Wnt Signaling Regulates the Invasion Capacity of Human Mesenchymal Stem Cells - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:03 |只看该作者 |倒序浏览 |打印

作者：Peter Neth, Marisa Ciccarella, Virginia Egea, Juergen Hoelters, Marianne Jochum, Christian Ries作者单位：Division of Clinical Chemistry and Clinical Biochemistry, Department of Surgery, Ludwig-Maximilians-University, Munich, Germany 3 L5 H7 t( g$ t' P7 X! u

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      【摘要】1 A+ a( Y6 c8 p  i: M/ I. H- C
   Human mesenchymal stem cells (hMSCs) exhibit the potential to contribute to a wide variety of endogenous organ tissue repair. However, the signals governing hMSC mobilization out of the bone marrow, release into the bloodstream, and migration/invasion into the target tissue are largely unknown. Since canonical Wnt signaling regulates not only tumor but also various stem cell attributes, we hypothesized that this signal transduction pathway might also be involved in governing the transmigration of hMSCs through human extracellular matrix (ECM). Stimulation of hMSCs with recombinant Wnt3a or LiCl resulted in the accumulation of the transcriptional activator &szlig;-catenin, its translocation into the nucleus, and the upregulation of typical Wnt target genes such as cyclin D1 and membrane-type matrix metalloproteinase-1 (MT1-MMP). Moreover, both stimuli significantly enhanced hMSC proliferation up to 40%. In addition, an increase of more than twofold in the ability of hMSCs to transmigrate through Transwell filters coated with human ECM was observed. In a reverse approach, Wnt signaling in hMSCs was inhibited by knocking down the expression of either &szlig;-catenin or low-density lipoprotein receptor-related protein 5 using RNA interference technology. These inhibition strategies resulted in downregulation of the Wnt target genes cyclin D1 and MT1-MMP, in a reduced proliferation rate, and in a strikingly diminished invasion capacity (64% and 52%). Taken together, this study provides for the first time decisive evidence that canonical Wnt signaling is critically involved in the regulation of the proliferation, as well as of the migration/invasion capacity of hMSCs, representing essential stem cell features indispensable during tissue regeneration processes. , I1 c2 {3 f# z: o
      【关键词】 Mesenchymal stem cells Migration Invasion Wnt signaling -Catenin Low-density lipoprotein receptor-related protein RNA interference Small interfering RNA' V, ]4 _& C) O9 A. w, o6 I
   INTRODUCTION; U6 J! u5 I9 W
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Human mesenchymal stem cells (hMSCs) are pluripotent bone marrow cells that can be expanded ex vivo and differentiated into several mesodermal lineages, such as cartilage, bone, and fat .

Studies on regulative elements of stem cell mobilization have led to several reports demonstrating that hMSCs express a restricted set of chemokine receptors and respond to the respective chemokines with targeted migration . Although considerable research has been devoted to the effectors of stem cell migration, rather less attention has been paid to the signal transduction pathways eliciting these mechanisms in hMSCs. In particular, the identification of molecular signaling cascades governing hMSC migration/invasion is of major importance to conduct hMSC-mediated therapy, either through mobilization of these cells out of the bone marrow or after exogenous application of this adult stem cell population for tissue repair.

One of the major signal transduction pathways that has been associated with various stem cell attributes is the Wnt signaling pathway. In this respect, it has been demonstrated that hematopoietic stem cells maintain an undifferentiated self-renewing state through constitutive activation of the canonical Wnt signaling pathway with Wnt3a, a prominent member of the Wnt family ." x$ @+ j8 k  I5 _( W
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Recently, it has been reported that the activation of the Wnt signal transduction pathway by Wnt3a-conditioned medium stimulates not only hematopoietic but also mesenchymal stem cell proliferation, while retaining pluripotency . Taken together, these data favor the assumption that the Wnt signaling pathway plays an important role in controlling typical stem cell attributes of hMSCs.# N; r0 r2 J# ]2 g! R" {- ?

