©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Hepatocyte Growth Factor Releases Epithelial and Endothelial Cells from Growth Arrest Induced by Transforming Growth Factor-1 (*)

(Received for publication, June 19, 1995; and in revised form, October 27, 1995)

Jussi Taipale (1)(§) Jorma Keski-Oja (1) (2)(¶)

From the  (1)Departments of Virology, the Haartman Institute and (2)Dermatology and Venereology, University of Helsinki, FIN-00014 Helsinki, Finland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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-beta (TGF-beta). 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-beta1. 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-beta1 inhibited Mv1Lu cells was also induced by HGF. In addition, HGF induced anchorage independent growth of Mv1Lu cells that was refractory to TGF-beta1 growth inhibition. Immunoprecipitation analysis indicated that HGF prevented the suppression of Cdk4 and Cdk2, but not the induction of p21, by TGF-beta1. Since both TGF-beta1 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.


INTRODUCTION

Transforming growth factors-beta 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-beta (^1)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-beta 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-betas 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-beta 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-beta potently suppresses these events(18, 19, 20) . At tissue level HGF stimulates branching morphogenesis and tissue regeneration(12, 22, 23, 24, 25) , while TGF-beta inhibits ductal growth and induces fibrosis(26, 27, 28, 29) .

Both HGF and TGF-beta are secreted from cells in a latent form(1, 8, 30, 31) , and associate with extracellular matrices(8, 32, 33, 34) . TGF-beta associates with extracellular matrix via latent TGF-beta binding proteins(35) , while HGF associates with heparan sulfate proteoglycans (8, 36) . TGF-beta 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-beta. However, the responsiveness of epithelial cells, such as keratinocytes, to TGF-beta 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-beta1, and to understand the role of extracellular matrix derived growth factors and growth inhibitors in mesenchymal-epithelial interactions.


MATERIALS AND METHODS

Reagents and Antibodies

Recombinant human TGF-beta1 was a gift of Dr. P. Puolakkainen (Oncogen, Seattle, WA). Sf9 recombinant human HGF was from R& Systems (Minneapolis, MN). The majority of the HGF preparation is in a latent single chain form, requiring serum derived factors for activation. Mouse monoclonal and goat anti-human HGF neutralizing antibodies were from R& Systems. Nonimmuno-goat IgG was from Jackson Immunoresearch Laboratories (West Grove, PA). Neutralizing antibodies to TGF-beta1 (AB101NA), TGF-beta2 (AB12NA), and keratinocyte growth factor (MAB251) were from R& Systems. Mouse monoclonal anti-thrombospondin (C6.7) antibody was from Life Technologies (Gaithersburg, MD). Polyclonal affinity purified antibodies to Cdk2 (M2), Cdk4 (C22), p21 (L17), Met (C28), and type II TGF-beta receptor were from Santa Cruz Biotechnology (Santa Cruz, CA). GammaBind G and gelatin-Sepharoses were from Pharmacia (Uppsala, Sweden). Radiochemicals were purchased from Amersham (Amersham, UK), except for I-labeled TGF-beta1 (Bolton-Hunter, specific activity 0.1 mCi/µg) which was from DuPont NEN.

Cell Culture

Human embryonic lung fibroblasts (CCL-137; American Type Culture Collection, Rockville, MD) and Madin-Darby canine kidney epithelial (MDCK) cells were grown in Eagle's modification of minimal essential medium (MEM) containing 10% heat inactivated fetal calf serum (FCS), 100 IU/ml penicillin, and 50 µg/ml streptomycin. FCS was replaced by 10% newborn calf serum or 10% noninactivated fetal calf serum for bovine capillary endothelial cells and mink lung epithelial Mv1Lu cells (CCL-64), respectively. Because latent HGF requires serum-derived factors for activation(8, 30, 31, 38) , all experiments were carried out in medium containing 10% FCS.

