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Aspirin-triggered lipoxin A₄ and lipoxin A₄ up-regulate transcriptional corepressor NAB1 in human neutrophils ¹

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA

³Correspondence: Center for Experimental Therapeutics and Reperfusion Injury, Thorn Building for Medical Research, Brigham and Women’s Hospital, 75 Francis St., Boston, MA 02115, USA. E-mail: cnserhan{at}zeus.bwh.harvard.edu

SPECIFIC AIMS

The aim of this study was to identify in human neutrophils a subset of lipoxin A₄/aspirin-triggered 15-epi-lipoxin A₄ (LXA₄/ATL) -selective early response genes with the hypothesis that such proteins might be key regulators of downstream events associated with endogenous anti-inflammation and protective actions of LXA₄/ATL in acute inflammation and resolution.

PRINCIPAL FINDINGS

1. ATLa modulates a distinct set of transcripts in human neutrophils
A three-way differential display RT-PCR was used to identify genes that specifically respond to ATLa (aspirin-triggered lipoxin analog, 15-epi-16-(para-fluoro)-phenoxy-LXA₄) in human neutrophils. Direct comparisons were performed with human neutrophils (PMN) exposed to the chemoattractant LTB₄, vehicle alone, or an anti-inflammatory LXA₄/ATL analog (see Fig. 1 A for structures), since LTB₄ is a potent chemotactic activator and both LXA₄ and ATL inhibit PMN functional responses in vitro and in vivo. Results in Fig. 1B indicate that several bands representing cDNA fragments were differentially modulated. The LXA₄ and ATL analogs, which both compete for ³H-LXA₄-specific binding to the LXA₄ receptor, were prepared by total organic synthesis. To identify genes positively and selectively regulated by ATLa, the prominent bands enhanced in the ATLa DDPCR were excised, cloned, sequenced, and analyzed with the BLAST program.

View larger version (20K): [in this window] [in a new window]   Figure 1. Identification of LXA4-selective early response genes. A) Structures of eicosanoid signaling molecules. Leukotriene B4 (LTB4), a 5-lipoxygenase derived product, is a potent chemotactic agent. The anti-inflammatory lipoxin A4 (LXA4) and 15-epi-LXA4 are products of transcellular biosynthesis in human tissues and can be described as counter-regulatory or ‘STOP signals’. LXA4 is the product of lipoxygenase-initiated pathways, and 15-epi-LXA4 is generated via aspirin-triggered acetylated-COX-2. ATLa, the aspirin-triggered analog 15-epi-16-(para-fluoro)-phenoxy-LXA4, is a synthetic stable mimetic that acts via the LXA4 receptor in leukocytes. B) Representative DDRT-PCR analysis. Profiles were obtained from incubations of PMN (37°C, 60 min) with either vehicle alone (control), 100 nM LTB4, or 100 nM ATLa. Bands selectively induced by ATLa were excised, amplified, cloned, and sequenced.

2. ATLa rapidly induces NAB1 transcript levels
We focus on one of these prominent cDNA fragments (denoted L8B2) that appeared to be selectively induced in freshly isolated human neutrophils exposed to 100 nM of ATLa. This fragment was expressed much less, if at all, in neutrophils from the same donor incubated with either vehicle or LTB₄ (right lane, lower band in Fig. 1B ). GenBank sequence similarity searches revealed that the cDNA fragment denoted L8B2 (300 nucleotides) was 100% complementary to the 3' untranslated region of the transcriptional corepressor NAB1 mRNA and gave a high homology with the 3' untranslated region of TA16 mRNA. Semiquantitative RT-PCR using a pair of oligonucleotide primers designed to the 3' end of the NAB1 coding sequences confirmed that the NAB1 gene was indeed differentially up-regulated as an early response of human PMN exposed to ATLa.

3. ATLa up-regulates NAB1 partly via a GPCR-mediated pathway
We next evaluated involvement of the cell surface receptor ALX in the rapid induction of NAB1. Lipoxin A₄ and aspirin-triggered LXA₄ are endogenous ligands identified for ALX. 15-(R/S)-methyl-LXA₄ is a synthetic analog that acts as a mimetic of both LXA₄ and 15-epi-LXA₄. Like ATLa, 15-(R/S)-methyl-LXA₄ competes with ³H-labeled LXA₄ for specific binding to ALX and displays bioactions of endogenous LXA₄ and 15-epi-LXA₄. Rapid induction of NAB1 was observed when PMN were exposed to 15-(R/S)-methyl-LXA₄. Since the LXA₄ receptor responses are pertussis toxin (PTX) sensitive, we investigated whether up-regulation of NAB1 is a downstream event in the ALX signaling circuit. Freshly isolated human PMN were used to modulate the rapid response of NAB1 to ATLa. As expected, exposure of 100 nM ATLa gave induction of NAB1. This up-regulation of NAB1 transcripts by ATLa was dramatically decreased by treating the PMN with PTX, an inhibitor of G_i-coupled receptors. These results suggest that ATLa selectively increases NAB1 transcript levels via a PTX-sensitive GPCR present on human neutrophils.

