(Received for publication, June 19, 1995; and in revised form, October 27, 1995)
From the
Human lung fibroblasts and Mv1Lu mink lung epithelial cells were
used as a model to study the role of extracellular matrix in
epithelial-mesenchymal interactions. Extracellular matrices of
fibroblasts were found to contain growth promoting activity that
reduced the sensitivity of Mv1Lu cells to the growth inhibitory effects
of transforming growth factor- (TGF-
). The majority of the
activity was identified as hepatocyte growth factor/scatter factor
(HGF) by inhibition with specific antibodies and by reconstitution of
the effect by recombinant HGF. HGF induced cell proliferation when
contact-inhibited Mv1Lu cells were trypsinized and plated in the
presence of TGF-
1. The effect was valid also in assays where
Madin-Darby canine kidney epithelial cells or bovine capillary
endothelial cells were used. The multiplication of chronically
TGF-
1 inhibited Mv1Lu cells was also induced by HGF. In addition,
HGF induced anchorage independent growth of Mv1Lu cells that was
refractory to TGF-
1 growth inhibition. Immunoprecipitation
analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2,
but not the induction of p21, by TGF-
1. Since both TGF-
1 and
HGF require proteolysis for activation, the results imply that
proteolytic activity of epithelial and endothelial cells directs their
responses to signals from mesenchymal-type extracellular matrices, and
that during development, matrix-bound growth and invasion promoting and
suppressing factors are activated in a coordinated manner.
Transforming growth factors- are well characterized
proteins that are proteolytically activated. They induce growth arrest
in epithelial and endothelial cells and increase the synthesis of
extracellular matrix components(1, 2) . Hepatocyte
growth factor is a prototype of an emerging family of epithelial and
hematopoietic growth factors which are also activated by
proteolysis(3, 4, 5) . Receptors for
TGF-
(
)are transmembrane proteins with serine-threonine
kinase function(6, 7) . HGF receptor is a tyrosine
kinase product of the c-met proto-oncogene(8, 9) . TGF-
is produced and
its receptors are expressed by both epithelial and mesenchymal
cells(1, 10) . HGF expression is, in turn, typically
restricted to mesenchymal cells while its receptor expression is
epithelial(3, 11, 12) .
Both TGF-s
and HGF are expressed in a wide variety of
tissues(3, 11, 12, 13) , and their
effects on epithelial cells in vitro and in vivo are
largely reciprocal. HGF induces the conversion of mesenchymal cells to
epithelia(14) , while members of the TGF-
family induce
transdifferentiation of epithelia to
mesenchyme(1, 15, 16) . HGF stimulates the
proliferation, motility, and invasiveness of epithelial and endothelial
cells(11, 17, 21) , while TGF-
potently
suppresses these events(18, 19, 20) . At
tissue level HGF stimulates branching morphogenesis and tissue
regeneration(12, 22, 23, 24, 25) ,
while TGF-
inhibits ductal growth and induces
fibrosis(26, 27, 28, 29) .
Both
HGF and TGF- are secreted from cells in a latent
form(1, 8, 30, 31) , and associate
with extracellular
matrices(8, 32, 33, 34) . TGF-
associates with extracellular matrix via latent TGF-
binding
proteins(35) , while HGF associates with heparan sulfate
proteoglycans (8, 36) . TGF-
is activated by
plasmin(37) , and HGF can be activated directly by u-PA (8, 30) or a factor XII related
proteinase(31, 38) .
Pure cultures of epithelial
cells are strongly inhibited by TGF-. However, the responsiveness
of epithelial cells, such as keratinocytes, to TGF-
growth
inhibition can be altered by the presence of mesenchymal feeder cells.
This study was initiated to identify factors in fibroblast
extracellular matrix that could modulate the sensitivity of epithelial
cells to TGF-
1, and to understand the role of extracellular matrix
derived growth factors and growth inhibitors in mesenchymal-epithelial
interactions.
