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Aspirin (ASA) regulates 5-lipoxygenase activity and peroxisome proliferator-activated receptor -mediated CINC-1 release in rat liver cells: novel actions of lipoxin A₄ (LXA₄) and ASA-triggered 15-epi-LXA₄ ¹

ANNA PLANAGUMÀ, ESTHER TITOS, MARTA LÓPEZ-PARRA, JOAN GAYA^*, GLORIA PUEYO, VICENTE ARROYO and JOAN CLÀRIA²

DNA Unit,
^* Hormonal Laboratory and
Liver Unit, Hospital Clínic, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona; and
Química Farmacéutica Bayer S.A. (C.C. Division), Barcelona 08036, Spain

²Correspondence: DNA Unit, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain. E-mail: jclaria{at}clinic.ub.es

SPECIFIC AIM

The aim of this study was to explore new mechanisms of action of aspirin (ASA) other than cyclooxygenase (COX) and prostaglandin (PG) inhibition that may contribute to its anti-inflammatory and antithrombotic properties. We assessed the effects of ASA on eicosanoid-generating pathways and its effect on peroxisome proliferator-activated receptor (PPAR) and cytokine-induced neutrophil chemoattractant-1 (CINC-1) levels in rat liver cells.

PRINCIPAL FINDINGS

1. ASA switches arachidonic acid metabolism from PGE₂ to leukotriene B₄ (LTB₄) and 15-epi-lipoxin A₄ (15-epi-LXA₄) biosynthesis in rat Kupffer cells
The liver is the first organ to receive the bulk of absorbed ASA from the gut and plays a major role in the hydrolysis of acetylsalicylic acid to salicylate. For this reason, hepatic cells are probably the cell types that encounter most of ASA’s active compound. We focused our interest in Kupffer cells because these liver resident macrophages express COX-1, COX-2, 5-LO, and 5-LO-activating protein (FLAP) mRNA (Fig. 1 A) and constitute the most important source of eicosanoids within the liver sinusoid. In Kupffer cells, we found that the inhibitory effect of ASA on COX-derived products (e.g., PGE₂) was associated with a dose-dependent increase of the 5-LO product LTB₄ and the endogenous anti-inflammatory eicosanoid 15-epi-LXA₄ (Fig. 1B ). ASA also dose-dependently stimulated 5-LO activity and LTB₄ production in rat CRL-2192 macrophages. Together, these findings are consistent with the notion that although the primary effect of ASA is the inhibition of COX with a consequent reduction of PG formation, this nonsteroidal anti-inflammatory drug (NSAID) exerts significant effects on other pathways for arachidonic acid metabolism (i.e., LTB₄ and 15-epi-LXA₄ biosynthesis).

View larger version (15K): [in this window] [in a new window]   Figure 1. A) Detection of COX-1, COX-2, 5-LO, and FLAP mRNA expression by RT-PCR in rat Kupffer cells. m, molecular weight DNA ladder. B) Effect of ASA on PGE2, LTB4 and 15-epi-LXA4 biosynthesis in rat Kupffer cells. Results represent the mean ± SE of 11 different experiments. *P < 0.05, **P < 0.025, and ***P < 0.005 vs. vehicle.