4. NAB1 is a target gene of two anti-inflammatory agents
Glucocorticoids and ATLa have anti-inflammatory properties in vivo. We investigated whether NAB1 exhibits similar or differential tissue-selective responsiveness to these agents. Incubations of freshly isolated human PMN with 100 nM glucocorticoid results in increased levels of NAB1 transcripts. Temporal expression patterns are consistent with previous reports characterizing NAB1 as an immediate-early response gene to glucocorticoids in leiomyosarcoma cells. Our results indicate that the glucocorticoid pathway leading to NAB1 up-regulation is functional in human PMN, and the temporal patterns suggest it is distinct from the ATLa route to NAB1 induction.

NAB1 has been identified as a glucocorticoid-responsive gene in murine smooth muscle cells. These observations suggested that ATLa and glucocorticoids, which act via different receptors, might regulate the same gene target—namely, NAB1—not only in neutrophils, but also in other types of cells. Since i.v. injection of ATL protects lung tissues from PMN-mediated damage, we examined NAB1 expression using in situ hybridization in lung tissues of mice treated with or without i.v. injection of ATLa (5 µg/mouse tail vein injection). The results localize ATLa-induced NAB1 transcripts to smooth muscle cells, most evident within microvessels of the lungs.

CONCLUSIONS AND SIGNIFICANCE

Many key events of acute inflammatory reactions are neutrophil-driven responses involving orchestrated regulation by small lipid and protein signaling molecules. There is appreciation of the biosynthetic capacity of PMN not only as sources of bioactive lipid mediators, but also for their expression of new transcripts as well as proteins such as chemokines/cytokines. This is relevant because PMN can be present in high numbers at sites of acute inflammation within tissues. Recent findings indicate temporal regulation of eicosanoid profiles, where switching to protective eicosanoids such as lipoxin correlates with the onset of resolution of the acute inflammatory reaction. So it was of interest to investigate the early actions of LXA₄/ATL on PMN gene expression.

Aspirin-triggered 15-epi-lipoxin A₄ (ATL) is an endogenous lipid-derived mediator that mimics the actions of native lipoxin A₄, a putative ‘stop signal’ involved in regulating inflammation and resolution. A metabolically more stable analog of ATL, 15-epi-16-(para-fluoro)-phenoxy-LXA₄ analog (ATLa), displays potent anti-inflammatory actions and inhibits neutrophil recruitment, neutrophil-mediated tissue injury and acute inflammation in vitro and in vivo. These include a novel protective action of ATLa that dampens PMN-mediated lung damage from second organ reperfusion injury and LXA₄/ATL analog-accelerated edema resolution. When compared with aspirin in vivo, ATLa is > 100-fold more potent and selective as an inhibitor of neutrophil infiltration, suggesting that the generation of ASA-triggered lipid mediators contributes to the anti-PMN recruitment profile associated with ASA’s therapeutic actions in humans.

Several research groups recently added to the knowledge of the ATLa signaling pathway. Initial attention focused on the very rapid GPCR-mediated events of lipoxin and ATL that occur within seconds or minutes. Other reports have evaluated contribution of lipoxin signaling to the overall resolution process by evaluating very late responses. Little is known about the molecular mechanisms that link the events triggered within minutes by ALX to those observed many hours after exposure. In vivo, LXA₄, ATL, and their analogs are potent inhibitors of PMN responses to proinflammatory eicosanoids (e.g., LTB₄) and bioactive peptides (e.g., tumor necrosis factor ). We exploited the now-appreciated counter-regulation of PMN by two separate eicosanoid signaling pathways, namely, LTB₄ and LXA₄, and identified, using DDPCR, a set of ATLa-selective early response genes in human PMN. ATLa target genes include the transcription factor Sp3, a nuclear corepressor NAB1, and at least three genes of unidentified function. Similar analyses with LTB₄-responsive transcripts (see Fig. 1B ) could be helpful in determining their potential role(s) in functional responses of PMN during host defense and inflammation.

