Control of Type II Transforming Growth Factor-beta Receptor Expression by Integrin Ligation*

DanHui WangDagger §, LuZhe Sun, Elizabeth Zborowskaparallel , James K. V. Willsonparallel , Jiangen GongDagger §, Janaki Verraraghavan**, and Michael G. Brattain**Dagger Dagger

From the ** Departments of Surgery and Biochemistry, University of Texas Health Science Center, San Antonio, Texas 78284, the  Department of Pharmacology, University of Kentucky, Lexington, Kentucky 40536, the parallel  Department of Medicine, Case Western Reserve University Ireland Cancer Center and School of Medicine, Cleveland, Ohio 44106, and the Dagger  Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Ectopic expression of the alpha 5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha 5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta ) activity. MCF-7 breast cancer cells do not express either alpha 5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha 5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha 5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha 5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha 5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha 5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha 5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha 5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha 5beta 1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

As mediators of cell matrix-cell interaction, integrins impart diverse biological properties to the cells that express them. A number of studies have demonstrated that integrin expression affects tumor cell proliferation and progression (1-3). Immunohistochemical analysis showed that the expression of integrins is altered in human tumors compared with corresponding normal cells. Many neoplastic cells show reduced expression of integrins (4-9). Overexpression of certain integrins, such as alpha 5beta 1, can reverse tumorigenicity and anchorage-independent growth in some transformed cells (10, 11). Perturbation of integrin alpha 5beta 1binding to its ligand stimulates the growth of a variant of the K562 cells (12). Recently, we showed expression of integrin alpha 5subunit selectively blocks DNA synthesis (13) and disruption of alpha 5beta 1ligation enhanced DNA synthesis in a mitogen-activated protein kinase-dependent but epidermal growth factor receptor-independent manner (14). Extracellular matrix recognition by integrin alpha 5beta 1 may, therefore, play a role in the negative control of cell growth, which may be lost in some cancer cells.

Loss of negative growth factors can also lead to abnormal growth in transformed cells. Transforming growth factor-beta (TGF-beta )1 has been identified as a potent growth inhibitor in various normal as well as transformed cell types (15, 16). Escape from the negative growth control by TGF-beta is an important step during oncogenic transformation (15) because the growth of normal mammary epithelial cells is inhibited by TGF-beta , whereas their transformed counterparts are often resistant to its inhibitory effects (17-19). Loss of TGF-beta sensitivity in estrogen receptor + breast cancer cells has frequently been associated with loss of the TGF-beta type II receptor (RII), which, along with the TGF-beta type I receptor (RI), is necessary for TGF-beta signal transduction (20, 21). The importance of loss of TGF-beta signaling in breast cancer cells was demonstrated by differences in tumorigenicity of MCF-7 breast cancer clones with and without RII expression (22) and the ability of re-expression of RII in MCF-7 cells to inhibit tumorigenicity in athymic mice (21). RII has been shown to be a tumor suppressor gene by the criteria that its mutational inactivation is associated with a hereditary form of colon cancer and that RII ectopic expression in cancer cells from individuals with this form of cancer reverses malignancy in athymic mice (24, 25). Re-expression of RI in cancer cells that are deficient in this receptor also reverses tumorigenicity (26).

Two lines of evidence indicate that autocrine negative TGF-beta may be more important than response to exogenous TGF-beta in controlling tumor growth. The first of these was the demonstration that removal of autocrine TGF-beta activity by stable transfection of a TGF-beta antisense expression vector leads to malignant progression of cancer cells in athymic mice (27, 28). This approach blocked autocrine TGF-beta activity because endogenous TGF-beta was removed from these cells but did not affect the expression of TGF-beta receptors and, therefore, permitted the retention of response to exogenously produced TGF-beta in the tumor environment from nonmalignant cells. The occurrence of tumor formation indicated that exogenous TGF-beta produced by nonmalignant cells was insufficient to achieve tumor suppression. The second line of evidence involved the re-expression of RII in a cell line that was homozygous for mutational inactivation of the gene. Re-expression of RII regenerated autocrine negative TGF-beta but did not regenerate an inhibitory response to exogenous TGF-beta (25). However, reversion of tumorigenicity did occur, indicating that autocrine TGF-beta was critical. Thus, the available evidence indicates that mechanisms for regeneration of RII expression and autocrine negative TGF-beta will be of importance in our understanding of controls of TGF-beta signal transduction in malignancy and may lead to novel treatment or prevention approaches for cancer.

