Effects of Hypoxia on Monocyte Inflammatory Mediator Production
2003; Elsevier BV; Volume: 278; Issue: 40 Linguagem: Inglês
10.1074/jbc.m305944200
ISSN1083-351X
AutoresMaryanne Demasi, Leslie G. Cleland, Rebecca J. Cook‐Johnson, Gillian E. Caughey, Michael J. James,
Tópico(s)Immune cells in cancer
ResumoBlood-derived monocytes are found at sites of inflammation as well as in solid tumors and atherosclerotic arteries. They are an abundant source of inflammatory eicosanoids such as prostaglandin E2 (PGE2) and thromboxane A2, which are formed via arachidonic acid (AA) metabolism by cyclooxygenase-1/2 (COX-1/2). In vitro studies of inflammatory mediator production are conducted invariably in room air, which does not reflect the oxygen tensions found in monocyte-containing lesions, which are frequently hypoxic. In this work we examined the effects of hypoxia at levels reported in these lesions, on monocyte COX-2 expression, the related events that lead to eicosanoid synthesis, and relationships with tumor necrosis factor (TNF)-α synthesis. In fresh human monocytes exposed to hypoxia (1% O2), there was an increase in COX-2 protein compared with cells in normoxia, and this was attributable to increased transcription and mRNA stability. However, the synthesis of PGE2 and thromboxane A2 was reduced in hypoxia and did not reflect the increased level of COX-2. Monocytes prelabeled with [3H]AA followed by lipopolysaccharide stimulation in the presence of hypoxia showed a reduced release of AA compared with cells in normoxia. In addition, hypoxia resulted in decreased phosphorylation of the p44/42 mitogen-activated protein kinase and of cytosolic phospholipase A2. Hypoxia also increased TNF-α synthesis, which appeared to play a role in COX-2 expression, and the observed increase TNF-α synthesis appeared to result from reduced PGE2 synthesis. Overall, the results suggest the existence of an autocrine loop of regulation between monocyte eicosanoid and TNF-α production, which is dysregulated in hypoxia and establishes hypoxia as being an important environmental determinant of inflammatory mediator production. Blood-derived monocytes are found at sites of inflammation as well as in solid tumors and atherosclerotic arteries. They are an abundant source of inflammatory eicosanoids such as prostaglandin E2 (PGE2) and thromboxane A2, which are formed via arachidonic acid (AA) metabolism by cyclooxygenase-1/2 (COX-1/2). In vitro studies of inflammatory mediator production are conducted invariably in room air, which does not reflect the oxygen tensions found in monocyte-containing lesions, which are frequently hypoxic. In this work we examined the effects of hypoxia at levels reported in these lesions, on monocyte COX-2 expression, the related events that lead to eicosanoid synthesis, and relationships with tumor necrosis factor (TNF)-α synthesis. In fresh human monocytes exposed to hypoxia (1% O2), there was an increase in COX-2 protein compared with cells in normoxia, and this was attributable to increased transcription and mRNA stability. However, the synthesis of PGE2 and thromboxane A2 was reduced in hypoxia and did not reflect the increased level of COX-2. Monocytes prelabeled with [3H]AA followed by lipopolysaccharide stimulation in the presence of hypoxia showed a reduced release of AA compared with cells in normoxia. In addition, hypoxia resulted in decreased phosphorylation of the p44/42 mitogen-activated protein kinase and of cytosolic phospholipase A2. Hypoxia also increased TNF-α synthesis, which appeared to play a role in COX-2 expression, and the observed increase TNF-α synthesis appeared to result from reduced PGE2 synthesis. Overall, the results suggest the existence of an autocrine loop of regulation between monocyte eicosanoid and TNF-α production, which is dysregulated in hypoxia and establishes hypoxia as being an important environmental determinant of inflammatory mediator production. Retraction: Effects of hypoxia on monocyte inflammatory mediator production: Dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesis.Journal of Biological ChemistryVol. 