supply, triggering anti-inflammatory responses, preconditioning, and promoting angiogenesis [16 ]. The results of the present study demonstrate that adenosine also modulates macrophage functions by means of the normoxic induction of HIF-1 in the J774A.1 cell line and in peritoneal macrophages. Exposure of J774A.1 macrophages to adenosine receptor agonists and antagonists showed that A2A receptors initiate the signaling pathway leading to HIF-1 activation. It has been shown recently that adenosine interaction with the A3 receptor stimulates HIF-1 expression in melanoma and glioblastoma cells incubated under hypoxic conditions [39 40 ] but that stimulation of the A3 receptor alone was not sufficient to induce HIF-1. Conversely, we demonstrate here that in different experimental settings, adenosine is able to trigger HIF-1 expression and activity under normoxic conditions.

Although hypoxia is the main activating factor of HIF-1, an increasing body of evidence indicates that this transcription factor may also be up-regulated by a number of nonhypoxic stimuli, such as stimulation with growth factors, cytokines, hormones, or exposure to NO and LPS [9 13 41 42 ]. It is interesting that the mechanisms involved in activating HIF-1 under hypoxic and nonhypoxic conditions are strikingly different, insofar as unlike hypoxia, nonhypoxic stimuli do not induce HIF-1 by means of HIF-1{alpha} stabilization. Studies carried out by a number of laboratories have found that the predominant mechanism in this induction is an increase in HIF-1{alpha} protein translation [9 ], and it has also been demonstrated that HIF-1{alpha} mRNA levels can be increased by angiotensin II, hepatocyte growth factor, and LPS [10 12 13 43 ].

The same transcriptional and post-transcriptional mechanisms also seem to play a role in adenosine-mediated HIF-1 activation, although we did not investigate the mechanisms involved in HIF-1 degradation and hence, cannot exclude that adenosine also affects HIF-1 protein stability. Indeed, our Northern blot analysis revealed that CGS21680 exposure increased HIF-1 mRNA levels, and transfection experiments with luciferase under the control of the HIF-1 promoter region showed that the adenosine/A2A receptor pathway increases HIF-1 transcription. Comparison of the adenosine-mediated activation of luciferase activity in cells transfected with two different constructs (with or without the 5'-UTR region, which enhances the translation efficiency of HIF-1 mRNA [12 ]) indicates that a further mechanism leading to the full activation of HIF-1 in cells exposed to CGS21680 is an increased translation of HIF-1 mRNA molecules, which may account for the presence of active HIF-1 at 2 h of CGS21680 treatment when HIF-1 mRNA up-regulation is still absent. Cooperation of these two molecular mechanisms may lead to the strong up-regulation of nuclear accumulation, DNA-binding activity, and transactivation capacity reported here. Our findings are in line with recent results showing that adenosine stimulates the translation of IL-10 mRNA [44 ], which indicates that post-transcriptional mechanisms are important in the adenosine-mediated modulation of gene expression. In a recent work, demonstrating that the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) and LPS stimulate VEGF expression synergestically in macrophages by activating HIF-1, NECA alone did not transcriptionally induce HIF-1 levels [45 ]. The discrepancy between these results and those reported in the present study may possibly arise from the different time length of exposure to the adenosine receptor agonists. In fact, Ramanathan and colleagues [45] examined HIF-1 expression and activity in macrophages treated for 12–24 h, whereas we found that the A2A receptor-mediated effects on HIF-1 are transient and peak at 2–4 h (see Figs. 1 2 3 ).

The role of iron availability in HIF-1 regulation has been demonstrated by studies of the effect of its removal by exogenous chelators [30 ] and of the consequences of blocking iron uptake via the transferrin receptor [46 ]. However, on the basis of our determinations of the activity of the "iron sensor" IRPs (which is related closely and inversely to iron levels in the labile pool) [34 35 ] and the additive effects of CGS21680 and DFO in our transfection experiments, adenosine-mediated HIF-1 induction does not involve any changes in intracellular iron levels. This seems to be different from the decreased iron availability, which has been reported recently to play a role in the normoxic induction of HIF-1 in differentiating U937 cells [47 ].

