Myeloma Cells Inhibit Non-Canonical Wnt Co-Receptor Ror2

ORIGINAL ARTICLE Myeloma cells inhibit non-canonical wnt co-receptor ror2 expression in human bone marrow osteoprogenitor cells: effect of wnt5a/ror2 pathway activation on the osteogenic differentiation impairment induced by myeloma cells

M Bolzoni1, G Donofrio2, P Storti1, D Guasco1, D Toscani1, M Lazzaretti3, S Bonomini1, L Agnelli4, A Capocefalo2, B Dalla Palma1, A Neri4, F Nicolini5, G Lisignoli6, F Russo1, S Colla7, F Aversa1 and N Giuliani1

Multiple myeloma (MM) is characterized by the impaired osteogenic differentiation of human mesenchymal stromal cells (hMSCs). Canonical Wnt signaling is critical for the regulation of bone formation, however, recent evidence suggests that the non-canonical Wnt agonist Wnt5a stimulates human osteoblastogenesis through its co-receptor Ror2. The effects of MM cells on non-canonical Wnt signaling and the effect of the activation of this pathway on MM-induced osteoblast exhaustion are not known and were investigated in this study. We found that the osteogenic differentiation of bone marrow hMSCs toward osteoprogenitor cells (PreOB) significantly increased Ror2 expression, and that MM cells inhibit Ror2 expression by PreOB in co-culture by inhibiting the non-canonical Wnt5a signaling. The activation of the non-canonical Wnt pathway in hMSCs by means of Wnt5a treatment and the overexpression of Wnt5 or Ror2 by lentiviral vectors increased the osteogenic differentiation of hMSCs and blunted the inhibitory effect of MM in co-culture. Consistently, Wnt5a inhibition by specific small interfering RNA reduced the hMSC expression of osteogenic markers. Our findings demonstrate that the Wnt5a/Ror2 pathway is involved in the pathophysiology of MM-induced bone disease and that the activation of the non-canonical Wnt5a/Ror2 pathway in hMSCs increases osteogenic differentiation and may counterbalance the inhibitory effect of MM cells.

Leukemia (2013) 27, 451–463; doi:10.1038/leu.2012.190 Keywords: multiple myeloma; microenvironment; mesenchymal cells; non-canonical Wnt signaling

INTRODUCTION patients.13,14 However, our previous in vitro data indicate that MM Multiple myeloma (MM) is characterized by the presence of cells inhibit canonical Wnt signaling in murine pluripotential 15 osteolytic bone lesions because of a severe imbalance in bone mesenchymal cells but not in bone marrow (BM) hMSCs and, in remodeling.1,2 The osteogenic differentiation of human mesen- line with this, canonical Wnt signaling was not deregulated in the 15 chymal stromal cells (hMSCs) is typically impaired in MM patients bone microenvironment of MM patients. Furthermore, it has and leads to osteoblast deficiency.2–4 Both in vitro and in vivo recently been shown that the activation of the canonical Wnt evidences indicated that the canonical Wnt signaling pathway is pathway by Wnt3a in hMSCs stimulates cell proliferation and critical for the regulation of bone formation.5–7 In MM mouse inhibits osteogenic differentiation in vitro.16,17 model, its activation leads to bone mass improvement by Together with canonical Wnt signaling, a non-canonical Wnt reducing the development of osteolytic lesions.8–10 Specifically, pathway has been identified that is not dependent on b-catenin C57BL/KaLwRij mice were inoculated i.v. with murine 5TGM1 cells, activation. Non-canonical Wnt signaling by Wnt5a is transduced resulting in tumor growth in bone and development of MM bone through frizzled (FZD) receptors and the Ror2 co-receptor to disease; activated Wnt signaling in osteoblasts by treatment with various cascades, including the disheveled pathways involving the lithium chloride inhibited myeloma bone disease and decreased Rho family of small GTPase, JNK and Rho-associated kinase (ROCK). tumor burden in bone.8 Other in vivo mouse models were used to Wnt5a can also trigger intracellular calcium flux, which can lead to evaluate the role of osteoblastic cells in the development of the activation of phospho-protein kinase C (P-PKC), calcium- osteolytic lesions such as SCID-hu mice, showing that stimulation dependent molecules such as calmodulin-dependent protein of osteoblastic cells reduced in vitro MM cell growth in the kinase II or calcineurin, and the nuclear factor of activated presence of osteoclasts11 and blocking of DKK-1 increased bone T cells (NFATc1).18–20 formation and bone mass.12 Finally, it has also been demonstrated Interestingly, recent evidence suggests that, unlike canonical that the expression of the DKK-1 inhibitor of the canonical Wnt activation by Wnt3a, non-canonical activation by Wnt5a or pathway correlates with the presence of focal bone lesions in MM Wnt4, stimulates the osteogenic properties of BM hMSCs20–23

