From the Center for Gene Therapy, Tulane University Health Sciences Center, New Orleans, Louisiana 70112
Received for publication, January 13, 2003 , and in revised form, April 30, 2003.
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ABSTRACT |
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INTRODUCTION |
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Preliminary observations (15) suggested to us that conditioned medium from cultures of hMSCs increased the rate of proliferation when added to freshly plated cultures of hMSCs. In the experiments described here, we demonstrate that hMSCs in the early log phase of growth synthesize and secrete dickkopf-1 (Dkk-1), an inhibitor of the canonical Wnt pathway (1618).
The Wnt signaling pathway controls patterning and cell fate determination
in the development of a wide range of organisms, from Drosophila to
mammals (19). The signaling
can occur by at least three different pathways
(20). In the canonical
pathway, Wnt ligands bind to the transmembrane receptor frizzled and the
co-receptor lipoprotein-related proteins 5 and 6 (LRP-5/6). Activation of
frizzled recruits the cytoplasmic bridging molecule, disheveled, so as to
inhibit glycogen synthetase kinase 3. Inhibition of glycogen synthetase kinase
3 decreases phosphorylation of -catenin, preventing its degradation by
the ubiquitin-mediated pathway
(20,
21). The stabilized
-catenin translocates to the nucleus, where it complexes with
transcription factors that promote either differentiation or proliferation
(22,
23). Also, the
-catenin
stabilizes adherens junctions
(24). The binding of Dkk-1 to
LRP-5/6 and to the associated protein kremen dissociates LRP-5/6 from frizzled
and thereby prevents the formation of a functional Wnt receptor complex
(2529).
The decrease in Wnt signaling destabilizes
-catenin and inhibits
Wnt-induced transcriptional regulation. This in turn can influence cell fate
and cell growth and may predispose cells to apoptosis
(17,
30). Signaling through Wnt
ligands has been shown to have a role in differentiation of neural systems
(31,
32), skeletal muscle
(33), cardiac cells
(34), endoderm
(35), cartilage
(36), and limbs
(37). The effects of Wnt
signaling, however, are complex in part because some members of the family of
Wnt ligands can trigger signaling through noncanonical pathways involving
Jun/Jun kinase (JNK) or calcium ion regulation
(20). For example, alternative
signaling through the canonical Wnt/
-catenin and the noncanonical
Wnt/Ca2+ pathways in Xenopus produced
antagonistic regulation of covergent extension movements during gastrulation.
Also, alternative signaling through Wnt and two different frizzled receptors
in Drosophila drove either epithelial planar polarity or
morphogenesis by activating different regions of the same disheveled bridging
molecule (39). In addition, a
second dickkopf-2 (Dkk-2) activated instead of inhibited Wnt/
-catenin
signaling in Xenopus
(40) through a pathway that
was independent of disheveled
(41).
The results presented here demonstrate that after stationary phase, when
hMSCs are replated at clonal densities, synthesis of the Wnt inhibitor Dkk-1
allows cells to reenter the cell cycle by inhibiting the canonical
Wnt/-catenin signaling pathway.
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EXPERIMENTAL PROCEDURES |
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Preparation of Labeled Media and Cell ExtractsTo remove unwanted cells and debris, the media were filtered through a 0.22-µm2 pore size membrane (Millipore Corp., Bedford, MA). To remove unincorporated [35S]methionine, the medium was diafiltered against 10 volumes of PBS (Sigma) using a tangential flow filtration system fitted with 150-cm2 polyvinylidene difluoride 5-kDa filters (Millipore). Cells were counted with a hemacytometer, followed by lysis in PBS containing 0.01% (w/v) SDS (Sigma). The cell lysates were dialyzed against 1000 volumes of 1x PBS for 24 h using 3500-Da limiting dialysis cassettes (Pierce). Radioactivity was assayed by liquid scintillation counting using 30% scintillant (Scintisafe; Fisher).
Electrophoretic Analysis and Western BlottingUnless
otherwise stated, electrophoresis was carried out using commercial reagents
and systems (Novex; Invitrogen). Two µl of medium were added to 5 µl of
SDS-PAGE sample buffer and 1 µl of 2-mercaptoethanol (Sigma). The samples
were heated at 100 °C for 2 min and electrophoresed on a 412%
NuPage bis-Tris gel using the MES buffering system. In some experiments,
samples were loaded in triplicate and at different dilutions to assess
aberrant migration due to the presence of excessive serum albumin. Gels were
either silver-stained (Silver Quest Staining Kit; Invitrogen) or blotted onto
polyvinylidene difluoride filters for autoradiography and immunoblotting. For
autoradiographic analysis, filters were air-dried and exposed to
autoradiography film (Kodak Biomax MR; Sigma). After a 2-day exposure, the
film was automatically developed using a commercial instrument and reagents
(AGFA Corp.). For immunoblotting, filters were blocked in PBS containing 0.1%
(v/v) Tween 20 (Sigma) for 1 h. For detection of -catenin, blots were
probed with an anti-
-catenin monoclonal antibody at a dilution of 1:1000
(clone 5H10; Chemicon International, Temecula, CA). For detection of Dkk-1,
blots were probed in 1 µgml1 of anti-Dkk-1
polyclonal antibody (see below) followed by an anti-rabbit
peroxidase-conjugated monoclonal antibody (clone RG 96; Sigma). For detection
of the polyhistidine tag, blots were probed with a peroxidase-conjugated
monoclonal antibody at a dilution of 1:1000 (clone his-10; Sigma). For
detection of Jun and phospho-Jun polyclonal antibodies, SC 45 and SC 16312
were used (Santa Cruz Biotechnology, Santa Cruz, CA) at a 1:100 dilution. VCAM
was detected with the monoclonal antibody (clone MAB2143; Chemicon
International). For detection of actin, blots were probed with a monoclonal
antibody at a dilution of 1:1000 (clone AC-10; Sigma). For detection of GAPDH,
blots were probed with a monoclonal antibody at a dilution of 1:1000 (clone
6C5; Chemicon International). All unconjugated monoclonal antibodies were
detected with an anti-mouse horse-radish peroxidase-conjugated rabbit serum
(Sigma), and unless otherwise stated, polyclonal antisera were detected by
either goat anti-rabbit or rabbit anti-goat horseradish peroxidase conjugates
(Sigma). Positive bands were detected by chemiluminescence in accordance with
a previously described procedure
(42) using an imaging system
to detect and quantify the signal (Typhoon Imaging System; Amersham
Biosciences). Equal sample loading for Western blots was confirmed by Bradford
protein assay (Sigma) and measuring cell number. Equal transfer to the
membrane was confirmed by Ponceau S staining (Sigma). Western blots and
autoradiographic analyses were repeated using hMSCs from at least two donors.
