 |
INTRODUCTION |
During the initial phases of carcinoma cell invasion, as tumor
cells begin to spread and infiltrate into the surrounding normal tissues, these cells must first degrade the basement membrane and other
elements of the extracellular matrix
(ECM),1 including type IV
collagen, laminin, and fibronectin (FN) (1). Multiple protease
families, including the matrix metalloproteinases (MMPs), serine
proteases, and cysteine proteases, are suspected of contributing to the
invasive and metastatic abilities of a variety of malignant tumors
(2-5), but the specific biochemical mechanisms that facilitate these
invasive behaviors remain elusive.
More than 23 human MMPs, and numerous homologues from other
species, have been reported (5), and matrix metalloproteinase-26 (MMP-26)/endometase/matrilysin-2 is a novel member of this
enzyme family that was recently cloned and characterized by our group (6) and others (7-9). MMP-26 mRNA is primarily
expressed in epithelial cancers, such as lung, breast, endometrial, and
prostate carcinomas, in their corresponding cell lines (6-9), and in a very limited number of normal adult tissues, such as the uterus (6, 8),
placenta (7, 8), and kidney (9). Recently, we have found that the
levels of MMP-26 gene and protein expression are higher in a
malignant choriocarcinoma cell line (JEG-3) than in normal human
cytotrophoblast cells (10). Our preliminary studies indicate that
expression of MMP-26 may be correlated with the malignant
transformation of human prostate and breast epithelial cells. The
specific expression of MMP-26 in malignant tumors and the
proteolytic activity of this enzyme against multiple components of the
ECM, including fibronectin, type IV collagen, vitronectin, gelatins,
and fibrinogen, as well as non-ECM proteins such as insulin-like growth
factor-binding protein 1 and
1-protease inhibitor (6-9), indicate
that MMP-26 may possess an important function in tumor progression.
Another member of the MMP family considered to be an
important contributor to the processes of invasion, metastasis, and
angiogenesis exhibited by tumor cells is gelatinase B
(MMP-9) (11-14). Uría and López-Otín
(8) have demonstrated that MMP-26 is able to cleave MMP-9, and here we
examine the possibility that MMP-26 facilitates tumor cell invasion
through the activation of pro-MMP-9. The highly invasive and metastatic
cell line utilized for this study, an androgen-repressed human prostate
cancer (ARCaP), was derived from the ascites fluid of a patient with
advanced prostate cancer that had metastasized to the lymph nodes,
lungs, pancreas, liver, kidneys, and bones (15). This cell line
produces high levels of MMP-9 and gelatinase A (MMP-2) (15, 16).
In this study, we provide evidence that MMP-26 is capable of activating
pro-MMP-9, and that once activated, MMP-9 cleaves fibronectin, type IV
collagen, and gelatin with great efficiency. Both the MMP-26 and MMP-9
proteins were highly expressed in the ARCaP cells, and co-localization
of their expression patterns was consistently observed. The
invasiveness of ARCaP cells through FN or type IV collagen was
significantly decreased in the presence of antibodies specifically
targeting MMP-26 or MMP-9. In addition, cells transfected with
antisense MMP-26, showing significant reduction of MMP-26 at
the protein level, exhibited a reduction of invasive potential in
vitro in addition to a significant diminution in observed levels
of active MMP-9 protein. These results support the hypothesis that
activation of MMP-9 by MMP-26 may promote the in vitro
invasiveness of ARCaP cells through FN or type IV collagen, whereas the
co-expression of MMP-26 and MMP-9 in many human prostate carcinoma
tissues indicates that this relationship may also occur in
vivo.
 |
MATERIALS AND METHODS |
Cell Culture--
ARCaP, DU145, PC-3, and LNCaP, which are all
established human prostate carcinoma cell lines, were routinely grown
in low-glucose Dulbecco's modified Eagle's medium supplemented
with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml
streptomycin in a humidified atmosphere containing 5% CO2
at 37 °C.
Silver Stain and Gelatin Zymography--
Purified recombinant
MMP-26 (6) or MMP-7 were incubated with purified pro-MMP-9 (17) or
pro-MMP-2 (18) in HEPES buffer (50 mM HEPES, pH 7.5, 200 mM NaCl, 10 mM CaCl2, and 0.01%
Brij-35) at 37 °C. For the dosage dependence of MMP-9 activation,
MMP-9 (0.2 µM, final concentration) was incubated with
MMP-7 and MMP-26 at the indicated molar concentration ratio (2:1, 4:1,
and 8:1) for 24 h. The MMP-9 activation was quenched by 2×
SDS-PAGE sample buffer containing 50 mM EDTA. The resulting
solution was further diluted five times and 5 µl of the diluted
sample was loaded onto SDS-polyacrylamide gels (8%). For the time
dependence of MMP-9 activation, MMP-9 (0.2 µM) was
incubated with MMP-7 (0.05 µM) and MMP-26 (0.05 µM) for the indicated time periods (0, 4, 8, 24 and
48 h) before quenching with the sample buffer. For FN cleavage assays, 2 µl of FN (0.25 mg/ml) were incubated with 30 µl of MMP-26 (final concentration 0.05 µM), pro-MMP-9 (final
concentration 0.2 µM), or MMP-26-activated MMP-9
solutions in 1× HEPES buffer at 37 °C for 18 h. For silver
staining, the reaction was stopped by adding 4× reducing sample buffer
(6% SDS, 40% glycerol, 200 mM Tris-HCl, pH 6.8, 5%
-mercaptoethanol, 200 mM EDTA, and 0.08% bromphenol
blue) and boiled for 5 min. Following electrophoresis on a 9%
SDS-polyacrylamide gel, the protein bands were visualized by silver
staining (19). For gelatin zymogram, the gel was incubated for 3 h
at 37 °C before it was stained with 0.1% Coomassie Blue solution
(17, 20, 21).
