The role of the GPR91 ligand succinate in hematopoiesis

Uncorrected Version Published on February 9, 2009 as DOI:10.1189/jlb.1008618 The role of the GPR91 ligand succinate in hematopoiesis Yaron Hakak,1 Karin Lehmann-Bruinsma, Shirley Phillips, Thuy Le, Chen Liaw, Daniel T Connolly, and Dominic P Behan Arena Pharmaceuticals, Inc., San Diego, California, USA Abstract: Regulation of cellular metabolism by the citric acid cycle occurs in the mitochondria However, the citric acid cycle intermediate succinate was shown recently to be a ligand for the G-protein-coupled receptor GPR91 Here, we describe a role for succinate and its receptor in the stimulation of hematopoietic progenitor cell (HPC) growth GPR91 mRNA and protein expression were detected in human bone marrow CD34؉ progenitor cells, as well as in erythroid and megakaryocyte cultures and the erythroleukemic cell line TF-1 Treatment of these cell cultures with succinate resulted in increased proliferation rates The proliferation response of TF-1 cells was pertussis toxin (PTX)-sensitive, suggesting a role for Gi signaling Proliferation was also blocked when TF-1 cells were transfected with small interfering RNA specific for GPR91 Succinate stimulated activation of the Erk MAPK pathway and inositol phosphate accumulation in a PTX-sensitive manner Pretreatment of TF-1 cells with the Erk1/2 kinase (MEK) inhibitor PD98059 blocked the proliferation response Succinate treatment additionally protected TF-1 cells from cell death induced by serum deprivation Finally, in vivo administration of succinate was found to elevate the levels of hemoglobin, platelets, and neutrophils in a mouse model of chemotherapy-induced myelosuppression These results suggest that succinate GPR91 signaling is capable of promoting HPC development J Leukoc Biol 85: 000 – 000; 2009 Key Words: hematopoietic progenitor cells ⅐ erythroid ⅐ megakaryocyte of the liver and kidney, succinate levels have been reported to increase significantly in tissues up to millimollar concentrations [4 – 8] Cellular responses to succinate administration include increased ion transport and gluconeogenesis in the proximal tubules of the kidney [9, 10] and the potentiation of the actions of platelet agonists [11] A recent report identified succinate as a ligand for the G-protein-coupled receptor (GPCR) GPR91 [12] In a recombinant system overexpressing GPR91, succinate was shown to stimulate calcium mobilization and inositol phosphate (IP) accumulation with EC50 values of 56 ␮M and 391 ␮M, respectively Succinate treatment also activated the Erk1/2 MAPK pathway and inhibited forskolin-stimulated cAMP accumulation GPR91 was not activated by other citric acid cycle intermediates, and a panel of other GPCRs tested was not activated by succinate GPR91 mRNA was detected in mouse kidney, liver, and spleen In vivo, administration of succinate in mice raised mean arterial pressure This effect was a result of an increase in renin secretion from the kidney and was absent in GPR91-deficienct mice Other studies have also suggested a role for GPR91 in retinal angiogenesis [13] and dendritic cell function [14] Here, we report that GPR91 mRNA expression is also enriched in early- and late-stage hematopoietic progenitor cells (HPCs), which reside within the bone marrow, where they undergo regulated stages of proliferation and differentiation This maturation process leads to the production of the various immune cell types [15] In vitro studies have shown that erythropoietin (EPO) and other hematopoietic growth factors activate several signaling pathways in HPCs, including the MAPK Erk1/2 and calcium mobilization [16 –22] Activation of these pathways is relevant for HPC proliferation, differentiation, and survival Given the reported capacity of GPR91 to activate these pathways as well, the receptor may likewise modulate HPC biology We therefore studied succinate GPR91 signaling in HPCs and have identified a role for it in the stimulation of progenitor cell growth INTRODUCTION Succinate, an intermediate of the citric acid cycle, plays a key role in energy metabolism It is synthesized in the mitochondria by the oxidation of succinyl-CoA and is itself converted to fumarate by succinate dehydrogenase Aside from its conventional role in energy metabolism, other functions for this intermediate have been reported Cytosolic succinate has been found to inhibit hypoxia-inducible factor (HIF)-␣ prolyl hydroxylase, resulting in the stabilization of the transcription factor HIF-1␣ [1, 2] Extracellular succinate is found in blood at concentrations up to 20 ␮M [3] Under physiological and pathological conditions such as during exercise and ischemia 0741-5400/09/0085-0001 © Society for Leukocyte Biology MATERIALS AND METHODS Reagents Sodium succinate dibasic hexahydrate and pertussis toxin (PTX) were purchased from Sigma-Aldrich (St Louis, MO, USA) EPO, thrombopoietin (TPO), Correspondence: Arena Pharmaceuticals, Inc., 6166 Nancy Ridge Drive, San Diego, CA 92121, USA E-mail: yhakak@arenapharm.com Received October 14, 2008; revised November 18, 2008; accepted January 13, 2009 doi: 10.1189/jlb.1008618 Journal of Leukocyte Biology Volume 85, May 2009 Copyright 2009 by The Society for Leukocyte Biology IL-3, stem cell factor (SCF), and GM-CSF were purchased from Stem Cell Technologies (Vancouver, BC, Canada) IL-11 (Oprelvekin) was purchased from Wyeth (Madison, NJ, USA) PD98059 was purchased from Calbiochem (San Diego, CA, USA) Antiphospho (p)- and total Erk1/2 ELISA kits were purchased from BioSource International, Inc (Camarillo, CA, USA) Cell culture Human bone marrow and cord blood CD34ϩ cells were purchased from Stem Cell Technologies Megakaryocyte and erythroid progenitor cells were derived in vitro as described previously with some modifications [23–25] Briefly, CB CD34ϩ cells were cultured in StemSpan (Stem Cell Technologies) medium with 50 ng/ml TPO and 1% penicillin/streptomycin for days to generate megakaryocyte cells Erythroid progenitor cultures were derived by culturing CB CD34ϩ cells in StemSpan medium with U/ml EPO, 20 ng/ml SCF, 20 ng/ml IL-3, 20 ng/ml GM-CSF, and 1% penicillin/streptomycin for days FACS analysis of the megakaryocyte culture indicated that 42% of the cells were CD41ϩ, and 64% of the cells in the erythroid culture were glycophorin Aϩ (Supplemental Fig 1) TF-1, Hel92.1, and K562 cell lines were obtained from American Type Culture Collection (Manassas, VA, USA) TF-1 cells were cultured in RPMI 1640, 10% FBS (Invitrogen, Carlsbad, CA, USA), ng/ml GM-CSF, and 1% penicillin/streptomycin Bone marrow stromal cell cultures were purchased from Lonza (Basel, Switzerland) All cells were cultured at 37°C in 5% CO2 Quantitative RT-PCR (qRT-PCR) analysis Total RNA was isolated from cultured cells using an RNEasy kit (Qiagen, Valencia, CA, USA) by following the manufacturer’s protocol Total RNA for human tissues and primary cells was purchased from Clontech Laboratories, Inc (Mountain View, CA, USA) The quality of total RNA was assessed using an Agilent 2100 bioanalyzer (visible absence of significant 28S and 18S band degradation) and by spectrophotometry qRT-PCR was performed using the iScript cDNA synthesis kit and iTaq Supermix kit (Quanta BioSciences, Inc., Gaithersburg, MD, USA) Reactions were run and analyzed on an ABI Prism 7900HT machine (Applied Biosystems, Foster City, CA, USA) Immunoblot analysis Cells were lysed in cell lysis buffer (Cell Signaling Technology, Boston, MA, USA), supplemented with a protease inhibitor cocktail (Sigma-Aldrich) Lysates were cleared by centrifugation (12,000 g for 10 min) and resolved by SDS-PAGE Immunoblot analysis was then performed with antibodies directed against GPR91 (Abcam Inc., Cambridge, MA, USA) and GAPDH (Cell Signaling Technology) Proliferation assays Primary cell cultures were plated at 50,000 cells/well in a 96-well plate Megakaryocyte- and erythroid-cultured cells were starved overnight in StemSpan medium containing ng/ml TPO or 0.5 U/ml EPO, respectively Cells were then treated with succinate for days TF-1 cells were plated at 30,000 cells/well in a 96-well plate Cells were cultured overnight in RPMI 1640 containing 2% FBS and 100 ng/ml PTX where indicated Cells were then treated with compounds for 24 h Addition of succinate to the cell cultures did not alter the pH of the medium GPR91 expression was inhibited by transfecting TF-1 cells with small interfering (si)RNA specific for the receptor (Dharmacon, Chicago, IL, USA) using oligofectamine reagent (Invitrogen) prior to cell starvation No cell death was observed under these depleted medium culture conditions as measured by FACS analysis for 7-amino-actinomycin (7-AAD) staining (for details, see below) Cell proliferation was measured using the colorimetric MTT assay (Promega, Madison, WI, USA), which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells The absorbance of the formazan product at 490 nm is measured directly from 96-well assay plates A linear relationship between cell number and absorbance was established (Supplemental Fig 2) Similar results were also observed when using a tritiated thymidine incorporation assay and p-Erk1/2 was detected by ELISA kits according to the manufacturer’s guidelines OD was measured at 450 nm IP accumulation assay IP accumulation was determined as described previously [26] with minor modifications TF-1 cells were plated at 50,000 cell/well in a 96-well plate Cells were cultured in inositol-free media (Invitrogen) containing 0.4 ␮Ci H-myoinositol/well in a total of 100 ␮l/well PTX (100 ng/ml) was included where indicated Cells were cultured overnight at 37°C in 5% CO2 The H-myoinositol-containing media were then replaced with inositol-free medium with the indicated concentration of succinate Cells were incubated for h followed by medium removal and addition of 200 ␮l ice-cold 0.1 M formic acid Cells were lysed by a freeze/thaw cycle Labeled IP was purified using a resin-based filtration system (Bio-Rad Laboratories, Hercules, CA, USA) Eluates were counted in a liquid scintillation counter Cell death assay TF-1 cells were plated at 30,000 cells/well in a 96-well plate Cells were cultured overnight in RPMI 1640 and 100 ng/ml PTX where indicated Cells were then treated with succinate or EPO for 48 h FACS analysis of cells for staining with the nucleic acid dye 7-AAD (BD Biosciences, San Jose, CA, USA) was used as a measure of cell death [27] Mouse myelosuppression study Seven-week-old male BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA, USA) Animal studies were performed according to the 1996 Guide for the Care and Use of Laboratory Animals published by the National Academy of Sciences (Washington, DC, USA) All study protocols were reviewed and approved by the Arena Pharmaceuticals Institutional Animal Care and Use Committee (San Diego, CA, USA) Mice were housed under controlled temperature and humidity and a lighting cycle of 12 h/day Mice were fed a laboratory diet and distilled water ad libitum Myelosuppression was induced by s.c administration of the DNA alkylating agent busulfan (ESP Pharma Inc., Edison, NJ, USA) at 15 mg/kg on Days and Starting on Day 4, saline or succinate (30 mg/kg) was administered twice/day or IL-11 (500 ug/kg) once/day s.c for consecutive days On Day 17, the mice were anesthetized and eye bleeds performed Blood samples were analyzed on a Cell Dyn 3700 (Abbott, Abbott Park, IL, USA) At Day 17 of the chemotherapy cycle, platelet, hemoglobin, and neutrophil levels were reduced significantly in the busulfan-treated mice relative to nonchallenged mice RESULTS GPR91 is expressed in HPCs qRT-PCR analysis of GPR91 mRNA expression was performed for a panel of human tissues and cell types (Fig 1A) GPR91 mRNA was expressed in CD34ϩ HPCs, megakaryocyte and erythroid progenitor cultures, and two of three erythroleukemic cell lines tested Receptor mRNA was also detected in bone marrow-derived stromal cell cultures These results indicate that GPR91 is expressed transcriptionally by early and lineage-committed HPCs as well as the stromal cells that support them Consistent with the reported expression in mouse tissues, GPR91 mRNA was detected in human kidney, liver, and spleen GPR91 protein levels were also investigated in the hematopoietic samples Immunoblot analysis confirmed that the receptor is expressed in CD34ϩ progenitor cells, erythroid and megakaryocyte cultures, and TF-1 cells, but not K562 cells Erk1/2 ELISA assay Succinate stimulates the proliferation of cultured megakaryocyte and erythroid progenitor cells TF-1 cells were serum-deprived overnight and then treated with the indicated compounds for 10 Cells were washed with ice-cold PBS and lysed Total GPR91 has been shown to activate MAPK signaling in a recombinant system [12] As this pathway has been reported to Journal of Leukocyte Biology Volume 85, May 2009 http://www.jleukbio.org Fig Expression of GPR91 in human HPCs (A) Total RNA was collected from human bone marrow (BM) CD34ϩ cells and in vitro-derived megakaryocyte and erythroid progenitor cells qRTPCR analysis of GPR91 mRNA levels was performed for HPC samples as well as peripheral tissues Results are normalized to the housekeeping gene ribosomal protein S9 mRNA expression levels Data are the mean and SE of three replicate measurements (B) Immunoblot analysis of lysates of cell lines and primary cells using antibodies directed against GPR91 and GAPDH be involved in HPC growth, the effect of succinate treatment on progenitor cell proliferation was investigated CD34ϩ cells were cultured in TPO or EPO and other cytokines to derive megakaryocyte or erythroid progenitor cells Cell cultures were then depleted of cytokine growth factors and stimulated with succinate for days Treatment of megakaryocytes with succinate resulted in a concentration-dependent increase in cell growth by up to 62% relative to vehicle-treated cells (Fig 2A) Cell growth of megakaryocytes cultured in complete growth medium increased by 87% relative to vehicle-treated cells Treatment of erythroid progenitor cultures with succinate induced up to a 44% increase in cell growth relative to the vehicle treatment, as compared with over a 200% increase when cells were given complete growth medium (Fig 2B) expression levels were decreased substantially (Fig 3C) Furthermore, the citric acid cycle intermediate ␣-ketogluterate, which does not activate GPR91 [12], did not stimulate TF-1 cell proliferation (data not shown) Taken together, these data indicate that GPR91 mediates the proliferation response to succinate by coupling to the Gi signal transduction pathway To further define the molecular mechanism mediating the proliferation response, cellular signaling induced by succinate Succinate stimulates TF-1 cell proliferation through GPR91 and requires activation of the Erk MAPK pathway The erythroleukemic cell line TF-1 was used to further study the mechanism of succinate-mediated cell proliferation These cells express HPC markers, are responsive to hematopoietic growth factors, and can be differentiated to the erythroid or megakaryocyte lineage [28, 29] Treatment of TF-1 cells with succinate resulted in a threefold increase in cell growth (EC50 270 ␮M), a response that was approximately twice that induced by EPO (Fig 3A) The proliferation response was sensitive to pretreatment of cells with PTX, indicating that Gi signaling was required Succinate treatment of the leukemic cell line K562, which does not express GPR91, did not stimulate proliferation (Supplemental Fig 3) To determine if GPR91 mediates the succinate response, TF-1 cells were transfected with siRNA specific for GPR91 or cyclophilin B as a negative control TF-1 cell proliferation induced by succinate administration was diminished significantly in cells transfected with siRNA specific for GPR91 but not in cells transfected with cyclophilin B-specific siRNA (Fig 3B) The proliferation response to EPO or GM-CSF treatment was unaffected by GPR91 expression levels qRT-PCR analysis of the transfected cells confirmed that the receptor mRNA Fig Succinate stimulates the proliferation of cultured megakaryocyte and erythroid progenitor cells (A) Megakaryocyte and (B) erythroid progenitor cells were cultured medium depleted of serum and growth factors and subsequently treated with succinate for days Cellular proliferation was assessed using a MTT colorimetric assay with an absorbance (O.D.) measurement at 490 nm Cell growth in complete growth medium (CGM) was also measured Data are the mean and SE of three replicate measurements *, P Ͻ 0.05; **, P Ͻ 0.001, versus vehicle-treated using the Student-Newman-Keuls test Hakak et al Succinate-mediated hematopoiesis Fig Succinate stimulates TF-1 cell proliferation via GPR91 (A) The erythroleukemic cell line TF-1 was cultured under serum-deprived conditions in the presence or absence of PTX Cells were then treated with EPO or succinate for 24 h and cell proliferation assessed using a MTT colorimetric assay with an absorbance (O.D.) measurement at 490 nm (B) TF-1 cells were transfected with siRNA specific for GPR91 or cyclophilin B (CycloB) as a control Cells were then cultured as described above and assayed for cell proliferation (C) GPR91 mRNA expression was assessed by qRT-PCR analysis in the transfected TF-1 cells Data are the mean and SE of three replicate measurements *, P Ͻ 0.05; **, P Ͻ 0.001, compound versus vehicle-treated; †, P Ͻ 0.05; ‡, P Ͻ 0.001, PTX versus non-PTX-treated succinate group; #, P Ͻ 0.05; &, P Ͻ 0.001, GPR91 siRNA versus untransfected group using the Student-Newman-Keuls test was investigated An ELISA-based assay was used to monitor the activation of the MAPKs Erk1/2 in TF-1 cells Erk1/2 activation was increased by succinate treatment in a concentration-dependent manner by up to fivefold relative to a vehicle control (Fig 4A) These levels of stimulation were comparable with that of EPO and like the proliferation response, were PTX-sensitive Succinate treatment of TF-1 cells also increased IP accumulation (EC50 365 ␮M) in a PTX-sensitive manner (Fig 4B) A similar increase in IP levels was observed upon succinate treatment of human embryo kidney (HEK) 293 cells overexpressing GPR91 (EC50 360 ␮M; data not shown) The EC50 concentrations for IP elevation were similar to the EC50 concentrations obtained in proliferation assays To assess whether Erk1/2 activation mediates the proliferation response, PD98059, a selective pharmacological inhibitor of the Erk1/2 kinase MEK, was used Pretreatment of TF-1 cells with PD98059 inhibited succinate-induced cell proliferation completely (Fig 4C) EPO-induced cell proliferation was also blocked by PD98059 pretreatment Succinate protects TF-1 cells from cell death induced by serum starvation The Erk MAPK pathway is known to stimulate prosurvival signaling under conditions of cellular stress Therefore, the protective effect of succinate was studied in a cell death assay TF-1 cells were induced to undergo apoptosis by extended incubation in serum-depleted culture medium Under these conditions, 47% of the cells were apoptotic, whereas cells cultured in complete growth medium had only 2% apoptotic cells EPO and succinate treatments decreased the percentage of apoptotic cells to 12–5%, respectively (Fig 5) The protec4 Journal of Leukocyte Biology Volume 85, May 2009 tive effect of succinate was ablated by preincubation of cells with PTX, and the EPO response was relatively unaffected Succinate enhances multilineage blood cell recovery upon chemotherapy-induced myelosuppression Given the proliferation and survival responses observed in vitro, the capacity of succinate to stimulate blood cell development in vivo was assessed using a mouse model of chemotherapy-induced myelosuppression This model, which uses busulfan as the chemotherapeutic agent that causes depletion of blood cells, allows for the assessment of agents that stimulate blood cell development or that display HPC chemoprotective properties Blood samples were analyzed 17 days after the initial chemotherapy injection At this point, in the blood cell recovery cycle, hemoglobin, platelet, neutrophil, and lymphocyte levels were depressed significantly in busulfan-treated mice relative to the levels in control mice Administration of the hematopoietic growth factor IL-11 following chemotherapy increased hemoglobin and platelet levels significantly by Day 17 (Fig 6) Succinate treatment for days following chemotherapy stimulated the Day 17 levels of hemoglobin (15.0Ϯ0.3 g/dL vs 13.6Ϯ0.2 g/dL in vehicle), platelets (248Ϯ22ϫ103/␮l vs 147Ϯ15ϫ103/␮l in vehicle), and neutrophils (0.43Ϯ0.08 ϫ103/␮l vs 0.20Ϯ0.04ϫ103/␮l in vehicle) significantly Lymphocyte levels were unaffected by succinate treatment DISCUSSION The recent finding that the citric acid cycle intermediate succinate is a ligand for GPR91 provides additional means by http://www.jleukbio.org Fig Activation of Erk1/2 by succinate mediates the TF-1 cell proliferation response (A) TF-1 cells were cultured in serum-deprived medium in the presence or absence of PTX Cells were then stimulated with succinate or EPO for 10 Erk1/2 activation was monitored using a p-Erk ELISA assay Erk1/2 activation is normalized to total Erk1/2 protein levels and fold activation calculated as a ratio to the vehicle control Data are the mean and SE of three replicate measurements *, P Ͻ 0.05; **, P Ͻ 0.001, compound versus vehicle-treated; †, P Ͻ 0.05; ††, P Ͻ 0.01; ‡, P Ͻ 0.001, PTX versus non-PTX-treated succinate group using the Student-Newman-Keuls test (B) TF-1 cells were cultured in inositol-free medium containing 3H-myoinositol in the presence or absence of PTX Cells were then washed and treated with succinate for h and lysed IP accumulation was measured using a liquid scintillation counter Data are the mean and SE of three replicate measurements (C) TF-1 cells were serum-deprived and subsequently treated with the MEK inhibitor PD98059 Cells were then treated with EPO or succinate for 24 h and cell proliferation assessed using a MTT colorimetric assay with an absorbance (O.D.) measurement at 490 nm Data are the mean and SE of three replicate measurements **, P Ͻ 0.001, compound versus vehicle-treated; ‡, P Ͻ 0.001, PTX versus non-PTX-treated compound group using the StudentNewman-Keuls test which to elucidate the cellular functions of the receptor Here, we have examined the role of succinate GPR91 signaling in HPC development Interrogation of GPR91 mRNA expression in a diverse panel of cell and tissue types indicated that the receptor is enriched in hematopoietic progenitor samples GPR91 was found to be expressed in isolated bone marrow CD34ϩ cells, erythroid and megakaryocyte cell cultures, and the erythroleukemic cell line TF-1 The receptor was addition- ally found to be expressed in bone marrow stromal cells, cell types that are constituents of the stromal niche [30] It is therefore possible that GPR91 also regulates stromal niche biology, as has been suggested for other GPCRs [31, 32] Investigation of succinate GPR91 signaling in HPCs revealed a functional role in cellular growth Treatment with succinate stimulated the proliferation of erythroid and megakaryocyte progenitor cell cultures A similar response was observed in TF-1 cells, an erythroleukemic cell line that maintains hematopoietic, progenitor-like properties Stimulation of TF-1 cell proliferation by succinate was sensitive to PTX pretreatment and was reduced when GPR91 expression was down-regulated using siRNA Erk1/2 was activated in TF-1 cells treated with succinate in a PTX-sensitive manner, and pharmacological inhibition of MEK blocked succinateinduced cell proliferation These results indicate that GPR91 mediates the proliferation response induced by succinate through Gi pathway activation of Erk1/2 Succinate was observed additionally to protect TF-1 cells from apoptosis induced by serum deprivation This effect too was PTX-sensitive, suggesting that Gi signaling also mediates this response In a previous study, He et al [12] had shown that GPR91 induced IP accumulation, calcium mobilization, and MAPK activation in Chinese hamster ovary and HEK 293 cell lines overexpressing the receptor Activation of these pathways was only inhibited partially by pretreatment with PTX These results suggest that recombinant GPR91 is able to activate Gi and Gq pathways This is in contrast to our findings with an endogenously expressed receptor, in which Gi coupling was observed exclusively in functional and signaling assays As recombinant systems are known to allow for promiscuous Gprotein coupling, the results presented here may be more reflective of natural GPR91 signaling Our observation that Erk1/2 activation is dependent on Gi signaling also suggests a possible role for the G-␤ ␥ subunit in GPR91-mediated responses [33–36] The hematopoietic response to succinate was also investigated in vivo Succinate administration was found to stimulate multilineage blood cell recovery after chemotherapy treatment Fig Succinate inhibits cell death in serum-deprived TF-1 cells, which were cultured in serum-deprived medium in the presence or absence of PTX Cells were then treated with U/ml EPO or 0.5 mM succinate for 48 h Cells were analyzed for 7-AAD staining using FACS as a measure of cell death Analysis of cells cultured in complete growth medium is also shown Results are representative of two independent studies Hakak et al Succinate-mediated hematopoiesis Fig Succinate stimulates multilineage blood cell recovery from chemotherapy-induced myelosuppression Balb/C mice were treated with the chemotherapy drug busulfan followed by saline, succinate (30 mg/kg), or IL-11 (500 ug/kg) administration for days Orbital bleeds were collected 17 days after the initiation of the study and analyzed for the levels of (A) hemoglobin (Hg), (B) platelets, (C) neutrophils, and (D) lymphocytes Data are the mean and SE using eight animals/group *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001, versus vehicle-treated using the Student-NewmanKeuls test of mice Interestingly, this effect was restricted to cell types derived from the myeloid lineage, without affecting the lymphoid lineage The mechanism by which succinate stimulates hematopoiesis in vivo is unclear Given that GPR91 is expressed on human myeloid precursor cells, the increase in blood cell levels induced by succinate in mice may occur through direct GPR91-mediated stimulation of those cells Verification of GPR91 expression on mouse HPCs would support this notion However, limitations, such as the lack of an available mouse GPR91-specific antibody applicable for FACS analysis and the minute number of HPCs that can be collected from mice, make such efforts technically challenging Succinate could likewise stimulate supportive stromal cells, also expressing GPR91 In addition, succinate-mediated GPR91 activation in the kidney has been shown to stimulate the renin-angiotensin system in vivo [12, 37], thereby resulting in elevated levels of circulating angiotensin II In vitro, angiotensin II can stimulate HPC colony formation [23], and in a mouse model of chemotherapy-induced myelosuppression, it can enhance blood cell levels [38] It is also possible that succinate administration increases circulating levels of EPO, although it is generally thought that Gs-coupled receptors enhance EPO synthesis in the kidney [39] However, succinate may stimulate EPO synthesis independently of GPR91 by the stabilization of HIF-1␣ [1, 2] This transcription factor induces the expression of target genes, including EPO, in response to hypoxic conditions [40, 41] In conclusion, we report that the GPR91 ligand succinate can stimulate HPC growth in vitro and blood cell development in vivo under conditions of myelosuppression Future studies should elucidate the mechanisms involved in this process It is noteworthy that low oxygen tension conditions, which are believed to exist within the bone marrow compartment [42], have Journal of Leukocyte Biology Volume 85, May 2009 been shown to stimulate HPC growth and differentiation in vitro [43– 46] As extracellular succinate accumulates under hypoxic conditions [4, 5, 7, 8], it is interesting to speculate that succinate GPR91 signaling may in part mediate the stimulatory effects of low oxygen content It would therefore also be of interest to investigate whether oxygenation regulates the levels of succinate within the bone marrow or in cultured HPCs Information from these studies would further our understanding of the significance of succinate GPR91 signaling in HPC biology ACKNOWLEDGMENTS We thank Dana Buckman for FACS analysis support and Ling Liu and Kevin Creehan for in vivo support REFERENCES Koivunen, P., Hirsila, M., Remes, A M., Hassinen, I E., Kivirikko, K I., Myllyharju, J (2007) Inhibition of hypoxia-inducible factor (HIF) hydroxylases by citric acid cycle intermediates: possible links between cell metabolism and stabilization of HIF J Biol Chem 282, 4524 – 4532 Selak, M A., Armour, S M., MacKenzie, E D., Boulahbel, H., Watson, D G., Mansfield, K D., Pan, Y., Simon, M C., Thompson, C B., Gottlieb, E (2005) Succinate links TCA cycle dysfunction to oncogenesis by inhibiting HIF-␣ prolyl hydroxylase Cancer Cell 7, 77– 85 Kushnir, M M., Komaromy-Hiller, G., Shushan, B., Urry, F M., Roberts, W L (2001) Analysis of dicarboxylic acids by tandem mass spectrometry High-throughput quantitative measurement of methylmalonic acid in serum, plasma, and urine Clin Chem 47, 1993–2002 Correa, P R., Kruglov, E A., Thompson, M., Leite, M F., Dranoff, J A., Nathanson, M H (2007) Succinate is a paracrine signal for liver damage J Hepatol 47, 262–269 Hochachka, P W., Dressendorfer, R H (1976) Succinate accumulation in man during exercise Eur J Appl Physiol Occup Physiol 35, 235–242 http://www.jleukbio.org Sadagopan, N., Li, W., Roberds, S L., Major, T., Preston, G M., Yu, Y., Tones, M A (2007) Circulating succinate is elevated in rodent models of hypertension and metabolic disease Am J Hypertens 20, 1209 –1215 Sanborn, T., Gavin, W., Berkowitz, S., Perille, T., Lesch, M (1979) Augmented conversion of aspartate and glutamate to succinate during anoxia in rabbit heart Am J Physiol 237, H535–H541 Taegtmeyer, H (1978) Metabolic responses to cardiac hypoxia Increased production of succinate by rabbit papillary muscles Circ Res 43, 808 – 815 Gullans, S R., Kone, B C., Avison, M J., Giebisch, G (1988) Succinate alters respiration, membrane potential, and intracellular Kϩ in proximal tubule Am J Physiol 255, F1170 –F1177 10 Gullans, S R., Brazy, P C., Dennis, V W., Mandel, L J (1984) Interactions between gluconeogenesis and sodium transport in rabbit proximal tubule Am J Physiol 246, F859 –F869 11 Huang, E M., McGowan, E B., Detwiler, T C (1984) Succinate potentiates the action of platelet agonists Thromb Res 36, 1– 12 He, W., Miao, F J., Lin, D C., Schwandner, R T., Wang, Z., Gao, J., Chen, J L., Tian, H., Ling, L (2004) Citric acid cycle intermediates as ligands for orphan G-protein-coupled receptors Nature 429, 188 –193 13 Sapieha, P., Sirinyan, M., Hamel, D., Zaniolo, K., Joyal, J S., Cho, J H., Honore, J C., Kermorvant-Duchemin, E., Varma, D R., Tremblay, S., Leduc, M., Rihakova, L., Hardy, P., Klein, W H., Mu, X., Mamer, O., Lachapelle, P., Di Polo, A., Beause´jour, C., Andelfinger, G., Mitchell, G., Sennlaub, F., Chemtob, S (2008) The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis Nat Med 14, 1067– 1076 14 Rubic, T., Lametschwandtner, G., Jost, S., Hinteregger, S., Kund, J., Carballido-Perrig, N., Schwarzler, C., Junt, T., Voshol, H., Meingassner, J G., Mao, X., Werner, G., Rot, A., Carballido, J M (2008) Triggering the succinate receptor GPR91 on dendritic cells enhances immunity Nat Immunol 9, 1261–1269 15 Wilson, A., Trumpp, A (2006) Bone-marrow hematopoietic-stem-cell niches Nat Rev Immunol 6, 93–106 16 Smithgall, T E (1998) Signal transduction pathways regulating hematopoietic differentiation Pharmacol Rev 50, 1–19 17 Klingmuller, U (1997) The role of tyrosine phosphorylation in proliferation and maturation of erythroid progenitor cells—signals emanating from the erythropoietin receptor Eur J Biochem 249, 637– 647 18 Hara, T., Miyajima, A (1996) Function and signal transduction mediated by the interleukin receptor system in hematopoiesis Stem Cells 14, 605– 618 19 Carroll, M P., Spivak, J L., McMahon, M., Weich, N., Rapp, U R., May, W S (1991) Erythropoietin induces Raf-1 activation and Raf-1 is required for erythropoietin-mediated proliferation J Biol Chem 266, 14964 –14969 20 de Groot, R P., Coffer, P J., Koenderman, L (1998) Regulation of proliferation, differentiation and survival by the IL-3/IL-5/GM-CSF receptor family Cell Signal 10, 619 – 628 21 Ren, H Y., Komatsu, N., Shimizu, R., Okada, K., Miura, Y (1994) Erythropoietin induces tyrosine phosphorylation and activation of phospholipase C-␥ in a human erythropoietin-dependent cell line J Biol Chem 269, 19633–19638 22 Miller, B A., Cheung, J Y (1994) Mechanisms of erythropoietin signal transduction: involvement of calcium channels Proc Soc Exp Biol Med 206, 263–267 23 Rodgers, K E., Xiong, S., Steer, R., diZerega, G S (2000) Effect of angiotensin II on hematopoietic progenitor cell proliferation Stem Cells 18, 287–294 24 Tao, H., Gaudry, L., Rice, A., Chong, B (1999) Cord blood is better than bone marrow for generating megakaryocytic progenitor cells Exp Hematol 27, 293–301 25 Schattner, M., Lefebvre, P., Mingolelli, S S., Goolsby, C L., Rademaker, A., White, J G., Foster, D., Green, D., Cohen, I (1996) Thrombopoietinstimulated ex vivo expansion of human bone marrow megakaryocytes Stem Cells 14, 207–214 26 Chengalvala, M., Kostek, B., Frail, D E (1999) A multi-well filtration assay for quantitation of inositol phosphates in biological samples J Biochem Biophys Methods 38, 163–170 27 Schmid, I., Krall, W J., Uittenbogaart, C H., Braun, J., Giorgi, J V (1992) Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry Cytometry 13, 204 –208 28 Kitamura, T., Tange, T., Terasawa, T., Chiba, S., Kuwaki, T., Miyagawa, K., Piao, Y F., Miyazono, K., Urabe, A., Takaku, F (1989) Establishment and characterization of a unique human cell line that proliferates dependently on GM-CSF, IL-3, or erythropoietin J Cell Physiol 140, 323– 334 29 Murate, T., Saga, S., Hotta, T., Asano, H., Ito, T., Kato, K., Tsushita, K., Kinoshita, T., Ichikawa, A., Yoshida, S (1993) The close relationship between DNA replication and the selection of differentiation lineages of human erythroleukemia cell lines K562, HEL, and TF1 into either erythroid or megakaryocytic lineages Exp Cell Res 208, 35– 43 30 Mayani, H., Guilbert, L J., Janowska-Wieczorek, A (1992) Biology of the hemopoietic microenvironment Eur J Haematol 49, 225–233 31 Richmond, R S., Tallant, E A., Gallagher, P E., Ferrario, C M., Strawn, W B (2004) Angiotensin II stimulates arachidonic acid release from bone marrow stromal cells J Renin Angiotensin Aldosterone Syst 5, 176 –182 32 Manske, J M., Sullivan, E L., Andersen, S M (1995) Substance P mediated stimulation of cytokine levels in cultured murine bone marrow stromal cells Adv Exp Med Biol 383, 53– 64 33 Crespo, P., Xu, N., Simonds, W F., Gutkind, J S (1994) Ras-dependent activation of MAP kinase pathway mediated by G-protein ␤ ␥ subunits Nature 369, 418 – 420 34 Koch, W J., Hawes, B E., Allen, L F., Lefkowitz, R J (1994) Direct evidence that Gi-coupled receptor stimulation of mitogen-activated protein kinase is mediated by G ␤ ␥ activation of p21ras Proc Natl Acad Sci USA 91, 12706 –12710 35 Faure, M., Voyno-Yasenetskaya, T A., Bourne, H R (1994) cAMP and ␤ ␥ subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells J Biol Chem 269, 7851–7854 36 Cabrera-Vera, T M., Vanhauwe, J., Thomas, T O., Medkova, M., Preininger, A., Mazzoni, M R., Hamm, H E (2003) Insights into G protein structure, function, and regulation Endocr Rev 24, 765–781 37 Toma, I., Kang, J J., Sipos, A., Vargas, S., Bansal, E., Hanner, F., Meer, E., Peti-Peterdi, J (2008) Succinate receptor GPR91 provides a direct link between high glucose levels and renin release in murine and rabbit kidney J Clin Invest 118, 2526 –2534 38 Rodgers, K E., Xiong, S., diZerega, G S (2002) Accelerated recovery from irradiation injury by angiotensin peptides Cancer Chemother Pharmacol 49, 403– 411 39 Fisher, J W., Nakashima, J (1992) The role of hypoxia in renal production of erythropoietin Cancer 70, 928 –939 40 Fisher, J W (2003) Erythropoietin: physiology and pharmacology update Exp Biol Med (Maywood) 228, 1–14 41 Ratcliffe, P J., O’Rourke, J F., Maxwell, P H., Pugh, C W (1998) Oxygen sensing, hypoxia-inducible factor-1 and the regulation of mammalian gene expression J Exp Biol 201, 1153–1162 42 Chow, D C., Wenning, L A., Miller, W M., Papoutsakis, E T (2001) Modeling pO(2) distributions in the bone marrow hematopoietic compartment I Krogh’s model Biophys J 81, 675– 684 43 Bradley, T R., Hodgson, G S., Rosendaal, M (1978) The effect of oxygen tension on hemopoietic and fibroblast cell proliferation in vitro J Cell Physiol 97, 517–522 44 Katahira, J., Mizoguchi, H (1987) Improvement of culture conditions for human megakaryocytic and pluripotent progenitor cells by low oxygen tension Int J Cell Cloning 5, 412– 420 45 LaIuppa, J A., Papoutsakis, E T., Miller, W M (1998) Oxygen tension alters the effects of cytokines on the megakaryocyte, erythrocyte, and granulocyte lineages Exp Hematol 26, 835– 843 46 Mostafa, S S., Miller, W M., Papoutsakis, E T (2000) Oxygen tension influences the differentiation, maturation and apoptosis of human megakaryocytes Br J Haematol 111, 879 – 889 Hakak et al Succinate-mediated hematopoiesis ... elucidate the cellular functions of the receptor Here, we have examined the role of succinate GPR91 signaling in HPC development Interrogation of GPR91 mRNA expression in a diverse panel of cell... addition, succinate- mediated GPR91 activation in the kidney has been shown to stimulate the renin-angiotensin system in vivo [12, 37], thereby resulting in elevated levels of circulating angiotensin... of low oxygen content It would therefore also be of interest to investigate whether oxygenation regulates the levels of succinate within the bone marrow or in cultured HPCs Information from these