FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2116821504> ?p ?o ?g. }

• W2116821504 endingPage "2844" @default.
• W2116821504 startingPage "2837" @default.
• W2116821504 abstract "Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR α and β but not γ mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E2 accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation. Hypoxic injury provokes inflammation of many tissues including the ocular surface. In rabbit corneal epithelial cells, both peroxisome proliferator-activated receptor (PPAR)-inducible cytochrome P450 4B1 and cyclooxygenase-2 (COX-2) mRNAs were increased by hypoxia. PPAR α and β but not γ mRNAs were detected in these cells. The PPAR activator, WY-14,643 increased COX-2 expression. Similarly, non-steroidal anti-inflammatory drugs with the ability to activate PPARs induced COX-2 independently of prostaglandin synthesis inhibition. COX-2 protein overexpression by hypoxia and PPAR activation was not associated with a parallel increase in prostaglandin E2 accumulation. However, the enzyme regained full catalytic activity when: 1) hypoxic cells were re-exposed to normoxic conditions in the presence of heme and arachidonic acid, and 2) WY-14,643-treated cells were depleted of intracellular GSH. Consistent with previous observations showing that the corneal production of cytochrome P450-derived inflammatory eicosanoids is elevated by hypoxia and inflammation, the current data suggest that hypoxic injury is a model of inflammation in which molecules other than COX-derived arachidonic acid metabolites play a major proinflammatory role. This study also suggests that increased cellular GSH may be the mechanism responsible for the characteristic dissociation of PPAR-induced COX-2 expression and activity. Moreover, we provide new insights into the commonly observed lack of efficacy of classical non-steroidal anti-inflammatory drugs in the treatment of hypoxia-related ocular surface inflammation. cyclooxygenase prostaglandin peroxisome proliferator-activated receptors cytochrome P450 rabbit corneal epithelium heme oxygenase hydroxyeicosatetraenoic acid hydroxyeicosatrienoic acid leukotriene non-steroidal anti-inflammatory drug reverse transcription polymerase chain reaction l-buthionine-[S,R]sulfoximine maleic acid diethylester Eye closure during sleep as well as contact lens wear creates an environment that has been described as a state of subclinical inflammation characterized by corneal swelling, conjunctival vasodilation, and significant levels of polymorphonuclear leukocytes in the tear film. These changes are attributed to reduced oxygen and carbon dioxide exchange, leading to corneal hypoxia and subsequent acidosis (1.Sack R.A. Tan K.O. Tan A. Invest. Ophthalmol. Vis. Sci. 1992; 33: 626-640PubMed Google Scholar). There exist a host of mediators that have been implicated in the development and progression of corneal inflammation (2.Bruce A.S. Brennan N.A. Surv. Ophthalmol. 1990; 35: 25-58Abstract Full Text PDF PubMed Scopus (113) Google Scholar, 3.Smolin G. Eye. 1989; 3: 167-171Crossref PubMed Scopus (12) Google Scholar). The source of these mediators may be the injured tissue or infiltrating inflammatory cells. Although the progression of an inflammatory response is generally dependent on the inflammatory infiltrate, its initiation is usually the result of inflammatory mediators released from the damaged tissue. Among the endogenous corneal inflammatory mediators released from the epithelium are the arachidonic acid-derived eicosanoids, which have been implicated in the initiation, development, and progression of an inflammatory response (4.Williams K.I. Higgs G.A. J. Pathol. 1988; 156: 101-110Crossref PubMed Scopus (89) Google Scholar, 5.Williams R.N. Delamere N.A. Paterson C.A. Exp. Eye Res. 1985; 41: 733-738Crossref PubMed Scopus (20) Google Scholar). The relative contribution of each eicosanoid to this reaction is unclear. These eicosanoids are produced by the activity of cyclooxygenase (COX)1 (primarily PGE2), lipoxygenase (12(S)-HETE and leukotrienes, e.g. LTB4) and cytochrome P450 monooxygenase (12(R)-HETE and 12(R)-HETrE) (6.Laniado Schwartzman M. Green K. Edelhauser H.F. Hackett R.B. Hull D.S. Potter D.E. Tripathi R.C. Advances in Ocular Toxicology. Plenum Press, New York1997: 3-20Crossref Google Scholar). We have previously demonstrated that 12(R)-HETE and 12(R)-HETrE possess potent inflammatory properties with 12(R)-HETrE being a powerful angiogenic factor and that both metabolites assume the role of inflammatory mediators in hypoxia- and alkali-induced injury in the cornea, in vivo (7.Conners M.S. Stoltz R.A. Webb S.C. Rosenberg J. Dunn M.W. Abraham N.G. Schwartzman M.L. Invest. Ophthalmol. Vis. Sci. 1995; 36: 828-840PubMed Google Scholar, 8.Conners M.S. Urbano F. Vafeas C. Stoltz R.A. Dunn M.W. Laniado Schwartzman M. Invest. Ophthalmol. Vis. Sci. 1997; 38: 1963-1971PubMed Google Scholar). Additional studies using in vitro models of corneal epithelial injury further support a relationship between injury, hypoxia, and the production of the CYP-derived eicosanoids (9.Vafeas C. Mieyal P.A. Urbano F. Falck J.R. Chauhan K. Berman M. Laniado Schwartzman M. J. Pharmacol. Exp. Ther. 1998; 287: 903-910PubMed Google Scholar). Studies to identify the CYP isoform responsible for the production of 12(R)-HETE and 12(R)-HETrE revealed that a CYP4B1 isoform is readily induced in response to hypoxia in vitroand in vivo and that antibodies against this isoform inhibited the synthesis of these eicosanoids (10.Mastyugin V. Aversa E. Vafeas C. Mieyal P. Laniado Schwartzman M. J. Pharmacol. Exp. Ther. 1999; 289: 1611-1619PubMed Google Scholar). Hypoxia and cellular injury provoke the induction of COX-2 in many cell types (11.Schmedtje 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, 12.Chida M. Voelkel N.F. Am. J. Physiol. 