293Issue 52PreviewVOLUME 278 (2003) PAGES 38607–38616 Full-Text PDF Open AccessEffects of hypoxia on monocyte inflammatory mediator production: Dissociation between changes in cyclooxygenase-2 expression and eicosanoid synthesis.Journal of Biological ChemistryVol. 292Issue 38PreviewVOLUME 278 (2003) PAGES 38607–38616 Full-Text PDF Open Access The histopathology of inflamed lesions shows infiltration with monocytes, which are an abundant source of clinically important inflammatory mediators. The eicosanoids, prostaglandin E2 (PGE2) 1The abbreviations used are: PGE2, prostaglandin E2; AA, arachidonic acid; cPLA2, cytosolic phospholipase A2; COX, cyclooxygenase; ELISA, enzyme-linked immunosorbent assay; ERK, extracellular signal-regulated kinase; FCS, fetal calf serum; LPS, lipopolysaccharide;MAP, mitogen-activated protein; MAPK, MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PEI, polyethyleneimine; RIA, radioimmunoassay; TNF-α, tumor necrosis factor-α; TXA2, thromboxane A2; TXB2, thromboxane B2. and thromboxane A2 (TXA2), are produced via the cyclooxygenase (COX) activity of monocytes. Both have roles in inflammation. PGE2 can cause hyperalgesia and vasodilation, and TXA2 is a facilitator of inflammatory cytokine production (1Zhang Y. Shaffer A. Portanova J. Seibert K. Isakson P.C. J. Pharmacol. Exp. Ther. 1997; 283: 1069-1075PubMed Google Scholar, 2Caughey G.E. Pouliot M. Cleland L.G. James M.J. 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This work uses fresh human monocytes to examine this issue and focuses on COX-2 expression, eicosanoid production, and possible autocrine relationships with TNF-α synthesis. LPS (Escherichia coli serotype 0111:B4), E-Toxa-Clean, heme, and zinc-protophorhyrin IX were from Sigma. Minisorp polyethylene tubes were from Nunc, Roskilde, Denmark. Polyvinylidene difluoride and nitrocellulose protein transfer membranes were from Bio-Rad. Mouse anti-β-actin, rabbit polyclonal anti-COX-2, PGE2 standard, TXB2 standard, and NS398 were from Cayman Chemicals, Ann Arbor, MI. TXB2 antiserum was prepared from a rabbit immunized with TXB2-thyroglobulin conjugates (18James M.J. Walsh J.A. Prostaglandins Leukotrienes Essent. Fatty Acids. 1988; 31: 91-95Abstract Full Text PDF PubMed Scopus (17) Google Scholar). Peroxidase-labeled donkey anti-rabbit and goat anti-mouse antibodies and [3H]PGE2 and [3H]TXB2 were from Amersham Biosciences. Pyrogen-free Lymphoprep was from Nycomed Pharma, Oslo, Norway. Low LPS FCS was from Invitrogen. Antibodies to the phosphorylated forms of cytosolic phospholipase A2 (cPLA2), p38 and p44/42 MAP kinases, and the MEK-1 inhibitor PD98059 were purchased from New England Biolabs, Beverly, MA. Monocyte Isolation—Buffy coats were obtained fresh from the Red Cross Blood Center, Adelaide, South Australia. Mononuclear cells were isolated from the buffy coats by centrifugation (800 × g, 30 min) on pyrogen-free Lymphoprep. After two washes, the mononuclear cells were suspended in 10 ml of running buffer (Hanks' balanced saline solution, 0.21% trisodium citrate). In this solution, the cells underwent countercurrent centrifugal elutriation (J-6 M/E Elutriation System, Beckman, Palo Alto, CA) with a constant rotor speed (2,000 rpm) and a constant flow rate of 11 ml/min for 30 min. Purity of the obtained monocyte fraction (>85%) was assessed by fluorescence-activated cell sorter analysis. Contaminant cells were essentially all lymphocytes. For the maintenance of minimal LPS contamination, mononuclear cell isolation procedure was performed under sterile conditions, and elutriator tubing was treated with E-Toxa-Clean, 70% ethanol, and Milli-Q water before each elutriation. Cell Stimulation—Elutriated monocytes were resuspended (2 × 106/ml) in RPMI 1640 medium supplemented with 10% (v/v) heat-inactivated low LPS FCS, l-glutamine, Hepes, 100 units/ml penicillin, and 100 μg/ml gentamycin. One-ml aliquots were incubated in Minisorp tubes at 37 °C in 5% CO2 as indicated with either 10 μm arachidonic acid (AA) or 1 μm calcium ionophore A23187 for 15 min or 200 ng/ml LPS for 0–18 h. All cell stimulations with AA or A23187 were performed in RPMI (FCS-free). Incubations with LPS were performed in complete medium (10% FCS) at 37 °C. Cell suspensions were centrifuged, and cell-free supernatants were stored at -20 °C until eicosanoid determination. Hypoxia—Ambient oxygen concentrations of 1% were maintained using a controlled incubator with CO2/O2 monitoring and CO2/N2 gas sources (Edwards Instrument Co., Wilmington, MA). CO2 was maintained at 5%. Culture medium was preequilibrated overnight before cell exposure and maintained at a pH of 7.3. The pO2 of the medium was 33 mm Hg in hypoxia and 154 mm Hg in normoxia. The appearance of cells in hypoxia was indistinguishable from those maintained in normoxic conditions by light microscopy. Eicosanoid Measurement—TXA2 has a t ½ of ∼30 s under physiological conditions and is hydrolyzed to the stable metabolite TXB2, which was measured. PGE2 and TXB2 levels were determined by RIAs. The TXB2 assay used rabbit antiserum raised against thyroglobulin-conjugated TXB2 (18James M.J. Walsh J.A. Prostaglandins Leukotrienes Essent. Fatty Acids. 1988; 31: 91-95Abstract Full Text PDF PubMed Scopus (17) Google Scholar). TNF-α Measurement by ELISA—Nunc plates were coated with mouse monoclonal coating antibody against human TNF-α (5 μg/ml in 0.2 mol/liter Na2CO3, pH 9.4) overnight at 4 °C. The plate was then blocked by the addition of 200 μl of 0.5% bovine serum albumin for 1 h at 37 °C. Serial dilutions of human recombinant TNF-α (ranging from 20 to 0.312 ng/ml) or 50-μl samples diluted 1:3 were added together with 50 μl of mouse monoclonal (matched pair antibody) against 0.05 μg/ml human TNF-α for 2 h at room temperature. Plates were washed between steps with phosphate-buffered saline containing 0.05% Tween 20. 100 μl of Extravidin® peroxidase (1:4,000 dilution in 0.5% bovine serum albumin) was added for 15 min at 37 °C. Finally, 100 μl of the peroxidase substrate, TMB (Sigma), in 0.5 m phosphate citrate buffer (according to manufacturer's protocol) was added. The reaction was stopped by the addition of 100 μl of 2 m H2SO4. Absorbance was measured at 450 nm in a microplate reader (model 450, Bio-Rad). Western Immunoblot—Monocyte pellets (5 × 106) were washed twice in phosphate-buffered saline before the addition of 60 μl of ice-cold lysis buffer (Hepes-buffered Hanks' balanced saline solution, pH 7.4, 0.5% Triton X-100, 10 μg/ml leupeptin, 10 μg/ml aprotinin) and 60 μl of 2× sample buffer (0.125 m Trizma base, pH 6.8, 20% glycerol, 4% SDS, 10% 2-mercaptoethanol). Samples were heated at 95 °C for 7 min before storing at -20 °C. Proteins (50 μg/lane) were separated on 9% SDS-PAGE and then transferred onto either a Sequi-Blot™ polyvinylidene difluoride membrane or a nitrocellulose membrane at -4 °C for 16 h at 300 mA. The membrane was blocked for 1 h at 25 °C in Tris-buffered saline (25 mm Tris-HCl, 0.2 m NaCl, 0.15% Tween 20, pH 7.6) containing 5% (w/v) dried