In this study we demonstrate that canonical Wnt signaling induced by recombinant Wnt3a or LiCl is of pivotal importance not only in the regulation of hMSC proliferation but also in the capacity of these cells to migrate through human extracellular matrix (ECM). Moreover, inhibition of canonical Wnt signaling by RNA interference (RNAi) against either ß-catenin or low-density lipoprotein receptor-related protein 5 (LRP5) resulted in the repression of Wnt target genes, such as cyclin D1 and MT1-MMP, in a reduced proliferation rate, as well as in a drastically diminished invasion capacity of hMSCs. Thus, our data suggest for the first time that canonical Wnt signaling is critically involved in mediating the invasive characteristics of hMSCs, which represent an essential feature of activated stem cells during tissue repair processes.* T  a: }( v0 y6 ]" @
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MATERIALS AND METHODS
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Cell Culture and Proof of Stem Cell Features% D. Z6 T: `) {" N# A- O9 l3 j
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Cryopreserved hMSCs from three donors were purchased from Cambrex (Walkersville, MD, http://www.cambrex.com). Donor information is summarized in supplemental online Table 1. The hMSC isolates had been characterized by the manufacturer using flow cytometry. Cells were positive with regard to CD105, CD166, CD29, and CD44, and negative for CD14, CD34, and CD45. The cultivation of hMSCs was performed in mesenchymal stem cell growth medium (Cambrex), consisting of basal medium and growth supplements (50 ml of mesenchymal cell growth supplement, 10 ml of 200 mM L-glutamine, 0.5 ml of penicillin-streptomycin mixture). Cells were grown at 37¡ãC in a humidified atmosphere containing 95% air and 5% CO2 according to the supplier¡¯s instructions. Medium was refreshed twice a week, and cells were used for further subculturing or cryopreservation prior to reaching confluence. hMSCs used in our experiments were at the fifth or sixth passage of cultivation. For serum-free conditions, hMSCs were incubated in Dulbecco¡¯s modified Eagle¡¯s medium (DMEM) (PAA Laboratories, Linz, Austria, http://www.paa.at) supplemented with 1% Nutridoma SP (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com). Stimulation studies were carried out using recombinant mouse Wnt3a (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) or LiCl (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Their differentiation capacity along the mesodermal lineages was demonstrated as described previously .
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Small Interfering RNA Transfection of hMSCs, e( W+ D* g0 v* r0 t/ \" }

Small interfering RNAs (siRNAs) targeted against ß-catenin, LRP5, cyclin D1, and MT1-MMP were designed in our laboratory according to the protocol developed by Reynolds et al. . Sense and antisense oligonucleotides were synthesized by Qiagen (Hilden, Germany, http://www1.qiagen.com). Non-target-directed siRNA used as a negative control was also purchased from Qiagen. The siRNA sequences were as follows:

Negative control siRNA (sense): 5'-r(UUCUCCGAACGUGUCACGU)d(TT)-3'7 Z0 ^4 r* i5 a; v4 M
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Negative control siRNA (antisense): 5'-r(ACGUGACACGUUCGGAGAA)d(TT)-3', c" ^% U; ^' j, h

ß-catenin siRNA (sense): 5'-r(UGGUUGCCUUGCUCAACAA)d(TT)-3'

ß-catenin siRNA (antisense): 5'-r(UUGUUGAGCAAGGCAACCA)d(TT)-3'3 j3 z2 l- x, b! E) o
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LRP5 siRNA (sense): 5'-r(CCAACGACCUCACCAUUGA)d(TT)-3'
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LRP5 siRNA (antisense): 5'-r(UCAAUGGUGAGGUCGUUGG)d(TT)-3'/ q' ~3 E3 ?- ~  Y' |9 \+ y3 E

Cyclin D1 siRNA (sense): 5'-r(CCACAGAUGUGAAGUUCAU)d(TT)-3'

Cyclin D1 siRNA (antisense): 5'-r(AUGAACUUCACAUCUGUGG)d(TT)-3'2 Y; i* Z  \+ y2 Z; Y, D5 B7 g

MT1-MMP siRNA (sense): 5'-r(CCAGAAGCUGAAGGUAGAA)d(TT)-3'1 N! F* w: v# c) q6 s0 }& T

MT1-MMP siRNA (antisense): 5'-r(UUCUACCUUCAGCUUCUGG)d(TT)-3'.( ^. ^, h  Z6 H' H
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The negative control siRNA exhibits no target in the human transcriptome, whereas ß-catenin siRNA is directed against the region 842¨C860 of the human ß-catenin mRNA (NM_001904  ).3 P- w8 d3 e& `/ ?6 D
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One day before siRNA transfection, 48,000 hMSCs were plated in a cavity of a six-well plate by adding 2 ml of hMSC medium, resulting in 30% confluence after 24 hours of incubation. For preparing the transfection mixtures, the respective siRNAs were added to 250 µl of serum-free DMEM in a final concentration of 25 nM. In a separate tube, 5 µl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, http://www. invitrogen.com) was diluted in 250 µl of serum-free DMEM. After unifying both solutions, the final transfection mixture was incubated for 20 minutes at room temperature. This transfection solution was applied to the cells, and after an incubation period of 6 hours, it was replaced by 2 ml of hMSC medium.( D8 B5 Y: l8 a0 A. C" [: w0 d  H$ |

RNA Isolation and Quantitative Reverse Transcription-Polymerase Chain Reaction) L) C# H# _' ?- y; {9 }5 Z, J

DNase-digested total RNA was extracted from cultured cells using the RNeasy Mini Kit (Qiagen) according to the manufacturer¡¯s protocol. cDNA synthesis was performed from 100¨C500 ng of total RNA using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA, http://www.bio-rad.com) according to the instructions of the supplier. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) was carried out on a LightCycler (Roche) using LightCycler-FastStart DNA Master SYBR Green I Kit (Roche). Primer sets for the quantification of ß-catenin, cyclin D1, LRP5, and MT1-MMP transcripts were designed and purchased from Search-LC (Heidelberg, Germany, http://www.search-lc.de). PCR was performed with 1 µl of cDNA as a template, 2 µl of primer set (Search-LC), 2 µl of LC-FastStart DNA Master SYBR Green I mix, and 15 µl of H2O. Thermocycling was performed as follows: initial denaturation was carried out at 95¡ãC for 10 minutes, and amplification was achieved by 45 cycles at 95¡ãC for 10 seconds, at 68¡ãC for 10 seconds, and at 72¡ãC for 16 seconds. By comparison of the number of cycles with a given standard, the exact quantity of mRNA equivalents was determined. To demonstrate the fidelity of the primers and lack of secondary amplification, each amplicon was analyzed by melting curve analysis combined with agarose gel electrophoresis.# O7 w# V( {6 w! ^; Y5 ?

