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Published ahead of print on August 31, 2005
J Am Soc Nephrol 16: 2897-2905, 2005
© 2005 American Society of Nephrology
doi: 10.1681/ASN.2004121051
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Cell Biology

Hypoxia Increases Group IIA Phospholipase A2 Expression under Inflammatory Conditions in Rat Renal Mesangial Cells

Claudia Petry, Andrea Huwiler, Wolfgang Eberhardt, Marietta Kaszkin and Josef Pfeilschifter

Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany

Address correspondence to: Dr. Josef Pfeilschifter, Pharmazentrum Frankfurt, Klinikum der Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. Phone: +49-69-6301-6951; Fax: +49-69-6301-7942; E-mail: pfeilschifter{at}em.uni-frankfurt.de

Received for publication December 7, 2004. Accepted for publication July 25, 2005.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Hypoxia evokes a common mechanism of oxygen sensing mediated by hypoxia-inducible transcription factors (HIF) in many mammalian cells. This study investigated the effect of hypoxia on group-IIA secretory phospholipase A2 (sPLA2-IIA) expression in renal mesangial cells. Stimulation of cells with IL-1{beta} under normoxic conditions (21% O2) is known to induce expression and secretion of the group sPLA2-IIA. This induction is further enhanced by constantly reducing the O2 concentration to 1% O2, and is accompanied by increased sPLA2 activity. To see whether hypoxia potentiates IL-1{beta}–induced sPLA2-IIA gene expression, a 2.67-kb fragment of the rat sPLA2-IIA promoter was fused to a luciferase reporter construct and used to transfect mesangial cells. Hypoxia alone is not able to activate the sPLA2 promoter, whereas it significantly enhances IL-1{beta}–stimulated promoter activity. A deletion mutant of the promoter that lacks the two putative hypoxia responsive elements (HRE) is devoid of the potentiating effect of hypoxia. Moreover, site-directed mutagenesis of either of the two HRE is sufficient to abolish the potentiating effect of hypoxia. Electrophoretic mobility shift assays show that HIF-2{alpha}, which is the only HIF subtype expressed in mesangial cells, binds to both HRE in the sPLA2-IIA promoter. In summary, the data show that in an inflammatory setting hypoxia is able to potentiate sPLA2-IIA expression and activity in renal mesangial cells, and thereby may critically contribute to enhanced formation of inflammatory lipid mediators seen in a diverse range of kidney diseases.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To ensure survival in a variety of environments, mammalian cells have evolved mechanisms to respond to decreased oxygen availability (hypoxia) and to restore oxygen homeostasis. Many physiologic responses to hypoxia are molecularly controlled by the transcription factors hypoxia-inducible factor (1) (HIF) -1, HIF-2 and HIF-3, heterodimeric proteins composed of HIF-1{alpha}, HIF-2{alpha} or HIF-3{alpha} subunits, which are the oxygen-sensitive components, and HIF-1{beta}, respectively. The latter, also denoted as the aryl hydrocarbon receptor nuclear translocator (ARNT), is constitutively expressed and, like the {alpha}-subunits of HIF, is a member of the basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS) family (1,2). Under ambient oxygen conditions, HIF-1{alpha} is continuously hydroxylated on a critical proline residue, and subsequently associates with the von Hippel Lindau (VHL) protein, which directs HIF-1{alpha} to degradation by the ubiquitin-proteasome pathway. Importantly, hypoxia slows down the degradation of HIF-1{alpha} because the enzyme responsible for HIF-1{alpha} hydroxylation, a prolyl hydroxylase, is oxygen-dependent and nonhydroxylated HIF-1{alpha} is not recognized by VHL protein. As a result HIF-1{alpha} accumulates and translocates into the nucleus where it binds to hypoxia-responsive elements (HRE) in the promoter of hypoxia target genes and activates their expression (3).

Usually, inflamed tissues have lower levels of oxygen than healthy tissues—they are hypoxic. This causes activation of HIF-1{alpha} and helps establish a rapid and fully developed inflammatory response. In this context, it is known that leukocytes, the key players of the innate immune system, have adapted to hypoxia and exhibit high degrees of lactate formation, even under normoxic conditions. In a recent study using conditional knockouts of HIF-1{alpha}, it was shown that HIF-1{alpha} controls the redness and swelling of injured tissues, as well as the ability of leukocytes to enter these sites (4).