Previously, we showed that vitamin D3 was inhibitory to wild type MCF-7 clones that expressed RII and to RII-null clones expressing ectopic RII (29). In contrast, RII-null clones were refractory to inhibition (29). Response to vitamin D3 was associated with induction of higher RII levels and enhanced TGF-beta autocrine negative activity, suggesting that other growth modulators may cause inhibition by inducing TGF-beta autocrine negative activity. Given the ability of alpha 5 integrin to affect growth in a negative fashion (13, 14), we hypothesized that re-expression of cell surface alpha 5beta 1 integrin in cancer cells deficient in the expression of the alpha 5 subunit would lead to regeneration of RII expression and autocrine negative TGF-beta activity. This hypothesis was tested by stable transfection of a MCF-7 breast cancer clone lacking both RII and alpha 5 integrin expression with an alpha 5 integrin cDNA. Transfection resulted in re-expression of RII and regeneration of autocrine TGF-beta activity, as well as response to exogenous TGF-beta . RII expression and TGF-beta responses were dependent upon alpha 5beta 1 ligation to endogenous MCF-7 fibronectin (FN) and were enhanced when exogenous FN was used to coat culture plates, indicating that growth inhibition by alpha 5beta 1 ligation involves induction of autocrine TGF-beta activity.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Cell Culture-- MCF-7 cells were originally obtained from American Type Culture Collection and adapted to McCoy's 5A medium supplemented with 10% fetal bovine serum (FBS), pyruvate, vitamins, amino acids, and antibiotics (30). Working cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and routinely checked for mycoplasma contamination as described previously (21). MCF-7 limiting dilution clones were obtained by diluting the parental cells in 96-well tissue culture plates at 0.5 cell/well as described previously (21). The strain of MCF-7 cells utilized in this study is insensitive to TGF-beta because it lacks RII (21).

Integrin alpha 5 Stable Transfection-- An integrin alpha 5 expression vector for mammalian cells was used for stable transfection as described previously (13). The plasmid was linearized and transfected into a typical MCF-7 limiting dilution clone (designated MCF-7 LDC4) by electroporation with a Bio-Rad Gene Pulser at 250 V and 960 microfarads. Control cells were transfected with a Neo-containing plasmid. The transfected cells were plated in 100-mm culture dishes in 10% FBS medium for 2 days. Selection of stable transfectants was carried out by adding Geneticin (600 µg/ml; Life Technologies, Inc.) into the medium. After three weeks, Geneticin-resistant clones were ring-cloned and expanded for screening of alpha 5 expression. The control clones were pooled and designated the MCF-7 LDC4 Neo pool.

RNA Analysis-- Total RNA was isolated from cultured cells by the guanidine isothiocyanate method (31). For detection of FN mRNA levels, cells (106) were plated in 100-mm culture dishes coated with poly-L-lysine (10 µg/ml; Sigma) or FN (10 µg/ml; Collaborative Biomedical Products) for 4 days in 10% FBS medium, and total RNA was then isolated. For detection of RII mRNA levels, cells (106) were plated in 100-mm culture dishes coated with poly-L-lysine (10 µg/ml) or FN (10 µg/ml) for 1 and 2 h in McCoy's 5A medium supplemented with 2% bovine serum albumin (Sigma) and 24 and 96 h in McCoy's 5A medium supplemented with 10% FBS.

The construction of the integrin alpha 5 subunit and RII antisense probes has been described (21, 32). The FN riboprobe plasmid was constructed by subcloning a 232-base pair BamHI-PvuII fragment of the human FN cDNA into a pBSK(-) vector (Stratagene Cloning System). T7 RNA polymerase was used to synthesize the FN antisense probe (13, 32). The RNase protection assay was performed by hybridization of the radioactive riboprobes with total RNA (20 µg) isolated from the control or alpha 5-transfected cells as described previously (32).

Immunoprecipitation-- To determine cell surface integrin alpha 5beta 1 expression, cell surface proteins were labeled with biotin, immunoprecipitated with an anti-alpha 5 subunit monoclonal antibody (Life Technologies, Inc.), and analyzed by SDS-polyacrylamide gel electrophoresis as described previously (32).