For analysis of Cdk2,
Cdk4, p21, and Met, the cells were labeled for 6 h with 150 µCi/ml
[S]methionine in methionine-free medium
containing 10% dialyzed FCS. Subsequently, the cells were lysed in a
buffer containing 0.5% Nonidet P-40, 50 mM NaCl, 4 mM Na
VO
, 20 mM NaF, and 20 mM Tris-HCl buffer, pH 7.5, for 30 min at 0 °C, and the lysate
was clarified at 2000
g for 1 h. Aliquots of cell
lysates were incubated with affinity purified antibodies (1 µg/ml)
for 1 h at 0 °C, and the immune complexes were precipitated as
above. The beads were washed four times with the lysis buffer and twice
with PBS, followed by SDS-PAGE analysis under reducing conditions.
Further experiments
indicated that the plating of the Mv1Lu cells on fibroblast matrices
reduced their sensitivity to TGF-1. A small decrease in number of
Mv1Lu cells was observed when the cells were cultured on plastic in the
presence of TGF-
1 (10 ng/ml) and 10% FCS for 7 days (Fig. 1A; see also (40) ). In contrast, a
2-3-fold increase in cell number was observed in Mv1Lu cells
cultured on lung fibroblast matrices in the presence of TGF-
1 and
10% FCS (Fig. 1A), indicating that the responsiveness
was unexpectedly altered.
Figure 1:
Fibroblast
extracellular matrix-derived hepatocyte growth factor reduces the
sensitivity of epithelial cells to TGF-1 growth inhibition. A, analysis of Mv1Lu cell growth on fibroblast matrices.
Extracellular matrices were extracted from confluent 12-well plate
cultures of human lung fibroblasts by sodium deoxycholate extraction
(see ``Materials and Methods''). Mink lung epithelial Mv1Lu
cells (cells seeded, 3.6
10
) were plated on the
matrices (lung fibroblast matrix) or plain tissue culture plastic
(Plastic) in Dulbecco's MEM containing 10% FCS and 10 ng/ml
TGF-
1 (triplicate wells). The matrices were washed twice with 1.2 M NaCl in phosphate buffer prior to plating to remove
heparin-bound material where indicated. Neutralizing polyclonal
antibody to HGF was added to cultures as indicated (8 µg/ml;
anti-HGF+). After 7 days incubation at 37 °C, the cells were
trypsinized and counted in triplicate by a Coulter counter. Bars represent one standard deviation. B, immunoblotting
analysis of purified fibroblast extracellular matrices. Fibroblast
extracellular matrix-derived proteins were separated on a 4-15%
gradient SDS-PAGE under nonreducing conditions. Proteins were
transferred to nitrocellulose membrane, followed by immunoblotting
using a monoclonal antibody to HGF. The matrices were washed twice with
1.2 M NaCl where indicated. Recombinant HGF was used as a
standard. The migration of molecular mass markers is shown on the left.
Figure 2:
Hepatocyte growth factor prevents
TGF-1-induced growth arrest. A, confluent culture of
Mv1Lu cells was trypsinized and plated in MEM + 10% FCS on 12-well
plate at a density of 10
cells/cm
. Increasing
concentrations of HGF and TGF-
1 were added to the wells, and the
cultures were incubated at 37 °C for 90 h. The cells were
trypsinized and counted with a Coulter counter (in triplicate). Cell
numbers are expressed as apparent number of cell divisions =
log
(number of cells after culture/number of cells plated).
Points at x axis are at arbitrary positions for clarity. B, MDCK cells and bovine capillary endothelial (BCE)
cells were plated in the presence or absence of TGF-
1 (1 ng/ml)
and HGF (20 ng/ml). The cells were counted after 4 (MDCK) or 7 days (BCE). Cell numbers are expressed as
above.
Figure 3:
HGF
induces thymidine incorporation in Mv1Lu cells growth inhibited by
TGF-1. Mv1Lu cells were plated on 24-well plates (30,000
cells/well) as described in the legend to Fig. 2. Cells were
growth arrested with 10 ng/ml TGF-
1 for HGF induction and washing
experiments, or with low serum (0.2%) for serum induction experiments
immediately after plating (90 h). At times indicated on the figure, the
cells were washed and incubated in fresh medium (
), treated with
HGF (20 ng/ml) without removing TGF-
1 containing culture medium
(
), or induced with addition of FCS to 10% final concentration
(
). Cells were subsequently labeled with 1 µCi of
[6-
H]thymidine for 2 h. Incorporated
radioactivity is expressed as times control (cpm/cpm of the respective
control at 0 h). Bars represent 1 standard deviation of
triplicate wells.
