HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/9/1099 02-0485fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZERNECKE, A. Articles by WEBER, C. Search for Related Content PubMed PubMed Citation Articles by ZERNECKE, A. Articles by WEBER, C.

Suppression of endothelial adhesion molecule up-regulation with cyclopentenone prostaglandins is dissociated from IB- kinase inhibition and cell death induction¹

ALMA ZERNECKE^*,2, WOLFGANG ERL^*,2, LINE FRAEMOHS^,2, MICHAEL LIETZ^,2 and CHRISTIAN WEBER^,3

^* Institut für Prophylaxe der Kreislaufkrankheiten, Ludwig-Maximilians-Universität München, 80336 München, Germany; and
Department of Molecular Cardiovascular Research, Rheinisch-Westfälische-Technische-Hochschule Aachen, 52074 Aachen, Germany

³Correspondence: Kardiovaskuläre Molekularbiologie, Universitätsklinikum Aachen, Pauwelstrasse 30, 52074 Aachen, Germany. E-mail: cweber{at}ukaachen.de

SPECIFIC AIMS

The cyclopentenone prostaglandins (cPG) 15-deoxy-^12,14-prostaglandin J₂ (dPGJ₂) and prostaglandin A₁ (PGA₁) can inhibit multiple steps in nuclear factor (NF) -B signaling and induce apoptosis in different cell types. We explored the differential mechanisms underlying dose-dependent effects of cPG on the inflammatory up-regulation of cell adhesion molecules (CAM) and induction of cell death by tumor necrosis factor (TNF-) in endothelial cells.

PRINCIPAL FINDINGS

1. Inhibition of endothelial CAM induction with cPG
Pretreatment of human umbilical vein endothelial cells (HUVEC) with dPGJ₂ or PGA₁ for 2 h dose-dependently inhibited the up-regulation of intercellular adhesion molecule 1 (ICAM-I) and vascular cell adhesion molecule-1 (VCAM-1) surface expression induced by stimulation with TNF-, with dPGJ₂ being more effective than dPGA₁. The inhibition was most pronounced for VCAM-1 up-regulation at 6 h, whereas ICAM-1 expression at 24 h was attenuated only at the higher concentrations. Moreover, cPG suppressed monocyte arrest on TNF--stimulated HUVEC in shear flow. In contrast, the structurally related analogs dPGA₁ and dPGA₂ had no effect on CAM up-regulation at similar concentrations, revealing a feature specific to dPGJ₂ and PGA₁.

2. Enhancement of endothelial cell death by cPG
We next tested whether effects of cPG on CAM up-regulation were due to induction of cell death. Trypan blue exclusion assays revealed that pretreatment of HUVEC with dPGJ₂ at 10 µmol/L and PGA₁ at 25 µmol/L for 2 h markedly enhanced cell death induced by stimulation with TNF- for 24 h, whereas pretreatment at lower concentrations effective in inhibiting CAM up-regulation did not significantly affect TNF--induced death of HUVEC. This was confirmed by other specific assays of apoptosis: nuclear fragmentation and annexin V staining.

3. Inhibition of IKK, IB- degradation, and p65 translocation by high cPG concentrations
Transcriptional activation of VCAM-1 and ICAM-1 involves mobilization and binding of NF-B to motifs in the promoter regions of these genes that follows phosphorylation and degradation of its inhibitor IB-. Since dPGJ₂ can directly inhibit IB- kinase (IKK) activity by interactions of its reactive carbonyl group in the cyclopentenone ring in HeLa cells, we tested TNF--induced IKK activation in HUVEC challenged with TNF- for 15 min. As confirmed by densitometrical analysis, dPGJ₂ at 10 µmol/L and PGA₁ at 25 or 50 µmol/L clearly inhibited TNF--induced IKK activity whereas dPGJ₂ at 5 µmol/L or PGA₁ at 12.5 µmol/L did not (Fig. 1 A). We then tested the effects of cPG on TNF--induced IB- degradation by immunoblotting. Basal levels of IB- displayed by resting HUVEC were completely degraded after stimulation with TNF- for 15 min (Fig. 1B ). Degradation of IB- was prevented by PGA₁ (at 25 µmol/L but not at 12.5 µmol/L) and dPGJ₂ (at 10 µmol/L but not at 5 µmol/L) and unaffected by dPGA₁ or dPGA₂ (Fig. 1B ). In line with these effects, cPG at high but not low concentrations reduced nuclear translocation of NF-B, as evident by p65 immunoblotting of nuclear extracts (Fig. 1C ). At concentrations inhibiting CAM up-regulation, cPG did not alter expression of the NF-B subunit p65, which can be subject to caspase-mediated cleavage in TNF--stimulated HUVEC (Fig. 1C ) and enhanced AP-1 activation, as evident by c-jun phosphorylation (Fig. 1D ). The NF-B-dependent expression of inhibitor of apoptosis proteins (iap) has been shown to contribute to the protection from apoptosis, and its inhibition increases the susceptibility to cell death. cPG at low concentrations did not reduce iap expression in activated HUVEC (Fig. 1E, F ). These data suggest that effects of dPGJ₂ or PGA₁ at concentrations not causing HUVEC death are not due to inhibition of IKK or IB- degradation.