Cell Adhesion and 3-(4,5-Dimethyl Thiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide Assays-- A 96-well Corning tissue culture plate was coated overnight at room temperature with FN at concentrations of 0, 1.0, 2.5, 5.0, and 10.0 µg/ml, blocked with 3% bovine serum albumin for 3 h, and then rinsed once with phosphate-buffered saline. Confluent cells were trypsinized, plated at 4 × 104 cells/well on FN-coated plates in serum-free medium, and incubated for 15 min. Unattached cells were gently washed away by three rinses with serum-free medium. The relative number of attached cells was determined by the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay as described previously (33).

The specificity of cell adhesion to FN was determined using a monoclonal anti-human integrin alpha 5 subunit antibody (Life Technologies, Inc.). The cells were incubated in the absence or presence of the antibody (1:100 dilution) for 30 min at 4 °C and then plated at 4 × 104 cells/well in 96-well plates coated with FN (10 µg/ml). Determination of cell adhesion and the 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay were performed as described above.

Tumorigenicity-- Exponentially growing cells of MCF-7 Neo pool and alpha 5 sense clone 15 were inoculated subcutaneously as described previously (21). Growth curves for xenografts were determined by measuring the volume (V) of tumors. V is expressed as V = (L × W2) × 0.5, where L is the length and W is the width of the xenograft.

Mitogenesis Assay-- Inhibition of cell proliferation by exogenous TGF-beta 1 in Neo and alpha 5-transfected cells was determined by measuring [3H]thymidine incorporation as described previously (21). Briefly, cells were plated in 24-well plates at 1.5 × 104 cells/well in the presence of various concentrations of TGF-beta 1 (0.2-10 ng/ml). After 4 days of incubation, cells received a 1-h pulse with [3H]thymidine (25 µCi) and were washed with supplemental McCoy's 5A medium three times, and DNA was precipitated with 10% trichloroacetic acid and then solubilized with 0.2 M sodium hydroxide. The amount of [3H]thymidine incorporated was analyzed by liquid scintillation counting in a Beckman LS7500 scintillation counter. Growth inhibition by TGF-beta 1 is represented as the percentage of [3H]thymidine incorporation of TGF-beta 1-treated cells relative to untreated cells.

Plating Efficiency Assay-- The effect of TGF-beta 1 neutralizing antibody on the clonogenic potential of control and alpha 5-transfected cells was compared to determine autocrine TGF-beta activity as described previously (25, 26). Cells were seeded at low density (400 cells/well) in 24-well plates in the presence of control IgG (10 µg/ml; R & D Systems, Inc.) or TGF-beta 1 neutralizing antibody (10 µg/ml; R & D Systems, Inc.). After 8 days of incubation without medium change, cell colonies were fixed with 1% glutaraldehyde, stained with 0.1% crystal violet, and dissolved in 1% Triton X-100 as described by Westergren-Thorsson et al. (34).

Transient Transfection and Luciferase Assay-- The TGF-beta -responsive luciferase expression plasmid (p3TP-Lux) was used for transient transfections, and luciferase assays were performed as described previously (35). MCF-7 Neo control and alpha 5 sense-transfected cells were transfected with 30 µg of p3TP-Lux and 10 µg of beta -galactosidase plasmid by electroporation with a Bio-Rad Gene Pulser at 250 V and 960 microfarads. The electroporated cells were plated onto 6-well tissue culture plates. After the cells attached, control IgG (10 µg/ml) and TGF-beta 1 neutralizing antibody (10 µg/ml) were added. The cells were harvested with 200 µl of lysis buffer (luciferase assay system; Promega). Luciferase activity was measured in the first 10 s after substrate addition using a luminometer (Berthold Lumat LB 9501) and expressed as arbitrary units after normalization with beta -galactosidase activity.