1Hematology and BMT Center, University of Parma, Parma, Italy; 2Infectious Diseases Section, Department of Animal Health, University of Parma, Parma, Italy; 3Department of Genetics, Micro-organism Biology, Anthropology and Evolution, University of Parma, Parma, Italy; 4Department of Clinical Sciences and Community Health, University of Milan, and Hematology 1, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy; 5Department of heart, University of Parma, Parma, Italy; 6Laboratorio di Immunoreumatologia e Rigenerazione Tissutale, Istituto Ortopedico Rizzoli, Bologna, Italy and 7MD Anderson Cancer Center, Houston, TX, USA. Correspondence: Professor N Giuliani, Hematology and BMT Center, Department of Internal Medicine and Biomedical Science, University of Parma, Via Gramsci 14, 43126 Parma, Italy. E-mail: [email protected] Received 13 January 2012; revised 20 June 2012; accepted 4 July 2012; accepted article preview online 11 July 2012; advance online publication, 31 July 2012 Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 452 and other sources.24 It has also been demonstrated that the phosphatase (ALP) þ colony-forming unit-fibroblasts (CFU-F), and the pro-osteoblastogenic effects of Wnt5a occur through the activa- formation of bone nodules was recorded. tion of its co-receptor Ror2,25–27 and the critical role of Ror2 in osteoblastogenesis has been recently demonstrated in vitro and Wnt5a and Ror2 overexpression by lentiviral vectors in BM stromal in vivo.28,29 cell lines and primary hMSCs. Wnt5a and Ror2 expression was enforced In this study we investigated the still unknown effects of MM by specific lentiviral GFP vectors in the HS-5, hMSC and PreOB cell lines. cells on the non-canonical Wnt pathway, and the effects of the The empty vector pWPI was used as control. Plasmids encoding both activation of this pathway in hMSCs on the impaired osteogenic Wnt5a and Ror2 cDNA were used to obtain the vectors. The amplified differentiation induced by MM cells. cDNA sequence of Wnt5a/Ror2 was cloned in a lentiviral vector pWPI. The recombinant lentiviruses were obtained by transiently transfecting 293T cells, then concentrated by means of ultracentrifugation at 26 000 r.p.m. for 90 min at 4 1C. The cells were infected with lentiviral GFP vectors encoding MATERIALS AND METHODS the cDNA sequence of Wnt5a or Ror2 or pWPI. After 12–24 h, the efficiency Reagents of cell transduction was evaluated by quantifying GFP fluorescence by Recombinant murine (rm) Wnt3a and Wnt5a were obtained from R&D means of flow cytometry, and its efficacy was evaluated by means of real- System (Minneapolis, MN, USA); dexamethasone (Dex), ascorbic acid and time PCR and western blotting. beta-glycerophosphate were purchased from Sigma-Aldrich (St Louis, MO, USA); RPMI 1640, a-MEM (a-modified essential medium), Dulbecco Small interfering RNA (siRNA) transfection. Wnt5a in hMSCs and PreOB modified Eagle’s medium and culture media with glutamine, penicillin, cells was silenced using specific siRNA (Dharmacon, ThermoScientific, streptomycin and fetal bovine serum were purchased from Invitrogen Life Lafayette, CO, USA). Cells were electroporated with 2 nM of Wnt5a siRNA Technologies (Milan, Italy). or non-specific siRNA using a one-target protocol and the Gene Pulser System (Bio-Rad Laboratoires Inc., Herculex, CA, USA). After 24–48 h, mRNA Cell purification and cell-culture conditions and proteins were extracted for the evaluation of osteogenic marker expression. Cell lines. The human myeloma cell lines (HMCLs) KMS12 and KMS27 were a kind gift from Dr A Neri (IRCCS, Milan, Italy), whereas the XG-1 cells came from Dr M Amiot (INSERM, Nantes, France). The JJN3 cells were Co-cultures. Confluent, adherent primary hMSC or PreOB or hOB-01 osteoblastic cells were co-cultured with JJN3 or XG-1 or KMS12 or obtained from the DMSZ (Braunschweig, Germany), and the HS-5 human þ stromal cell line from the American Type Culture Collection (Rockville, MD, KMS27 HMCLs or freshly purified MM CD138 cells added directly or USA). The pre-osteocytic cell line hOB-01 was a kind gift from Dr Julia placed in a transwell insert (pore size 0.4 mM; Corning, Life Sciences, Billiard (Collegeville, PA, USA). Amsterdam, The Netherlands) for 24–48 h. In some long-term (2–3 weeks) experiments, primary human BM mononuclear cells were co-cultured with HMCLs placed in a transwell insert in the presence or absence of Purification of primary CD138 þ BM cells from MM patients and þ rmWnt5a (500 mg/ml) or rmWnt3a (500 mg/ml) in osteogenic differentiation controls. Primary CD138 cells were isolated from mononuclear cells medium. obtained from BM aspirates of 40 consecutive newly diagnosed or relapsed Transduced hMSCs were cultured with Wnt5a or Ror2 and HMCLs in MM patients, 7 patients with monoclonal gammopathy of uncertain þ osteogenic differentiation medium in the presence or absence of a significance (MGUS) and 2 healthy donors underwent to surgery. CD138 transwell insert in short-term (48–72 h) co-cultures to evaluate osteogenic normal plasma cells (PCs) cells were also purified from three tonsils of markers and long-term (2–3 weeks) co-cultures to evaluate ALP staining subjects who underwent tonsillectomy for non-neoplastic diseases. and bone nodule formation. Informed consent was obtained in accordance with the Declaration of Helsinki, and approval was obtained from the Institutional Review Board of the University Hospital of Parma. All of the MM patients ALP and bone nodule assays. ALP and bone nodule formation in underwent total body radiography to identify the presence of osteolytic primary hMSCs were assayed for 2 and 3 weeks, respectively. Primary bone lesions. Patient characteristics are summarized in Supplementary hMSC were incubated in osteogenic differentiation medium in order to Table S1. obtain PreOB cells under the different experimental conditions, and in The CD138 þ MM cells were purified immunomagnetically using anti- hMSCs transduced with lentiviral vectors (pWPI, Wnt5a and Ror2) and CD138 monoclonal antibody-coated magnetic microbeads (MACS, Miltenyi differentiated towards PreOB cells for 2–3 weeks. Biotec, Bergisch-Gladbach, Germany). Samples with a purity of more than For the ALP assay, the cells after 2 weeks were fixed with cold formalin at 90% via flow cytometry (BD biosciences, San Jose`, CA, USA) were analyzed. 4 1C, washed three times with PBS, and stained with a semi-quantitative These samples were evaluated immediately after purification. ALP histochemical kit (Sigma-Aldrich, Milan, Italy) in accordance with the manufacturer’s procedures. The ALP staining was quantified using ID image analysis software (Kodak Digital Science, Rochester, NY, USA). For Primary hMSCs and osteoprogenitor (preOB) cell cultures. Primary À the evaluation of bone nodule formation, the cells after 3 weeks were fixed hMSCs were obtained from the CD138 fraction of BM mononuclear cells with a 1:1 mixture (vol/vol) of 37% formaldehyde and ethanol for 5 min, taken either from MM patients (10 cases) or healthy donors—including washed three times with PBS, and stained for 10 min with a 2% solution of matching subjects for sex and age—who underwent orthopedic or heart alizarin red, pH 4.2 (Sigma-Aldrich). The nodules were quantified by means surgery without neoplastic disease (10 cases), incubated in a-MEM medium of directly counting all of the alizarin red-stained nodules under light supplemented with 15% fetal bovine serum and 2 mM of glutamine until microscopy. confluence, and then analyzed at the first passage. To induce the osteoblast phenotype of the hMSCs towards preOB cells, we incubated BM mononuclear cells in the presence or absence of osteogenic differentiating RNA isolation and reverse-transcriptase polymerase chain reaction a-MEM medium supplemented with 15% fetal bovine serum and 2 mM of (RT-PCR) amplification À 8 glutamine in the presence of ascorbic acid (50 mg/ml) and Dex (10 M) for Total RNA was extracted from the cells using the RNeasy total RNA 2 weeks. isolation kit (Qiagen, Valencia, CA, USA), and then quantified using a Nanodrop ND-100 (Celbio SpA, Milan, Italy). One microgram of RNA was Wnt5a treatment of BM mesenchymal stromal cells. Confluent HS-5 reverse transcribed with 400 U Moloney murine leukemia reverse- stromal cells were treated with increasing doses of rmWnt5a (R&D system) transcriptase (Applied Biosciences, Life Sciences, Carlsbad, CA, USA) in (100–500 ng/ml) for 20 min at 37 1C (in order to evaluate intracellular accordance with the manufacturer’s protocol. The cDNAs were amplified calcium-influx) using the fluorescent calcium indicator Fluo-4AM (Mole- by means of PCR using the following specific primer pairs: cular Probes, Invitrogen) and flow cytometry (BD biosciences), or for 2 h in WNT5a:F:50-GAGTTCGTGCACGCCCGCGA-30;R:50-CCGCCAGGAACGTGC order to evaluate the effect on NFATc1 and ROCK activity. TTG-30; To evaluate the effects of Wnt5a on osteogenic differentiation, BM ROR2:F:50-CCTGGACACGACAGACACTG-30;R:50-AAGTTATGATTTGGGTG mononuclear cells were seeded in osteogenic differentiation medium in TGC-30; the presence of rmWnt5a (500 ng/ml) or rmWnt3a (500 ng/ml) or vehicle FZD5: F: 50-TTCATGTGCCTGGTGGTGGGC-30;R:50-TACACGTGCGACAGGG for 2–3 weeks, and the presence of early PreOB cells such as alkaline ACACC-30;

