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From the
Transforming growth factor
Cell adhesion to the extracellular matrix (ECM),
Integrins have an important
biological role as mediators of cell matrix-cell interaction. A number
of studies have demonstrated that integrins are associated with
transformation and differentiation of certain cell types. For example,
overexpression of integrin
The role of integrins in a wide
array of biological processes underlines the significance of
determining the mechanisms by which particular cell types control the
steady-state expression of these receptors as well as the mechanisms by
which the extracellular environment can alter their expression.
Extensive studies have shown that the expression of integrins is under
the control of several growth factors and cytokines. For example,
platelet-derived growth factor BB was found to induce integrin
As indicated above, most studies of TGF-
The specificity of cell adhesion
to FN was determined using the monoclonal anti-human integrin
It has been shown that exogenous TGF-
A large bank of
colon carcinoma cell lines with a broad spectrum of biological
properties has been characterized in our laboratory
(22) . We
have previously shown that sensitivity to exogenous TGF-
The integrin
In conclusion, our data show that the autocrine expression of
TGF-
We thank Jenifer Zak for the skillful preparation of
this manuscript.
Volume 270,
Number 23,
Issue of June 9, pp. 14154-14159, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Modulates the Expression
of Integrin
in Human Colon Carcinoma
FET Cells (*)
ABSTRACT
INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(TGF-) has been
extensively studied as an exogenous agent that stimulates the
expression of extracellular matrix proteins and their cell-surface
integrin receptors in a variety of cell types. However, the recent
demonstration of autocrine TGF- growth effects in a number of cell
types suggests that the steady-state expression of extracellular matrix
and integrin proteins and their biological activity may also be under
autocrine TGF- control. Previously, we reported that repression of
autocrine TGF-
activity by constitutive expression of
a full-length TGF- antisense cDNA led to abrogation of
autocrine negative TGF- and, as a result, increased tumorigenicity
and anchorage-independent growth of a poorly tumorigenic,
well-differentiated colon carcinoma cell line designated FET (Wu, S.,
Theodorescu, D., Kerbel, R. S., Willson, J. K. V., Mulder, K. M.,
Humphrey, L. E., and Brattain, M. G.(1992) J. Cell Biol. 116,
187-196). Consequently, we have used this model system to study
the effects of repression of autocrine TGF- activity
on the expression of integrin 
and
integrin -mediated cell adhesion to
fibronectin. The expression of the integrin subunit
was reduced in TGF- antisense transfected FET cells at
both mRNA and protein levels as determined by RNase protection assays
and immunoprecipitation, respectively. Autocrine TGF- had no effect on the transcription of integrin and subunits, indicating that autocrine
TGF- may regulate integrin
expression at the
post-transcriptional level. The diminished expression of integrin
on the cell surface led to the
reduced adhesion of TGF- antisense transfected cells
to fibronectin. This phenomenon could be reversed by treatment with
exogenous TGF-.
()
mediated in part by the integrin family of cell-surface
glycoproteins, plays an important role in the control of cell
migration, proliferation, differentiation, and invasion (1, 2).
Integrins are formed by the noncovalent association of an subunit
with a subunit. The subunit dimerized by at
least six subunits, termed
-
, constitutes the largest
component of the integrin family. Among them,
, 
,
and 
are the receptors for collagen,
laminin, and other ECM proteins, whereas is the receptor specific for FN.
reverses
tumorigenicity and anchorage-independent growth in transformed Chinese
hamster ovary cells (3). Similar results were obtained in a stable
variant of the K562 erythroleukemia cell line that overexpresses
integrin (4) . In contrast,
loss of integrin expression is
associated with a transformed phenotype of fibroblasts
(5) , and
increased tumorigenicity was shown in integrin
-deficient Chinese hamster ovary cell
variants
(6) . The integrins also have a signal transduction
function that affects the expression of many essential genes relevant
to matrix remodeling during differentiation. For example, monoclonal
antibody to integrin induces the
expression of two extracellular matrix-degrading metalloproteinases,
collagenase and stromelysin
(7) . Recently, we showed that
antibody to leads to increased DNA synthesis in
quiescent HT1080 cells
(8) .