Protein N-terminal Sequencing--
Samples were separated by
SDS-PAGE and transferred to ProBlottTM polyvinylidene
difluoride membranes (Applied Biosystems) using CAPS buffer (10 mM CAPS, pH 11, 0.005% SDS). Proteins were visualized by
staining with Coomassie Brilliant Blue R-250 solution (0.1% Coomassie
Brilliant Blue R-250, 40% methanol, 1% acetic acid) and excised
fragments were sent for sequencing. N-terminal sequencing was performed
at the Bioanalytical Core Facility, Florida State University.
Reverse Transcriptase-PCR Analysis--
RNA was extracted from
the original cells by Trizol according to manufacturer protocols
(Invitrogen, Carlsbad, CA), and 2 µg of total RNA were
subjected to reverse transcriptase-PCR according to the standard
protocol provided with the PCR kit (Invitrogen Corp., Carlsbad, CA).
The MMP-26 forward primer was
5'-ACCATGCAGCTCGTCATCTTAAGAG-3'; the reverse primer was
5'-AGGTATGTCAGATGAACATTTTTCTCC-3'; for glyceraldehyde-3-phosphate
dehydrogenase the forward primer was 5'-ACGGATTTGGTCGTATTGGG-3'; the
reverse primer was 5'-TGATTTTGGAGGGATCTCGC-3'. PCR reactions were
performed using a Biometra Personal Cycler (Biometra, Germany) with 30 thermal cycles of 10 s at 94 °C denaturing, 30 s at
60 °C annealing, and 1 min at 72 °C elongation. Ten µl of the
amplified PCR products were then electrophoresed on a 1.0% agarose gel
containing 0.5 mg/ml ethidium bromide for analysis of size differences.
To confirm the amplification of the required cDNA sequences, PCR
products were digested with a restriction enzyme as directed by the manufacturer.
Generation and Characterization of Polyclonal
Antibodies--
Specific antigen peptides corresponding to unique
sequences in the pro-domain and metalloproteinase domain of MMP-26 were synthesized by Dr. Umesh Goli at the Biochemical Analysis, Synthesis and Sequencing Services Laboratory of the Department of Chemistry and
Biochemistry, Florida State University (Tallahassee, FL). The sequence
selected from the pro-domain was
Thr50-Gln-Glu-Thr-Gln-Thr-Gln-Leu-Leu-Gln-Gln-Phe-His-Arg-Asn-Gly-Thr-Asp67,
and the sequence selected from the metalloproteinase domain was
Asp188-Lys-Asn-Glu-His-Trp-Ser-Ala-Ser-Asp-Thr-Gly-Tyr-Asn201
of the prepro-enzyme. Using the BLAST search method at the National Center for Biotechnology Information web site against all of the sequences in the data banks, no peptide with >45% level of identity was found (6), predicting the antibodies directed against these two
peptides should be specific. The purity of these peptides was verified
by reverse-phase high performance liquid chromatography and mass
spectrometry. Rabbit anti-human antibodies were then generated,
purified, and characterized as described previously (19, 21). Western
blot analyses have demonstrated that these two antibodies are highly
specific for MMP-26 because they do not cross-react with human
matrilysin (MMP-7), stromelysin (MMP-3), gelatinase A (MMP-2),
gelatinase B (MMP-9), and some other proteins tested (data not shown).
Western Blotting--
Western blotting for MMP-26 was performed
by lysing the cells with Tris-buffered saline (50 mM Tris
and 150 mM NaCl, pH 7.4) containing 1.5% (v/v) Triton
X-114 as described previously (21). Aliquots (20 µl) of cell
lysate and media containing equal volumes (20 µl) from each treatment
treated with SDS sample buffer were then loaded onto an
SDS-polyacrylamide gel. Samples were electrophoresed and then
electroblotted onto a nitrocellulose membrane. Immunoreactive MMP-26
bands were visualized using a horseradish peroxidase or alkaline phosphatase-conjugated secondary antibody (Jackson
ImmunoResearch, West Grove, PA). Western blot analysis for
MMP-9 was performed with a 1 µg/ml dilution of polyclonal anti-MMP-9
antibody (Oncogene Science, Cambridge, MA). MMP-9 bands were visualized
using an alkaline phosphatase-conjugated secondary antibody (Jackson
ImmunoResearch) followed by the addition of 5-bromo-4-chloro-3-indoyl
phosphate and nitro blue tetrazolium. The blot membranes were then
scanned, and the signal intensities were measured by integrated
morphometry analysis (IMA) (Metamorph System, version 4.6r8, Universal
Imaging Corporation, Inc., West Chester, PA). The signal intensities
obtained were expressed as integrated optical density (the sum of the
optical densities of all pixels that make up the object). All the bands used the same exclusive threshold for analysis.
Immunocytochemistry and Immunohistochemistry--
Cells were
fixed in 50% methanol, 50% acetone for 15 min and permeated
with 1% Triton X-100 in Tris-buffered saline for 15 min.