1996; 270: L872-L878PubMed Google Scholar). Moreover, induction of COX-2 mRNA and protein together with increasing levels of prostanoids, mainly PGE2, is the hallmark of inflammation in many tissues (13.DuBois R.N. Abramson S.B. Crofford L. Gupta R.A. Simon L.S. Van De Putte L.B. Lipsky P.E. FASEB J. 1998; 12: 1063-1073Crossref PubMed Scopus (2231) Google Scholar,14.Vane J.R. Bakhle Y.S. Botting R.M. Annu. Rev. Pharmacol. Toxicol. 1998; 38: 97-120Crossref PubMed Scopus (2620) Google Scholar). Among the factors associated with the transcriptional activation of COX-2 are the PPARs. These are nuclear hormone receptors that regulate gene transcription in response to peroxisome proliferators and fatty acids, including certain PGs, HETEs, as well as some of the commonly used non-steroidal anti-inflammatory drugs (NSAIDs) (15.Willson T.M. Wahli W. Curr. Opin. Chem. Biol. 1997; 1: 235-241Crossref PubMed Scopus (183) Google Scholar, 16.Forman B.M. Chen J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4312-4317Crossref PubMed Scopus (1870) Google Scholar). PPAR ligands activate transcription of COX-2 as well as the transcription of several CYP4 genes, including CYP4B1 (17.Meade E.A. McIntyre T.M. Zimmerman G.A. Prescott S.M. J Biol. Chem. 1999; 274: 8328-8334Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar, 18.Claire A.E. Simpson M. Gen. Pharmacol. 1997; 28: 351-359Crossref PubMed Scopus (253) Google Scholar, 19.Zeldin D.C. Plitman J.D. Kobayashi J. Miller R.F. Snapper J.R. Falck J.R. Szarek J.L. Philpot R.M. Capdevila J.H. J. Clin. Invest. 1995; 95: 2150-2160Crossref PubMed Scopus (105) Google Scholar). Hence, we carried out studies to determine the involvement of COX-2 in hypoxia-induced injury of the corneal epithelium and to examine its relation to PPAR activation and CYP4B1 expression. The RCE cell line was obtained from Dr. Kaoru Arakai-Sasake (Osaka University, Osaka, Japan) (20.Araki K. Ohashi Y. Kinoshita S. Hayashi K. Yang X.Z. Hosaka Y. Aizawa S. Handa H. Invest. Ophthalmol. Vis. Sci. 1993; 34: 2665-2671PubMed Google Scholar). Cells were grown to confluence in Petri dishes (60 mm) using medium containing a mixture of Dulbecco's modified Eagle's medium/F-12 (1:1) in the presence of 10% fetal bovine serum and 1% antibiotic/antimycotic mixture (Fungizone, Life Technologies, Inc.); 48 h before the experiment, the medium was changed to Dulbecco's modified Eagle's medium/F-12 containing 0.4% fetal bovine serum and antibiotics. Cells were then washed twice with 2 ml of sterile phosphate-buffered saline and incubated with 2 ml of serum-free medium containing antibiotics with and without PPAR ligands or COX inhibitors under normoxia (5% CO2, 95% air (∼20% O2)) or in a modular tissue culture chamber (Billups-Rothenburg, Del Mar, CA) supplied continuously with 5% CO2, 0% O2, 95% N2 (hypoxia) and bubbled through deionized H2O into the chamber within a 37 °C incubator for an additional 2–24 h. In some experiments, cells were incubated withl-buthionine-[S,R]sulfoximine (BSO, 1 mm) and maleic acid diethylester (DEM, 1 mm) for 18 h to deplete intracellular GSH. Protein content was determined using the DC Protein Assay (Bio-Rad) with bovine serum albumin as the standard. Cell lysates (30 μg of protein) were mixed with Laemmli reagent (final concentration 1%, w/v, sodium dodecyl sulfate; 10%, v/v, glycerol; 0.5%, w/v, bromphenol blue) under reducing conditions (4%, v/v, β-mercaptoethanol) and heated for 5 min at 85 °C. SDS-polyacrylamide gel electrophoresis was performed using 10% (w/v) and 3% (w/v) acrylamide for separating gel and stacking gel, respectively. Protein transfer onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech) was performed at 16 V for 1 h. Membranes were saturated in blocking buffer containing 5% nonfat dry milk and incubated for 2 h at room temperature with the following polyclonal antibody preparations: rabbit anti-murine COX-2, goat anti-human COX-1, rabbit anti-human HO-1, or rabbit anti-human HO-2. After washing with buffer (Tris/HCl, 0.42%, pH 7.4, containing 0.1% Tween 20), membranes were further incubated with an anti-rabbit or anti-goat IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at dilution 1:4,000 for 45 min at room temperature. Immunoreactive proteins were detected using chemiluminescence substrates according to the manufacturer's instructions and were visualized after exposure to HyperfilmTM ECL (Amersham Pharmacia Biotech). Quantitation was performed by scanning the blot into Photoshop and performing densitometry with NIH Image. At the end of the incubation period, the medium was collected and stored at −80 °C. Solid phase enzyme immunoassay (Cayman Chemical, Ann Arbor, MI) was performed as suggested by the manufacturer. PGE2 levels were determined using a standard curve and a linear log-logit transformation. Cells were incubated under normoxia or hypoxia or treated with WY-14,643 (100 μm) for 12 and 24 h in a serum-free medium. Cells were then washed twice with phosphate-buffered saline and further incubated with arachidonic acid (10 μm) in the presence or absence of heme (2 μm), NS398 (1 μm), indomethacin (5 μm), or GSH (10–50 mm) for 1 h in an oxygenated incubator. PGE2 levels in the medium were measured by enzyme immunoassay as