and subunit expression in rabbit
vascular smooth muscle cells
(9) , and interleukin-1 was
shown to increase integrin and subunit mRNA levels, but to decrease integrin 
subunit mRNA levels in MG-63 human osteosarcoma cells (10). Among
the growth factors, TGF- has been the most extensively studied
exogenous modulator of the expression of ECM proteins and their
integrin receptors
(11) . TGF- has been shown to stimulate
the expression of FN and collagen and their incorporation into the ECM
(12). A number of integrin subunits in the subfamily have been shown to be stimulated by TGF- in human
fibroblasts
(13, 14) . Treatment of MG-63 human
osteosarcoma cells with TGF- markedly decreases integrin
subunit mRNA and protein levels with concomitant
increases in , , and subunit expression
(15) . Interestingly, the
Engelbreth-Holm-Swarm matrix was found to down-regulate TGF- synthesis at the transcriptional level in mouse epithelial
cells
(16) .
function and mechanism have centered around treatment of cells with one
of the TGF- isoforms. However, it is generally believed that
TGF- exerts its role in an autocrine as well as a paracrine
fashion
(17) . The autocrine negative growth activity of
TGF- was demonstrated by the ability of neutralizing
TGF- and TGF- antibodies to increase
proliferation, DNA synthesis, and anchorage-independent
growth
(18, 19, 20, 21) . We have used
stable transfection of a TGF- antisense cDNA to
demonstrate autocrine TGF- activity in a colon
carcinoma cell line designated FET
(21) . Characterization of
transfected FET cells with constitutively repressed TGF- expression showed high cloning efficiency in
anchorage-independent assays and enhanced tumorigenicity in athymic
nude mice. These studies suggested that loss of autocrine TGF-
activity may be an important step in progression of malignancy. Given
the effects of exogenous TGF- treatment on the expression of
integrins in a wide variety of model systems, we hypothesized that
autocrine TGF- may control biological function by
regulating steady-state integrin receptor expression and consequently
cellular interactions with the ECM. We have tested this hypothesis
using the FET model system, described above, in which autocrine
TGF- activity was constitutively repressed
(21) .
Cells and Culture
The FET human colon carcinoma
cell line was originally established in vitro from a primary
human colon tumor
(22) . FET TGF- antisense
transfected cells (FET B) and FET control cells transfected with the
neomycin-resistant gene were established and characterized as described
previously
(21) . Cells were maintained in chemically defined
McCoy's 5A serum-free medium (Life Technologies, Inc.)
supplemented with 20 µg/ml insulin (Sigma), 4 µg/ml transferrin
(Sigma), and 10 ng/ml epidermal growth factor (Sigma) at 37 °C in a
humidified atmosphere of 5% CO as described
previously
(23) . Subcultures were obtained by treatment with
0.125% trypsin in Joklik's tissue culture medium (Life
Technologies, Inc.) containing 0.1% EDTA.
RNase Protection Assays
Total RNA was isolated
from cultured cells by the guanidine isothiocyanate method
(24) .
RNase protection assays were performed as described by Wu et al. (25). The subunit template was constructed by
subcloning a 405-base pair BamHI-AccI fragment of the
human subunit cDNA into plasmid pBSK (Stratagene
cloning system). A 214-base pair SpeI-PstI fragment
of integrin cDNA was also inserted into the pBSK
vector. Both and subunit antisense
probes were prepared using T RNA polymerase. The
construction of the TGF- antisense probe has been
described previously by Wu et al.(21) . Determinations
of the integrin subunit and TGF- mRNA levels in total
RNA samples (40 µg) were performed with 1 10
cpm of each of the labeled probes. The hybridization mixture was
digested with RNase A and RNase T, followed by treatment
with proteinase K. The protected fragment of the probe was analyzed by
urea-polyacrylamide gel electrophoresis and visualized by
autoradiography.
Immunoprecipitation
To quantitate the cell-surface
subunit protein level, 10
cells were
resuspended in 1 ml of phosphate-buffered saline (pH 7.4), and
cell-surface proteins were labeled with 0.1 mg/ml sulfosuccinimidyl
6-(biotinamido)hexanoate (Pierce). The labeled cells were washed with
phosphate-buffered saline and solubilized with 1 ml of extraction
buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1
mM CaCl, 1 mM MgCl, 1%
Nonidet P-40). Cell extract (300 µl) was precleared with 30 µl
of protein G-agarose suspension (Pierce) for 8 h at 4 °C.
