Inhibition of Tumor Necrosis Factor alpha -mediated NFkappa B Activation and Leukocyte Adhesion, with Enhanced Endothelial Apoptosis, by G Protein-linked Receptor (TP) Ligands*

Anthony W. AshtonDagger , Gabriel M. WareDagger , Dhananjaya K. KaulDagger , and J. Anthony WareDagger §

From the Departments of Dagger  Medicine and § Molecular Pharmacology, Albert Einstein College of Medicine, Yeshiva University, Bronx, New York 10461

Received for publication, October 21, 2002, and in revised form, January 6, 2003

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Tumor necrosis factor (TNF) alpha  is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A2 (TXA2) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA2 mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta . The potential interaction between TXA2 and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA2 mimetic, attenuated TNFalpha -induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha -enhanced gene expression, and also reduced TNFalpha -induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha -induced NFkappa B activation in endothelial cells, as determined by reduced Ikappa B phosphorylation and NFkappa B nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha . This inhibition resulted from the Galpha q-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha -mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA2, in combination with TNFalpha , may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

A critical mediator of several inflammatory and ischemic conditions (1), tumor necrosis factor alpha  (TNFalpha )1 promotes leukocyte adhesion to vascular endothelium both in vitro and in vivo; such adhesion is mediated by the complex interplay of adhesion receptors on both leukocytes and endothelial cells. TNFalpha induces the transcription of the leukocyte adhesion molecules ICAM, VCAM, and E-selectin by activating the transcription factor NFkappa B. Multiple vascular cell types, including macrophages, cardiac myocytes, mast cells, and neutrophils release TNFalpha during ischemia and inflammation (1-3). In addition to its proinflammatory effect, TNFalpha can cause cell death by apoptosis and promote cell survival (4). These diverse effects can result in apparently contradictory experimental findings, as experimental data support both deleterious and beneficial roles for TNFalpha in, for example, preservation of myocardial function following ischemia and reperfusion (5-8).

The complexity of the physiologic effects of TNFalpha is mimicked by the mechanisms by which it generates signals to produce those effects. TNFalpha interacts with two cell surface receptors, TNFR1 and TNFR2, ubiquitously expressed on cells of the cardiovascular system. TNFR1 is dominant in most cell types; an independent role for TNFR2 is yet to be established (4). After ligand binding, a conformational change in the pretrimerized TNF receptor complex causes dissociation of the SODD protein and, in a series of protein-protein interactions, intracellular mediators are recruited to the cytosolic portion of the receptor (9). TNFR1 activates two broad signaling pathways, both initiated by the recruitment of TRADD to TNFR1. One pathway, mediated by the subsequent recruitment of FADD, results in caspase 8 activation and initiation of apoptosis. Conversely, the survival pathway involves association of RIP and TRAF-2 with TRADD. The TRADD·RIP·TRAF-2 complex recruits the IKK signalosome to TNFR1 thus activating NFkappa B. In addition, the TRADD·RIP·TRAF-2 complex also activates MAPK pathways that lead to AP-1 activation (4, 10).

During inflammation, multiple cytokines and stimulants of vascular endothelium are released. Thus, any pathophysiological effect produced by TNFalpha must take place in the context of "cross-talk" with those mediators. The most common class of receptor for these mediators are G protein-linked receptors, multiple forms of which are expressed on vascular cells. One mediator of particular importance in ischemia and inflammation is the eicosanoid thromboxane A2 (TXA2), released from activated platelets and damaged vessel walls. Local and systemic elevations in TXA2 are reported in several thrombotic and vascular diseases (11). TXA2 mediates vascular damage by inducing platelet activation, vasoconstriction, vascular smooth muscle hypertrophy/hyperplasia, and by converting the endothelial surface to a "prothrombotic" state. These biological actions of TXA2 are mediated by a G protein-linked receptor (TP) of which two alternatively spliced subtypes, TPalpha and TPbeta , exist in humans (12, 13). Although the intracellular tail of TPbeta differs from that of TPalpha , these isoforms couple to 80% of the same G proteins (14). Differences in signaling reported so far mediate receptor trafficking and desensitization (15-17).

In this study, we sought to investigate whether stimulation of TP, a prototypical G protein-coupled receptor that is activated during ischemia and inflammation, could alter TNFalpha -induced endothelial cell apoptosis and leukocyte adhesion in vivo. Furthermore, we tested the possibility that such an effect resulted from a selective effect on specific signal transduction pathways employed by TNFalpha . The data present a currently unrecognized interaction between TNFalpha and ligands for G protein-coupled receptors that may provide a mechanism by which endothelial cell death occurs during inflammation.

    EXPERIMENTAL PROCEDURES
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INTRODUCTION
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Chemicals-- The G protein inhibitors NF023 (Galpha i), NF449 (Galpha s), GP antagonist 2A (Galpha q), pertussis toxin, and GDP were all purchased from Calbiochem-Novabiochem. All other chemicals were of suitable grade and purchased from Sigma unless otherwise stated.

Cell-based Receptor Enzyme-linked Immunosorbent Assay and Fluorescence-activated Cell Sorter-- Human endothelial cells (HEC) isolated from umbilical veins were cultured as reported previously (18). HEC were stimulated for up to 24 h with media alone (Control), recombinant human TNFalpha or IL-1beta (100 units/ml in all experiments unless otherwise stated), the TXA2 mimetic IBOP (0-200 nM), or the TP blocker SQ29548 (5 µM) (Cayman Chemical, Ann Arbor, MI). Expression of ICAM-1, VCAM-1, and E-selectin was detected by enzyme-linked immunosorbent assay (19) or flow cytometry (18) using commercially available monoclonal antibodies (Dako Corp., Carpinteria, CA).

