Specific oxylipins enhance vertebrate hematopoiesis via the receptor GPR132 Jamie L. Lahvica,b, Michelle Ammermana,b, Pulin Lia,b, Megan C. Blaira,b, Emma R. Stillmana,b, Eva M. Fastc,d, Anne L. Robertsona,b, Constantina Christodouloua,b, Julie R. Perlina,b, Song Yanga,b, Nan Chiange, Paul C. Norrise, Madeleine L. Dailya,b, Shelby E. Redfielda,b, Iris T. Chana,b, Mona Chatrizeha,b, Michael E. Chasea,b, Olivia Weisa,b, Yi Zhoua,b, Charles N. Serhane, and Leonard I. Zona,b,c,d,f,1 aStem Cell Program, Boston Children’s Hospital, Boston, MA 02115; bDivision of Hematology/Oncology, Boston Children’s Hospital, Boston, MA 02115; cHarvard Stem Cell Institute, Harvard University, Cambridge, MA 02138; dDepartment of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA 02138; eCenter for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Boston, MA 02115; and fHoward Hughes Medical Institute, Boston Children’s Hospital, Boston, MA 02115 Edited by Gregg L. Semenza, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 30, 2018 (received for review April 11, 2018) Epoxyeicosatrienoic acids (EETs) are lipid-derived signaling mole- require G protein signaling components to elicit many phenotypes cules with cardioprotective and vasodilatory actions. We recently (10–12). For instance, we previously showed that EET’sen- showed that 11,12-EET enhances hematopoietic induction and hancement of zebrafish hematopoiesis requires signaling via engraftment in mice and zebrafish. EETs are known to signal via G Gα12/13 (5). protein-coupled receptors, with evidence supporting the existence As traditional biochemical methods have so far failed to of a specific high-affinity receptor. Identification of a hematopoietic- identify EET receptors, here we used bioinformatic techniques specific EET receptor would enable genetic interrogation of EET to identify candidate receptors and assayed those candidates for signaling pathways, and perhaps clinical use of this molecule. We EET responsiveness in vitro. Only GPR132 (G2A), a previously developed a bioinformatic approach to identify an EET receptor described fatty acid receptor (13–16), showed responsiveness to based on the expression of G protein-coupled receptors in cell lines EET. We demonstrated that GPR132 is required for EET- with differential responses to EETs. We found 10 candidate EET induced hematopoietic stem cell specification in the zebrafish BIOLOGY receptors that are expressed in three EET-responsive cell lines, but and for normal hematopoietic stem cell transplant in the mouse. DEVELOPMENTAL not expressed in an EET-unresponsive line. Of these, only recombinant Previously described fatty acid activators of GPR132 induced GPR132 showed EET-responsiveness in vitro, using a luminescence- hematopoietic phenotypes in the zebrafish essentially identical to based β-arrestin recruitment assay. Knockdown of zebrafish gpr132b those observed with EET, further confirming that these molecules prevented EET-induced hematopoiesis, and marrow from GPR132 activate the same pathway. We performed structure–activity re- knockout mice showed decreased long-term engraftment capability. lationship analyses to determine the full range of GPR132 acti- In contrast to high-affinity EET receptors, GPR132 is reported to re- vators. Rather than a regiospecific, high-affinity receptor, our data spond to additional hydroxy-fatty acids in vitro, and we found that show that GPR132 is likely a low-affinity, promiscuous receptor these same hydroxy-fatty acids enhance hematopoiesis in the zebra- for a select panel of oxygenated polyunsaturated fatty acids, whose fish. We conducted structure–activity relationship analyses using both activity drives hematopoiesis in embryonic and adult vertebrate cell culture and zebrafish assays on diverse medium-chain fatty acids. contexts. Certain oxygenated, unsaturated free fatty acids showed high activa- tion of GPR132, whereas unoxygenated or saturated fatty acids had Significance lower activity. Absence of the carbon-1 position carboxylic acid pre- vented activity, suggesting that this moiety is required for receptor Small-molecule enhancers of hematopoietic stem cell trans- activation. GPR132 responds to a select panel of oxygenated polyunsat- plant could improve the safety of this treatment and expand urated fatty acids to enhance both embryonic and adult hematopoiesis. the pool of eligible patients. We previously showed that the lipid 11,12-epoxyeicosatrienoic acid (EET) enhanced transplant hematopoiesis | free fatty acid | GPCR | zebrafish in zebrafish and mice. We use a bioinformatic approach to identify candidate EET receptors and demonstrate that EET icosanoids are endogenous bioactive mediators derived from activates GPR132. We find that this receptor is important in Earachidonic acid, and are responsible for a variety of