described above. Total RNA was isolated with TRIzol reagent (Life Technologies, Inc.) and quantified spectrophotometerically. RT reaction was performed with 5 μg of total RNA and an oligo(dT) primer using the First-Strand cDNA synthesis kit (Amersham Pharmacia Biotech). For amplification of 28 S rRNA and PPARs (α, β, γ), a specific backward primer was used in the single reaction. The reaction mixture was then subjected to brief incubation at 65 °C in order to inactivate the enzyme. PCR reactions were carried out in a final volume of 100 μl consisting of 20 mm Tris-HCl, pH 9.0, 2 mm MgSO4, 200 μm amounts of each deoxyribonucleoside triphosphate, 4 μl of the RT first-strand cDNA product, 0.2 μm of each forward and reverse primer, and 2.5 units of Pfu Turbo DNA polymerase (Stratagene, La Jolla, CA). The reactions were heated to 97 °C for 1 min and then immediately cycled 30 or 35 times through a 1-min denaturing step, 1.5-min annealing step at 50 or 55 °C, and a 2-min extension step at 72 °C. After the cycling procedure, a final 10-min elongation step at 72 °C was performed. The following primers were used: CYP4B1 primers, 4B1-F (5′-TCTCTGGGTTGGACAGTTCATTG) and 4B1-R (5′-TGTCTCCTTTGCCAAACGTACAC); COX-2 primers, COX-2F (5′-GCCCTTCCTCCTGTGGCTGAT) and COX-2R (5′-TTGAGCACATCGCACACTCT); 28 S rRNA primers, 28 S rRNA-F (5′-AAACTCTGGTGGAGGTCCGT) and 28 S rRNA-R (5′-CTTACCAAAAGTGGCCCACTA). Primers for the different PPARs were designed as described previously (21.Guan Y. Zhang Y. Davis L. Breyer M.D. Am. J. Physiol. 1997; 273: F1013-F1022Crossref PubMed Google Scholar). Total RNA (10 μg) was separated on a 1% agarose gel containing formaldehyde, visualized by ethidium bromide staining, and transferred overnight to Hybond N plus membranes (Amersham Pharmacia Biotech) through capillary blotting. The membranes were probed with 32P-labeled cDNA probes in Rapid-Hyb hybridization buffer (Amersham Pharmacia Biotech). The rabbit COX-2 cDNA probe was obtained from Dr. M. Breyer, Vanderbilt University (22.Chang M.S. Tsai J.C. Yang R. DuBois R.N. Breyer M.D. O'Day D.M. Curr. Eye Res. 1997; 16: 1147-1151Crossref PubMed Scopus (7) Google Scholar). The rabbit 28 S rRNA probe was generated by PCR as described above. The resulting blots were subjected to autoradiography with NEF reflection autoradiography films (NEN Life Science Products). Densitometry analysis was done as described for the Western blot. Analysis of variance and Student'st test for unpaired data were used to evaluate differences among control and treated samples. A p value less than or equal to 0.05 was considered significant. Unless stated otherwise, results are presented as means ± S.E. COX-2 is induced in many cell types in response to injury, growth factors and cytokines. In this respect, RCE cells exhibit a similar response; COX-2 protein levels are markedly increased by 2–3-fold in response to these stimuli (data not shown). Hypoxic injury is considered an inflammatory stimulus in many tissues. In RCE cells, hypoxia, caused a 3-fold increase in COX-2 protein levels within 24 h (Fig. 1 A). The effect of hypoxia on COX-2 protein was time-dependent and correlated with the increasing levels of COX-2 mRNA (Fig.1 B). CYP4B1, a cytochrome P450 protein whose expression has been correlated with the synthesis of the inflammatory eicosanoids 12(R)-HETE and 12(R)-HETrE by the corneal epithelium (10.Mastyugin V. Aversa E. Vafeas C. Mieyal P. Laniado Schwartzman M. J. Pharmacol. Exp. Ther. 1999; 289: 1611-1619PubMed Google Scholar), showed a similar time course of expression in response to hypoxia (data not shown). The transcriptional activation of COX-2 and CYP4B1 following hypoxia may share a common mechanism; both have been shown to be activated by PPAR ligands. As seen in Fig.2 A, RT-PCR of RCE cell mRNA revealed the presence of PPARα and PPARβ transcripts but no transcript for PPARγ. Control experiments indicated that the expression of PPARs α, β, and γ was readily detected in rabbit liver RNA (Fig. 2 B). The specificity of these PCR products (corneal epithelial PPAR α and β) was further demonstrated by restriction analysis based on the published sequences of the rabbit PPARs (21.Guan Y. Zhang Y. Davis L. Breyer M.D. Am. J. Physiol. 1997; 273: F1013-F1022Crossref PubMed Google Scholar). We further examined the effect of common peroxisome proliferators as well as NSAIDs that have been shown to act as PPAR ligands (23.Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. J Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar) on COX protein levels. As seen in Fig.3 A, the commonly used PPAR ligand, WY-14,643, greatly increased COX-2 protein levels. WY-14,643 was more potent than clofibrate; its addition to the medium caused a 2-fold increase in COX-2 protein (Fig. 3 A), while clofibrate increased COX-2 protein by about 30% (data not shown). Under hypoxic conditions, addition of WY-14,643 did not produce an additional significant increase in COX-2 levels (Fig. 3 A), suggesting that hypoxia and the PPAR ligand may share a common mechanism for induction of COX-2. Indomethacin as well as flurbiprofen significantly increased COX-2 protein levels by about 2-fold. Aspirin at a concentration of 100 μm also increased COX-2 protein levels (data not shown). Similar to WY-14,643, addition of these NSAIDs to hypoxia-treated cells did not cause a further increase in COX-2 protein levels (data not shown). Neither hypoxia nor WY-14,643 or NSAIDs changed the level of the constitutively expressed COX-1 protein (Fig. 3 A). Actinomycin D inhibited COX-2 expression