Soft Agar Assays

Bottom agarose (0.7%, Compactigel, FMC Bioproducts, Rockland, ME) containing Dulbecco's MEM and 10% fetal calf serum was cast on plastic 6-well plates. Subsequently, 2.5 times 10^4 cells in Dulbecco's MEM were mixed with bottom agarose medium 1:1 (final agarose concentration 0.35%) at 37 °C and plated on the bottom agarose and allowed to gel on ice for 30 min. Where indicated, HGF (20 ng/ml) or TGF-beta1 (10 ng/ml) were added to both agarose layers. Subsequently, top agarose was overlaid with 200 µl of Dulbecco's MEM and cells were incubated at 37 °C for 9 days before counting of the colonies.

Extracellular Matrix Preparations

Extracellular matrices were prepared according to Hedman et al.(39) . Briefly, confluent cultures of fibroblasts cultured on plastic were washed once with PBS (0.14 M NaCl in 10 mM sodium phosphate buffer, pH 7.4) and extracted three times with 0.5% sodium deoxycholate in 10 mM Tris-HCl buffer, pH 7.8, for 20 min on ice. Subsequently, the purified extracellular matrices were washed once with PBS. Where indicated, the preparations were washed twice with PBS containing 1.2 M NaCl for 5 min at room temperature. For cell culture, the extracellular matrices were washed three times with PBS and once with MEM containing 10% fetal calf serum.

Thymidine Incorporation Assays

To determine the rate of DNA synthesis, cells were labeled for 2-4 h with 1 µCi/ml [6-^3H]thymidine. Subsequently, the cells were washed and fixed with methanol. Radioactivity incorporated to DNA was solubilized by 0.2 M NaOH and determined by a liquid scintillation counter.

Cross-linking Analysis of the TGF-beta Receptors

Cells cultured on 6-well plates were washed four times with PBS, and incubated with 20 pMI-labeled TGF-beta1 in PBS at room temperature for 20 min. Cells were then cross-linked with 1 mM BS(3) for 30 min, and the reaction quenched by the addition of glycine to 20 mM. After washing 3 times with PBS, the cells were lysed at 0 °C with 2 ml of 1% Triton X-100 in PBS containing 1 mM phenylmethylsulfonyl fluoride, 200 IU/ml aprotinin, 100 µg/ml antipain, soybean trypsin inhibitor, and leupeptin, each (Sigma). The lysates were subsequently clarified by centrifugation at 2000 times g for 40 min and used for immunoprecipitation as described below for cell lysates.

Metabolic Labeling and Immunoprecipitation

For precipitation of fibronectin and thrombospondin, Mv1Lu cells were cultured to 50% confluence and labeled for 18 h in MEM containing 10% dialyzed FCS, 50 µCi/ml [S]methionine (>1000 Ci/mmol), and 5 µM non-radioactive methionine. Culture medium was clarified at 10,000 times g for 5 min in a microcentrifuge. Aliquots of medium (400 µl) were reacted with monoclonal anti-thrombospondin antibody (2 µg/ml) for 2 h at 0 °C. Subsequently, antibodies were precipitated by treatment with 30 µl of GammaBind G-Sepharose in an end-over shaker at 4 °C for 30 min. Fibronectin was affinity purified using 30 µl of gelatin-Sepharose. The beads were washed three times with PBS, and bound proteins were eluted to 50 µl of SDS-PAGE sample buffer containing 2% SDS and 10% 2-mercaptoethanol.

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(3)VO(4), 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 times 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.

SDS-PAGE, Immunoblotting, and PhosphorImager Quantitation

Gradient (4-15%) SDS-PAGE was carried out using the Laemmli buffer system under nonreducing conditions (Bio-Rad minigels without a stacking gel). Immunodetection of antigens was performed using biotin-avidin amplification and enhanced chemiluminescence detection as described(32, 34) . For PhosphorImager analysis, all samples were run on a 8-20% ExcelGel (Pharmacia; 48 samples of 10 µl). The gel was subsequently air-dried and imaged for 40 h at room temperature using Fuji BAS-IIIs imaging plate. The results were subsequently quantitated using Fuji BAS 1000 Bio-Imaging Analyzer and MacBAS 1.0 software.