milk. Subsequently, membranes were treated with the relevant antibodies at the following dilutions: polyclonal COX-2, 1:10,000; anti-TX synthase, 1:10,000; polyclonal phospho-p38 MAPK, 1:1,000; phospho-p44/42 MAPK, 1:1,000; and monoclonal β-actin antibody. These were followed by horseradish peroxidase-conjugated donkey anti-rabbit or sheep anti-mouse antibodies. Bound antibodies were revealed with the Supersignal WestPico chemiluminescent system following the manufacturer's protocol (Pierce). Northern Blot—Total RNA was isolated using TriZol (Invitrogen) according to manufacturer's protocol. Total RNA (10 μg/lane) was heated at 68 °C for 10 min, electrophoresed on a 1% agarose-formaldehyde gel, transferred to a positively charged nylon membrane (Hybond N+, Amersham Biosciences), and UV cross-linked. Membranes were prehybridized for 3 h at 55 °C and subsequently hybridized overnight at 43 °C with random primer [32P]dCTP-labeled human COX-2 cDNA or glyceraldehyde-3-phosphate dehydrogenase probe using a GIGAprime DNA Labeling Kit (Bresatec, Adelaide, Australia). The COX-2 cDNA probe was prepared by reverse transcription-PCR as described (19Pouliot M. Baillargeon J. Lee J.C. Cleland L.G. James M.J. J. Immunol. 1997; 158: 4930-4937PubMed Google Scholar). Equal RNA loading efficiency was determined by visualization of 28 S and 18 S bands over UV light or glyceraldehyde-3-phosphate dehydrogenase. COX-2 Promoter-Reporter Construct—A vector containing a 7-kb fragment (GenBank accession number AF044206) of the COX-2 promoter region was the gift of Dr. Steven Prescott of the Huntsman Cancer Institute (University of Utah) (20Meade E.A. Smith W.L. DeWitt D.L. J. Biol. Chem. 1993; 268: 6610-6614Abstract Full Text PDF PubMed Google Scholar). This was used as the template for amplification by PCR of a fragment containing bases -531 through +65 relative to the COX-2 transcriptional start site. A reporter construct driven by this segment was responsive to hypoxia in endothelial cells (21Schmedtje Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar). Briefly, the conditions for the PCR were 0.2 unit/reaction AmpliTaq Gold® (Applied Biosystems), 1.5 mm MgCl2, 1× (10×) buffer (100 mm Tris-HCl, pH 8.3, 500 nm KCl, 15 mm MgCl2, 0.01% (w/v) glycerin), 0.2 mm dNTPs (New England Biolabs), 100 ng/reaction each primer and high performance liquid chromatography grade water (Sigma) to a total volume of 20 μl. Conditions were 95 °C for 10 min, 30 cycles of (94 °C for 30 s, 50 °C for 30 s, 72 °C for 1 min), and 72 °C for 10 min. The primers used had specific restriction sites built into the 5′-most ends to facilitate ligation in a specific orientation into pGL3-Basic (Promega). This vector contains the gene coding for the firefly luciferase gene but no promoter. The specific primers were, with the restriction sites in bold italics, fpro-531COX-2 (5′-GCGGTACC GTTACTCGCCCCAGTCTGTC-3′) and rpro+65COX-2 (5′-GGCTCGAG CGAGGCGCTGCTGAGGAG-3′). The PCR product was purified using the MinElute™ PCR Purification Kit (Qiagen), restricted in separate reactions with KpnI and XhoI (New England Biolabs) according to manufacturer's instructions, and then purified further for transformation using the MinElute™ Reaction Cleanup Kit (Qiagen). The isolated restricted PCR product was then ligated at the KpnI and XhoI sites located in the multiple cloning site of the pGL3-Basic vector using T4 DNA Ligase (Promega). This vector pGL3-COX-2-531 was then transformed into MAX Efficiency® DH5α™-competent cells (Invitrogen) according to the manufacturer's instructions. Sequencing (ABI Prism® model 3700) confirmed the orientation and sequence of COX-2-531. Overnight cultures were then grown and plasmid isolated using the Endofree® Plasmid Maxi Kit (Qiagen) to ensure minimal LPS contamination. Transient Transfection—U937 monocytic cells were plated in 12-well plates (2 × 106 cells/2 ml) in RPMI with 10% FCS and 50 ng/ml phorbol 12-myristate 13-acetate, which promotes differentiation after 3–5 days of treatment (22Pedrinaci S. Ruiz-Cabello F. Gomez O. Collado A. Garrido F. Int. J. Cancer. 