when used in combination with hypoxia, indomethacin, and WY-14,643 (Fig.3 B), further indicating that COX-2 is transcriptionally activated under these conditions. Indeed, Northern analysis demonstrated that addition of WY-14,643 resulted in a 2-fold increase in COX-2 mRNA (Fig. 4). Indomethacin at 30 μm, while increasing COX-2 protein levels (Fig.3 A) and COX-2 mRNA (data not shown), inhibited PGE2 levels by 60% and 80% in cells grown under normoxic and hypoxic conditions, respectively. The effect of indomethacin on COX-2 protein levels was not reversed by addition of exogenous PGE2. In fact, addition of exogenous PGE2 to normoxia- and indomethacin-treated cells had little or no effect on COX-2 protein levels (Fig. 5), suggesting a dissociation between COX-2 expression and PGE2 levels. Similar results were obtained in hypoxia/indomethacin-treated cells (data not shown).Figure 4Effect of WY-14,643 on COX-2 mRNA in RCE cells. Quiescent cells were incubated under normoxic conditions in the presence or absence of WY-14,643 for 8, 12, and 24 h. Northern blot analysis of total RNA was performed using the rabbit COX-2 cDNA and 28 S rRNA probes. Results are the mean ± S.E.,n = 3; *, p < 0.05 from control normoxia; ○, control; ▾, WY-14,643.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Effect of indomethacin and PGE2on COX-2 protein levels. Quiescent cells were incubated under normoxia in the presence and absence of PGE2 (1 μm) and indomethacin (30 μm) for 12 h. At the end of the incubation period, cells were harvested and COX-2 protein levels determined as described under “Experimental Procedures.” The results are the mean ± S.E., n= 3, *p < 0.05 from control normoxia.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The increase in COX-2 protein levels in response to hypoxia or WY-14,643 was not associated with a comparable increase in PGE2 levels (Fig.6 A). In fact, marked 65% and 64% decreases in PGE2 levels were observed in cells grown under hypoxic conditions for 12 and 24 h, respectively. In WY-14,643-treated cells for 12 h PGE2 levels were similar to the control levels; however, at 24 h the levels were significantly reduced by 25% as compared with control cells (Fig.6 A). Cells grown under hypoxic conditions and treated with WY-14,643 showed a similar decrease in PGE2 levels to that in hypoxia, namely 701 ± 53, 448 ± 63, and 438 ± 61 pg PGE2/ml in normoxia, hypoxia, and hypoxia+WY-14,643, respectively. These results are similar to that obtained for the protein where no additive effect for hypoxia and WY-14,643 was observed (Fig. 3 A). There are several possibilities that could account for the decreased PGE2 levels. One is a lack of substrate, arachidonic acid, which has been long considered as the rate-limiting step in prostaglandin synthesis. Addition of 10 μm arachidonic acid to hypoxia or WY-14,643-treated cells during the culture period restored the levels of PGE2accumulation to that seen in cells incubated under normoxic conditions (Fig. 6 B). However, the increase in PGE2 levels in cells supplemented with arachidonic acid did not reflect the 2–4-fold increase in COX-2 protein seen in cells incubated under the same conditions (Figs. 1 and 3). Another possibility is that the induced COX-2 protein is defective or is inactive due to a lack of cofactors such as heme. In many cells, the level of heme is regulated by the activity of heme oxygenase (HO), specifically the inducible isoform, HO-1. Western blot analysis revealed that only hypoxic conditions caused a marked increase in HO-1 protein levels (Fig. 7). WY-14,643 and indomethacin, while increasing COX-2 protein, had no effect on HO-1 protein level. In all experimental conditions, the protein levels of the constitutively expressed isoform, HO-2, remained unchanged (Fig.7). These results suggest that, at least in hypoxia, a shortage of cellular heme may contribute to the decrease in PGE2levels. The reduced PGE2 levels in hypoxia-treated cells may also stem from lack of oxygen. However, neither heme nor oxygen can account for the relatively depressed PGE2 in WY-14,643-treated cells. It is well documented that the catalytic activity of COX isoforms requires the presence of hydroperoxides (24.Kulmacz R.J. FEBS Lett. 1998; 430: 154-157Crossref PubMed Scopus (43) Google Scholar). On the other hand, conditions of oxidative stress such as those associated with hypoxia and inflammatory processes are associated with increased expression of GSH-dependent peroxidase (25.Mates J.M. Sanchez-Jimenez F. Frontiers Biosci. 1999; 4: D339-D345Crossref PubMed Google Scholar). Glutathione peroxidase as well as GSH itself have been shown to inhibit COX-1 and COX-2 catalytic activity (26.Capdevila J.H. Morrow J.D. Belosludtsev Y.Y. Beauchamp D.R. DuBois R.N. Falck J.R. Biochemistry. 