Osteopontin Enhances the Expression and Activity of
MMP-2 via the SDF-1/CXCR4 Axis in Hepatocellular
Carcinoma Cell Lines
Rihua Zhang1, Xiaolin Pan1, Zuhu Huang2, Georg F. Weber3, Guoxin Zhang1*
1Department of Gastroenterology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, China, 2Department of Infection Diseases, The First Affiliated
Hospital of Nanjing Medical University, Nanjing, China, 3University of Cincinnati Academic Health Center, College of Pharmacy, Cincinnati, Ohio, United States of America
Background and Aims: Osteopontin, SDF-1a, and MMP-2 are important secreted molecules involved in the
pathophysiology of human hepatocellular carcinoma (HCC). This study investigates the effect of the SDF-1a/CXCR4 axis
on expression and activity of MMP-2 induced by osteopontin.
Methods: The expression of CXCR4, SDF-1a, MMP-2 and their associated cellular signaling cascades, involving Akt and MAP
Kinases, were determined by Western blotting. The activities of MMP-2 and MMP-9 were assayed by gel zymography. The
role of the osteopontin receptors integrin avb3 and CD44v6 was evaluated using neutralizing antibodies. We also
established CXCR4-deficient SMMC7721 cell lines by transfection with miRNA-CXCR4 plasmids and determined cell invasion
activity in a transwell assay.
Results: In comparison with untreated cells, recombinant human osteopontin (rhOPN) up-regulated CXCR4, SDF-1a, and
MMP-2 expression about 5-, 4-, and 6-fold on the protein levels through binding to integrin avb3 and CD44v6 in
hepatocellular carcinoma cells (SMMC7721 and HepG2). Inhibition of the SDF-1a/CXCR4 axis down-regulated the rhOPN-
induced MMP-2 expression and activity. rhOPN also activated Akt, p38 and JNK. Down-regulation of CXCR4 decreased the
rhOPN-induced invasion in SMMC7721 cells.
Conclusion: These results indicate that rhOPN up-regulates MMP-2 through the SDF-1a/CXCR4 axis, mediated by binding to
integrin avb3and CD44v6 and activating the PI-3K/Akt and JNK pathways in HepG2 and SMMC7721 cells. Therefore, the
osteopontin-SDF-1a/CXCR4-MMP-2 system may be a new therapeutic target for treating HCC progression.
Citation: Zhang R, Pan X, Huang Z, Weber GF, Zhang G (2011) Osteopontin Enhances the Expression and Activity of MMP-2 via the SDF-1/CXCR4 Axis in
Hepatocellular Carcinoma Cell Lines. PLoS ONE 6(8): e23831. doi:10.1371/journal.pone.0023831
Editor: Jean-Marc Vanacker, Institut de Ge ´nomique Fonctionnelle de Lyon, France
Received March 8, 2011; Accepted July 26, 2011; Published August 31, 2011
Copyright: ? 2011 Zhang et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from National Natural Science Foundation of China (No. 81072032 and 30770992), and Social Development Funds
of Jiangsu Province and from Jiangsu Health Department, China (No. B52007070 and H200702). GW is founder and CEO of MetaMol Theranostics. The funders had
no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Many experimental and clinical studies have demonstrated that
a substantial number of secreted factors are involved in the
pathophysiology of human hepatocellular carcinoma (HCC) .
Among them, the cytokine osteopontin, the SDF-1a/CXCR4 axis
(stromal cell derived factor-1/ CXC chemokine receptor 4), and
MMP enzymes are thought to play key roles in invasion and
Osteopontin is an aspartate-rich protein expressed by various
tissues and cell types. The existence of variant forms of osteopontin,
been described. sOPN interacts with integrins and variant CD44. It
contains several cell binding domains, including an arginine-
glycine-aspartate (RGD)-motif that engages a subset of cell surface
integrins (avb3, avb1, avb5, and a8b1), a serine-valine-valine-
tyrosine-glutamate-leucine-arginine (SVVYGLR)-containing do-
main that interacts with other integrins (a9b1, a4b1and a4b7),
and an ELVTDFTDLPAT domain that has been reported to bind
to integrin a4b1.The CD44-bindingsite hasbeen mappedto the
C-terminal portion of osteopontin. The cytokine activates various
signaling pathways to mediate multiple functions such as inflam-
mation, cell adhesion, migration and tumor invasion. Osteopontin
up-regulates matrix metalloproteinase 2 (MMP-2). In MDA-MB-
231 human breast cancer cells, MMP-2 was significantly decreased
following exposure to an inhibitor of osteopontin . Further study
hasshown that osteopontin activatesthe phosphoinositide 3-kinase/
Akt survival pathway [7,8].