Figure 4:
HGF induces anchorage independent growth
of Mv1Lu cells. Mv1Lu cells were plated at a density of 1500
cells/cm on Compactigel-agarose (see ``Materials and
Methods'') in MEM + 10% FCS containing HGF (20 ng/ml) and/or
TGF-
1 (10 ng/ml) where indicated (+). Cells were cultured for
9 days, and colonies >15 µm and >45 µm were then counted. Bars represent 1 standard deviation.
4`,6-Diamino-2-phenylindole (DAPI)-stained colonies are shown on the right. Bar = 250 µm (inset magnified 5
).
Figure 5:
HGF does not affect TGF-1 receptor
levels in Mv1Lu cells. Subconfluent (
50%) cultures of Mv1Lu cells
were treated with HGF (20 ng/ml) for 16 h. Expression of TGF-
receptors was analyzed by cross-linking with
I-labeled
TGF-
1, followed by immunoprecipitation with type II TGF-
receptor antibodies (see ``Materials and Methods''). The
immunoprecipitates were analyzed by 4-15% gradient SDS-PAGE
followed by PhosphorImager quantitation. A PhosphorImager image of the
gel is shown. Identification of the bands as type I and II TGF-
receptors, and results from PhosphorImager quantitation are shown on
the right. Migration of molecular mass markers is shown on the left.
Figure 6:
HGF does not interfere with
TGF-1-induced fibronectin or thrombospondin gene expression.
Subconfluent (
50%) cultures of Mv1Lu cells were treated with
increasing concentrations of TGF-
1 in the absence (
) or
presence (
) of HGF (20 ng/ml) in MEM containing 10% dialyzed FCS,
50 µCi/ml [
S]methionine, and 5 µM nonradioactive methionine (2.5% of usual) for 18 h. Fibronectin
and thrombospondin were precipitated from cell conditioned medium with
gelatin-Sepharose, or monoclonal antibodies followed by GammaBind
G-Sepharose, respectively. Samples were separated on 8-20%
SDS-PAGE under reducing conditions followed by PhosphorImager analysis
and autoradiography. The analysis of cell proliferation is shown for
comparison. Points represent averages of duplicate samples. Left, PhosphorImager analysis of the induction of fibronectin
by TGF-
1. x axis, TGF-
1 (ng/ml); y axis,
fold induction. Inset, autoradiography. Middle,
PhosphorImager analysis of the induction of thrombospondin by
TGF-
1. x axis, TGF-
1 (ng/ml); y axis, fold
induction. Inset, autoradiography. Right, analysis of
cell proliferation. Mv1Lu cells (10
/cm
) were
plated and treated with TGF-
1 and HGF as above. Cells were
trypsinized and counted in a Coulter counter after 3 days. The rate of
cell proliferation was analyzed and given as log
(cells at 3
days) normalized to control (100%) and TGF-
1 (16 ng/ml; 0%). x axis, TGF-
1 (ng/ml); y axis, rate of cell
proliferation, % of control.
Figure 7:
HGF prevents the suppression of Cdk2 and
Cdk4 by TGF-1. Subconfluent (
50%) cultures of Mv1Lu cells
were treated with HGF (20 ng/ml) and/or TGF-
1 (10 ng/ml) in the
presence of 10% FCS. After 12 h incubation, the cells were labeled for
6 h in fresh medium containing TGF-
1 and/or HGF, 10% dialyzed FCS,
and 150 µCi/ml [
S]methionine. Subsequently,
the cells were lysed to 0.5% Nonidet P-40 containing buffer followed by
immunoprecipitation with specific antibodies to Cdk2, Cdk4, p21, and
Met as indicated. Immunoprecipitates were analyzed by SDS-PAGE followed
by autoradiography shown. Constant 15% polyacrylamide gels were used
for Cdk's and p21, while the Met immunoprecipitate was run on a
4-15% gradient gel. Molecular mass markers are shown on the left.