RESULTS

Fibroblast Extracellular Matrix Contains Factors That Reduce the Sensitivity of Epithelial Cells to TGF-beta1 Growth Inhibition

To study the activation of extracellular matrix bound TGF-beta1(32, 33, 34, 35) during epithelial-mesenchymal interactions, Mv1Lu mink lung epithelial cells were cultured on purified human embryonic lung fibroblast matrices (see ``Materials and Methods''). Mv1Lu cells are extremely sensitive to growth inhibition by TGF-beta1, even in the presence of factors supporting rapid proliferation, such as 10% FCS (40) or 5 ng/ml EGF(32) . To minimize the effect of growth factors contained in the matrix, the cells were plated in a medium containing 10% FCS that supports maximal proliferation. Although fibroblast extracellular matrix contains biologically significant amounts of TGF-beta1 (approx100 pg/cm^2; (32) ), the growth of Mv1Lu cells was not severely decreased when plated on fibroblast matrices. In addition, neutralizing antibodies to TGF-beta1 had no effect on the growth of Mv1Lu cells plated on fibroblast matrices (Table 1). Two possibilities remained, namely that either Mv1Lu cells do not activate matrix-bound TGF-beta1, or that matrix reduces the sensitivity of Mv1Lu cells to TGF-beta1.



Further experiments indicated that the plating of the Mv1Lu cells on fibroblast matrices reduced their sensitivity to TGF-beta1. A small decrease in number of Mv1Lu cells was observed when the cells were cultured on plastic in the presence of TGF-beta1 (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-beta1 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-beta1 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 times 10^4) 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-beta1 (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.



Identification of HGF as a Factor in the Fibroblast Matrix That Desensitizes Epithelial Cells to TGF-beta1

Human lung fibroblasts secrete soluble factors that are mitogenic and morphogenic to MDCK canine kidney epithelial cells(41) . The morphogenic activity was assigned to hepatocyte growth factor/scatter factor(21) . Since HGF can associate with extracellular matrices(8) , we tested the ability of anti-HGF antibodies to inhibit the growth stimulatory effect of fibroblast extracellular matrix. The ability of fibroblast matrix to support Mv1Lu cell growth in the presence of TGF-beta1 (10 ng/ml) was significantly reduced by neutralizing goat anti-HGF antibodies (8 µg/ml; Fig. 1A), or by washing of the matrices with 1.2 M NaCl, which dissociates HGF from heparan sulfate proteoglycans in the extracellular matrix(8) . Neutralizing antibodies to another mesenchymally derived epithelial growth factor, keratinocyte growth factor, or non-immune goat IgG (8 µg/ml) did not affect the growth of Mv1Lu cells in the presence of TGF-beta1 and fibroblast extracellular matrix (data not shown). Approximately 0.5-1 ng of HGF/cm^2 of matrix was detected in the matrix preparations by immunoblotting, and washing of the matrices with 1.2 M NaCl reduced the amount of matrix-associated HGF over 50% (Fig. 1B).

HGF Prevents TGF-beta1-mediated Growth Arrest

We studied next the effect(s) of recombinant HGF on TGF-beta1 induced growth arrest in epithelial cells cultured on plastic. Mv1Lu cells were plated in the presence of 10% FCS and increasing concentrations of TGF-beta1 and HGF (added simultaneously). The cell number was determined at 90 h in a Coulter counter. TGF-beta1 alone at 0.3 ng/ml induced approx90% decrease in cell number, and growth inhibition was approx100% at concentrations of 1 ng/ml and higher (Fig. 2A; note logarithmic scale). HGF at 2 ng/ml did not alter the growth of the cells, while at 20 ng/ml it caused a slight decrease in cell number (approx30%). When the cells were cultured with both HGF and TGF-beta1, HGF attenuated the growth inhibitory effect of TGF-beta1 in a dose-dependent manner. Importantly, HGF at 20 ng/ml induced the growth of Mv1Lu cells in the presence of 10 ng/ml TGF-beta1, a concentration that alone caused complete growth arrest (Fig. 2A). However, the proliferation of the cells was approximately 50% slower than in control cultures. Similar results were obtained using bovine capillary endothelial cells, and a second epithelial cell line, MDCK (Fig. 2B). The effect of recombinant HGF could be blocked by a neutralizing monoclonal antibody (data not shown).