1995; 34: 3325-3337Crossref PubMed Scopus (105) Google Scholar). To evaluate whether changes in GSH levels underlie the decrease in PGE2 levels, cells were incubated with BSO and DEM to deplete endogenous GSH under hypoxia and with the addition of WY-14,643. As seen in Fig. 8, depletion of GSH increased PGE2 levels by 8-, 2- and 14-fold in normoxia-, hypoxia-, and WY-14,643-treated cells, respectively. Moreover, PGE2 accumulation in WY-14,643-treated cells was well correlated with the increased COX-2 protein expression. In contrast, measurement of COX activity as the conversion of arachidonic acid to PGE2 in 1 h in the presence of exogenous GSH demonstrated a concentration-dependent inhibition of COX activity (30% and 40% inhibition at 10 and 50 mm GSH, respectively). We further examined COX activity in cells grown for 12 or 24 h in hypoxia or normoxia and in cells treated with WY-14,643 and subsequently incubated under normoxia with 10 μmarachidonic acid and 2 μm heme for 1 h. As seen in Fig. 9, in 12- and 24-h hypoxia-treated cells COX activity was 2 and 5 times higher than that of the control cells, respectively, further indicating that the increased immunoreactive COX-2 protein is an active enzyme and that oxygen is a major limiting factor. However, COX activity in cells treated with WY-14,643 remained low and was about 50% of the activity measured in control cells (Fig. 9). Finally, addition of the selective COX-2 inhibitor, NS398, to the incubation medium revealed that about 50% of the COX activity in cells incubated under normoxic condition is driven by COX-2, while in hypoxia-treated cells or cells treated with WY-14,643, this activity accounts for almost 100% (Fig.10) suggesting that, although COX-1 protein level remains unchanged, its activity was diminished following hypoxia and WY-14643 treatment.Figure 10Effect of NS-398 and indomethacin on COX activity. RCE cells were grown for 24 h under normoxic or hypoxic conditions or treated with WY-14,643 (100 μm), washed twice, and incubated under normoxia with arachidonic acid (10 μm) and heme (2 μm) in the presence and absence of indomethacin (5 μm) or NS-398 (1 μm) for 1 h at 37 °C. Samples of the medium were collected and PGE2 levels measured. Results are the mean ± S.E., n = 4; *, p < 0.05 from normoxia indomethacin-treated cells; †,p < 0.05 from normoxia NS-398-treated cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Early prostaglandin research strove to establish a link between their synthesis and ocular inflammation. It is well documented that corneal surface injury increases production of cyclooxygenase-derived eicosanoids (PGI2, PGF2α, PGE2, PGD2, and thromboxane A2) by the corneal epithelium (27.Bazan H.E.P. Birkle D.L. Beuerman R.W. Bazan N.G. Curr. Eye Res. 1985; 4: 175-179Crossref PubMed Scopus (67) Google Scholar). There are, however, several observations that challenge the significance of their role in the inflammatory response. The increase in the production of these eicosanoids correlates poorly to the inflammatory sequelae (28.Gluud B.S. Jensen O.L. Krogh E. Birgens H.S. Acta Ophthalmol. 1985; 63: 375-379Crossref Scopus (12) Google Scholar). Moreover, while topical applications of micromolar concentrations of these eicosanoids are required to elicit inflammatory effects, much lower concentrations are usually recovered at the site of surface injury (29.Napoli S.A. Helm C. Insler M.S. Ensley H.E. Pretus H.A. Feigen L.P. Ann. Ophthalmol. 1990; 22: 30-34PubMed Google Scholar, 30.Srinivasan B.D. Kulkarni P.S. Invest. Ophthalmol. Vis. Sci. 1980; 19: 1087-1093PubMed Google Scholar). Furthermore, the use of metabolic inhibitors against the cyclooxygenase pathway is not as efficacious as corticosteroids in the treatment of ocular surface inflammation (31.Srinivasan B.D. Kulkarni P.S. Bito L.Z. Stjernschantz J. The Ocular Effect of Prostaglandins and Other Eicosanoids. Alan R. Liss, New York1989: 229-249Google Scholar, 32.Haynes W.L. Proia A.D. Klintworth G.K. Invest. Ophthalmol. Vis. Sci. 1989; 30: 1588-1593PubMed Google Scholar). These inhibitors tend to have partial efficacy at best, indicating that other corticosteroid-sensitive mediators may participate in the corneal inflammatory response. Studies in our laboratory identified the formation of the CYP-derived arachidonic acid metabolites 12(R)-HETE, a Na,K-ATPase inhibitor with chemotactic activity, and 12(R)-HETrE, a powerful proinflammatory mediator that has been shown to stimulate vasodilation, increase anterior chamber proteins, chemoattract polymorphonuclear leukocytes, and elicit angiogenesis (see Ref. 6.Laniado Schwartzman M. Green K. Edelhauser H.F. Hackett R.B. Hull D.S. Potter D.E. Tripathi R.C. Advances in Ocular Toxicology. Plenum Press, New York1997: 3-20Crossref Google Scholar and references therein). The endogenous conversion of arachidonate to these metabolites is increased following hypoxia in vitro andin vivo and after chemical injury in vivo. Their production rate correlates with the inflammatory response in vivo and inhibition of their formation attenuates the inflammation (reviewed in Ref. 6.Laniado Schwartzman M. Green K. Edelhauser H.F. Hackett R.B. Hull D.S. Potter D.E. Tripathi R.C. Advances in Ocular Toxicology. Plenum Press, New York1997: 3-20Crossref Google Scholar). In all, these studies clearly place the CYP-derived 12-hydroxyeicosanoids as corneal epithelial-derived mediators of ocular surface inflammation. However, the discovery of COX-2 as the inducible isoform of cyclooxygenase that is differently affected by common NSAIDs brought back the question of the involvement of prostaglandins in the ocular inflammatory process. The contemporary view is that prostaglandins are mediators of numerous biological processes including inflammation (33.Bazan N.G. Allan G. Surv. Ophthalmol. 1997; 41 Suppl. 2: S23-S34Abstract Full Text PDF PubMed Google Scholar). In the present study we examined the presence and inducibility of COX-2 in corneal epithelial cells following hypoxia-induced injury; such injury has been associated with a severe inflammatory response in vivo and with increased production of 12-HETE and 12-HETrEin vivo and in vitro (7.Conners M.S. Stoltz R.A. Webb S.C. Rosenberg J. Dunn M.W. Abraham N.G. Schwartzman M.L. Invest. Ophthalmol. Vis. Sci. 1995; 36: 828-840PubMed Google Scholar). We found that COX-2, but not COX-1, mRNA and protein levels are markedly increased in RCE cells following hypoxia. Hypoxia has been shown to stimulate the expression of COX-2 in the lung and endothelial cells (11.Schmedtje 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, 12.Chida M. Voelkel N.F. Am. J. Physiol. 