Receptor Cross-linking-- Human TGF-beta 1 was purified and iodinated by the chloramine T method as described previously (36). Equal numbers of the cells (105) were plated in 6-well plates, and after 5 days, binding and cross-linking of 200 mM 125I-TGF-beta 1 to the cell monolayer were performed as described by Segarini et al. (37). Labeled cells were solubilized in 200 µl of 1% Triton X-100. Equal amounts of cell lysate protein were electrophoresed by 4-10% gradient SDS-polyacrylamide gel electrophoresis under reducing conditions and exposed for autoradiography.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Expression of alpha 5beta 1 Integrin in MCF-7 Cells-- We transfected the alpha 5 subunit into a typical limiting dilution clone, MCF-7 LDC4. The alpha 5-positive transfectants were initially screened for increased expression of alpha 5 mRNA by RNase protection assays (Fig. 1). Two positive clones (designated cl.5 and cl.15) were isolated that expressed higher levels of alpha 5 subunit mRNA than the untransfected MCF-7 LDC4 and Neo-transfected (Neo pool) control cells. Increased alpha 5 mRNA expression was accompanied by increased cell surface expression (Fig. 2). The levels of cell surface integrin beta 1 subunit dimerized by alpha 5 subunit were also increased, indicating that the expression of beta 1 subunit might be up-regulated by increased alpha 5 subunit expression or that excess beta 1 subunit already present could complex with the increased levels of alpha 5 subunit. In contrast to alpha 5beta 1, the levels of alpha 2beta 1 and alpha 3beta 1 were unchanged in alpha 5 transfectants (data not shown), ruling out the possibility that increased alpha 5 expression might compete with other alpha  subunits for dimerizing with the beta 1 subunit. We next determined whether the increased alpha 5beta 1 enhanced adhesion to FN (Fig. 3A). Both alpha 5 transfectants showed 7-8-fold increased adhesion to FN-coated plates at FN concentrations ranging from 5 to 10 µg/ml, whereas Neo control cells showed only 3-4-fold enhancement. In addition, both alpha 5 transfectants showed ~5-fold increases of binding at FN concentrations ranging from 0 to 2.5 µg/ml, relative to Neo control cells. The specificity of cell adhesion was shown by blocking the attachment to FN with an anti-alpha 5 subunit antibody (Fig. 3B), thus indicating that enhanced cell attachment to FN was due to increased cell surface alpha 5beta 1expression.


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Fig. 1.   Integrin alpha 5 mRNA levels in alpha 5 sense-transfected MCF-7 LDC4. MCF-7 limiting dilution clone 4 was stably transfected with the integrin alpha 5 sense expression vector (alpha 5S.) and selected with Geneticin. Total RNA from two positive clones (cl.5 and cl.15) was isolated and compared with untransfected (MCF-7 LDC4) and Neo-transfected (Neo pool) control cells for alpha 5 mRNA levels by RNase protection assay. Actin mRNA levels were used to normalize loading of the samples. M.W. std., molecular weight standard.


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Fig. 2.   Cell surface integrin alpha 5 and beta 1 protein levels in alpha 5 sense-transfected MCF-7 LDC4. Cell surface proteins from MCF-7 LDC4 Neo pool, alpha 5 sense clone 5, and alpha 5 sense clone 15 were labeled with biotin. The biotinylated integrin from equal cell numbers of each cell type was immunoprecipitated with an anti-human alpha 5 subunit antibody and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting.


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Fig. 3.   Adhesion of MCF-7 LDC4 integrin alpha 5 sense transfectants to FN. A, MCF-7 LDC4, Neo, alpha 5S.5, and alpha 5S.15 cells were incubated in 96-well plates coated with different concentrations of FN for 15 min. Unattached cells were washed away, and the number of adherent cells determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. B, specificity of adhesion to FN by alpha 5beta 1was ascertained by preincubating cells either with (+) or without (-) alpha 5beta 1monocloned antibody (1:100) dilution for 30 min at 4 °C prior to adhesion on FN (10 µg/ml). C, DNA synthesis was measured by [3H]thymidine incorporation in cells plated for growth curves. Cells were seeded into 24-well plates (1.5 × 104 cells/well), and DNA incorporation determined at the indicated times as described under "Materials and Methods." Each point is the mean ± S.E. of nine determinations from three separate experiments. Ab, antibody.

Ectopic alpha 5 expression leading to enhanced alpha 5beta 1 ligation blocked DNA synthesis, whereas disruption of ligation led to increased DNA synthesis in other model systems (13, 14). If our hypothesis that enhanced alpha 5beta 1 function leads to induction of autocrine negative TGF-beta activity was correct, ectopic expression of alpha 5 should result in reduced cell proliferation. Growth curves of alpha 5-transfected clones showed more than 50% inhibition of proliferation relative to wild type cells (Fig. 3C).