Conversely, and as demonstrated in the case of other nonhypoxic, HIF-1-inducing stimuli [9 ], the up-regulation of HIF-1 by adenosine depends largely on the activation of the PKC and PI-3K pathways and their downstream effectors. We found increased levels of membrane PKC translocation and greater Akt activity soon after the CGS21680 stimulation of J774A.1 cells. Moreover, the transactivating capacity of HIF-1 was lost when specific inhibitors of these kinases were included in transfection experiments. Activation of these pathways through the A2A receptor seems to play a role in adenosine-mediated signaling, also in other cell types; in fact, it has been demonstrated that PKC [27 ] and PI-3K [28 ] are involved in adenosine-induced hepatocyte preconditioning and in the transduction of signals mediated by A2A receptors in neuronal cells [48 49 50 ].

Analysis of adenosine effect on gene expression allowed us to show that typical HIF-1 target genes, such as the glycolytic enzymes, are up-regulated in macrophages after CGS21680 treatment, and so, adenosine-mediated HIF-1 induction may play a role in the adaptation of macrophages to hypoxic conditions; similarly, the up-regulation of VEGF may make an important contribution to macrophage-dependent angiogenic activity. Our findings generally agree with the demonstration that adenosine strengthens the HIF-1-mediated activation of VEGF expression in hypoxic glioblastoma cells via the A3 receptor [40 ] but show that the macrophage response, which is mediated through a different signaling pathway and by means of different molecular mechanisms, also takes place under normoxic conditions.

The present study contributes to the increasing recognition of a link between HIF-1 and the metabolism of adenine nucleotides. In fact, the HIF-1-dependent activation of CD73 [51 ] and repression of equilibrative nucleoside transporters [52 ], which generate and clear extracellular adenosine, respectively, may lead to an expansion of the extracellular adenosine pool. Moreover, HIF-1-mediated induction of the adenosine A2B receptor [53 ] and the adenosine A2A receptor-dependent induction of HIF-1 reported here may represent examples of complementary mechanisms aimed at amplifying adenosine signaling. Our findings are consistent with the view that HIF-1 is essential for myeloid cells, as is demonstrated by the impaired myeloid functions and deficient inflammatory response found in macrophages and neutrophils after targeted HIF-1 inactivation [14 15 ]. Consistent with this, increased HIF-1 expression during monocyte differentiation to macrophages [54 ] may promote the adaptation to low oxygen levels found frequently at sites of inflammation.

Conversely, our limited analysis of inflammation-related HIF-1 target genes showed that TNF-{alpha} and CXCR4 are not responsive to HIF-1 in CGS21680-treated macrophages. In this regard, it is still not clear whether hypoxia is pro- or anti-inflammatory, as its potent anti-inflammatory effects in the case of lung injury [55 ] contrast with the demonstration that the appropriate expression of HIF target genes is essential for mounting a powerful inflammatory response [14 15 ]. Conversely, adenosine is generally considered a protective and anti-inflammatory molecule, as it inhibits the signaling pathways required for the synthesis and secretion of proinflammatory cytokines and attenuates the inflammatory process [17 ].

Although not conclusive, our data suggest that adenosine-mediated HIF-1 activation also plays an anti-inflammatory role, as it did not activate TNF-{alpha}, a typical product of stimulated macrophages, which is in line with previous evidence showing that adenosine prevents LPS-induced TNF-{alpha} formation [17 ]. Furthermore, the expression of the CXCR4 chemokine receptor, which is involved in leukocyte recruitment, was unchanged in the cells exposed to the adenosine receptor agonist CGS21680, a finding that is in line with the recent demonstration that adenosine desensitizes chemokine receptors and thus inhibits leukocyte trafficking [56 ].

In conclusion, the results of our study demonstrate a connection between HIF-1 activation and the accumulation of extracellular adenosine, two events that have been predicted to play a role in triggering the biochemical changes in immune cells, which are crucial for regulating the immune response [1 57 ]. Adenosine is released by hypoxic/damaged parenchymal cells and acts on neighboring inflammatory cells to activate HIF-1 and may act as a means of amplifying the role of local hypoxia as a driving force in the down-modulation of immune and inflammatory responses.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from Ministero dell’Istruzione, dell’Università e della Ricerca (FIRB, Cofin), to G. C. and R. C., from MIUR-FISR to G. C., and from Regione Piemonte to R. C. We gratefully acknowledge D. Taramelli and N. Basilico for help with the peritoneal macrophage preparation. We thank D. E. Richard, P. J. Ratcliffe, M. Schwarz, R. D. Thornton, and L. Kuhn for providing the plasmids.