Figure 2: Hepatocyte growth factor prevents TGF-beta1-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 10^4 cells/cm^2. Increasing concentrations of HGF and TGF-beta1 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(2)(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-beta1 (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.



HGF Induces Proliferation of Epithelial Cells Growth Arrested by TGF-beta

To study whether HGF can induce the growth of Mv1Lu cells that are under TGF-beta1 growth arrest, we seeded Mv1Lu cells on plastic 24-well plates in the presence of 10 ng/ml TGF-beta1 and 10% FCS, and treated the cells with HGF after a minimum of 56 h of growth arrest. For comparison, similarly treated cells were washed twice and incubated in 10% serum containing medium. Mv1Lu cells, growth inhibited by incubation in low serum (0.2%) and induced to grow by addition of FCS (10% final concentration), were used as a control. TGF-beta1 growth inhibited cells that were stimulated with HGF displayed slightly faster kinetics of [^3H]thymidine incorporation than serum-deprived cells treated with 10% FCS, maximal thymidine incorporation occurring at 20 h and geq24 h, respectively. Importantly, washing of the cells with MEM containing 10% FCS and incubation in fresh medium containing serum was not sufficient to induce [^3H]thymidine incorporation (Fig. 3).


Figure 3: HGF induces thymidine incorporation in Mv1Lu cells growth inhibited by TGF-beta1. 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-beta1 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 (bullet), treated with HGF (20 ng/ml) without removing TGF-beta1 containing culture medium (box), or induced with addition of FCS to 10% final concentration (circle). Cells were subsequently labeled with 1 µCi of [6-^3H]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.



HGF Induces Anchorage Independent Growth of Mv1Lu Cells

To study whether HGF can also bypass growth inhibition caused by loss of adhesion to substratum, we seeded Mv1Lu cells in soft agar in the presence of 10% FCS and HGF and/or TGF-beta1. Mv1Lu cells did not form colonies in soft agar in the presence of 10% FCS with or without TGF-beta1. Unexpectedly, HGF induced very efficiently anchorage independent growth, and TGF-beta1 did not inhibit HGF-induced colony formation (Fig. 4). In visual inspection, colonies induced by HGF together with TGF-beta1 were smaller, but slightly more numerous than colonies induced by HGF alone (Fig. 4).


Figure 4: HGF induces anchorage independent growth of Mv1Lu cells. Mv1Lu cells were plated at a density of 1500 cells/cm^2 on Compactigel-agarose (see ``Materials and Methods'') in MEM + 10% FCS containing HGF (20 ng/ml) and/or TGF-beta1 (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 times).



HGF Does Not Alter the Expression Level of TGF-beta Receptors

To address the possibility that the effect of HGF is caused by loss or down-regulation of TGF-beta receptor function, we analyzed the expression of TGF-beta receptors in HGF-treated Mv1Lu cells. Subconfluent cultures of Mv1Lu cells were treated for 16 h with HGF, and the expression of TGF-beta receptors was analyzed by cross-linking with I-labeled TGF-beta1 followed by immunoprecipitation with antibodies specific for the type II TGF-beta receptor. PhosphorImager analysis of the immunoprecipitates run in SDS-PAGE indicated that the expression levels of type I or II TGF-beta receptors were not significantly altered by HGF (Fig. 5).


Figure 5: HGF does not affect TGF-beta1 receptor levels in Mv1Lu cells. Subconfluent (50%) cultures of Mv1Lu cells were treated with HGF (20 ng/ml) for 16 h. Expression of TGF-beta receptors was analyzed by cross-linking with I-labeled TGF-beta1, followed by immunoprecipitation with type II TGF-beta 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-beta receptors, and results from PhosphorImager quantitation are shown on the right. Migration of molecular mass markers is shown on the left.