Expression of RII in alpha 5 Transfectants-- Previously, we showed that MCF-7 cells were insensitive to TGF-beta 1 because they expressed nearly undetectable levels of RII (21). Therefore, if alpha 5beta 1-mediated growth inhibition was associated with autocrine negative TGF-beta activity, RII expression must be restored. To test this hypothesis, we examined RII mRNA levels in alpha 5 transfectants by RNase protection assay (Fig. 4). High steady state RII mRNA levels were induced in alpha 5-transfected cells compared with Neo control cells when the cells were cultured on poly-L-lysine. This was probably due to enhanced production of endogenous FN, as described below. Growth on exogenous FN further increased RII mRNA levels to 2.5-fold (as determined by densitometry) in alpha 5-transfected cells, whereas it had no effect in Neo control cells. RI mRNA levels remained the same in alpha 5-transfected and Neo-transfected cells (data not shown). TGF-beta receptor cross-linking with 125I-TGF-beta 1 showed little binding to RII of control cells, whereas substantially higher binding of TGF-beta 1 was observed in the alpha 5 transfectants (Fig. 5). Interestingly, binding of TGF-beta 1 to RI was fairly prominent in control cells, as was binding to RIII. This has been observed in previous studies of this cell line (21, 22), as well as in other cell lines (38). Transfection of alpha 5 subunit resulted in a substantial increase in RI binding. This is in accordance with the increased RII expression because this receptor is thought to be responsible for RI recruitment to the cell surface (35).


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Fig. 4.   TGF-beta RII mRNA levels in MCF-7 LDC4 alpha 5 sense transfectants. Total RNA (20µg) harvested from MCF-7 LDC4 Neo pool, alpha 5S.5 and alpha 5S.15 cells, plated either on poly-L-lysine or FN at the indicated time points was used in RNase protection assays with an antisense RII probe. Actin was used to normalize RNA loading.


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Fig. 5.   Cell surface expression of TGF-beta receptors in MCF-7 LDC4 alpha 5 sense transfectants. 125I-Labeled TGF-beta was cross-linked to its cell surface receptor on the control MCF7 LDC4 Neo pool, alpha 5S.5, and alpha 5S.15 cells as described under "Materials and Methods."

Induction of Autocrine Negative TGF-beta Activity by alpha 5beta 1 Transfection-- To determine whether autocrine negative TGF-beta activity was induced as a result of alpha 5beta 1 expression, TGF-beta 1 neutralizing antibody blockade of endogenously produced TGF-beta 1 was employed, utilizing a previously described clonal assay (25, 26). Cells expressing autocrine negative TGF-beta activity will show enhanced colony formation and growth as a result of the antibody-mediated neutralization of TGF-beta 1, whereas those cells that do not express autocrine negative activity will be unaffected by antibody treatment. Cell growth and colony formation are determined by crystal violet staining. Standard curves were performed measuring crystal violet levels with known numbers of MCF-7 cells to ensure that the assay was performed over a linear range of MCF-7 cells. As shown in Fig. 6, TGF-beta 1 neutralizing antibody stimulated colony formation in alpha 5 transfectants, whereas it had no effect on Neo control cells (Fig. 6A). The percentage of stimulation by the antibody was calculated and plotted in Fig. 6B. TGF-beta 1 neutralizing antibody treatment resulted in 25% stimulation for clone 5 and 55% for clone 15. 


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Fig. 6.   Effect of TGF-beta 1 neutralizing antibody on the plating efficiency of MCF-7 LDC4 integrin alpha 5 sense transfectants. A, cells (400) were plated on FN-coated (10 µg/ml) 24-well plates either in the presence or absence of neutralizing TGF-beta antibody (1:100). Cell growth was quantitated by crystal violet staining 8 days later. B, after staining, cell colonies were dissolved in 1% Triton X-100, and absorbance measurements at 595 nm and cell number are expressed as the percentage of stimulation. Each value is the mean ± S.E. of four replicates.

To confirm the enhancement of autocrine TGF-beta activity after alpha 5 expression, we compared the activity of a TGF-beta -responsive promoter in control cells with that in alpha 5-transfected cells. The p3TP-Lux promoter contains a TGF-beta response element from the plasminogen activator inhibitor gene inserted upstream of the luciferase reporter gene and has been extensively utilized as a marker for TGF-beta responsiveness (23, 35). Therefore, it would be expected that induction of autocrine TGF-beta activity would result in enhanced expression of the p3TP-Lux construct in alpha 5 transfectants relative to Neo control cells. Fig. 7 shows that both alpha 5 transfectants expressed 5-10-fold higher levels of luciferase activity than Neo control. If increased luciferase activity of p3TP-Lux construct was due to autocrine TGF-beta , neutralizing antibody treatment would reduce expression of the reporter construct. As shown in Fig. 7A, TGF-beta 1 neutralizing antibody treatment resulted in a substantial decrease in luciferase reporter activity in both alpha 5-transfected clones, whereas it had no effect on Neo control cells. This experiment was repeated four times, and similar results were obtained. Similarly, an alpha 5 neutralizing antibody was used to show that disruption of alpha 5beta 1 ligation to FN resulted in approximately a 60% reduction in the enhanced luciferase activity associated with the alpha 5 clone 15 transfectant (Fig. 7B), thus confirming that the enhanced endogenous TGF-beta activity was dependent on the ectopic alpha 5 expression.