Phospholipase A2 (PLA2) constitutes a superfamily of enzymes that specifically release fatty acids, often arachidonic acid, from the sn-2 position of membrane phospholipids for production of important lipid mediators such as prostaglandins, leukotrienes or platelet-activating factor (5). Eicosanoids largely contribute to the cardinal symptoms of inflammation and the rate-limiting step in their synthesis is the availability of free arachidonic (6,7). High levels of PLA2 have been found in serum and exudates from patients suffering from inflammatory diseases, like rheumatoid arthritis or acute pancreatitis and trauma (8,9). Particularly in diseases such as rheumatoid arthritis, atherosclerosis or glomerulonephritis hypoxia is a prevalent factor that impacts the recruitment and function of immune cells and other aspects of disease progression (4,10).

Mesangial cells are highly specialized smooth muscle–like cells located in the renal glomerulus (11) and are active players in the inflammatory response to glomerular injury. They cross-communicate with invading immune cells such as neutrophils or macrophages, and are involved in most pathologic processes of the renal glomerulus (6,12). Quiescent mesangial cells do not produce any inflammatory mediators constitutively. But upon exposure to proinflammatory stimuli, e.g., IL-1{beta} or TNF-{alpha} produced by professional immune cells, mesangial cells respond with three major events: (1) formation of proinflammatory mediators including IL-1{beta} and TNF-{alpha}, (2) increased proliferation, and (3) increased production of extracellular matrix components. Mesangial cells express at least four PLA2 subtypes either constitutively or after induction, including the cytosolic cPLA2 (13), the calcium-independent iPLA2 (14), the secretory group IIA sPLA2 (15,16) and the group V sPLA2 (17).

Previously, we reported that prostaglandin formation is amplified by hypoxia in mesangial cells (18,19). In this study, we show that hypoxia accelerates cytokine-induced sPLA2-IIA expression and activity in mesangial cells. The effect of hypoxia is mediated by HIF-2{alpha}, the predominant form of HIF subtypes in the glomerulus, which binds to two HRE elements in the sPLA2-IIA promoter sequence and enhances its gene transcription induced by IL-1{beta}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals
Redi prime II random prime labeling system, Nick columns, the prostaglandin E2 (PGE2) enzyme immunoassay, [{alpha}-32P]-CTP, [{gamma}-32P]-ATP, PGE2-ELISA, Hyperfilm MP and horseradish-coupled secondary antibodies were from Amersham Pharmacia Biotech (Freiburg, Germany); Trizol and all cell culture nutrients were from Invitrogen (Karlsruhe Germany); Effectene Transfection Reagent was from Qiagen (Hilden, Germany); the Dual Luciferase Reporter Assay System was provided by Promega (Mannheim, Germany); the QuikChange Site-Directed Mutagenesis kit was from Stratagene (Amsterdam, The Netherlands); 12-epi-scalaradial was obtained from Biomol GmbH (Hamburg, Germany); IL-1{beta} and CGP43182 (2-hydroxy-4-oxo-1,5-di-oxyspiro-5,5-undeco-2-ene-3-N-(2,4-dichlorophenyl)-carboxamide) were kindly provided by Novartis Pharma Ltd. (Basel, Switzerland); the polyclonal antibody against HIF-2{alpha} was from Novus Biologicals, (Littleton, CO); the monoclonal antibody against rat group IIA sPLA2 was generated as described (20).

Cell Culture
Cultivation and characterization of rat renal mesangial cells were performed as described previously (21). For the experiments in this study, passages 10 to 20 were used.

Cell Stimulation and Western Blot Analysis
Confluent cells were stimulated for the indicated time periods in DMEM containing 0.1 mg/ml fatty acid-free BSA. To stop the reaction, the medium was removed, and the cells were washed and homogenized exactly as described previously (14). The homogenate was centrifuged for 10 min at 14,000 x g, and the supernatant was taken for protein determination. Equal amounts of protein (50 to 100 µg) were subjected to SDS-PAGE. Secreted sPLA2-IIA was enriched from the culture supernatant by precipitation with 7% (weight/volume) TCA and dissolved in SDS-sample buffer without dithiothreitol. Protein transfer and Western blot analysis was performed as described previously (14) using polyclonal antibodies against HIF-2{alpha} at a dilution of 1:1000 or a monoclonal antibody against sPLA2-IIA at a dilution of 1:200.