Formalin-fixed paraffin-embedded human prostate cancer tissues were
sectioned to 4 µm thickness and fixed on slides. The sections were
dewaxed with xylene and rehydrated in 100 and 95% ethanol. Nonspecific
antibody binding in cells and sections was blocked with blocking buffer
(0.2% Triton X-100, 5% normal goat serum, and 3% bovine serum
albumin in Tris-buffered saline) for 1 h at room temperature prior
to overnight incubation with affinity-purified specific rabbit
anti-human MMP-26 antibody in the same buffer (5 µg/ml for
immunocytochemistry and 10 µg/ml for inmmunohistochemistry) or goat
anti-human MMP-9 antibody (25 µg/ml for immunohistochemistry, R&D
Systems, Minneapolis, MN) at 4 °C. Cells and sections were incubated
with alkaline phosphatase-conjugated secondary antibody (Jackson
ImmunoResearch) diluted (1:5000) in the blocking buffer for 4 h at
room temperature. The signals were detected by adding Fast-Red (Sigma).
Purified preimmune IgGs from the same animal were used as negative
controls for MMP-26. Normal goat serum was used as a negative control
for MMP-9. The sections were counterstained lightly with hematoxylin
for viewing negatively stained cells.
Preparation of MMP-26 Constructs--
Full-length cDNA of
MMP-26 was amplified by PCR according to published sequences
(6) and cloned into modified mammalian expression vector
pCRTM3.1-Uni with a FLAG tag at its C-terminal as described
(22). Following confirmation of cDNA sequencing, plasmids
containing correct inserts were used as sense vectors and plasmids with
reversibly inserted cDNA were used as antisense vectors (22).
Transfections of ARCaP Cells and Isolation of MMP-26 Sense and
Antisense Construct Stably Transfected Clones--
ARCaP cells were
transfected with sense and antisense MMP-26
cDNA-containing vectors using LipofectAMINE 2000 (Invitrogen) as
described earlier (22, 23). Sense- and antisense-transfected cell lines
were treated identically with regard to transfection conditions and
maintenance in the selection medium. Stable transfectants were selected
by growing the cells in 400 µg/ml Geneticin (G418; Invitrogen). Cells
that survived were then expanded in the absence of G418 for additional
studies. Stable transfectants were screened on the basis of FLAG and
MMP-26 expression. Clones with MMP-26 sense- and
antisense-integrated constructs were selected and analyzed for MMP-26
expression, invasive capabilities in modified Boyden chamber invasion
assays, and co-localization with MMP-9. Parental ARCaP cells served as controls.
Cell Invasion Assay--
The invasiveness of ARCaP cells
cultured in the presence of MMP-26 or MMP-9 functional blocking
antibodies, parental ARCaP cells, sense MMP-26- and
antisense MMP-26-transfected cells through reconstructed ECM
was determined as per our previous report (24). The final
concentrations of MMP-26 antibody were 10 and 50 µg/ml. The preimmune
IgG from the same animal was used as control for MMP-26 antibody, and
the final concentration was 50 µg/ml. The mouse anti-human MMP-9
monoclonal antibody is Ab-1, clone 6-6B, which is a functional
neutralizing antibody that inhibits the enzymatic activity of MMP-9
(25) (Oncogene Research Products, CalBiochem, La Jolla, CA). The final
concentrations of MMP-9 monoclonal antibody were 10 and 25 µg/ml. The
preimmune mouse IgG (Alpha Diagnostic Intl. Inc., San Antonio, TX) was
used as control, and the concentration was 25 µg/ml. Briefly,
modified Boyden chambers containing polycarbonate filters with 8-µm
pores (Becton Dickinson, Boston, MA) were coated with 0.5 mg/ml human
plasma FN (Invitrogen) or 0.5 mg/ml type IV collagen (Sigma).
Three-hundred µl of prepared cell suspension (1 × 106 cells/ml) in serum-free medium was added to each
insert, and 500 µl of media containing 10% fetal bovine serum was
added to the lower chamber. After 60 h of incubation, invasive
cells that had passed through the filters to the lower surface of the
membrane were fixed in 4% paraformaldehyde (Sigma). The cells were
then stained with 0.1% crystal violet solution and photographed with an Olympus DP10 digital camera (Melville, NY) under a Nikon FX microscope (Melville, NY). The cells were then counted by IMA. For
statistical analyses, the number of invasive cells treated with
preimmune IgG was assumed to reflect 100% cell invasion. The ratio of
the number of invaded cells that were treated with antibody or the
MMP-26 gene-transfected cells to preimmune IgG or parental
cells, respectively, was used for subsequent comparative analyses by
analysis of variance (ANOVA). Media from each insert was collected for
Western blot and gelatin zymogram analyses.
Immunofluorescence and Confocal Laser Scanning
Microscopy--
Cells were cultured on 8-well slides for 24 h,
then fixed in fresh 4% paraformaldehyde for 15 min at room temperature
and permeabilized with 0.2% Triton X-100 in 10% normal
goat serum in phosphate-buffered saline. The fixed, permeabilized cells
were stained for 1 h at room temperature with anti-human MMP-26
(25 µg/ml) or a goat anti-human antibody targeting MMP-9 (R&D
Systems, Minneapolis, MN) (1:200 dilution). Secondary rhodamine
red-X-conjugated mouse anti-rabbit IgG for MMP-26 or
fluorescein-conjugated donkey anti-goat IgG (Jackson ImmunoResearch)
for MMP-9 were subsequently applied at a 1:200 dilution for 1 h at
room temperature. Slow Fade mounting medium was added to the slides,
and fluorescence was analyzed using a Zeiss LSM510 laser scanning
confocal microscope (Carl Zeiss, Germany) equipped with a multiphoton
laser according to our previous report (23). Images were processed for
reproduction using Photoshop software version 6.0 (Adobe Systems,
Mountainview, CA). Purified preimmune IgGs from the same animal were
used as negative controls for MMP-26, and normal goat serum was used as a negative control for MMP-9.