Received January 24, 2007; revised March 28, 2007; accepted April 13, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Sitkovsky, M., Lukashev, D. (2005) Regulation of immune cells by local-tissue oxygen tension: HIF1 {alpha} and adenosine receptors Nat. Rev. Immunol. 5,712-721[CrossRef][Medline]
  2. Semenza, G. L. (2001) Hypoxia-inducible factor 1: oxygen homeostasis and disease pathophysiology Trends Mol. Med. 7,345-350[CrossRef][Medline]
  3. Berra, E., Ginouves, A., Pouyssegur, J. (2006) The hypoxia-inducible-factor hydroxylases bring fresh air into hypoxia signaling EMBO Rep. 7,41-45[CrossRef][Medline]
  4. Fandrey, J., Gorr, T. A., Gassmann, M. (2006) Regulating cellular oxygen sensing by hydroxylation Cardiovasc. Res. 71,642-651[Abstract/Free Full Text]
  5. Masson, N., Ratcliffe, P. J. (2003) HIF prolyl and asparaginyl hydroxylases in the biological response to intracellular O(2) levels J. Cell Sci. 116,3041-3049[Abstract/Free Full Text]
  6. Kaelin, W. G. (2005) Proline hydroxylation and gene expression Annu. Rev. Biochem. 74,115-128[CrossRef][Medline]
  7. Semenza, G. L. (2003) Targeting HIF-1 for cancer therapy Nat. Rev. Cancer 3,721-732[CrossRef][Medline]
  8. Guzy, R. D., Hoyos, B., Robin, E., Chen, H., Liu, L., Mansfield, K. D., Simon, M. C., Hammerling, U., Schumacker, P. T. (2005) Mitochondrial complex III is required for hypoxia-induced ROS production and cellular oxygen sensing Cell Metab. 1,401-408[CrossRef][Medline]
  9. Dery, M. A., Michaud, M. D., Richard, D. E. (2005) Hypoxia-inducible factor 1: regulation by hypoxic and non-hypoxic activators Int. J. Biochem. Cell Biol. 37,535-540[CrossRef][Medline]
  10. Tacchini, L., De Ponti, C., Matteucci, E., Follis, R., Desiderio, M. A. (2004) Hepatocyte growth factor-activated NF-{kappa}B regulates HIF-1 activity and ODC expression, implicated in survival, differently in different carcinoma cell lines Carcinogenesis 25,2089-2100[Abstract/Free Full Text]
  11. Fukuda, R., Hirota, K., Fan, F., Jung, Y. D., Ellis, L. M., Semenza, G. L. (2002) Insulin-like growth factor 1 induces hypoxia-inducible factor 1-mediated vascular endothelial growth factor expression, which is dependent on MAP kinase and phosphatidylinositol 3-kinase signaling in colon cancer cells J. Biol. Chem. 277,38205-38211[Abstract/Free Full Text]
  12. Page, E. L., Robitaille, G. A., Pouyssegur, J., Richard, D. E. (2002) Induction of hypoxia-inducible factor-1{alpha} by transcriptional and translational mechanisms J. Biol. Chem. 277,48403-48409[Abstract/Free Full Text]
  13. Blouin, C. C., Page, E. L., Soucy, G. M., Richard, D. E. (2004) Hypoxic gene activation by lipopolysaccharide in macrophages: implication of hypoxia-inducible factor 1{alpha} Blood 103,1124-1130[Abstract/Free Full Text]
  14. Cramer, T., Yamanishi, Y., Clausen, B. E., Forster, I., Pawlinski, R., Mackman, N., Haase, V. H., Jaenisch, R., Corr, M., Nizet, V., Firestein, G. S., Gerber, H. P., Ferrara, N., Johnson, R. S. (2003) HIF-1{alpha} is essential for myeloid cell-mediated inflammation Cell 112,645-657[CrossRef][Medline]
  15. Peyssonnaux, C., Datta, V., Cramer, T., Doedens, A., Theodorakis, E. A., Gallo, R. L., Hurtado-Ziola, N., Nizet, V., Johnson, R. S. (2005) HIF-1{alpha} expression regulates the bactericidal capacity of phagocytes J. Clin. Invest. 115,1806-1815[CrossRef][Medline]
  16. Linden, J. (2005) Adenosine in tissue protection and tissue regeneration Mol. Pharmacol. 67,1385-1387[Abstract/Free Full Text]