1990; 45: 294-298Crossref PubMed Scopus (30) Google Scholar). After differentiation, cells were transfected using Jet PEI (PolyTransfection), according to the manufacturer's instructions. Briefly, 4 μg of the pGL3-COX-2-531 construct was suspended in 75 μlof150mm sterile NaCl solution. Also 4 μl of Jet PEI solution was suspended in 75 μl of 150 mm sterile NaCl solution. The Jet PEI/NaCl solution was then added to the DNA/NaCl solution and incubated at room temperature for 30 min. The medium in the wells was then changed to fresh medium, and 150 μl of the DNA/Jet PEI was added to each well. The transfection was allowed to proceed for 5 h, and the medium replaced again with either hypoxic or normoxic medium. The cells were then stimulated with 100 ng/ml serum-treated zymosan for specified times. Following the transfection period, the medium was removed and discarded and the cells lysed with Passive Lysis Buffer supplied in the Dual Luciferase™ Reporter Assay Kit. The lysate was then assayed for luciferase activity. Statistical Analysis—Results are expressed as the mean ± S.E. of triplicate incubations. Analysis of variance followed by the Newman-Keuls multiple comparisons test was used to identify the statistically significant differences between treatments using WINKS (Texasoft, Cedar Hill, TX). Effect of Hypoxia on Monocyte COX-2 Message and Protein— LPS (200 ng/ml) induced COX-2 mRNA and protein in fresh human monocytes in a time-dependent manner over 18 h. The up-regulation of COX-2 mRNA and protein was greatly potentiated by hypoxia (1% O2) (Fig. 1). This augmentation of COX-2 expression by hypoxia was observed with a variety of costimuli (Fig. 2).Fig. 2Effect of hypoxia on COX-2 protein induction in monocytes with various stimuli. Monocytes (5 × 106) were stimulated with 200 ng/ml LPS, 1 ng/ml TNF-α, or 2 ng/ml interleukin (IL)-1β for 24 h in normoxia or hypoxia. Cells were processed for Western blot analysis as described under "Methods." Blots are representative of three separate experiments, the mean values of which are shown in the graph. * p < 0.05 compared with the same stimuli in normoxic monocytes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) It was reported that hypoxia can increase transcription of COX-2 in endothelial cells (21Schmedtje Jr., J.F. Ji Y.S. Liu W.L. DuBois R.N. Runge M.S. J. Biol. Chem. 1997; 272: 601-608Abstract Full Text Full Text PDF PubMed Scopus (638) Google Scholar), and therefore this mode of regulation in monocytes was examined. Many attempts to transfect fresh human monocytes transiently with a COX-2 promoter/luciferase reporter construct were unsuccessful. However, the human monocytic cell line U937 was transfectable, and these cells were used. Hypoxia augmented activity of the 531-bp segment of the COX-2 promoter in U937 cells (Fig. 3). Another mode of regulation of COX-2 levels can occur post-transcriptionally with stabilization of mRNA in response to LPS or interleukin-1β (23Barrios-Rodiles M. Tiraloche G. Chadee K. J. Immunol. 