Hypoxia
Oxygen concentrations of 4% were maintained using an incubator with CO2/O2 monitoring and CO2/N2 gas sources (Hereaus Instruments, Osterode, Germany). Hypoxic conditions of 1% were achieved in a humidified variable aerobic workstation (IN VIVO2 400, Ruskinn Technologies, Leeds, United Kingdom). The IN VIVO2 contains an oxygen sensor that continuously monitors the chamber oxygen tension. The appearence of the cells in hypoxia was indistinguishable from those maintained in normoxia by light microscopy.

Transfection and Luciferase Reporter Gene Assay
For transfection, rat renal mesangial cells were cultured in 6-well dishes and incubated for 24 h at 37°C as described previously (22). Thereafter, the cells were incubated in DMEM containing 0.1 mg/ml fatty acid-free BSA. In parallel, they were transfected with 400 ng of plasmid DNA (a 2.67-kb rat sPLA2-IIA promoter fragment fused into a luciferase reported gene-containing vector as described previously [23]) plus 100 ng Renilla luciferase DNA per well by use of the Effectene transfection reagent following the manufacturer’s recommendations. Values for the relative sPLA2-IIA promoter activity were calculated from the ratio of firefly/Renilla luciferase activities.

Site-Directed Mutagenensis
Mutations of the first HRE sequence at –490 to –494 and the second HRE sequence at –2225 to –2229 of the sPLA2-IIA promoter (accession number AF375595) (23) were introduced by replacing the CG in the ACGTG motifs by an A as described (24) using a site-directed mutagenesis kit and following the manufacturer’s instructions. The point mutations were verified by sequencing. The 0.405-kb deletion mutant comprising nucleotides –405 to +1, which lacks both HRE sites, was generated as described previously (25).

Electrophoretic Mobility Shift Assay
Preparation of nuclear extracts from cultured mesangial cells and subsequent electrophoretic mobility shift assay (EMSA) was performed as described (22). The primers used for EMSA are: for wild-type (wt)-HRE-1: 5'-ATGACCAAGTTCTATGAAG-3'; and for wt-HRE-2: 5'-ATGACTGACAATTAAGCAGC-3' (the underlined sequences represent the consensus sites). Competition experiments were performed by coincubation with different dilutions of a primer stock solution corresponding to 1000-, 5000-, and 10,000-fold excess (1000, 5000, 10,000 pmol) of unlabeled double-stranded oligonucleotide in the DNA-protein binding reaction. For the supershift experiments, 2 µl of a specific polyclonal HIF-2{alpha} antibody was preincubated for 20 h at 4°C before the binding reaction.

sPLA2 Activity Assay
Equal volumes of supernatants were taken for an in vitro assay using [14C]oleic acid-labeled Escherichia coli as a substrate (26) in a total volume of 0.2 ml including 20 mM Tris/HCl, pH 8.5, and 10 mM CaCl2. Samples were incubated for 30 min at 37°C and stopped by addition of 2.5 ml Dole reagent. Liberated [14C]-labeled fatty acids were extracted by adding 1.5 ml heptane and 1 ml water and then vigorously vortexed. The heptane phase was loaded onto a silica gel column and [14C]-labeled free fatty acids were eluted with diethylether and counted in a {beta}-counter.

PGE2 Determination
Equal volumes of supernatants were subjected to a PGE2-ELISA (Amersham) according to the manufacturer’s instructions.

Northern Blot Analysis
Total cellular RNA was extracted from mesangial cells using the Trizol reagent. 15 µg total RNA was separated on a 1.4% agarose/formaldehyde gel. Membranes used for RNA transfer were obtained from Biorad (Munich, Germany). After ultraviolet cross-linking, membranes were hybridized. The cDNA of the sPLA2-IIA fragment was radioactively labeled with [{alpha}32P]dCTP using the Redi prime II random prime labeling system.