  17. Hasko, G., Cronstein, B. N. (2004) Adenosine: an endogenous regulator of innate immunity Trends Immunol. 25,33-39[CrossRef][Medline]
  18. Pinhal-Enfield, G., Ramanathan, M., Hasko, G., Vogel, S. N., Salzman, A. L., Boons, G. J., Leibovich, S. J. (2003) An angiogenic switch in macrophages involving synergy between Toll-like receptors 2, 4, 7, and 9 and adenosine A(2A) receptors Am. J. Pathol. 163,711-721[Abstract/Free Full Text]
  19. Forsythe, J. A., Jiang, B. H., Iyer, N. V., Agani, F., Leung, S. W., Koos, R. D., Semenza, G. L. (1996) Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1 Mol. Cell. Biol. 16,4604-4613[Abstract]
  20. Carmeliet, P., Dor, Y., Herbert, J. M., Fukumura, D., Brusselmans, K., Dewerchin, M., Neeman, M., Bono, F., Abramovitch, R., Maxwell, P., Koch, C. J., Ratcliffe, P., Moons, L., Jain, R. K., Collen, D., Keshert, E. (1998) Role of HIF-1{alpha} in hypoxia-mediated apoptosis, cell proliferation and tumor angiogenesis Nature 394,485-490[CrossRef][Medline]
  21. Taramelli, D., Recalcati, S., Basilico, N., Olliaro, P., Cairo, G. (2000) Macrophage preconditioning with synthetic malaria pigment reduces cytokine production via heme iron-dependent oxidative stress Lab. Invest. 80,1781-1788[Medline]
  22. Tacchini, L., Dansi, P., Matteucci, E., Desiderio, M. A. (2001) Hepatocyte growth factor signaling stimulates hypoxia inducible factor-1 (HIF-1) activity in HepG2 hepatoma cells Carcinogenesis 22,1363-1371[Abstract/Free Full Text]
  23. Bianchi, L., Tacchini, L., Cairo, G. (1999) HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation Nucleic Acids Res. 27,4223-4227[Abstract/Free Full Text]
  24. Tacchini, L., Matteucci, E., De Ponti, C., Desiderio, M. A. (2003) Hepatocyte growth factor signaling regulates transactivation of genes belonging to the plasminogen activation system via hypoxia inducible factor-1 Exp. Cell Res. 290,391-401[CrossRef][Medline]
  25. Cairo, G., Tacchini, L., Pogliaghi, G., Anzon, E., Tomasi, A., Bernelli-Zazzera, A. (1995) Induction of ferritin synthesis by oxidative stress. Transcriptional and post-transcriptional regulation by expansion of the "free" iron pool J. Biol. Chem. 270,700-703[Abstract/Free Full Text]
  26. Mullner, E. W., Neupert, B., Kuhn, L. C. (1989) A specific mRNA binding factor regulates the iron-dependent stability of cytoplasmic transferrin receptor mRNA Cell 58,373-382[CrossRef][Medline]
  27. Carini, R., De Cesaris, M. G., Splendore, R., Vay, D., Domenicotti, C., Nitti, M. P., Paola, D., Pronzato, M. A., Albano, E. (2001) Signal pathway involved in the development of hypoxic preconditioning in rat hepatocytes Hepatology 33,131-139[CrossRef][Medline]
  28. Carini, R., Grazia De Cesaris, M., Splendore, R., Baldanzi, G., Nitti, M. P., Alchera, E., Filigheddu, N., Domenicotti, C., Pronzato, M. A., Graziani, A., Albano, E. (2004) Role of phosphatidylinositol 3-kinase in the development of hepatocyte preconditioning Gastroenterology 127,914-923[CrossRef][Medline]
  29. Schioppa, T., Uranchimeg, B., Saccani, A., Biswas, S. K., Doni, A., Rapisarda, A., Bernasconi, S., Saccani, S., Nebuloni, M., Vago, L., Mantovani, A., Melillo, G., Sica, A. (2003) Regulation of the chemokine receptor CXCR4 by hypoxia J. Exp. Med. 198,1391-1402[Abstract/Free Full Text]
  30. Wang, G. L., Semenza, G. L. (1993) Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: implications for models of hypoxia signal transduction Blood 82,3610-3615[Abstract/Free Full Text]
