From the Division of Pulmonary and Critical Care
Medicine, Departments of Medicine, ** Environmental
Medicine, and
Microbiology, New York University, New York,
New York 10016, the § Institute for Advanced Study, School
of Natural Science, Princeton University, New Jersey 08540, and
¶ Hoffman-La Roche Inc., Nutley, New Jersey 07110
Received for publication, October 25, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Wnt-induced-secreted-protein-1 (WISP-1) is a
cysteine-rich, secreted factor belonging to the CCN family. These
proteins have been implicated in the inhibition of metastasis; however,
the mechanisms involved have not been described. We demonstrated that overexpression of WISP-1 in H460 lung cancer cells inhibited lung metastasis and in vitro cell invasion and motility. We
investigated the possibility that WISP-1 may regulate activation of
Rac, a small GTPase important for cytoskeletal reorganizations during motility. In an indirect assay, WISP-1-expressing cells exhibited marked reduction in Rac activation compared with control cells. Blocking antibodies to Wnt-induced secreted protein-1
(WISP-1)1 is a member of a
family of cysteine-rich proteins called CCN factors that include connective tissue growth factor (CTGF), cysteine-rich 61 (CYR61), nephroblastoma overexpressed (Nov), WISP-2, and WISP-3 (1-5). CCN
proteins are modular in structure, consisting of an N-terminal signal
sequence followed by domains with sequence similarity to insulin-like growth factor-binding protein, von Willebrand
factor C, thrombospondin type 1, and cysteine knot at the C terminus with the exception of WISP-2, which lacks the C terminus region (6).
WISP-1 and WISP-2 were isolated as differentially expressed genes from
a subtractive hybridization of transcripts between Wnt-1-overexpressing
C57MG mammary epithelial cells and control cells (5). Studies on the
promoter of WISP-1 showed that cAMP-response element-binding protein
binding sites played an important role in the transcriptional
activation of WISP-1, while lymphoid enhancer factor/T cell
factor binding sites characteristic of CCN proteins exhibit a variety of biological activities. CYR61 induces
cell proliferation, angiogenesis, and cell adhesion through activation
of CCN family members have also been implicated in tumorigenesis and
metaplasia. Genomic copies and mRNA of WISP-1 and mRNA of WISP-3 were found to be significantly increased in colon carcinoma cell
lines and in colon tumors compared with normal mucosa (4). The
oncogenic properties of WISP-1 were apparent in normal rat kidney
fibroblasts overexpressing the gene, which demonstrated morphological
transformation and increased rates of cell proliferation and
tumorigenicity in nude mice (10). WISP-1 also protected cells from
p53-dependent apoptosis through activation of
AKT, up-regulation of Bcl-XL, and inhibition of cytochrome
c release (16).
While these proteins exert transforming and growth stimulatory effects
on some cell types, they can also cause inhibition of growth and
metastasis of tumor or transformed cells. The mouse equivalent of
WISP-1, mELM1, was shown to be down-regulated in highly metastatic
mouse melanoma cells. Consistently, upon mELM1 transfection and
expression, the cells became less metastatic (17). In a differential
display study, the expression of rCOP-1, the rat equivalent of WISP-2,
was significantly down-regulated in cells transformed by both activated
H-Ras and an inactivated mutant of p53. When rCOP-1 was
reintroduced into transformed cells, a high incidence of death occurred
in the transfected cells (18). In Src-transformed chicken embryo
fibroblasts, Nov was shown to be down-regulated in a transcriptional
and also post-transcriptional manner (11). In addition, the expression
of Nov was found to inhibit the growth of glioma cells (19). In
non-small-cell lung cancer cells, CYR61 was shown to have tumor
suppressor effects, and its expression was repressed in lung cancer
samples compared with normal counterparts (20).
In the present paper, we showed that overexpression of WISP-1 in H460
cells reduced metastasis of these cells to the lungs in nude mice.
In vitro studies demonstrated reduced invasion of WISP-1-expressing cells through MatrigelTM (BD
Biosciences), a derivative of basement membranes. Metastasis and
invasion require, in part, the migration of cells across the substratum. We showed that WISP-1-expressing cells were impaired in
migration within Boyden chambers in response to serum. Biochemical pathways that regulate cell migration invoke the activation of small
Rho-like GTPases, which function to reorganize cytoskeletal elements.
WISP-1-expressing cells demonstrated marked inhibition of Rac
activation. In the presence of blocking antibodies to integrins and
WISP-1, Rac activation was restored. Transfection of a constitutively active Rac construct, RacG12V, into WISP-1 cells augmented the invasion
and motility of these cells. Microarray analysis and real-time PCR
showed that metalloproteinase-1 (MMP-1) expression was reduced in
WISP-1 cells but was increased above control levels in
RacG12V-transfected WISP-1 cells. These cells also showed increased invasion through collagen I, a substrate of MMP-1. These results were
discussed in the context of WISP-1/integrin interactions and the
effects on cell invasion and metastatic potential of lung cancer cells.
