From the Department of Obstetrics and Gynecology,
Hamamatsu University School of Medicine, Handayama 1-20-1, Hamamatsu,
Shizuoka 431-3192, Japan, the ¶ Department of Obstetrics and
Gynecology, Jichi Medical School, Tochigi 329-0498, Japan, and the
Second Department of Pathology, Miyazaki University School of
Medicine, Miyazaki 889-1692, Japan
Received for publication, January 9, 2003, and in revised form, February 3, 2003
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ABSTRACT |
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Bikunin, a Kunitz-type protease inhibitor, could
potentially suppress tumor cell invasion and metastasis. Our
previous study revealed that overexpression of bikunin in a human
ovarian cancer cell line, HRA, resulted in a down-regulation in
uPA and uPAR gene expression. For identifying
the full repertoire of bikunin-regulated genes, a cDNA microarray
hybridization screening was conducted using mRNA from
bikunin-treated or bikunin-transfected HRA cells. A number of
bikunin-regulated genes were identified, and their regulation was
confirmed by Northern blot analysis. Our screen identified 11 bikunin-stimulated genes and 29 bikunin-repressed genes. The identified
genes can indeed be classified into distinct subsets. These include
transcriptional regulators, oncogenes/tumor suppressor genes, signaling
molecules, growth/cell cycle, invasion/metastasis, cytokines,
apoptosis, ion channels, extracellular matrix proteins, as well as some
proteases. This screen identified suppression of several genes such as
CDC-like kinase, LIM domain binding, Ets domain transcription
factor, Rho GTPase-activating protein, tyrosine
phosphorylation-regulated kinase, hyaluronan-binding protein,
matriptase, and pregnancy-associated plasma protein-A (PAPP-A), which
have previously been implicated in enhancing tumor promotion. Northern
blot analysis confirmed that several genes including
matriptase and PAPP-A were down-regulated by
bikunin by ~9-fold. Further, genetic inhibition of matriptase or
PAPP-A could lead to diminished invasion. These results show
that bikunin alters the pattern of gene expression in HRA cells leading
to a block in cell invasion.
Cancer metastasis is a complex multistep process involving
sequential interactions between the disseminating tumor cells and a
continuously changing host microenvironment. These interactions include
cell-cell and cell-extracellular matrix
(ECM)1 communication, which
regulate tumor cell attachment, spreading, and migration, the
dissolution of tissue barriers through the degradative activity of
enzymes such as metallo and serine proteinases, and growth modulation
by autocrine or paracrine growth factors (1).
Although bikunin was originally identified as a Kunitz-type protease
inhibitor essential for inhibition of trypsin and plasmin, there is now
increasing evidence to indicate a role for this glycoprotein in the
regulation of cell biology (2). Bikunin may be important in inhibiting
the inflammatory response: it has been shown to inhibit the induction
of pro-inflammatory cytokines in several types of cells (2, 3). In
animal models of inflammatory status, mice treated with bikunin exhibit
decreases in several indices of inflammation (3, 4).
Interestingly, in the case of neoplastic cells, exposure of bikunin to
cancer cells induces suppression of invasion and metastasis (5, 6). The
bikunin molecule has effects independent of its ability to inhibit
trypsin and plasmin (7). A number of high affinity specific receptors
and binding proteins exist for bikunin on the tumor cell surface (8).
It binds cartilage link protein (Crtl1), one of the hyaluronan-binding
proteins (8). Bikunin also interacts with the 45-kDa bikunin receptor,
a putative CD44 accessory protein (8). Bikunin may cause inhibition of ECM invasion by interacting both with its receptor and Crtl1 in various
cell types (8). It is possible that bikunin is associated as a
heterotrimer with the bikunin receptor and Crtl1.
It has been established that uPA and uPAR have been shown to modulate
the invasion of tumor cells derived from several organs (9). We have
previously reported that bikunin down-regulates genes involved in tumor
aggressiveness (6, 7). For example, bikunin target genes uPA
and uPAR are central components of the tumor invasion
program (9). We provide evidence that bikunin repressed uPA
and uPAR expression by inhibiting the MAP kinase pathway
(10). Further, transfection of HRA cells with the bikunin gene caused
pronounced inhibition of uPA expression and drastic suppression of ECM
invasion (11). Exogenously added bikunin could suppress peritoneal
disseminated metastasis and intraperitoneal ovarian tumor growth, and
expression of uPA in tumors of bikunin-injected nude mice was
significantly lower than in
controls.2 Similarly, when
bikunin-transfected cells were intraperitoneally injected into the
mice, the size of the intraperitoneal tumors and the levels of uPA were
also suppressed compared with luciferase-transfected control cells
(11). These data strongly support that uPA is mechanistically involved
in the regulation of these processes. Why bikunin treatment is capable
of inducing inhibition of cancer cell invasion is not fully known.
Presumably, there are select target genes that bikunin is capable of
regulating. The physiological function of bikunin still remains to be established.
To understand better how bikunin regulates different facets of tumor
cell biology, we sought to identify bikunin-regulated genes in a human
ovarian cancer cell line, HRA, using microarray technology. There has
not been any attempt to systematically define the full repertoire of
bikunin-regulated genes. Identification of these genes is required not
only for revealing the nature of all signaling pathways used by bikunin
but also for defining the set of proteins that are induced or repressed
by this inhibitor. In the current study, we started this investigation
using a cDNA microarray hybridization analysis of RNA isolated from
bikunin-treated and untreated HRA cells. In other experiments, we used
HRA cells transfected with the bikunin gene. Here we show, using two
different approaches, that we have identified at least 40 bikunin
target genes, and that bikunin-selective targets include genes involved in the regulation of transcription factors, oncogenes/tumor suppressor genes, signal transduction modifiers, cell growth, cell cycle, inflammation, invasion/metastasis, apoptosis, ion channels,
extracellular matrix proteins, and several proteases. The encoded
proteins are involved in a broad range of cellular functions and
signaling pathways. Interestingly, two different members of the
protease (matriptase and PAPP-A) were markedly repressed by bikunin. We confirm that, consistent with this, exposure of ovarian cancer cells to
bikunin or bikunin gene transfection suppresses a
matriptase/PAPP-A-dependent invasion, suggesting a
potential role for bikunin in modulating tumor biology.
Materials--
Purified human bikunin was obtained from Mochida
Pharmaceutical Co. (Gotenba, Japan). LipofectAMINE Plus reagent was
purchased from Invitrogen. Mouse monoclonal anti-PAPP-A antibodies
(IgG2b) were obtained from CosmoBio Inc. (Tokyo, Japan). Boyden-type
cell invasion chambers (BioCoat MatrigelTM invasion
chambers) were obtained from Collaborative Biomedical (Franklin Lakes,
NJ). The nude mice (Balb-c, nu/nu) were obtained from SLC (Hamamatsu,
Japan). All other chemicals were analytical grade.
Cell Culture--
The ovarian cancer cell line, HRA, was grown
in Dulbecco's modified Eagle's medium (Sigma Chemical Co.)
supplemented with 10% fetal bovine serum (Invitrogen), penicillin (100 units/ml), and streptomycin (100 µg/ml) under a 5% CO2
atmosphere with constant humidity (6). For all experiments in which
bikunin was added, cells were incubated in serum-free medium. Cells
were disaggregated routinely with 0.1% trypsin/EDTA solution and
replated at a split ratio of 1:10. Total RNA isolations were performed
using the Trizol reagent (Invitrogen). Protein concentrations in the
supernatants of cell extracts were measured by the Bio-Rad protein assay.
Microarray Probe Labeling and Hybridization--
The cDNA
microarrays (TaKaRa Human 3K CHIP Ver. 3.0; TaKaRa Bio Inc., Otsu,
Japan; www.takara-bio.co.jp) were spotted with 3,126 sequence-verified
clones that are available from GenBankTM
(www.ncbi.nlm.nih.gov/Genbank/index.html). Probes for cDNA
microarrays were generated using total RNA from cells either exposed to
bikunin (1 µM, 6 h; Exp. 1) or transfected with the
bikunin gene (Exp. 2) in a standard reverse transcriptase reaction in
which some of the dTTP was replaced with either Cy3-labeled dUTP or
Cy5-labeled dUTP. In some experiments, the bikunin sample was labeled
with Cy3; and in others, it was labeled with Cy5, with essentially identical results. Hybridization of both Cy3- and Cy5-labeled probes to
the same microarray was carried out in a sealed, humid hybridization
cassette for about 14 h at 65 °C. Microarrays slides were
washed and then dried by centrifugation at room temperature. Nine genes
were tested for confirmation by Northern blot hybridization.
Microarray Data Analysis--
To determine the fluorescent
intensities of the two dyes for each spot, the fluorescence signals of
Cy3- and Cy5-tagged cDNA spots on arrays at 532 nm (Cy3) and 635 nm
(Cy5) simultaneously were scanned immediately with an Affymetrix 428 Array Scanner and quantitated using a BioDiscovery ImaGene Ver. 4.2 (TaKaRa Bio Inc.). The background-subtracted median ratio value was
calculated for each spot, and replicate spots on each slide were
averaged. The fluorescence intensities were normalized by applying a
scaling factor so that the median fluorescence ratio of all spots with detectable signals above background on each microarray was 1.0. The
spots that displayed a 2-fold or greater difference in fluorescence intensities between the two dyes were used to generate gene clusters. Poor quality spots were removed if they were very small, irregularly shaped, or with pixels that were not uniformly distributed throughout the spot.
Northern Blot Hybridization with cDNA Probes--
Northern
blot hybridization was carried out as described previously (5). Samples
of total RNA (10 µg) were separated by electrophoresis through
denaturing 1.2% agarose gels containing 1% formaldehyde and
transferred onto nylon or nitrocellulose membranes (Hybond
N+, Amersham Biosciences) using standard molecular
biological techniques. Hybridization was carried out with
[ Bikunin Transfection--
The bikunin expression vector
pCMV-bikunin-IRES-bsr and the control vector pCMV-luciferase-IRES-bsr
(16) encoding luciferase (luc) were transfected into HRA
cells by the standard calcium phosphate precipitation method (17) as
described previously (11). The cells were selected in the presence of
10 mg/ml blasticidin S hydrochloride (Funakoshi Co. Ltd., Tokyo,
Japan). Resistant clones were obtained after 4 weeks, and
bik+ transfectants and a
luc+ transfectant were obtained. The cells were
subsequently maintained in the presence of 10 mg/ml blasticidin S
hydrochloride. The initial bik+ mass cultures
were subjected to at least two rounds of subcloning in order to obtain
stable bik+ clones (Clones 1-5). DNA sequencing
verified the correct insertion of the bik cDNA. Finally,
HRA bik+ tumor cell clones with inducible bik
protein expression were confirmed by immunocytochemical staining and
Western blot analysis (18). luc+ transfectants
were used as a control (11).
Antisense MT-SP1 and PAPP-A Oligodeoxynucleotides and Cell
Transfection--
Antisense 18-base phosphorothioate
oligodeoxynucleotides corresponding to the human MT-SP1
and PAPP-A mRNA were synthesized and consisted of the
antisense sequences of 5'-AGC TGC TCA TCC TAG GCA-3'
(AS-MT-SP1) and 5'-GCC CAA CTC CTG CTG GAA-3'
(AS-PAPP-A), respectively. Oligonucleotides mixed with
lipofectin reagent were incubated for 15 min at room temperature.
Thereafter, the oligonucleotide-liposome complexes were then added to
cells and washed twice with medium (19). After 4 h, fresh normal
growth medium containing 10% fetal bovine serum was added. Forty-eight
hours later the cells were analyzed for PAPP-A protein expression by
Western blot, and the cells were used for ECM invasion experiments as
described below.
Western Blot Analysis--
HRA cells transiently transfected
with either AS-PAPP-A as well as stably transfected with
bikunin were harvested in ice-cold 1× phosphate-buffered saline, and
cell pellets were lysed in radioimmune precipitation assay buffer.
Centrifuged lysates (50 µg) from each cell line were analyzed by
SDS-polyacrylamide gel electrophoresis on 15% gels and transferred to
a polyvinylidene difluoride membrane by semi-dry transfer. Membranes
were blocked for 1 h at room temperature in Tris-buffered saline
containing 0.1% Tween 20 and 2% bovine serum albumin. Blots were
probed with the following primary antibodies overnight at 4 °C:
rabbit polyclonal anti-bikunin (1:1000) or monoclonal anti-PAPP-A
(1:1000). This was followed by incubation with the appropriate
horseradish peroxidase-conjugated secondary antibody (Dako, Copenhagen,
Denmark) at a dilution of 1:50,000 for 1 h. Detection was achieved
by enhanced chemiluminescence (Amersham Biosciences).
Cell Growth Assay--
To examine the proliferation of each cell
line, 5 × 103 cells were seeded, and the number of
cells in each cell line was counted in triplicate after 24 h to
assess plating efficiency. Each experiment was done in triplicate.
ECM Invasion Assay--
Chemoinvasion assays were carried out in
a Boyden chamber as described (10). The upper surface of chamber was
precoated with a layer of artificial basement membrane, Matrigel. The
cell suspension (1 × 105 cells/well) was added to the
upper chamber. The lower chamber was filled with fibroblast-conditioned
medium, which acted as a chemoattractant. To measure invasion,
incubation was at 37 °C for 24 h. The invaded cells in the
lower side of the filter were stained with hematoxylin. Triplicate
filters were used for each cell type and assay condition, and 10 random
fields were counted per filter under a microscope (×400). The
experiments were repeated in duplicate.
The primary goal of this study was to identify genes regulated at
the transcriptional level by bikunin signaling in human ovarian cancer
cells. HRA cells were evaluated for its suitability in studying gene
expression changes in response to bikunin stimulation. The cell line
was found not to express mRNA and protein for bikunin (11).
Exogenously added bikunin induced a suppression of tumor cell invasive
ability in a modified Boyden chamber assay compared with control cells
(5-8,10). Furthermore, transfection of bikunin gene in these cells
also induces a specific and significant decrease in cell invasiveness
(11). Bikunin had negligible effects on the in vitro growth
of the HRA cells (5-8, 10, 11). Previous work has shown that bikunin
could down-regulate uPA and uPAR expression in
some of ovarian cancer cell lines (5-8, 10, 11). Thus, this decrease
in cell invasiveness was not due to a decrease in cell proliferation
but rather a suppression of uPA and uPAR gene expression. Therefore, two different methods (bikunin exposure and
bikunin gene transfection) were used to identify genes induced or
repressed in the HRA cells in microarray technologies.
Microarray Analysis of HRA Cells, Identification of Target Genes in
HRA Cells Transfected with the Bikunin Gene--
In the first set of
experiments, we tried to achieve the overexpression of the bikunin gene
in HRA cells (Exp. 1, Table I). To test
whether bikunin transfection could activate or inactivate potential
target genes, HRA cells were stably transfected with bikunin or
luciferase control (luc+). Of the stable bikunin
transfectants, the clone 1 (C1) cells were selected for the microarray
analysis because it showed the greatest bikunin expression (11). Total
RNA was purified from these samples, labeled with either Cy3 or Cy5
fluorescence, and then hybridized to the TaKaRa Human 3K CHIP. Each
slide contained duplicate sets of samples, and the colors of the two
cDNA probes were reversed in duplicate assays. Quantitation of the
signals produced two kinds of information: the intensity of the signal was proportional to the abundance of the corresponding mRNA, and the degree of redness or greenness indicated the fold induction or
repression of mRNA by bikunin transfection of the cells. The average hybridization intensity across all probe sets from C1 cells was
normalized to that obtained from a control luc+.
For each gene, the intensity ratio of C1/luc+
was calculated. Ratios of 1 indicate equal intensities and, therefore, no change in gene expression between the two cell lines. Ratios below
0.5 indicate down-regulation of gene expression in C1 cells. In
contrast, ratios above 2.0 indicate an up-regulation of gene expression
in C1 cells. Using this criterion, we found that 12 and 31 genes were
up- and down-regulated in C1 cells, respectively. Further, our analyses
revealed that 3 mRNAs (D30612, BC012816, and NM005399) were induced
by 3.0-fold or more, and 7 mRNAs (NM030941, NM021978, NM003992,
AB028983, NM006482, NM002375, and U28727) were repressed by
3.0-fold or more (Table I, Exp. 1).
Identification of Target Genes in HRA Cells Exposed to
Bikunin--
Many extracellular stimuli are known to activate or
suppress multiple and dependent signaling pathways leading to
transcriptional activation or suppression of different families of
genes. Bikunin transfection experiments in our studies have effects on
gene expression (see Table I, Exp. 1). We examined whether each of the
identified target genes might also be regulated by exogenously added
bikunin. Thus, in the second setting of experiments, for undertaking a systematic analysis of bikunin-regulated genes, HRA cells were exposed
to a control serum-free medium or the serum-free medium supplemented
with purified bikunin (40 µg/ml or 1 µM) for 6 h, and total RNA was isolated from treated and untreated cells (Table I,
Exp. 2). We chose the length of treatment to be 6 h, because our
previous studies have shown that this is the optimum time for
suppression of uPA and uPAR (5-8, 10). The two
sets of RNA from bikunin-treated and untreated cells were then used for microarray analysis. For each gene, HRA cells treated with bikunin (HRA(+)) was compared with untreated cells (HRA(
Taken together, there were 40 gene targets commonly identified by both
methods (bikunin exposure and bikunin transfection), in which 11 or 29 genes were induced or repressed, respectively. The majority of
target genes observed in the C1 cells were also affected by exogenous
bikunin (Table I). This suggests that the ability of bikunin to
regulate the genes identified in this study are likely caused by their
ability to bind to cells via specific bikunin receptors and not via a
nonspecific target. The putative function of genes selectively
regulated by bikunin may correlate with the biological phenotype
affected by bikunin in ovarian cancer cells.
Summary of Genes Induced or Repressed by Bikunin--
Analysis of
the results indicated several genes that were commonly altered in
response to both bikunin exposure and bikunin overexpression (Table I),
most of which had not previously been identified as bikunin target
genes. Cellular functions of many but not all of the bikunin-regulated
genes identified by our screen are known. In Table I, the proteins
encoded by these genes are grouped according to their functions. The
identified bikunin-stimulated or repressed genes can indeed be
classified into distinct subsets, each of which is probably induced by
a distinct bikunin-elicited signaling pathway. These include
transcriptional regulators, oncogenes/tumor suppressor genes, signaling
molecules, growth, proliferation, and cell cycle, invasion/metastasis,
cytokines, apoptosis, ion channels, extracellular matrix proteins, as
well as several proteases.
In the previous experiments, several proteins (for examples, uPA and
uPAR) known to be involved in invasion and metastasis were
significantly suppressed by bikunin. It is interesting to note that, in
this microarray analysis, uPA and uPAR were at
levels 1.87- and 1.79-fold lower in bikunin-transfected cells than in control cells. A repeat of the cDNA microarray with mRNA
samples obtained in cells exposed to bikunin for 6 h confirmed the
down-regulation of both uPA (
In general, bikunin is able to repress several genes related to tumor
aggressiveness. For example, TAF11 (20), GA-binding protein (21),
GTF2H2 (22), E74-like factor 2 (13), LIM domain binding 2 (23), and
CDC-like kinase 3 (CLK3) (24) are related to transcription molecules.
The repressed signaling molecules are the Rho GTPase-activating protein
5 (25), dual-specificity tyrosine phosphorylation-regulated kinase 2 (12), and four-and-a-half LIM domain proteins (FHL) (26). Examples of
oncogenes/tumor suppressor genes are Rho GTPase-activating protein 5 (25), zinc finger protein 282 (27), and RAB11A (28). In addition,
bikunin modulates a large number of cellular regulatory proteins
affecting cell growth such as hyaluronan-binding protein 2, CD81
antigen, and fatty acid-binding protein 3. An example of the
invasion/metastasis-related group is suppression of tumorigenicity 14 (matriptase, epithin). This protease has been shown to activate uPA.
Bikunin also represses pro-inflammatory cytokine-associated signaling
and enhances apoptosis. Two genes encode a voltage-independent
calcium-activated channel and a voltage-dependent chloride
channel (29) were also repressed. Syndecan has a role in cell adhesion,
maturation, proliferation, and prognosis (30). PAPP-A is the
insulin-like growth factor-binding protein protease (31). Therefore,
most of the bikunin target genes are involved in modulation of cell
proliferation, invasion, and tumor metastasis.
Validation of Microarray Results Using Northern Blot
Analysis--
To validate the data obtained in the microarray
analysis, we have subjected the RNA samples from the cells that were
originally used for microarray analysis to Northern blot analysis. Fold
induction calculated from the microarray data was compared with that
obtained using Northern blot analysis (Fig.
1). We initially measured expression levels of two bikunin target genes, uPA and uPAR,
that have been identified in other systems (5-8, 10, 11). The
hybridization signals for uPA and uPAR were significantly higher for
the RNA isolated from the control-treated cells, and the intensity of hybridization signal was very faint for the RNA isolated from bikunin-transfected cells. Quantitation of the signal revealed that the
expression of uPA and uPAR was 6.3- and 2.1-fold
lower in cells transfected with bikunin over the control cells. Thus, Northern blot analysis revealed that uPA and uPAR
were both reduced in response to bikunin expression.
The other six genes were selected for secondary confirmation based on a
combination of cDNA probe availability and putative gene function
because of interesting properties of the encoded proteins. As shown in
Fig. 1, all of the five candidate bikunin-repressed genes were strongly
reduced by bikunin, although the level of GAPDH mRNA was
unchanged. For all of the genes examined, the actual fold induction by
Northern blot analysis was significantly greater than that derived from
the microarray analysis. Quantitation of the Northern signals revealed
that the fold changes observed in the microarray analysis were in
general underestimates. Thus, even a relatively small difference noted
in the microarray analysis may be physiologically significant. This
analysis cannot exclude the possibility that stimulation/repression
occurs indirectly through activation/inactivation of one or more
intermediary molecules.
Kinetics of Gene Induction and Repression by Bikunin--
PAPP-A
mRNA was strongly repressed within 3 h of bikunin treatment
(Fig. 2). Thus, this mRNA represents
the early response gene. The level of this mRNA was recovered to
almost untreated levels after 12-24 h of bikunin treatment, indicating
that the suppression process is transient. For the other repressed gene MT-SP1 (matriptase), the reduction in the mRNA level had
slow kinetics. Interestingly, even after 24 h of bikunin
treatment, the level of this mRNA was very low, indicating that the
repressing effects lasted a long time. Thus, these two mRNAs
represent the early and late response genes whose suppressions are most
likely mediated by two different pathways. The levels of
GAPDH mRNA remained unchanged during the 24-h treatment
with bikunin.
Functions of Bikunin-repressed Genes, PAPP-A and MT-SP1
(Matriptase)--
It is clear that an exposure of cells to bikunin or
transfection of cells with the bikunin gene profoundly changes the
cellular abundance of a large number of mRNAs whose products are
essential to every aspect of cell physiology. Because PAPP-A
and MT-SP1 (matriptase) were identified as the strongly
repressed genes in response to treatment with bikunin (Table I and
Figs. 1 and 2), we selected them for further functional studies. We
determined whether genetic inhibition of PAPP-A or
MT-SP1 (matriptase) could lead to diminished invasion that
is dependent on PAPP-A or MT-SP1 (matriptase) protein. The
AS-PAPP-A or AS-MT-SP1 constructs were transfected into HRA cells for transient down-expression by using the
LipofectAMINE Plus reagent. The conditioned medium was collected after
transfection, and the level of PAPP-A was analyzed by Western blot. As
expected, PAPP-A protein expression was reduced when the cells were
transfected with AS-PAPP-A (Fig.
3, A and B). Since we could not obtain anti-MT-SP1 antibody, down-regulation of MT-SP1 expression was not determined in this study. Cells that down-express PAPP-A or possibly MT-SP1 were cultured for determining their invasive
ability in a modified Boyden chamber assay. Wild-type HRA cells,
LipofectAMINE Plus-alone cells, and luciferase-transfected cells
(luc+) served as controls. As shown in Fig.
3C, cell invasion was significantly reduced in
AS-MT-SP1 cells or to a lesser degree in
AS-PAPP-A cells. Cell growth was down-regulated in
AS-PAPP-A cells by ~20%, whereas MT-SP1 down-expression
failed to affect cell proliferation. These results demonstrated that
MT-SP1 and PAPP-A have a specific effect on the cell invasiveness
in HRA cells.
Current investigations have focused on the understanding of the
molecular mechanism(s) by which bikunin controls invasiveness in human
ovarian cancer HRA cells. To address the mechanisms that explain these
functions, we sought to identify bikunin-regulated genes using
microarray analysis in cultured cells. Here we show that either
overexpression of bikunin or treatment of HRA cells with
bikunin-altered gene expression in a similar fashion, suggesting that
the actions of bikunin occur extracellularly or at the plasma membrane.
Altered gene expression was confirmed by Northern blot analysis. Most
significantly, antisense for either of two proteins (PAPP-A and MT-SP1)
down-regulated by bikunin, mimics the loss of invasive ability caused
by bikunin.
Of the 43 bikunin-regulated genes reported in this study, 12 genes were
induced or 31 genes were repressed. The genes selectively regulated by
bikunin have a role in functions such as tumor biology. All of the
transcriptional factor- and signaling molecule-related genes were
repressed by bikunin. Bikunin also modulates a large number of
oncogenes/tumor suppressor genes, cytokines, and (extra)cellular regulatory proteins affecting cell adhesion, growth, and apoptosis (32-34). In addition, the NM002249 encoded protein is an integral membrane protein that forms a voltage-independent calcium-activated channel with three other calmodulin-binding subunits (35). The chloride
channel 6 encodes a voltage-dependent chloride channel gene
(29). These data may be related to the fact that bikunin can inhibit
cytokine-induced calcium influx in several types of cells (6, 36).
An example of the invasion/metastasis-related group is suppression of
tumorigenicity 14 (37). The protein encoded by suppression of
tumorigenicity 14 (matriptase, epithin) gene is strongly repressed. Epithin was originally identified as a mouse type II membrane serine
protease, which plays important roles in cell migration and tumor cell
metastasis (37). The human orthologues of epithin, MT-SP1 and its
N-terminal-truncated form, matriptase, have also been reported (38,
39). Matriptase is an 80-kDa matrix-degrading protease (40), and is
complexed with a Kunitz-type serine protease inhibitor, hepatocyte
growth factor activator inhibitor-1 (HAI-1) (41). The isolated cDNA
for matriptase appears to be a truncated form of MT-SP1, lacking the
N-terminal 172 amino acids of the coding sequence (39). Matriptase can
convert hepatocyte growth factor (HGF)/scattering factor to its active
form, and can activate c-Met tyrosine phosphorylation (42). Further,
protease-activated receptor 2 (PAR2) and single-chain uPA (sc-uPA) were
identified as substrates of matriptase (43). Studies with
MT-SP1/matriptase suggest that epithin and its human orthologue may
play important roles in cell migration as well as cancer invasion and
metastasis. In the previous experiments, several proteins including uPA
and uPAR known to be involved in invasion and metastasis were
significantly suppressed by bikunin (5-8, 10, 11). Although the
changes in mRNA levels detected by microarray hybridization were
generally moderate, in each case where the changes were confirmed by
Northern analysis, they were shown to be much more dramatic than
corresponding microarray results (Fig. 1). A similar underestimate of
the degree of changes of mRNA expression by microarray analysis has
been reported in other studies (44).
It would be particularly interesting to know whether the two genes,
MT-SP1 (matriptase) and PAPP-A, are
strongly repressed by bikunin treatment. In this study we showed that
MT-SP1 down-expression by an antisense strategy can specifically reduce
cell invasion (Fig. 3). Therefore, we speculate that bikunin can
inhibit tumor cell invasion possibly via suppression of MT-SP1
(matriptase)-dependent activation of HGF, c-Met, and uPA.
PAPP-A recently has been identified as an insulin-like growth factor
(IGF)-binding protein (IGF-BP)-4 protease (31). IGFs are mitogenic
peptides that regulate cell proliferation. IGF-BP-4 is an inhibitor of
IGF action (45), and proteolysis of IGF-BP-4 enhances IGF
bioavailability (46). Thus, it is likely that PAPP-A plays an important
role in regulating the availability of IGFs and regulating tumor cell
functions. In this study we also showed that HRA cells transiently
transfected with an AS-PAPP-A are less invasive than control
cells. Altogether, these results suggest that MT-SP1 (matriptase) and
PAPP-A may have a direct pro-invasive effect in HRA cells.
A certain gene (PAPP-A) is affected as early as 3 h
following bikunin treatment. It seems likely that PAPP-A is
an early response gene among putative bikunin target genes. Another
gene (MT-SP1, matriptase) is a late response gene (Fig. 2).
The molecular basis for this type of specificity may be due to some
combination of bikunin-dependent interactions with other
cofactors or pathways. There may also be unique cell cofactors that
play a role in dictating bikunin specificity. Bikunin is a protease
inhibitor rather than a transcription factor. Thus the changes in gene
expression are probably caused by some form of altered signaling.
Taken together, most of the bikunin down-regulated genes are shown to
inhibit cell proliferation, invasion, and tumor metastasis, whereas
most of the up-regulated genes promote apoptosis and tumor suppression.
Therefore, bikunin may negatively regulate possible cross-talk between
tumor aggressiveness and extracellular signaling actions. Experiments
reported here support our previous data (5-8, 10, 11). It is not
known, however, how many of these genes identified in this study
contain functional response elements. It is possible that bikunin may
repress these genes via a transrepression mechanism that involves
competition for limiting amounts of co-activators.
In conclusion, we have delineated for the first time the biochemical
mechanism by which bikunin reduces ECM invasion by modulating specific
gene expression. Decreased expression of specific genes such as
matriptase by bikunin may reflect down-regulation of uPA activation,
implying that bikunin facilitates a shift in balance toward decreasing
proteolytic activity of uPA. The results presented here should alert us
to the fact that bikunin will have additional global effects on
neoplastic cells by modulating the expression of a large number of
cellular genes. Clearly, the genomic response to bikunin signaling is
complex. Future studies focused on the regulation and functional
significance of the target genes reported here should increase our
knowledge of the biological activity of bikunin in non-neoplastic and
neoplastic cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dCTP by random oligonucleotide priming to
specific activities of 0.4-0.9 × 109 cpm/µg. The
following cDNA sequences were used as probes: DYRK2, bp 1344-2154
of the human cDNA (12); E74-like factor 2 (new Ets-related factor;
NERF), NERF cDNA (13); triple functional domain
(PTPRF-interacting), Trio cDNA (encoding amino acids 2249-2861 plus 140 bp of 3' nontranslated sequence) (14); suppression of
tumorigenicity 14 (MT-SP1[matriptase]), the full-length MT-SP1 cDNA (pcDNA3/MT-SP1) ligated into the pcDNA3 vector
(Invitrogen) under the control of a cytomegalovirus promoter (15); a
partial human PAPP-A cDNA (553 bp), a PCR product
(GenBankTM U28727; nucleotides 5593-6145) generated from
human ovaries using PAPP-A-specific primers (F: 5'-TGA GTC TCT GAC CAT
TTG GGT GAC-3', B: 5'-TTG GCT GCA GAA AAG GGA GCA G-3'). uPA cDNA
and uPAR cDNA were prepared as described previously (5). Filters were reprobed with the cDNA for glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) to correct for the amount of RNA loaded onto the
filters (5). After hybridization, the membranes were washed and exposed on Kodak BioMax MS-1 film at
70 °C. Filters were quantitated by
scanning densitometry using a Bio-Rad model 620 Video Densitometer with
a 1-d Analyst software package from Macintosh.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Microarray analysis of HRA cells
)), and the intensity ratio of HRA(+)/HRA(
) was calculated. Using the above criterion, we
found that 11 and 29 genes were up- and down-regulated in HRA(+), respectively. If ±3.0 is a conservative estimate for determining the
minimum magnitude of real ratios, we found up-regulation of 7 genes
(3.14-8.57-fold higher in bikunin treatment), whereas 17 genes were
down-regulated upon bikunin treatment (3.01-7.12-fold lower).
1.74-fold) and
uPAR (
1.95-fold) but not significantly.
View larger version (32K):
[in a new window]
Fig. 1.
Northern analysis of selected
bikunin-repressed genes. The regulation of uPA and
uPAR mRNA as well as five bikunin target genes
identified in the microarray screen was independently tested using
Northern blot analysis. Blots were probed with GAPDH to normalize for
differences in RNA loading. All reactions were performed in duplicate,
and the average -fold repression is shown. 10 µg of total RNA from
luciferase transfectant control cells ( ) or bikunin transfectant C1
cells (+) were analyzed. The numbers on the top compare the
folds of induction/repression of each mRNA as measured by
microarray and Northern blot analysis. Panel 1, uPA;
panel 2, uPAR; panel 3, E74-like factor 2;
panel 4, dual-specificity tyrosine phosphorylation-regulated
kinase 2; panel 5, triple functional domain; panel
6, suppression of tumorigenicity 14; panel 7, PAPP-A;
panel 8, GAPDH.
View larger version (39K):
[in a new window]
Fig. 2.
Kinetics of PAPP-A and
MT-SP-1 (matriptase) gene repression by bikunin.
10 µg of total RNA prepared from HRA cells treated with 1 µM bikunin for indicated time periods were analyzed by
Northern blot. Kinetics of induction of an early bikunin-down-regulated
gene (PAPP-A) and a late bikunin-repressed gene
(MT-SP1, matriptase) are shown.
View larger version (28K):
[in a new window]
Fig. 3.
Suppression of cell invasion in
AS-MT-SP1 or AS-PAPP-A cells.
A, detection of PAPP-A protein expression in PAPP-A-specific
antisense-transfected cells by Western blot. The conditioned media were
collected and used for 8-16% SDS-polyacrylamide gel electrophoresis
under reducing conditions and Western blot. The arrow
indicates the ~200-kDa PAPP-A band. Cells were transfected with
LipofectAMINE Plus alone (lane 1) or with
AS-PAPP-A (lane 2). Lane 3, wild-type
HRA cells; lane 4, bikunin-transfected C1 cells.
B, the levels of PAPP-A were quantified by densitometric
analysis and are represented in the form of a bar graph. The
mean value of triplicate experiments is indicated. Lane 1 serves as a control (100%). C, cells were transiently
transfected with AS-PAPP-A or AS-MT-SP1. Both
non-transfected and transfected cells were used for the cell invasion
assay. Equal numbers of cells were plated in the upper chamber. After
24 h of incubation, invaded cells were counted. The cells
transfected with AS-PAPP-A or AS-MT-SP1 showed
significant reduction of cell invasion compared with non-transfected
cells or cells transfected with LipofectAMINE Plus alone or with
luciferase-transfected cells (luc+). Values
indicate the number of invasive cells/filter and are expressed as
mean ± S.D. (n = 3). D, for cell
growth assay, each cell line was counted 24 h after plating. Each
experiment was done in triplicate. Results are the mean ± S.D. of three different determinations. Each superscript letter
(a, b, and c) is significantly
different (p < 0.05).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Drs. M. Fujie, K. Shibata, T. Noguchi, and A Suzuki (Equipment center and Photo center, Hamamatsu University School of Medicine) for helping with the biochemical analysis. We are also thankful to Drs. H. Morishita, Y. Kato, K. Kato, and H. Sato (BioResearch Institute, Mochida Pharmaceutical Co., Gotenba, Shizuoka), Drs. Y. Tanaka and T. Kondo (Chugai Pharmaceutical Co. Ltd., Tokyo), and Drs. S. Miyauchi and M. Ikeda (Seikagaku Kogyo Co. Ltd., Tokyo) for their continuous and generous support of our work.
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FOOTNOTES |
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* This work was supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan (to H. K. and Y. H.) and by a grant from the Yamanouchi Foundation for Research on Metabolic Disorders.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 81-53-435-2309; Fax: 81-53-435-2308; E-mail: hirokoba@hama-med.ac.jp.
Published, JBC Papers in Press, February 5, 2003, DOI 10.1074/jbc.M300239200
2 M. Suzuki and H. Kobayashi, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: ECM, extracellular matrix; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MT-SP1, membrane type serine protease 1; luc, luciferase; PAPP-A, pregnancy-associated plasma protein-A; IGF, insulin-like growth factor; Crtl1, cartilage link protein 1.
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1. |
Brodt, P.,
Fallavollita, L.,
Khatib, A. M.,
Samani, A. A.,
and Zhang, D.
(2001)
J. Biol. Chem.
276,
33608-33615 |
2. | Fries, E., and Blom, A. M. (2000) Int. J. Biochem. Cell Biol. 32, 125-137[CrossRef][Medline] [Order article via Infotrieve] |
3. | Nakamura, H., Abe, S., Shibata, Y., Sata, M., Kato, S., Saito, H., Hino, T., Takahashi, H., and Tomoike, H. (1997) Int. Arch. Allergy Immunol. 112, 157-162[Medline] [Order article via Infotrieve] |
4. |
Futamura, Y.,
Kajikawa, S.,
Kaga, N.,
and Shibutani, Y.
(1999)
Obstet. Gynecol.
93,
100-108 |
5. |
Kobayashi, H.,
Suzuki, M.,
Kanayama, N.,
Nishida, T.,
Takigawa, M.,
and Terao, T.
(2002)
Eur. J. Biochem.
269,
3945-3957 |
6. | Kobayashi, H., Suzuki, M., Tanaka, Y., Kanayama, N., and Terao, T. (2002) J. Biol. Chem. PMID: 12496270 |
7. | Suzuki, M., Kobayashi, H., Tanaka, Y., Hirashima, Y., and Terao, T. (2001) Biochim. Biophys. Acta 1547, 26-36[Medline] [Order article via Infotrieve] |
8. |
Hirashima, Y.,
Kobayashi, H.,
Suzuki, M.,
Tanaka, Y.,
Kanayama, N.,
Fujie, M.,
Nishida, T.,
Takigawa, M.,
and Terao, T.
(2001)
J. Biol. Chem.
276,
13650-13656 |
9. | Andreasen, P. A., Egelund, R., and Petersen, H. H. (2000) Cell. Mol. Life Sci. 57, 25-40[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Kobayashi, H.,
Suzuki, M.,
Tanaka, Y.,
Hirashima, Y.,
and Terao, T.
(2001)
J. Biol. Chem.
276,
2015-2022 |
11. | Suzuki, M., Kobayashi, H., Tanaka, Y., Hirashima, Y., Kanayama, N., Takei, Y., Saga, Y., Suzuki, M., Itoh, H., and Terao, T. (2003) Int. J. Cancer 104, 289-302[CrossRef][Medline] [Order article via Infotrieve] |
12. |
Becker, W.,
Weber, Y.,
Wetzel, K.,
Eirmbter, K.,
Tejedor, F. J.,
and Joost, H. G.
(1998)
J. Biol. Chem.
273,
25893-25902 |
13. | Oettgen, P., Akbarali, Y., Boltax, J., Best, J., Kunsch, C., and Libermann, T. A. (1996) Mol. Cell. Biol. 16, 5091-5106[Abstract] |
14. |
Debant, A.,
Serra-Pages, C.,
Seipel, K.,
O'Brien, S.,
Tang, M.,
Park, S. H.,
and Streuli, M.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
5466-5471 |
15. |
Cho, E. G.,
Kim, M. G.,
Kim, C.,
Kim, S. R.,
Seong, I. S.,
Chung, C.,
Schwartz, R. H.,
and Park, D.
(2001)
J. Biol. Chem.
276,
44581-44589 |
16. | Urabe, M., Hasumi, Y., Ogasawara, Y., Matsushita, T., Kamoshita, N., Nomoto, A., Colosi, P., Kurtzman, G. J., Tobita, K., and Ozawa, K. (1997) Gene (Amst.) 200, 157-162[CrossRef][Medline] [Order article via Infotrieve] |
17. | Wigler, M., Pellicer, A., Silverstein, S., and Axel, R. (1978) Cell 14, 725-731[Medline] [Order article via Infotrieve] |
18. | Chen, Y. T., Holcomb, C., and Moore, H. P. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6508-6512[Abstract] |
19. | Felgner, P. L., Gadek, T. R., Holm, M., Roman, R., Chan, H. W., Wenz, M., Northrop, J. P., Ringold, G. M., and Danielsen, M. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 7413-7417[Abstract] |
20. | Mengus, G., May, M., Jacq, X., Staub, A., Tora, L., Chambon, P., and Davidson, I. (1995) EMBO J. 14, 1520-1531[Abstract] |
21. | Watanabe, H., Sawada, J., Yano, K., Yamaguchi, K., Goto, M., and Handa, H. (1993) Mol. Cell. Biol. 13, 1385-1391[Abstract] |
22. | Humbert, S., van Vuuren, H., Lutz, Y., Hoeijmakers, J. H., Egly, J. M., and Moncollin, V. (1994) EMBO J. 13, 2393-2398[Abstract] |
23. |
Retaux, S.,
Rogard, M.,
Bach, I.,
Failli, V.,
and Besson, M. J.
(1999)
J. Neurosci.
19,
783-793 |
24. | Hanes, J., von der Kammer, H., Klaudiny, J., and Scheit, K. H. (1994) J. Mol. Biol. 244, 665-672[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Burbelo, P. D.,
Miyamoto, S.,
Utani, A.,
Brill, S.,
Yamada, K. M.,
Hall, A.,
and Yamada, Y.
(1995)
J. Biol. Chem.
270,
30919-30926 |
26. | Lee, S. M., Tsui, S. K., Chan, K. K., Kotaka, M., Li, H. Y., Chim, S. S., Waye, M. M., Fung, K. P., and Lee, C. Y. (1998) Somat. Cell. Mol. Genet. 24, 197-202[Medline] [Order article via Infotrieve] |
27. |
Okumura, K.,
Sakaguchi, G.,
Naito, K.,
Tamura, T.,
and Igarashi, H.
(1997)
Nucleic Acids Res.
25,
5025-5032 |
28. | Drivas, G. T., Shih, A., Coutavas, E. E., D'Eustachio, P., and Rush, M. G. (1991) Oncogene 6, 3-9[Medline] [Order article via Infotrieve] |
29. | Nomura, N., Nagase, T., Miyajima, N., Sazuka, T., Tanaka, A., Sato, S., Seki, N., Kawarabayasi, Y., Ishikawa, K., and Tabata, S. (1994) DNA Res. 1, 223-229[Medline] [Order article via Infotrieve] |
30. | Wiksten, J. P., Lundin, J., Nordling, S., Kokkola, A., and Haglund, C. (2000) Anticancer Res. 20, 4905-4907[Medline] [Order article via Infotrieve] |
31. |
Conover, C. A.,
Faessen, G. F.,
Ilg, K. E.,
Chandrasekher, Y. A.,
Christiansen, M.,
Overgaard, M. T.,
Oxvig, C.,
and Giudice, L. C.
(2001)
Endocrinology
142,
2155 |
32. | Choi-Miura, N. H., Tobe, T., Sumiya, J., Nakano, Y., Sano, Y., Mazda, T., and Tomita, M. (1996) J. Biochem. 119, 1157-1165[Abstract] |
33. | Oren, R., Takahashi, S., Doss, C., Levy, R., and Levy, S. (1990) Mol. Cell. Biol. 10, 4007-4015[Medline] [Order article via Infotrieve] |
34. | Spener, F., Unterberg, C., Borchers, T., and Grosse, R. (1990) Mol. Cell. Biochem. 98, 57-68[Medline] [Order article via Infotrieve] |
35. |
Kohler, M.,
Hirschberg, B.,
Bond, C. T.,
Kinzie, J. M.,
Marrion, N. V.,
Maylie, J.,
and Adelman, J. P.
(1996)
Science
273,
1709-1714 |
36. | Kanayama, N., el Maradny, E., Halim, A., Liping, S., Maehara, K., Kajiwara, Y., and Terao, T. (1995) Am. J. Obstet. Gynecol. 173, 192-199[CrossRef][Medline] [Order article via Infotrieve] |
37. |
Hooper, J. D.,
Clements, J. A.,
Quigley, J. P.,
and Antalis, T. M.
(2001)
J. Biol. Chem.
276,
857-860 |
38. |
Takeuchi, T.,
Shuman, M. A.,
and Craik, C. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11054-11061 |
39. |
Lin, C. Y.,
Anders, J.,
Johnson, M.,
Sang, Q. A.,
and Dickson, R. B.
(1999)
J. Biol. Chem.
274,
18231-18236 |
40. |
Lin, C. Y.,
Wang, J. K.,
Torri, J.,
Dou, L.,
Sang, Q. A.,
and Dickson, R. B.
(1997)
J. Biol. Chem.
272,
9147-9152 |
41. |
Lin, C. Y.,
Anders, J.,
Johnson, M.,
and Dickson, R. B.
(1999)
J. Biol. Chem.
274,
18237-18242 |
42. |
Lee, S. L.,
Dickson, R. B.,
and Lin, C. Y.
(2000)
J. Biol. Chem.
275,
36720-36725 |
43. |
Takeuchi, T.,
Harris, J. L.,
Huang, W.,
Yan, K. W.,
Coughlin, S. R.,
and Craik, C. S.
(2000)
J. Biol. Chem.
275,
26333-26342 |
44. |
Soukas, A.,
Cohen, P.,
Socci, N. D.,
and Friedman, J. M.
(2000)
Gene Dev.
14,
963-980 |
45. |
Hwa, V.,
and Oh, Y.
(1999)
Endocrinol. Rev.
20,
761 |
46. |
Fowlkes, J. L.,
Serra, D. M.,
Nagase, H.,
and Thrailkill, K. M.
(1999)
Ann. N. Y. Acad. Sci.
878,
696-699 |