Statistical Analyses
Statistical analyses were performed using one-way ANOVA, followed by a Bonferroni post hoc test for multiple comparisons (GraphPad InStat version 3.00 for Windows NT, GraphPad Software, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of Hypoxia on sPLA2 -IIA Expression and Activity
Exposure of renal mesangial cells to the proinflammatory cytokine IL-1{beta} is well known to trigger expression and secretion of sPLA2-IIA, which reaches a plateau after 24 h (14,16). Accordingly, mesangial cells were treated for 24 h in the presence or absence of IL-1{beta} (1 nM) at normoxic (21% O2) or hypoxic (10% to 1%) conditions. As shown in Figure 1, the induction of sPLA2-IIA protein expression by IL-1{beta} under normoxic conditions (21% O2) was significantly increased in a concentration-dependent manner when lowering the O2 content to 10%, 7%, 4%, and 1% (Figure 1A). A similar enhancement of the IL-1{beta} effect is also observed in the presence of cobalt chloride, which is known to mimic the effect of hypoxia and has been shown to induce HIF-1 expression (27). At concentrations of 10 µM to 150 µM, cobalt chloride increases the IL-1{beta} effect on sPLA2 protein expression in a dose-dependent manner (Figure 2). In the absence of IL-1{beta} neither hypoxia (Figure 1A) nor cobalt chloride (data not shown) have an effect on sPLA2-IIA protein expression.



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  		Figure 1. Effect of hypoxia on secretory phospholipase A2 (sPLA2-IIA) protein secretion in mesangial cells. Quiescent mesangial cells were stimulated for 24 h at 21%, 10%, 7%, 4% or 1% oxygen (O2) either in the absence (–) or presence (+) of IL-1{beta} (1 nM) as indicated. Thereafter, secreted proteins were separated by SDS-PAGE (13% acrylamide gel) and subjected to Western blot analysis using a monoclonal anti–sPLA2-IIA antibody at a dilution of 1:200 (A). Bands were visualized by the enhanced chemiluminescence (ECL) method according to the manufacturer’s instructions. Blots are representative of 3 independent experiments giving similar results. Bands were densitometrically evaluated (B) and data expressed as % of normoxic IL-1{beta} stimulation and are means ± SEM (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001 compared with the normoxic IL-1{beta} stimulated values.

 


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  		Figure 2. Effect of cobalt chloride (CoCl2) on sPLA2-IIA protein secretion in mesangial cells. Quiescent mesangial cells were stimulated for 24 h with either vehicle (control; –) or cobalt chloride in the absence (–) or presence (+) of IL-1{beta} (1 nM) as indicated. Thereafter, secreted proteins were concentrated by TCA precipitation, separated by SDS-PAGE (13% acrylamide gel) and subjected to Western blot analysis using a monoclonal anti–sPLA2-IIA antibody at a dilution of 1:200. Equal loading of proteins was ascertained by Ponceau S staining. Data are representative of three independent experiments giving similar results.

 
In parallel to the induced sPLA2-IIA protein expression, PGE2 formation is also increased under IL-1{beta} + hypoxia treatment compared with IL-1{beta} treatment alone (Table 1). However, when reducing the O2 content to 1%, PGE2 synthesis is completely abolished (Table 1), due to the fact that cyclooxygenases require a certain amount of O2 for their action.


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  		Table 1. Effect of hypoxia on prostaglandin E2 formation in mesangial cells.