HGF Does Not Inhibit TGF-beta1-induced Extracellular Matrix Protein Expression

We analyzed next the dose responses of fibronectin and thrombospondin induction in TGF-beta1-treated Mv1Lu cells(42, 43, 44) . Subconfluent Mv1Lu cells were treated with increasing concentrations of TGF-beta1 in the presence or absence of HGF for 18 h. Immunoprecipitation analysis of the cell conditioned medium indicated that HGF had no effect on fibronectin expression, but induced the expression of thrombospondin approximately 2-fold. HGF did not interfere with the induction of thrombospondin or fibronectin by TGF-beta1 (Fig. 6). These data indicate that HGF-treated cells are responsive to TGF-beta1, and that HGF does not inhibit the extracellular matrix protein inducing signal transduction mechanisms in Mv1Lu cells.


Figure 6: HGF does not interfere with TGF-beta1-induced fibronectin or thrombospondin gene expression. Subconfluent (approx50%) cultures of Mv1Lu cells were treated with increasing concentrations of TGF-beta1 in the absence (box) 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-beta1. x axis, TGF-beta1 (ng/ml); y axis, fold induction. Inset, autoradiography. Middle, PhosphorImager analysis of the induction of thrombospondin by TGF-beta1. x axis, TGF-beta1 (ng/ml); y axis, fold induction. Inset, autoradiography. Right, analysis of cell proliferation. Mv1Lu cells (10^4/cm^2) were plated and treated with TGF-beta1 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(2)(cells at 3 days) normalized to control (100%) and TGF-beta1 (16 ng/ml; 0%). x axis, TGF-beta1 (ng/ml); y axis, rate of cell proliferation, % of control.



HGF Blocks TGF-beta1-induced Suppression of Cyclin-dependent Kinase-4

As another approach to analyze the mechanism of HGF induced refractoriness to growth suppression, we studied the effects of HGF on cell cycle proteins that are regulated by TGF-beta1. Subconfluent (50%) cultures of Mv1Lu cells were treated with TGF-beta1 and/or HGF in the presence of 10% FCS for 18 h. The cells were labeled with [S]methionine for the last 6 h, followed by immunoprecipitation analysis of Cdk2, Cdk4, p21, and Met (see ``Materials and Methods'' for details). Treatment of Mv1Lu cells with TGF-beta1 led to down-regulation of immunoprecipitated Cdk4 and Cdk2, and in an induction of p21. Simultaneous treatment of the cells with HGF prevented the suppression of Cdk4 and Cdk2, but had no effect on p21 levels. TGF-beta1 alone or in combination with HGF did not have major effects on the levels of the HGF receptor Met (Fig. 7).


Figure 7: HGF prevents the suppression of Cdk2 and Cdk4 by TGF-beta1. Subconfluent (50%) cultures of Mv1Lu cells were treated with HGF (20 ng/ml) and/or TGF-beta1 (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-beta1 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.




DISCUSSION

In this report, we find that culturing of epithelial cells on fibroblast extracellular matrix reduces their sensitivity to growth inhibition by TGF-beta. Hepatocyte growth factor/scatter factor is identified as a major factor capable of mediating this effect. Recombinant HGF inhibited TGF-beta1 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-beta is commonly used as a basis for a biological assay of TGF-beta1 activity(45) . It is often found that TGF-beta activity cannot be detected from samples unless TGF-beta 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-beta-induced growth arrest in Mv1Lu cells severely compromises this assay, when used to detect active TGF-beta1. TGF-beta could thus be inadvertently classified as ``latent'' due to interference of this assay by HGF, and increases in ``active TGF-beta'' could in fact represent down-regulation of HGF activity.