Leukocyte Adhesion Assays-- Confluent monolayers of first passage HEC were stimulated with TNFalpha , IBOP, or the two in combination for 8 h and were washed extensively before the addition of leukocytes. U937 cells, a leukocyte cell line, were labeled with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (2 µM) for 30 min and 1 × 106 cells were added to stimulated HEC monolayers for 2 h. At the conclusion, cells were washed with phosphate-buffered saline and incubated with lysis buffer (50 mM HEPES, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.1 mM phenylmethylsulfonyl fluoride) for 30 min. U937 attachment was quantitated in a fluorometric plate reader using fluorescein isothiocyanate filters.

Analysis of Leukocyte Adhesion and Extravasation in Vivo-- The modulation of TNFalpha by TXA2 in vivo was analyzed by monitoring the passage of leukocytes along postcapillary venules in the externalized cremaster muscle as previously described (20). C57-black mice were injected intraperitoneally with murine TNFalpha (150 pM), the TXA2 mimetic IBOP (50 nM), or the two together and prepared for intravital microscopy after 4 h. Adherent leukocytes were quantified per 100 µm of vessel length. Extravasated leukocytes were determined as the number of interstitial leukocytes adjacent (within 30 µm) to venules.

Immunoprecipitation and Immunoblotting-- Confluent HEC were treated with IBOP, IL-1beta , or TNFalpha alone or in combination for 5 min. Monolayers were washed twice in phosphate-buffered saline containing 1 mM phenylmethylsulfonyl fluoride and scraped into immunoprecipitation lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate, 0.1 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin). Cell suspensions were sonicated, incubated on ice for 30 min, and clarified by centrifugation. For immunoprecipitation, protein content was determined and 300 µg of total protein was incubated for 16 h at 4 °C with protein G-agarose beads coated with saturating amounts of antibodies to TNFR1, TNFR2, NFkappa B (p65RelA) (Santa Cruz Biotechnology, Santa Cruz, CA), RIP, or TRADD (Transduction Laboratories). The resulting immune complexes were recovered after centrifugation by boiling for 10 min in SDS-PAGE loading buffer. Nuclear proteins were isolated as previously reported (21).

For immunoblotting, aliquots of whole cell lysates (30 µg) or isolated immune complexes were separated by SDS-PAGE under reducing conditions using 10% acrylamide gels. Proteins were transferred onto polyvinylidene difluoride membrane and analyzed by immunoblotting as previously described (18) using antibodies against MADD, STAT1, NIK (Pharmingen), Ask1, MEKK1, FADD, TRAF-2, Ikappa B, IKKgamma , and the phosphorylated forms of p38, ERK1/2, JNK (Santa Cruz), STAT1 (Tyr701) (Upstate Biotechnology), and Ikappa B (Calbiochem-Novabiochem Corp.) in addition to those described above. An alpha -tubulin monoclonal antibody was used to control for loading.

Annexin V Staining-- For annexin V staining, HEC stimulated with IBOP, TNFalpha , or the two in combination (16 h) were harvested using trypsin/EDTA and stained for annexin V using the Pharmingen annexin-fluorescein isothiocyanate staining kit (Pharmingen). Annexin V staining was quantitated using flow cytometry.

Caspase Activity Assays-- Following treatment with TNFalpha , IBOP, or the two in combination for 16 h, HEC were scraped into lysis buffer and incubated on ice for 30 min. Lysates were clarified by centrifugation and protein concentration was estimated. Protease reactions utilized cleavage of the chromogenic peptide substrates Z-DEVD-p-nitroanilide and Ac-IETD-p-nitroanilide for caspase 3- and 8-like activity, respectively (Biomol Research Laboratories, Plymouth Meeting, PA). Caspase activity assays were carried out in reaction buffer (100 mM HEPES, pH 7.5, 5 mM dithiothreitol, 20% (v/v) glycerol, 0.5 mM EDTA, 0.1% (w/v) bovine serum albumin, 10 mM caspase substrate) using 300 µg of total protein. Reactions were incubated at 30 °C for 30 min and cleavage of colorimetric substrates quantitated at 415 nm.

Cell Transfection and Luciferase Assays-- HEC were transfected with the NFkappa B-dependent luciferase reporter construct pBII-Luc(2× Igk kappa B-fos-luciferase) (22) or STAT1 oligonucleotides using the GenePORTER transfection reagent (Gene Therapy Systems, San Diego, CA). Transfected HEC were stimulated with TNFalpha or IL-1beta alone or in combination with IBOP for 16 h and relative luciferase activity was determined using the luciferase gene reporter kit (Roche Molecular Biochemicals). STAT1 sense (5'-ggTggCAggATgTCTCAgTgG 3') and antisense (5'-CCACTgAgACATCCTgCCACC-3') oligonucleotides were transfected into cells 24 h prior to assay; preliminary experiments established that this time was sufficient for the ablation of STAT1 expression.

RNA Isolation and Northern Blot Analysis-- Confluent HEC were exposed to TNFalpha or IBOP or both agents for up to 8 h. HEC were washed twice with phosphate-buffered saline and total mRNA was isolated using TRIzol reagent (Invitrogen). RNA (5 µg of each sample) was separated on a denaturing 1% agarose gel, transferred to nylon membrane (Amersham Biosciences), and blotted for leukocyte adhesion molecule (LAM) expression as previously described (23). Full-length human cDNA probes to ICAM-1, VCAM-1, E-selectin, and GAPDH mRNAs were made from all cDNAs by random priming using the Megaprime Labeling kit (Amersham Biosciences). After blotting, membranes were dried and autoradiographed for the appropriate period of time with changes in mRNA levels quantitated by scanning densitometry.