2. ASA modulates peroxisome proliferator-activated receptor (PPAR) in rat hepatocytes
To ascertain the biological significance of ASA actions in rat macrophages, we put our findings into the context of the liver sinusoid where Kupffer cells are in close contact with hepatocytes. We wanted to examine the influence of ASA and Kupffer cell-derived arachidonic acid products on gene transcriptional factors implicated in the control of inflammation. We focused our investigation on one group of such transcriptional factors—the PPAR family—and specifically in PPAR, the predominant PPAR subtype in hepatocytes. As shown in Fig. 2 A, ASA significantly reduced PPAR levels in rat hepatocytes. The 5-LO-derived product LTB₄, a member of the novel class of ASA-triggered 15R containing LX (15-epi-LXA₄), and the selective PPAR agonist activator Wy-14643 decreased PPAR protein expression in rat hepatocytes (Fig. 2A ). The observation that ASA decreases PPAR protein expression in rat hepatocytes was further confirmed in nuclear extracts from a human mononuclear leukocyte cell line (THP-1 cells) (Fig. 2B ), indicating that this ASA property is not restricted to the liver scenario. Conversely and as shown in Fig. 2C , PGE₂ alone and PGE₂ in association with LTB₄ did not induce any change in PPAR protein expression. Similarly, PPAR levels in rat hepatocyte cultures were not modified by the selective COX-1 and COX-2 inhibitors SC-560 and celecoxib, respectively (Fig. 2C ).

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of ASA and Kupffer cell-derived arachidonic acid metabolites on PPAR protein expression. A) Rat hepatocytes were exposed to either vehicle (0.1% ethanol), 1 mM ASA, 1 µM LTB4, 1 µM 15-epi-LXA4-methyl ester, or 0.1 µM Wy-14643 for 24 h at 37°C and nuclear extracts were probed with a specific anti-murine PPAR antiserum. B) Human mononuclear leukocytes (THP-1 cells) were exposed to vehicle (0.1% ethanol) or 1 mM ASA for 24 h at 37°C and nuclear extracts analyzed by Western blot. C) Rat hepatocytes were exposed to either vehicle (0.1% ethanol), 0.3 µM PGE2, 0.3 µM PGE2 in association with 1 µM LTB4, 3 µM celecoxib, or 3 µM SC-560 for 24 h at 37°C and nuclear extracts were probed as described above. Results are representative of 12 separate experiments. *P < 0.05, **P < 0.005, and ***P < 0.001 vs. vehicle.

3. ASA modulates the secretion of cytokine-induced neutrophil chemoattractant-1 (CINC-1) by rat hepatocytes
Because the PPAR family of transcriptional regulators is involved in the modulation of various inflammatory genes, including members of the interleukin-8 (IL-8) family, we next evaluated the effects of ASA on the secretion of CINC-1 [the rat counterpart of human IL-8 and/or growth-regulated gene product (GRO)] by rat hepatocytes. Incubation of rat hepatocyte cultures with ASA resulted in decreased levels of CINC-1. The mechanism by which ASA down-regulates CINC-1 secretion is unknown, although the selective PPAR agonist activator Wy-14643 reduced CINC-1 production in rat hepatocytes, implying an action of ASA at the level of PPAR. In contrast, addition of LTB₄ in association with PGE₂ significantly increased CINC-1 secretion by rat hepatocytes.

4. LXA₄ and ASA-triggered 15-epi-LXA₄ modulate 5-LO activity, PPAR protein expression and CINC-1 release
LXA₄, an endogenous eicosanoid that carries "stop signals" for inflammation and is generated via biosynthetic pathways that involve the dual lipoxygenation of arachidonic acid by either 5- and 15-LO or 5- and 12-LO, significantly inhibited 5-LO activity in rat macrophages. PGE₂ alone and PGE₂ in association with LXA₄ significantly decreased macrophage 5-LO activity. It was recently reported in human neutrophils that PGE₂ induces a switch in eicosanoid biosynthesis from the predominantly LTB₄- and 5-LO-initiated pathway to LXA₄. Our findings demonstrating that in rat macrophages LXA₄ inhibits 5-LO activity to a similar extent as PGE₂ suggest that this endogenous anti-inflammatory eicosanoid plays an active role in the switching of eicosanoid classes possibly during resolution of inflammation. On the other hand, ASA-triggered 15-epi-LXA₄, an endogenous lipid-derived mediator that mimics the action of native LXA₄, significantly reduced PPAR and CINC-1 levels in hepatocytes. Taken together and because 5-LO activity and PPAR and CINC-1 levels are involved in the extent and duration of the inflammatory response, these findings provide additional molecular mechanisms for these putative endogenous stop signals of inflammation.