Cell Invasion Assay
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The invasive capacity of hMSCs was analyzed using Costar Transwell invasion chambers with polycarbonate membrane filters of 6.5 mm diameter and 8 µm pore size (Corning, Corning, NY, http://www.corning.com) to form dual compartments in a 24-well tissue culture plate. The membranes were coated with 10 µg of human ECM purified from human placenta (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) and dried overnight at room temperature under sterile conditions. The dried membranes were reconstituted with serum-free medium (DMEM) for 2 hours at 37¡ãC prior to the experiment. Samples each containing 5 x 103 cells in 200 µl of serum-free medium were added to the upper compartments. The lower compartments were filled with 600 µl of DMEM containing 10% human serum (PAA Laboratories) as a source of chemoattractants that had been depleted from gelatinases by gelatin-Sepharose affinity chromatography as described previously . The invasion chambers were incubated for 48 hours at 37¡ãC and 5% CO2. After incubation, cells and ECM on the top surface of the filters were wiped off with cotton swabs. Cells that had migrated into the lower compartment and attached to the lower surface of the filter were counted after staining with Diff Quick (Medion Diagnostics, D¨¹dingen, Switzerland, http://medion-diagnostics.com). The invasion rate was expressed as percentage of migrated cells compared with the total cell number seeded in the upper compartment of the chamber at the beginning of the experiment. Each measurement was performed in triplicate.
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SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis
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After stimulation with Wnt3a or transfection with siRNAs, cells were cultivated for 1, 3, or 7 days and subsequently trypsinized (0.5% trypsin, 5.3 mM EDTA). Protein isolation was carried out using lysis buffer (50 mM Na2HPO4; 50 mM NaH2PO4; 0.2 M NaCl; 5 mM EDTA; 1% Triton X-100, pH 6.0). After a 30-minute incubation on ice, samples were centrifuged (13.000g, 20 minutes, 4¡ãC), and 2x dithiothreitol (DTT) loading buffer (0.4 M Tris, pH 6.8; 4% SDS; 20% glycerol; 10% DTT) was added to the sample supernatants, with subsequent incubation for 5 minutes at 95¡ãC. Following electrophoretic separation by SDS-polyacrylamide gel electrophoresis, proteins were electroblotted on nitrocellulose membranes (Whatman, Brentford, U.K., http://www.whatman.com). The membranes were blocked in NET buffer (150 mM NaCl; 5 mM EDTA, pH 8.0; 50 mM Tris/HCl, pH 7.5; 0.05% Triton X-100) containing 2.5% gelatin (Merck) for 1 hour at room temperature. Polyclonal antibodies against ß-catenin (goat anti-ß-catenin sc-1496; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), LRP5 (goat anti-LRP5 sc-21389; Santa Cruz Biotechnology), and ß-actin (goat anti-ß-actin sc-1616; Santa Cruz Biotechnology) were used at dilutions of 1:750 (ß-catenin) and 1:400 (LRP5, ß-actin). Incubation was performed for 1 hour at room temperature. Thereafter, membranes were washed in NET buffer, and a further incubation was carried out with a peroxidase-conjugated antibody (sheep anti-goat IgG; Sigma-Aldrich) at a dilution of 1:20,000. For visualization of the protein bands, the enhanced chemiluminescence system was used as recommended by the manufacturer (GE Healthcare, Little Chalfont, U.K., http://www.amershambiosciences.com). Semiquantitative evaluation of the bands was performed by densitometric analysis. The protein expression levels of ß-catenin and LRP5 were thereby normalized to that of the housekeeping gene ß-actin.- K- p- g5 p( i  R7 d. f& D4 ]2 C

CyQuant Cell Proliferation Assay/ E- w8 N+ V2 |$ f, W

Cell proliferation was quantified using the CyQuant cell proliferation assay kit (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com) according to the manufacturer¡¯s protocol. For fixation, cells were washed in phosphate-buffered saline (PBS) and stored at ¨C80¡ãC until further processing. After thawing, cell lysis was performed in 250 µl of lysis buffer. Two 100-µl aliquots were transferred into the cavities of a 96-well microtiter assay plate (Corning), and each was mixed with 100 µl of cell lysis buffer containing CyQuant GR dye. After 5 minutes of incubation, fluorescence was measured using a fluorescence microplate reader (HTS 7000 Bio Assay Reader; PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) at 480 nm excitation and 530 nm emission. For each experiment, a standard calibration curve was generated by plotting the measured fluorescence values of the samples versus the respective cell number that had been determined before using a hematocytometer.6 ?# r2 R, G9 h" Z: s
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Immunocytochemistry

For subcellular localization of ß-catenin by immunocytochemistry, hMSCs were plated onto chamber slides (BD Biosciences, San Diego, http://www.bdbiosciences.com), resulting in 25% confluence, and were treated with 150 ng/ml Wnt3a 24 hours before paraformaldehyde fixation (4%) was performed for 30 minutes at 37¡ãC. Permeabilization of the cells was achieved by a 30-minute incubation at 37¡ãC with PBS containing 0.2% Triton X-100. To minimize unspecific binding of the antibody, blocking was carried out with buffer containing 1% bovine serum albumin (Sigma-Aldrich) for 1 hour. The ß-catenin antibody (goat anti-ß-catenin sc-1496; Santa Cruz Biotechnology) was applied at a 1:10 dilution for 30 minutes. As a specificity control, PBS was used instead of the primary antibody to exclude unspecific binding of the secondary antibody. After repeated washing with PBS, the cells were incubated with a donkey anti-goat antibody labeled with fluorescein isothiocyanate (Santa Cruz Biotechnology) for an additional 30 minutes. Finally, cells were mounted with ProLong AntiFade Kit (Molecular Probes) by a cover slip. Analysis was performed by fluorescence microscopy.4 I% y2 d2 b- }
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Osteogenic and Adipogenic Differentiation and Histochemical Staining of hMSCs

Osteogenic and adipogenic differentiation of hMSCs were carried out as described previously  FCS, 40 IU/ml Pen/Strep, 4 mM L-glutamine, 0.01 mg/ml insulin). To monitor osteogenic differentiation in hMSCs, Alizarin red staining was performed. For this purpose, hMSCs were washed in PBS three times and fixed in 70% ethanol at ¨C20¡ãC for 1 hour. The cells were then stained by the addition of 40 mM Alizarin red S (Sigma-Aldrich) for 10 minutes and washed three times with PBS. Adipogenic differentiation was assayed using oil red O staining for the accumulation of lipid droplets. For this purpose, cells were rinsed in PBS and fixed in prechilled 4% paraformaldehyde for 5 minutes at room temperature. Thereafter, the cell layers were stained with 0.2% oil red O in 60% isopropanol and washed with 50% ethanol to remove excess stain.

Statistical Analysis
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Statistical significance was assessed by comparing mean values (¡À SD) using Student¡¯s t test for independent groups. Significance was assumed for p
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RESULTS
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All of the following detailed results (graphical representation) are derived from hMSCs that had been isolated from donor I (supplemental online Table 1). Data obtained from this hMSC lot are representative of the results collected from three different donor cell populations that were analyzed in this study (data summarized in supplemental online Tables 2, 3).

Activation of the Wnt Pathway  v# S7 \) s  n+ G1 t6 Y! q

Activation of the Wnt Pathway Results in the Accumulation of ß-Catenin, Its Translocation into the Nucleus, and the Transcriptional Activation of Wnt Target Genes. To investigate whether treatment of hMSCs with recombinant Wnt3a (150 ng/ml) influences ß-catenin in hMSCs on the protein level, we monitored the expression of ß-catenin by semiquantitative Western blot analysis during a time period of 7 days. An accumulation of ß-catenin protein (1.7-fold) was observed 1 day after addition of Wnt3a. This effect remained stable until day 3 (1.5-fold) and day 7 (2.2-fold) during incubation with Wnt3a (Fig. 1A).2 H$ g0 Y4 ?* {& J% j6 j
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Figure 1. Effects of Wnt3a on ß-catenin expression, its subcellular localization, and the induction of typical Wnt target genes. (A): Stimulation of human mesenchymal stem cells (hMSCs) with 150 ng/ml Wnt3a revealed a clear increase of the ß-catenin protein level, as shown by Western blot analysis. For quantification, ß-catenin protein signals were scanned by densitometric analysis and normalized to ß-actin. (B): Immunocytochemistry analysis of ß-catenin exhibited mainly a perinuclear staining of ß-catenin in unstimulated hMSCs (upper panels), whereas addition of 150 ng/ml Wnt3a for 1 day (lower panels) evoked a clear nuclear staining of ß-catenin. Immunofluorescence was performed using a polyclonal antibody against ß-catenin (left panels). In addition, the nucleus was stained with 4,6-diamidino-2-phenylindole (right panels). Scale bars = 50 µm. (C): The mRNA expression levels of the known Wnt target genes cyclin D1 and MT1-MMP were quantified at days 1 and 7 during stimulation with Wnt3a (150 ng/ml) and normalized to the expression levels in unstimulated hMSCs (set as 100%). Both genes were upregulated under stimulating conditions on days 1 and 7, with a more prominent effect in the case of cyclin D1. (D): Similar induction rates for cyclin D1 and MT1-MMP were observed when hMSCs were stimulated with 1 mM LiCl. The expression levels were normalized to that of hMSCs treated with 1 mM NaCl (control). Data are presented as mean ¡À SD of one triplicate experiment that is representative of three independent experiments (*, p & Z' Y% p. \! d) I
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To confirm the data obtained by Western blot analysis and to prove whether Wnt3a stimulation has any influence on the subcellular localization of ß-catenin, immunocytochemistry was performed. Untreated hMSCs (control) exhibited perinuclear staining for ß-catenin. In contrast, treatment with Wnt3a (150 ng/ml) for 1 day revealed a clear nuclear staining of ß-catenin, suggesting that Wnt3a stimulation is responsible for the accumulation of ß-catenin and for its translocation from the cytoplasm into the nucleus (Fig. 1B).

To evaluate whether Wnt3a-mediated accumulation of ß-catenin would result in the activation of typical Wnt target genes, the mRNA expression levels of cyclin D1 and MT1-MMP were quantified by quantitative RT-PCR (qRT-PCR) 1 and 7 days after Wnt3a stimulation (150 ng/ml). At both time points, a significant induction of cyclin D1 and a slight increase of MT1-MMP mRNA was observed (Fig. 1C). In addition, we examined the influence of the GSK-3ß inhibitor LiCl as an alternative activator of the Wnt signaling pathway. Incubation of hMSCs with LiCl (1 mM) led to an upregulation of cyclin D1, as well as of MT1-MMP (Fig. 1D), similar to that observed under Wnt3a stimulatory conditions.+ v' |) T9 B2 Z' _3 G

Taken together, these findings demonstrate that the activation of the Wnt pathway in hMSCs results in an accumulation of ß-catenin, its translocation into the nucleus, and, furthermore, in an enhanced expression of Wnt target genes.

Wnt3a and LiCl Induce hMSC Proliferation Without Affecting the Progenitor Cell Phenotype. To investigate whether the application of recombinant Wnt3a would exhibit any effect on the proliferative behavior of hMSCs, we assayed different concentrations of Wnt3a. We observed an increase in cell proliferation with a maximum (140%) at a concentration of 150 ng/ml Wnt3a (Fig. 2A). In a similar manner, stimulation of hMSCs by LiCl (1 and 4 mM) resulted in a comparable increase in cell proliferation. To exclude the possibility that this effect was caused by Cl¨C ions, controls were performed with the corresponding concentrations of NaCl, indicating that only Li  ions mediate the enhanced proliferation (Fig. 2B). The data sets for all three donor cell populations are given in supplemental online Table 2.% G' J! }3 s( Y
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Figure 2. Proliferation behavior of human mesenchymal stem cells (hMSCs) upon treatment with Wnt3a or LiCl. Cell numbers were quantified using the CyQuant cell proliferation assay kit. (A): One week of stimulation with recombinant Wnt3a significantly enhanced the proliferation of hMSCs in a dose-dependent manner. (B): A similar increase in hMSC numbers was induced by 1 and 4 mM LiCl as compared with control or NaCl-treated cells. Data are presented as mean ¡À SD of one triplicate experiment. Similar results were obtained in three independent experiments. *, p
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To evaluate whether the Wnt3a- or LiCl-treated cells maintain their undifferentiated hMSC progenitor cell character and function, we induced differentiation along the osteogenic and adipogenic lineage 7 days after the addition of these stimuli. The cells were still able to undergo the typical morphological changes during osteogenic, as well as adipogenic, differentiation (supplemental online Fig. 1). In addition, these cells revealed Ca2  deposition (Alizarin red staining) or accumulation of fat vacuoles (oil red O staining), reflecting a primarily unchanged progenitor cell phenotype during Wnt3a or LiCl stimulatory conditions. Furthermore, we analyzed whether treatment with Wnt3a or LiCl might influence the induction of hMSC differentiation. Neither osteogenic nor adipogenic differentiation was observed (supplemental online Fig. 2).7 f, c  v$ e/ s, F

Wnt3a and LiCl Enhance the Invasion Capacity of hMSCs Through Human ECM. Based on the fact that activated mesenchymal stem cells have to emigrate out of the bone marrow to reach areas of tissue defects, we analyzed whether the activation of the Wnt signaling pathway would influence the invasive capacity of hMSCs. For this purpose, Transwell filters coated with human ECM were used. In the presence of 150 and 200 ng/ml Wnt3a, the transmigration rate was increased 2.3- and 1.9-fold, respectively (Fig. 3A). In addition, application of LiCl at concentrations of 1 and 4 mM enhanced hMSC invasion in a dose-dependent manner compared with control and NaCl-treated cells (Fig. 3B). Supplemental online Table 2 summarizes the corresponding data of all three donor cell populations.0 \& P3 p# @" C" D! K4 ]
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Figure 3. Invasion rates of human mesenchymal stem cells (hMSCs) upon stimulation with Wnt3a or LiCl. Transwell filters coated with human extracellular matrix were used, and absolute cell invasion rates were calculated in the lower compartment 48 hours after seeding the cells in the upper compartment (Materials and Methods). (A): The addition of 150 and 200 ng/ml Wnt3a resulted in an increase of more than twofold of cell invasion. **, p : q# N, B( O% T
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Inhibition of Wnt Signaling: Knockdown of Wnt Pathway Components
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RNA Interference Against ß-Catenin and LRP5 Induces an Efficient and Long-Lasting Knockdown on mRNA and Protein Level and Impairs Wnt Signaling. Since hMSCs showed an increased proliferation as well as invasion capacity upon activation of Wnt signaling, we analyzed whether inhibition of this pathway by knocking down ß-catenin as a downstream mediator or LRP5 as a coreceptor of Wnt ligands would affect these characteristics in the opposite way. Therefore, hMSCs were transfected with siRNAs either against ß-catenin or LRP5. The knockdown efficiency was examined on mRNA and protein level by quantitative RT-PCR and semiquantitative Western blot analysis, respectively. On mRNA level knockdown efficiencies of 80%¨C95% for ß-catenin and 70%¨C81% for LRP5 were observed during a time period of 7 days after transfection as compared with control cells treated with non-target-directed siRNA (Fig. 4A). Consistently, the amounts of the respective proteins were also significantly reduced under specific knockdown conditions in comparison with the controls (Fig. 4B). Interestingly, the reduction of ß-catenin and LRP5 on mRNA level was already observed at day 1 after siRNA transfection (Fig. 4A), whereas the knockdown on protein level became most obvious on days 3 and 7 in the case of ß-catenin and on day 7 with regard to LRP5 (Fig. 4B). As shown in supplemental online Table 3, similar data were obtained for the mRNA and protein reduction of ß-catenin and LRP5 using RNAi approaches in hMSCs of all three donors.2 U' \2 {0 C; M
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Figure 4. siRNA-mediated knockdown of ß-catenin and LRP5 in human mesenchymal stem cells (hMSCs) on transcriptional and protein level. (A): The mRNA transcripts were quantified after hMSC transfection with the respective siRNAs. Compared with the non-target-directed nc-siRNA (set as 100%), the mRNA levels were decreased to 5%¨C20% for ß-catenin and to 19%¨C30% for LRP5 during a time period of 7 days. (B): The knockdown efficiencies on the protein level of ß-catenin (left panel) and LRP5 (right panel) were normalized to that of unaffected ß-actin. A substantial reduction of ß-catenin was observed at day 3 (31%) and was most distinctive at day 7 (67%). A diminished LRP5 protein expression (47%) was observed 7 days after transfection. nc-siRNA-transfected cells were used as a control to evaluate the respective knockdown efficiencies. Results are mean ¡À SD of one triplicate approach that is representative of three independent experiments. **, p 9 J7 L& E9 J; }" o
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To evaluate whether silencing of ß-catenin would affect the basal activity of the Wnt signal transduction pathway in hMSCs associated with an impaired transcription of Wnt target genes, we quantified cyclin D1 and MT1-MMP mRNA expression by qRT-PCR on days 1 and 7 after induction of RNAi. Most notably, a prominent reduction in cyclin D1 and MT1-MMP mRNA levels was detected 7 days after siRNA transfection (Fig. 5A).7 r6 b6 D  }$ Q* E, z1 I

Figure 5. Transcriptional changes of Wnt target genes after silencing of ß-catenin and/or LRP5. (A): Quantification of mRNA expression of cyclin D1 and MT1-MMP was performed 1 and 7 days after transfection with ß-catenin siRNA. A statistically significant decrease of the respective mRNA transcripts could be detected for both genes at day 7 post-transfection. (B): Quantitative reverse transcription-polymerase chain reaction analysis of cyclin D1 was carried out in ß-cat-siRNA- or LRP5-siRNA-transfected human mesenchymal stem cells during stimulation with Wnt3a (150 ng/ml). Due to the fact that the lowest protein levels for ß-catenin and LRP5 were observed 1 week after the respective siRNA transfection, Wnt3a stimulation was performed on day 6 after transfection for 24 hours. The expression levels of cyclin D1 were quantified 7 days after siRNA transfection. In both cases, a significant decrease in cyclin D1 expression was observed compared with the controls. Data are mean ¡À SD of one triplicate approach that is representative of three independent experiments. *, p ' x, C9 f( R$ K6 v% g
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To study whether gene silencing of either ß-catenin or LRP5 would diminish Wnt3a response on Wnt target genes, the mRNA expression level of cyclin D1 was monitored by qRT-PCR. In both siRNA transfection approaches, a significantly reduced expression of cyclin D1 could be observed in comparison with negative control siRNA-transfected hMSCs upon treatment with 150 ng/ml Wnt3a (Fig. 5B).
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Knockdown of Both ß-Catenin and LRP5 Reduces hMSC Proliferation and Invasion. As Wnt3a and LiCl application resulted in an increase of cell proliferation, we were interested whether inhibition of Wnt signaling accomplished by the knockdown of either ß-catenin or LRP5 would lead to a diminished growth behavior of hMSCs. Proliferation studies performed 7 days post-siRNA transfection revealed that the knockdown of either ß-catenin or LRP5 resulted in a significant reduction of cell proliferation (Fig. 6A).$ h' f0 A) b' H5 H

Figure 6. Proliferation and invasion behavior of human mesenchymal stem cells (hMSCs) after RNA interference against ß-catenin and LRP5. (A): Transfection with ß-cat-siRNA and LRP5-siRNA resulted in a significant decrease in cell proliferation compared with nc-siRNA-transfected hMSCs. All proliferation assays were carried out at day 7 after siRNA transfection. (B): Invasion assays were performed within a time frame of 48 hours between day 5 and day 7 after transfection of the respective siRNAs. The knockdown of ß-catenin resulted in a 64% reduced cell invasion. LRP5-siRNAs caused a drop in the invasion rate of 52%. Data are mean ¡À SD of one triplicate approach representative of three individual experiments. **, p + a9 x4 Q7 G8 ]( z) D

On the basis of these results, we analyzed whether blocking of Wnt signaling would diminish hMSC invasion as well. As described above, we observed a highly efficient knockdown of ß-catenin and LRP5 on mRNA and with a delay of 3¨C7 days post-transfection on protein level. For this reason, invasion assays were performed for 48 hours between day 5 and day 7 after transfection with the respective siRNAs. The experiments revealed a highly significant reduction of cell invasion to 36% ¡À 7% and 48% ¡À 9% for ß-catenin and LRP5, respectively (Fig. 6B).
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Taken together, these results strongly suggest that inhibition of Wnt signaling in hMSCs¡ªaccomplished by the knockdown of either ß-catenin or LRP5¡ªresults not only in a downregulation of typical Wnt target genes but also in decreased cell proliferation, as well as in a strikingly diminished invasion capacity.
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Interference of Wnt Target Gene Expression Exhibits a Direct Connection to the Proliferation and Invasion Behavior of hMSCs. To evaluate whether there exists a direct link between the expression of the Wnt target genes cyclin D1 and MT1-MMP and the proliferation or invasion capacity of hMSCs, we performed RNA interference against these two transcripts. The knockdown efficiency was examined on mRNA level by quantitative RT-PCR and ranged from 90% to 61% for cyclin D1 and from 79% to 41% for MT1-MMP during a time period of 7 days (Fig. 7A).

Figure 7. RNA interference against cyclin D1 and MT1-MMP exhibits a direct connection to proliferation and invasion of human mesenchymal stem cells (hMSCs). (A): Compared with the non-target-directed nc-siRNA (set as 100%), transfection of hMSCs with the respective target-directed siRNAs decreased cyclin D1 mRNA to 10%¨C39% and MT1-MMP mRNA to 21%¨C59% during a time period of 7 days. (B): Cell proliferation studies were performed 7 days after transfection of hMSCs with cyclin D1-siRNA. A significant reduction in cell proliferation by 25% was observed as a consequence of the knockdown of cyclin D1 compared with nc-siRNA-transfected hMSCs (set as 100%). Interestingly, this effect was even more pronounced upon activation of the Wnt pathway by either Wnt3a (37%) or LiCl (51%). (C): Invasion assays were accomplished for 48 hours directly after transfection of hMSCs with nc-siRNA (set as 100%) or MT1-MMP-siRNA in the presence or absence of Wnt3a or LiCl. The knockdown of MT1-MMP in nonstimulated hMSCs (control) resulted in a 53% reduced cell invasion rate. hMSCs transfected with MT1-MMP-siRNA and treated with either Wnt3a or LiCl exhibited a less distinctive reduction in their invasive behavior (33% and 34%). Results are mean ¡À SD of one triplicate approach that is representative of three independent experiments. **, p

Proliferation studies performed 7 days post-cyclin D1 siRNA transfection revealed a significant reduction in proliferation compared with negative control siRNA-transfected hMSCs (Fig. 7B). Interestingly, this phenomenon was even more pronounced upon activation of the Wnt pathway either by Wnt3a or LiCl. With respect to the analysis of the invasive behavior of hMSCs, the knockdown of MT1-MMP resulted in a dramatic decrease of their ability to transmigrate through human ECM (Fig. 7C). This effect was also observed upon Wnt3a or LiCl treatment. Taken together, these results strongly suggest that cyclin D1 and MT1-MMP have a major impact on the proliferation and invasion capacity of hMSCs, respectively, and that the expression of both genes is primarily regulated by the canonical Wnt signal transduction pathway.
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DISCUSSION

The regulative network conducting adult stem cells in endogenous tissue repair is of prime interest for understanding organ regeneration and preventing degenerative diseases .- k+ H+ o# I+ M

To activate canonical Wnt signaling, hMSCs were stimulated with different amounts of recombinant Wnt3a, which resulted in a dose-dependent increase of cellular proliferation. This observation is in line with the findings that Wnt3a-conditioned medium promotes proliferation in hMSCs  were induced in hMSCs as well. Moreover, activation of the Wnt signal transduction cascade resulted in an enhanced migration of hMSCs through human ECM, whereas inhibition of Wnt signaling by RNAi either against ß-catenin or LRP5 diminished the invasive capacity of hMSCs.  R5 p: n1 a' w' O

Based on the fact that activated stem cells have to emigrate from the bone marrow to reach the designated location for tissue repair .8 ]9 c3 j' b$ P. g$ `. ]+ t3 Q

To our knowledge, the present study is the first experimental evidence that the activation of the canonical Wnt signal transduction pathway either by Wnt3a or LiCl results in an enhanced invasion capacity of hMSCs promoting their ability for transmigration of human ECM. So far, an association of Wnt and cellular invasion has only been reported for tumor cells. In a recent study, conditioned medium containing Wnt3a promoted transmigration of myeloma plasma cells through endothelial cells and reconstituted mouse basement membranes (Matrigel)  may also suggest that ß-catenin signaling is involved in mediating an invasive cellular phenotype.; S" B& s# U/ z# W! }4 @
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To further prove our hypothesis that the Wnt signal transduction cascade is associated with the proliferation as well as invasion behavior of hMSCs, we used a reverse approach to block canonical Wnt signaling. We performed a dualistic strategy by knocking down either the upstream mediator LRP5 or the downstream mediator ß-catenin to see whether both interventions would result in a similar hMSC phenotype. For this purpose, we used our recently established nonviral siRNA transfection approach, which enables a highly efficient and long-term RNA interference in hMSCs . Both RNAi attempts resulted in a prominent knockdown of ß-catenin and LRP5 on mRNA level on day 1 after transfection, remaining highly efficient until day 7. In contrast, the reduction of ß-catenin and LRP5 on protein level was most obvious between day 3 and day 7 following RNAi. These data suggest that the half-life of both target proteins is significantly longer than that of the corresponding mRNA molecules. Therefore, the following proliferation and invasion assays on hMSCs were performed in the time frame of the lowest protein levels upon RNAi.
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The knockdown of ß-catenin resulted in a clearly diminished proliferation rate, as well as in a drastically reduced invasive behavior of hMSCs. Similar findings have been observed in colon cancer cells, where RNAi against ß-catenin evoked a markedly decreased ß-catenin-dependent gene expression and a reduction of cellular proliferation in vitro, as well as in vivo  also exhibited a significant downregulation in hMSCs in our experiments applying RNAi against ß-catenin.
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To investigate whether there exists a direct correlation between the expression levels of cyclin D1 and hMSC proliferation, we performed RNAi against cyclin D1, which plays a fundamental role in the cell cycle during the G1-S-phase transition .

With respect to the regulation of hMSC migration/invasion by Wnt signaling, it is of particular importance that the variety of Wnt target genes encompasses several members being involved in the process of cancer cell invasion, such as urokinase-type plasminogen activator (uPA) .. f+ ~$ ?* p* h; @9 {0 {; a

Although proteases such as MMPs seem to play a major role in the migration/invasion process, we cannot exclude the possibility that the diminished invasion capacity as a consequence of ß-catenin downregulation by RNAi is, at least in part, a result of a reduced cadherin/ß-catenin interaction. Basically, ß-catenin regulates cell functions not only by its interaction with nuclear transcription factors of the TCF/LEF family but also through its important role in cellular architecture. Via binding to the cytoplasmic domain of type I cadherins, ß-catenin assists the structural organization and function of cadherins by linking cadherins through -catenin to the actin cytoskeleton .
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In an additional approach to study Wnt-dependent effects, we performed RNAi against LRP5, since this coreceptor is also involved in the activation of the canonical Wnt pathway . To confirm that the cellular effects observed after LRP5 and ß-catenin knockdown are due to a diminished Wnt3a binding capacity and ß-catenin-mediated signal transduction, respectively, LRP5- and ß-catenin-siRNA-transfected hMSCs were stimulated with Wnt3a. These experiments clearly showed that the upregulation of the Wnt target gene cyclin D1 is strongly influenced by Wnt3a binding to LRP5 and that the resulting signal transduction is dependent on the presence of ß-catenin.
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Moreover, the knockdown of LRP5 not only resulted in a reduced proliferation rate but also dramatically diminished hMSC invasion through human ECM, indicating that this process is clearly dependent on canonical Wnt signaling mediated via LRP5. In this regard, our findings on canonical Wnt signaling processes in hMSCs are closely related to published data in tumor biology, especially concerning ß-catenin involvement in cancer metastasis , which promotes the invasive cellular behavior.
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Taken together, our study indicates for the first time that hMSCs, similar to tumor cells, use canonical Wnt signaling to mediate their migration/invasion behavior. The acquisition of such an ability for invasion and movement through tissues might represent part of a program during stem cell activation required for optimal execution of tissue repair and regeneration  may require careful consideration in future tumor therapies, since this approach may negatively affect characteristic stem cell features such as self-renewal and invasiveness during physiological tissue repair processes.  f: r' [! e4 D1 L1 M* m/ d7 {1 D

DISCLOSURES; f* J7 @  ]; G5 }9 ^# }: i
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS; k& [/ R' e4 g
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This work was supported by Grant 260 of the Medical Faculty (Förderprogramm f¨¹r Forschung und Lehre) (P.N.). In addition, we greatly appreciate partial financial support by Prof. Dr. W. Mutschler (Director of the Surgical Department, City Center, Ludwig-Maximilians-University of Munich). We thank Claudia Geissler for excellent technical assistance. P.N. and M.C. contributed equally to this work.2 ?$ [- H$ `7 _1 f
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