Statistical Analysis-- Data were pooled and statistical analysis was performed using the Mann-Whitney U test.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

TXA2 Mimetics Stimulate LAM Expression on HEC-- Stimulation with the TXA2 mimetic IBOP (50 nM) increased expression of LAM molecules on HEC, with maximal induction after 16 h for ICAM-1 and 6-8 h for VCAM-1 and E-selectin (p <=  0.001, Fig. 1A). Induction of LAM expression by IBOP was concentration-dependent. ICAM-1 and VCAM-1 expression on HEC were maximally induced at IBOP concentrations >= 25 nM (IC50 12 nM, p <=  0.005) at 16 and 6 h, respectively. E-selectin was maximally induced at IBOP concentrations >= 50 nM (IC50 25 nM) after 6 h (p <=  0.01, Fig. 1B). U46619, another TP agonist, also stimulated LAM expression on HEC with maximal induction at 400 nM and time courses similar to those shown for IBOP (data not shown). Inclusion of the TP antagonist SQ29548 (5 µM) abrogated IBOP-induced LAM expression (data not shown), indicating the effect was mediated by the TXA2 receptor (TP).


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Fig. 1.   Effect of the TXA2 mimetic IBOP on LAM expression by HEC. A, representative flow cytometer tracing indicating the levels of ICAM staining on HEC treated with media alone (), IBOP (black-square), or IBOP in the presence of the TP blocker SQ29548 (). Staining with preimmune IgG is also shown (). B, time course for the induction of LAM by IBOP. HEC were stimulated with IBOP (50 nM) for up to 16 h and probed for E-selectin (Delta ), VCAM (), and ICAM (black-diamond ) expression. Control cultures were incubated with media alone. C, cells were stimulated for the appropriate time with IBOP (0-400 nM) and the expression of E-selectin (triangle ), VCAM (), and ICAM (black-diamond ) was determined. All data generated using the flow cytometric assay are expressed as geometric mean of the curve (mean ± S.D.) from three different experiments. * indicates significance (p <=  0.05) from control. D, NFkappa B in nuclear extracts of IBOP-stimulated cell lines expressing TPalpha or TPbeta . To determine the G protein(s) involved in activation of NFkappa B, cells were incubated with GDP (50 µM) or antagonists of specific G proteins (10 µM, Galpha q, Galpha i, Galpha s, and pertussis toxin (Galpha iPT)) for 30 min prior to the addition of stimulants. Histone H1 is shown as a loading control. These data are representative of three individual experiments.

LAM expression is regulated by increased transcription, which in turn is mediated by NFkappa B (24). HEC express both TP isoforms, TPalpha and TPbeta . In cell lines overexpressing each of the two TP isoforms on a null background, NFkappa B nuclear translocation (Fig. 1C) and LAM expression (data not shown) were found to follow ligation of TPbeta , but not of TPalpha . NFkappa B nuclear translocation in response to IBOP in TPbeta expressing cells was inhibited by loading the cells with GDP, indicating the process was mediated by G proteins, and by two pharmacological inhibitors of the heterotrimeric G protein Galpha i (Fig. 1C). Antagonists of Galpha q and Galpha s were ineffective, consistent with the concept that this process was mediated by Galpha i signaling.

IBOP Prevents the Induction of LAM on HEC by TNFalpha , but Not IL-1beta -- We hypothesized that TXA2 may regulate expression of LAM induced by other proinflammatory mediators, such as IL-1beta or TNFalpha . TNFalpha and IL-1beta increased expression of ICAM-1, VCAM-1, and E-selectin by an average of 5.5-, 12-, and 15-fold, respectively (p <=  0.001) (Fig. 2A). Inclusion of IBOP with IL-1beta did not significantly alter the induction of LAMs, suggesting that IL-1beta and TP stimulation were neither synergistic nor additive. In contrast, stimulation with both TNFalpha and IBOP abrogated the induction of LAM (p <=  0.001 versus TNFalpha ) (Fig. 2A). IBOP-mediated inhibition of TNFalpha -induced expression of ICAM-1 (p <=  0.001, Fig. 2B), VCAM-1, and E-selectin (data not shown) was blocked by SQ29548 (5 µM), indicating that TP binding was a requirement for this effect. SQ29548 did not alter basal or TNFalpha -induced LAM expression (Fig. 2B).


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Fig. 2.   Inhibition of the induction of LAM on TNFalpha , but not by IL-1beta , stimulated HEC by TXA2 mimetics. A, HEC were stimulated with IBOP (100 nM), TNFalpha or IL-1beta (100 units/ml) alone or in combination and LAM expression was quantitated by flow cytometry after 2, 6, or 16 h for expression of E-selectin (), VCAM (), and ICAM (black-square), respectively. B, HEC were stimulated with IBOP, TNFalpha , or the TP blocker SQ29548 (5 µM) either alone or in combination and ICAM-1 expression was measured. C, HEC were stimulated with TNFalpha and increasing concentrations of IBOP (0-200 nM) and ICAM-1 expression was quantitated after 16 h. D, HEC were stimulated with media alone (), TNFalpha (), IBOP (black-square), or both agents together (open circle ) and ICAM-1 expression was quantitated over 24 h. Control cultures were incubated with media alone. Data (B-D) are expressed as OD measured at 415 nm (mean ± S.D.) from four different experiments. * indicates significance (p <=  0.05) from control, # indicates significance (p <=  0.05) from TNFalpha or IBOP treatment.

The inhibition of LAM expression by IBOP in TNFalpha -stimulated HEC was also concentration-dependent (Fig. 2C). TNFalpha treatment induced a 4.5-fold increase in ICAM-1 expression (Fig. 2C), above that seen with untreated HEC. Increasing concentrations of the TXA2 mimetic IBOP attenuated the induction of ICAM-1 by TNFalpha with inhibition greatest at IBOP concentrations >= 100 nM (p <=  0.01, IC50 = 50 nM) (Fig. 2C). The induction of E-selectin and VCAM-1 expression were similarly inhibited by IBOP in TNFalpha -stimulated HEC (data not shown). Inhibition of LAM expression on TNFalpha -stimulated HEC was not because of alterations in the time course for maximal expression, as cells stimulated with both agents displayed only basal expression of ICAM-1 (Fig. 2D), VCAM-1, and E-selectin (data not shown) at all time points. Thus, the prevention of TNFalpha -induced LAM expression by TXA2 mimetics did not result from cross-talk between the two agents in which the time to peak expression was altered.