In this report, we find that culturing of epithelial cells on
fibroblast extracellular matrix reduces their sensitivity to growth
inhibition by TGF-. Hepatocyte growth factor/scatter factor is
identified as a major factor capable of mediating this effect.
Recombinant HGF inhibited TGF-
1 mediated growth arrest of
epithelial cells, and biologically significant amounts of HGF were
found in fibroblast extracellular matrix. In addition, antibodies to
HGF inhibited the effect of fibroblast matrix by 50-70%. The fact
that the inhibition was not complete suggests that additional factors
may be involved in the full activity of fibroblast matrix.
The
growth inhibition of mink lung epithelial cell line Mv1Lu by TGF-
is commonly used as a basis for a biological assay of TGF-
1
activity(45) . It is often found that TGF-
activity cannot
be detected from samples unless TGF-
is activated prior to the
assay. Routinely used methods for activation include heating to 80
°C for 15 min, or acidification to pH 2(45) . HGF is
completely inactivated both by acid and heat treatment at 70 °C for
20 min(46) . The fact that HGF inhibits TGF-
-induced
growth arrest in Mv1Lu cells severely compromises this assay, when used
to detect active TGF-
1. TGF-
could thus be inadvertently
classified as ``latent'' due to interference of this assay by
HGF, and increases in ``active TGF-
'' could in fact
represent down-regulation of HGF activity.
In the mouse, TGF-1
is found around developmentally stabilized ducts of mammary epithelium,
but not close to invading end buds(26) . Similarly, embryonic
lung synthesizes a TGF-
1-rich collagenous matrix around developing
alveoli(47) . Latent TGF-
binding protein and TGF-
1
are also found in the subendothelial
matrix(32, 48, 49) . In addition, mutations
in endoglin, a component of the TGF-
receptor system in
endothelial cells, cause hereditary hemeorrhagic telangiectasia type
1(50) . These results suggest that TGF-
has a role in the
formation and maintenance of epithelial and endothelial structures. The
fact that HGF prevents TGF-
1 induced growth arrest in both
epithelial and endothelial cells suggests that angiogenic and branching
morphogenesis promoting effects of HGF (17, 21) could
result from its ability to suppress the growth inhibitory effects of
subepithelial or endothelial matrix-derived TGF-
. The ability of
TGF-
1 and glucocorticoids to block mammary ductal growth (26) could in turn be explained by the ability of TGF-
and
glucocorticoids to down-regulate the expression of HGF(51) . In
addition, the induction of thrombospondin by HGF (Fig. 5) is
likely to further suppress HGF expression, since thrombospondin
positively regulates the activation of TGF-
(52) .
The
fact that a fibroblast-derived epithelial cell growth stimulator (HGF)
can override the effects of a growth inhibitor (TGF-) is
unexpected, since epithelial cells are growth inhibited in mixed
cultures, and primary cultures of animal cells are typically dominated
by fibroblastic cells after a few passages. TGF-
1 is known to be
activated during co-culture of endothelial and smooth muscle cells (20) , and HGF is down-regulated when fibroblasts are cultured
with epithelial cells(53) . Our data suggests that the
mechanism of TGF-
1 induced fibrosis and inhibition of epithelial
regeneration is not directly attributable to the induction of
epithelial growth inhibition and extracellular matrix synthesis, but
involves prior suppression of epithelial growth factors, such as
HGF(51) . These results imply that the inhibitory effects of
TGF-
superfamily members on epithelial regeneration can be
counteracted by exogenous HGF.
TGF- arrests the cell cycle of
epithelial cells in mid to late
G
(18, 54) . Certain viral transforming
proteins, such as simian virus-40 large T, abrogate growth inhibitory
response of epithelial cells to TGF-
1 without interfering with the
induction of extracellular matrix gene
expression(43, 55, 56, 57) . We find
here that HGF acts like viral transforming proteins in desensitizing
cells to TGF-
1 growth inhibition without affecting the induction
of extracellular matrix proteins.