Leukemia (2013) 451 – 463 & 2013 Macmillan Publishers Limited Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 453 ALP: F: 50-ACGTGGCTAAGAATGTCATC-30;R:50-CTGGTAGGCGATGTCC healthy controls was assessed with oligonucleotide arrays. Both TTA-30; cell types expressed the activator of non-canonical Wnt pathways COL1A1:F:50-GCAACATGGAGACTGGTGAG-30;R:50-GGATGGAGGGAGT WNT5A, but lacked the main activators of canonical Wnt signaling TTACAGG-30; 0 0 0 (WNT1, WNT3A and WNT8); on the other hand, both expressed the BGLAP:F:5-GTAGTGAGGAGACCCAGGCG-3 ;R:5-GGGAAGAGGAAAGA canonical inhibitor DKK-1 and the receptor LRP5 and LRP6 AGGGTG-30; GAPDH F: 50-CAACGGATTTGGTCGTATTG-30;R:50-GGAAGATGGTGATGGG (Table 1). Ror2 co-receptors and FZD receptors (FZD2, FZD3 and ATTT-30. FZD5) were also detected in both cell types (Table 1). PCR reactions were performed in a thermal cycler (MiniCycler, My The osteogenic differentiation of hMSC towards preOB upre- Research, Watertown, MA, USA) for 30 cycles. Cycle number, melting gulated Ror2, FZD5 and the canonical inhibitor FRZB, but not temperature and product size for the PCR reactions were: 30 cycles; 55 1C WNT5A mRNA expression (Table 1 and Figure 1a). The stimulatory and 561 bp for WNT5A; 32 cycles, 56 1C and 120 bp for ROR2; 27 cycles, effect of osteogenic differentiation on Ror2 expression was also 56 1C and 475 bp for FZD5 and ALP; 23 cycles; 60 1C 537 bp for COL1A1; and demonstrated at the protein level by means of western blotting 25 cycles, 58 1C and 209 bp for GAPDH. Subsequently, 10 ml of each amplified (Figure 1b). reaction were run on a 1.5% agarose gel and then stained with Gel Red The modulation of all these Wnt-related genes by the (Biotium Inc., Sic, Rome, Italy) (1 mg/ml). Images were recorded using a digital Kodak DC 120 camera (Kodak Digital Science-Eastman Kodak). osteogenic differentiation process was observed in the samples obtained from MM patients as well as healthy controls (Table 1). Interestingly, the comparison of the mRNA expression of the Wnt- Real-time PCR and PCR array related molecules between healthy donors and MM patients Real-time PCR was performed using the TaqMan Gene Expression Assay shows that ROR2 mRNA in both hMSCs and PreOB was (Applied Biosystems) for the following genes: WNT5A: Hs00998537_m1, downregulated in MM patients as compared with healthy controls WNT11: Hs00182986_m1, WNT4: Hs01573504_m1, ROR2: Hs00171695_m1, (Table 1). Among the remaining Wnt-related genes evaluated FZD5: Hs00361869_g1, ALP: Hs00758162_m1, COL1A1: Hs01076777_m1, herein, we observed a significant upregulation of DKK-1 mRNA in and ABL Hs01104728_m1. The PCR amplifications were performed in duplicate wells in an iCycler iQ Real-Time Detection System (Bio-Rad, Milan, MM samples in comparison with healthy controls in both hMSC Italy). Using the endogenous reference gene ABL, the comparative Ct and PreOBs (Table 1). method was applied to normalize the differences in the quantity and quality of RNA, and mRNA was quantified using the comparative DCt Expression of non-canonical WNT signaling molecules by HMCLs method (DCt ¼ mean Ct gene À mean Ct ABL); DDCt was evaluated as the and CD138 þ MM cells difference between the DCt of a sample and the DCt of the control. The fold-change in mRNA expression was calculated as 2 À DDCt. The expression of Wnt5a and its receptors was also evaluated in þ For the PCR array, 1 mg of total RNA was reverse transcribed using RT2 several HMCLs as well as in purified CD138 cells from a cohort First Stand Kit (Qiagen), and cDNA was directly added to PCR Master mix of MM patients, MGUS and control subjects (Figures 1c–e and containing SYBR green. The mixtures were then aliquoted into 96-well PCR Supplementary Table S1). PCR demonstrated that JJN3 and XG-1 array plates, which profile the expression of 84 Wnt signaling-specific cells did not express WNT5A mRNA, whereas the KMS12 and genes (PAHS-043; SA Biosciences, Qiagen). Thermal cycling was done in KMS27 did. However, WNT5A mRNA levels were lower in the accordance with the manufacturer’s protocol, and the fold-change in CD138 þ MM cells compared with hMSCs and PreOB cells expression was assessed by means of DDCt. (Figure 1c). The presence of Wnt5a protein was only demonstrated in KMS27 cells by means of western blotting, which had sensitivity Western blotting around 50–100 ng (Figure 1e). JJN3 cells significantly expressed Nuclear and cytosolic extracts were obtained from the cells and evaluated the Ror2 co-receptors (Figures 1c and 1d), and all of the HMCLs by western blotting as previously described.3 The nuclear extracts were tested were positive for FZD5 mRNA. prepared using the Nuclear Extraction Kit (Active Motif, Carlsbad, CA, USA), The CD138 þ cells of 27 of the 40 analyzed MM patients (67.5%) and tested using the following primary antibodies: anti-total-b-catenin expressed WNT5A mRNA, and western blotting demonstrated (1:1000) (Chemicon International, Temecula, CA, USA), anti-active-anti- Wnt5a protein expression in 7 out of 28 cases (25%). There was no Phospho-PKC (1:1000) (Cell Signaling Technology, Euroclone, Milan, Italy), anti-human Ror2 (1:200) (R&D System Inc.), anti-Wnt5a (1:1000) and anti- significant relationship between the presence or absence of calcineurin (1:1000) (Cell Signaling Technology). Anti-b-actin (1:5000) WNT5A mRNA transcript or WNT5A mRNA levels (DCt) and the (Sigma-Aldrich, Inc.) and anti-histone H1 antibodies (1:500) (Upstate, Lake disease state (diagnosis or relapse), prognostic staging (ISS), or the Placid, NY, USA) were, respectively, used as internal controls for cytosol and radiographic presence or absence of osteolytic bone lesions (P ¼ nuclear proteins. The secondary antibodies were peroxidase conjugated, NS) (Supplementary Table S1). The median WNT5A mRNA levels in anti-rabbit, (Chemicon), anti-goat, (Rockland Immunochemicals, Gilbertsville, CD138 þ of MM patients were significantly higher than those of PA, USA) and anti-mouse antibodies (BD Pharmingen, Franklin Lakes, NJ, USA). normal PC (median À DCt: 0.1 vs À 4.4, P ¼ 0.04) (Figure 1d), Chemiluminescence was detected after immunoblotting by incubating the whereas the difference between MM and MGUS patients did not membranes for 5 min with luminol solution (ECL Plus Western Blotting reach statistical significance (median DCt: 0.1 vs 3, P 0.14) Detection System, GE Healthcare Amersham, Milan, Italy). The immunoreactive À À ¼ (Figure 1d). ROR2 mRNA levels were significantly higher in bands were visualized using an exposure time of five minutes (Kodak X-OMAT) þ and quantified by the Image J software (http://rsbweb.nih.gov/ij/). CD138 MM cells than normal PC (Po0.0001), but not than CD138 þ MGUS cells (P ¼ 0.33) (Figure 1d). Finally, FZD5 mRNA levels were higher in MM patients as compared with both MGUS NFATc1, ROCK and JNK assays. patients and normal PC (Po0.0001) (Figure 1d). NFATc1, ROCK and JNK activity were, respectively, evaluated by means of an ELISA-based method (Trans AM NFATc1, Active Motif, Vinci Biochem, Vinci, Firenze, Italy), using commercially available kits (Rock Activity Assay HMCLs and primary CD138 þ MM cells inhibit Ror2/FZD5 expression Kit, Cell Biolabs, Inc., San Diego, CA, USA), and FACE JNK (Active Motif), all and the non-canonical Wnt signaling pathway in human BM PreOB used in accordance with the manufacturers’ procedures. cells (PreOBs) To investigate whether MM cells affect the non-canonical Wnt5a pathway in osteprogenitor cells, we co-cultured PreOB cells or the RESULTS osteogenic cell line HOB-01 with the JJN3, XG-1, KMS27 or KMS12, Expression of non-canonical WNT signaling molecules by hMSCs: or purified CD138 þ MM cells, in the presence or absence of a effect of osteogenic differentiation transwell insert for 24–48 h. In these co-culture systems, HMCLs The expression of non-canonical Wnt-related molecules by BM and primary CD138 þ MM cells reduced ROR2 mRNA expression in hMSCs and PreOB cells obtained from both MM patients and co-cultured PreOB cells in the presence or absence of a transwell

& 2013 Macmillan Publishers Limited Leukemia (2013) 451 – 463 Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 454 insert after 24 h (as shown for XG-1 in Figure 2a). This effect was þ þ 5.3 / / observed with PreOB obtained either from MM patients or from 9.38 5.62 3.45 À À À À healthy controls. The level of FZD5 mRNA also decreased in co- cultured XG-1 and PreOB cells in the presence of a transwell insert but, in the cell-to-cell contact co-cultures the level of FZD5 mRNA 0.58 1.84 1.37 2.54 increased likely due to the high expression of FZD5 in XG-1 cells À À À À (Figure 2a). The inhibitory effect of XG-1 on Ror2 expression in PreOB cells was conﬁrmed at the protein level (Figure 2b) after 1.11 2.49 1.97 3.19 48 h of co-culture. Similarly, other cell lines such as JJN3 inhibited À À À À Ror2 protein expression in HOB-01 cells (Figure 2b) in the presence or absence of a transwell insert. In addition to inhibiting þþ þ À þþ þÀ Ror2, the HMCLs decreased the active form of PKC (P-PKC) in HOB- / / 4.08 4.84 1.34 2.59 01 cells (Figure 2c). Interestingly, the inhibitory effect of HMCLs on À À À À P-PKC expression in PreOB cells was independent of the production of Wnt5a by HMCLs cells because this effect was þÀ þþ þ þÀ þþ þ þ þ þÀ 4.2 3.3 / / / / þ 4.64 3.94 observed in both Wnt5a-negative cells such as XG-1 and Wnt5a À À À À cells such as KMS27 (Figure 2c). Similarly we found that puriﬁed PCs from primary MM tumors inhibit P-PKC in both PreOB and HOB-01 with a more pronounced effect in HOB-01 (Figure 2d). 0.59

À In line with the Ror2 and P-PKC inhibition, we found that HMCLs and primary CD138 þ MM cells reduced activated p-JNK in co- cultured PreOB cells (data not shown) as well as NFATc1 activity as 0.09 0.26 1.34 shown for XG-1 and two representative MM patients in Figure 2e. À À Finally, HMCLs also had an inhibitory effect on ROCK activity in co-cultured PreOB cells, as shown for XG-1 (Figure 2f). 0.63 À Activation of the non-canonical Wnt pathway with Wnt5a treatment increases human BM osteoblastogenesis and reduces the inhibitory

þþþþÀ þ / 5.32 0.95 1.65 2.35 5.35 0.16 4.38 0.75 0.65 1.8 3.74 effect of both HMCLs and primary CD138 MM cells in long-term À À À À co-cultures We ﬁrst evaluated the capacity of Wnt5a treatment to activate the 4.4 þÀ þþþþþÀ þþþþþÀ non-canonical signaling pathway in vitro using the human BM 5.3 / / / 2.98 4.04

À stromal cell line HS-5. Stimulation of HS-5 cells with rmWnt5a À À ÀÀ (100–500 ng/ml) for 20 min at 37 1C led to a dose-dependent increase in Ca þþ inﬂux, as evaluated by means of a 4-Fluo AM assay (Figure 3a), which was signiﬁcant effect at the highest 1.14 0.74 2.08 À À À concentration (500 ng/ml). In line with this observation we found that Wnt5a treatment increased P-PKC and calcineurin expression (Figure 3b). Consequently, increased NFATc1 and ROCK activity 7 0.05 8 7.74 9.54 À À was observed in HS-5 cells upon treatment with Wnt5a 500 ng/ml À À (Figure 3c). The effect of Wnt5a treatment on the osteogenic differentiation of BM cells was evaluated by measuring CFU-F and bone 11.4 11.2 12.19 12.14 À À nodule formation in long-term cultures. Stimulation with rmWnt5a À À (500 ng/ml) increased ALP þ CFU-F levels and bone nodule formation in BM mononuclear cells as compared with non-treated

0.13 controls, whereas Wnt3a (500 ng/ml) did not have this effect

À (Figure 3d). To investigate whether the activation of the non-canonical pathway can revert the inhibitory effect of MM cells on osteo- 9.25 1.8 9.77 1.15 10.04 0.66 10.03 blastogenesis, CFU-F levels and bone nodule formation were À À À À evaluated in long-term co-cultures of BM mononuclear cells and either XG-1 or CD138 þ MM cells maintained in osteogenic differentiation medium, with or without canonical (Wnt3a) and 12.2 12.1 11.38 11.24 non-canonical Wnt molecules (Wnt5a). Wnt5a (but not Wnt3a) À À À À treatment blunted the inhibitory effect of HMCLs on the CFU-F (Figure 3e) and bone nodule formation (Figure 3f), as well as the inhibitory effect of CD138 þ MM cells on CFU-F formation À À ÀþÀÀþÀ À À ÀþÀÀþÀ À À ÀþÀÀþÀ À À ÀþÀÀþþþþþþÀ 35 35 32.85 22.15 35 30.55 23.95 27.2 27.25 22.65 22.9 22.55 27.4 27.65 25.3 24.65 27.64 35 35 32.9 23 35 32.4 24.95 28.15 25.7 23.5 24.2 23.45 26.8 25.45 26.05 25.4 26.3 10.58 12.19 10.78 12.14 34.23 35 32.87 21.8 35 30.63 23.57 26.6 26.55 22.7 21.97 21.27 27.8 27.7 24.73 24.2 30.6 34.53 35 33.53 22.6 35 31.77 24.5 27.8 26.45 23.7 23.1 21.93 27.1 25.1 25.73 25.13 27.5 WNT1 WNT3A WNT4 WNT5A WNT8A WNT11 FZD2 FZD3 FZD5 FZD6 FZD7 DKK1 SFRP-1 FRZB(Figure LRP5 LRP6 3e). ROR2 À À À À Ct Ct Ct Ct D D D D Modulation of Wnt5a expression by hMSCs affects their À À À À osteogenic properties and blocks the inhibitory effect of HMCLs in co-culture : Expression of the main Wnt signaling activators, receptors and inhibitors by hMSC and PreOB in both healthy and MM samples To investigate the effect of the activation of the non-canonical Median Ct Median Median Ct Median Median Ct Median Median Ct Median Wnt5a pathway in mesenchymal stromal cells further, Wnt5a hMSC PreOB PreOB hMSC expression was enforced by speciﬁc lentivirus GFP vectors in HS-5 Healthy MM Table 1. Abbreviations: hMSC, human mesenchymal stromal cells, MM, multiple myeloma; PreOB, osteoprogenitor cells. cells and primary hMSCs. The empty vector pWPI was used as the

Leukemia (2013) 451 – 463 & 2013 Macmillan Publishers Limited Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 455

Figure 1. Expression of Wnt5a, Ror2 and FZD5 in human BM mesenchymal and PreOB cells, and in HMCLs and in CD138 þ cells. The expression of Wnt5a, Ror2 and FZD5 was evaluated with real-time PCR in BM mesenchymal (hMSC) and PreOB cells (PreOB) obtained after the 2 weeks’ culture of hMSCs in osteogenic differentiation medium. The graphs show the median fold-change in mRNA expression (n-fold calculated as 2 À DDCt) in PreOB cells and hMSCs (*Po0.01) (a). Ror2 protein expression in hMSC and PreOB as revealed by western blotting; actin was used as the internal control (b). The expression of Wnt5a, Ror2 and FZD5 was evaluated by means of real-time PCR in HMCLs (c) and primary purified CD138 þ cells obtained from MM patients, MGUS and normal PC (d). The graphs show the median À DCt values in HMCLs (c) and primary CD138 þ cells (d) (hMSCs and PreOB cells used as controls). The presence of Wnt5a and Ror2 transcripts in the KMS12, KMS27, XG-1 and JJN3 was revealed by qualitative PCR. PreOB cells and the HOB-1 pre-osteocytic cell line were, respectively, used as the positive controls (Con þ ) for Wnt5a and Ror2 (c). Wnt5a and Ror2 protein expression in KMS27, JJN3 and XG-1 as shown by western blotting (Con þ : PreOB cells for Wnt5a and HOB-01 cells for Ror2). Different doses of recombinant Wnt5a were used as a control to quantify the sensitivity of the western blot analysis (e). control. The efficiency of cell transduction was evaluated by flow osteogenic differentiation medium for 2–3 weeks. ALP staining cytometry, and was consistently 90% or more (Figure 4a). The and bone nodule formation (detected by means of alizarin efficacy of Wnt5 transduction at the mRNA level was evaluated by red staining) were greater in the primary PreOB cells over- real-time PCR, and demonstrated significantly higher Wnt5a expressing Wnt5a (PreOB-Wnt5a) than in those transduced mRNA levels in the Wnt5-transfected HS-5 and hMSC cells as with the control vector pWPI (PreOB-pWPI) (Figure 4f). Consistent compared with those transfected with the control vector pWPI with this, mRNA expression of the osteogenic markers ALP and (Figure 4b). Wnt5a overexpression was also demonstrated at the COL1A1 was upregulated in PreOB-Wnt5a in comparison with protein level in the 48-h conditioned media of the transduced PreOB-pWPI, as shown by qualitative (Figure 4g) and real-time cells (Figure 4c). As a result of the Wnt5 overproduction, there was PCR (Figure 4h). an increased level of active P-PKC and a reduced level of total Finally, in order to investigate whether Wnt5a overexpression b-catenin (Figure 4d), as well as upregulation of cytoplasmic ROCK can revert the inhibitory effects of HMCLs on the expression of and nuclear NFTAc1 activities (Figure 4e). These findings suggest osteogenic markers in co-culture, we created co-culture systems that the overexpression of Wnt5a in mesenchymal stromal cells using PreOB-Wnt5a or PreOB-pWPI cells and XG-1 cells in the induces the non-canonical Wnt pathway and inhibits the absence and presence of a transwell insert. The presence of XG-1 canonical pathway. in the co-cultures with PreOB-Wnt5a cells did not inhibit ALP or The effect of Wnt5a overexpression in hMSCs on osteogenic COL1A1 mRNA levels regardless of the absence or presence of a differentiation was investigated by plating transduced hMSCs in transwell insert in comparison with the control PreOB-pWPI cells.

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Figure 2. Inhibition of Ror2 expression in human BM PreOB cells and myeloma cell co-cultures. Confluent human BM PreOB cells obtained by culturing first-passage adherent hMSCs in osteogenic differentation medium for 2 weeks were co-cultured with XG-1, KMS27 or primary purified CD138 þ cells for 24–48 h in the presence or absence of a transwell insert. Ror2 and FZD5 mRNA expression was evaluated by means of real-time PCR after 24 h of co-culture. The graphs show the median fold-change (2 À DDCt) in three independent experiments (a). Ror2 protein expression was evaluated by means of western blotting in PreOB and HOB-01 after 48 h of co-culture with XG-1 or JJN3 in the presence or absence of a transwell insert (b). P-PKC expression was evaluated by means of western blotting in the cytoplasm of HOB-01 cells or PreOBs co-cultured with either HMCLs in the presence or absence of a transwell insert (c) or primary CD138 þ cells of a representative MM patient (d). Graphs show the optical density (OD) ratio of P-PKC normalized for the internal control actin in both HOB-01 and PreOB (e). NFATc1 activity was evaluated using an ELISA-based method according to manufacturer’s procedures. The graph±bars indicate the mean normalized OD related to NFATC1 activity (±s.d.) (*P ¼ 0.05). ROCK activity in the cytoplasm was evaluated as described in Materials and methods. The graph±bars show the mean OD related to ROCK activity (±s.d.) (*Po0.05) (f).

In all cases, ALP mRNA levels increased and COL1A1 mRNA levels Wnt5a and PreOB siRNA Wnt5a) in comparison with the control remained unchanged (Figure 4h). siRNA (hMSC siRNA Cy and PreOB siRNA Cy) (Figure 5a). To confirm the potential role of Wnt5a as a target in regu- Qualitative PCR showed that Wnt5a silencing reduced the lating the osteogenic properties of hMSCs, we silenced Wnt5a expression of the osteogenic markers ALP, COL1A1 and OC with a specific siRNA in both hMSCs and PreOB cells. There was mRNA (Figure 5b), and real-time PCR confirmed the reduction in a significant reduction in Wnt5a transcript levels after 24 h in ALP mRNA levels induced by siRNA anti-Wnt5a in PreOB cells the cells treated with specific anti-Wnt5a siRNA (hMSC siRNA (Figure 5c).

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Figure 3. Wnt5a treatment activates the non-canonical Wnt pathway in human BM mesenchymal cells, and increases their osteogenic properties by counterbalancing the inhibitory effect of myeloma cells. The human BM stromal cell line was treated with rmWnt5a at different concentrations (100–500 ng/ml). Intracellular calcium-influx was evaluated by means of flow cytometry after 20 min of treatment using the fluorescent calcium indicator Fluo-4AM (Molecular Probes, Invitrogen) (a). P-PKC and calcineurin expression was evaluated in HS-5 after 20 min of treatment with rmWnt5a at 200 and 500 ng/ml (b). NFATc1 and ROCK activity was checked after 2 h: the graph±bars represent mean optical density (±s.d.) (*Po0.05) (c). Evaluation of ALP þ CFU-F, and bone nodule formation as indicated by alizarin red staining, in human BM mononuclear cells seeded in osteogenic differentiation medium in the presence of rmWnt5a (500 ng/ml), rmWnt3a (500 ng/ml) or vehicle for 2–3 weeks. The graph±bars indicate the mean number of ALP þ CFU-F and bone nodules (±s.d.) (*Po0.05) (d). Human BM mononuclear cells were co-cultured with XG-1 or primary purified CD138 þ for 2–3 weeks in osteogenic differentiation medium, in a transwell insert in the presence or absence of rmWnt5a (500 ng/ml) or rmWnt3a (500 ng/ml). Media was replaced every 3 days. At the end of culture period, ALP þ CFU-F (e) and bone nodules (f) were quantified as described in the Materials and methods. The photographs show the ALP (e) and alizarin red staining (f) of a representative experiment (original magnification Â 1). The graph±bars indicate the mean number of ALP þ CFU-F and bone nodules (±s.d.) per well (*Po0.05) (e and f).

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Leukemia (2013) 451 – 463 & 2013 Macmillan Publishers Limited Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 459 Ror2 overexpression induced by a lentiviral vector in hMSCs than in PreOB-pWPI cells in osteogenic differentiation medium increases their osteogenic properties and counterbalances the (Figure 6c). In line with this, the expression of the osteogenic inhibitory effect of HMCLs markers ALP and COL1A1 mRNA was higher in PreOB-Ror2 than in As we found that both HMCLs and primary CD138 þ MM cells PreOB-pWPI cells (Figures 6d and e). Finally, the stimulatory effect inhibit the expression of the Ror2 co-receptor of non-canonical on ALP and COL1A1 mRNA expression level was also maintained Wnt5a Wnt signaling in hMSCs differentiated towards PreOB, we in the co-culture system with HMCLs, as shown for XG-1 in the next investigated the effect of Ror2 overexpression on the presence or absence of a transwell insert (Figure 6e). osteogenic properties of hMSCs in the presence of HMCLs. We first enforced Ror2 expression using a lentiviral vector in HOB-01 PreOB cells, HS-5 BM mesenchymal stromal cells, and primary DISCUSSION hMSCs. The efficiency and efficacy of cell transduction were, The non-canonical Wnt pathway acts in a b-catenin-independent respectively, evaluated by means of flow cytometry and real-time manner as Wnt only binds to the FZD receptor and not to the LRP PCR (Figure 6a). Consistent with the Ror2 overexpression, an 5/6 co-receptor.18,19 The signal transduction of non-canonical Wnt increase in P-PKC protein levels and a decrease in b-catenin levels activators also requires the presence of co-receptors such as were observed, supporting the activation of the non-canonical Ror2.26 The most widely known routes of non-canonical signaling pathway and the inhibition of the canonical pathway (Figure 6b). are the planar cell polarity pathway and the Wnt/Ca þþ signaling We then investigated the effect of primary hMSC Ror2 over- pathway.18,19 The planar cell polarity signal is transduced by expression on osteogenic differentiation towards PreOB cells, and the RhoA-ROCK pathway and the small GTPases RAC1, which found that ALP staining was significantly greater in PreOB-Ror2 activates JNK signaling, whereas the Wnt/Ca þþ signal leads to

Figure 5. Wnt5a suppression by siRNA in human BM PreOB cells inhibits the mRNA expression of osteogenic markers. Wnt5a was silenced by specific siRNA in hMSCs and PreOB cells obtained as described in the Materials and Methods. (a) The cells were electroporated with 2 nmol of anti-Wnt5a siRNA (siRNA Wnt5a) or non-specific siRNA (siRNA Cy) for 24 h before mRNA extraction. Wnt5a mRNA was evaluated to verify the efficacy of siRNA transfection. The graph±bars represent the mean Wnt5a mRNA levels (±s.d.) expressed as À DCt (**Po0.01) of three independent experiments. (b) ALP (ALP), collagen I (COL1A1) and osteocalcin (OC) mRNA expression were evaluated in PreOB siRNA Wnt5a and PreOB siRNA Cy by means of PCR. (c) ALP mRNA levels were evaluated by real-time PCR in PreOB siRNA Wnt5a and PreOB siRNA Cy. The graph±bars represent the mean ALP mRNA levels (±s.d.) expressed as À DCt of three independent experiments (*Po0.05).

Figure 4. Lentiviral vector induced overexpression of Wnt5a in human BM mesenchymal stromal cells activates non-canonical Wnt signaling, and stimulates their osteogenic properties by counterbalancing the inhibitory effect of myeloma cells on the expression of osteogenic markers in co-culture systems. Wnt5a overexpression was induced by means of lentiviral vectors in the human BM cell line HS-5 and primary hMSCs as described in the Materials and Methods. (a) The efficacy of cell transduction was evaluated by flow cytometry detection of GFP expression in the cells transduced with Wnt5a or the control vector pWPI. (b) The efficiency of cell transduction at mRNA level was evaluated by means of real-time PCR: the graph±bars indicate the mean À DCt (±s.d.) of Wnt5a mRNA levels in three independent experiments (**Po0.01). (c) Western blot of the conditioned media of transduced cells. (d) P-PKC and b-catenin expression evaluated in HS-5 cells infected with control vector pWPI or Wnt5a. (e) ROCK and NFATc1 activity measured as described in Materials and methods. The graph±bars indicate the mean optical density (OD) related to ROCK and NFATc1 activity (±s.d.) (*Po0.05). (f) Primary hMSCs transduced with pWPI or Wnt5a vectors were plated in osteogenic differentiation medium and allowed to differentiate towards PreOB. ALP was evaluated after 2 weeks of culture, and bone nodule formation (alizarin red staining) after 3 weeks. The photographs show ALP staining (original magnification Â 1) and bone nodules (alizarin red) (original magnification Â 40) in a representative experiment. The graph±bars indicate the mean number of ALP þ CFU-F and bone nodules (±s.d.) per well (*Po0.05). (g) ALP, COL1A1 and GAPDH mRNA expression by PreOB-pWPI and PreOB-Wnt5a was evaluated by means of PCR after 2 weeks of culture in osteogenic differentiation medium. (h) ALP and COL1A1 mRNA expression was evaluated by means of real-time PCR in PreOB-pWPI or PreOB-Wnt5a co-cultured with or without XG-1 in the presence or absence of a transwell insert. The graph±bars indicate the mean fold-change in mRNA levels (2 À DDCt) in the different samples and controls (**Po0.01; *Po0.05) (h).

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Figure 6. Overexpression of Ror2 induced in human BM mesenchymal stromal cells by means of a lentiviral vector increases the early osteogenic capacity and the expression of osteogenic markers in co-cultures with myeloma cells. Ror2 overexpression was induced using lentiviral vectors in human PreOB HOB-01 cells, human BM stromal HS-5 cells, and primary hMSCs as described in Materials and Methods. (a) The efficacy of cell transduction was evaluated by flow cytometry detection of GFP expression in the cells transduced with Ror2 or the control vector pWPI. (b) The efficiency of cell transduction was evaluated at mRNA level by means of real-time PCR. The graph±bars indicate the mean À DCt (±s.d.) of Ror2 mRNA levels in three independent transfections (**Po0.01). (c) Ror2, P-PKC and b-catenin expression were evaluated at the protein level in HOB-01 transfected with Ror2 (HOB-01-Ror2) or the control vector pWPI (HOB-01-pWPI). (d) Primary hMSCs transduced with Ror2 and pWPI were plated in osteogenic differentiation medium to differentiate towards PreOB, and ALP was evaluated after 2 weeks of culture. The photographs show ALP staining (original magnification Â 40) in a representative experiment. The graph±bars indicate the mean number (±s.d.) of ALP þ CFU-F per well (*Po0.05). (e) ALP, COL1A1 and GAPDH mRNA expression in PreOB-pWPI and PreOB- Ror2 was evaluated by means of PCR after 2 weeks of culture in osteogenic differentiation medium. (f) ALP and COL1A1 mRNA expression was evaluated by means of real-time PCR in PreOB-pWPI and PreOB-Ror2 co-cultured or not with XG-1 cells in the presence or absence of a transwell insert. The graph±bars indicate the fold-change in mRNA levels (2 À DDCt) in the different samples and controls (*Po0.05).

Leukemia (2013) 451 – 463 & 2013 Macmillan Publishers Limited Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 461 the activation of PKC, and an increase in intracellular Ca þþ and The capacity of HMCLs and primary CD138 þ MM cells to inhibit NFATc1 activation.18,19 It has recently been shown that activation Ror2 expression and the non-canonical Wnt signaling pathway in of the non-canonical signal pathway by Wnt5a rather than human PreOB cells was observed in the presence and absence of a activation of the canonical pathway by Wnt3a stimulates more transwell insert, as we have previously reported in the case of differentiated osteoblast CFU-OB precursors in human BM Runx2 activity.3 This suggests that both soluble factors and cell cultures,21,22 and that the pro-osteogenic effect of Wnt5a is contacts may be involved in these effects. Interestingly, it has mediated by the activation of Ror2 receptors.26 These data been reported that the Runx2 and Wnt5a/Ror2 pathways have suggest that the non-canonical signal pathway may have a role in a cooperative rather than a direct relationship in controlling the osteogenic differentiation of hMSCs. osteoblastogenesis,31,32 which supports the hypothesis that both On the basis of these findings, we first investigated the systems are probably involved in the MM-induced suppression of expression of canonical and non-canonical Wnt signaling activa- the osteogenic differentiation of hMSCs. tors, receptors and inhibitors in hMSCs, and the impact of Interleukin-7 has previously been identified as a possible osteogenic differentiation on their expression. Our data indicate soluble factor involved in the MM-induced inhibition of Runx2 that non-canonical rather than canonical Wnt signaling is and blockade of osteogenic differentiation,3,33 but there is no activated in hMSCs, because the hMSCs expressed the Wnt5a evidence concerning its involvement in suppressing the non- activator of the non-canonical Wnt pathway, but not the Wnt3a canonical signal pathway. Further studies are therefore necessary activator of the canonical pathway, and do express the canonical to identify the soluble factors involved in the MM-induced inhibitor Dkk-1. We also found that osteogenic differentiation inhibition of the non-canonical Wnt signaling pathway in human induced an increase in the expression of both FZD5 and Ror2 in PreOB cells, especially as it has been found that HMCLs and hMSCs, suggesting that osteogenic differentiation is accompanied primary CD138 þ MM cells do not express inhibitors of the non- by the overexpression of the receptor and co-receptor systems of canonical Wnt pathway such as sFRP-1,15 but may produce the non-canonical Wnt pathway, together with an overexpression inhibitors of the canonical pathway, such as DKK-1, sFRP-2 and of the inhibitor of Wnt canonical signaling FRZB. Interestingly, sFRP-3.13,15,34 the critical role of the co-receptor Ror2 in osteoblast formation As we found that HMCLs and primary CD138 þ MM cells inhibit has recently been underlined.28,29 Furthermore, we demonstrated Ror2 and the non-canonical Wnt5a signaling pathway in human that Ror2 mRNA expression was lower in both hMSC and PreOB BM PreOB cells, we next investigated whether activation of the from MM patients than those obtained from healthy controls, non-canonical pathway can stimulate osteogenic differentiation in suggesting a critical role of Ror2 in the pathophysiology of hMSCs and blunt the inhibitory effect of MM cells. Several osteoblast suppression in MM. approaches were used: (i) stimulation with recombinant Wnt5a Thus, on the basis of our experimental evidence and published treatment; (ii) enforced Wnt5a expression by lentiviral vectors; findings, we investigated the potential effect of both HMCLs and and (iii) enforced Ror2 expression by lentiviral vectors. Treatment primary CD138 þ MM cells on the non-canonical Wnt signaling with Wnt5a activated the non-canonical pathway and increased pathway in PreOB cells, and found that they inhibit FZD5 and Ror2 both CFU-F and CFU-OB formation in BM cultures and, more expression in both human BM PreOB cells and HOB-01 cells in co- importantly, blunted the inhibitory effect of HMCLs and primary culture systems, and also inhibit P-PKC expression and NFATc1 CD138 þ MM cells. In contrast, Wnt3a treatment did not led to any and ROCK activity. This suggests that HMCLs and primary CD138 þ changes in ALP expression or bone nodule formation, and did not MM cells inhibit the non-canonical Wnt signaling pathway in block the suppressive effect of HMCLs and primary CD138 þ MM PreOB cells, including the Wnt5a/Ca þþ/PKC and Wnt5a/Ror2 cells on CFU-F and CFU-OB in co-cultures. Accordingly, it has been transduction pathways, thus inhibiting the activity of both NFATc1 reported by others that Wnt5a stimulates more differentiated and ROCK. We have previously found that HMCLs do not inhibit CFU-OB osteoblast precursors in human BM cultures,21,22 whereas the expression and nuclear translocation of b-catenin in human stimulation with canonical Wnt3a increases the proliferation of BM PreOB cells in the same co-culture systems despite their early undifferentiated mesenchymal progenitor cells but inhibits production of canonical Wnt inhibitors such as Dkk-1 and their osteogenic differentiation.16,17 sFRP-3,15 though it has been previously reported that primary Secondly, we induced Wnt5a overexpression by a lentiviral CD138 þ MM cells inhibit canonical Wnt signaling in murine vector in BM stromal cell lines and primary BM hMSCs from pluripotential MSCs.13,15 Interestingly recent data show that MM patients, and found that Wnt5a overexpression increased SFRP-3 produced by HMCLs stimulate canonical Wnt signaling the levels of active P-PKC and NFATc1 and ROCK activity, in PreOB cells, in contrast to Dkk-1, and increases the level thus demonstrating the activation of the non-canonical Wnt of active b-catenin30 indicating that the lack of effect of MM pathway. Interestingly, Wnt5a overexpression decreased the level cells on canonical Wnt signaling in BM opsteoprogenitor cells of b-catenin, the main component of the canonical pathway, could be due to the opposing effects of Dkk-1 and sFRP-3 both suggesting that Wnt5a inhibits canonical signaling and the produced by MM cells. Overall our evidence and that of others presence of regulatory cross-talk between the canonical and suggests that HMCLs and primary CD138 þ MM cells inhibit non-canonical pathways. In line with this hypothesis, it has been non-canonical rather than canonical Wnt signaling in human BM previously reported that the canonical and non-canonical Wnt PreOB cells. pathways have an antagonistic relationship insofar as Wnt5a Interestingly, the inhibitory effect of HMCLs on the inhibits the effects of Wnt3a on MSCs, whereas Wnt3a suppresses non-canonical Wnt signal pathway in human PreOB cells the Wnt5a-mediated enhancement of osteogenesis.21,22 We was observed regardless of their production of Wnt5a, as it was found that Wnt5a overexpression stimulates the osteogenic found in Wnt5a-negative and Wnt5a þ HMCLs. This is not capacity of primary hMSCs by increasing ALP expression and surprising as the level of Wnt5a produced by HMCLs such as bone nodule formation, and that Wnt5a suppression by siRNA KMS27 is low; o500 ng/ml, which stimulates non-canonical Wnt5a inhibits the expression of osteogenic markers. signaling and osteogenic differentiation in PreOB cells. Further- The overexpression of Wnt5a in hMSCs also blunted the more, we failed to find a relationship between Wnt5a expression inhibitory effect of HMCLs on the expression of osteogenic in primary CD138 þ MM cells and the presence of osteolytic bone markers in co-cultures. In line with our findings, others have lesions in a large cohort of MM patients. Taken together, these demonstrated that non-canonical Wnt-4 overexpression in MSCs findings suggest that Wnt5a production by MM cells is not enhances the osteogenic differentiation of MSCs in vitro and bone involved in inhibiting osteogenic differentiation in a paracrine formation in vivo.23 Taken together, these findings suggest that fashion. that Wnt5a-mediated non-canonical Wnt pathway is a potential

& 2013 Macmillan Publishers Limited Leukemia (2013) 451 – 463 Non-canonical Wnt5a signaling in myeloma bone microenvironment M Bolzoni et al 462 target for stimulating the osteogenic and bone regenerative novel culture systems and a myelomatous mouse model. Haematologica 2006; 91: properties of human MSCs. Accordingly, it has been reported that 192–199. anabolic agents, such as strontium ranelate, stimulate osteoblast 12 Yaccoby S, Ling W, Zhan F, Walker R, Barlogie B, Shaughnessy Jr JD. Antibody- differentiation by activating NFAT-mediated non-canonical Wnt based inhibition of DKK1 suppresses tumor-induced bone resorption and multi- signaling.35 Interestingly, by means of gene expression profiling ple myeloma growth in vivo. Blood 2007; 109: 2106–2111. analysis on GeneChip HG-U133Plus2.0 arrays, we also found that 13 Tian E, Zhan F, Walker R, Rasmussen E, Ma Y, Barlogie B et al. The role of the Wnt- Wnt5a or Ror2 overexpression mediated by lentiviral vectors signaling antagonist DKK1 in the development of osteolytic lesions in multiple upregulated several chemokines, including CXCL10 and CXCL11 myeloma. N Engl J Med 2003; 349: 2483–2494. 14 Pinzone JJ, Hall BM, Thudi NK, Vonau M, Qiang YW, Rosol TJ et al. The role of (Agnelli L et al., unpublished data) that are known to be involved 36 Dickkopf-1 in bone development, homeostasis, and disease. Blood 2009; 113: in osteoblast differentiation and in the inhibition of osteo- 517–525. 37 clastogenesis. Similarly, recently published data indicated that 15 Giuliani N, Morandi F, Tagliaferri S, Lazzaretti M, Donofrio G, Bonomini S treatment of osteoblasts with Wnt5a induces the production of et al. Production of Wnt inhibitors by myeloma cells: potential effects on several chemokines involved in the regulation of inflammatory canonical Wnt pathway in the bone microenvironment. 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Organogenesis 2008; 4: lines and PreOB cells, as well as in primary hMSCs, activated the 68–75. non-canonical Wnt pathway and inhibited the canonical pathway 19 Ku¨hl M. Non-canonical Wnt signaling in Xenopus: regulation of axis formation and (and consequently the expression of osteogenic markers) even in gastrulation. Semin Cell Dev Biol 2002; 13: 243–249. the presence of co-cultured MM cells. These results support the 20 Liu F, Kohlmeier S, Wang CY. Wnt signaling and skeletal development. Cell Signal pathophysiological role of Ror2 in the MM-induced inhibition of 2008; 20: 999–1009. osteogenic differentiation and further suggest that non-canonical 21 Baksh D, Boland GM, Tuan RS. Cross-talk between Wnt signaling pathways in human mesenchymal stem cells leads to functional antagonism during osteo- Wnt signaling in hMSCs may be a therapeutic target for increasing genic differentiation. J Cell Biochem 2007; 101: 1109–1124. their osteogenic properties in MM-related bone disease. 22 Baksh D, Tuan RS. Canonical and non-canonical Wnts differentially affect the development potential of primary isolate of human bone marrow mesenchymal stem cells. J Cell Physiol 2007; 212: 817–826. CONFLICT OF INTEREST 23 Chang J, Sonoyama W, Wang Z, Jin Q, Zhang C, Krebsbach PH et al. Noncanonical Wnt-4 signaling enhances bone regeneration of mesenchymal stem cells in The authors declare no conflict of interest. craniofacial defects through activation of p38 MAPK. J Biol Chem 2007; 282: 30938–30948. 24 Santos A, Bakker AD, de Blieck-Hogervorst JM, Klein-Nulend J. WNT5A induces ACKNOWLEDGEMENTS osteogenic differentiation of human adipose stem cells via rho-associated kinase This study was supported by grants from the International Myeloma Foundation, the ROCK. Cytotherapy 2010; 12: 924–932. 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