SDF-1 and its receptors, such as CXC chemokine receptor 4
(CXCR4), are thought to play critical roles in motility, homing,
and proliferation of many cancer cells . SDF-1, which belongs
to the CXC chemokine subfamily, is produced in two forms, SDF-
1a (CXCL12a) and SDF-1b (CXCL12b), by alternative splicing of
the SDF-1 gene. The binding of SDF-1a to its receptor CXCR4
stimulates receptor dimerization and activates downstream
PLoS ONE | www.plosone.org1August 2011 | Volume 6 | Issue 8 | e23831
signaling to play an important role in a wide array of disease
We thus assessed the role of the SDF-1a/CXCR4 axis in the
process of OPN mediated MMP-2 up-regulation in the two
cell lines,HepG2 and
Materials and Methods
rhOPN (Recombinant human Osteopontin/his) (#1433-OP/CF)
was purchased from R&D Systems (USA). PD98059 (#9900),
LY294002 (#9901), MAPKFamily Antibody Sampler
(#9926), Phospho-Akt (Ser473), Antibody (#9271) and SDF-1
antibody (#3530) were purchased from Cell Signaling Technology
(USA). Rabbit polyclonal to CXCR4 (#ab2074) was obtained from
(#MAB4073), Anti-integrin aV clone AV1 monoclonal antibody
affinity purified polyclonal antibody (#AB1868P) came from
Millipore (USA). SB203580 (#S8307), SP600125 (#S5567) and
ECM gel (#e1270) were obtained from Sigma-Aldrich (USA).
AMD3100 (#10011332) was purchased from Cayman Chemical
and Functional Grade Purified anti–human CXCR4 (12G5) (#16-
9999) from eBioscience (USA).
The human hepatocellular carcinoma cell lines SMMC7721
and HepG2 cells  were cultured in DMEM supplemented with
10% fetal bovine serum (FBS), penicillin (100 U/ml), streptomycin
sulfate (100 mg/ml), and maintained at 37uC with 5% CO2in a
Construction of miRNA-CXCR4 expression plasmids and
stable clone selection
Four distinct domains within the coding region of the human
CXCR4 cDNA were targeted for RNA interference. For this
purpose, four pairs of reverse complementary oligonucleotides
were designed and synthesized as Table 1.
The oligonucleotides were annealed and inserted into the
#K4936-00) to create pcDNA6.2-GW/EmGFP–miR -CXCR4-
1-4, 2-4, 3-1, and 4-4. A control construct was also created.
We used lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA)
to separately transfect the five kinds of plasmids into SMMC7721
cells. To select for successful transfectants, the cells were cultured
48 hours after transfection in selection medium containing 3 mg/
ml blasticidin (Sigma-Aldrich, Saint Louis, MO, USA). Blasticidin-
resistant cells were maintained in culture medium supplemented
with 3 mg/ml blasticidin for further analysis.
Gel zymography for evaluation of gelatinolytic activity
In this study, the human hepatocellular carcinoma cell lines
SMMC7721 and HepG2 (16106) were seeded in 6-cm (diameter)
dishes containing complete growth medium. After 12 hours
incubation in DMEM with 0.1% BSA, the medium was changed
to DMEM with 0.1% BSA in the absence or presence of rhOPN
(50 nM) for 60 hours. The rhOPN concentration is in the range
commonly associated with cancer . We then collected the
supernatant and centrifuged it at 12,000 rpm for 10 min to pellet
insoluble material. The protein concentration in the supernatant
was determined using a Protein Assay Rapid Kit (Bio-Rad, Osaka,
Japan). Samples containing 40 mg total protein in sample buffer
(10% SDS, 4% sucrose, 0.25 M Tris-HCl, pH 6.8 and 0.1%
bromophenol blue) were used in gelatin zymography. The
samples, diluted 1:1 with 26 sample buffer, were not boiled but
warmed in a water bath (55uC) for 3–5 min before being subjected
to electrophoresis in a 10% SDS-polyacrylamide gel (SDS-PAGE)
containing 0.1% gelatin under non-reducing conditions. The gel
was washed twice for 30 min in 2.5% Triton X-100 at room
temperature to remove the SDS. After the second wash, all but 2–
3 ml of the Triton X-100 was removed, and 100 ml of
development buffer (0.05 M Tris-HCl pH 8.8, 5 mM CaCl2,
0.02% NaN3, 0.02% Brij) was added for further incubation for
24 hours at 37uC. The gel was then stained for 3 hours in
Coomassie blue (0.1% Coomassie brilliant blue R250 (w/v) in
fixing/destaining solution) and destained in fixing/destining
solution (methanol: acetic acid: water, 4.5:1:4.5) until clear bands
of gelatinolysis appeared on a dark background. Total activity was
analyzed using a scanning densitometer with molecular analysis
software (Bio-Rad) .