Several mechanisms for
TGF--mediated cell cycle arrest have been postulated. These
include down-regulation of c-myc proto-oncogene
expression(56) , suppression of retinoblastoma-protein
phosphorylation(54) , suppression of the activity of
cyclin-dependent kinases Cdk2 and
Cdk4(58, 59, 60) , and induction of
cyclin-dependent kinase inhibitors p15
,
p21
, and p27
(61, 62, 63) . TGF-
growth
inhibition is likely to be mediated by the retinoblastoma protein (54) and/or related proteins p107 and p130(64) . Mv1Lu
cells overexpressing Cdk4(60) , viral transforming proteins
with pRb binding domains, or the transcription factor E2F1 (65) are refractory to TGF-
1-induced growth arrest.
Hypophosphorylated Rb binds to E2F1 and suppresses its
activity(66) , and Cdk4 is capable of phosphorylating
Rb(67) . Our results suggest that HGF acts upstream of pRb like
proteins, by inhibiting the suppression of Cdk4 by TGF-
1. In
contrast, HGF has no effect on the TGF-
1 mediated increase in
immunoprecipitated p21. These results suggest that the level of p21
induced by TGF-
1 is incapable of inducing growth arrest, and that
TGF-
induced growth arrest of epithelial cells requires both
suppression of G
Cdks and induction of Cdk inhibitors. The
failure of HGF to suppress p21 induction by TGF-
1 may contribute
to the slow growth phenotype of Mv1Lu cells treated with both factors
(see (63) and (68) ).
Several reports have
suggested that the loss of growth suppression by TGF- contributes
to the malignant phenotype. In several cases, the resistance of tumor
cells to TGF-
can be explained in terms of reduced expression
level or mutation of the type II TGF-
receptor (33, 69, 70, 71) . Present results
indicate that the loss of genes required for the maintenance of
mutually exclusive c-met/HGF expression (72) could
also contribute to TGF-
resistance and malignancy (see (73) ).
Loss of cell adhesion to the substratum blocks the
cell cycle of anchorage dependent cell lines at the G-S
boundary. Cell adhesion controls the G
-S transit
independently of growth factor-mediated mitogenic events(74) .
We find here that HGF induces both TGF-
1 resistance and anchorage
independent growth in Mv1Lu cells. The mechanism of these two effects
may be similar. Latent TGF-
1 is deposited to the extracellular
matrix(32, 33, 34, 35) . Loss of
adhesion could result in a failure to deposit matrix, leading to
resorption of matrix components and activation of sufficient amounts of
TGF-
-like factors to effect growth inhibition. In addition, the
fact that two extracellular matrix-associated growth
factors, TGF-
and HGF, are capable of inducing anchorage
independent growth (this report; Refs. 8, 32, and 75) suggests
that anchorage dependence of normal cells may not be mediated directly
by adhesion, but in part by extracellular matrix-associated growth
factors.
The present results indicate that mesenchymal extracellular
matrices contain closely balanced amounts of latent growth and invasion
promoting and suppressing factors, HGF and TGF-1, and that the
proteolytic state of epithelial cells can dictate how they respond to
extracellular matrices. Since both TGF-
and HGF are found in
platelets(46, 76) , it is likely that this balance
exists also in fibronectin-fibrin matrices during wound healing. HGF is
activated by cultured cells, and little difference is observed in
biological activity between active and latent forms in the presence of
serum(8, 31) . In contrast, TGF-
is not normally
activated by cultured cells, and latent TGF-
1 is at least 100-fold
less efficient in inducing Mv1Lu growth arrest than activated
TGF-
1. (
)It is thus tempting to speculate that two
proteolytic states of cells exist: ``TGF-
activating''
quiescent state (mature epithelium; see (77) ) and ``HGF
activating'' proliferative or invasive state (cultured cells,
invading cells, cancer). These states could be controlled by growth
factors whose expression pattern is more restricted, such as the
members of the fibroblast growth factor family. Since neither
TGF-
1 nor HGF are required for normal development of most internal
organs(78, 79, 80) , it is likely that the
inducer/suppressor balance can be maintained by other members of the
TGF-
and HGF families, such as BMP-6 (81) and
MSP(4, 5) .