In the mouse, TGF-beta1 is found around developmentally stabilized ducts of mammary epithelium, but not close to invading end buds(26) . Similarly, embryonic lung synthesizes a TGF-beta1-rich collagenous matrix around developing alveoli(47) . Latent TGF-beta binding protein and TGF-beta1 are also found in the subendothelial matrix(32, 48, 49) . In addition, mutations in endoglin, a component of the TGF-beta receptor system in endothelial cells, cause hereditary hemeorrhagic telangiectasia type 1(50) . These results suggest that TGF-beta has a role in the formation and maintenance of epithelial and endothelial structures. The fact that HGF prevents TGF-beta1 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-beta. The ability of TGF-beta1 and glucocorticoids to block mammary ductal growth (26) could in turn be explained by the ability of TGF-beta 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-beta(52) .

The fact that a fibroblast-derived epithelial cell growth stimulator (HGF) can override the effects of a growth inhibitor (TGF-beta) 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-beta1 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-beta1 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-beta superfamily members on epithelial regeneration can be counteracted by exogenous HGF.

TGF-beta arrests the cell cycle of epithelial cells in mid to late G(1)(18, 54) . Certain viral transforming proteins, such as simian virus-40 large T, abrogate growth inhibitory response of epithelial cells to TGF-beta1 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-beta1 growth inhibition without affecting the induction of extracellular matrix proteins.

Several mechanisms for TGF-beta-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-beta 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-beta1-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-beta1. In contrast, HGF has no effect on the TGF-beta1 mediated increase in immunoprecipitated p21. These results suggest that the level of p21 induced by TGF-beta1 is incapable of inducing growth arrest, and that TGF-beta induced growth arrest of epithelial cells requires both suppression of G(1) Cdks and induction of Cdk inhibitors. The failure of HGF to suppress p21 induction by TGF-beta1 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-beta contributes to the malignant phenotype. In several cases, the resistance of tumor cells to TGF-beta can be explained in terms of reduced expression level or mutation of the type II TGF-beta 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-beta resistance and malignancy (see (73) ).

Loss of cell adhesion to the substratum blocks the cell cycle of anchorage dependent cell lines at the G(1)-S boundary. Cell adhesion controls the G(1)-S transit independently of growth factor-mediated mitogenic events(74) . We find here that HGF induces both TGF-beta1 resistance and anchorage independent growth in Mv1Lu cells. The mechanism of these two effects may be similar. Latent TGF-beta1 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-beta-like factors to effect growth inhibition. In addition, the fact that two extracellular matrix-associated growth factors, TGF-beta 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-beta1, and that the proteolytic state of epithelial cells can dictate how they respond to extracellular matrices. Since both TGF-beta 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-beta is not normally activated by cultured cells, and latent TGF-beta1 is at least 100-fold less efficient in inducing Mv1Lu growth arrest than activated TGF-beta1. (^2)It is thus tempting to speculate that two proteolytic states of cells exist: ``TGF-beta 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-beta1 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-beta and HGF families, such as BMP-6 (81) and MSP(4, 5) .


FOOTNOTES

*
This work was supported by the Academy of Finland, Sigrid Juselius Foundation, Biocentrum Helsinki, Finnish Cancer Organizations, The Research and Science Foundation of Farmos, Novo Nordisk Foundation, and the University of Helsinki. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Predoctoral fellow of the Academy of Finland.

To whom correspondence should be addressed: Dept. of Virology, University of Helsinki, P. O. Box 21 (Haartmaninkatu 3), FIN-00014, University of Helsinki, Helsinki, Finland. Tel.: 358-0-434-6476; Fax: 358-0-434-6491.

(^1)
The abbreviations used are: TGF-beta, transforming growth factor-beta; HGF, hepatocyte growth factor/scatter factor; MDCK, Madin-Darby canine kidney cells; MEM, minimal essential medium; FCS, fetal calf serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis.

(^2)
J. Taipale and J. Keski-Oja, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Pauli Puolakkainen for recombinant active TGF-beta1, Dr. Kari Alitalo for critical review of the manuscript, and Sami Starast for fine technical assistance.


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