Cell Culture and Transfections--
H460 and H1299 cells as well
as their transfectants were cultured in DMEM supplemented with 10%
fetal bovine serum, penicillin, and streptomycin. H460 and H1299 cells
were transfected with HA- and FLAG-tagged WISP-1 cDNA, and clonal
cell lines were established as described previously (16). The
constitutively active Rac mutant, RacG12V, was subcloned into the
retrovirus vector pBabe and transfected into the packaging cell line,
phoenix A. The supernatant containing viable, non-replicative viruses
was collected and used to infect H460-WISP1 cells. Stable cell
populations were selected using puromycin. Control cell lines were also
generated by infection with the pBabe empty vector. Conditioned medium
was generated by incubating cells in serum-free medium. Three days
later, the medium was collected, cleared of cell debris by
centrifugation, concentrated ~10-fold through a Centriplus YM-100
column (Millipore, Bedford, MA), and sterilized using a
0.45-µM polyvinylidene difluoride filter (Whatman,
Clifton, NJ) before use.
Western Blotting and Immunoprecipitation--
Cells were
trypsinized and counted, and 3 × 106 cells were
seeded in 10-cm dishes for 24 h. The cells were then starved in serum-free DMEM for 16 h followed by induction with serum for various periods of time. Serum stimulation was stopped by the addition
of cold PBS, and the cells were lysed in RIPA buffer containing 1% SDS
and 1% deoxycholate. Protein assay was used to determine equivalent
amounts of protein for gel loading, and the lysate was added to sample
buffer and boiled for 8 min. Proteins were fractionated by
electrophoresis on SDS gels and transferred onto polyvinylidene
difluoride immobilon membranes. The membranes were blocked with 5%
milk and incubated with primary antibodies followed by washes and
staining with secondary antibodies. Detection and visualization was by
chemiluminescence (PerkinElmer Life Sciences) followed by exposure to
X-OMAT film (Kodak, Rochester, NY).
In Vivo Lung Metastasis Assay--
In an in vivo lung
metastasis experiment, vector control or WISP-1-expressing H460 cells
(106 cells/0.1 ml of DPBS) were injected into the lateral
tail veins of nude mice (Taconic, Germantown, NY). Eight weeks later,
the animals were sacrificed, and the lungs were fixed in formalin. Lung
sections were processed, stained with hematoxylin and eosin, and
observed for tumor cell colony formation.
Migration and Invasion Assays--
Cells were trypsinized and
counted, and 2 × 104 cells were placed in upper
migration chambers (BD Biosciences), which were then rested in wells
containing 1.5% FBS in DMEM. Incubation was carried out for 8 h
in a humidified CO2 chamber. Non-migratory cells in the
upper chamber were removed by scraping, and migrated cells on the lower
surface were fixed in methanol and stained with methylene blue. The
number of migrated cells were then counted from a total of nine regions
of the filter and calculated as numbers/cm2. For the
invasion assays, 4 × 104 cells were placed in upper
chambers precoated with a layer of Matrigel with 5% FBS present in the
lower chamber. The cells were incubated for 48 h and then
processed as described above. Collagen I-coated wells were made by
adding 2 µg/µl of collagen I (Chemi-Con, Temecula, CA) to 19 µg
in upper chambers and drying for a few hours at room temperature. Cell
invasion assays in collagen I-coated filters were performed as
described above.
Rac Activation Assay--
Equal numbers of cells were seeded and
incubated for 24 h in a 5% CO2 chamber, serum-starved
for 16 h, and treated with DMEM containing 10% FBS or no serum
for 15 min. The reaction was stopped with cold DPBS, the cells were
lysed with lysis buffer containing Mg2+, and the lysate was
centrifuged for 10 min. The supernatant (0.5 µg of proteins) was
added to 10 µl of p21-activated kinase (PAK)-agarose conjugates (UBI,
Lake Placid, NY), rotated at 4 °C for 75 min, and followed by three
washes of the protein complexes with lysis buffer. PAK-bound proteins
were dissociated and denatured by heating in sample buffer at 98 °C
for 8 min and subjected to gel electrophoresis. Rac proteins were
visualized using anti-Rac antibody (UBI) and chemiluminescence
techniques. Inhibitory antibodies to the integrins Microarray Analysis and Real-time PCR--
H460 control,
H460/WISP-1, and H460/WISP-1/RacG12V cells were seeded to equal density
and incubated for 24 h in a 5% CO2 chamber. The cells
were washed twice with DPBS and lysed for RNA precipitation using
RNeasy mini-columns (Qiagen, Valencia, CA). The RNA was used to
generate double-stranded cDNA (Invitrogen), which was then
transcribed into biotinylated cRNA (Affymetrix, Santa Clara, CA). The
fragmented products were hybridized to U133A gene chips and processed
in the fluidics station (Affymetrix). The probe array was scanned, and
the data analyzed using the Microarray Suite Software (Affymetrix).