1996; 270: L872-L878PubMed Google Scholar, 34.Ji Y.S. Xu Q. Schmedtje Jr., J.F. Circ. Res. 1998; 83: 295-304Crossref PubMed Scopus (80) Google Scholar). While acute exposure (2 h) to hypoxia increased COX activity in endothelial cells (35.Michiels C. Arnould T. Knott I. Dieu M. Remacle J. Am. J. Physiol. 1993; 264: C866-C874Crossref PubMed Google Scholar), chronic exposure (24 h) in these cells and in alveolar macrophages resulted in decreased COX-2 protein and PGE2 synthesis (36.Farber H.W. Barnett H.F. Circ. Res. 1991; 68: 1446-1457Crossref PubMed Scopus (50) Google Scholar, 37.Hempel S.L. Monick M.M. Hunninghake G.W. Am. J. Respir. Cell Mol. Biol. 1996; 14: 170-176Crossref PubMed Scopus (92) Google Scholar). In our study, 12–24 h of hypoxia markedly decreased PGE2 accumulation while increasing COX-2 expression. The use of the selective COX-2 inhibitor NS-398 further suggested that COX activity in hypoxic cells is essentially that of COX-2, whereas in control (normoxia) cells, both COX-1 and COX-2 contributed equally to the production of PGE2. Recent studies demonstrated that a CYP4B1 isoform is induced in the corneal epithelium in response to hypoxia and is involved in the production of 12-HETE and 12-HETrE (10.Mastyugin V. Aversa E. Vafeas C. Mieyal P. Laniado Schwartzman M. J. Pharmacol. Exp. Ther. 1999; 289: 1611-1619PubMed Google Scholar). A similar pattern of induction was observed in RCE cells. The increased CYP4B1 and COX-2 mRNA levels suggest a common mechanisms for regulation. Indeed, the rabbit lung CYP4B1 has been shown to be activated by clofibrate, a PPAR activator (15.Willson T.M. Wahli W. Curr. Opin. Chem. Biol. 1997; 1: 235-241Crossref PubMed Scopus (183) Google Scholar). Likewise, COX-2 expression has been shown to be induced by numerous PPAR ligands (17.Meade E.A. McIntyre T.M. Zimmerman G.A. Prescott S.M. J Biol. Chem. 1999; 274: 8328-8334Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Moreover, clofibrate increased conversion of arachidonic acid to 12-HETE and 12-HETrE in the corneal epithelium (10.Mastyugin V. Aversa E. Vafeas C. Mieyal P. Laniado Schwartzman M. J. Pharmacol. Exp. Ther. 1999; 289: 1611-1619PubMed Google Scholar). These findings indicate the possibility that PPAR activation may underlie the increased expression of COX-2 and CYP4B1 in hypoxia-treated cells. RT-PCR analysis clearly detected signals for PPARα and PPARβ, but not PPARγ, in hypoxia-treated cells. Treatment of cells with WY-14,643, a selective PPARα and β activator, markedly increased COX-2 expression. Furthermore, NSAIDs known to act as peroxisome proliferators such as indomethacin, flurbiprofen, and aspirin (23.Lehmann J.M. Lenhard J.M. Oliver B.B. Ringold G.M. Kliewer S.A. J Biol. Chem. 1997; 272: 3406-3410Abstract Full Text Full Text PDF PubMed Scopus (1061) Google Scholar) also increased COX-2 protein and mRNA. Interestingly, several NSAIDs have been shown to inhibit NFκB activation (38.Kopp E. Ghosh S. Science. 1994; 265: 956-959Crossref PubMed Scopus (1621) Google Scholar); the latter may be an important transcriptional activator of COX-2 expression in response to hypoxia (11.Schmedtje 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). The observation that these NSAIDs did not decrease COX-2 expression under hypoxic conditions argues against a dependence on NFκB in the hypoxia-induced expression of COX-2. In all, these findings suggest the possibility that PPAR activation, at least in part, contributes to the hypoxia-induced expression of COX-2 and CYP4B1. To this end, addition of PPAR ligands to hypoxia-treated cells did not yield a further increase in either COX-2 protein, mRNA, or PGE2accumulation, suggesting that hypoxia and PPAR ligands may share a similar mechanism, e.g. PPAR activation, by which they transcriptionally activate COX-2 expression. Noteworthy is the fact that the increased expression of COX-2 after addition of a PPAR ligand was, as in hypoxia, associated with a decrease in PGE2 accumulation. However, while COX enzymatic activity in hypoxic cells could be fully expressed, when oxygen is replenished, to reflect the 3–4-fold increase in protein levels, it was just about 50% of control in cells treated with WY-14,643. Ledwithet al. (39.Ledwith B.J. Pauley C.J. Wagner L.K. Rokos C.L. Alberts D.W. Manam S. J. Biol. Chem. 1997; 272: 3707-3714Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) have shown that PPAR ligands, including WY-14643, cause little or no increase in PGE2 levels in mouse liver cells, while markedly increasing COX-2 expression. These authors demonstrated that PPAR ligands do not inhibit COX enzymatic activity and suggested that these agents exhibit an indirect inhibitory effect on COX activity. Our findings concur with this report; however, they also offer a potential mechanism for WY-14,643. As indicated before, GSH peroxidase exerts a regulatory control on COX activity (26.Capdevila J.H. Morrow J.D. Belosludtsev Y.Y. Beauchamp D.R. DuBois R.N. Falck J.R. Biochemistry. 