 
PLA2 activity measured in the supernatant of cells also increases upon IL-1{beta} treatment and is further accelerated by combined IL-1{beta} + hypoxia (Figure 3, black bars). Surprisingly, a PLA2 activity in the supernatant also increases under hypoxia alone (Figure 3), suggesting that another PLA2 different from the sPLA2-IIA is released. In an attempt to characterize this PLA2 activity, various inhibitors were employed in an in vitro activity assay using hypoxia-stimulated supernatants. Because all subtypes of sPLA2 contain several disulfide bridges their activities can be blocked by reducing agents such as dithiothreitol (DTT), whereas cPLA2 is insensitive to DTT (28). As shown in Figure 3, the hypoxia-induced PLA2 activity is sensitive to 10 mM of DTT, suggesting that the enzyme belongs to the class of sPLA2. An inhibitory effect is also obtained in the presence of CGP43182, which inhibits sPLA2 activities without affecting the cPLA2 activity (29,30) (Figure 3). CGP43182 acts as a scavenger for the calcium ion located at the catalytic site of the sPLA2. Similarly, the potent and selective sPLA2 inhibitor 12-epi-scalaradial (31) reduces the hypoxia-induced PLA2 activity in vitro (Figure 3). When chelating the Ca2+ ions in the assay system with EGTA, the hypoxia-induced PLA2 activity is completely lost, which excludes a Ca2+-independent PLA2 (data not shown). Moreover, we found no change in cPLA2 protein expression in cell lysates exposed to hypoxia, further excluding the possibility that this enzyme could have been released (data not shown).



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  		Figure 3. Effects of various inhibitors on hypoxia-stimulated PLA2 activity in supernatants of mesangial cells. Supernatants from cells exposed for 24 h to 21% O2 or 1% O2 in the absence (control) or presence of IL-1{beta} (1 nM) were preincubated in vitro for 30 min at 37°C with either vehicle, dithiothreitol (DTT) (10 mM), CGP43182 (15 µM) or epi-scalaradial (15 µM) before the activity assay. Data are expressed as % of normoxic control and are means ± SEM (n = 3). ***P < 0.001 compared with the appropriate control values as indicated.

 
In the next step, sPLA2-IIA mRNA levels were investigated. Incubation of mesangial cells for 24 h with IL-1{beta} under normoxic conditions increases sPLA2-IIA mRNA steady-state levels, and this was further augmented under 4% O2 (Figure 4A) and 1% O2 (Figure 4B). Similarly, a marked dose-dependent enhancement of IL-1{beta}–induced sPLA2-IIA mRNA expression occurs in the presence of cobalt chloride (Figure 4C). Neither hypoxia nor cobalt chloride induced sPLA2-IIA mRNA levels in the absence of IL-1{beta} (Figure 4).



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  		Figure 4. Effects of hypoxia and CoCl2 on sPLA2-IIA mRNA steady-state levels in mesangial cells. Quiescent mesangial cells were stimulated for 24 h at 21% O2 (A and B), 4% O2 (A), 1% O2 (B), or with CoCl2 (C) in the absence (control, –) or presence (+) of IL-1{beta} (1 nM). RNA was extracted and Northern blot analysis was performed using a probe for rat sPLA2-IIA as described in Materials and Methods. Equal loading was ascertained by a final comparison to an 18S ribosomal RNA probe. The data shown are representative for three independent experiments giving similar results.

 
Effect of Hypoxia on sPLA2-IIA Promoter Activity
It has been reported that HIF-2{alpha} is the predominant form of the HIF subtypes found in glomerular cells and peritubular fibroblasts in the kidney (32,33). Consequently, HIF-2{alpha} is the responsible regulator of erythropoietin expression, which mainly takes place in the peritubular fibroblasts. Under normoxic conditions our cultures of rat mesangial cells only express a minor amount of HIF-2{alpha} protein constitutively, due to the rapid turnover and degradation of HIF-2{alpha} under normoxia. However, exposure of cells for 24 h to hypoxia (1% O2) leads to a pronounced increase of HIF-2{alpha} protein expression (Figure 5). HIF-1{alpha} is not detectable under any conditions in mesangial cells (data not shown). Furthermore, IL-1{beta} does not increase HIF-2{alpha} expression neither under normoxic nor hypoxic conditions (Figure 5).



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  		Figure 5. Effect of hypoxia on hypoxia-inducible factor (HIF)-2{alpha} protein expression in mesangial cells. Quiescent mesangial cells were exposed for 24 h to 21% O2 or 1% O2 in the absence (–) or presence (+) of IL-1{beta} (1 nM). Thereafter, cells were lysed and protein extracts were separated on SDS-PAGE (8% acrylamide gel) and subjected to Western blot analysis using a specific polyclonal antiserum against HIF-2{alpha} at a dilution of 1:1000. Bands were visualized by the ECL method according to the manufacturer’s instructions. Data are representative of at least three independent experiments giving similar results.