CONCLUSIONS AND SIGNIFICANCE

ASA is an NSAID used to treat a wide variety of medical conditions. Besides to its well-known anti-inflammatory, analgesic, and anti-pyretic properties, ASA inhibits platelet aggregation, is useful in preventing myocardial infarction and stroke, has neuroprotective actions, and decreases the incidence of cancer. Although most pharmacological properties of ASA are related to its ability to acetylate COX leading to irreversible inhibition of PG synthesis, the complete mechanism of action of ASA is still a subject of debate. At high doses, for instance, some properties of ASA are not mediated by inhibition of COX and PGs. ASA is able to modulate activity of the nuclear factor-B, activator protein 1, the heat shock transcriptional factor, and the p38 and p44Erk1 and p42Erk2 mitogen-activated protein kinases. In the current study, we provide new molecular mechanisms underlying the pleiotropic response to ASA by demonstrating that this NSAID promotes the formation of endogenous anti-inflammatory compounds (e.g., 15-epi-LXA₄) and modulates 5-LO activity and PPAR and CINC-1 levels in rat liver sinusoidal cells.

A hypothetical scheme for the actions of ASA on arachidonic acid metabolism in rat liver cells is illustrated in Fig. 3 . In Kupffer cells, ASA inhibits COX and PGE₂ biosynthesis and favors the oxidation of arachidonic acid via the 5-LO pathway (e.g., LTB₄). The increase in LTB₄ formation after ASA treatment may be secondary to either the shunting of arachidonic acid to the 5-LO pathway or abolishment of the inhibitory action exerted by COX-derived products (including PGE₂) on LTB₄ biosynthesis. Before ASA is extensively hydrolyzed to salicylate in the liver and enters systemic circulation, this NSAID triggers the release of potent biologically active eicosanoids (i.e.,15-epi-LXA₄) by Kupffer cells. Since Kupffer cells have COX-2 and 5-LO in place, the formation of ASA-triggered 15-epi-LXA₄ in these liver resident macrophages may occur by interaction of ASA-acetylated COX-2 with 5-LO or conversion via transcellular routes of 15R-HETE released by underlying hepatocytes. Unlike endothelial and epithelial cells, the biosynthesis of 15R-HETE in hepatocytes is initiated by COX-2-independent pathways. Regardless of their cellular origin, liver cells are a rich source of 15-epi-LXA₄ during ASA treatment. These ASA-triggered 15-epi-LXA₄ may exert diverse anti-inflammatory activities in autocrine and paracrine fashion, contributing to the broad range of beneficial actions of ASA. In the current investigation, we demonstrate that besides decreasing 5-LO activity in macrophages (to an extent similar to PGE₂), ASA-triggered 15-epi-LXA₄ significantly inhibit PPAR and CINC-1 levels in underlying hepatocytes. In rat hepatocytes, ASA directly reduces PPAR protein levels and the secretion of CINC-1 (Fig. 3) . In contrast, LTB₄ together with PGE₂ generated in Kupffer cells stimulates CINC-1 production in adjacent hepatocytes.

View larger version (51K): [in this window] [in a new window]   Figure 3. Hypothetical scheme of ASA actions in rat liver sinusoidal cells.

Because arachidonic acid-derived products along with gene transcriptional regulators such as PPAR and target genes such as CINC-1 play a central role in the control of inflammation, our findings provide an important contribution to understanding the mechanism of action of one of the most widely used NSAIDs: ASA. The link between membrane-derived bioactive lipids such as LXA₄ and ASA-triggered 15-epi-LXA₄ and the modulation of 5-LO, PPAR, and CINC-1 provides new opportunities for investigations into cell signaling and new targets for drug design.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0224fje; to cite this article, use FASEB J. (October 4, 2002) 10.1096/fj.02-0224fje

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