View larger version (38K): [in this window] [in a new window]   Figure 1. Effects of cPG on IKK activity, IB- expression, p65 translocation, JNK activity, and iap expression. HUVEC were left untreated or pretreated with dPGJ2, PGA1, dPGA1, or dPGA2 (in µmol/L) for 2 h and stimulated with TNF- for 15 min. IKK activity in cell lysates was analyzed by kinase assays and equal loading was confirmed by blotting of IKK- (A). To study IB- degradation, cells lysates were analyzed by immunoblotting for IB- (B). Nuclear translocation of NF-B was assessed by immunoblotting of nuclear extracts with a mAb to p65 subunit (C) and phosphorylation of c-jun was studied by immunoblotting with mAb to p-c-jun vs. total c-jun (D). Iap expression was studied by immunoblotting with mAb to iap-1 (E) or iap-2 (F). Blots are representative of at least 3 independent experiments.

4. Dissociation of cPG effects on endothelial CAM but not cell death from IKK inhibition
To confirm that the suppression of endothelial CAM up-regulation with cPG is independent from effects on IKK activity, we used the C179A mutant of IKK-ß, which does not interact with a reactive carbonyl group in the cPG cyclopentenone ring and so is resistant to inhibition by cPG. Transient expression of the C179A IKK-ß mutant in HUVEC as assessed by green fluorescence protein (GFP) coexpression (Fig. 2 A) substantially reduced the enhancement of cell death induced by TNF- seen with high concentrations of PGA₁ (Fig. 2B ). In contrast, suppression of TNF--induced up-regulation of CAM by cPG was unaffected by the C179A mutant (Fig. 2C ). This indicates that IKK inhibition was not required for the suppression of endothelial CAM induction with low concentrations of cPG.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effects of a IKK mutant resistant to cPG interaction on cell death and CAM regulation. HUVEC cotransfected with eGFP and vector alone or IKK-ß C179A mutant cDNA were left untreated (control) or pretreated with PGA1 at 10 or 25 µmol/L for 2 h and stimulated with TNF- for 24 h (A–C). GFP expression of transfectants was analyzed by flow cytometry (A). Trypan blue exclusion assays were performed and expressed as % of Trypan blue-positive cells (B). VCAM-1 surface expression was analyzed by flow cytometry and given as % of control sMFI (C). Data represent mean ±SD of at least 3 independent experiments.

5. Effects of cPG on CAM induction are associated with inhibition of NF-B DNA binding
We used EMSA and a novel kit for analyzing p65 binding to an immobilized oligonucleotide to study the effect of cPG on DNA binding of NF-B to its consensus sequence after its nuclear translocation stimulated by TNF-. dPGJ₂ and PGA₁ at concentrations not affecting IKK activity, but not dPGA₁ or dPGA₂, dose-dependently blocked DNA binding of NF-B in accordance with effects on CAM expression. As confirmed by FACS analysis, NF-B-dependent transactivation of a reporter gene was suppressed by cPG at similar concentrations.

CONCLUSIONS AND SIGNIFICANCE

Monocyte infiltration mediated by cytokine-inducible endothelial CAM is pivotal for inflammatory reactions but also crucially involved in atherogenesis. The results of the present study demonstrate that cPG dose-dependently suppressed the TNF--induced up-regulation of ICAM-1 and VCAM-1 expression and monocyte arrest on HUVEC, corroborating evidence for anti-inflammatory effects of cPG. Multiple mechanisms differentially affecting signaling pathways of NF-B (e.g., inhibition of IKK or DNA binding) have been invoked in actions of cPG, but their contribution to the suppression of CAM expression and function in endothelial cells had not been elucidated. At low concentrations, cPG inhibited TNF--induced DNA binding of NF-B while increasing c-jun phosphorylation. This suggests that cPG inhibit endothelial CAM induction by impairing NF-B DNA binding but not AP-1 activation. In contrast, cPG enhanced TNF--induced cell death, IKK activity and IB- degradation only at high concentrations. The effects on CAM are dissociated from proapoptotic signals that are due to suppression of IKK activation. The synthetic analogs dPGA₁ or dPGA₂ were ineffective in all assays. Hence, we have characterized a structurally confined mechanisms accounting for anti-inflammatory effects of low-dose cPG in endothelial cells.