 
To evaluate whether the hypoxia-induced potentiation of IL-1{beta}–triggered sPLA2-IIA mRNA steady-state levels results from an increased rate of sPLA2-IIA gene transcription mediated by HIF-2{alpha}, we investigated the promoter activity of sPLA2-IIA. In addition to a multitude of putative cis-acting elements involved in regulation of sPLA2-IIA expression, we found two regions that show a high homology to HRE motifs in the sPLA2-IIA gene promoter, denoted as HRE-1 (–490 to –494) and HRE-2 (–2225 to –2229).

Transient transfection of mesangial cells with a 2.67-kb fragment of the rat sPLA2-IIA promoter fused into a luciferase reporter gene construct (23) reveals that IL-1{beta} under normoxic conditions increases the promoter activity as described previously (23). Under hypoxic conditions (1% O2) alone, no enhanced promoter activity is detected (Figure 6A). However, when hypoxia is combined with IL-1{beta} treatment, a significant increase of promoter activity is observed compared with normoxic IL-1{beta}–treated samples (Figure 6A).



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  		Figure 6. Effect of hypoxia on IL-1{beta}–induced sPLA2-IIA promoter activity in mesangial cells. Subconfluent mesangial cells were co-transfected with the wild-type 2.67-kb sPLA2-IIA promoter DNA (A), with the {Delta}HRE-1 DNA (B), the {Delta}HRE-2 DNA (C), or with the 0.405-kb deletion mutant ({Delta}HRE-1 + {Delta}HRE-2) (D), plus 0.2 µg of a plasmid containing the Renilla luciferase gene coupled with a cytomeglovirus promoter (pRL-CMV). After overnight transfection, cells were exposed for an additional 24 h to either 21% O2 (black bars) or 1% O2 (open bars) in the absence (Co) or presence of IL-1{beta} (1 nM). The values for the beetle luciferase activity were related to values for Renilla luciferase activity and are depicted as relative luciferase activities. Values are means ± SEM (n = 3). HRE indicates hypoxia responsive elements. *P < 0.05, compared with the normoxic IL-1{beta} value.

 
To test for a functional role of the two putative HRE in the amplification of the IL-1{beta}–induced sPLA2-IIA promoter activity, each HRE was mutated as described (24). Previous studies have documented that similar mutations within the HIF-1 binding site of the hypoxia-reponsive erythropoietin enhancer abolish both binding of and transcriptional activation by HIF-1 (24,34). For both mutations the IL-1{beta}–stimulated promoter activity is still seen, but the enhancement by combined hypoxia is lost (Figure 6, B and C), suggesting that each of the HRE sites is functional and essential to obtain an amplified sPLA2-IIA promoter activity in response to hypoxia.

Furthermore, a 0.4-kb deletion mutant of the sPLA2-IIA promoter ({Delta}HRE-1 + {Delta}HRE-2), consisting only of nucleotides –405 to +1 (23) and thus lacking both HRE sites (Figure 6D), is still activated by IL-1{beta} but shows no enhancement in the presence of hypoxia (Figure 6D).

Electrophoretic Mobility Shift Analysis of HRE Binding Sites of sPLA2 -IIA Promoter
To further determine whether hypoxia triggers binding of HIF-2{alpha} to the putative HRE in the sPLA2-IIA promoter, we performed EMSA using radioactively-labeled oligonucleotides recognizing the putative HRE-1 or HRE-2 binding sites, respectively. Cells were exposed to either 21% O2 or 1% O2 for 4 h and EMSA were performed with nuclear extracts. At 21% O2 (control cells), several complexes were obtained for both HRE oligonuleotides as shown in Figure 7. Importantly, the intensity of DNA binding seen for IL-1{beta} is strongly increased under hypoxic conditions compared with normoxic conditions. Hypoxia selectively increases two main complexes formed with the HRE-1 oligonucleotide (Figure 7A, arrows) and also with the HRE-2 oligonucleotide (Figure 7B, arrows). Addition of cold HRE-1 or HRE-2 oligonucleotides competes concentration-dependently for binding and prevents complex formation. To determine whether HIF-2{alpha} is part of these complexes, we performed supershift analysis. Incubation of extracts with a rabbit anti-HIF-2{alpha} antibody completely blocks complex formation (Figure 7, A and B). However, there was no appearance of a supershifted complex. This may be explained by the fact that the anti-HIF-2{alpha} antibody, when binding HIF-2{alpha}, masks the DNA-binding site of HIF-2{alpha} and thus prevents the formation of labeled supershifted complexes. In summary, these data strongly suggest that HIF-2{alpha} is the primary hypoxia-induced factor that binds to HRE-1 and HRE-2 in the sPLA2-IIA promoter.