1999; 163: 963-969PubMed Google Scholar), although this has not been examined in hypoxia. Therefore, the effect of hypoxia on COX-2 mRNA stability in monocytes was examined. Monocytes were transiently stimulated with LPS for 15 min in normoxia or hypoxia and then washed and incubated in fresh normoxic or hypoxic medium for 3 h to allow synthesis of COX-2 mRNA. Actinomycin D was added to inhibit further transcription, and the level of COX-2 mRNA was measured for a further 3 h. COX-2 mRNA levels decreased in normoxia by more than 90% within 3 h after the addition of actinomycin D (Fig. 4). By comparison, COX-2 mRNA levels decreased in hypoxia by less than 20% within 3 h after the addition (Fig. 4).Fig. 4Effect of hypoxia on COX-2 mRNA stability. Monocytes (5 × 106) were transiently stimulated with 200 ng/ml LPS for 3 h (37 °C) in normoxia or hypoxia. Actinomycin D (AD) (5 μg/ml) was then added, and the level of COX-2 mRNA was assessed for a further 3 h by Northern analysis. The blot is representative of three separate experiments, the mean values of which are shown in the graph as percent change from time 0 h. * p < 0.05 compared with the equivalent times in hypoxic monocytes. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of Hypoxia on Monocyte COX-2 Activity—Fresh monocytes were treated with LPS for 18 h at 37 °C in normoxia or hypoxia, and the accumulation of PGE2 and TXA2 in the cell supernatants was measured. There was a marked reduction in the accumulation of PGE2 and TXA2 synthesis in hypoxia (Fig. 5). Similar time courses and reduced eicosanoid synthesis in hypoxia were also observed when LPS was transient, with LPS removal after 15 min (data not shown). The reduced synthesis of these eicosanoids in hypoxia did not correlate with the increased expression of COX-2 protein in hypoxia, described above. Possible explanations for the disparate hypoxia-induced changes in COX-2 expression and eicosanoid synthesis were sought. Effect of Heme on COX-2 Activity in Hypoxia—COX-2 is a heme-containing enzyme, and cellular heme levels can be reduced by heme oxygenase, including the inducible isoform, heme oxygenase-1, which may be up-regulated during hypoxia (24Lee P.J. Jiang B.H. Chin B.Y. Iyer N.V. Alam J. Semenza G.L. Choi A.M. J. Biol. Chem. 1997; 272: 5375-5381Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 25Bonazzi A. Mastyugin V. Mieyal P.A. Dunn M.W. Laniado-Schwartzman M. J. Biol. 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Chem. 2000; 275: 2837-2844Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar) did not affect the amount of COX-2 protein (Fig. 6a) or the production of PGE2 and TXA2 (Fig. 6b) protein in normoxia or hypoxia under these experimental conditions, suggesting that heme oxygenase activity or heme levels are not responsible for reduced COX-2 activity in hypoxia. Dependence of COX-2 Activity on O 2 as a Cosubstrate in Hypoxia—COX-2 utilizes oxygen as a cosubstrate during the conversion of AA to prostaglandin H2, the common precursor of PGE2 and TXA2. In this study, O2 in the incubation chamber was set at 1%; cf. ∼20% for air at sea level. This level of hypoxia reduced dissolved oxygen in the incubation medium to 33 mm Hg. To determine whether these levels of O2 were rate-limiting for eicosanoid synthesis, monocytes were first incubated in hypoxia with LPS to induce COX-2. After 18 h, cells were washed twice and incubated in fresh hypoxic or normoxic medium with 10 μm exogenous AA for 15 min. Oxygenation of the medium had no effect on the production of PGE2 and TXA2 (Fig. 7). These results indicated that dissolved O2 at the levels of hypoxia used in this study were not rate-limiting for COX activity. Effect of Exogenous AA on COX-2 Activity in Hypoxia—Monocytes were incubated with LPS for 18 h in the absence or presence of hypoxia to induce COX-2. The following day cells were washed twice and incubated with fresh normoxic or hypoxic medium and 10 μm exogenous AA for 15 min. In hypoxia, there was an increase in PGE2 and TXA2 synthesis (Fig. 8). This