Consistent with these observations, IBOP-mediated inhibition of LAM expression prevented leukocyte adhesion to TNFalpha -stimulated HEC. Stimulation with either the TXA2 mimetic IBOP or TNFalpha induced a 3-4-fold increase in adhesion of the monocytic cell line U937 to HEC (p <=  0.005, Fig. 3A). Simultaneous treatment with both agents resulted in U937 adhesion similar to that in untreated controls (p >=  0.4, Fig. 3A). Similar results were observed when the adhesion of leukocytes to the vessel wall and their extravasation from the vasculature into surrounding tissues was examined in vivo. Injection of recombinant murine TNFalpha (150 pM) into mice resulted in a 7- and 11-fold increase in leukocyte adhesion to and extravasation from, respectively (p <=  0.0001), the venous network of cremaster muscle preparations after 4 h (Fig. 3B). Co-stimulation with IBOP (50 nM) resulted in an attenuation of TNFalpha -mediated leukocyte adhesion and extravasation (p <=  0.005 versus TNFalpha alone), with leukocyte rolling increased 3-fold (data not shown). Consistent with the observation above that ligation of TPbeta induces LAM expression in EC, IBOP stimulation did not cause an increase in leukocyte trafficking in vivo (Fig. 3B), as a mouse homologue for TPbeta has not been identified, and the murine TP resembles the human TPalpha isoform.


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Fig. 3.   Prevention of the adhesion of leukocytes to TNFalpha -stimulated endothelium both in vitro and in vivo through TP-stimulated inhibition of transcription. A, adhesion of labeled U937 cells to HEC stimulated with IBOP (100 nM), TNFalpha (100 units/ml), or the two in combination was quantitated after 2 h by measuring the fluorescence of the resulting lysates. Data (mean ± S.D.) are from four different experiments. B, effect of TNFalpha and IBOP alone and in combination on leukocyte adhesion and extravasation in the mouse cremaster muscle in vivo. Vessel segments were examined by video microscopy and the number of leukocytes adherent to the vessel wall (black-square) or in the extravascular tissue () were counted. Data are expressed as cells per 100-µm vessel length and are the average (mean ± S.E.) of five mice for each group. * indicates significance (p <=  0.01) from control, # indicates significance (p <=  0.01) from TNFalpha . C, HEC were stimulated for 3 h (E-selectin and VCAM-1) or 8 h (ICAM-1) with IBOP (100 nM), TNFalpha (100 units/ml), or the two in combination. Total RNA was extracted and E-selectin, VCAM-1, and ICAM-1 expression were analyzed by Northern blotting. GAPDH mRNA was probed as a loading control. Autoradiographs are from a single experiment and are representative of three experiments.

Regulation of ICAM-1, VCAM-1, and E-selectin by TNFalpha depends upon increased transcription (24). To determine how TP inhibits LAM expression in response to TNFalpha , we examined the pattern of LAM mRNA expression. Fig. 3C shows that only ICAM-1 mRNA was detectable in untreated endothelial cells by Northern blot analysis. TNFalpha and IBOP both induced ICAM-1 expression 9-fold over controls after stimulation for 8 h (Fig. 3C). Both agents also induced robust expression of VCAM-1 and E-selectin after 3 h. The equivalent induction of ICAM-1, VCAM-1, and E-selectin mRNA by TNFalpha and TP stimulation is consistent with the pattern of protein expression determined by flow cytometry (Fig. 2A). Consistent with the results in panels A and B, incubation with both TNFalpha and IBOP prevented the increased mRNA expression for ICAM-1, VCAM-1, and E-selectin observed with either agent alone (Fig. 3C).

TP Stimulation Prevents Activation of NFkappa B by TNFalpha -- Comparison of the ICAM-1, VCAM-1 promoters, and E-selectin promoters indicated that NFkappa B and AP1 were factors that possibly indicated transcriptional regulation by TNFalpha . To examine whether TNFalpha -stimulated NFkappa B activity is regulated by TXA2 mimetics, we transfected HEC with a NFkappa B-sensitive luciferase reporter construct. TNFalpha , IL-1beta , and IBOP treatment induced luciferase activity in transfected HEC to similar levels when used individually (Fig. 4A, p <=  0.005). In contrast, IBOP abrogated the transcriptional activity of NFkappa B when used in combination with TNFalpha , but not IL-1beta (Fig. 4A).


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Fig. 4.   Prevention of gene expression in response to TNFalpha by TP-mediated inhibition of the nuclear translocation of NFkappa B. A, HEC were treated with IBOP, IL-1beta , or TNFalpha alone or in combination for 16 h and luciferase activity in transfected HEC lysates was determined. Data are from four different experiments (mean ± S.D.). * and # indicate significance (p <=  0.01) from control and TNFalpha , respectively. B, Ikappa B phosphorylation and NFkappa B nuclear translocation in response to TNFalpha and IL-1beta , with or without IBOP co-stimulation, after 30 min, was determined using specific antibodies for p65RelA and phosphorylated Ikappa B. In panels B and C, histone H1 is shown as a loading control. C, NFkappa B in nuclear extracts of HEC stimulated with TNFalpha and IBOP was measured. To determine the G protein(s) involved in the inhibition of TNFalpha induced activation of NFkappa B by IBOP, cells were incubated with GDP (50 µM), or antagonists of specific G proteins (10 µM, Galpha q, Galpha i, and Galpha s) for 30 min prior to the addition of stimulants. D, IBOP reverses the activation of NFkappa B by TNFalpha . HEC were incubated with IBOP or TNF alone or in combination for 15 or 30 min. In addition, HEC were incubated with IBOP 15 min after TNFalpha treatment had begun (lane 6); in the reverse experiment, TNFalpha was added 15 min after treatment with IBOP (lane 8). The activation of NFkappa B was examined by nuclear translocation of NFkappa B, phosphorylation of Ikappa B, and complex formation between NFkappa B and Ikappa B. In the bottom two subpanels, immunoprecipitation (IP) of NFkappa B is followed by immunoblotting (WB) with Ikappa B or NFkappa B, the latter as a control. Histone H1 blotting was used to control for loading on nuclear protein preparations. The blots shown are representative of four individual experiments.