Blots were stripped using the Blot Restore kit (Chemicon International).
Electroelution and Tryptic Fingerprinting of Bands200 µl
of 5-fold concentrated radiolabeled medium were separated by electrophoresis
on a 420% polyacrylamide Tris-glycine preparative gel (Invitrogen). 15
fractions were laterally electroeluted into 1 ml of 100 mM ammonium
bicarbonate (pH 8.0) using a whole gel eluter system (Bio-Rad). The fractions
were analyzed by SDS-PAGE followed by 10-fold concentration by rotary
evaporation (Savant AES 2010 Rotary Evaporation System; Savant Inc., Holbrook,
NY). Samples were proteolytically digested in 50-µl reactions containing
100 mM ammonium bicarbonate (pH 8.0) in the presence of 5 ng of
agarose-coupled trypsin (Sigma). The reaction was incubated at 37 °C for
16 h followed by removal of the trypsin by centrifugation. Analysis by mass
spectrometry was carried out using commercial instruments and reagents
(Ciphergen Biosystems Inc., Freemont, CA). Aliquots (2 µl) of digested
samples were mixed with 2 µl of a saturated solution of
-cyano-4-hydroxycinnamic acid in acetonitrile. The mixture was
air-dried onto silica-coated aluminum mass spectrometry chips and analyzed
using a PBS II SELDI-TOF chip reader. The program PeptIdent
(43) was used to analyze
triplicate data sets and appropriate controls with settings for the detection
of acrylcisteinyl groups and oxidized methionine residues. Both the Swiss Prot
and TREMBL databases were searched. Data were reproduced using samples from
three donors.
Antibody Production and PurificationA 15-residue peptide corresponding to a sequence in the second cysteine-rich domain of Dkk-1 (17), ARHFWSKICKPVLKE, was synthesized and conjugated to key-hole limpet hemocyanin (Sigma). The conjugated peptide was used to immunize two New Zealand White rabbits. Antibodies were purified from 20-ml aliquots of postimmune serum by affinity chromatography against the immunizing peptide. Briefly, 5 mg of peptide at a concentration of 1 mg ml1 in 100 mM sodium bicarbonate (pH 8.2) was cycled through a 1-ml N-hydroxysuccinimide-activated Sepharose column (Amersham Biosciences) for 16 h at a flow rate of 1 ml min1. The column was then blocked with 500 mM Tris-HCl (pH 8.0) and washed with PBS. For antibody purification, 50 ml of a 5 mg ml1 solution of postimmune rabbit serum was cycled through the peptide-coupled column for 5 h. The column was then washed with 50 ml of PBS following elution of the polyclonal antibodies in 0.5-ml fractions with 100 mM glycine, pH 2.0. The fractions were adjusted to pH 7.4 with 100 mM Tris-HCl and then visualized by SDS-PAGE prior to use. Using a similar protocol, Dkk-1 was immunoaffinity-purified from 50 ml of conditioned medium by affinity chromatography using antibody-coupled N-hydroxysuccinimide-activated Sepharose.
Production of Recombinant Dkk-1The cDNA encoding human
Dkk-1 (17) was prepared by
RT-PCR using mRNA from hMSCs (see below). The cDNA was cloned into the
prokaryotic expression vector pET 16b using standard protocols and reagents
(New England Biolabs, Beverly, MA). The construct was transformed into BL 21
(DE3) Escherichia coli. Unless otherwise stated, all
biochemical reagents for the production of recombinant Dkk-1 were acquired
from Fisher. A saturated culture of the transformed bacteria was prepared in
50 ml of LB broth containing 100 µg ml1
ampicillin. The overnight culture was added to 1 liter of fresh LB medium with
ampicillin and allowed to grow to an optical density (600 nm) of 0.6.
