Resolvin D1 limits 5-lipoxygenase nuclear localization and leukotriene B4 synthesis by inhibiting a calcium- activated kinase pathway Gabrielle Fredmana,1, Lale Ozcana, Stefano Spolitua, Jason Hellmannb, Matthew Spiteb, Johannes Backsc, and Ira Tabasa,1 aDepartment of Medicine, Department of Pathology and Cell Biology, and Department of Physiology, Columbia University, New York, NY 10032; bInstitute of Molecular Cardiology, University of Louisville, Louisville, KY 40202; and cLaboratory for Cardiac Epigenetics, Department of Cardiology, Heidelberg University, and German Centre for Cardiovascular Research (DZHK), 69120 Heidelberg, Germany Edited by Mauro Perretti, Queen Mary University of London, London, United Kingdom, and accepted by the Editorial Board August 26, 2014 (received for review June 13, 2014) Imbalances between proinflammatory and proresolving mediators critical because although LTB4 is crucial for host defense, exu- can lead to chronic inflammatory diseases. The balance of arach- berant production underlies the basis for several inflammatory idonic acid-derived mediators in leukocytes is thought to be achieved diseases and impairs endogenous resolution programs (11, 18). through intracellular localization of 5-lipoxygenase (5-LOX): nuclear Moreover, complete blockade of LTB4 biosynthetic enzymes may 5-LOX favors the biosynthesis of proinflammatory leukotriene B4 compromise host defense; thus, understanding new mechanisms (LTB4), whereas, in theory, cytoplasmic 5-LOX could favor the bio- that temper LTB4 production is essential for translational research synthesis of proresolving lipoxin A4 (LXA4). This balance is shifted in this area (19). in favor of LXA4 by resolvin D1 (RvD1), a specialized proresolving Here, we report that RvD1, by suppressing the activation of the mediator derived from docosahexaenoic acid, but the mechanism is calcium-sensing kinase calcium-calmodulin-dependent protein ki- not known. Here we report a new pathway through which RvD1 nase II (CaMKII), decreases the phosphorylation and nuclear promotes nuclear exclusion of 5-LOX and thereby suppresses LTB4 localization of 5-LOX and thereby limits LTB4 biosynthesis. and enhances LXA4 in macrophages. RvD1, by activating its receptor These results provide a mechanistic understanding of how RvD1 formyl peptide receptor2/lipoxin A4 receptor, suppresses cytosolic tempers proinflammatory responses to facilitate a rapid transi- calcium and decreases activation of the calcium-sensitive kinase cal- tion to resolution. cium-calmodulin-dependent protein kinase II (CaMKII). CaMKII inhi- bition suppresses activation P38 and mitogen-activated protein Results kinase-activated protein kinase 2 kinases, which reduces Ser271 RvD1 Suppresses AA-Stimulated LTB4 by Blocking P38/MK2-Mediated phosphorylation of 5-LOX and shifts 5-LOX from the nucleus to the 5-LOX Phosphorylation and Nuclear Localization. We first showed ’ cytoplasm. As such, RvD1 s ability to decrease nuclear 5-LOX and the that 1 nM RvD1 enhanced AA-stimulated LXA4 generation (Fig. LTB4:LXA4 ratio in vitro and in vivo was mimicked by macrophages 1A) and blocked LTB4 formation in both bone marrow-derived lacking CaMKII or expressing S271A-5-LOX. These findings provide macrophages and zymosan-elicited peritoneal macrophages (Fig. mechanistic insight into how a specialized proresolving mediator 1B and SI Appendix, Fig. S1 and S2A). We conducted an RvD1 from the docosahexaenoic acid pathway shifts the balance toward dose–response experiment and found that the suppression of AA- resolution in the arachidonic acid pathway. Knowledge of this mech- stimulated LTB4 generation was close to maximal at 1 nM RvD1 anism may provide new strategies for promoting inflammation res- olution in chronic inflammatory diseases. Significance ersistent inflammation and its failed resolution underlie the Specialized proresolving mediators, such as resolvin D1 (RvD1), Ppathophysiology of prevalent human diseases, including cancer, are endogenous molecules that both dampen inflammation diabetes, and atherosclerosis (1). Hence, uncovering mechanisms without compromising host defense and promote tissue reso- to suppress inflammation and enhance resolution is of immense lution. A prime example is RvD1’s ability to decrease the ratio of – interest (2 5). Resolution is orchestrated in part by specialized proinflammatory leukotriene B4 (LTB4) to proresolving lipoxin A4 proresolving mediators (SPMs), including lipoxins, resolvins, pro- (LXA4), but the mechanism is not known. We have discovered tectins, and maresins (2), and by protein and peptide mediators (6). a new calcium kinase signaling pathway through which RvD1 A common protective function of SPMs is their ability to limit lowers the nuclear:cytoplasmic ratio of 5-lipoxygenase (5-LOX), excessive proinflammatory leukotriene formation without being the common enzyme for LTB4 and LXA4 biosynthesis This shift in immunosuppressive (2, 7). Specifically, resolvin D1 (RvD1) is 5-LOX localization dampens LTB4 production and enhances LXA4 