physi- zebrafish and mouse hematopoiesis, and we further show that ological phenotypes (1). Epoxyeicosatrienoic acids (EETs), a GPR132 has responsiveness to additional oxygenated poly- major class of eicosanoids formed by cytochrome P450 enzymes, unsaturated fatty acids such as EET. Thus, GPR132 receives play critical roles in endothelial migration, monocyte adhesion, lipid-derived signals to regulate hematopoiesis and is a thera- tumor metastasis, and vasodilation, among other cell-type- peutic target for enhancing HSC transplant. specific effects (2–4). We recently showed that 11,12-EET enhances the specification of hematopoietic stem and progenitor Author contributions: J.L.L., P.L., J.R.P., N.C., Y.Z., C.N.S., and L.I.Z. designed research; cells (HSPCs) in developing zebrafish embryos, as well as the J.L.L., M.A., P.L., M.C.B., E.R.S., E.M.F., A.L.R., C.C., J.R.P., N.C., P.C.N., M.L.D., S.E.R., transplant of HSPCs in both fish and mice (5). Despite the im- I.T.C., M.C., M.E.C., and O.W. performed research; J.L.L., E.M.F., C.C., S.Y., C.N.S., and portant physiological roles of EETs, their direct protein target(s) L.I.Z. analyzed data; and J.L.L., P.L., E.M.F., C.N.S., and L.I.Z. wrote the paper. remain unknown. The authors declare no conflict of interest. Previous results indicate that EETs bind to at least one spe- This article is a PNAS Direct Submission. cific G protein-coupled receptor (GPCR). Nanomolar or even Published under the PNAS license. picomolar concentrations of 11,12- or 14,15-EET can elicit spe- Data deposition: The data reported in this paper have been deposited in the Gene Ex- cific cellular phenotypes (6, 7), and a bead-tethered EET without pression Omnibus (GEO) database, https://www.ncbi.nlm.nih.gov/geo (accession no. the ability to cross the plasma membrane maintained its activity GSE113550). (8), suggesting the existence of a high-affinity, membrane-bound 1To whom correspondence should be addressed. Email: [email protected]. EET receptor. Chen et al. (9) demonstrated that U937 and other This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. EET-responsive cell lines express a single high-affinity EET re- 1073/pnas.1806077115/-/DCSupplemental. ceptor of about 47 kDa in size. This is likely a GPCR, as EETs www.pnas.org/cgi/doi/10.1073/pnas.1806077115 PNAS Latest Articles | 1of6 Downloaded by guest on September 24, 2021 Results A B Identification of Candidate EET Receptors. EETs elicit phenotypes 5 GPR132 ce 11,12-EET only in specific cell types, suggesting that a putative EET re- 4 14,15-EET b-arrestin PGE2 ceptor might be selectively expressed in these cell types. We gal b-gal 3 performed RNAseq profiling in duplicate on three previously minescen 2 b-arrestin Lu b- identified EET-responsive human cell lines [U937 monocytes (17), e 1 EaHy endothelial cells (18), and PC3M-LN4 prostate cancer cells liligandgand GPCRGPCR lativ e 0 (4)], two of which show binding to a radiolabeled EET analog (9). R -9 -8 -7 -6 -5 -4 -3 We profiled a fourth cell line (HEK293) that has no known re- Ligand Conc (log10M) C D sponsiveness to EET and shows no such binding (9). Although we ADRB2 10 HRH1 20 ce detected reads for hundreds of GPCRs (SI Appendix, Fig. S1), only Isoproterenol Histamine 37 GPCRs were expressed in common in all three EET-responsive 11,12-EET 8 11,12-EET 15 14,15-EET 14,15-EET nescence cell lines above 0.3 fragments per kilobase per million reads i 6 10 minescen (FPKM) (SI Appendix,Fig.S2A), a conservative threshold for 4 Lu physiologically meaningful abundance. Of these, 27 were also e 5 2 expressed at moderate to high levels (FPKM > 0.9) in our EET 0 0 Relativ nonbinding cell line (SI Appendix,Fig.S2B). This left 10 candidate Relative Lum -10 -8 -6 -4 -2 -10 -8 -6 -4 -2 EET receptors that were expressed only in EET binding cell lines Ligand Conc (log10M) Ligand Conc (log10M) and were missing from the nonbinding cell line. All candidates had E PTGER2 F PTGER4 8 e 6 predicted molecular weights within 20% of the predicted 47-kDa PGE2 PGE2 size of an EET receptor (9), and candidates included both well- 11,12-EET 5 11,12-EET 6 scenc β 4 studied GPCRs such as the -adrenergic receptor and prosta- ne 4 3 glandin receptors and orphan GPCRs such as GPR132 and umi L 2 GPR135 (SI Appendix,Fig.S2C). 2 ive t 1 a 0 0 Relative Luminescence β Rel EET Activates -Arrestin Recruitment via GPR132 in Vitro. GPCR -10 -8 -6 -4 -2 -10 -8 -6 -4 -2 activation causes recruitment of β-arrestin, which can be mea- Ligand Conc (log10M) Ligand Conc (log10M) sured with the luminescence-based PathHunter assay (13, 15, G H e 4 CCRL2 5 GPR135 19). We tested for EET-induced β-arrestin recruitment via each 11,12-EET nc Chemerin e MIP-3beta ence 14,15-EET candidate GPCR with the exception of GPR68, PTGER1, and c 4 3 11,12-EET nes i 3 LPAR6, which have no available PathHunter assays (Fig.