contrasted with results above (Fig. 5) where eicosanoid synthesis from endogenous AA was reduced in hypoxia. This suggested that the reduced accumulation of eicosanoids observed in hypoxia, despite the up-regulated levels of COX, was caused by the decreased availability of endogenous AA substrate. Effect of Hypoxia on Endogenous AA Release—Monocytes were incubated with [3H]AA in normoxia for 18 h at 37 °C to incorporate labeled AA into cell membranes. After 18 h, cells were washed three times and incubated with LPS in normoxia or hypoxia for 30 h. In addition, [3H]AA-prelabeled cells that had been hypoxic for 9 h were returned to oxygenated conditions for the next 21 h. In normoxia, there was a time-dependent increase in the release of labeled AA from monocytes when stimulated with LPS (Fig. 9). By comparison, there was a marked reduction in the release of AA from monocytes stimulated with LPS in hypoxia (Fig. 9). Reoxygenation after 9 h of hypoxia resulted in a gradual restoration of AA release from cells to rates that were similar to those observed in normoxic cells (Fig. 9). Because cPLA2 is prominently involved in the release of AA from membrane phospholipids, the effects of hypoxia on cPLA2 phosphorylation were examined. Effect of Hypoxia on the Phosphorylation of cPLA 2 —After stimulation with 1 μm A23187, the phosphorylation of cPLA2 appeared to be maximal at 30 min, and dephosphorylation occurred at times after 30 min (Fig. 10). In contrast, phosphorylation of cPLA2 in hypoxia appeared to be reduced at 10 min and showed an accelerated dephosphorylation of the enzyme at later times (Fig. 10). MAP kinases may regulate the phosphorylation and activation of cPLA2 (27Gijon M.A. Spencer D.M. Siddiqi A.R. Bonventre J.V. Leslie C.C. J. Biol. Chem. 2000; 275: 20146-20156Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 28Hiller G. Sundler R. Cell. Signal. 1999; 11: 863-869Crossref PubMed Scopus (43) Google Scholar, 29Fouda S.I. Molski T.F. Ashour M.S. Sha'afi R.I. Biochem. J. 1995; 308: 815-822Crossref PubMed Scopus (51) Google Scholar, 30Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar, 31Syrbu S.I. Waterman W.H. Molski T.F. Nagarkatti D. Hajjar J.J. Sha'afi R.I. J. Immunol. 1999; 162: 2334-2340PubMed Google Scholar, 32Miura K. Schroeder J.T. Hubbard W.C. MacGlashan Jr., D.W. J. Immunol. 1999; 162: 4198-4206PubMed Google Scholar). Therefore, the effects of hypoxia on the phosphorylation of p44/42 MAPK (ERK1/2) and p38 MAPK were examined. Effect of Hypoxia on the MAPK Pathways—In normoxia, the phosphorylation of p44/42 was maximal at 30 min followed by dephosphorylation up to 240 min (Fig. 11). In hypoxia, there was a reduction in the phosphorylation at 30 min and accelerated dephosphorylation at later times (Fig. 11). In normoxia, the phosphorylation of p38 MAPK was maximal at 30–60 min followed by dephosphorylation at later times. Hypoxia had no effect on the time course of phosphorylation of p38 MAPK or the time course of decay in the amount of phosphorylated enzyme (Fig. 11). Effect of Inhibition of p44/42 MAPK Activation on AA Release in Hypoxia—Monocytes were incubated with [3H]AA in normoxia for 18 h at 37 °C for incorporation into cell membranes. After 18 h, cells were washed three times and incubated with LPS in normoxia or hypoxia for 30 h. In addition, some prelabeled cells that had been hypoxic for 9 h were returned to oxygenated conditions with or without PD98059, an inhibitor of p44/42 MAPK phosphorylation. PD98059 inhibited the restoration of AA release from reoxygenated cells to levels similar to those observed in cells maintained in hypoxia (Fig. 12). Effect of Hypoxia on Monocy
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