Nuclear translocation and full transcriptional activity of NFkappa B require phosphorylation of the NFkappa B inhibitor protein, Ikappa B (4). Treatment with TNFalpha , IL-1beta , or IBOP for 20 min increased Ikappa Balpha phosphorylation in HEC lysates 10-12-fold and induced nuclear translocation of p65RelA, the most common NFkappa B subunit. Untreated endothelial cells had little or no phosphorylated Ikappa B or nuclear p65RelA as assessed by immunoblotting (Fig. 4B). In accordance with the luciferase data (showing a lack of NFkappa B-mediated transcriptional activity), co-stimulation with IBOP prevented the nuclear translocation of p65RelA and the enhanced Ikappa Balpha phosphorylation observed with TNFalpha , but not IL-1beta , alone (Fig. 4B). The inhibition of TNFalpha -induced NFkappa B activation by TP was prevented both in GDP-loaded cells, showing that the effect was G protein-mediated, and was also blocked by an antagonist of Galpha q signaling, but not that of Galpha s or Galpha i (Fig. 4C). IBOP stimulation also abrogated TNFalpha -induced NFkappa B activation in cell lines expressing TPalpha or TPbeta alone (data not shown), consistent with the previously reported data that both TP isoforms are coupled to Galpha q (14).

Interestingly, IBOP not only inhibited TNFalpha -induced NFkappa B nuclear translocation but also reversed the process (Fig. 4D). Simultaneous addition of TNF and IBOP (30 min) inhibited the nuclear accumulation of NFkappa B observed with either agent alone. TNFalpha stimulation for 15 min induced nuclear NFkappa B staining and Ikappa B phosphorylation similar to that obtained after 30 min (Fig. 4D, lane 3 versus lane 5). Addition of IBOP (to TNFalpha -stimulated HEC) for a further 15 min resulted in an absence of Ikappa B phosphorylation and nuclear NFkappa B staining (lane 6). In addition, NFkappa B was found to be reassociated with Ikappa B in HEC treated with TNFalpha for 15 min prior to the addition of the TP agonist for a further 15 min, indicating that the levels of free NFkappa B were returned to baseline (Fig. 4D, lane 6 versus lane 5). The reciprocal was also true, as addition of TNFalpha after IBOP stimulation reversed the activation of NFkappa B, decreased Ikappa B phosphorylation, and increased Ikappa B·NFkappa B complexes (Fig. 4D, lane 8 versus lane 7). These data indicate that TP agonists both inhibit the activation of NFkappa B by TNFalpha and also reverse previously activated NFkappa B.

TP Specifically Inhibits the Activation of NFkappa B, but Not Other TNFalpha Pathways, by Disrupting Early Events in Receptor Signaling-- NFkappa B activation by IL-1beta and TNFalpha converge at the IKK signalosome and share all distal elements. The inability of TXA2 mimetics to prevent IL-1beta -mediated NFkappa B activation indicates that the pathways involved may be specific to TNFalpha . Stimulation with either IBOP or TNFalpha alone, or together, did not alter the cellular expression of any of the proteins investigated (data not shown). Thus, we immunoprecipitated TNFR1 and TNFR2 and blotted for the expected proteins to investigate whether TP stimulation altered the protein-protein interactions evoked by TNFalpha signaling. None of the proteins tested were recruited to the cytoplasmic tail of TNFR1 or TNFR2 in the absence of TNFalpha (Fig. 5, lanes 1 and 2). TNFalpha stimulation induced robust association of TRADD, RIP, TRAF-2, and IKKgamma with TNFR1. TRADD associated with TNFR1 to a similar degree in the presence or absence of IBOP. In contrast, IBOP co-stimulation prevented association of RIP, TRAF-2, and IKKgamma with TNFR1 (Fig. 5A, lane 4). Furthermore, neither TNFR1 nor TRADD co-immunoprecipitated with either anti-RIP or TRAF-2 antibodies in the presence of TNFalpha and IBOP (data not shown). This indicates that the interaction of RIP and TRAF-2 with TRADD, which occur independently, were both inhibited by IBOP. STAT-1 phosphorylation at Tyr701 was recently reported to be part of the TNFR1·TRADD complex, and inhibited TRADD-TRAF-2/RIP interactions without influencing TRADD-FADD interactions (25). Interestingly, the TNFalpha -induced STAT1-TNFR1 interaction was increased 3-fold by co-stimulation with the TXA2 mimetic IBOP (Fig. 5A). TNFalpha was found to increase STAT1 phosphorylation (Fig. 5B), which was augmented 3-fold by co-stimulation with IBOP. IBOP alone did not stimulate STAT1 phosphorylation (Fig. 5B), however, suggesting that the enhancement of TNF2-induced STAT1 phosphorylation by IBOP and TNFalpha is likely a result of another mechanism, such as inhibition of phosphatase activity. As is the case with inhibition of NFkappa B, the IBOP-enhanced phosphorylation of STAT1 was reduced by inhibition of Galpha q signaling (Fig. 5B). Thus, the abrogation of TNFR1 NFkappa B signaling by IBOP may result from a TP-mediated enhancement of STAT1 activation by TNFalpha .