Real-time PCR reactions involved first-strand synthesis from RNA by
oligo(dT) priming (Ambion, Austin, TX), addition of template to a PCR
mix containing the SYBR® green reporter molecule (Applied Biosystems,
Foster City, CA), and running the PCR reaction in the ABI Prism 7000 Sequence Detector (Applied Biosystems). The comparative cycle threshold
(CT) method was used to analyze the data by
generating relative values of the amount of target cDNA. CT represents the number of cycles for the
amplification of target to reach a fixed threshold and correlates with
the amount of starting material present. To obtain relative values, the
following arithmetic formula was used:
2 WISP-1 Expression Inhibits Metastasis and Invasion of H460
Cells--
The large-cell lung cancer line, H460, exhibits properties
of neuroendocrine cells and is highly metastatic. To investigate the
role of WISP-1 in metastasis, H460 cells transfected with the human
WISP-1 gene or vector control were injected into the tail vein of nude
mice and monitored for tumor growth in the lungs. Eight weeks after
injection, vector-transfected cells were found to be metastatic to the
lung and formed large tumor clusters, whereas WISP-1-expressing cells
had markedly reduced numbers and size of tumor colonies, indicating
that WISP-1 expression inhibited lung metastasis (Fig.
1A). H460 transfectants were
also used in an in vitro assay for invasion using
Matrigel-coated chambers. WISP-1-expressing cells demonstrated
considerably reduced invasiveness across Matrigel compared with vector
control cells (Fig. 1B). An invasion assay using conditioned
medium generated from either H460 vector control (vectorCM) or
WISP-1-expressing cells (WISP-1CM) was conducted (Fig. 1C).
The invasion of H460 cells incubated with vectorCM was significantly
greater than that with WISP-1CM, indicating inhibition of invasion by
soluble WISP-1 secreted into the conditioned medium (Fig.
1C). Data represent the mean ± S.D. of triplicate
determinations.
WISP-1 Down-regulates the Motility of Lung Cancer Cells in Boyden
Chamber Assays--
The motility of cells incorporates changes in the
cytoskeleton that affect cellular polarization and adhesion. A
component of the process of invasion is the motility of cells across a
substratum. Since WISP-1 in H460 cells inhibited metastasis and
invasion, we tested whether the expression of WISP-1 may reduce the
motility of cells in vitro using Boyden chamber assays. A
total of 2 × 104 cells in serum-free medium were
placed in the upper Boyden Chamber and induced to move across a
gradient of serum factors (1.5% FBS). Following incubation for 8 h in a 5% C02 incubator, H460/WISP-1 cells were
significantly less motile compared with vector transfectants. A
checkerboard analysis was performed that confirmed that H460 cells
preferentially migrated across a gradient of chemotactic serum factors
(data not shown). Similar inhibition by WISP-1 in the motility of H1299
lung cancer cells was also observed (Fig. 2).
Rac Activation Is Inhibited in WISP-1-expressing Cells--
Cell
migration is regulated by pathways that involve cytoskeletal
rearrangements resulting in membrane ruffling, lamellae formation,
and cell protrusions. These cellular activities are controlled by
Rho-like GTPases such as Rho, Rac, and Cdc42. To investigate whether
functional changes in cell motility by WISP-1 expression in lung cancer
cells may be related to the activity of Rho GTPases, WISP-1 expressing
and vector control cells were subjected to an indirect assay for Rac
activation. The results showed that even though equivalent amounts of
total Rac protein were present, WISP-1-expressing cells exhibited
reduced binding of GTP-bound Rac (activated Rac) to PAK/agarose
conjugates compared with control cells. Similar results were obtained
for WISP-1-expressing H1299 cells that exhibited diminished
precipitation of activated Rac compared with control cells, indicating
a reduction in Rac activity (Fig. 3).
Restoration of Rac Activation in the Presence of Inhibitory
Antibodies--
CCN family members have been shown to bind and
activate integrins. To determine whether the inactivation of Rac by
WISP-1 may involve upstream components such as integrins,
inhibitory antibodies to integrins
To further ascertain the role of WISP-1 in the inhibition of Rac
activation, affinity-purified antibodies previously shown to have a
blocking effect on WISP-1 (16) were used in a Rac activation assay.
WISP-1 blocking antibodies inhibited the effect of WISP-1 on Rac and
restored Rac activation, indicating specificity of function of WISP-1
(Fig. 4B).
Reconstitution of Rac Activity in H460-WISP1 Cells--
To
determine whether Rac activity is responsible for the reduction in cell
motility and invasion in WISP-1-expressing cells, an activated mutant
form of Rac, RacG12V, was expressed in H460-WISP-1 cells by retroviral
infection. A control WISP-1 cell line was also generated using the
vector alone. The transfectants were subjected to a Rac activation
assay where H460/WISP-1/RacG12V cells showed greater Rac activity
compared with control transfectants (Fig.
5). These cells were used for subsequent
microarray studies and bioassays.