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Fig. 7.   Effect of TGF-beta 1 neutralizing antibody on the transcription of a TGF-beta -responsive promoter. A, 3TP-Lux and pSVbeta -galactosidase plasmids were transiently transfected into Neo, alpha 5S.5, and alpha 5S5.15 cells by electroporation, and luciferase activity was measured 48 h after transfection in cells treated with IgG or TGF-beta neutralizing antibody (10µg/ml). Values are means of duplicate samples. B, 3TP-Lux transfectants were treated with either alpha 5 antibody or TGF-beta neutralizing antibody (10 µg/ml).

We previously showed that autocrine TGF-beta controlled steady state levels of FN in both native and RII-transfected cells (25, 32). Consequently, induction of autocrine TGF-beta should be associated with increased endogenous FN expression by alpha 5 transfectants. The alpha 5-transfected cells showed a 3-fold increase (as determined by densitometry) in FN mRNA levels compared with Neo control cells (Fig. 8A). FN mRNA levels were further increased in alpha 5-transfected cells when the cells were plated on FN, whereas the levels in Neo control cells still remained the same. The enhanced FN expression was due to autocrine TGF-beta as shown by the ability of TGF-beta neutralizing antibody treatment to repress FN expression in alpha 5 transfectant cells to a level comparable to that of NEO controls (Fig. 8B).


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Fig. 8.   Fibronectin mRNA levels in MCF-7 LDC4 alpha 5 sense transfectants. A, RNase protection assay was performed to determine FN mRNA levels in Neo and alpha 5S.15 cells grown either on poly-L-lysine or FN-coated plates for 2 days. Actin mRNA was used to normalize samples. B, Cells were plated on poly-L-lysine and treated with IgG (-) or TGF-beta neutralizing antibody (Ab) (+), and FN mRNA levels were measured, as described above.

Growth Inhibitory Effects of TGF-beta 1 on alpha 5 Transfectants-- Induction of autocrine TGF-beta activity suggested that response to exogenous TGF-beta effects should also result from alpha 5 transfection. The MCF-7 LDC4 Neo pool was insensitive to TGF-beta 1 in the absence or presence of exogenous FN (Fig. 9). The alpha 5 transfectants showed reduced basal proliferation relative to NEO controls as indicated above in Fig. 3C. DNA synthesis in the alpha 5 transfectants was further inhibited by TGF-beta 1 in a dose-dependent manner (Fig. 9). When the two alpha 5 transfectants were plated in 24-well culture plates coated with exogenous FN (10 µg/ml), increased sensitivity to TGF-beta 1 was demonstrated. Increased sensitivity on FN was likely due to the increased RII expression when cells were grown on FN as demonstrated in Fig. 4, above.


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Fig. 9.   Growth inhibition of integrin alpha 5 sense transfectants by TGF-beta 1. MCF-7 LDC4 Neo pool and the two alpha 5 sense transfectants were plated in 24-well plates coated with or without FN (10 µg/ml) at 1.5 × 104 cells/well in the presence of various concentrations of TGF-beta 1. DNA synthesis was assayed 4 days later by measuring [3H]thymidine incorporation. Thymidine incorporation in the presence of TGF-beta 1 was calculated as percentage of the thymidine incorporation in the absence of TGF-beta 1 for each clone. Each point is the mean ± S.E. of nine determinations from three separate experiments.

Effect of Integrin Expression on Tumorigenicity-- To assess the effect of alpha 5 expression on the malignant properties of MCF-7 cells, we inoculated Neo control and alpha 5-transfected clone 15 into ovariectomized, estrogen-supplemented nude mice as described previously (21). The size of xenografts formed was monitored with time (Fig. 10). Initially, MCF-7 LD 4 Neo pool and alpha 5 clone 15 formed similar sized xenografts (< 200 mm3) until day 8 after inoculation. After day 8, growth was delayed in alpha 5 clone 15 compared with Neo control. At day 28, Neo controls formed ~2.2-fold larger xenografts than alpha 5 clone 15. This result indicates that integrin alpha 5beta 1 expression in MCF-7 cells can partially reverse the malignant properties of the cell line.