Biotinylated integrin was
immunoprecipitated with 2 µl of monoclonal anti-human integrin
antibody (Life Technologies, Inc.) and 50 µl of
protein G-agarose after overnight incubation at 4 °C. The
precipitates were washed six times with buffer containing 50
mM Tris (pH 7.5), 0.5 M NaCl, 1 mM
CaCl, 1 mM MgCl, and 0.1% Tween 20.
The biotinylated and subunits were
dissociated from the immune complex by heating the samples in SDS
nonreducing sample buffer at 95 °C for 10 min and were analyzed by
SDS-polyacrylamide gel electrophoresis using a Bio-Rad Mini-PROTEAN II
gel apparatus. The proteins were electrotransferred onto an Immun-Lite
blotting membrane (Bio-Rad), which was then blocked with 5% nonfat dry
milk and incubated for 1 h in the presence of streptavidin-alkaline
phosphatase (Life Technologies, Inc.). A chemiluminescent substrate,
3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryloxy)phenyl-1,2-dioxetane
disodium salt (Bio-Rad), for alkaline phosphatase was then added to the
blot. The and subunits were
visualized after autoradiography.
Transient Transfection and CAT Assay
The
subunit promoter-CAT constructs used in this study
have been reported previously
(26) . Promoter activity was
measured using the P-924 CAT and
P-1300 CAT constructs containing 924 and 1300 base
pairs, respectively, 5` of the transcription start
site. FET Neo control and TGF- antisense transfected
cells were transfected with 30 µg of promoter-CAT plasmids together
with 10 µg of -galactosidase plasmid by electroporation with a
Bio-Rad Gene Pulser at 250 V and 960 microfarads. The electroporated
cells were plated onto 10-cm culture dishes in the serum-free medium
and harvested after 48 h. The expression of the CAT reporter gene was
assayed to determine the promoter activity in these
cells. The cell pellets were resuspended in 0.25 M Tris (pH
7.8) and lysed by three cycles of freeze-thaw. -Galactosidase
activity in the cell extracts was quantified to normalize the amounts
of extracts for CAT assay. Aliquots representing equal amounts of
-galactosidase activity were incubated with
[
C]chloramphenicol (0.25 µCi) and acetyl
coenzyme A (114 µg) for 6 h at 37 °C, extracted with ethyl
acetate, and separated by thin-layer chromatography as described by
Sambrook et al.(27) . The levels of acetylated
chloramphenicol were quantitated by the AMBIS image acquisition and
analysis system. Isolation of nuclei and nuclear run-on assays were
performed essentially as described by Greenberg and Ziff
(28) with some modifications
(29) .
Cell Adhesion and MTT Assays
Ninety-six-well
Corning tissue culture plates were coated with FN at concentrations of
0, 0.1, 0.3, 0.6, 1.2, 2.4, 5.0, and 10.0 µg/ml for 2 h at 37
°C and then rinsed once with phosphate-buffered saline. Confluent
cells were detached by treatment with trypsin, plated at 6
10
cells/well on FN-coated plates, and incubated for 90 min
in a humidified incubator. Unattached cells were gently washed away by
four rinses with phosphate-buffered saline, and attached cells were
determined by the MTT assay as described
previously
(30, 31) .
subunit antibody. The antibody was added to FN-coated
plates at dilutions of 1:50-1:500 and incubated for 30 min at 37
°C. Determination of cell adhesion and the MTT assay were performed
as described above.
Exogenous TGF-
The FET cell line is a poorly tumorigenic,
well-differentiated human colon carcinoma cell line that was previously
established in vitro from a primary tumor and has been
extensively characterized
(22, 32) . FET cells expressed
high levels of TGF-Increases Integrin
Subunit mRNA Steady-state Level in FET Parental
Cells
mRNA, but no detectable
TGF- and TGF- mRNAs by Northern blot
analysis
(21) . To determine whether exogenous TGF- can stimulate integrin subunit expression in
FET parental cells, the steady-state levels of mRNA from the cells
treated with TGF- (5 ng/ml) at different time points
were compared by RNase protection assay (Fig. 1). The
steady-state mRNA level of integrin subunit was
increased after 12 h of exposure to TGF-, while a
maximal increase was observed after 24 h of TGF- treatment.