  31. Kvietikova, I., Wenger, R. H., Marti, H. H., Gassmann, M. (1995) The transcription factors ATF-1 and CREB-1 bind constitutively to the hypoxia-inducible factor-1 (HIF-1) DNA recognition site Nucleic Acids Res. 23,4542-4550[Abstract/Free Full Text]
  32. Agani, F., Semenza, G. L. (1998) Mersalyl is a novel inducer of vascular endothelial growth factor gene expression and hypoxia-inducible factor 1 activity Mol. Pharmacol. 54,749-754[Abstract/Free Full Text]
  33. Nemeth, Z. H., Leibovich, S. J., Deitch, E. A., Sperlagh, B., Virag, L., Vizi, E. S., Szabo, C., Hasko, G. (2003) Adenosine stimulates CREB activation in macrophages via a p38 MAPK-mediated mechanism Biochem. Biophys. Res. Commun. 312,883-888[CrossRef][Medline]
  34. Hentze, M. W., Kuhn, L. C. (1996) Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide, and oxidative stress Proc. Natl. Acad. Sci. USA 93,8175-8182[Abstract/Free Full Text]
  35. Cairo, G., Pietrangelo, A. (2000) Iron regulatory proteins in pathobiology Biochem. J. 352,241-250[CrossRef][Medline]
  36. Staller, P., Sulitkova, J., Lisztwan, J., Moch, H., Oakeley, E. J., Krek, W. (2003) Chemokine receptor CXCR4 downregulated by von Hippel-Lindau tumor suppressor pVHL Nature 425,307-311[CrossRef][Medline]
  37. Guida, E., Stewart, A. (1998) Influence of hypoxia and glucose deprivation on tumor necrosis factor-{alpha} and granulocyte-macrophage colony-stimulating factor expression in human cultured monocytes Cell. Physiol. Biochem. 8,75-88[CrossRef][Medline]
  38. Demasi, M., Cleland, L. G., Cook-Johnson, R. J., Caughey, G. E., James, M. J. (2003) Effects of hypoxia on monocyte inflammatory mediator production: dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesis J. Biol. Chem. 278,38607-38616[Abstract/Free Full Text]
  39. Merighi, S., Benini, A., Mirandola, P., Gessi, S., Varani, K., Leung, E., MacLennan, S., Baraldi, P. G., Borea, P. A. (2005) A3 adenosine receptors modulate hypoxia-inducible factor-1{alpha} expression in human A375 melanoma cells Neoplasia 7,894-903[CrossRef][Medline]
  40. Merighi, S., Benini, A., Mirandola, P., Gessi, S., Varani, K., Leung, E., Maclennan, S., Borea, P. A. (2006) Adenosine modulates vascular endothelial growth factor expression via hypoxia-inducible factor-1 in human glioblastoma cells Biochem. Pharmacol. 72,19-31[CrossRef][Medline]
  41. Sandau, K. B., Fandrey, J., Brune, B. (2001) Accumulation of HIF-1{alpha} under the influence of nitric oxide Blood 97,1009-1015[Abstract/Free Full Text]
  42. Jung, Y., Isaacs, J. S., Lee, S., Trepel, J., Liu, Z. G., Neckers, L. (2003) Hypoxia-inducible factor induction by tumor necrosis factor in normoxic cells requires receptor-interacting protein-dependent nuclear factor {kappa} B activation Biochem. J. 370,1011-1017[CrossRef][Medline]
  43. Frede, S., Stockmann, C., Freitag, P., Fandrey, J. (2006) Bacterial lipopolysaccharide induces HIF-1 activation in human monocytes via p44/42 MAPK and NF-{kappa}B Biochem. J. 396,517-527[CrossRef][Medline]
  44. Nemeth, Z. H., Lutz, C. S., Csoka, B., Deitch, E. A., Leibovich, S. J., Gause, W. C., Tone, M., Pacher, P., Vizi, E. S., Hasko, G. (2005) Adenosine augments IL-10 production by macrophages through an A2B receptor-mediated posttranscriptional mechanism J. Immunol. 175,8260-8270[Abstract/Free Full Text]