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  		Figure 7. Electrophoretic mobility shift assay of HIF-2{alpha} binding to HRE-1 and HRE-2 in mesangial cells. Nuclear extracts of mesangial cells exposed for 4 h to 21% O2 or 1% O2 in the absence (–) or presence (+) of IL-1{beta} (1 nM) were taken for electrophoretic mobility shift assay using primers for HRE-1 (A) or HRE-2 (B) as described in Materials and Methods. Where indicated, competition experiments were performed by using different molar excess of unlabeled wild-type (wt)-HRE-1 or wt-HRE-2 consensus oligonucleotides (depicted as different dilutions of an oligonucleotide stock solution). Where indicated, nuclear extracts were preincubated for 20 h at 4°C with an anti–HIF-2{alpha} antibody before the addition of the labeled oligonucleotides. Data are representative for three independent experiments giving similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Sufficient oxygen supply is vital for development and physiology of mammalian cells. When simple diffusion of oxygen becomes inadequate to provide the necessary flow of substrate, evolution has provided cells with tools to register and respond to hypoxia by upregulation of expression of specific genes, allowing an adaptation to hypoxia-induced stress conditions (1,2,35). The most important factor in these adaptational changes of gene transcription is the HIF.

In this study we show for the first time that sPLA2-IIA is a hypoxia-regulated gene. Interestingly, hypoxic conditions per se are not able to upregulate the expression of sPLA2-IIA. Only in the presence of an additional stimulus, like the proinflammatory cytokine IL-1{beta}, does hypoxia accelerate sPLA2-IIA expression and activity. This coordinated action of two signaling pathways seems to be a typical feature in IIA-sPLA2 induction and, similarly, IL-1{beta} + nitric oxide (NO) (36) or IL-1{beta} + PPAR{alpha} agonists (23) show additive or synergistic effects on IIA-sPLA2 mRNA expression compared with IL-1{beta} alone, whereas neither NO nor PPAR{alpha} have effects by themselves. The requirement of two input signals could guarantee that sPLA2 is only induced under inflammatory conditions and not under physiologic conditions when NO or PPAR{alpha} levels may increase, or when O2 levels drop. In this context, it is worth noting that hypoxia was also found to be a co-stimulus with IFN-{gamma}–induced inducible NO synthase expression in macrophages without showing an effect alone (37).

The enhancing effect of hypoxia found in this study is due to a direct binding of HIF-2{alpha} to both identified HRE sites in the promoter of sPLA2-IIA and a subsequent activation of gene transcription. This conclusion is based on the following findings: (1) HIF-2{alpha} is the only subtype of HIF expressed in mesangial cells (Figure 5); (2) under hypoxia, HIF-2{alpha} accumulates in mesangial cells (Figure 5); (3) HIF-2{alpha} binds to both HRE elements in the sPLA2 promoter as seen in EMSA (Figure 7); and (4) mutation of either of the two HRE in the promoter abolishes the enhancing effect of hypoxia (Figure 6, B and C).

Whether there is an additional effect of hypoxia on sPLA2-IIA mRNA stability as has been shown for cyclooxygenase-2 mRNA (10) has not been investigated. However, when considering the long half-life of sPLA2-IIA mRNA of >12 h (38) under normoxic conditions and the absence of stability-regulating AUUUA motifs in the 3'-untranslated region, the latter possibility seems rather unlikely.