Figure 1:
Effect of exogenous
TGF- on integrin subunit mRNA levels
in FET cells. Total RNA was extracted from FET cells treated with
TGF- (5 ng/ml) for 0, 2, 6, 12, 24, and 48 h. Integrin
subunit mRNA and actin mRNA were detected in 40
µg of total RNA using a RNase protection assay. Actin mRNA levels
are shown to indicate equal loading of the samples. Molecular weight
markers are shown in lane M.
Repression of Autocrine TGF-
Repressed autocrine TGF- Activity Reduces
Integrin Subunit mRNA Steady-state Level and
Cell-surface Integrin Expression
activity was
obtained by constitutive expression of a TGF- antisense cDNA in FET cells. A typical stable clone, designated
FET B, that expresses high levels of TGF- antisense
cDNA was selected for these studies
(21) . The steady-state mRNA
levels of integrin and subunits in
FET B and FET Neo control cells were determined by RNase protection
analysis (Fig. 2). FET B cells showed an 5-fold reduction of
the TGF-
mRNA level, as published
previously
(21) . The expression of integrin subunit mRNA was reduced 2.5-fold in FET B cells compared
with FET Neo cells. The modulation of
expression was
relatively specific in that integrin subunit mRNA
expression was not altered by repression of autocrine TGF-. In
addition, there was no change in the mRNA levels of integrin
and subunits (data not shown).
Reduced mRNA expression suggested a corresponding
reduction of receptor expression on
the cell surface of FET B cells compared with FET Neo cells. This was
confirmed by immunoprecipitation experiments examining the cell-surface
expression of integrin (Fig. 3).
Figure 2:
Integrin and
mRNA levels in FET cells transfected with
TGF- antisense expression plasmid (FET B) and control
plasmid (FET Neo). Total RNA was isolated from both cell lines.
Integrin and subunit mRNAs,
TGF- mRNA, and actin mRNA were detected in 40 µg
of total RNA by a RNase protection assay. The two photographs are from
one gel. Because of the low expression of the integrin subunit, the upper photograph was exposed for a longer
period of time than the lower one. Actin mRNA levels are shown
to indicate equal loading of the samples.
Figure 3:
Cell-surface integrin
protein levels in FET cells
transfected with TGF- antisense cDNA (FET B) and
control plasmid (FET Neo). Cell-surface proteins from equal numbers of
cells were labeled with biotin. Biotinylated integrin
was immunoprecipitated with an
anti-human subunit antibody and analyzed by
SDS-polyacrylamide gel electrophoresis and Western blotting. The
negative control did not receive anti- antibody during
immunoprecipitation.
Autocrine TGF-
To examine whether the
down-regulation of integrin Modulates Integrin
Expression at the
Post-transcriptional Level
expression by repression of autocrine TGF- was due to
transcriptional regulation, we transiently transfected integrin
subunit promoter-CAT constructs
(26) into FET
B and FET Neo cells. The levels of CAT enzyme, expressed under the
control of the integrin promoter, were quantitated
using the standard assay for [C]chloramphenicol
conversion (Fig. 4). There was no difference in the CAT activity
in FET B and FET Neo cells after transfection with the CAT constructs.
A nuclear run-on assay was performed to confirm these results (data not
shown). Again, no difference in the subunit
transcription rate was observed in FET B and FET Neo cells. These
results indicate that autocrine TGF- does not regulate integrin
expression at transcriptional
levels.
Figure 4:
Integrin promoter (924-
and 1300-base pair fragments) activity in FET cells transfected with
TGF- antisense expression plasmid (FET B) and control
plasmid (FET Neo). A, FET B and FET Neo cells were transfected
with P-924 CAT and P-1300 CAT
plasmids. Cells were harvested 48 h after transfection.