Densitometric and Statistical Analysis--
Samples were
simultaneously stained with antibody and preimmune IgG on the same
slide, and the areas of MMP-26 immunostaining were quantified by IMA.
Four photographs were taken from each sample with an Olympus DP10
digital camera under a Nikon FX microscope. An appropriate color
threshold was determined (color model, HSI; hue, 230-255; saturation
and intensity, full spectrum), the glandular epithelia from each image
was isolated into closed regions, and all areas of staining in
compliance with these specific parameters were measured by IMA. The
total area of these closed regions was determined by region
measurement, and the ratio of signal area to total area was then
determined. The average of the four ratios obtained from each sample
was then used for subsequent analysis. The same color threshold was
maintained for all samples. The preimmune staining ratio was subtracted
from the antibody-staining ratio, and this value was then divided by
the preimmune staining ratio to yield the reduced signal to background
ratios used for subsequent comparative analyses by ANOVA. Statistical
analysis of all samples was performed with the least significant
difference correction of ANOVA for multiple comparisons. Data represent
the mean ± S.D. from three experiments where differences with
p < 0.05 were considered to be significant.
 |
RESULTS |
Activation of Pro-MMP-9 by MMP-26 and Cleavage of Substrates by
Activated MMP-9--
Gelatin zymography was utilized for determination
of MMP-9 activity levels following cleavage by MMP-26. Zymography
revealed that pro-MMP-9 presented as 225-, 125-, and 94-kDa
gelatinolytic bands under non-reducing conditions (Fig.
1, A, lane 1, and
B, lanes 1 and 6). The 225-kDa band is
a homodimer of pro-MMP-9, the 125-kDa band is a heterodimer of
pro-MMP-9 and neutrophil gelatinase-associated lipocalin, and the
94-kDa band is a monomer of pro-MMP-9 (17, 26, 27). New 215-, 115-, and
86-kDa bands were generated after incubation with MMP-26 (Fig. 1,
A and B), and their activities were increased in
a dose- and time-dependent manner (Fig. 1, A and
B). Compared with MMP-7, the cleavage products generated by
MMP-26 at the concentrations tested appear more stable (Fig. 1,
A and B). However, pro-MMP-2 was not activated
after incubation with identical concentrations of MMP-26 (data not
shown).

View larger version (44K):
[in this window]
[in a new window]
|
Fig. 1.
Activation of pro-MMP-9 by MMP-26.
A and B, gelatin zymogram of MMP-9 activity
before and after activation of pro-MMP-9 by MMP-26 under non-reducing
conditions. The 225-kDa band is a homodimer of pro-MMP-9, the 125-kDa
band is a heterodimer of pro-MMP-9 and neutrophil gelatinase-associated
lipocalin, and the 94-kDa band is a monomer of pro-MMP-9 (17, 26, 27).
The activation reactions were incubated at 37 °C. A,
dose-dependent analysis of pro-MMP-9 activation by MMP-26
(lanes 1-4) and MMP-7 (lanes 6-8). The
activation reaction was incubated 37 °C for 24 h. The gelatin
zymogram reaction was incubated at 37 °C for 3 h. The 86-kDa
band was sequenced and the sequence is MRTPRXG, which is a
product cleaved at the Ala93-Met94 site.
B, time-dependent analysis of pro-MMP-9
activation by MMP-26 (lanes 1-5) and MMP-7 (lanes
6-10). The gelatin zymogram reaction was incubated at 37 °C
for 3 h. The ratio labeled in A and B is the
molar concentration ratio. C, pro-MMP-9 activated by MMP-26
and cleavage of FN by MMP-26 and MMP-9 as detected by a silver-stained
gel under reducing conditions. The molar concentration ratio for
pro-MMP-9:MMP-26 is 4:1 and the reaction was incubated at 37 °C for
24 h. The molecular mass standards are labeled on the
left and the estimated molecular masses of the FN cleavage
products are labeled on the right.
|
|
MMP-26 cleaved pro-MMP-9 (94 kDa) to yield a new 86-kDa band on a
silver-stained gel under reducing conditions (Fig. 1C,
lane 4). N-terminal sequencing showed that the 86-kDa
protein had the sequence of MRTPRXG, which is the same N
terminus as reported during activation of pro-MMP-9 by
HgCl2 (28), human fibroblast-type collagenase (MMP-1) (17),
phenylmercuric acid (29), and aminophenylmercuric acid (30). For
further confirmation of MMP-9 activity, digestive assays were performed
utilizing FN as a substrate. MMP-26 alone demonstrated weak cleavage of
FN (Fig. 1C, lane 6), whereas pro-MMP-9 exhibited
no cleavage of FN (Fig. 1C, lane 7). Once
activated by MMP-26, MMP-9 cleaved FN very effectively, generating at
least 6 new bands (Fig. 1C, lane 8).
Expression of MMP-26 in Human Prostate Gland and ARCaP
Cells--
Immunohistochemistry staining revealed that the intensity
of MMP-26 staining was the highest in human prostate carcinoma (15 patient cases, Gleason grades 5-7), was low in prostatitis (9 cases),
and was very low in benign prostate hyperplasia (BPH) (12 cases) and
normal prostate gland tissues (7 cases) (Fig.
2A). Densitometric and
statistical analysis (Fig. 2B) showed that the intensities
of the immunostaining signals were significantly different between
normal prostate gland and prostate cancer samples (p = 0.0007), between BPH and prostate cancer (p = 0.0025),
and also between prostatitis and prostate cancer (p = 0.0043). However, there were no significant differences between normal
and BPH (p > 0.05), normal and prostatitis
(p > 0.05), or BPH and prostatitis tissues
(p > 0.05) (Fig. 2B).