protective in several disease models (8) and limits excessive production. By providing a new mechanistic understanding of leukotriene B4 (LTB4) production without compromising host how RvD1 tempers inflammation to facilitate resolution, these defense (7, 9). However, the mechanism underlying these actions findings can help devise new therapeutic strategies for diseases of RvD1 is not well understood. driven by nonresolving inflammation. Arachidonic acid (AA) is first converted into 5-hydro- peroxyeicosatetraenoicacid (5-HPETE) and then into leukotri- Author contributions: G.F., L.O., and I.T. designed research; G.F., S.S., J.H., and M.S. per- formed research; J.B. and I.T. contributed new reagents/analytic tools; G.F., S.S., J.H., M.S., ene A4 (LTA4) by 5-lipoxygenase (5-LOX) (10, 11). Subsequent and I.T. analyzed data; and G.F., L.O., and I.T. wrote the paper. hydrolysis of LTA4 by LTA4 hydrolase yields LTB4 (10, 11). The authors declare no conflict of interest. During inflammation, 5-LOX is phosphorylated and translocates This article is a PNAS Direct Submission. M.P. is a Guest Editor invited by the Editorial to the nuclear membrane, which favors the biosynthesis of LTB4 Board. – (12 17). However, major gaps remain in our understanding of the Freely available online through the PNAS open access option. relevance of this pathway to primary cells and animal models and 1To whom correspondence may be addressed. Email: [email protected] or gf2269@ how they are regulated by SPMs. Further, it is currently not known columbia.edu. how this pathway may influence the biosynthesis of lipoxin A4 This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (LXA4), which is a SPM that also requires 5-LOX. These gaps are 1073/pnas.1410851111/-/DCSupplemental. 14530–14535 | PNAS | October 7, 2014 | vol. 111 | no. 40 www.pnas.org/cgi/doi/10.1073/pnas.1410851111 Downloaded by guest on September 29, 2021 activation state (Fig. 2 A and B). FPR2/ALX is a G protein- coupled receptor, which led us to explore the possible role of cAMP. We found that 8-bromo-cAMP blocked RvD1’s ability to decrease p-MK2, whereas the Rp-cAMP mimicked RvD1’s actions (SI Appendix, Fig. S4). These results are consistent with a receptor-mediated response involving Gi proteins, which is in line with previous studies exploring the mechanism of action of the FPR2/ALX ligand LXA4 (26, 27). Most important, the P38 in- hibitor SB20380 blocked both 5-LOX nuclear localization (Fig. 2C and SI Appendix, Fig. S5) and AA-stimulated LTB4 production (Fig. 2D) to a similar extent as RvD1, and the fact that SB20380 and RvD1 were not additive in blocking LTB4 suggested that RvD1 and P38 were in the same pathway. As more direct proof, we showed that RvD1 decreased AA-stimulated phospho-Ser271-5- − − LOX (Fig. 3A). We then transfected 5-LOX-deficient (Alox5 / ) Fig. 1. RvD1 (D1) suppresses AA-stimulated LTB4 a in a receptor-dependent manner upstream of LTA4 hydrolase. (A, B, and D) BMDMs were pre- incubated with vehicle control, 1 nM RvD1, or 1 nM LXA4 for 15 min, fol- lowed by incubation with 10 μM AA for 40 min. The media were then assayed for LXA4 by liquid chromatography (LC)-MS/MS (A) and LTB4 by ELISA (n = 6 for LC-MS/MS and n = 3 for ELISA; mean ± SEM; *P < 0.05 versus AA). In the experiment in A, LXB4 was not detected by LC-MS/MS. (C)AsinB, but the cells were pretreated with IgG control (black bars) or anti-ALX/FPR2 IgG (gray bars) for 1 h at 37 °C (n = 3; mean ± SEM; *P < 0.05 versus all other groups). (E) Scheme of LTB4 biosynthesis. (F)AsinB,except10μMLTA4 was used instead of AA (n = 3; mean ± SEM; **P < 0.05 versus vehicle; n.s., non- significant). LTA4 showed no cross reactivity with the LTB4 ELISA. MEDICAL SCIENCES (SI Appendix,Fig.S3). These actions of RvD1 were mediated through its receptor formyl peptide receptor2/lipoxin A4 receptor (FPR2/ALX) (20), as both an FPR2/ALX blocking antibody (Fig. 1C) and the FPR2/ALX antagonist WRW4 (SI Appendix, Fig. S2B) blocked the ability of RvD1 to reduce AA-stimulated LTB4 generation. LXA4, another ligand for FPR2/ALX (21, 22), also blocked LTB4 generation (Fig. 1D). LTB4 is generated from LTA4, a product of 5-LOX, through the action of LTA4 hydrolase Fig. 2. RvD1 decreases nuclear localization of 5-LOX through inhibition of (Fig. 1E) (11). To help pinpoint the step in this pathway at which p38-MAPKAPK2 (MK2) signaling. (A and B) BMDMs were preincubated with RvD1 was acting, we circumvented the 5-LOX step by incubating vehicle control or RvD1 and then incubated with 10 μM AA for 5 min. Cell macrophages with LTA instead of AA. As expected, LTA sig- lysates were immunoblotted for phospho- and total p38 or MK2 and quanti- 4 4 fied by densitometry (n = 3; mean ± SEM; ***P < 0.001 versus AA). (C) BMDMs nificantly increased LTB4 level, but RvD1 did not block this in- treated similar to those in C were permeabilized and stained with Alexa-488 crease (Fig. 1F), indicating that RvD1 was acting upstream of the anti-5-LOX antibody (green) and counterstained with the nuclear stain DAPI LTA4 hydrolase.