-Galactosidase activity in cell extracts was normalized for CAT
assay. B, the amounts of product and remaining substrate were
quantitated by scanning the autoradiographic film. The graph was
plotted by the ratio of product to
substrate.
Reduction of Cell-surface Integrin
Next, it was determined
whether the down-regulation of Expression Leads to Reduced
Cell Adhesion to FN That Can be Reversed by Exogenous
TGF-
Treatment
expression affected biological function as reflected by
alterations of cell adhesion to FN (Fig. 5). Levels of
enhancement ranged from 1.4 to 2.8-fold for FET Neo cells when
coating was performed at FN concentrations ranging from 0.1 to 10
µg/ml, while FN coating had little effect on FET B cell adhesion as
an enhancement of only 1.4-fold over bovine serum albumin coating was
obtained at a FN concentration of 10 µg/ml
(Fig. 5A). Thus, adhesion to FN-coated culture plates
relative to bovine serum albumin-coated plates was enhanced
4-5-fold for FET Neo cells relative to FET B cells in the FN
range between 1.2 and 10 µg/ml. Treatment of both FET Neo and FET B
cells with 5 ng/ml TGF-
increased the level of binding
to FN-coated plates over bovine serum albumin controls
(Fig. 5B). After TGF- treatment, the
level of enhancement of adhesion to FN for both cell types was
approximately equal. These results indicate that repression of
autocrine TGF- leads to reduced adhesion to FN. Moreover,
treatment with exogenous TGF- led to restoration of
binding to FN. The reduced levels of adhesion in FET B cells were
consistent with the lower steady-state levels of cell-surface integrin
in these cells relative to control
FET Neo cells. We hypothesized that restoration of adhesion after
TGF- treatment of FET B cells was due to induction of
integrin expression. Consequently,
the concentration-dependent effects of TGF- treatment
on cell-surface integrin expression
were determined by immunoprecipitation in both types of cells.
TGF- treatment increased both FET Neo and FET B
expression of cell-surface integrin (data not shown).
Figure 5:
Adhesion of FET control () and FET
TGF-
antisense transfected () cells as a
function of FN concentration. Substrates were prepared by coating
96-well Corning tissue culture plates with FN at concentrations of 0,
0.1, 0.3, 0.6, 1.2, 2.4, 5.0, and 10.0 µg/ml for 2 h. Cells from
confluent cultures were seeded at 6
10
/well onto
FN-coated plates and allowed to adhere for 90 min. After removal of
unattached cells, adhesion was determined by the MTT assay. The effects
of TGF- were determined by treating the cells for 48 h
prior to the adhesion assay. A, no TGF-;
B, 5 ng/ml TGF-. Each point is the average of
two individual experiments determined from triplicate
wells.
Since repression of autocrine
TGF- activity down-regulated cell-surface integrin
expression and cell adhesion to FN,
it was of importance to determine whether the reduced cell adhesion to
FN was mediated by loss of cell-surface expression of integrin
. Therefore, blocking of adhesion to
FN by monoclonal antibody against the subunit was
performed (Fig. 6). Blocking with antibody
showed 60 to 20% inhibition of adhesion to FN in FET Neo control cells
at antibody dilutions of 1:50-1:500. In contrast, FET B cells
showed very little dependence upon integrin
for binding to FN as a 1:50 dilution
of antibody resulted in only a 20% reduction of
binding. Lower antibody concentrations did not give significant levels
of inhibition of binding to FN. Antibodies to and
subunits had little or no effect on binding to FN at
1:50 dilutions (Fig. 6A). Furthermore, blocking of
adhesion to FN by antibody was also performed to
determine whether restoration of cell adhesion to FN by treatment with
exogenous TGF- was due to increased expression of
cell-surface integrin (Fig. 6B). After treatment of FET B cells with 5
ng/ml TGF-, the levels of integrin
-mediated adhesion to FN in FET B
cells were similar to those in FET Neo cells at the same antibody
dilution as shown in Fig. 6B. Therefore, it appears that
repression of autocrine TGF- selectively reduces integrin
-mediated adhesion to FN. Treatment
with exogenous TGF- selectively restores the relative
degree of integrin -mediated adhesion
to FN in FET B cells.