View larger version (64K):
[in this window]
[in a new window]
|
Fig. 2.
Comparison of MMP-26 expression in human
normal and pathological prostate tissues. A,
immunohistochemistry and localization of MMP-26 in human prostate
carcinoma (15 patient cases), prostatitis (9 cases), benign prostate
hyperplasia (12 cases), and normal prostate gland tissues (7 cases).
Cells stained red indicate MMP-26 expression. Photographs
were taken under a microscope with ×400 magnification. B,
densitometric analysis of MMP-26 expression in human prostate tissues.
The quantification analysis was described under "Materials and
Methods." Four pictures were taken from each sample with ×200
magnification. The epithelial regions were selected and the staining
area and total selected area were obtained by IMA and analyzed by
one-way ANOVA with LSD correction. Data shown are the mean ± S.D.
values from the different prostate tissues. *, p < 0.01. BPH, benign prostate hyperplasia; Normal,
normal prostate tissue; Carcinoma, prostate
adenocarcinoma.
|
|
For selection of a prostate cancer cell line that expressed
MMP-26 for use as a working model, reverse transcriptase-PCR
and Western blot analyses were used to detect MMP-26 expression in four
human prostate cancer cell lines. MMP-26 mRNA was
identified in the ARCaP, DU145, and LNCaP cell lines, but not in the
PC-3 cell line (Fig. 3A).
Whereas the 20-kDa form of MMP-26 was detected in the ARCaP detergent
phase, a doublet between 30 and 40 kDa of pro-MMP-26 was located in the
ARCaP aqueous phase (Fig. 3B). This doublet might be two
N-glycosylated forms of pro-MMP-26 predicted according to
the ScanProsite program, with two possible N-glycosylation sites at N64GTD67 and
N221QSS224. MMP-26 may have N-linked
sugars according to the results obtained from N-glycosidase
F (PNGase F, Roche Molecular Biochemicals) digestion experiments (data
not shown). MMP-26 protein was not detected in the DU145, LNCaP, or
PC-3 cell lines (Fig. 3B), or in the ARCaP media under these
experimental conditions (data not shown). Immunocytochemistry data
confirmed that MMP-26 was localized inside the ARCaP cells (Fig.
3C) in a polarized manner.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 3.
MMP-26 mRNA and protein
expression in ARCaP cells. A, reverse transcriptase-PCR
analysis of MMP-26 mRNA in ARCaP, DU145, LNCaP, and PC-3
cell lines. MMP-26 plasmid is used as control (top panel,
lane 1). The mRNA levels of a glycolysis pathway enzyme,
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), are shown
in the bottom panel as a positive control to normalize
cellular mRNA concentration. B, Western blot analysis of
MMP-26 protein in ARCaP, DU145, LNCaP, and PC-3 cell lines. The
last lane is recombinant pro-MMP-26 as a control.
C, immunocytochemistry localization of MMP-26 in ARCaP
cells. Left panel, the primary antibody is rabbit
anti-MMP-26 antibody; right panel, the primary antibody is
preimmune IgG from the same rabbit. Red staining indicates
MMP-26 expression. Scale bars = 12 µm.
Arrows show the positive staining signals. The cells were
counterstained with hematoxylin for viewing of negatively stained cells
(purple).
|
|
Inhibitory Effects of Anti-MMP-26 and Anti-MMP-9 Antibodies on the
Invasiveness of ARCaP Cells--
To determine the role of MMP-26 and
MMP-9 in ARCaP cell invasiveness, antibodies targeting the
metalloproteinase domain of MMP-26 and targeting MMP-9 were utilized
during in vitro cell invasion assays. We found significant
(p < 0.01) reduction in the invasive potential of
ARCaP cells through FN at concentrations of 10 (62.4%) and 50 µg/ml
(46.0%) for the MMP-26 antibody (Fig. 4A), and at concentrations of
10 (55.9%) and 25 µg/ml (53.1%) for the MMP-9 antibody (Fig.
4B), when compared with the preimmune IgGs. We also found
significantly (p < 0.01) reduced invasive potential in
the movement of ARCaP cells through type IV collagen at concentrations
of 10 (29.3%) and 50 µg/ml (18.8%) for the MMP-26 antibody (Fig.
4A), and at concentrations of 10 (52.2%) and 25 µg/ml
(28.0%) for the MMP-9 antibody (Fig. 4B), when compared with the preimmune IgG. Antibody targeting the pro-domain of MMP-26 also significantly decreased the invasive potential of ARCaP cells through FN and type IV collagen (data not shown). These results show
that both anti-MMP-26 and anti-MMP-9 antibodies significantly inhibit
ARCaP cell invasion through FN and type IV collagen.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 4.
Blocking of ARCaP cell invasion through FN
and type IV collagen by MMP-26 and MMP-9 antibodies. The invasion
assay was performed with modified Boyden chambers. The MMP-26 antibody
is a rabbit anti-human MMP-26 metallo-domain antibody. The MMP-9
antibody is a mouse anti-human MMP-9 monoclonal antibody. The
percentage of invading cells was quantified as described under
"Materials and Methods." A, comparison of the invaded
cell number in the presence of MMP-26 antibody/preimmune IgG.
Control, preimmune rabbit IgG and the final concentration is
50 µg/ml. Ten and 50 µg of IgG means the final concentrations are
10 and 50 µg/ml, respectively. B, comparison of the
invaded cell number in the presence of MMP-9 antibody/preimmune IgG.