1995; 34: 3325-3337Crossref PubMed Scopus (105) Google Scholar). Depletion of GSH in cells treated with WY-14,643 restored COX activity and increased it to levels that better reflected the increase in protein expression induced by WY-14,643. It is possible that the indirect inhibitory activity of WY-14,643 referred to in the study of Ledwith et al. (39.Ledwith B.J. Pauley C.J. Wagner L.K. Rokos C.L. Alberts D.W. Manam S. J. Biol. Chem. 1997; 272: 3707-3714Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) is an effect on GSH metabolism leading to increased GSH levels that have been shown in our study as well as others to inhibit COX activity. Indeed, there are numerous reports demonstrating the effect of peroxisome proliferators including WY-14,643 on GSH metabolism (40.Cai Y. Appelkvist E.L. DePierre J.W. J. Biochem. Toxicol. 1995; 10: 87-94Crossref PubMed Scopus (43) Google Scholar). The dissociation between COX-2 expression and PGE2 levels in cells that were subjected to hypoxia could be the result of several factors operating under these conditions. One such factor is the availability of the substrate, arachidonic acid. However, experiments in which cells were prelabeled with arachidonic acid indicate that the levels of free arachidonic acid are, in fact, increased during hypoxia (data not shown). This is in accordance with reports which demonstrated that in many tissues ischemia/hypoxia leads to increased fatty acid release, including arachidonic acid, subsequent to activation of phospholipase A2 (41.Mukherjee A.B. Miele L. Pattabiraman N. Biochem. Pharmacol. 1994; 48: 1-10Crossref PubMed Scopus (190) Google Scholar). One may argue that, when COX is inhibited, such increases may be shifted to other metabolic enzymes such as lipoxygenases and CYP monooxygenase. However, these cells do not express significant levels of either lipoxygenase or cytochrome P450 monooxygenase activity in culture under normoxia or hypoxia (data not shown). The small increase in PGE2 accumulation may be due to the fact that the addition of exogenous arachidonic acid resulted in increased hydroperoxide levels, an essential requirement for COX catalytic activity (42.Marshall P.J. Kulmacz R.J. Lands W.E. J. Biol. Chem. 1987; 262: 3510-3517Abstract Full Text PDF PubMed Google Scholar). There are several reports indicating the presence of hydroperoxides with various preparations of arachidonic acid (43.Allen K.G. Huang C.J. Morin C.L. Anal. Biochem. 1990; 186: 108-111Crossref PubMed Scopus (17) Google Scholar). It is noteworthy that PGE2 levels in hypoxia-treated cells were essentially the same when either arachidonic acid was added or GSH was depleted. This suggests that the increased hydroperoxide level rather than substrate is the major factor contributing to the increased PGE2 levels. With regard to hypoxia, factors such as lack of oxygen and increasing heme degradation due to rapid induction of HO-1 (44.Lee P.J. Jiang B.-H. Chin B.Y. Iyer N.V. Alam J. Semenza G.L. Choi A.M.K. J. Biol. Chem. 1997; 272: 5375-5381Abstract Full Text Full Text PDF PubMed Scopus (653) Google Scholar) are likely to be the major factors in limiting COX activity. Indeed, reoxygenation in the presence of heme restored COX activity in hypoxia-treated cells. On the other hand, the WY-14,643 inhibitory effect on COX activity was not removed by reoxygenation and heme. The apparent disparity between expression of COX-2 and endogenous levels of PGE2 in the injured cells may have important pathophysiological and therapeutic implications. It is well documented that prolonged exposure of the corneal epithelium to hypoxia in the form of eye closure or contact lens wear leads to a pronounced inflammatory response (7.Conners M.S. Stoltz R.A. Webb S.C. Rosenberg J. Dunn M.W. Abraham N.G. Schwartzman M.L. Invest. Ophthalmol. Vis. Sci. 1995; 36: 828-840PubMed Google Scholar). It is also known that NSAIDs are only partially effective in treating ocular surface inflammation. Inasmuch as COX-2 is defined as an inflammatory gene, in the context of this type of injury and this tissue, it seems likely that its rapid induction does not lead to increased levels of PGE2,i.e. an increase in its expression does not yield a parallel increase in PGE2, further indicating that the inflammatory response in the corneal epithelium is not prostanoid-mediated under these conditions. Moreover, under these conditions, NSAIDs may intensify the inflammatory response by activating PPARs and subsequently amplifying the response via induction of CYP4B1 and production of the proinflammatory eicosanoids, 12-HETE and 12-HETrE. The increased production of the latter in the presence of NSAIDs may also result from shunting arachidonic acid to this pathway. To this end, preliminary studies have demonstrated induction of PPARs α and β, COX-2, and CYP4B1 expression in corneal epithelium from rabbit eyes that were subjected to hypoxia via eye closure and contact lens wear; in this model the inflammatory response including edema, vasodilation, and neovascularization correlates with the duration of contact lens wear and is associated with increased synthesis of 12-HETE and 12-HETrE. The events demonstrated in the cultured corneal epithelial cells may also occur in vivo not only in the cornea but also in other tissues where hypoxia produces an inflammatory response such as in vascular occlusion diseases. Moreover, the results of this study suggest that the therapeutic action of peroxisome proliferators such as fibrates may involve an increase in GSH levels and a decrease in the level of peroxidation. Similarly, NSAIDs may act in such a way by activating PPARs and thus indirectly promoting antioxidant mechanisms. We thank Dr. Matthew Breyer (Department of Medicine, Vanderbilt University) for the rabbit COX-2 cDNA." @default.