Figure 6:
Inhibition of adhesion of FET control
(
) and FET TGF- antisense transfected
(&cjs2110;) cells to FN by antibodies to integrin receptors.
Ninety-six-well Corning tissue culture plates were coated with FN (10
µg/ml). Monoclonal antibodies to integrin subunits were added
at dilutions of 1:50-1:500 and incubated for 30 min at 37 °C.
A, no TGF-; B, 5 ng/ml
TGF-. 1, no antibody; 2, antibody; 3, antibody;
4-6, antibody. Each point is the
average of two individual experiments determined from triplicate
wells.
is able to
regulate the expression of integrins
(13, 14, 15, 33) . These
results indicated that the biological functions of TGF- and
integrin-mediated cell-matrix interaction overlap in terms of
regulation of cell growth, migration, and differentiation. However,
demonstration that autocrine TGF- in a native cell
line controls the steady-state expression of integrin receptors and
their biological functions has not been reported.
and TGF- is dependent upon the progression
status of colon carcinoma cell lines
(34) . Poorly tumorigenic
cell lines that retain differentiated characteristics in tissue
culture, such as basolateral polarity and transport function, respond
to exogenous TGF- treatment with decreased proliferation, loss of
anchorage independence, and increased expression of carcinoembryonic
antigen and ECM proteins, including basement membrane components such
as laminin and collagen IV in addition to
FN
(34, 35, 36) . Cell lines sensitive to
exogenous TGF- were shown to have an autocrine negative TGF-
function
(21, 25, 29) . Interestingly, the ECM
material produced by the cells with autocrine negative TGF- is
able to induce a differentiation-like response in another colon
carcinoma cell line, designated MOSER, whereas the ECM material from
highly progressed colon carcinoma cell lines without autocrine negative
TGF- is not able to induce this response
(37) . Moreover, we
recently showed that the expression of the subunit
selectively blocks DNA synthesis in HT1080 fibrosarcoma
cells
(8) . Taken together, these lines of evidence suggest that
autocrine TGF- might play an important role in controlling the
biological properties through autocrine control of integrin and/or ECM
protein expression and that the malignant progression seen in FET B
cells might in part be due to loss of steady-state control of integrin
function. Previously, it had been shown that tumor progression is
associated with loss of TGF-
responsiveness
(17, 21, 34, 38) . Given
the role of in the tumorigenicity of
other cell types, it is possible that the steady-state levels of
under autocrine TGF- control
are important in suppressing the tumorigenic potential of FET cells.
subunit mRNA steady-state level and
cell-surface integrin expression
were down-regulated in FET TGF- antisense transfected
cells compared with the control cells, whereas there was no change in
the mRNA levels of integrin and subunits, indicating that the modulation of expression is relatively specific in this cell line. The decrease
in cell-surface integrin expression
and cell adhesion to FN by repression of autocrine TGF- in FET
cells was reversed by treatment with exogenous TGF-,
further confirming that the expression of integrin
is under the control of autocrine
TGF- regulation. A number of mechanisms for increased integrin
expression resulting from TGF- treatment
have been observed, including increased and subunit
synthesis
(14) and decreased decay
(13) . We studied
whether autocrine TGF- regulates integrin
expression at a transcriptional
level. The negative results indicate that autocrine TGF- does not appear to control integrin expression at the transcriptional level in FET cells. Thus,
autocrine TGF- may function by increasing the
stability of integrin mRNA, facilitating integrin protein incorporation
into the cell membrane, and/or inhibiting the protein degradation.
has a role in modulating the steady-state expression of
integrin and, as a result, controls
integrin -mediated cell adhesion.
Repression of autocrine TGF- activity in the FET colon
carcinoma cell line resulted in diminished integrin
expression and cell adhesion to FN.
Exogenous TGF- treatment restored cell-surface
integrin expression and integrin
-mediated adhesion in the
TGF- antisense transfected cells to the same level as
in control cells. These results indicate that autocrine TGF- may
affect cell growth and differentiation in part via modulation of the
expression of ECM proteins and integrins.
, transforming growth factor ; CAT, chloramphenicol
acetyltransferase; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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