Control, preimmune mouse IgG and the concentration is 25 µg/ml. Ten and 25 µg of IgG means that the concentrations are 10 and 25 µg/ml, respectively. The invaded cell numbers of the preimmune
IgG treatment were used as the 100% invasiveness. Type IV,
type IV collagen. Data shown are the mean ± S.D. values from four
separate experiments for each group. *, p < 0.01; **,
p < 0.001.
|
|
MMP-26 Protein Expression in Stable Transfectants by
Immunocytochemistry and Western Blotting--
To further confirm the
role of MMP-26 in ARCaP cell invasion, we transfected pCR 3.1 vectors
containing full-length MMP-26 cDNA in both sense and
antisense orientations into ARCaP cells. Immunocytochemistry and
Western blotting were performed to determine MMP-26 protein expression
levels in the parental cells in addition to the sense and antisense
MMP-26 construct-transfected cells. Immunocytochemistry
showed very strong MMP-26 staining in both the parental ARCaP and sense
MMP-26 construct-transfected cells, whereas the antisense
MMP-26 construct-transfected cells exhibited only minimal
staining for MMP-26 (Fig. 5A).
Western blotting revealed strong MMP-26 bands in the parental ARCaP and
sense MMP-26 construct-transfected cells, whereas only a
very faint band was detected in the antisense MMP-26
construct-transfected cells. No MMP-26 was detected in the cell
culture media (Fig. 5B).

View larger version (67K):
[in this window]
[in a new window]
|
Fig. 5.
MMP-26 protein expression in parental ARCaP,
sense MMP-26 construct, and antisense
MMP-26 construct stably transfected cells.
A, immunocytochemistry of MMP-26 expression in parental
ARCaP, sense MMP-26 construct, and antisense
MMP-26 construct stably transfected cells. Red
staining indicates MMP-26 expression. Arrows show examples
of the positive staining signals. The cells were counterstained with
hematoxylin for viewing of MMP-26 negative cells (purple).
B, Western blot analysis of MMP-26 protein expression.
Parental ARCaP, sense MMP-26 construct, and antisense
MMP-26 construct stably transfected cells were cultured
utilizing an equivalent number of cells. Conditioned medium samples
were collected prior to cell lysis.
|
|
Reduction of Invasiveness of MMP-26 Antisense Stable
Transfectants--
Both the parental ARCaP and sense MMP-26
construct-transfected cell lines invaded through either FN or type IV
collagen in vitro during cell invasion assays (Fig.
6A), but without a marked difference (p > 0.05) in their invasive potentials
(Fig. 6B). Antisense MMP-26 construct-transfected
cells showed a significant (p < 0.01) decrease in
invasive potential through the same materials (44.0 and 23.5%,
respectively) when compared with parental ARCaP cells (Fig. 6,
A and B). A significant (p < 0.01) difference between the sense and antisense MMP-26
construct-transfected cells was also noted (Fig. 6, A and
B).

View larger version (75K):
[in this window]
[in a new window]
|
Fig. 6.
Invasion of parental ARCaP, sense, or
antisense MMP-26 construct stably transfected cells
through FN and type IV collagen. A, cells that invaded
to the lower surface of the membrane were photographed under a
microscope with ×40 magnification. B, the percentage of
invading cells in parental and MMP-26 sense or antisense
construct stably transfected ARCaP cells. The cell numbers of the
invaded parental cells were used as 100% invasiveness. The cells were
counted and analyzed as described under "Materials and Methods."
Data shown are the mean ± S.D. values from three separate
experiments for each group. *, p < 0.01; **,
p < 0.001.
|
|
Reduced Levels of Active MMP-9 in MMP-26 Antisense Stably
Transfected Cells--
To determine the role of MMP-26-mediated MMP-9
activation in ARCaP cell invasion, the level of MMP-9 in conditioned
media samples collected from the Boyden chambers during in
vitro cell invasion assays was detected. Western blotting revealed
a strong 86-kDa band of active MMP-9 in the conditioned media from
parental ARCaP and sense MMP-26 construct-transfected cells.
A similar band, but of weaker intensity, was detected in the
conditioned media collected from the antisense MMP-26
construct-transfected cells (Fig.
7A). Semiquantitative analysis
revealed that the active form of MMP-9 was significantly decreased
(p < 0.01) in both the FN and type IV collagen
invasive assay media from the antisense MMP-26
construct-transfected cells (Fig. 7B).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 7.
Detection of MMP-9 in the invasive assay
media from parental ARCaP, sense MMP-26 construct, or
antisense MMP-26 construct stably transfected
cells. A, Western blot of the invasive assay media.
Media samples were collected from the upper compartments of Boyden
chambers during cell invasion assays. B, densitometry
scanning and semiquantitative analysis of the levels of MMP-9 in the
invasive assay media. Data shown are the mean ± S.D. values from
four separate experiments for each group. *, p < 0.001.
|
|
Co-localization of MMP-26 with MMP-9 in Parental and MMP-26 Sense
Gene Stably Transfected ARCaP Cells, and Co-expression of MMP-26 and
MMP-9 in Human Prostate Carcinoma Tissue Samples--
Double
immunofluorescence experiments were performed in parental ARCaP and
MMP-26 stably transfected cells with human MMP-26 sense or antisense genes. The red color indicates MMP-26 and the green
color indicates MMP-9 protein staining. Merged images show a color
shift to orange-yellow, indicating co-localization between MMP-26 and
MMP-9. Confocal laser scanning microscopic analysis revealed
co-localization of both proteins in the cytoplasm of parental ARCaP
(Fig. 8A,
a-d) and sense-transfected cells (Fig. 8A, e-h), but not in the
antisense-transfected cells (Fig. 8A, i-l). Very weak signals were detected in
parental ARCaP control cells using purified preimmune IgG for the
detection of MMP-26 and nonimmune goat sera for the detection of MMP-9
(Fig. 8A, m-p). MMP-26 and MMP-9
proteins were also found to be co-expressed in human prostate carcinoma
tissue samples (Fig. 8B).