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Fig. 5.   IBOP-mediated prevention of the association of proteins responsible for NFkappa B signaling with TNFR1 and TNFR2 upon TNFalpha stimulation. The binding of downstream effectors to TNFR1 (A) and TNFR2 (C) was analyzed after stimulation for 5 min with IBOP, TNFalpha , or the two in combination. TNFR1 or TNFR2 were immunoprecipitated and immune complexes were probed with antibodies to the proteins as indicated in the figure. In addition, lysates from stimulated HEC were probed using antibodies against STAT1 phosphorylated at Tyr701 (B). Blots were reprobed with a monoclonal STAT1 antibody to assess loading (n = four experiments). The role of STAT1 in regulating the association of proteins with TNFR1 and TNFR2 was assessed using an antisense oligonucleotide to deplete HEC of STAT1 with the sense oligonucleotides used as a control (panels A and C). The blots shown are representative of three individual experiments.

When potential alterations in TNFR2 signaling were examined, we observed that TRAF-2 bound to TNFR2 and associated with NIK in TNFalpha -stimulated HEC (Fig. 5C). Co-stimulation with IBOP and TNFalpha prevented association of NIK and TRAF-2, but not TRAF-2 and TNFR2. Furthermore, TP stimulation induced the association of RIP with TNFR2, which does not occur following TNFalpha alone, in contrast to the case with TNFR1 (Fig. 5C). Thus, TXA2 mimetics may enhance those aspects of TNFR2 signaling that result in apoptosis, as well as inhibit NFkappa B activation, by redirecting RIP to associate with TNFR2.

In this model, the effects of TP stimulation on the recruitment of signaling intermediates to TNFR1 and TNFR2 depend upon the increased association of STAT1 with TNFR1. To explore further the causative role of STAT1, we used antisense oligonucleotides to antagonize STAT1 expression. Transfection of HEC with the STAT1 antisense oligonucleotides abrogated STAT1 expression over a period of 16 h, and did not affect HEC viability. STAT1 sense oligonucleotides did not affect STAT1 expression during the same time course (data not shown). When STAT1 oligonucleotide-treated HEC were stimulated, those HEC treated with sense oligonucleotides were not found to differ from untransfected cells (Fig. 5, A and C). In contrast, STAT1 antisense oligonucleotides ablated the interfering effects of TP stimulation, thus allowing RIP, TRAF-2, and IKKgamma to be recruited to TNFR1. Similarly, the recruitment of NIK to TNFR2 was also restored and the recruitment of RIP did not occur. Thus, the enhanced phosphorylation and recruitment of STAT1 to TNFR1 in the presence of IBOP is a crucial step in the antagonism of TNFalpha -induced NFkappa B activation by TP stimulation.

The importance of MAPK activation to LAM transcription has recently become apparent (26-29). To determine whether the effects of TXA2 were specific for NFkappa B, we probed for MAPK activation in extracts of HEC stimulated with IBOP, TNFalpha , or both agents with phosphospecific antibodies. TNFalpha treatment induced a 4-, 8.5-, and 13-fold increase in p38, ERK1/2, and JNK phosphorylation, respectively (Fig. 6A). The increased MAPK phosphorylation was not diminished by IBOP co-stimulation. Because IBOP disrupts TNFalpha -mediated TRAF-2-TNFR1 interactions, we posited that MAPK activation following TNFalpha was a response to either MADD/TNFR1 or TRAF-2/TNFR2 signaling. TRAF-2 associated with Ask1 and MAKK1 in TNFalpha -stimulated HEC (Fig. 6B) and co-stimulation with IBOP did not influence their recruitment. Similarly, the association of MADD with TNFR1 in response to TNFalpha was unaffected by co-stimulation with IBOP (Fig. 6C). Thus, TP stimulation induces a functional bifurcation in TNFR2 signaling that maintains MAPK activation but inhibits NFkappa B activation.


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Fig. 6.   Lack of inhibition of TNFalpha -mediated MAPK activation by TP stimulation in HEC. In panel A, lysates from stimulated HEC were probed using monoclonal antibodies to the phosphorylated forms of p38, ERK1/2, and JNK following stimulation for 5 min with IBOP, TNFalpha , or the two in combination. Blots were reprobed with a monoclonal alpha -tubulin antibody to assess loading (n = four experiments). The interaction of TNFR2 (B) and TNFR1 (C) with downstream effectors that influence MAPK activation were determined after TNFR1 or TNFR2 were immunoprecipitated and immune complexes were probed with antibodies against MADD, Ask1, or MEKK1.

TP Does Not Inhibit Caspase Activation by TNFalpha and Enhances Endothelial Cell Apoptosis-- Concurrent TP stimulation inhibits TNFalpha -induced activation of a major cell survival pathway. TNFalpha also promotes cell death by activating caspases. Thus, we examined the effect of TXA2 mimetics on TNFalpha -mediated caspase activation and cell survival. TNFalpha -mediated caspase activation causes the association of TRADD and FADD (4). Immunoprecipitation of TNFR1 showed that FADD associated with the receptor when TNFalpha was present; this association was not altered by co-stimulation with IBOP (Fig. 7A). Association of FADD and TNFR1 activates caspase 8 and subsequently caspase 3. Thus, we next examined caspase activity in stimulated HEC lysates. Control cells contained little caspase 3- or 8-like enzyme activity; however, robust caspase 3 and 8 activation were observed in TNFalpha -treated cells, which was not altered by IBOP (Fig. 7B). Thus, proapoptotic signaling from TNFR1 remained intact in the presence of TXA2. We next examined the effect of simultaneous TNFalpha and TP stimulation on endothelial cell survival. Stimulation with IBOP or TNFalpha alone increased annexin V staining by 2-fold in untreated HEC after 16 h (p <=  0.01, Fig. 7C). In contrast, the use of both agents induced a 5-fold increase in endothelial cell apoptosis (p <=  0.001). This synergistic increase in endothelial cell death is likely the result of the preservation of the proapoptotic cascade of TNFR1, with the inhibition of prosurvival pathways of both TNFR1 and TNFR2, such as NFkappa B activation. Indeed, when HEC that had been transfected with oligonucleotides with the NFkappa B consensus binding site were treated with TNFalpha , endothelial cell apoptosis was increased 2-fold and ICAM-1, VCAM-1, and E-selectin expression was ablated (data not shown). Thus, the inhibition of TNFalpha -induced NFkappa B activity by TXA2 stimulation appears to be sufficient to promote endothelial cell apoptosis.