Interestingly, NO has been reported to amplify eicosanoid synthesis by interfering with both cyclooxygense-2 (39) and sPLA2-IIA expression and activity (36). The mechanism of this NO-mediated enhancement of sPLA2-IIA expression remains unclear. Very recent data suggest that HIF is not exclusively induced by hypoxia, but can also be activated by reactive oxygen species (40) and NO (41,42). In contrast, during hypoxia NO inhibits mitochondrial respiration and redirects oxygen toward HIF prolyl hydroxylases to prevent the stabilization of HIF-1{alpha} (43). Thus, NO may profoundly modify the hypoxia signaling pathway. In this context it is noteworthy that there is a marked overlap between hypoxia- and NO-regulated target genes (44). Inflammatory conditions are characterized by sustained, high-output NO production via inducible NO-synthase (6,45) and consequently this setting may impact hypoxia signaling.

Furthermore, we show that hypoxia alone is able to increase a PLA2 activity in the supernatants of mesangial cell cultures, which is not identical to the sPLA2-IIA. This subtype was excluded because under hypoxia alone no sPLA2-IIA mRNA or protein is found. Nevertheless, based on various inhibitor studies, we were able to pinpoint the secreted enzyme to the subgroup of sPLA2 that is DTT- and Ca2+-sensitive and inhibited by CGP43182 (29,30) and 12-epi-scalaradial (31), two potent and selective sPLA2 inhibitors. iPLA2 was excluded because of the Ca2+-sensitivity of the secreted enzyme. cPLA2 was excluded because no change of cPLA2 protein expression in lysates nor in the supernatants was detected upon hypoxia (data not shown), which could have taken place if hypoxia had led to increased apoptosis or necrosis of a subpopulation of the cells.

Mesangial cells had previously been shown to secrete, in addition to group IIA, group V sPLA2 (17). However, we were unable to detect an effect of hypoxia on group V sPLA2 and can presently only speculate that an additional low molecular weight PLA2 might be regulated by hypoxia, which certainly deserves further investigation.

In this context, it has been reported that, during ischemia, endothelial cells also respond with increased PLA2 activity, which was suggested to belong to the group of secreted PLA2, but was not further specified (46). Furthermore, an increased amount of arachidonic acid is liberated from neuroblastoma cells (47) and from cultures of rat hippocampal slices (48) upon ischemic injury. However, these PLA2 activities were appointed to the group of cytosolic PLA2 rather than to the secreted PLA2 activities (48). Especially in the neuronal system, the inflammatory response occuring upon ischemic injury may contribute to neuronal degeneration and the clinical manisfestation of Alzheimer disease.

Collectively, hypoxia amplifies arachidonic acid release by an upregulation of sPLA2-IIA expression (this study) and simultaneously increases the capacity of cells to metabolize free arachidonic acid via an enhanced expression of cyclooxygenase-2 (10,49,50). It is tempting to speculate that hypoxia may also affect the more downstream enzymes like PGE synthase or prostacyclin synthase to consistently and coordinately upregulate eicosanoid biosynthesis. Finally, this concerted action of hypoxia on the eicosanoid-generating cascade of enzymes might explain the well-documented increase in prostaglandin synthesis in conditions of limited oxygen availability in brain, kidney, and heart, as well as in cell culture of endothelial and mesangial cells (18,19,51,52).

Pharmacologic strategies for treating diseases associated with pathologically increased formation of prostaglandins have a lucrative market, as just recently proven by the so-called cyclooxygenase-2–selective inhibitors. Linking eicosanoid biosynthesis to the biochemical systems by which cells register and respond to hypoxia will provide novel targets and eventually drugs that lead to better therapies of inflammatory diseases. This again would prove that the path to a new drug does not necessarily need to be a linear process.


   Acknowledgments
 
This work was supported by the German Research Foundation (SFB 553; PF361/1-2, PF 361/2-1; HU842/2-3) and a fellowship from the International Graduate School: "Roles of Eicosanoids in Biology and Medicine" (C.P.). We thank Dr. Soheyla Shabahang for helpful discussions, as well as Roswitha Müller and Silke Kusch for excellent technical assistance.


   Footnotes
 
Published online ahead of print. Publication date available at www.jasn.org.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received for publication December 7, 2004. Accepted for publication July 25, 2005.


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