Metalloproteinase-1 Is Down-regulated by WISP-1 but Up-regulated by
RacG12V--
The inhibition of cell invasion by WISP-1 may involve
changes in the expression of genes that regulate matrix turnover or degradation. To determine how the expression of WISP-1 may alter the
expression of genes involved in cellular invasion, microarray analysis
was performed using cRNA derived from H460/vector, H460/WISP-1, and
H460/WISP-1/RacG12V transfectants. The biotin-labeled cRNA was
hybridized to gene chips, which were then processed and scanned. The
data obtained were used in binary comparative analyses using the
Microarray Suite Software. Genes significantly different in expression
were identified based on calculated p values. Among the
genes of interest, MMP-1 was identified as a gene down-regulated in
WISP-1-expressing cells but up-regulated in the WISP-1/RacG12V cells,
which may play role in cell invasion.
To verify this data, real-time PCR was conducted using primers specific
to MMP-1 and actin. Target amounts were normalized against actin, and
the H460 control target was used as a calibrator (see "Materials and
Methods"). As shown in Fig. 6, WISP-1
cells showed ~3-fold lower expression of MMP-1 than control cells.
WISP-1/RacG12V cells, on the other hand, had increased values of about
3-fold higher than control cells. These results indicated that WISP-1 inhibited the expression of MMP-1, whereas activation of Rac restored the level of MMP-1 in WISP-1-expressing cells (Fig. 6).
Migration and Invasion of RacG12V-transfected WISP-1-expressing
Cells--
To test whether increased Rac activity in WISP-1/RacG12V
cells may restore motility and invasion in these cells, a modified Boyden Chamber assay was conducted. The assay showed that
reconstitution of Rac activity in H460/WISP-1/RacG12V cells increased
the motility of these cells compared with WISP-1 cells (Fig.
7A). Similarly, WISP-1/RacG12V
cells also exhibited higher levels of invasion than WISP-1 cells,
indicating that activation of Rac promotes cellular invasion in
vitro (Fig. 7B). MMP-1 proteolyses several types of
substrate including collagen type I. A collagen I invasion assay was
used to determine whether reduced expression of MMP-1 by WISP-1 may
affect the invasion of cells across a target substrate. The upper wells
of migration chambers were coated with 19 µg of collagen I, and the
lower chambers were filled with 5% FBS-containing medium. Consistent
with the reduced expression of MMP-1 (Fig. 6), WISP-1 cells were
impaired in the invasion of collagen I compared with control cells.
WISP-1/RacG12V cells were, however, as efficient as control cells in
the invasion assay (Fig. 7C), which correlated with
increased MMP-1 expression by RacG12V transfection.
Members of the CCN family play important roles in development
during chondrogenesis, skeletogenesis, and neurogenesis. In adults,
they may serve to maintain matrix structure and composition and tissue homeostasis. How the natural functions of CCN proteins relate to cancer and other diseases is not well understood. In addition, the correlation between the incidence and progression of
cancer with the expression of CCN proteins cannot be generalized across
different types of cancer. This may be due to the presence of distinct
cohorts of receptors and/or variable regulation of the ligands at the
cell surface, such as cleavage by proteases, in a cell type-specific
manner. In addition, the response of CCN proteins to a given
environment is strongly influenced by its multidomain structure, which
may regulate the formation of variable complexes of binding partners.
In cancer, a destructive turn occurs when tumor cells acquire the
ability to metastasize by spreading and colonizing host tissues. Loss
of cadherins and cell adhesion contribute to the breakdown of a
coherent epithelial organization. Of the two forms of highly metastatic
cancer, small-cell lung cancer (SCLC) and non-small-cell lung cancer
(NSCLC), the latter conforms to this model and demonstrates a tendency
to down-regulate the expression of cadherins (21). Tumor cells invade
and migrate across the extracellular matrix and blood vessels,
processes involving chemotactic and chemokinetic responses. While
chemotaxis relies mainly on the paracrine expression of growth factors,
chemokinesis occurs through autocrine stimulation. NSCLC cells, for
example, respond to a number of growth factors such as epidermal growth
factor, insulin-like growth factor 1, insulin-like growth factor
2, and hepatocyte growth factor, which play a role in inducing
chemotaxis (22). In a checkerboard analysis, H460, a NSCLC cell line
with some neuroendocrine characteristics, responded in a chemotactic manner under low serum conditions (data not shown). SCLC, on the other
hand, does not appear to be chemotactic but is chemokinetic through
autocrine expression of neuroendocrine regulatory factors, such as
bombesin (gastrin-releasing peptide), and stimulation of endogenous
receptors (23).
The mechanism of cell invasion involve both the secretion
metalloproteinases, which digest basement membrane (24), and the motility of cells. Motile cells adopt a polar morphology in the form of
distinctive front and trailing edges. The motility of cells also
require the cycling of localized attachment and detachment from the
surface of matrices. The Rho family of GTPases has been characterized
for their involvement in cellular actin reorganizations during the
motility of cells (25). Small GTPases cycle between an active and
inactive state through the hydrolysis of GTP. Cdc42 regulates pseudopod
extensions, whereas Rac and Rho initiate the formation of lamellae and
the establishment of stress fibers, respectively. The activities of
Cdc42 and Rac propel cell motion by polarizing cells into a motile
phenotype. Rho, on the other hand, generally, stabilized stress fibers
and focal adhesions, thereby inhibiting cell motility (25).
In this paper, we studied the effects of WISP-1, a Wnt-1-inducible
factor belonging to the CCN family, on the metastasis and invasion of
lung cancer cells. When H460 and H1299 lung cancer cells were
transfected with WISP-1, lamellar structures were largely reduced, and
the cells were less spread on the surface of tissue culture plates
(16).2 In vitro
motility and invasion of WISP-1-expressing H460 and H1299 as well as
the in vivo metastasis of H460/WISP-1 cells were significantly reduced compared with vector control cells. These results
prompted the hypothesis that the activation of Rac may be inhibited by
WISP-1, resulting in the reduction of lamellar structures, cell
motility, and invasion.
In an indirect assay for Rac activation, we observed a marked reduction
in the activation of Rac for WISP-1 transfectants of both H460 and
H1299 cells compared with control cells. Upon transfection of a
constitutively active Rac mutant, RacG12V, increases in the motility
and invasion of cells were observed for H460/WISP-1/RacG12V cells,
demonstrating that the down-regulation of Rac activity by WISP-1 was
responsible for the inhibition of cell motility and invasion.
The activation of Rac occurs downstream of several pathways including
integrin/Src kinase (26) and platelet-derived growth factor signaling
(27). In addition, the pertussis toxin-sensitive G protein,
Gi (28), and cAMP-dependent pathways (29) have also been implicated in Rac-mediated cell spreading and migration. To
understand how WISP-1 may be inhibiting Rac, a number of inhibitors were used in the Rac activation assay. We found that none of the inhibitors to phosphatidylinositol 3-kinase, Src, G-proteins, and
calmodulin were able to inhibit endogenous Rac activity significantly in control cells (data not shown). On the other hand, when inhibitory antibodies to several integrins were used, Rac activation in WISP-1 cells was restored to almost wild-type levels, while there was no
effect on control cells. Similar observations were made using blocking
antibodies to WISP-1. Therefore, the down-regulation of Rac activation
in WISP-1 cells may be attributed to the interactions of WISP-1 with integrins.
In addition, a consequence of WISP-1 overexpression is the
down-regulation of MMP-1, a metalloproteinase involved in lung cancer
metastasis, which may occur through WISP-1 inhibition of Rac
activation. Consistently, the invasion of cells across collagen I, a
substrate of MMP-1, was compromised in WISP-1-transfected cells but was
increased in the WISP-1/RacG12V transfectants.
It is unclear how WISP-1 may regulate Rac function through integrins.
The mechanism may involve a shift in the balance of activatory
and inhibitory signals. Mechanosensory effects mediated through
integrins may produce cytoskeletal-mediated signaling operating through
actin-myosin and microtubular dynamics (30). However, it is possible
that the inhibition may occur through changes in activity of Rac
regulators. As an example, mechanical stresses applied to fibroblasts
resulted in the inhibition of Rac activity and lamellipodia formation,
with implications that a guanine exchange factor may be responsible
(31). Therefore, the effect of WISP-1 on integrin signaling may be
multitiered. On one level, the activating functions of integrins may be
altered or absent. On another level, a set of alternative reactions may occur, triggered by cell shape and cytoskeletal changes.
Integrins are the only known receptors for CCN proteins, and receptor
activation may produce a variety of effects. For example, interaction
of CYR61 with integrin Similarly, WISP-1 was reported to have tumorigenic properties
when expressed in normal rat kidney fibroblasts (7), whereas in
melanoma cells, contrasting effects were observed and WISP-1-expression resulted in the inhibition of tumorigenesis and metastasis. Consistent with the latter, we demonstrated that expression of WISP-1 in H460 lung
cancer cells inhibited lung metastasis. In mammary cancer tissue from
Wnt-1 transgenic mice, mesenchymal cells demonstrated high levels of
expression of WISP-1, whereas in cancer cells, low levels or negative
staining was observed. This suggests that the secretion of WISP-1 by
stromal cells may have positive regulatory effects on cancer cells
through paracrine signaling (5). On the other hand, autocrine WISP-1
production by cancer cells may result in the inhibition of
tumorigenesis and metastasis, and therefore, its expression may be
down-regulated in cancer. It is plausible that there may be differences
between cancer and normal cells in the processing and presentation of
WISP-1 on the cell surface. This in turn may cause variability in
WISP-1 interactions with integrins that result in specific signaling
and biological outcomes in their respective cell types. Some support
for this comes from our unpublished data showing that the secreted form of WISP-1 from H460 lung cancer cells is highly glycosylated and that
the pattern of glycosylation differs between cancer cells and normal
fibroblasts. Investigations are being carried out to evaluate the
functional significance of the data. Finally, further studies will be
needed to determine if the expression or processing of WISP-1 in tumors
may have prognostic value in predicting cancer metastasis.
v
5 and
1 integrins restored Rac activation in WISP-1 cells,
suggesting that the inhibitory effect of WISP-1 on Rac lies downstream
of integrins. Constitutively activated Rac mutant (RacG12V) was
transfected into WISP-1 cells to restore Rac activation and these
WISP-1/RacG12V transfectants were used for further studies. We
performed microarray and real-time PCR analyses to identify genes
involved in invasion that may be differentially regulated by WISP-1.
Here, we showed decreased expression of metalloproteinase-1 (MMP-1) in
WISP-1 cells compared with controls but increased expression in
WISP-1/RacG12V cells. In an invasion assay across collagen I, an MMP-1
target matrix, WISP-1 cells were significantly less invasive compared
with controls, whereas WISP-1/RacG12V cells showed elevated invasion
levels. This work illustrates a negatively regulated pathway by WISP-1
involving integrins and Rac in the down-regulation of invasion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-catenin signaling played a
minor role (7). WISP-3 was identified by homologous sequence searches;
however, the regulation of its gene expression remains unknown (5).
CYR61 is an immediate-early gene inducible by several growth factors
including TGF-
, fibroblast growth factor, (8) and platelet-derived
growth factor (2, 9), whereas CTGF appears to be regulated mainly by
TGF-
(10). Nov, on the other hand, is down-regulated under serum
conditions (11).
V
3 integrins in endothelial cells (4). CTGF plays a key role downstream of TGF-
and SMAD signaling and stimulates production of fibronectin and collagen, important for wound
closure (10, 12). Several members of this family of growth factors
appear to have important roles in the development and homeostasis of
bone and cartilage. The expression of CYR61 correlates with the early
stage chondrocytic differentiation of limb bud mesenchyme (11), whereas
CTGF and Nov are expressed at a later stage during cartilage
development. In addition, CYR61 promotes chondrogenesis of limb bud
mesenchyme in in vitro cultures (13). Wnts are important
developmental genes, known to regulate chondrocytic differentiation
(14), and WISP-1 and WISP-2 may play a role downstream of Wnt signaling
in regulating this process. CCN proteins are also linked to
degenerative cartilage diseases. Mutations in the gene of WISP-3 are
associated with progressive pseudorheumatoid dysplasia, a disease
characterized, in part, by the degeneration of cartilage (15).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6,
v
5, and
1 (Chemi-Con) were
added to cell cultures 24 h prior to treatment with serum and
harvesting for activation assays.
CT,
where
CT = difference between the threshold
cycles of the target and an endogenous reference (actin), and
CT = difference between
CT of the target sample and a designated
calibrator (vector control). The calculated result represents the
amount of normalized target relative to the calibrator.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Overexpression of WISP-1 inhibits
in vivo metastasis and in vitro
invasion of H460 lung cancer cells. A, H460/vector
control formed large and extensive colonies of tumors in the lungs of
nude mice. In contrast, tumor size was reduced in WISP-1 expressors.
Arrows indicate tumor masses, which stained darker than the
normal lung parenchyma. B, in an in vitro
invasion assay, H460/WISP-1 cells demonstrated reduced invasiveness
across Matrigel compared with control cells. C, H460 cells
were less invasive in the presence of WISP-1-conditioned medium
(WISP-1CM) compared with vector control conditioned medium
(vectorCM).
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Fig. 2.
WISP-1 down-regulates motility of H460 and
H1299 lung cancer cells. Vector (Vec)- and WISP-1
(W1)-expressing cells were evaluated for migration in the
presence of 1.5% serum in Boyden Chambers. WISP-1 transfectants
demonstrated reduced levels of motility compared with vector control
for both H460 and H1299 cell lines.
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Fig. 3.
WISP-1 down-regulates the activation of Rac
in H460 and H1299 cells. PAK-agarose conjugates were used to
precipitate GTP-bound Rac (activated Rac), and the pulled-down proteins
were electrophoresed, transferred to a polyvinylidene difluoride
membrane, and immunoblotted with anti-Rac antibody. The assay showed a
strong reduction in the precipitation of activated Rac in
WISP-1-transfected H460 and H1299 cells compared with vector controls.
Cell lysate controls demonstrated equal amounts of total Rac.
1 caused an
increase in Rac activation in WISP-1-expressing cells but not control
cells (Fig. 4A). When
v
5 and
1 antibodies were
added together, the effect on the activation assay was greater than
either antibodies alone (Fig. 4A). On the other hand, there
was no effect of these antibodies on vector-expressing cells (Fig.
4B).
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Fig. 4.
Inhibitory antibodies to integrins and WISP-1
restore Rac activation in WISP-1 cells. A, antibodies
to integrin v
5 and
1
alleviated inhibition of Rac activation by WISP-1. B, in
contrast, control cells showed no effect on Rac activation in the
presence of inhibitory antibodies to integrins. Blocking antibodies to
WISP-1 also reversed the effect of WISP-1 on the inhibition of Rac
activation.
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Fig. 5.
Reconstitution of Rac activity by
transfection of a constitutively active Rac mutant, RacG12V.
WISP-1-expressing cells (W1) were infected with virus
particles carrying the gene for an activated form of RacG12V
(W1/Rac), and Rac activity was assayed using the PAK-agarose
pull-down method. The amount of GTP-bound Rac was increased in
RacG12V-expressing WISP-1 cells indicating that Rac activity has been
restored.
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Fig. 6.
MMP-1 expression is down-regulated by WISP-1
but up-regulated by RacG12V. Real-time PCR was conducted to verify
data obtained from microarray analyses. CT
values were used to calculate fold change of MMP-1 levels from each
sample over that of the vector control. The data showed that the
expression of MMP-1 was reduced in WISP-1-expressing H460 cells
(W1) by 3-fold over control cells (Vec). In
WISP-1/RacG12V (W1/Rac) co-transfectants,
however, the MMP-1 transcript was increased by about 3-fold over the
levels of control cells.
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Fig. 7.
Expression of RacG12V induces motility and
invasion of H460/WISP-1 cells across both Matrigel and collagen I. In a modified Boyden Chamber assay, reconstitution of Rac activity in
H460/WISP-1/RacG12V cells increased both cell motility (A)
and invasion across Matrigel (B). C, similarly,
when collagen I was used as a barrier, WISP-1-expressing cells
(W1) were less invasive compared with control cells (I).
Transfection of activated Rac restored cell invasion in WISP-1/RacG12V
cells (W1/Rac) to the level in control cells.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6
1 promotes cell
adhesion and chemotaxis in vascular smooth muscle cells (32). In human skin fibroblasts, CYR61 stimulates migration and proliferation through
integrin
v
5 and integrin
v
3, respectively (33). In contrast, when
CYR61 was overexpressed in lung cancer cells, tumor growth was
suppressed (20).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. William N. Rom for helpful advice and discussions, Dr. Long Cui for assistance with animal work, and Chen-Lu Li and Arvin Ajamian for technical assistance.
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FOOTNOTES |
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* This work was supported by General Clinical Research Center, the National Institutes of Health Grants MO1-RR00096 and RO1-ES0916, and Charles E. Culpeper Biomedical Pilot Initiative from the Rockefeller Brothers Fund (to K.-M. T.-W.).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: Division of
Pulmonary and Critical Care Medicine, New York University, 550 First Ave. MSB141, New York, NY 10016. Tel.: 212-263-0243; Fax: 212-263-8902; E-mail: tchouk02@endeavor.med.nyu.edu.
Published, JBC Papers in Press, January 15, 2003, DOI 10.1074/jbc.M210945200
2 L. L. Soon, T.-A. Yie, A. Shvarts, A. J. Levine, F. Su, and K.-M. Tchou-Wong, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: WISP, Wnt-induced secreted protein; CTGF, connective tissue growth factor; CYR61, cysteine-rich 61; Nov, nephroblastoma overexpressed; TGF, transforming growth factor; DMDM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; PBS, phosphate-buffered saline; DPBS, Dulbecco's PBS; MMP, metalloproteinase; PAK, p21-activated kinase; SCLC, small-cell lung cancer; NSCLC, non-small-cell lung cancer.
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REFERENCES |
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---|
1. | Bradham, D. M., Igarashi, A., Potter, R. L., and Grotendorst, G. R. (1991) J. Cell Biol. 114, 1285-1294[Abstract] |
2. | O'Brien, T. P., Yang, G. P., Sanders, L., and Lau, L. F. (1990) Mol. Cell. Biol. 10, 3569-3577[Medline] [Order article via Infotrieve] |
3. | Martinerie, C., Viegas-Pequignot, E., Guenard, I., Dutrillaux, B., Nguyen, V. C., Bernheim, A., and Perbal, B. (1992) Oncogene. 7, 2529-2534[Medline] [Order article via Infotrieve] |
4. |
Babic, A. M.,
Kireeva, M. L.,
Kolesnikova, T. V.,
and Lau, L. F.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
6355-6360 |
5. |
Pennica, D.,
Swanson, T. A.,
Welsh, J. W.,
Roy, M. A.,
Lawrence, D. A.,
Lee, J.,
Brush, J.,
Taneyhill, L. A.,
Deuel, B.,
Lew, M.,
Watanabe, C.,
Cohen, R. L.,
Melhem, M. F.,
Finley, G. G.,
Quirke, P.,
Goddard, A. D.,
Hillan, K. J.,
Gurney, A. L.,
Botstein, D.,
and Levine, A. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
14717-141722 |
6. |
Perbal, B.
(2001)
Mol. Pathol.
54,
57-79 |
7. |
Xu, L.,
Corcoran, R. B.,
Welsh, J. W.,
Pennica, D.,
and Levine, A. J.
(2000)
Genes Dev.
14,
585-595 |
8. |
Sampath, D.,
Zhu, Y.,
Winneker, R. C.,
and Zhang, Z.
(2001)
J. Clin. Endocrinol. Metab.
86,
1707-1715 |
9. | Latinkic, B. V., O'Brien, T. P., and Lau, L. F. (1991) Nucleic Acids Res. 19, 3261-3267[Abstract] |
10. | Igarashi, A., Okochi, H., Bradham, D. M., and Grotendorst, G. R. (1993) Mol. Biol. Cell 4, 637-645[Abstract] |
11. | Scholz, G., Martinerie, C., Perbal, B., and Hanafusa, H. (1996) Mol. Cell. Biol. 16, 481-486[Abstract] |
12. |
Duncan, M. R.,
Frazier, K. S.,
Abramson, S.,
Williams, S.,
Klapper, H.,
Huang, X.,
and Grotendorst, G. R.
(1999)
FASEB J.
13,
1774-1786 |
13. | Wong, M., Kireeva, M., Kolesnikova, T., et al.. (1997) Dev. Biol. 192, 492-508[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Hartmann, C.,
and Tabin, C. J.
(2000)
Development
127,
3141-3159 |
15. | Hurvitz, J. R., Suwairi, W. M., Van Hul, W., El-Shanti, H., Superti-Furga, A., Roudier, J., Holderbaum, D., Pauli, R. M., Herd, J. K., Van Hul, E. V., Rezai-Delui, H., Legius, E., Le Merrer, M., Al-Alami, J., Bahabri, S. A., and Warman, M. L. (1999) Nat. Genet. 23, 94-98[CrossRef][Medline] [Order article via Infotrieve] |
16. |
Fei, S.,
Overholtzer, M.,
Besser, D.,
and Levine, A. J.
(2002)
Genes Dev.
16,
46-57 |
17. |
Hashimoto, Y.,
Shindo-Okada, N.,
Tani, M.,
Nagamachi, Y.,
Takeuchi, K.,
Shiroishi, T.,
Toma, H.,
and Yokota, J.
(1998)
J. Exp. Med.
187,
289-296 |
18. |
Zhang, R.,
Averboukh, L.,
Zhu, W.,
Zhang, H,
Jo, H.,
Dempsey, P. J.,
Coffey, R. J.,
Pardee, A. B.,
and Liang, P.
(1998)
Mol. Cell. Biol.
18,
6131-6141 |
19. |
Gupta, N.,
Wang, H.,
McLeod, T. L.,
Naus, C. C.,
Kyurkchiev, S.,
Advani, S., Yu, J.,
Perbal, B.,
and Weichselbaum, R. R.
(2001)
Mol. Pathol.
54,
293-299 |
20. |
Tong, X.,
Xie, D.,
O'Kelly, J.,
Miller, C. W.,
Muller-Tidow, C.,
and Koeffler, H. P.
(2001)
J. Biol. Chem.
276,
47709-47714 |
21. |
Garber, M. E.,
Troyanskaya, O. G.,
Schluens, K.,
Petersen, S.,
Thaesler, Z.,
Pacyna-Gengelbach, M.,
van de Rijn, M.,
Rosen, G. D.,
Perou, C. M.,
Whyte, R. I.,
Altman, R. B.,
Brown, P. O.,
Botstein, D.,
and Petersen, I.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
13784-13789 |
22. | Bredin, C. G., Liu, Z., Hauzenberger, D., and Klominek, J. (1999) Int. J. Cancer 82, 338-345[CrossRef][Medline] [Order article via Infotrieve] |
23. | Wistuba, I. I., Gazdar, A. F., and Minna, J. D. (2001) Semin. Oncol. 28, 3-13 |
24. | Noël, A., Gilles, C., Bajou, K., Devy, L., Kebers, F., Lewalle, J. M., Maquoi, E., Munaut, C., Remacle, A., and Foi-dart, J. M. (1997) Invasion Metastasis 17, 221-239[Medline] [Order article via Infotrieve] |
25. | Ridley, A. J. (1995) Curr. Opin. Genet. Dev. 5, 24-30[Medline] [Order article via Infotrieve] |
26. |
Suzuki-Inoue, K.,
Yatomi, Y.,
Asazuma, N.,
Kainoh, M.,
Tanaka, T.,
Satoh, K.,
and Ozaki, Y.
(2001)
Blood
98,
3708-3716 |
27. | Chiariello, M., Marinissen, M. J., and Gutkind, J. S. (2001) Nat. Cell Biol. 3, 580-586[CrossRef][Medline] [Order article via Infotrieve] |
28. |
Ueda, H.,
Morishita, R.,
Yamauchi, J.,
Itoh, H.,
Kato, K.,
and Asano, T.
(2001)
J. Biol. Chem.
276,
6846-6852 |
29. |
O'Connor, K. L.,
and Mercurio, A. M.
(2001)
J. Biol. Chem.
276,
47895-47900 |
30. |
Chen, C. S.,
Mrksich, M.,
Huang, S.,
Whitesides, G. M.,
and Ingber, D. E.
(1997)
Science.
276,
1425-1428 |
31. |
Katsumi, A.,
Milanini, J.,
Kiosses, W. B.,
Del Pozo, M. A.,
Kaunas, R.,
Chien, S.,
Hahn, K. M.,
and Schwartz, M. A.
(2002)
J. Cell Biol.
158,
153-164 |
32. |
Grzeszkiewicz, T. M.,
Lindner, V.,
Chen, N.,
Lam, S. C.,
and Lau, L. F.
(2002)
Endocrinology
143,
1441-1450 |
33. |
Grzeszkiewicz, T. M.,
Kirschling, D. J.,
Chen, N.,
and Lau, L. F.
(2001)
J. Biol. Chem.
276,
21943-21950 |