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Fig. 7.   Lack of inhibition of TNFalpha -mediated caspase activation by TP stimulation and promotion of endothelial cell apoptosis. A, binding of FADD to TNFR1 was determined by Western blotting of immune complexes from HEC stimulated with IBOP, TNFalpha , or the two in combination for 5 min (n = three experiments). B and C, TP stimulation and TNFalpha both activate caspases in HEC, which results in increased apoptosis. After stimulation with IBOP, TNFalpha , or the two in combination for 16 h, cell lysates were assayed for caspase 3 (black-square) and 8 () activity (B) or stained using fluorescein isothiocyanate-conjugated annexin V as a marker of apoptosis (C). The percentage of cells binding annexin V was determined by flow cytometry. Data are expressed as A415 nm (B) or percentage of cells binding annexin V (C) (mean ± S.D.) and represent the average of three experiments. * and # indicate significance (p <=  0.01) from control and TNFalpha , respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study we found that ligands for TP, the G protein-coupled receptor for TXA2, abrogated the enhanced expression of proinflammatory markers on TNFalpha -stimulated endothelial cells and inhibited leukocyte adhesion to TNFalpha -treated endothelium in vitro and in vivo. Furthermore, in endothelial cells incubated with both agents, apoptosis was markedly increased, as TXA2 mimetics prevented TNFalpha from initiating pathways linked to cell survival, but not those linked to apoptosis.

TP stimulation prevented NFkappa B activation by both TNFalpha receptors. The cross-talk between the TXA2 and TNFalpha receptors is unlikely to occur at a point downstream of the convergence of the TNFR1 and TNFR2 signaling pathways, as activation of NFkappa B by IL-1beta , which shares these pathways, was unaffected by TP stimulation. Thus, mechanisms that universally inhibit NFkappa B activation, such as induction of A20 and inhibition of Ikappa B kinase (4), do not appear to be involved. In addition, TRAF-2 bound to TNFR2, but not TNFR1, in HEC stimulated with TNFalpha and IBOP. Together these data indicate that TP-mediated inhibition of both TNFR1 and TNFR2 is facilitated via novel mechanisms.

Association of RIP and TRAF-2 with TNFR1 are necessary for NFkappa B activation and cells deficient in either protein do not activate NFkappa B fully in response to TNFalpha (30, 31). Overexpression of STAT1 inhibits the association of RIP and TRAF-2 with TNFR1·TRADD complexes, but does not affect FADD·TRADD complex formation, after TNFalpha stimulation (25). Concurrent stimulation with TNFalpha and IBOP also interfered with TRADD-RIP/TRAF-2 interactions but not FADD binding or caspase activation. Furthermore, stimulation with TNFalpha and IBOP increased STAT1 phosphorylation on Tyr701 and the association of TNFR1 and STAT1. Thus, TP enhanced TNFalpha -mediated STAT1 activation; this may inhibit NFkappa B activation by TNFR1, most likely by competitively inhibiting RIP/TRAF-2 binding to a TNFR1·TRADD complex. The TXA2- and Galpha q-mediated enhancement of TNFalpha -stimulated STAT1 activation is another novel finding of the present study. This model of antagonism by TP ligands is an extension of the findings of Wang et al. (25), who showed that STAT1 overexpression prevented the recruitment of RIP to TNFR1 in a concentration dependent fashion. Other G protein-linked receptor ligands, such as angiotensin II and alpha 1 adrenergic receptors, have also been reported to stimulate STAT1 (32, 33). Whereas the pathways of STAT1 activation may differ, all these receptors are linked to Galpha q, which suggests the possibility of a common mechanism of G protein-mediated inhibition of TNF-stimulated NFkappa B activation during inflammation and reperfusion.

TP stimulation also inhibited TNFR2 activation of NFkappa B. TP stimulation did not inhibit TRAF-2-TNFR2 interactions, however; TP induced a functional bifurcation in TNFR2 signaling, leading to activation of MAPK, but not NFkappa B. This division of TNFR2 signaling was mediated by selective inhibition of NIK-TRAF-2 interactions after TRAF-2 binds to TNFR2. We propose that recruitment of RIP to TNFR2 in the presence of IBOP mediates the selective inhibition of NIK binding. RIP overexpression alters the signaling pathway of TNFR2, "switching" the downstream effects from NFkappa B activation to a cascade inducing programmed cell death (34). TP signaling prevents the RIP-TRADD interaction in response to TNFalpha by inducing translocation of STAT1 to TNFR1. In this model, RIP displaced from TNFR1 by STAT1 binds to TNFR2. Thus, the enhanced association of RIP with TNFR2 may aid the induction of apoptosis and prevent NFkappa B activation by TNFalpha in the presence of TP stimulation. Indeed, the importance of STAT1 to this pathway of inhibition was highlighted by the experiments with antisense STAT1 oligonucleotides, indicating that the recruitment of RIP to TNFR2 was abrogated in the absence of STAT1.

These data present a model for the inhibition of TNFalpha -mediated NFkappa B activation by IBOP. The effects of IBOP are more complicated than just mere inhibition of the NFkappa B pathway, however. Initiating IBOP treatment after TNFalpha stimulation had achieved maximal expression reduced NFkappa B activation to baseline. This response is characterized by the movement of NFkappa B of the nucleus, decreased Ikappa B phosphorylation, and reassociation of the two proteins. Whereas the inhibition of proximal events in TNFalpha signaling by IBOP explain why no further NFkappa B activation occurs, it does not fully explain the rapid reversion to baseline for NFkappa B activated during the previous period. One possibility, proposed by Ghosh and Karin (35), is that Ikappa B may be recruited to the nucleus to recomplex to NFkappa B and cause its redistribution to the cytoplasm. In the case of the present experiments, TP might be suspected of triggering a phosphatase that leads to dephosphorylation of Ikappa B. The ability to inhibit previously activated NFkappa B is a powerful proapoptotic effect of TP stimulation; regardless of the order that HEC receive the stimuli, the result is the inhibition of NFkappa B and the induction of apoptosis.