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Fig. 10.   Xenograft growth of MCF-7 LDC4 Neo and integrin alpha 5 sense clone 15. Exponentially growing (1 × 107 cells) MCF-7 LDC4 Neo pool and alpha 5 sense clone 15 (cl15) were subcutaneously inoculated into ovariectomized athymic nude mice supplemented with 17beta -estradiol. Tumors were measured externally on the indicated days in two dimensions using a caliper. Xenograft volume was determined from the equation V = (L × W2) × 0.5, where L is the length and W is the width of the tumor. Each point represents the mean ± S.E. of 10 xenografts.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A number of studies have indicated that loss or reduced expression of integrin receptors results in abnormal cell growth (10, 11, 12). To test the hypothesis that alpha 5beta 1 ligation has tumor suppressor effects mediated through autocrine TGF-beta , we restored integrin alpha 5 expression in MCF-7 cells. The MCF-7 cell line provided a good model system for this study in that it expressed low amounts of alpha 5 integrin and was insensitive to the growth inhibitory effects of TGF-beta 1 due to repression of RII expression (21-23). Integrin alpha 5 transfectants expressed similar levels of cell surface integrin as Hs578T cells, another breast cancer cell line that was sensitive to growth inhibitory effects of TGF-beta 1.2 The alpha 5 transfection resulted in an increase in expression of RII, which was accompanied by increased autocrine TGF-beta activity as assessed by 1) enhanced clonality following TGF-beta neutralizing antibody treatment; 2) decreased endogenous activity of a TGF-beta -sensitive reporter system in response to TGF-beta neutralizing antibody treatment with either TGF-beta 1 or alpha 5 antibodies; and 3) stimulation of FN expression, which was reversed by TGF-beta neutralizing antibody treatment. Up-regulation of RII expression was also reflected by increased sensitivity to inhibition by treatment with exogenous TGF-beta .

Previously, we showed that blockade of FN/alpha 5beta 1 ligation by antibodies against FN or integrin alpha 5 subunit stimulated DNA synthesis in cancer cell lines with moderate to high alpha 5beta 1 cell surface expression (13, 14). These results are consistent with a previously described model that suggests that moderate adhesion to a loosely organized extracellular matrix facilitated both migration and growth, but strong adhesion to a fully organized extracellular matrix suppressed proliferation and contributed to inhibition of growth (4). Thus, the low level of expression of integrin alpha 5beta 1 on the cell surface of wild type MCF-7 cells could contribute to weak adhesion and hence abnormal growth in MCF-7 cells. Ectopic alpha 5 expression leads to results consistent with a model suggesting that higher alpha 5beta 1 surface expression allows for greater adhesion due not only to alpha 5, but to greater endogenous FN expression as well. Inhibition of proliferation is either due to or augmented by the generation of autocrine TGF-beta activity. Exogenous FN coating allows for even stronger adhesion and further enhancement of autocrine TGF-beta activity. Most importantly, the results indicate that alpha 5beta 1 ligation and autocrine TGF-beta interact in a reciprocal manner that is self-sustaining for both autocrine negative activity and cell-extracellular matrix interactions. Moreover, this interaction is tumor-suppressive. This model may well apply to other systems given that autocrine TGF-beta signaling and alpha 5beta 1 ligation have both been individually associated with tumor suppression in various model systems. Autocrine TGF-beta has been shown to control steady state alpha 5 expression (32) in model systems that also show tumor-suppressive TGF-beta function (28).

Our results suggest that alpha 5beta 1 may have a negative growth regulatory role in some cancer cells and normal cells through modulation of TGF-beta sensitivity. Apparently, signal transduction mechanisms for activating the TGF-beta pathway are essentially intact in MCF-7 cells because restoration of alpha 5beta 1 expression leads to autocrine as well as exogneous TGF-beta inhibitory responses. However, other types of cancer cells may be resistant to this mode of regulation despite alpha 5beta 1 expression because of perturbations of the TGF-beta pathway resulting from malignant transformation. For example, HCT116 colon carcinoma cells express high levels of integrins that mediate adhesion to FN, but this cell line still exhibits a highly malignant phenotype due to a mutated RII gene (25). It is also possible that downstream signaling messengers encoded by oncogenes or tumor suppressor genes that participate in either a primary or secondary manner in signal transduction are abnormally modulated in some cell types.