Enzyme-linked immunosorbent assay (ELISA) was done with a
human SDF-1 Quantikine kit (R&D), used in accordance with the
manufacturer’s protocol. In this study, the human hepatocellular
carcinoma cell lines SMMC7721 and HepG2 (16106) were seeded
in 6-cm (diameter) dishes containing complete growth medium.
After 12 hours incubation in DMEM with 0.1% BSA, the medium
was changed to DMEM with 0.1% BSA in the absence or
Table 1. Reverse complementary oligonucleotides.
OPN Induces MMP-2 through the SDF-1/CXCR4 Axis
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presence of rhOPN (50 nM) for 24, 48, 72 hours. We then
collected the supernatants and measured total protein content
using the BCA protein assay kit (Pierce) before analysis. Results are
representative of three independent experiments.
Western blotting analysis
The SMMC7721 and HepG2 cells (16106) were treated with
rhOPN (50 nM) for 48 hours, and then lysed in RIPA buffer.
Equal amounts of protein (60 mg) were electrophoresed on 12%
SDS-PAGE gels and electrophoretically transferred to Immobilon-
P membranes (Millipore, Bedford, MA, USA). The membranes
were probed overnight at 4uC with antibody to CXCR4 (1:1000),
MMP-2, SDF-1a and monoclonal anti-a-tubulin (1:5000) in
TBST containing 1% BSA (w/v). The blots were then incubated
for 2 hours with anti-rabbit or anti-mouse secondary antibodies,
the immune complex was detected using an ECL plus detection kit
(Pierce, Rockford, IL, USA), and analyzed using a scanning
densitometer with molecular analysis software (Bio-Rad).
Integrin avb3and CD44v6 neutralization
SMMC7721 and HepG2 cells were cultured as described above
in the presence of anti-integrin avb3or anti-CD44v6 neutralizing
antibodies, or of control IgG. After 60 hours, the cells were
collected and Western blotting was performed for the relevant
Cell invasion assay
Cell invasion was studied using 24-well transwell plates (Corning
Costar, Schiphol-Rijk, Netherland). 60 ml of the ECM gel solution
was added to the top compartment of each cell culture insert and
dried overnight under laminar air flow. The cells under study were
harvested, washed twice with PBS, resuspended in serum-free culture
medium with 0.2% BSA and adjusted to a final concentration of 106
per ml. 600 ml serum-free DMEM/ 0.2% BSA containing rhOPN
(50 nM) was added to the lower compartment of each well, and
200 ml of the cell suspension was added to the pre-coated upper
compartment. The plate with inserts was incubated for 48 hours in a
cell culture incubator at 37uC and 5% CO2. To determine the
background migration, some wells of the 24-well plate were prepared
without rhOPN in the lower compartment. Cells remaining on the
top side of the filter were removed by soft mechanical dislodging, and
the number of cells migrating to the bottom of the filter was counted
using a light microscope (in each chamber, six fields were counted at
2006magnification for each condition).
SMMC7721 and HepG2 cells were collected with trypsin/
EDTA, washed with fluorescence-activated cell sorting (FACS)
buffer (phosphate-buffered saline [PBS], 2 mM EDTA, and 0.5%
BSA), and then incubated in FACS buffer for 1 hour at 4uC in the
presence of monoclonal antibodies at the manufacturer’s recom-
Figure 1. SDF-1a, CXCR4 and MMP-2 expression are induced by rhOPN in SMMC7721 and HepG2 cells. SMMC7721 cells (A) or HepG2
cells (B) were stimulated with various concentrations of rhOPN for 48 hours, the cells were collected, and SDF-1a, CXCR4 and MMP-2 were detected
by Western blotting assay. (C) HepG2 cells were stimulated with 50 nM rhOPN for increasing time frames, the cells were collected, and SDF-1a, CXCR4
and MMP-2 were detected by Western blotting assay. (D) SDF-1 ELISA of culture supernatants (SMMC7721 and HepG2) after 0–72 hours of rhOPN
(50 nM). (E) MMP-2 activity was analyzed by gelatin zymography after stimulation with 50 nM rhOPN for 60 hours in the SMMC7721 and HepG2 cell
lines. *denotes P,0.05 versus control. The results presented are representative of at least three independent experiments.
OPN Induces MMP-2 through the SDF-1/CXCR4 Axis
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mended concentrations. Binding of anti-CD44var (v6) and anti-
integrin aV clone AV1 were visualized with FITC-conjugated
rabbit anti-mouse immunoglobulin (Chemicon, Temecula, CA).
The cells were washed, fixed with 1% paraformaldehyde and the
fluorescence was quantified on 10,000 cells using a FacsCalibur
with Cellquest software (BD Biosciences, PharMingen).
The data were analyzed by two-tailed Student’s t-test for single
comparisons and by one-way analysis of variance for multiple
group comparisons. Differences were considered significant at a
probability of error below 5% versus control.
Osteopontin up-regulates SDF-1a, CXCR4 and MMP-2
expression in hepatocellular carcinoma cells
To determine the effect of rhOPN on the SDF-1a/CXCR4
axis and MMP-2 expression, Western blotting analysis and gel
zymography were done in two human hepatocellular carcinoma
cell lines, SMMC7721 and HepG2. Figure 1A shows that the
expression of SDF-1a, CXCR4, and MMP-2 protein were
induced by rhOPN. There was an apparent increase in the
CXCR4 protein level when the concentration of rhOPN was
3.12 nM in SMMC7721 cells, and the same phenomenon was
also observed in HepG2 cells (Figure 1B). Figure 1C shows that
MMP-2 expression was detectable within 24 hours after the
addition of rhOPN, reached a maximum around 60 hours. The
MMP-2 levels increased in a time-dependent manner in HepG2
cells. SDF-1a and CXCR4 expression increased accordingly
(Figure 1C and 1D). Figure S1A and S1B are quantification of
expression described in Figure 1A and 1B based on grayscale
After identifying MMP-2 protein expression in SMMC7721 and
HepG2 cells, we further analyzed the MMP-2 activity in the two
cell lines by gelatin zymography. The results demonstrated that
the activity of MMP-2 but not MMP-9 was induced by rhOPN at
a dose of 50 nM (Figure 1E).
Figure 2. Effects of the SDF-1a/CXCR4 axis on rhOPN-induced MMP-2 expression and activity. (A) Verification by Western blotting of the
miRNA knockdown of CXCR4 showed a significant reduction of the CXCR4 protein in all clones (1-4, 2-4, 3-1, 4-4). After blocking the SDF-1a/CXCR4
axis with miRNA-CXCR4 and inhibitors (SDF-1 neutralizing antibody at 100 ng/ml, CXCR4 inhibitor 12G5 at 50 mg/ml, or CXCR4 inhibitor AMD3100 at
500 ng/ml), the cells were stimulated by rhOPN in serum-free medium for 60 hours, the cells were collected and analyzed by Western blotting in
SMMC7721 cells (C) and in HepG2 cells (F). The supernatants of SMMC7721 cells (B) and HepG2 cells (E) were analyzed by gelatin zymography. (D)
and (G) show the densitometric ratio of MMP-2 protein/a-tubulin. (H) Western blotting was used to assay the MMP-2 expression induced by rhOPN
(50 nM) or/and SDF-1 (30 nM) for 48 hours. * denotes P,0.05 versus control. The results presented are representatives of at least three independent
OPN Induces MMP-2 through the SDF-1/CXCR4 Axis
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The SDF-1a/CXCR4 axis is involved in osteopontin-
induced MMP-2 expression and activity
The MMPs are a large family of proteolytic enzymes, which play
an important role in cancer invasion and metastasis due to their
Among them, MMP-9 and MMP-2 have been found to be highly
associated with metastatic spread by various cancers. Therefore, to
determine whether the SDF-1a/CXCR4 axis mediates osteopon-
tin-induced MMP-2 expression and activity, we established
CXCR4-deficient SMMC7721 cell lines (clone 1-4, 2-4, 3-1 and
4-4) through the transfection of miRNA-CXCR4. SMMC7721-
vector was used as a control. The CXCR4 protein was detected by
Western blotting. CXCR4 expression was significantly down-
regulated in all of the four miRNA clones (Figure 2A).
The SMMC7721 cells and the miRNA transfectant clones were
stimulated with rhOPN for 60 hours. At that time, the cells and
their conditioned medium were collected for gelatin zymography
and Western blotting. Decreased amounts of MMP-2 proteins were
detected in CXCR4-deficient SMMC7721 cells (clones 1-4, 2-4, 3-1
and 4-4) (Figure 2C), compared to SMMC7721 and vector control.
The increased activity of MMP-2 but not MMP-9 was abolished in
the CXCR4-deficient SMMC7721 cells (Figure 2B).
Figure 3. Integrin avb3and CD44 mediated OPN-induced CXCR4 expression in SMMC7721 and HepG2 cells. FACS analysis using
monoclonal antibodies to avb3integrin (left) and CD44 (right) was done for SMMC7721 cells (A) and HepG2 cells (C), stimulated by rhOPN for
24 hours. The grey area represents isotype control, while the dark line represents the control and the grey line represents the experimental group.
SMMC7721 (E) and HepG2 (F) cells were treated with rhOPN (50 nM), in the presence of neutralizing antibodies to integrin avb3or CD44v6, or control
IgG. After 60 hours, the cells were collected and Western blotting was performed to detect CXCR4. (B), (D), (G) and (H) are quantitative evaluations.
The results are shown as mean 6 standard deviation (n=3). * denotes P,0.05 compared to rhOPN treatment in the absence of antibody.
OPN Induces MMP-2 through the SDF-1/CXCR4 Axis
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