• W2116821504 created "2016-06-24" @default.
• W2116821504 creator A5001385380 @default.
• W2116821504 creator A5011638826 @default.
• W2116821504 creator A5041210629 @default.
• W2116821504 creator A5044355307 @default.
• W2116821504 creator A5076127154 @default.
• W2116821504 date "2000-01-01" @default.
• W2116821504 modified "2023-10-15" @default.
• W2116821504 title "Regulation of Cyclooxygenase-2 by Hypoxia and Peroxisome Proliferators in the Corneal Epithelium" @default.
• W2116821504 cites W1596621826 @default.
• W2116821504 cites W1754314044 @default.
• W2116821504 cites W1981101682 @default.
• W2116821504 cites W1985238295 @default.
• W2116821504 cites W1990345525 @default.
• W2116821504 cites W1994821631 @default.
• W2116821504 cites W1999509419 @default.
• W2116821504 cites W2003536031 @default.
• W2116821504 cites W2007019274 @default.
• W2116821504 cites W2010237936 @default.
• W2116821504 cites W2015897844 @default.
• W2116821504 cites W2028754976 @default.
• W2116821504 cites W2038197749 @default.
• W2116821504 cites W2038537466 @default.
• W2116821504 cites W2040211695 @default.
• W2116821504 cites W2051720790 @default.
• W2116821504 cites W2053837499 @default.
• W2116821504 cites W2066534566 @default.
• W2116821504 cites W2081415790 @default.
• W2116821504 cites W2081635255 @default.
• W2116821504 cites W2087908472 @default.
• W2116821504 cites W2088404163 @default.
• W2116821504 cites W2089932791 @default.
• W2116821504 cites W2118232192 @default.
• W2116821504 cites W2118321652 @default.
• W2116821504 cites W2125471714 @default.
• W2116821504 cites W2149233326 @default.
• W2116821504 cites W2150854309 @default.
• W2116821504 cites W2155913257 @default.
• W2116821504 cites W2158943061 @default.
• W2116821504 cites W3063220799 @default.
• W2116821504 doi "https://doi.org/10.1074/jbc.275.4.2837" @default.
• W2116821504 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10644750" @default.
• W2116821504 hasPublicationYear "2000" @default.
• W2116821504 type Work @default.
• W2116821504 sameAs 2116821504 @default.
• W2116821504 citedByCount "117" @default.
• W2116821504 countsByYear W21168215042012 @default.
• W2116821504 countsByYear W21168215042013 @default.
• W2116821504 countsByYear W21168215042014 @default.
• W2116821504 countsByYear W21168215042016 @default.
• W2116821504 countsByYear W21168215042017 @default.
• W2116821504 countsByYear W21168215042018 @default.
• W2116821504 countsByYear W21168215042020 @default.
• W2116821504 countsByYear W21168215042021 @default.
• W2116821504 countsByYear W21168215042022 @default.
• W2116821504 countsByYear W21168215042023 @default.
• W2116821504 crossrefType "journal-article" @default.
• W2116821504 hasAuthorship W2116821504A5001385380 @default.
• W2116821504 hasAuthorship W2116821504A5011638826 @default.
• W2116821504 hasAuthorship W2116821504A5041210629 @default.
• W2116821504 hasAuthorship W2116821504A5044355307 @default.
• W2116821504 hasAuthorship W2116821504A5076127154 @default.
• W2116821504 hasBestOaLocation W21168215041 @default.
• W2116821504 hasConcept C127078168 @default.
• W2116821504 hasConcept C170493617 @default.
• W2116821504 hasConcept C178790620 @default.
• W2116821504 hasConcept C181199279 @default.
• W2116821504 hasConcept C185592680 @default.
• W2116821504 hasConcept C2779257441 @default.
• W2116821504 hasConcept C2779689624 @default.
• W2116821504 hasConcept C2909020919 @default.
• W2116821504 hasConcept C2910266098 @default.
• W2116821504 hasConcept C529295009 @default.
• W2116821504 hasConcept C540031477 @default.
• W2116821504 hasConcept C54355233 @default.
• W2116821504 hasConcept C55493867 @default.
• W2116821504 hasConcept C7836513 @default.
• W2116821504 hasConcept C86803240 @default.
• W2116821504 hasConcept C95444343 @default.
• W2116821504 hasConceptScore W2116821504C127078168 @default.
• W2116821504 hasConceptScore W2116821504C170493617 @default.
• W2116821504 hasConceptScore W2116821504C178790620 @default.
• W2116821504 hasConceptScore W2116821504C181199279 @default.
• W2116821504 hasConceptScore W2116821504C185592680 @default.
• W2116821504 hasConceptScore W2116821504C2779257441 @default.
• W2116821504 hasConceptScore W2116821504C2779689624 @default.
• W2116821504 hasConceptScore W2116821504C2909020919 @default.
• W2116821504 hasConceptScore W2116821504C2910266098 @default.
• W2116821504 hasConceptScore W2116821504C529295009 @default.
• W2116821504 hasConceptScore W2116821504C540031477 @default.
• W2116821504 hasConceptScore W2116821504C54355233 @default.
• W2116821504 hasConceptScore W2116821504C55493867 @default.
• W2116821504 hasConceptScore W2116821504C7836513 @default.
• W2116821504 hasConceptScore W2116821504C86803240 @default.
• W2116821504 hasConceptScore W2116821504C95444343 @default.
• W2116821504 hasIssue "4" @default.
• W2116821504 hasLocation W21168215041 @default.

• next