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 8.
Co-localization of MMP-26 and MMP-9 in
parental and sense MMP-26-transfected ARCaP cells and
an example of MMP-26 and MMP-9 co-expression in the same human prostate
carcinoma tissues. A, double immunofluorescence
staining and confocal laser scanning micrographs of parental, sense,
and antisense MMP-26-transfected ARCaP cells. Red
indicates MMP-26 signals and green indicates MMP-9 signals.
Yellow reveals the co-localization of MMP-26 and
MMP-9. Blue fluorescence represents the nuclei.
a-d, parental ARCaP cells. e-h, sense
MMP-26 construct-transfected cells. i-l,
antisense MMP-26 construct-transfected cells.
m-p, parental ARCaP cells with preimmune IgG and goat
sera as controls. Scale bars = 5 µm. B, an
example of co-expression of MMP-26 and MMP-9 proteins in the same human
prostate carcinoma tissues. a, strong positive MMP-26
protein staining in epithelial cells of human prostate carcinoma
(Gleason grade 3 + 3). b, positive MMP-9 protein staining in
epithelial cells of the same human prostate carcinoma. Cells stained
red indicate MMP-26 and MMP-9 expression. The sections were
counterstained with hematoxylin for viewing negatively stained cells.
Photographs were taken under a microscope with ×400
magnification.
|
|
 |
DISCUSSION |
MMP-26 is able to activate MMP-9 by cleavage at the
Ala93-Met94 site of the prepro-MMP-9, which is
the same cleavage site detected previously during activation with
HgCl2 (28), human fibroblast-type collagenase (17),
phenylmercuric acid (29), and aminophenylmercuric acid (30). This
activation was confirmed by the effective cleavage of FN using MMP-9
activated by MMP-26. These results indicate that the zymogen form of
MMP-9 can be transiently activated without the proteolytic loss of the
cysteine (Cys99)-switch residue, even though these findings
may appear to be in conflict with the original Cys-switch hypothesis
(31). The 86-kDa form of MMP-9 may also be further activated to produce lower molecular mass active species similar to the process activated by
other MMPs (17). Among all the MMPs, matrilysin (MMP-7) and MMP-26
share domain structures with pro- and metalloproteinase domains only
and are both expressed in epithelial cells (6-9). Therefore, MMP-26 is
also named as matrilysin-2 (8). Both MMP-26 and MMP-7 could activate
MMP-9 but their cleavage sites in pro-MMP-9 are different. Matrilysin
cleaved MMP-9 at two sites, Glu59-Met60 and
Arg106-Phe107 of the prepro-MMP-9 (17). Our
current results also demonstrated that the MMP-9 activation mediated by
MMP-26 is much slower than that mediated by MMP-7, but the activation
products are much more stable when compared with the products of
activation by MMP-7. This indicates that activation of MMP-9 by MMP-26
is prolonged but persistent, which is consistent with the process of
tumor cell invasion. MMP-26 did not cleave pro-MMP-2, another
gelatinase, indicating that pro-MMP-9 activation by MMP-26 is highly
selective. MMP-9 is a powerful enzyme, and is considered to be an
important contributor to the processes of invasion, metastasis, and
angiogenesis in various tumors (11-14, 32-36).
This work has tested the hypothesis that MMP-26 may enhance human
prostate cancer cell invasion via the activation of pro-MMP-9 using an
ARCaP cell line as a working model. The ARCaP cell line is a highly
invasive and metastatic human prostate cancer cell line that expresses
both MMP-9 (15) and MMP-26. We found that MMP-26 mRNA
was detected in the ARCaP cell line and two other human prostate
carcinoma cell lines, DU145 and LNCaP, but the MMP-26 protein was only
detected in ARCaP cells. More importantly, high levels of MMP-26
protein were also detected in human prostate carcinoma cells by
immunohistochemistry, but only low expression was seen in prostatitis,
benign prostate hyperplasia, and normal prostate tissues. This is in
agreement with reports of MMP-26 gene expression in
epithelial cancers (6-9). We have previously reported that the levels
of MMP-26 gene and protein expression are increased in a
malignant choriocarcinoma cell line (JEG-3) to levels that are well in
excess of that found in normal human cytotrophoblast cells (10). The
majority of MMP-26 protein detected was in the detergent phase of the
ARCaP cell lysates, not in the conditioned media, and only low levels
were observed in the aqueous phase. This is in accordance with recent
studies demonstrating that MMP-26-transfected COS-7 and
HEK293 cells secrete the protein poorly (7-9). As MMP-26 was found in
the detergent phase of the ARCaP cell lysates, it is possible that
MMP-26 may be associated with cell membrane components via an
unidentified mechanism. Membrane-associated MMP-26 may participate
directly in degradation of the ECM, activating pro-enzymes, and
releasing growth factors, partially accounting for the inhibition of
ARCaP cell invasion by the MMP-26 antibody tested. These reports
converge to suggest that MMP-26 may play an important role in human
carcinoma invasion and tumor progression.
MMP-26 exhibits wide substrate specificity, and is capable of degrading
many components of the basement membrane and other ECM components
(6-9, 37). Although MMP-26 can cleave type IV collagen, fibronectin,
and other proteins, it is a catalytically less powerful enzyme than
gelatinase B/MMP-9. The inhibition of ARCaP cell invasion by
MMP-26-specific antibodies suggests that MMP-26 may contribute to ARCaP
cell invasion by cleaving ECM components directly and/or by activating
pro-MMP-9 to cleave the ECM. Our FN cleavage assays with MMP-26 alone
and MMP-26-activated MMP-9 show that once activated by MMP-26, MMP-9
cleaves FN more efficiently. This indicates that the activation of
MMP-9 may be a major pathway for MMP-26 promotion of ARCaP cell
invasion. Indeed, this hypothesis was further verified by ARCaP cell
invasion inhibition in the presence of MMP-9 functional blocking
antibodies. When the proteolytic activity of MMP-26 is combined with
that of activated MMP-9, which digests ECM and basement membrane
proteins in an even more aggressive fashion than MMP-26 alone, this
hints at an amplification mechanism by which MMP-26 might contribute
significantly to the processes of tumor cell invasion and subsequent metastasis.
Several lines of evidence have demonstrated that biochemical activation
of pro-MMP-9 by MMP-26 may be a physiologically and pathologically
relevant event. Our results demonstrated that antibodies directed
against MMP-26 catalytic domain and prodomain both blocked the ARCaP
cell invasion. Equally as significant, a function blocking monoclonal
antibody that inhibits MMP-9 catalytic activity (25, 38) also prevented
the invasion of ARCaP cells in patterns similar to a MMP-26 antibody.
These results verify our hypothesis that activation of pro-MMP-9 by
MMP-26 promotes invasion of human prostate cancer cells.
Recently, our group has also determined that MMP-26 autodigested itself
during the folding process. Two of the major autolytic sites were
Leu49-Thr50 and
Ala75-Leu76, which left the
"cysteine-switch" sequence (PHC82GVPD) intact (37),
and suggests that Cys82 may not play a role in the latency
of the zymogen form. Another group has demonstrated that autolytic
activation of MMP-26 occurred at LLQ59
Q60FH, which is upstream from the cysteine residue known to
be responsible for the latency of many other MMPs (39). Interestingly,
our pro-domain antigen peptide mimics the Thr50 to
Asp67 region of MMP-26, and the resultant antibody complex
shields the autocleavage sites (data not shown). This fortunate
circumstance may account for the decreased invasiveness of ARCaP cells
treated with our antibody targeting the pro-domain, while also
suggesting that the catalytic activity of MMP-26 may not
require the highly conserved cysteine-switch
activation mechanism.
To further confirm the role of MMP-26 during ARCaP cell invasion, we
generated stably transfected ARCaP cells with vectors containing
full-length MMP-26 cDNA in both the sense and antisense orientations. Our results show that transfection of the ARCaP cells
with antisense MMP-26 constructs leads to decreased levels of MMP-26 protein expression when compared with parental and sense controls, suggesting that this antisense construct is responsible for
the observed decrease in MMP-26 protein expression, resulting in
profound biological consequences. In cell invasion assays, antisense-transfected cells show a marked reduction in invasiveness over those of parental ARCaP and MMP-26 sense
gene-transfected ARCaP cells, suggesting that the modulation of
MMP-26 in ARCaP cells altered the invasive potential of
these cells in our experimental model system, lending support to the
hypothesis that MMP-26 activity may play a crucial role in facilitating
the invasion of ARCaP cells through the ECM.
Western blotting of conditioned media collected from the upper
compartments of the Boyden chambers during invasion assays reveals that
the 86-kDa active form of MMP-9 is present in parental ARCaP and sense
MMP-26-transfected ARCaP cell media, but very little active
MMP-9 is present in the antisense MMP-26-transfected ARCaP
cell media. These findings suggest that MMP-26 activated MMP-9 in
parental ARCaP and sense MMP-26-transfected ARCaP cells, while very little activation took place in the antisense
MMP-26-transfected ARCaP cells. When present, active MMP-9
accumulates in the cytosol of human endothelial cells, where it is
eventually utilized by invading pseudopodia (40), and it is possible
that endogenous, self-activated MMP-26 acts as an activator for
intracellular pro-MMP-9. The active form of MMP-9 may then be stored
inside the cell, ready for rapid release when it is required to
facilitate the invasion of ARCaP cells.
Consistent with the above data, double immunofluorescence labeling and
confocal laser scanning microscopy reveal that MMP-26 and MMP-9 were
co-localized in parental ARCaP and sense MMP-26-transfected ARCaP cells, affording them ample opportunity to interact.
Co-localization was not observed in antisense
MMP-26-transfected ARCaP cells, as MMP-26 was not expressed
in these cells. Immunohistochemistry revealed a similar relationship in
human prostate tissue samples, demonstrating that MMP-26 and MMP-9 were
also co-expressed in prostate carcinomas. Recently, Nemeth et
al. (36) have reported that both MMP-9 mRNA and
protein were expressed in biopsy specimens from patients with
documented, bone-metastatic prostate cancer. Thus, the biochemical
activation mechanism of pro-MMP-9 that we observed in vitro
might well be applicable to prostate cancer in vivo.
Although direct degradation of the ECM by MMP-26 may contribute to the
processes of cell invasion and tumor metastasis, as the consequential
relationship between MMP-26 and MMP-9 begins to emerge, we find
evidence of coordination and a proteolytic cascade (activation of
MMP-9) that may be a major pathway to promote the invasion of human
prostate carcinoma. The specific expression of MMP-26 and its potential
role in the invasion of cancer cells suggest that MMP-26 may be a new
marker for certain types of prostate carcinomas, and perhaps a new
therapeutic molecular target for prostate cancer.