  45. Ramanathan, M., Pinhal-Enfield, G., Hao, I., Leibovich, S. J. (2007) Synergistic up-regulation of vascular endothelial growth factor (VEGF) expression in macrophages by adenosine A2A receptor agonists and endotoxin involves transcriptional regulation via the hypoxia response element in the VEGF promoter Mol. Biol. Cell 18,14-23[Abstract/Free Full Text]
  46. Jones, D. T., Trowbridge, I. S., Harris, A. L. (2006) Effects of transferrin receptor blockade on cancer cell proliferation and hypoxia-inducible factor function and their differential regulation by ascorbate Cancer Res. 66,2749-2756[Abstract/Free Full Text]
  47. Knowles, H. J., Mole, D. R., Ratcliffe, P. J., Harris, A. L. (2006) Normoxic stabilization of hypoxia-inducible factor-1{alpha} by modulation of the labile iron pool in differentiating U937 macrophages: effect of natural resistance-associated macrophage protein 1 Cancer Res. 66,2600-2607[Abstract/Free Full Text]
  48. Huang, N. K., Lin, Y. W., Huang, C. L., Messing, R. O., Chern, Y. (2001) Activation of protein kinase A and atypical protein kinase C by A(2A) adenosine receptors antagonizes apoptosis due to serum deprivation in PC12 cells J. Biol. Chem. 276,13838-13846[Abstract/Free Full Text]
  49. Mori, Y., Higuchi, M., Masuyama, N., Gotoh, Y. (2004) Adenosine A2A receptor facilitates calcium-dependent protein secretion through the activation of protein kinase A and phosphatidylinositol-3 kinase in PC12 cells Cell Struct. Funct. 29,101-110[CrossRef][Medline]
  50. Canals, M., Angulo, E., Casado, V., Canela, E. I., Mallol, J., Vinals, F., Staines, W., Tinner, B., Hillion, J., Agnati, L., Fuxe, K., Ferre, S., Lluis, C., Franco, R. (2005) Molecular mechanisms involved in the adenosine A and A receptor-induced neuronal differentiation in neuroblastoma cells and striatal primary cultures J. Neurochem. 92,337-348[CrossRef][Medline]
  51. Synnestvedt, K., Furuta, G. T., Comerford, K. M., Louis, N., Karhausen, J., Eltzschig, H. K., Hansen, K. R., Thompson, L. F., Colgan, S. P. (2002) Ecto-5'-nucleotidase (CD73) regulation by hypoxia-inducible factor-1 mediates permeability changes in intestinal epithelia J. Clin. Invest. 110,993-1002[CrossRef][Medline]
  52. Eltzschig, H. K., Abdulla, P., Hoffman, E., Hamilton, K. E., Daniels, D., Schonfeld, C., Loffler, M., Reyes, G., Duszenko, M., Karhausen, J., Robinson, A., Westerman, K. A., Coe, I. R., Colgan, S. P. (2005) HIF-1-dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia J. Exp. Med. 202,1493-1505[Abstract/Free Full Text]
  53. Kong, T., Westerman, K. A., Faigle, M., Eltzschig, H. K., Colgan, S. P. (2006) HIF-dependent induction of adenosine A2B receptor in hypoxia FASEB J. 20,2242-2250[Abstract/Free Full Text]
  54. Oda, T., Hirota, K., Nishi, K., Takabuchi, S., Oda, S., Yamada, H., Arai, T., Fukuda, K., Kita, T., Adachi, T., Semenza, G. L., Nohara, R. (2006) Activation of hypoxia-inducible factor 1 during macrophage differentiation Am. J. Physiol. Cell Physiol. 291,C104-C113[Abstract/Free Full Text]
  55. Thiel, M., Chouker, A., Ohta, A., Jackson, E., Caldwell, C., Smith, P., Lukashev, D., Bittmann, I., Sitkovsky, M. V. (2005) Oxygenation inhibits the physiological tissue-protecting mechanism and thereby exacerbates acute inflammatory lung injury PLoS Biol. 3,e174[CrossRef][Medline]
  56. Zhang, N., Yang, D., Dong, H., Chen, Q., Dimitrova, D. I., Rogers, T. J., Sitkovsky, M., Oppenheim, J. J. (2006) Adenosine A2a receptors induce heterologous desensitization of chemokine receptors Blood 108,38-44[Abstract/Free Full Text]
  57. Sitkovsky, M. V., Ohta, A. (2005) The ‘danger’ sensors that STOP the immune response: