Chemerin neutralization blocks hematopoietic ... - Stem Cells Journals

TISSUE-SPECIFIC STEM CELLS Chemerin Neutralization Blocks Hematopoietic Stem Cell Osteoclastogenesis SHANMUGAM MURUGANANDAN, HELEN J. DRANSE, JILLIAN L. ROURKE, NICHOLE M. MCMULLEN, CHRISTOPHER J. SINAL Department of Pharmacology, Faculty of Medicine, Dalhousie University, Halifax, Nova Scotia, Canada Key Words. Hematopoietic stem cell • Osteoclast • Chemerin • NFAT2 • CMKLR1 • Antibody

ABSTRACT Bone is a dynamic tissue that is continuously remodeled through the action of formative osteoblasts and resorptive osteoclasts. Chemerin is a secreted protein that activates chemokine-like receptor 1 (CMKLR1), a G proteincoupled receptor expressed by various cell types including adipocytes, osteoblasts, mesenchymal stem cells (MSCs), and macrophages. Previously, we identified chemerin as a regulator of adipocyte and osteoblast differentiation of MSCs. Herein we examined the role of chemerin in Lin2 Sca11 c-kit1 CD341 hematopoietic stem cell (HSC) osteoclastogenesis. We found that HSCs expressed both chemerin and CMKLR1 mRNA and secreted chemerin protein into the extracellular media. Neutralization of chemerin with a blocking antibody beginning prior to inducing osteoclast differentiation resulted in a near complete loss of osteoclastogenesis as evidenced by reduced marker gene expression and matrix resorption. This effect was conserved in an independent model of RAW264.7 cell

osteoclastogenesis. Reintroduction of chemerin by reversal of neutralization rescued osteoclast differentiation indicating that chemerin signaling is essential to permit HSC differentiation into osteoclasts but following blockade the cells maintained the potential to differentiate into osteoclasts. Mechanistically, neutralization of chemerin blunted the early receptor activator of nuclear factor-kappa B ligand induction of nuclear factor of activated T-cells 2 (NFAT2), Fos, Itgb3, and Src associated with preosteoclast formation. Consistent with a central role for NFAT2, induction or activation of NFAT2 by forced expression or stimulation of intracellular calcium release rescued the impairment of HSC osteoclastogenesis caused by chemerin neutralization. Taken together, these data support a novel autocrine/paracrine role for chemerin in regulating osteoclast differentiation of HSCs through modulating intracellular calcium and NFAT2 expression/activation. STEM CELLS 2013;31:2172–2182

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Constant remodeling of bone is essential for skeletal integrity as well as other vital aspects of physiology such as calcium and vitamin D homeostasis [1–3]. Osteoporosis and other bone loss disorders typically result from a dysregulation of bone turnover such that bone resorption exceeds bone formation leading to a deterioration of bone microarchitecture and reduction of bone mass and volume [4]. Loss of the normal balance of bone formation/resorption is most commonly associated with aging [5], hormonal changes in postmenopausal women [6], and pathogenic disorders such as diabetes mellitus [7]. Given that osteoporotic fractures are common (>9 million per year globally) and inflict a substantial economic burden in terms of lost productivity and health care expenditures [8], there is a present and growing need for more effective therapeutics to treat disorders of bone loss. In healthy individuals, coordinated development of bone forming osteoblasts and bone resorbing osteoclasts governs a homeostatic balance that ultimately maintains a constant bone

mass and volume [2]. Osteoblasts and osteoclasts originate from distinct stem cell types within the bone marrow—mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs), respectively [9–11]. Osteoblast differentiation of MSCs is orchestrated by a complex signaling cascade involving multiple pathways including the bone morphogenic proteins and winglesstype MMTV integration site (Wnt) proteins [10, 12]. Osteoclast differentiation of HSCs is largely under the control of immune modulators such as macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL; also known as TNFSF11), a tumor-necrosis factor (TNF)-family cytokine [13–15]. As disorders of bone loss are commonly associated with a relative increase in bone resorption, the potential to treat bone loss by inhibiting osteoclastogenesis has promise [16]. Osteoclast differentiation proceeds in a two stage differentiation process involving (a) induction of the FBJ osteosarcoma oncogene (Fos), nuclear factor of activated T-cells 2 (NFAT2 also called NFATc1 or NFATc) and nuclear factorkappa B (NFjB) resulting in preosteoclast formation [17, 18]

Author contributions: S.M., H.D., and J.R.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, and final approval of manuscript; N.M.: collection and/or assembly of data, data analysis and interpretation, and final approval of manuscript; C.S.: conception and design, data analysis and interpretation, manuscript writing, and final approval of manuscript. Correspondence: Christopher J. Sinal, Ph.D., Department of Pharmacology, Dalhousie University, 5850 College Street, Box 15000, Halifax, Nova Scotia, Canada B3H4R2. Telephone: 902-494-2347; e-mail: csinal@dal.ca Received December 20, 2012; accepted for C AlphaMed Press 1066-5099/2013/$30.00/0 doi: publication May 15, 2013; first published online in STEM CELLS EXPRESS June 14, 2013. V 10.1002/stem.1450

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Muruganandan, Dranse, Rourke et al.

and (b) osteoclast-specific robust induction of NFAT2 resulting in terminal differentiation [18, 19]. Although a complex network of signaling events are reported to mediate these two phases [17–19], the link between the early signaling pathways activated by RANKL and the NFAT2-induced terminal osteoclast differentiation is not fully established. Chemerin is a small (18 kDa) secreted protein that modulates inflammation, immune function, tumor progression, and recruitment of inflammatory cells through its interaction with chemokine-like receptor 1 (CMKLR1) [20–25]. Chemerin also serves as a ligand for two other receptors, G protein-coupled receptor 1 [26] and chemokine (C-C motif) receptor-like 2 (CCRL2) [27]. While CCRL2, the nonsignaling receptor for chemerin, can bind chemerin and increase local chemerin concentration to efficiently present it to CMKLR1 on nearby cells [28], all the biological effects ascribed to chemerin have been reported to be elicited through CMKLR1 activation [20–23]. In addition to the role in immune function, autocrine/paracrine activation of CMKLR1 by chemerin promotes adipogenesis of a variety of precursor cell types including 3T3-L1 fibroblasts and primary MSCs [29–31]. Given the importance of chemerin/CMKLR1 signaling to HSC-derived cells in the context of immune function, we hypothesized that this signaling pathway also regulates the differentiation of bone marrow HSCs to bone resorbing osteoclasts. Our experimental data provide evidence that supports a previously unrecognized autocrine/paracrine role for chemerin to regulate HSC differentiation and thereby, bone homeostasis.

MATERIALS

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METHODS

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the medium to neutralize extracellular chemerin activity. Goat IgG (catalog number AB-108-C, R&D Systems) was used as a control.

Osteoclast Differentiation and Detection of NFjB Activation in RAW264.7 Cells Osteoclast differentiation of monocyte RAW264.7 progenitors (American Type Culture Collection, Manassas, VA, www.atcc. org) was induced by 50 ng/ml RANKL. To monitor NFjB activity, the cells were stably transduced with the Cignal Lenti Reporter (luciferase) for NFjB (SABiosciences, Frederick, MD, www.sabiosciences.com). NFjB activity was assessed by measuring luciferase activity using a Luminoskan Ascent luminometer Luminoskan Ascent (Thermo Scientific, Waltham, MA, www.thermoscientific.com) and expressed as relative light units.

Osteoclast Detection and Characterization Osteoclasts were fixed and TRAP-stained using a leukocyte acid phosphatase kit (Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich. com) according to the manufacturer’s protocol. Images were captured using a Zeiss Axiovert 200 inverted microscope equipped with an AxioCam camera system (Zeiss Canada, Toronto, ON, Canada,www. corporate.zeiss.com). TRAP activity at 37 C was quantified with a substrate buffer (125 mg/ml Naphthol AS-BI phosphoric acid [Sigma] in diazotized fast garnet base chemical solution (Sigma-Aldrich) containing 6.7 mM sodium tartrate in 100 mM acetate buffer pH 5.2) at 560 nm and expressed as mOD/minute. Actin ring formation was visualized using tetramethylrhodamine-phalloidin (Life Technologies, Carlsbad, CA, www.invitrogen.com) staining for 1 hour. The cell nuclei were counterstained with 1 mg/ml Hoechst 33258 (SigmaAldrich) in phosphate buffered saline for 5 minute. The number of actin rings in each well was counted and expressed as actin rings per well. Image analysis of the total area of plate surface contained within actin rings was performed with Axiovision rel 4.8 (Zeiss Canada).

Animals All experiments were performed according to the guidelines of the Canadian Council on Animal Care and were approved by the Dalhousie University Committee on Laboratory Animals. The mice had free access to standard rodent chow and water, and were maintained at an ambient temperature of 20 C–22 C, relative humidity 18%–22%, and a 12 hours light/dark cycle.

Stem Cell Isolation and Differentiation Isolation, culture, and adipocyte or osteoblast differentiation of MSCs were performed as described previously [29, 30]. HSCs were isolated from mouse bone marrow using a modification of the method described by de Vries et al. [32]. Briefly, 2-month-old male C57BL/6J mice were euthanized with an overdose (90 mg/kg) of pentobarbital sodium (Ceva Sante Animale, LA Ballastiere, Libourne, France, http://www.ceva.com) and femurs and tibiae were removed. The marrow was flushed with HSC medium (aMEM containing 1% L-glutamine, 10% fetal bovine serum, 100 U/ ml penicillin, and 100 mg/ml streptomycin) and passed through a 23-gauge needle and filtered through a 70 mm pore size cell strainer (BD Biosciences, Bedford, MA, www.bdbiosciences. com). Lin2 Sca11 hematopoietic cells were isolated by sequential depletion of lineage positive (Lin1) cells (MagCellect Mouse Hematopoietic Cell Lineage Depletion Kit, R&D Systems) and isolation of Sca-1positive stem cells (Mouse Sca-11 Stem Cells kit, R&D Systems, Minneapolis, MN, www.rndsystems.com). The resulting Lin2 Sca11 cells were stained with 0.5 mg/ml fluorescein isothiocyanateconjugated rat anti-mouse c-kit (CD117) (BD Biosciences, catalog number 561680) and 10 mg/ml Alexa Fluor 647 (AF 647)-conjugated rat anti-mouse CD34 antibodies (BD Biosciences, catalog number 560230), and sorted by fluorescence-activated cell sorting to obtain Lin2 Sca11 c-kit1 CD341 pure HSCs. Osteoclast differentiation was induced by 30 ng/ml M-CSF (R&D Systems) and 50 ng/ml RANKL (R&D Systems) for 14 days. Undifferentiated control cells received M-CSF but no RANKL. Anti-chemerin antibody (CmAb) (catalog number AF2325, R&D Systems) was added to

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Matrix Resorption Assays HSCs were differentiated on calcified matrix using crystalline calcium phosphate coated polystyrene plates (Corning Osteo Assay Plate, Corning, NY, www.corning.com). After 14 days, cells were removed from the wells by washing with 10% bleach, followed by two washes in distilled water. The plates were air-dried and examined by light microscopy, in which resorption pits appear as light lacunae in the resorbed areas. Representative images were photographed under 103 magnification. The area of resorbed matrix was quantified using image analysis software (Axiovision rel 4.8, Zeiss Canada) and expressed as % resorbed area per well.

Western Blotting Cell lysates and media samples were separated by SDS-PAGE and transferred to nitrocellulose membranes as described previously [30]. Immunostaining was performed using goat anti-chemerin (1:1,000; catalog number AF2325, R&D Systems), goat anti-matrix metalloproteinase (MMP)29 (1:1,000; catalog number AF909, R&D Systems), rabbit anti-cathepsin K (1:500; catalog number ab19027, Abcam, Cambridge, MA, www.abcam.com), and mouse anti-b-actin (1:2,000; catalog number ab8226, Abcam). The appropriate secondary antibodies conjugated with a 680 nm or 800 nm infrared fluorophore (1:10,000, LI-COR Biosciences, Lincoln, NE, www.licor.com). The immunoreactive bands were detected by scanning at 700 nm or 800 nm, respectively, using a LI-COR Odyssey infrared scanner (LI-COR Biosciences).

Gene Expression Analysis Total RNA was isolated using an RNeasy Plus Minikit (Qiagen, Hilden, Germany, www.qiagen.com) according to the manufacturer’s instructions. Reverse transcription and quantitative PCR detection of genes were measured as described previously [29, 31, 33]. The Ct values for cyclophilin A were used to normalize the expression level of the gene of interest using the DDCt method [34]. Exon-spanning primers for quantitative real-time

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PCR (qPCR) were designed using the primerbank software algorithm (http://pga.mgh.harvard.edu/primerbank/) and are listed in supporting information Table S1.

Lentiviral Gene Transfer in Mouse HSCs Lentiviruses were generated according to previously established methods [35]. Briefly, HEK-293T/17 cells (ATCC CRL-11268) were transfected with lentiviral vectors, including psPAX2 (plasmid 12260, Addgene, Cambridge, MA, www.addgene.org) and pMD2.G (plasmid 12259, Addgene) along with the nonsilencing shRNA (Thermo Scientific GIPZ Lentiviral shRNAmir plasmid RHS_4346) a negative control that was verified to contain no homology to known mammalian genes, chemerin shRNA (Thermo Scientific GIPZ Lentiviral shRNAmir plasmid V2LMM_52383), or wild-type (WT) NFAT2 (plasmid 11101, Addgene), constitutively active (CA) mutant NFAT2 (plasmid 11102, Addgene), or green fluorescent protein (GFP) control (plasmid 11652, Addgene). Viral supernatants were collected at 24, 48, and 72 hours. The viruses for shRNA knockdown studies were purified by ViraBind Lentivirus Concentration and Purification kit (Cell Biolabs, San Diego, CA, www.cellbio labs.com). Viral titer was determined according to the procedure described for Open Biosystems Expression Arrest GIPZ Lentiviral shRNAmir (Thermo Scientific) using GFP as a marker for detection of transduced cells. Chemerin knockdown was assessed using chemerin expression plasmids in HEK-293T cell line as well as endogenous chemerin expression in HSCs. Transduction of mouse HSCs with the lentiviral shRNA was performed in HSC medium containing 5% fetal bovine serum, 10 ng/ml M-CSF, and 1024 M bmercaptoethanol. All transductions were carried out at a multiplicity of infection of 10 supplemented with 4 mg/ml protamine sulfate and 50 mM deoxynucleoside triphosphates for 96 hours and the transduction medium was changed every 24 hours. Lentiviruses generated for NFAT2 expression studies were pelleted by ultracentrifugation and viral titer was determined by quantitative PCR amplification. NFAT2 transduction ability of the viruses was assessed using a nonosteoclastogenic HEK-293A cell line.

Induction of Calcium Release To induce calcium release from intracellular stores, Thapsigargin (3 nM; catalog number T9033, Sigma-Aldrich) and Ionomycin (300 nM; catalog number I9657, Sigma-Aldrich) were used.

MTT Assay of Cellular Viability Cell viability after 8 days of treatment with CmAb was measured by a colorimetric 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) assay originally developed by Mosmann [36]. To measure cell viability, 100 ml MTT dye solution (0.5 mg/ml) was added to each well of a 96-well plate. The formazan product produced by viable cells was solubilized by the addition of 100 ml of dimethyl sulfoxide to the wells and the absorbance at 540 nm was determined. Absorbance values of control cells were considered as 100% cell viability.

Statistical Analyses Results are expressed as mean 6 SEM. Comparisons between values were performed using a Student’s t test (two-tailed) as in Figures 1, 6A. For comparison of multiple groups as in Figures 2–7, a one-way or two-way analysis of variance with Bonferroni post hoc analysis was used. For all statistical analyses, p < .05 was considered statistically significant.

RESULTS Chemerin/CMKLR1 Expression During HSC Osteoclast Differentiation Freshly isolated HSCs from mouse bone marrow, negative for markers of lineage commitment (Lin2) and positive for the

HSC markers [37, 38] Sca1 (Sca11), c-kit (c-kit1), and CD34 (CD341) expressed mRNA for both chemerin and the cognate receptor CMKLR1 at levels comparatively lower and higher, respectively, to bone marrow MSCs (supporting information Fig. S1). Following 14 days exposure to an osteoclastogenic medium containing M-CSF and RANKL, the Lin2 Sca11 ckit1 CD341 HSCs exhibited robust osteoclast differentiation, as evidenced by the presence of phalloidin-stained actin rings (Fig. 1A), TRAP-stained osteoclasts (Fig. 1B) and significant TRAP secretion (Fig. 1C). Consistent with this, qPCR analysis revealed increased mRNA levels for various osteoclast marker genes including TRAP, MMP-9, MMP-10, cathepsin K (CatK), calcitonin receptor (CalR), nuclear factor of activated T-cells 2 (NFAT2) also called NFATc1 or NFATc, osteoclast-associated receptor (OSCAR), and TNF receptor-associated factor 6 (TRAF6) (Fig. 1D). In contrast to the osteoclast marker genes, chemerin mRNA levels (Fig. 1E) were not significantly changed while CMKLR1 mRNA levels (Fig. 1F) decreased with osteoclast differentiation. Compared to other bone marrow cell types such as MSCs, adipocytes, or osteoblasts, the absolute amount of chemerin secreted by HSCs was lower (Fig. 1G). Moreover, in contrast to the differentiation dependent increase with MSC adipogenesis, chemerin protein secretion by HSCs did not increase with osteoclast differentiation (Fig. 1G).

Neutralization of Chemerin in HSCs To characterize the role of chemerin in the osteoclastogenic program of HSCs, we blocked chemerin/CMKLR1 signaling with a chemerin-neutralizing antibody (CmAb) beginning prior to application of osteoclastogenic stimulus (day 0) and persisting throughout 14 days of osteoclastogenic stimulation. Goat IgG was used as an isotype control for the goat antimouse chemerin-neutralizing antibody. Both vehicle (phosphate buffered saline) and IgG (50 mg/ml) treated cells exhibited robust osteoclast differentiation as evidenced by actin ring formation and the presence of large, TRAP positive osteoclasts (Fig. 2A). Neutralization of extracellular chemerin by CmAb markedly reduced actin ring formation and the presence of TRAP positive cells (Fig. 2A). Quantitative analysis verified 50% and 75% loss of actin ring number and area, respectively, with 25 mg/ml CmAb, while a higher concentration (50 mg/ml) caused >95% loss of both actin ring number and area (Fig. 2B). Consistent with this, TRAP secretion was reduced in a concentration-dependent manner with >50% loss at 25 mg/ml and >95% loss with 50 mg/ml CmAb compared to vehicle or IgG-treated cells (Fig. 2C). Both protein and mRNA levels for NFAT2, a critical transcription factor that regulates the expression of numerous osteoclastogenic genes [18], were markedly suppressed by chemerin neutralization (Fig. 2D, 2E). Moreover, chemerin neutralization resulted in a dramatic loss of MMP9 and CatK protein expression (Fig. 2D) and a concentration-dependent reduction of TRAP, MMP9, MMP10, CatK, CalR, OSCAR, and TRAF6 mRNA levels (Fig. 2E). To examine whether the loss of osteoclast differentiation would impact osteoclast-associated function, matrix resorption activity was measured. Direct or phasecontrast microscopic examination of the calcified matrix following 14 days of osteoclast differentiation revealed a near complete matrix loss in the wells treated with vehicle or IgG (50 mg/ml; Fig. 3A, 3B). In contrast, chemerin neutralization produced a marked decrease of matrix resorption with the highest concentration of 50 mg/ml CmAb reducing resorption activity to levels comparable to the undifferentiated M-CSF control (Fig. 3A, 3B). Quantitative analysis verified these qualitative observations and revealed >50% and >90% loss of matrix resorption with 25 and 50 mg/ml CmAb, respectively


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Figure 1. Modulation of Chemerin/CMKLR1 expression during HSC osteoclastogenesis. HSCs were induced to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand. Rhodamine-phalloidin staining of actin rings (A) and TRAP staining of osteoclasts (B) were used to detect osteoclastogenesis of Lin2 Sca11 c-kit1 CD341 HSCs. Kinetic analysis of extracellular TRAP activity during HSC osteoclastogenesis (C). qPCR analysis of mRNA levels for osteoclastogenic marker genes TRAP, MMP9, MMP10, CatK, CalR, NFAT2, OSCAR, and TRAF6 during HSC osteoclastogenesis (D). qPCR analysis of chemerin (E) and CMKLR1 (F) mRNA levels. To detect chemerin secretion, cell culture media were collected following 1-week (HSC, OCL, OBL) or 1-day (Adipo) of conditioning. Representative Western blot detection of chemerin protein secreted by HSCs, OCL, MSCs, adipocytes (Adipo), and OBL with b-actin as a loading control (G). All qPCR values are expressed relative to the mRNA levels measured on day 0. All values represent the mean 6 SEM, n 5 5. *, p < .05 versus undifferentiated. Abbreviations: CatK, cathepsin K; CalR, calcitonin receptor; HSC, hematopoietic stem cell; MSC, mesenchymal stem cell; OBL, osteoblasts; OCL, osteoclasts; OSCAR, osteoclast-associated receptor; TRAF6, TNF receptor-associated factor 6.

(Fig. 3C). Importantly, both the apparent potency and efficacy of CmAb with respect to inhibiting matrix resorption were very similar to that seen for other measures of osteoclast phenotype such as actin ring formation, TRAP secretion, and marker gene expression (Fig. 2). The mouse monocytic cell line, RAW 264.7, readily undergoes osteoclast differentiation upon exposure to RANKL and has been widely used to study the development and function of this cell type [39]. To determine whether the role for chemerin in osteoclastogenesis of primary HSCs was conserved in an independent model of osteoclast differentiation, chemerin neutralization was performed with RAW264.7 cells. RANKL treatment of RAW264.7 cells induced robust osteoclast differentiation, as evidenced by extensive actin ring formation (Fig. 4A, 4B) and TRAP secretion (Fig. 4C) under vehicle or IgG-treated condition. However, similar to HSCs, chemerin neutralization with 25 or 50 mg/ml CmAb markedly reduced actin ring formation (>50% loss with 25 mg/ml; >90% loss with 50 mg/ml) and TRAP secretion (>50% loss with 25 mg/ml; www.StemCells.com

>90% loss with 50 mg/ml) compared to vehicle treated cells (Fig. 4B, 4C). We developed lentiviral delivered shRNA to knockdown chemerin as a complementary experimental approach to study the role of this protein in HSC osteoclastogenesis. Transfection of the lentiviral plasmid achieved effective knockdown in HEK293T cells overexpressing chemerin mRNA (supporting information Fig. S2A). However, transduction with lentivirus particles expressing the same shRNA sequence (supporting information Fig. S2B) was less effective despite high transduction efficiency as assessed by a GFP marker (data not shown). Similarly, we were able to achieve only a partial knockdown (50%) of HSC chemerin mRNA or protein levels with lentivirus transduction (supporting information Fig. S2C, S2D). However, in agreement with the antibody neutralization studies, this incomplete knockdown resulted in a partial impairment of osteoclastogenesis as evidenced by reduced actin ring area and TRAP staining compared to vehicle and nonspecific shRNA expression (supporting information Fig. S2E). Moreover, while there was an overall trend toward reduced

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Figure 2. Neutralization of chemerin abrogates osteoclast differentiation of hematopoietic stem cells (HSCs). Lin2 Sca11 c-kit1 CD341 HSCs were induced to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand in the presence of phosphate buffered saline (Vehicle), IgG control, or anti-chemerin-neutralizing antibody (CmAb) for 14 days. Cell nuclei and actin ring formation were visualized using Hoechst (blue) and tetramethylrhodamine-phalloidin (red), respectively (A). TRAP staining was used as an additional approach to identify osteoclasts (A). Quantification of number and area of actin rings (B). Kinetic analysis of activity of osteoclast marker protein, TRAP (C). Representative Western blot detection of MMP9, CatK, and NFAT2 expression on day 14 osteoclast differentiation with b-actin as a loading control (D). Osteoclast marker genes, TRAP, MMP9, MMP10, CatK, CalR, NFAT2, OSCAR, and TRAF6 were measured using qPCR 14 days following initiation of osteoclast differentiation (E). All qPCR values are expressed relative to the mRNA levels measured in Veh control. All values represent the mean 6 SEM, n 5 5. *, p < .05 versus Veh. Abbreviations: CatK, cathepsin K; CalR, calcitonin receptor; OSCAR, osteoclast-associated receptor; TRAF6, TNF receptor-associated factor 6.

osteoclast marker gene expression, only MMP10 was reduced significantly (supporting information Fig. S2F).

Reversal of Chemerin Neutralization and Blockade of HSC Osteoclastogenesis In order to determine whether the inhibitory effect of chemerin neutralization on HSC osteoclastogenesis was reversible, after 7 days of blockade the differentiation medium containing CmAb was replaced with fresh differentiation medium containing IgG. Under this protocol, both actin ring number and area (Fig. 5A, 5B) as well as TRAP secretion (supporting information Fig. S3A) reached levels similar to that of IgG control and that was dramatically higher (50-fold) compared to cells that were continuously exposed to CmAb for 17 days (Fig. 5A). To assess the ability of chemerin to rescue the osteoclastogenic deficits induced by CmAb, the cells were exposed to a recombinant form of bioactive chemerin after 7 days antibody neutralization. Neither recombinant mouse chemerin (mCm) nor human chemerin (hCm) affected HSC osteoclastogenesis in the presence of IgG (Fig. 5C, 5E).

However, in the presence of CmAb both species of chemerin were able to increase actin ring formation (Fig. 5C--5F) and TRAP secretion (supporting information Fig. S3B) albeit not to levels observed in the absence of CmAb. Exposure of HSCs to CmAb at concentrations up to 100 mg/ml caused no significant cell toxicity (supporting information Fig. S4) indicating that the loss of osteoclastogenesis was not a consequence of cell death and that the recovery of osteoclast formation after removal of CmAb was a consequence of recruitment of viable multipotent HSCs to the osteoclastogenic program.

Regulation of Osteoclastogenesis by Chemerin Through NFAT2 The marked loss of NFAT2 expression with antibody neutralization suggested that modulation of the expression and/or activity of this transcription factor was relevant to the role of chemerin to osteoclastogenesis. However, it was not clear whether the defect in osteoclast differentiation with chemerin neutralization was mediated through a loss of NFAT2


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Figure 3. Neutralization of chemerin abrogates resorption of mineralized matrix. Representative images of matrix resorption (A, B) on day 14 and quantification of resorbed area (C) induced by osteoclast differentiation of HSCs in the presence of phosphate buffered saline vehicle (Veh), IgG control, or 10–50 mg/ml anti-chemerin neutralizing antibody (CmAb). All values represent the mean 6 SEM, n 5 3. *, p < .05 versus Veh. Abbreviation: HSC, hematopoietic stem cell.

Figure 4. Neutralization of chemerin abrogates osteoclast differentiation of monocyte RAW264.7 progenitors. RAW264.7 cells were induced to differentiate into osteoclasts with receptor activator of nuclear factor-kappa B ligand in the presence of phosphate buffered saline vehicle (Vehicle), IgG control, or anti-chemerin neutralizing antibody (CmAb) for 14 days. Cell nuclei and actin ring formation were visualized using Hoechst (blue) and tetramethylrhodamine-phalloidin (red), respectively (A). Quantification of number and area of actin rings (B). Kinetic analysis of activity of osteoclast marker protein, TRAP (C). All values represent the mean 6 SEM, n 5 5. *, p < .05 versus Veh.

expression or whether the loss of NFAT2 was a consequence of effects on later stages of differentiation. To investigate this, we assessed the RANKL-induced changes in early-stage gene expression. Following 72 hours of RANKL stimulation, an 8–10-fold induction of mRNA levels of NFAT2 and its www.StemCells.com

transcriptional partner, Fos, was observed (Fig. 6A). Moreover, NFAT2 target genes including integrin beta 3 (Itgb3) [40] and Src-kinase (Src) [41] were induced in HSCs within 72 hours of RANKL treatment (Fig. 6A). Consistent with the association of CMKLR1 with the early osteoclastogenic

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Figure 5. Reversal of chemerin neutralization restores osteoclastogenesis in Lin2 Sca11 c-kit1 CD341 hematopoietic stem cells (HSCs). Lin2 Sca11 c-kit1 CD341 HSCs were induced to differentiate into osteoclasts with macrophage colony-stimulating factor and receptor activator of nuclear factor-kappa B ligand for 17 days. Chemerin was neutralized using the anti-chemerin antibody (CmAb) at a final concentration of 50 mg/ ml beginning from day 0 and continuing to day 17 or until day 7 to reverse the neutralization effects from day 8 to 17 (A, B). Chemerin was restored in Lin2 Sca11 cells without the removal of CmAb by adding excessive amounts of mouse chemerin (mCm 5–10 mg/ml) (C, D) or small amounts of CmAb-insensitive human chemerin (hCm 0.05–0.1 mg/ml) (E, F) in the presence of CmAb from day 8 onward. Cell nuclei and actin ring formation were visualized using Hoechst (blue) and tetramethylrhodamine-phalloidin (red), respectively, at the end of 17 days (A, C, E). Quantification of number and area of actin rings (B, D, F). All values represent the mean 6 SEM, n 5 5. *, p < .05 versus IgG control day 0–17.

events, a loss of CMKLR1 was coincidental with these changes in the 72 hours period. NFjB is another important transcription factor that plays a pivotal role in osteoclast differentiation by activating NFAT2 and Fos [42–45]. RANKL increased NFjB luciferase reporter activity >20-fold within 24 hours after stimulation (Fig. 6A). To test whether chemerin mediated the RANKL-induced early-stage gene expression, we neutralized chemerin and assessed mRNA expression in the presence of RANKL for 72 hours. The CmAb markedly abrogated (>75% loss) the RANKLinduced mRNA expression of NFAT2, Fos, Itgb3, and Src in a dose-dependent manner (Fig. 6B). Moreover, the RANKLinduced loss of CMKLR1 expression was significantly reversed by CmAb (Fig. 6B). In contrast, chemerin neutralization did not significantly affect RANKL-induced NFjB reporter activation (Fig. 6B). To determine whether reduced NFAT2 expression was a major factor underlying the inhibition of osteoclastogenesis observed with chemerin neutralization, we tested whether NFAT2 expression alone was sufficient to rescue osteoclast differentiation of HSCs. Forced expression of WT-NFAT2 coincident with chemerin neutralization produced a partial rescue of expression of osteoclast markers such as MMP9 and CatK while the CA form restored the expression of these markers to levels similar in magnitude to that for phosphate buffered saline or IgG control (Fig. 6C). Consistent with this, CA-NFAT2 expression significantly restored actin ring formation (>75%) but

the WT failed (50% rescue for WT; total rescue for CA; Fig. 6F). The levels of NFAT2 protein were apparently higher with CA- versus WT-NFAT2 rescue (Fig. 6C). This was likely a consequence of a greater feed-forward amplification of endogenous NFAT2 expression provoked by CA-NFAT2 rather than differences in the inherent expression of lentivirus expressed WT-NFAT2 and CA-NFAT2 transgenes. In support of this, transduction of the nonosteoclastogenic 293A cell line resulted in similar levels of NFAT2 expression with both the WT-NFAT2 and CA-NFAT2 transgenes (supporting information Fig. S5). Intracellular calcium levels are an important factor governing the nuclear translocation of NFAT2 during osteoclastogenesis [46]. To determine whether changes in intracellular calcium contributed to the effects of chemerin neutralization on NFAT2 expression and osteoclastogenesis, we induced intracellular calcium release using the Ca21-ATPase inhibitor (thapsigargin) or the Ca21-ionophore (ionomycin). Stimulation of intracellular calcium release by either compound partially rescued (25%–50%) actin ring formation (Fig. 7A, 7B) as well as NFAT2, MMP9, and CatK protein expression (Fig. 7C) in the face of persistent chemerin neutralization. However, the rescue by calcium release was generally lower in magnitude compared to the rescue achieved with NFAT2


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Figure 6. Chemerin promotes NFAT2 expression during an early phase of osteoclast differentiation. Hematopoietic stem cells (HSCs) were treated with PBS or RANKL for a short period of 72 hours. mRNA levels of early phase osteoclast lineage genes, NFAT2, Fos, Itgb3, and Src along with CMKLR1 were measured using qPCR (A). NFjB activation was measured at 24 hours by a luciferase reporter assay (A). The early phase osteoclast lineage genes, NFAT2, Fos, Itgb3, and Src, and CMKLR1 mRNA levels and NFjB activation induced by RANKL were also assessed in the presence of PBS, IgG, or anti-chemerin neutralizing antibody (CmAb) at the indicated concentrations ranging from 10 to 100 mg/ ml (B). For NFAT2 rescue experiments, prior to initiating osteoclast differentiation, HSCs were transduced with GFP, WT-NFAT2-, or CANFAT2-overexpressing lentivirus. Representative Western blot detection of NFAT2, MMP9, and CatK expression after 14 days of osteoclast differentiation in the presence of 50 mg/ml IgG or CmAb with b-actin as a loading control (C). At the end of 14 days of osteoclast differentiation, cell nuclei and actin ring formation were visualized using Hoechst (blue) and tetramethylrhodamine-phalloidin (red), respectively, and the number and area of actin rings were measured (D, E). Kinetic analysis of activity of osteoclast marker protein, TRAP (F). All values represent the mean6 SEM, n 5 5. *, p < .05 versus PBS (A, B); versus PBS 1 GFP (E, F). #, p < .05 versus CmAb1GFP. Abbreviations: GFP, green fluorescent protein; NFAT2, nuclear factor of activated T cell; NFjB, nuclear factor-kappa B; PBS, phosphate buffered saline; RANKL, receptor activator of nuclear factor-kappa B ligand.

overexpression on MMP9, CatK, and NFAT2 expression (Fig. 7C). Taken together, these data indicate that chemerin regulates osteoclastogenesis primarily through modulating NFAT2 expression and activity, likely in part by affecting intracellular calcium levels.

DISCUSSION Our previous studies identified the contribution of chemerin/ CMKLR1 signaling as a determinant of the developmental fate of bone marrow-derived MSCs to the adipocyte and osteoblast lineage. In this study, we have identified a further role for this signaling pathway in regulating the osteoclastogenic differentiation of Lin2 Sca11 c-kit1 CD341 HSCs and thus revealed an expanded influence in regulating bone homeostasis. Similar to our previous findings [29, 30] that MSCs www.StemCells.com

are both a source and target of chemerin, we found that HSCsecreted chemerin also functions in an autocrine/paracrine fashion to promote osteoclastogenesis. Within the bone, crosstalk between osteoblast and osteoclast progenitors is essential for coupling of bone formation to bone resorption. One way this is achieved is through the paracrine actions of osteoblastsecreted RANKL to promote coordinated osteoclast differentiation of RANK receptor-expressing HSC progenitors [47, 48]. Following from this, chemerin secretion by other cell types within the bone marrow milieu may also influence HSC differentiation. For example, dynamic secretion of chemerin from MSCs and MSC-derived osteoblast may contribute to the homeostatic coupling of MSC osteoblastogenesis with HSC osteoclastogenesis through the paracrine actions of osteoblast-secreted chemerin on CMKLR1 receptor-expressing HSCs. Of all bone marrow cell types examined in this study, adipocytes secreted the highest amounts of chemerin. Many disorders of bone loss such as osteoporosis are characterized

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Figure 7. Thapsigargin- and ionomycin-induced calcium release partially rescues chemerin neutralization-induced loss of NFAT2 and osteoclastogenesis. Intracellular calcium release was induced by treatment with Thapsigargin (Thaps, 3 nM) or ionomycin (Iono, 300 nM). Cell nuclei and actin ring formation were visualized using Hoechst (blue) and tetramethylrhodamine-phalloidin (red) staining, respectively, 14 days following osteoclastogenic induction in the presence of 50 mg/ml IgG or CmAb (A). Quantification of number and area of actin rings (B). Representative Western blot detection of NFAT2, MMP9, and CatK expression 14 days following osteoclast differentiation in the presence of 50 mg/ml IgG or CmAb with b-actin as a loading control (C). All values represent the mean 6 SEM, n 5 5. *, p < .05 versus IgG. #, p < .05 versus CmAb. Abbreviations: CatK, cathepsin K; NFAT2, nuclear factor of activated T cell.

by both an increase in bone resorption and a preponderance of bone marrow adipocytes [4, 10]. Thus, given the proosteoclastogenic effect of chemerin/CMKLR1 toward HSCs, it is certainly possible that elevated levels of chemerin within the bone marrow milieu contribute to pathogenic disruptions of bone homeostasis that ultimately lead to the destruction of bone in osteoporotic and rheumatic diseases. It is generally believed that M-CSF and RANKL are both necessary and sufficient for osteoclast differentiation [14, 49– 52]. However, we have demonstrated that the autocrine/paracrine actions of chemerin are also essential for osteoclast differentiation of both HSCs and RAW264.7 cells. This was demonstrated most convincingly in the context of antibody neutralization of extracellular chemerin. When lentiviral shRNA-mediated knockdown of chemerin mRNA was used, the effects were qualitatively similar to that of antibody neutralization. The comparatively smaller impact of the latter approach on osteoclastogenesis was likely a consequence of the inability to achieve a substantial (>90%) loss of chemerin expression. Indeed, at lower antibody concentrations (e.g., 10 mg/ml) where only partial (30%–50%; data not shown) chemerin neutralization is achieved, incomplete inhibition of HSC osteoclastogenesis was observed (Figs. 2C, 3C, 4C, 6B). Importantly, the anti-osteoclastogenic effect of CmAb was not due to cytotoxicity and was almost completely reversible. This reversibility indicates that the cells did not lose the potential to differentiate but rather that chemerin was necessary for progression down the differentiation pathway. Unexpectedly, recombinant chemerin did not completely rescue the osteoclast differentiation blockade imposed by the CmAb. Chemerin is initially translated as a 163 aa preproprotein that is believed to be secreted as a relatively inactive 143 aa

proprotein (Chem163K) after removal of the N-terminal signal peptide sequence [23, 53]. Subsequently, extracellular proteolytic processing of the C-terminal is the major determinant of the biological activity of chemerin. This processing is complex and involves a number of enzymes including plasmin, carboxypeptidases, chymase, cathepsins, and elastase that are variously involved in bioactivating/deactivating chemerin to forms that vary with respect to intrinsic biological activity [54]. A limitation of this study is that the specific isoform(s) of chemerin present in the extracellular media of the HSCs and which promotes osteoclastogenesis is unknown. This is an important consideration as experimental data indicate that the actions of chemerin are highly contextual and that the abundance of chemerin isoforms varies based upon anatomical site. For example, while Chem163K is the major circulating form of chemerin in humans, the dominant isoform in synovial fluid is Chem158K [55]. A further consideration is that nothing is known regarding other post-translational modifications (e.g., glycosylation and oligomerization) that affect bioactivity, stability, or receptor selectivity of chemerin. Thus, supplementation of the media with bacterially expressed recombinant human Chem157S or murine Chem156S may not effectively reproduce the endogenous chemerin environment and thereby fully rescue osteoclastogenesis in the presence of the CmAb. Experimental evidence indicates that both HSCs and RAW264.7 cells exhibit two phases of osteoclast differentiation involving the formation of a progenitor preosteoclast cell prior to the development of a mature, multinucleated osteoclast [18, 56]. The majority of the genes required for osteoclast differentiation are induced during the early-stage of commitment to the osteoclast lineage [17]. An important early


event is the induction of NFAT2 expression, which is mediated through Fos and NFjB acting directly on the NFAT2 promoter [57, 58]. Our finding that chemerin neutralization substantially blunted the induction of NFAT2 and Fos gene expression by RANKL is consistent with a very early influence of chemerin/CMKLR1 signaling on HSC osteoclastogenesis. RANKL has also been shown to promote integrinmediated cell adhesion of HSCs during osteoclastogenesis [59]. Similarly, chemerin has previously been reported to promote adhesion of macrophage cell lines [22]. In this study, chemerin neutralization resulted in reduced levels of RANKLinduced itgb3 and Src expression, both of which are NFAT2 target genes [40, 41]. Taken together, these findings indicate that chemerin/CMKLR1 signaling is involved in key early events of osteoclastogenesis including NFAT2 induction and cell adhesion. RANKL-induced NFjB activation has also been shown to be a key early event in promoting NFAT2 and Fos expression and thereby, osteoclastogenesis. The absence of effect of the CmAb on NFjB activation indicates that chemerin modulates NFAT2 expression and osteoclastogenesis independent of this signaling pathway. Following the initial phase of NFAT2 induction, NFAT2 cooperates with Fos and NFjB to promote transcriptional activation of genes associated with terminal osteoclast differentiation. Also characteristic of this response is an auto-amplification of NFAT2 expression whereby NFAT2 itself binds and transactivates the NFAT2 promoter. Consistent with modulation of NFAT2 being an effect of chemerin/CMKLR1 signaling, forced expression of NFAT2 rescued osteoclastogenesis to varying degrees in the presence of CmAb. CA-NFAT2 was particularly effective and completely rescued HSC osteoclastogenesis in the face of persistent chemerin neutralization. Intriguingly, the protein level of NFAT2 in the osteoclasts expressing CA-NFAT2 was greater than the cells expressing WT-NFAT2. This difference in protein level was not related to differences in the inherent expression of the lentivirus transgenes but rather due to enhanced autoamplification of endogenous NFAT expression by CA-NFAT2. Consistent with this, previous studies have identified that FK506, an inhibitor of NFAT2 nuclear translocation, blocked the autoamplification of NFAT2 and that overexpression of NFAT2 induced the expression of endogenous NFAT2 gene [18]. The incomplete rescue of osteoclastogenesis achieved by expression of WT-NFAT2 also suggests that chemerin/ CMKLR1 is involved in modulating both the expression and activation of NFAT. In order to achieve nuclear translocation, NFAT2 must be first dephosphorylated by calcineurin, a calcium-dependent phosphatase [58]. Intriguingly, one of the earliest biological actions ascribed to chemerin was to promote calcium mobilization in immature dendritic cells and macrophages [23]. Thus, it is possible that chemerin promotes NFAT2 activation through increasing intracellular calcium concentrations. Our finding that increasing intracellular calcium levels with a Ca21-ATPase inhibitor (thapsigargin) or a Ca21ionophore (ionomycin) partially restored RANKL-induced osteoclastogenesis in the face of chemerin neutralization is

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CONCLUSION In summary, our data demonstrate that blockade of chemerin signaling abrogates osteoclastogenesis of HSCs and thereby reveals a hitherto unrecognized autocrine/paracrine role for this secreted protein. MSCs and MSC-derived cell types (e.g., osteoblasts and adipocytes) also express and secrete chemerin at levels higher than HSCs, while CMKLR1 receptor expression is comparatively highest in HSCs. Thus, chemerin may contribute, in a similar fashion to RANKL, to the paracrine cross talk between osteoblasts and osteoclasts and contribute to the homeostatic balance of bone formation/resorption. It is also possible that higher levels of chemerin secretion associated with changes in the bone marrow cell population (e.g., more adipocytes) may be pathogenic and contribute to the relative increase of bone resorption characteristic of disorders of bone loss. Denosumab is a therapeutic antibody that neutralizes RANKL and has been shown in clinical trials to improve bone mineral density and reduce the risk for vertebral fractures in postmenopausal women with osteopenia or osteoporosis [60, 61]. Our finding that chemerin neutralization reduces osteoclast differentiation reveals the possibility that chemerinneutralizing antibodies will also have therapeutic value for treating disorders of bone loss.

ACKNOWLEDGMENTS We are grateful for on-going collaborations with Eugene C. Butcher and Brian A. Zabel that were essential to this work. HJD is supported by a National Sciences and Engineering Research Council of Canada (NSERC) Canada Graduate Scholarship and HJD and JLR are supported by Izaak Walton Killam Predoctoral scholarships. This work was supported by funding from the Canadian Institutes of Health Research.

DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST The authors indicate no potential conflicts of interest.

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