Isopropyl-
-D-thiogalactopyranoside was added to a final
concentration of 0.4 mM to induce expression of Dkk-1. After 4 h,
the cells were harvested, resuspended in wash buffer (100 mM Tris,
pH 8.0, 100 mM KCl, 1 mM EDTA, 0.2% (w/v) deoxycholic
acid, and 10 mM phenylmethylsulfonyl fluoride), and then lysed by
sonication. Inclusion bodies were washed three times by centrifugation in wash
buffer and sonicated into 50 ml of 100 mM Tris, pH 8.0, containing
6 M urea, 10 mM phenylmethylsulfonyl fluoride, and 0.1
mM dithiothreitol. The inclusion body solution was added to 4
liters of refolding solution (100 mM Tris, pH 8.0, 100
mM KCl, 2% (w/v) N-lauryl sarcosine, 8% (v/v) glycerol,
100 µM NiCl2, 0.001% (v/v)
H2O2) and incubated for 48 h at 4 °C with vigorous
stirring. The sample was filtered through a 0.22-µm2 membrane
and concentrated to 200 ml by diafiltration using a tangential flow filtration
system fitted with 150-cm2 polyvinylidene difluoride 5-kDa filters
(Millipore Corp.). The sample was diafiltered against 40 volumes of 100
mM L-arginine HCl (pH 8.7). Histidine-tagged recombinant Dkk-1 was
purified by metal ion affinity chromatography as described elsewhere
(44) and then dialyzed into 20
mM ammonium carbonate at pH 8.7. The pure, dialyzed protein was
dried by rotary evaporation (Savant AES 2010 Rotary Evaporation System) in
10-µg aliquots and stored at 80 °C. For tissue culture studies,
each aliquot was resuspended in 1 ml of
-minimal essential medium
containing 10% (v/v) FCS.
Analysis of Colony Size and ProliferationhMSCs were plated at about 0.6 cells/cm2 and incubated in complete medium for 17 days. For direct visualization of colonies, a 5% (w/v) solution of crystal violet in methanol (Sigma) was added to tissue culture dishes previously washed twice with PBS. After 20 min, the plates were washed with distilled water and air-dried. Stained colonies with diameters of 2 mm or greater were counted. For assay of proliferation, cells were also quantified by fluorescent labeling of nucleic acids (CyQuant dye; Molecular Probes, Inc., Eugene, OR). hMSCs were plated at 100 cells/cm2 into 10-cm2 wells and allowed to grow for 4 days. The cells were washed with PBS, and medium was added containing the appropriate concentration of Dkk-1 and FCS. The cells were recovered by trypsinization as described above. Fluorescence analysis was carried out using a microplate fluorescence reader (FLX800; Bio-Tek Instruments Inc., Winooski, VT) set to 480-nm excitation and 520-nm emission. Data were statistically analyzed using the two-tailed Student's t test. Experiments were repeated using hMSCs from two donors.
Quantitative RT-PCR AnalysisExtraction of total mRNA was carried out from 1 million cells (High Pure; Roche Diagnostics). A one-tube RT-PCR (Titan; Roche Diagnostics) was employed for the synthesis of cDNA and PCR amplification. The following primers were designed for amplification: ccttctcatatgatggctctgggcgcagcggga (sense) and cctggaggtttagtgtctctgacaagtgtggaa (antisense) (for Dkk-1); catatggccgcgttgatgcggagcaaggat (sense) and cctaggtcaaattttctgacacacatggag (antisense) (for Dkk-2); agccatatgcagcggcttggggccacc (sense) and cctaggctaaatctcttcccctcccag (antisense) (for Dkk-3); tgacatatggtggcggccgtcctgctg (sense) and cctaggtttatagcttttctattttttggc (antisense) (for Dkk-4); and ccccttcattgacctcaact (sense) and cgaccgtaacgggagttgct (antisense) (for GAPDH). Reactions were carried out on a thermal cycler (Applied Biosystems 9700; PerkinElmer Life Sciences) to the following parameters: initial cDNA synthesis, 50 °C for 45 min; denaturation, 95 °C for 1 min; annealing, 52 °C for 1 min; and extension, 72 °C for 1 min for 28 cycles. Positive controls were total RNA extracted from human neuronal progenitor cells for Dkk-3 (Clonexpress, Gaithersburg, MD) and human placental RNA for other Dkk cDNAs (Stratagene, La Jolla, CA). The primers gtgcaatgtcttccaagttctt (sense) and atgagccggacaccccatggca (antisense) were used to amplify Wnt-5a under identical conditions to those used to amplify Dkk cDNA. Amplification of LRP-6 was achieved using the following primers: ccacaggccaccaatacagtt (sense) and tccggaggagtctgtacagggaga (antisense). Reactions were carried out to the following parameters on a thermal cycler (Applied Biosystems 9700): initial cDNA synthesis, 57 °C for 55 min; denaturation, 95 °C for 2 min; annealing, 55 °C for 1 min; and extension, 72 °C for 1 min for 30 cycles. For LRP cDNAs, control mRNA was prepared from JEG-3 choriocarcinoma cells. Samples were analyzed by Tris borate EDTA-PAGE using commercial systems and reagents (Novex; Invitrogen) followed by ethidium bromide staining (Sigma). VCAM-1 mRNA was detected by RT-PCR using a similar one-tube protocol to the one described above. The following amplimers were used: primer A, tgggaacgaacactcttacc (sense); primer B, aatccacgtggagatctact (sense); primer C, ttctcaaaactcacagggct (antisense); and primer D, tggattcccttttccagt (antisense). Combinations of the amplimers were used to amplify VCAM-1 cDNA to the following parameters on a thermal cycler (Applied Biosystems 9700): initial cDNA synthesis, 50 °C for 45 min; denaturation, 95 °C for 1 min; annealing, 50 °C for 1 min; and extension, 72 °C for 1 min for 30 cycles. The expected products are 869, 809, 929, and 869 bp for amplimer combinations AC, BC, AD, and BD, respectively. A previously described hybridization ELISA assay (45) was employed to compare the expression of Dkk-1, Wnt-5a, LRP-6, and VCAM-1 over time in culture. Where equal ratios of sample/GAPDH signal were achieved for different multiple different amounts of RNA template, the RT-PCR ELISA measurements were considered linear. The following biotinylated oligonucleotides were designed for the ELISA: Dkk-1, biotin-atagcaccttggatgggtatt; GAPDH, biotin-catgccatcactgccacccag; Wnt-5a, biotin-tcttggtggtcgctaggtat; LRP-6, biotin-gtgcctcttggttatgtgccact; and VCAM-1, biotin-cagggagggtctaccagctc. Similar results were generated using hMSCs from three donors.
Microarray Transcript AnalysisExperimental procedures for
GeneChip microarray were performed according to the Affymetrix GeneChip
Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA). In brief,
8 µg of total RNA was used to synthesize double-stranded DNA (Superscript
Choice System; Invitrogen). The DNA was purified by phenol/chloroform
extraction and concentrated by ethanol precipitation. In vitro
transcription was performed to produce biotin-labeled cRNA using a BioArray
HighYield RNA transcription labeling kit (Enzo Diagnostics, Farmingdale, NY).
Biotinylated cRNA was cleaned with the RNeasy minikit (Qiagen, Valencia, CA)
and quantified. 25 µg of biotinylated cRNA was fragmented to 50 200
nucleotides and hybridized for 16 h at 45 °C to HG-U133A array, which
contains sequences corresponding to 22,200 human genes. After washing,
the array was stained with streptavidin/phycoerythrin (Molecular Probes).
Staining signal was amplified by biotinylated anti-streptavidin (Vector
Laboratories Inc., Burlingame, CA) followed by secondary staining with
streptavidin/phycoerythrin. The array was then scanned on a Hewlett-Packard
GeneArray Scanner. The expression data were analyzed using the Affymetrix
MicroArray Suite version 5.0 and Affymetrix Data Mining Tool version 3.0.
Signal intensities of all probe sets were scaled to the target value of
2500.
Extraction of Cytoskeletal FractionsTriton-insoluble fractions were prepared in accordance with Ko et al. (46). Briefly, one-half million cells were suspended in 1 ml of ice-cold PBS containing a mixture of protease inhibitors (Roche Diagnostics) with 1% (v/v) Triton X-100 (Sigma). Lysis was allowed to proceed for 10 min on ice followed by a 60-s centrifugation at 800 x g to remove particulate bodies. The cytoskeletal pellet was separated from the cytoplasmic fraction by centrifugation at 14,000 x g for 15 min and resuspended in 1 ml x 1x SDS-PAGE loading buffer. The concentration of protein was measured by Bradford assay (Sigma) prior to immunoblotting.
ImmunocytochemistryhMSCs in tissue culture dishes were
fixed with 4% (v/v) paraformaldehyde (U.S. Biochemical Corp.) for 10 min at 4
°C and washed with PBS (Fisher). Sections (30 x 60 mm) of the dishes
containing the adherent cells were excised using a hot scalpel while under
constant hydration with PBS. The samples were blocked in PBS containing 0.4%
(v/v) Triton X-100 (Sigma) and 5% (v/v) goat serum (Sigma).
Anti--catenin (described above) was added in a 1:400 dilution to the
slides in block solution. An appropriate concentration of mouse
IgG1 (Cymbus Biotechnology (Chandlers Ford, UK) or Chemicon) was
used as an isotype control. The samples were incubated for 16 h at 4 °C
followed by washing in PBS. The samples were then incubated for 1 h in a 1:800
dilution of Alexa-Fluor 594-conjugated secondary antibody (Molecular Probes).
Slides were washed and mounted with medium containing
4',6-diamidino-2-phenylindole (Vector Laboratories). Immunofluorescence
microscopy and digital imaging were carried out using an upright fluorescent
microscope (Eclipse 800; Nikon). Similar distributions for
-catenin were
demonstrated using hMSCs from three donors.
Nuclear ExtractionThe extraction of nuclei was carried out
using a method involving Triton X-100 extraction and centrifugation in the
presence of divalent
cations.2 All reagents
were purchased from Sigma unless otherwise stated. Briefly, hMSCs were
cultured as described above in the presence of 0.1 µg
ml1 recombinant Dkk-1 or vehicle, recovered by
trypsinization, and washed twice in cold PBS. Cells were lysed in cold nuclei
extraction buffer (320 mM sucrose, 5 mM
MgCl2, 10 mM HEPES, pH 7.4, 1% Triton X-100), vortexed
for 10 s, and incubated on ice for 10 min. Intact nuclei were isolated by
centrifugation at 2000 x g for 10 min and resuspended in cold
nuclei wash buffer (320 mM sucrose, 5 mM
MgCl2, 10 mM HEPES, pH 7.4). Nuclei were then incubated
for 1 h at room temperature in blocking buffer (wash buffer containing 5%
(v/v) goat serum) and then immunolabeled with mouse anti-human -catenin
(Chemicon) at 1:1000 dilution for 1 h at room temperature. The nuclei were
then washed twice in nuclei wash buffer. The antibody was detected by a
secondary incubation with Alexa Flour 488-conjugated goat anti-mouse IgG1
(Molecular Probes) at 4 µg ml1 for 1 h at room
temperature. Following two washes, nuclei were resuspended in 500 µl of
DNA-prep stain containing propidium iodide (Coulter, Miami, FL), incubated for
30 min, and then analyzed on a Beckman Coulter Epics XL flow cytometer with
Expo 32 software (Coulter). Extracts were mounted onto a hemacytometer and
inspected by phase-contrast microscopy and epifluorescence using an upright
fluorescent microscope (Eclipse 800; Nikon) with digital imaging.
Cell Cycle AnalysisCells were seeded into
146-cm2 tissue culture plates at an initial seeding density of
100/cm2. After 4 days, the medium was replaced with fresh medium
with or without FCS, and the cultures were incubated for a further 24 h. Cells
were harvested by trypsinization and washed once with PBS, and then cell
pellets were frozen at 80 °C. For analysis, 500,000 cells were
incubated for 30 min on ice in a preparatory labeling reagent containing
propidium iodide, detergent, and RNase (New Concept Scientific, Niagara Falls,
NY). Fluorescent activated cell sorting was carried out using an automated
instrument (Epics XL; Beckman Coulter), and data were analyzed using ModFit LT
3.0 software (Verity Software House, Topsham, ME).
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RESULTS |
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Analysis of Secreted Proteins by [35S]Methionine LabelingTo identify newly synthesized proteins in the medium, hMSCs were plated at a density of 100 cells/cm2 and allowed to grow in medium containing 20% (v/v) FCS. Cells were labeled in the presence of 5 µCi ml1 of [35S]methionine for 48-h periods between days 5 and 7, days 10 and 12, or days 15 and 17. The early log phase of growth at days 57 was accompanied by the largest incorporation of radiolabel and the largest secretion of labeled protein (Fig. 1b). The most abundant labeled proteins were 185 and 100 kDa (Fig. 1b). Western blotting and immunoprecipitation demonstrated that these proteins were fibronectin and laminin, respectively (data not shown). An additional doublet of labeled protein was detected at 3035 kDa (Fig. 1b), a region that contained relatively little unlabeled protein (Fig. 1c). The radiolabeled 3035-kDa band was eluted from the gel (Fig. 1d) and examined by tryptic fingerprinting. 13 tryptic peptides were detected by surface-enhanced laser desorption/ionization mass spectrometry. The data were analyzed by the Pepmapper algorithm (43) with appropriate settings for detection of oxidized methionine and acryl-cysteine modifications. 7 of the 13 peptides were identical within 0.5 Da to tryptic peptides from Dkk-1 (Fig. 1i). The remaining six peptides corresponded to tryptic peptides from bovine prothrombin, also detectable in the appropriate fraction of control medium not conditioned by hMSCs (data not shown).
A rabbit polyclonal antibody was produced against a peptide corresponding to a 15-residue-long sequence in the second cysteine-rich domain of Dkk-1 (17) and used to probe Western blots of medium obtained from rapidly expanding hMSCs. The antibody detected a band of 30 kDa that was present in conditioned medium (Fig. 1e) but was absent in unconditioned medium (Fig. 1f). The intensity of the silver-stained band (Fig. 1e) suggested that the concentration of Dkk-1 in medium from rapidly expanding cultures of hMSCs was up to 50 ng ml1. Also, a small amount of Dkk-1 was recovered from conditioned medium by immunoaffinity chromatography using the same antibody (Fig. 1g). Western blotting of cells recovered from early log phase (5-day) and late stationary phase (15-day) cultures further indicated that Dkk-1 was expressed at a high levels during log phase and significantly down-regulated as the cultures became stationary (Fig. 1h).
Expression of Recombinant Dkk-1 in E. coliTo prepare
recombinant Dkk-1, the cDNA encoding the entire coding region of Dkk-1 was
cloned into the bacterial expression vector, pET 16b. The clone was
constructed to encode an in-frame hexahistidine tag at the amino terminus for
protein purification. Recombinant Dkk-1 was recovered in insoluble inclusion
bodies from the bacteria. The protein was solubilized, refolded, and purified.
The yield of protein was relatively low, at 100 µg of soluble protein
per liter of culture. Assays by SDS-PAGE under reducing and nonreducing
conditions indicated that about 60% of the protein had concatamerized through
intermolecular disulfide bond formation
(Fig. 2a). Circular
dichroism indicated that significant portions of the protein adopted
-sheet and
-helical structures (not shown), a conclusion that
agreed with the theoretical prediction of the secondary structure by the
PHDsec algorithm
(47). The recombinant Dkk-1
could be detected by the anti-Dkk-1 antiserum and a monoclonal antibody
against the hexahistidine tag (Fig.
2a).
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Effect of Recombinant Dkk-1 on hMSC ProliferationTo test the hypothesis that Dkk-1 increased proliferation of hMSCs, its effects on rate of growth were assayed. The hMSCs were plated at a density of 100 cells/cm2 in 6-well plates (10-cm2 surface area/well). After 4 days, when the cells were in early log phase of growth, the conditioned medium was removed and replaced with fresh medium containing either vehicle, 0.1 µg ml1 Dkk-1, or 0.01 µg ml1 Dkk-1. Fluorescence assays for cell number indicated that the recombinant Dkk-1 initially increased proliferation (Fig. 2b). The effect of Dkk-1 persisted for 30 h at 0.1 µg ml1 (Fig. 2b, top), whereas the effects of Dkk-1 were only significant for about 15 h when 10-fold less Dkk-1 was added (Fig. 2b, bottom), suggesting that the molecule had a short half-life.
To test the effect of recombinant Dkk-1 on the colony-forming potential of hMSCs, 100 hMSCs were plated onto a 176-cm2 tissue culture dish and allowed to form colonies in the absence or presence of Dkk-1 in medium supplemented with 10% (v/v) fetal calf serum instead of the optimal concentration of 20%. After 2.5 weeks, the recombinant Dkk-1 increased colony size (Fig. 2c, bottom). However, there was no statistically significant effect on colony number (Fig. 2c, top). The effects of Dkk-1 appeared to be biphasic in that concentrations as high as 0.5 µg ml1 failed to increase the rate of proliferation and reduced both the colony size and number (data not shown).
Microarray and RT-PCR Assays for Dkk-1 and LRP-6
Microarray assays on 5-day (early log), 10-day (late log), and 15-day
(stationary) cultures demonstrated that several components of the canonical
Wnt signaling pathway were expressed, including Dkk-1; Wnt-5a;
-catenin;
-catenin; frizzled 1, 4, 6, and 7; disheveled; glycogen
synthetase kinase 3
; and glycogen synthetase kinase 3
. To
investigate the mRNA profiles more closely, a quantitative RT-PCR and a
previously described ELISA-based assay was employed
(45). The level of Dkk-1 mRNA
was highest after 5 days in culture (early log), lower after 10 days (late
log), and not detectable at 15 days (stationary phase;
Fig. 3, a and
b). Expression of one of the Dkk-1 receptors, LRP-6,
paralleled expression of Dkk-1 with levels falling as hMSCs became confluent
(Fig. 3a). Multiple
attempts to amplify LRP-5 from hMSCs using different primers were
unsuccessful, but the same primers gave satisfactory signals when mRNA
generated from another source was assayed
(Fig. 3d). Further
RT-PCR assays demonstrated that there were no detectable levels of Dkk-2,
Dkk-3, and Dkk-4 transcripts (Fig.
3d). Also, RT-PCR assays for Wnt-5a demonstrated that the
gene was not expressed in early log cultures, expressed at moderate levels in
late log cultures, and expressed at high levels in stationary cultures
(Fig. 3, a and
b). To explore the observations further,
-catenin
levels were assayed based on the assumption that Dkk-1 expression early in
culture could inhibit the canonical Wnt pathway, leading to a destabilization
of
-catenin. As expected, Western blotting demonstrated that the
steady-state level of
-catenin was lower in early log phase cultures
than in late log or stationary phase cultures
(Fig. 3c). Also, the
-catenin molecules in the stationary phase were extensively
redistributed from the cytoplasmic pool to the detergent-insoluble actin-rich
cytoskeletal fraction (Fig.
3c), suggesting that
-catenin contributed to the
formation of actin-associated intracellular adherens junctions. Actin and
GAPDH were also assayed to serve as fractionation controls. It is noteworthy
that actin almost exclusively partitioned to the insoluble fraction of the
cell lysate, but GAPDH, which has been reported to exist in the cytoplasm and
associated with microtubules
(48), was almost completely
cytosolic.
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Recombinant Dkk-1 Decreases the Intracellular Concentration of
-CateninIn further experiments, the effects of
recombinant Dkk-1 on
-catenin levels in hMSCs were investigated. As
expected, treatment of stationary phase cultures of hMSCs with 0.1 µg
ml1 recombinant Dkk-1 reduced the levels of
-catenin (Fig.
4a).
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To examine effects of the recombinant Dkk-1 on the cellular distribution of
-catenin, monolayers were fixed with paraformaldehyde at the early log
phase (6 days) or stationary phase (15 days) of growth, and sections of the
dish were immunostained for
-catenin. In untreated early log phase
cultures,
-catenin was distributed throughout the cytoplasm and the
plasma membrane at sites of cell-cell contact
(Fig. 4b, i
and ii). In many instances of cell-cell contact, there appeared to be
a gradient of
-catenin distribution throughout the cytoplasm, with most
concentration proximal to the contact site
(Fig. 4b, i
and ii). In stationary cultures, the distribution of
-catenin
was similar, but the concentration at cell contacts was more apparent
(Fig. 4b, iii
and v). As expected, the addition of medium containing 0.1 µg
ml1 Dkk-1 produced a clearance of the cytoplasmic
pool of
-catenin (Fig.
4b, iv and vi). Low power images
confirmed that the effect of Dkk-1 was present throughout the monolayer
(Fig. 4b, v
and vi). The staining was specific for
-catenin, since extended
exposure of the control slides with an appropriate concentration of isotype
control did not give a fluorescent signal
(Fig. 4b,
vii).
Nuclear -Catenin Is Also Reduced by Recombinant
Dkk-1 Detailed inspection of stained hMSC monolayers at the late
log or stationary phase of growth was hindered by the high signal in untreated
cultures. This prevented accurate evaluation of nuclear levels of
-catenin, since it appeared indistinguishable from membrane-bound
fractions (exemplified in Fig.
5a). To test whether the recombinant Dkk-1 directly
affected nuclear
-catenin levels, late log phase cultures were treated
with medium in the presence or absence of 0.1 µg
ml1 Dkk-1. After an incubation period of 24 h,
the cells were recovered, and the nuclei were extracted in accordance with the
protocol described under "Experimental Procedures." The extracted
nuclei were stained for both
-catenin using a monoclonal antibody and an
Alexa 498-conjugated secondary antibody (green) and for DNA using
propidium iodide (red). Flow cytometric analysis of the extracted
nuclei demonstrated that there was an overall and highly significant
(p = 0.002, from 10,000 events) decrease of detectable
-catenin
in the nuclei of Dkk-1-treated cells (Fig.
5b). Furthermore, the nuclei from Dkk-1-treated cells
seemed to consist of two strikingly separate subpopulations: one with
relatively high levels of
-catenin, presumably derived from cells
expressing lower levels of LRP-6 on the membrane, and another with lower
levels of
-catenin, presumably derived from cells expressing more LRP-6.
It is probable that this partitioning of the nuclei from Dkk-1-treated cells
into two populations could reflect microscopic variations in cell density over
the monolayer, thus affecting LRP-6 expression. The isotype control yielded no
detectable signal (data not shown). The nuclear extraction protocol was
validated by microscopic inspection of wet-mounted nuclei on a hemacytometer
(Fig. 5c). The nuclei
were clearly visible by phase, and staining with propidium iodide confirmed
that the structures were chromatin-containing nuclei and not debris (see
merged image in Fig.
5c). In agreement with the flow cytometric data,
treatment with Dkk-1 markedly decreased the
-catenin in the nuclei. It
is noteworthy to add that upon closer inspection, there appeared to be uneven
-catenin staining of the nuclei, suggesting that the protein may deploy
itself to specific regions of the chromatin on entry into the nucleus.
|
Jun/JNK Signaling and Expression of VCAM-1Since Dkk-1 binds
to LRPs and the LRP complexes can regulate JNK
(49), we examined the pattern
of Jun/JNK signaling during the expansion of hMSCs cultures. Microarray data
for mRNAs (Table I) indicated
that three members of the Jun family (c-Jun, JunB, and JunD) were expressed
and that the levels of two (JunB and JunD) increased as the cultures entered
the stationary phase. JunD expression also increased during serum starvation
of the cells. Only one of three isoforms of JNK was detected, and the level
did not change as the cultures entered the stationary phase or underwent serum
starvation. Assays of the proteins by Western blotting indicated that c-Jun
was present but did not change during expansion
(Fig. 6a), an
observation that was consistent with the microarray data
(Table I). However, there was
an increase in c-Jun phosphorylated at serine 73 in stationary cultures that
coincided with the decrease of Dkk-1. There was no increase in c-Jun
phosphorylated at the serine 63 (not shown). The increase in phosphorylated
c-Jun was not reversed by the addition of recombinant Dkk-1. However, the
addition of Dkk-1 reduced the level of endogenous Dkk-1
(Fig. 6, a and
c), an observation consistent with the conclusion that
the recombinant Dkk-1 was active and previous observations indicating that
expression of Dkk-1 is down-regulated by inhibition of the Wnt/-catenin
pathway (16).
|
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Since phosphorylated c-Jun increased in the stationary phase, we searched the microarray data for downstream genes regulated by c-Jun. The second highest increase in signal intensity was found for VCAM-1, a gene that is up-regulated by c-Jun and c-Fos of the AP-1 class of transcription factors (50) and that regulates bone development (5153). Both RT-PCR (Fig. 6b) and Western blot (Fig. 6c) assays demonstrated a dramatic increase in VCAM-1 expression that coincided with the decrease in Dkk-1 and the increase in phosphorylated c-Jun. The addition of recombinant Dkk-1 to stationary cultures did not decrease the protein levels of VCAM-1 (Fig. 6c), but the levels of VCAM-1 mRNA were markedly increased by sequestering Dkk-1 from early log phase cultures by the addition of antibodies to Dkk-1 (Fig. 6d). The levels of VCAM-1 mRNA were also reduced in stationary cultures by the addition of nordihydroguatiaretic acid, an inhibitor of c-jun and/or c-fos (54, 55).
Dkk-1 Expression Is Concomitant with Cell Cycle Activity
Since Dkk-1 expression was highest in hMSCs during the early log phase of
growth, we tested the hypothesis that expression of Dkk-1 would decrease if
the cells were growth-arrested by serum starvation. Hybridization ELISA of
RT-PCR products indicated that Dkk-1, but not GAPDH levels, were significantly
reduced under serum-free conditions that inhibit division
(Fig. 7, a, b, c). In
addition, -catenin levels were increased in the growth-arrested hMSCs
(Fig. 7d), possibly in
response to the reduction of Dkk-1 synthesis. VCAM-1 levels remained unchanged
in response to serum starvation as assayed by microarray analysis (data not
shown).
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Effect of anti-Dkk-1 Antibodies on hMSCs and Malignant Cell LinesAntiserum to the synthetic peptide from Dkk-1 (Fig. 1e) was added to 5-day cultures of hMSCs in which a second lag period was induced by a change in medium (see Fig. 1a). As indicated in Fig. 8, a and b, the antiserum further decreased proliferation of cultures developed from two different donors. The addition of higher concentrations of the antiserum (25 and 50 µg ml1) had no effect on stationary cultures of hMSCs (Fig. 8c). Therefore, the effects were specific for rapidly proliferating hMSCs.
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Four lines of human malignant cells were assayed for expression of Dkk-1 by RT-PCR. The mRNA encoding Dkk-1 was present in both osteosarcoma lines tested and at much lower levels in one of two choriocarcinoma lines (Fig. 8d). The addition of anti-Dkk-1 antibodies to one osteosarcoma cell line increased the lag phase produced by a change of medium (Fig. 8f). The antibodies had no effect on the choriocarcinoma cell line (JEG-3), which did not express detectable levels of Dkk-1 mRNA. It is also noteworthy that the JEG-3 cell line also did not exhibit the lag phase in response to a change to fresh medium.
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DISCUSSION |
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The evidence that Dkk-1 inhibited the canonical Wnt signaling pathway was
based on several observations. Dkk-1 was a major component secreted into the
medium during the early log phase. The addition of recombinant Dkk-1 to
cultures increased proliferation during a second lag period that was induced
by replacing conditioned medium. Also, the addition of antibodies to Dkk-1
decreased proliferation of early log phase cultures. During expansion of the
cultures from the early log phase to the stationary phase, there was
coincidental down-regulation of expression of both Dkk-1 and its co-receptor,
and an up-regulation of Wnt-5a. At the same time, there was a marked increase
in total cellular levels of -catenin and of
-catenin in both the
nuclear and cytoskeletal fractions. The addition of recombinant Dkk-1
dramatically reduced the levels of
-catenin in the same compartments.
Since positive signaling through the canonical Wnt/
-catenin pathway is
generally linked to increased cell proliferation through activating c-Myc or
cyclin D1 (56), the increased
proliferation of hMSCs observed by inhibiting the pathway with Dkk-1 was
unexpected. The observations made here, however, are consistent with the model
for Wnt signaling during limb bud development proposed by Hartmann and Tabin
(37), in which positive
signaling through the canonical Wnt/
-catenin/Lef pathway drove
chondrocytes out the cell cycle and pushed them toward differentiation and a
postmitotic state.
The observations also raised the possibility that Dkk-1 down-regulates the AP-1/JNK pathway and thereby decreases expression of the cell adhesion protein VCAM-1. The levels of c-Jun remained constant as the culture passed from the early log to the stationary phase, but there was an increase in phosphorylated c-Jun that paralleled the decreased expression of Dkk-1. The increase in phosphorylated c-Jun coincided with a marked increase in expression of VCAM-1 that is positively regulated by the AP-1/JNK pathway (50). Sequestering endogenous Dkk-1 by the addition of antibodies to early log phase cultures markedly increased the expression of VCAM-1. In addition, expression of VCAM-1 in the cells was reduced by an inhibitor of the AP-1/JNK pathway (54, 55). Together, these observations strongly suggest a link between the canonical Wnt pathway and JNK-mediated transcriptional regulation of VCAM-1 expression in hMSCs.
The decrease in expression of Dkk-1 as the cells expanded may in itself explain why low density cultures approached a stationary phase without becoming confluent. However, the expression of Wnt-5a and reintroduction of positive signaling through a Wnt pathway may also contribute to the decreased proliferation. Wnt-5a was originally recognized as a ligand for a noncanonical Wnt signaling pathway, but the noncanonical Wnt pathways are still poorly understood. Also, a recent report indicates that Wnt-5a signaling can mimic positive signaling through the canonical pathway in one cell system (58). The expression of the Wnt-5a ligand in the stationary phase cultures is consistent with the extensive use of confluent cultures of hMSCs as feeder layers for the culture of hematopoietic stem cells and the recent reports that several Wnt ligands enhance hematopoiesis (5860).
The requirement for Dkk-1 to return hMSCs to the cell cycle is probably important for limiting replication of the cells in vivo, since, if replated at low density, the cells expand over a billion-fold over about 8 weeks (11), and such uncontrolled expansion in vivo would impose a considerable burden on the organism.
The effect of Dkk-1 in driving hMSCs into cell cycle are of interest in terms of the recent reports that two different mutations in LRP-5 that prevented Dkk-1 from inhibiting Wnt signaling caused high bone density in two unrelated kindreds (61, 62). In light of the observations made here, the effects of the mutations may be explained by increased Wnt signaling driving hMSCs out of the cell cycle and toward differentiation to osteoblast precursors. Judicious administration of Dkk-1 might be a useful means of stimulating expansion of hMSCs in vivo and thereby enhancing the tendency for the cells to home to sites of tissue damage and repair the tissues (63).
To explore the possibility that Dkk-1 might also decrease the lag phase observed in other cultured cells, we screened several malignant cell lines for expression of Dkk-1. Two osteosarcoma lines expressed the gene. Cultures of both exhibited a lag phase in culture, and the lag phase was prolonged by the addition of antibodies to Dkk-1. Therefore, synthesis of Dkk-1 may be a frequent means whereby cells that demonstrate a lag phase reenter the cell cycle. Also, the results raise the possibility that antibodies to Dkk-1 or antagonists may be a useful adjunct therapy for some malignancies.
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FOOTNOTES |
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To whom correspondence should be addressed: Center for Gene Therapy, Tulane
University Health Sciences Center, 1430 Tulane Ave., New Orleans, LA 70112.
E-mail:
dprocko{at}tulane.edu.
1 The abbreviations used are: hMSC, human mesenchymal stem cell; Dkk-1,
dickkopf-1; LRP, low density lipoprotein receptor-related protein; AP-1,
activating protein-1 class of transcription factors that includes dimers of
the Fos family (c-Fos, FosB, and Fra1) and the Jun family (c-Jun, JunD, and
JunB); JNK, c-Jun NH2-terminal kinase; VCAM-1, vascular cell
adhesion molecule-1; SELDI, surface-enhanced laser desorption/ionization; TOF,
time of flight; FCS, fetal calf serum; PBS, phosphate-buffered saline;
bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; MES,
4-morpholineethanesulfonic acid; RT, reverse transcriptase; DTT,
dithiothreitol; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ELISA,
enzyme-linked immunosorbent assay.
2 W. Telford, personal communication.
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REFERENCES |
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