An interesting aspect of this study was the enhancement of RII expression and TGF-beta function when alpha 5 transfectants were plated on FN-coated plates. These results, along with the demonstration that alpha 5 antibody treatment blocks autocrine TGF-beta activity, show that alpha 5beta 1 ligation to FN is critical to the generation of RII expression and hence TGF-beta -mediated signal transduction. Enhanced basal expression of FN in MCF-7 alpha 5 transfectants that were not grown on FN-coated plates was also observed. We postulate that increased steady state expression of FN after alpha 5 transfection allowed for alpha 5beta 1 ligation, which was critical to the basal RII expression and TGF-beta sensitivity associated with transfectant cells that were not exposed to FN-coated plates.

In a previous study, we reported that disruption of alpha 5beta 1/FN ligation resulted in stimulation of DNA synthesis (14). DNA synthesis was associated with up-regulated CDK2 activity without alterations of CDK inhibitors. DNA synthesis was also found to be dependent upon extracellular receptor kinase 1 and 2 activation. Thus, this previous study indicated alpha 5beta 1 ligation to FN had a repressive effect on cell cycle progression through repression of cyclin A and CDK2 expression. Exogenous treatment with TGF-beta has been shown to down-regulate cyclin A (39). Interestingly, TGF-beta has also been reported to repress ERK1/2 activation in some types of cells (40). This suggests that DNA synthesis resulting from disruption of alpha 5beta 1 ligation may be a reflection of the disruption of integrin related autocrine TGF-beta activity resulting in up-regulation of ERK activation and subsequent promotion of cell cycle transit.

There is evidence that integrins transduce signals cooperatively with other growth factor systems in the regulation of cell proliferation. The proliferative response of murine mammary carcinoma cells to platelet-derived growth factor-BB and basic fibroblast growth factor is dependent on the extracellular matrix environment, indicating that modification of extracellular matrix and/or surface integrin receptors may regulate responsiveness to these growth factors (41). Reciprocal enhancement of alpha 5beta 1-mediated adhesion by insulin and of insulin-mediated signal transduction by alpha 5beta 1 have recently been reported (42). However, alpha 5beta 1 ligation does not appear to modulate expression of insulin receptor in this system. Our results indicate that the TGF-beta signaling pathway can be rescued by re-expression of integrin in MCF-7 cells. Ligation of integrins with their extracellular matrix ligands has been shown to regulate gene expression in a number of studies (43). However, because studies on the regulation of RII mRNA transcription and stability are limited, it is not yet clear how ligation of alpha 5beta 1 integrin to FN increases RII mRNA level. Nevertheless, the induction of RII and autocrine TGF-beta activity by alpha 5beta 1 ligation suggests that integrin-mediated signal transduction plays a cooperative role with TGF-beta signal transduction in tumor suppression. Moreover, the results indicating reciprocal positive control of autocrine TGF-beta and alpha 5beta 1 ligation suggest that TGF-beta signal transduction and alpha 5beta 1 integrin signal transduction participate in a mutually self-sustaining tumor-suppressive autocrine loop.

    ACKNOWLEDGEMENTS

We thank Dr. J. Massague of Memorial Sloan-Kettering Cancer Center for the TGF-beta -responsive construct and Jenny Zak and Lorraine Gilmore for the skillful preparation of the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants CA 38173, CA 50457, and CA 72001 (to M. G. B.); CA 63480, CA 75253, and CA 79683 (to L. Z. S.); and CA 68316 and CA 43703 (to J. K. V. 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.

§ Performed in partial fulfillment of the requirements for the Ph.D. degree Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699

Dagger Dagger To whom correspondence should be addressed: Depts. of Surgery and Biochemistry, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78284. Tel.: 210-567-4524; Fax: 210-567-4664.

2 D. Wang and M. Brattain, unpublished results.

    ABBREVIATIONS

The abbreviations used are: TGF-beta , transforming growth factor-beta ; RI, RII, and RIII, TGF-beta type I, type II, and type III receptor, respectively; alpha 5, integrin alpha 5; alpha 5S.5 and alpha 5S.15, alpha 5 sense clones 5 and 15; cl., clone; FN, fibronectin; FBS, fetal bovine serum.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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