We found that TP stimulation does not affect TNFalpha -induced activation of JNK, p38, and ERK1/2. Activation of MAPK by TNFalpha is dependent upon TRAF-2 (36, 37). As the TNFR2-TRAF-2-Ask1, but not the TNFR1-TRADD-TRAF-2, pathway is preserved in the presence of TP stimulation, it is most likely the pathway that leads to MAPK activation. Induction of LAM requires MAPK activation and p38 is required for full transcriptional activity of NFkappa B (26, 38). The inhibition of TNFalpha -stimulated LAM expression, despite activation of all three MAPKs, indicates either that AP-1 and NFkappa B activation are both required for LAM expression, or instead that NFkappa B alone is necessary and sufficient. Interestingly, ERK, JNK, and p38 all promote EC apoptosis (39) and have direct links to caspase activation as well as other cell death pathways, such as the functional suppression of Bcl-2 (40). Thus, p38, JNK, and ERK activation in the presence of TNFalpha and TXA2 may enhance the caspase activation mediated by the TRADD-FADD pathway, thus leading to the enhanced apoptosis observed in endothelial cells treated with both agents.

Although not emphasized in this report, the inhibition of NFkappa B activation in HEC treated with TNFalpha and TXA2 mimetics is reciprocal. The ability of TXA2 mimetics to induce NFkappa B activation is specific for a single TP isoform; TPbeta , through a Galpha i-coupled signaling pathway not used by TPalpha , activates NFkappa B in response to IBOP and U46619. In contrast, the inhibition of TNFalpha signaling is potentially mediated through either isoform, as both couple to Galpha q. TNFalpha stimulation prevented the activation of NFkappa B in TP-stimulated HEC. Furthermore, TNFalpha could also reverse the activation of NFkappa B if cells were stimulated with IBOP prior to TNFalpha . The mechanism by which TNFalpha controls Galpha i-mediated NFkappa B activation is not yet identified.

Circulating levels of TXA2 and TNFalpha are increased during ischemia. In addition, ischemic episodes increase the number of circulating endothelial cells by stimulating apoptosis (41). These endothelial cells were macrovascular in origin and did not express markers of "activation" such as LAM. We have found that co-stimulation with TXA2 and TNFalpha produces a similar phenotype in macrovascular endothelial cells in vitro. Although TXA2 does not inhibit NFkappa B induction by all cytokines, e.g. IL-1beta , similar mechanisms might prevent activation of endothelial cell NFkappa B by such cytokines. The reciprocal inhibition of NFkappa B activation, such as that which occurs with TXA2 and TNFalpha , is sufficient for the induction of endothelial cell apoptosis and thus may increase the number of circulating endothelial cells during ischemia. Consistent with this hypothesis is the recent observation that blocking TNFalpha decreases the number of circulating endothelial cells (42).

Apoptosis mediates inflammation and leukocyte extravasation associated with renal injury by cleavage of the monocyte-activating polypeptide II protein (43). We speculate that apoptotic endothelial cells, exposed to TNFalpha and TXA2, could mediate leukocyte infiltration into ischemic tissues by a similar mechanism. Co-stimulation by TXA2 and TNFalpha may propagate an inflammatory response by perturbing the vessel wall, inducing injury to the vasculature, and exposing the subendothelial matrix following loss of endothelial cells because of apoptosis. In addition, the induction of endothelial apoptosis may attenuate an angiogenic response, thus reducing revascularization in inflamed or ischemic tissues.

Taken together, these data describe a previously unrecognized interaction between two mediators of inflammation and reperfusion injury that may provide a mechanism by which endothelial cell injury occurs during these conditions. In addition to elucidating this mechanism, a rationale is provided for the use of a TP antagonist to preserve the cell survival properties of TNFalpha during inflammation and reperfusion.

    ACKNOWLEDGEMENT

Full-length human cDNA probes to ICAM-1, VCAM-1, E-selectin, and GAPDH were a kind gift from Dr. J. Berman, Department of Pathology, AECOM.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants HL47032, HL51043, and HL55552 and American Heart Association postdoctoral fellowship 0020186T.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Vice President, Cardiovascular Research and Clinical Investigation, Eli Lilly and Co., Lilly Corporate Center, DC 0520, Indianapolis, IN 46285-0001. Tel.: 317-651-1034; Fax: 317-651-1033 E-mail: jaware@lilly.com.

Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M210766200

    ABBREVIATIONS

The abbreviations used are: TNFalpha , tumor necrosis factor alpha ; ICAM, intercellular adhesion molecule 1; TXA2, thromboxane A2; TNFR1, tumor necrosis factor alpha  receptor 1; TNFR2, tumor necrosis factor alpha  receptor 2; HEC, human endothelial cells; TP, thromboxane receptor; IBOP, [1S-(1alpha ,2beta (5Z), 3alpha (1E,3R),4alpha ]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5'-heptenoic acid; SQ29548, [1S]1alpha ,2beta (5Z), 3beta ,4alpha ]-7-[3[[2-[(phenylamino)carbonyl]-hydrazino]methyl]-7-oxabicyclo[2.2.1]-hept-2-yl; MAPK, mitogen-activated protein kinase; VCAM, vascular cellular adhesion molecule; IL-1beta , interleukin 1beta ; ERK1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun NH2-terminal kinase; STAT, signal transducers and activators of transcription; TRAF, TNF receptor-associated factor; LAM, leukocyte adhesion molecule; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

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