The RT-PCR analyses demonstrate that NAB1 is positively regulated by ATLa, involving a pertussis toxin-sensitive, GPCR-mediated pathway. The transcription factors involved in this up-regulation of NAB1 gene expression have yet to be determined. In epithelial cells, activation of ALX receptor by LXA₄/ATL analogs inhibits NF-B activity via modulation of its nuclear translocation. Schaldach et al. have reported an in vitro search for effectors of the arylhydrocarbon receptor where micromolar concentrations of LXA₄ stimulate transcription of a DRE-driven CAT reporter in Hepa-1 cells. LXA₄ and ATLa were evaluated for their ability to directly activate the nuclear eicosanoid receptors (PPARs) in reporter assays. In their bioactive concentration range (i.e., nanomolar), neither LXA₄ nor ATLa were activators of PPAR (B. Spiegelman, personal communication). Thus, our knowledge of LXA₄-responsive transcription factors is limited.

The present results further indicate that NAB1 is a common target of two anti-inflammatory agents: glucocorticoids and ATLa. Both can up-regulate NAB1 in PMN and smooth muscle cells. Differences in temporal expression patterns in PMN (Fig. 2) suggest that these agents act via distinct pathways. Here, we uncover a novel regulatory system shared by these two vastly different classes of anti-inflammatory molecules: aspirin-triggered lipoxin A₄ and glucocorticoids. Glucocorticoids are among the most effective anti-inflammatory drugs in clinical therapy, but side effects associated with steroid treatment limit their clinical use. Thus, new approaches are needed.

View larger version (76K): [in this window] [in a new window]   Figure 2. Involvement of NAB1 in the anti-inflammatory LXA4 signaling cascade. This model for counter-regulation is based on the demonstrated actions of lipoxin A4 and ATL as stop signals in inflammation and protection in lung. Aspirin-triggered lipoxin A4 analogs (ATLa) activate the LXA4 receptor, which down-regulates PMN responses in vitro and promotes resolution of inflammation in vivo. These extracellular signals are coupled to further downstream responses by up-regulation of protective cassette(s) of early response genes. NAB1 is induced via a pertussis toxin-sensitive, LXA4 receptor-mediated pathway and is a target gene of the glucocorticoids. Consistent with NAB-1’s function as a corepressor, NAB1 confers a repressive action on the adaptive program initiated by proinflammatory stimuli. NAB1 inhibits EGR1 activity resulting in down-regulation of EGR1 target genes. By counter-regulation or switching off of such proinflammatory programs, NAB1 can assist and further promote LXA4 and aspirin-triggered 15-epi-LXA4-mediated resolution of acute inflammation and tissue injury.

How might the function of NAB1 as a corepressor influence an inflammatory reaction? Nuclear cofactors are thought to be important in controlling cell fate decisions. Current findings suggest these proteins are involved in a general mechanism underlying the switch between transcriptionally active and repressive states. They are recruited to target gene promoters by direct binding to transcription factors, where they facilitate rapid changes in expression. The coactivator PGC-1 is thought be an intermediate protein that confers signal-dependent activation of an adaptive thermogenic program in brown adipocytes. The Groucho/TLE family of proteins provides critical repression of gene cassettes, facilitating the progression of many developmental processes including pattern formation and neurogenesis. Hence, transcriptional changes can be either inductive or repressive and can function in decisions of adaptive processes as well as cell specification.

A hypothetical scheme for counter-regulation is illustrated in Fig. 2 ;F2>. By counter-regulation or ‘switching off’ of proinflammatory programs such as those initiated by the early growth response gene 1, NAB1 can assist and further promote LXA₄ and aspirin-triggered, 15-epi-LXA₄-mediated resolution of acute inflammation and tissue injury. LXA₄, ATL, and their analogs inhibit cytokine gene expression in human neutrophils, fibroblasts, and epithelial cells. It is highly likely that additional yet unidentified interacting partners of NAB1 also participate in this process.

Taken together, our results demonstrate that ATLa, known to act via the ALXR, a G-protein-coupled receptor in mouse and human cells, up-regulates gene expression of the transcriptional repressor NAB1 in human neutrophils. ATLa and glucocorticoid targeting of transcription corepressors such as NAB1 opens the possibility of uncovering additional nuclear-related mechanisms with LX and ATL gene regulation and adds to the profile of LX rapid actions initiated at the surface membrane receptor that lead to and contribute to ‘stop signaling’.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0576fje; to cite this article, use FASEB J. (October 29, 2001) 10.1096/fj.01-0576fje

² P.R.D. and F.H.Q. share first authorship.

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