Trypan blue exclusion assays revealed that only at the highest concentrations used did dPGJ₂ and PGA₁ enhance HUVEC killing by TNF-. The apoptotic process is highly regulated by pro- and anti-apoptotic proteins partly under the control of NF-B, and inhibition of NF-B has been shown to induce apoptosis. For instance, NF-B-dependent expression of IAPs that interact with TNF-receptor-associated proteins and antagonize caspases can protect from apoptosis. In parallel, cPG inhibited TNF-induced IKK activity and IB degradation and iap expression in HUVEC only above threshold concentrations, an effect mediated by interaction of the reactive carbonyl group in the cPG ring with cysteine 179 of IKK. Thus, the induction of HUVEC death by cPG may be associated with an inhibition of IKK activity and iap expression, implying that a more profound and global inhibition of NF-B signaling is required to enhance cell death than for effects on CAM. This may be due to a difference in NF-B members involved in iap vs. CAM transcriptional regulation or in their susceptibility to cPG. Conversely, cPG did not inhibit TNF--induced IKK activity and IB- degradation at low concentrations despite suppressing CAM induction. Transfection of the C179A IKK mutant that impaired cell death induction but not suppression of CAM up-regulation by cPG confirmed this dissociation. Evidence in HeLa cells indicates that cPG directly interfere with DNA binding of NF-B subunits at lower concentrations than required for IKK inhibition. Gel shift analysis revealed that mechanisms for inhibiting CAM induction in HUVEC involves direct blockade of NF-B binding to its consensus sequence by cPG.

In conclusion, the suppression of Ig molecule up-regulation by TNF- in endothelial cells by cPG at low concentrations is dissociated from IKK inhibition and sensitization for cell death at higher concentrations and appears to be due to interference with DNA binding of NF-B (Fig. 3 ). These data define distinct mechanisms for anti-adhesive and proapoptotic effects of cPG, expand on the current knowledge on the mechanistic and structural aspects of cPG action that have been largely obtained in cell lines, such as HeLa cells, and extend these insights to a physiological cell type. Dose-dependent mechanisms of action in endothelial cells may enable a well-confined use of cPG as anti-inflammatory and possibly anti-atherosclerotic compounds while minimizing undesirable cell death-inducing side effects.

View larger version (17K): [in this window] [in a new window]   Figure 3. Schematic illustration depicting dose-dependent effects of cPG in endothelial cells.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0485fje; to cite this article, use FASEB J. (April 8, 2003) 10.1096/fj.02-0485fje

² A.Z., W.E., L.F., and M.L. contributed equally to this paper.

This article has been cited by other articles:

				O. Haworth and B. D. Levy Endogenous lipid mediators in the resolution of airway inflammation Eur. Respir. J., November 1, 2007; 30(5): 980 - 992. [Abstract] [Full Text] [PDF]

				Z.-H. Chen, Y. Yoshida, Y. Saito, A. Sekine, N. Noguchi, and E. Niki Induction of Adaptive Response and Enhancement of PC12 Cell Tolerance by 7-Hydroxycholesterol and 15-Deoxy-{Delta}12,14-Prostaglandin J2 through Up-regulation of Cellular Glutathione via Different Mechanisms J. Biol. Chem., May 19, 2006; 281(20): 14440 - 14445. [Abstract] [Full Text] [PDF]

				Z.-H. Chen, Y. Saito, Y. Yoshida, A. Sekine, N. Noguchi, and E. Niki 4-Hydroxynonenal Induces Adaptive Response and Enhances PC12 Cell Tolerance Primarily through Induction of Thioredoxin Reductase 1 via Activation of Nrf2 J. Biol. Chem., December 23, 2005; 280(51): 41921 - 41927. [Abstract] [Full Text] [PDF]

				U. Zeiffer, A. Schober, M. Lietz, E. A. Liehn, W. Erl, N. Emans, Z.-q. Yan, and C. Weber Neointimal Smooth Muscle Cells Display a Proinflammatory Phenotype Resulting in Increased Leukocyte Recruitment Mediated by P-Selectin and Chemokines Circ. Res., April 2, 2004; 94(6): 776 - 784. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/9/1099 02-0485fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by ZERNECKE, A. Articles by WEBER, C. Search for Related Content PubMed PubMed Citation Articles by ZERNECKE, A. Articles by WEBER, C.