Regulation of Bovine Bronchial Epithelial Cell Proliferation and Proto-oncogene Expression by Growth Factors Hajime Takizawa, Joe D. Beckmann, Masami Yoshida, Debra Romberger, and Stephen I. Rennard Pulmonary and Critical Care Medicine Section, Department of Internal Medicine, University of Nebraska Medical Center, Omaha, Nebraska

The proto-oncogenes are thought to play important roles in the regulation of cellular growth and differentiation. In order to evaluate the role of proto-oncogenes in the regulation of growth of bronchial epithelial cells, we studied steady-state levels offos, jun, and myc transcripts in response to fetal calf serum, bovine pituitary extract, and insulin. Extensively quiescent populations of bovine bronchial epithelial cells in growth factor-free medium were stimulated to divide by each of these three additives. We observed rapid but transient increases offos,jun, and myc expression in association with such growth stimulation. There were no changes in tubulin mRNA levels over the same time periods. Other "growth factors" (epidermal growth factor, hydrocortisone, epinephrine, triiodothyronine, and transferrin) were also studied and did not affect either cell growth or expression offos,jun, or myc. We further examined the effect oftransforming growth factor-S, (lOF-.Bl) on the above stimulatory effects. lOF-.B, consistently inhibited the growth induced by fetal calf serum, bovine pituitary extract, or insulin and, interestingly, reduced proto-oncogene myc mRNA level without altering that oi fo» andjun. In conclusion, proto-oncogenesfos, jun, and myc appear to play a role in the regulation of growth response in bovine bronchial epithelial cells. It is also possible that lOF-.Bl exerts its growth inhibitory effect, at least in part, through the processes that involve the regulation of proto-oncogene myc transcription in these cells.

There is increasing evidence that a variety of proto-oncogenes play an important role in the regulation of proliferation and differentiation of a number of cell types. Since it was reported that the proto-oncogene fos mRNA expression can be rapidly induced by growth factors in nontumor fibroblast cell lines (1, 2), data have been accumulating that suggest a crucial role of the proto-oncogenes in the proliferation and differentiation of mammalian cells (3, 4). In vivo studies have also suggested that changes of expression of proto-oncogenes occur during tissue regeneration (5, 6). Moreover, the protein products of some proto-oncogenes are structurally and functionally homologous to growth factors (e.g., sis for beta chain of platelet-derived growth factor) or growth factor receptor (e.g., erb B for epidermal growth factor [EGF] receptor) (7-10), further supporting the close relationship between the oncogenes and growth regulation. In fibroblasts, stimulation by growth factors such as (Received in originalform April 5, 1990 and in revised form May 10, 1991) Address correspondence to: Stephen I. Rennard, M.D., Pulmonary and Critical Care Medicine Section, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, NE 68198-2465. Abbreviations: cyclic adenosine monophosphate, cAMP; bovine pituitary extract, BPE; Dulbecco's modified Eagle's medium, DMEM; epidermal growth factor, EGF; fetal calf serum, FCS; Hanks' balanced salt solution, HBSS; interferon, IFN; minimal essential medium, MEM; sodium dodecyl sulfate, SDS; triiodothyronine, T3 ; tansforming growth factor-d. TGF-{3. Am. J. Respir. Cell Mol. BioI. Vol. 5. pp. 548-555, 1991

platelet-derived growth factor induces a rapid increase in expression of proto-oncogenes including c-fos and c-myc, as early responses to growth factors (1, 11). Products of both c-fos and c-myc are nuclear proteins that might be regulators of transcription (12). Moreover, recent reports suggest that products of c-fos and c-jun, a recently reported protooncogene (13, 14), are closely associated and act as a transcriptional activator complex (15, 16). Airway epithelial cells are known to respond to growth factors (17, 18). In order to better understand the role of proto-oncogenes in the regulation of the growth of these cells, we explored the expression of proto-oncogenes fos, myc, andjun in response to several growth factors in primary and passaged bovine bronchial epithelial cells. Insulin, bovine pituitary extract (BPE), and fetal calf serum (FCS) , additives that stimulate proliferation of bovine bronchial epithelial cells, all induced rapid, but transient, increases of mRNA levels for fos, jun, and myc proto-oncogenes. Transforming growth factor-.B (lOF-.B), which has been reported to inhibit the cell proliferation and to induce the terminal squamous differentiation in human bronchial (19) and rabbit tracheal epithelial cells (20), blocked the proliferative responses to insulin, BPE, and FCS. lOF-.Bl also reduced proto-oncogene myc expression in association with its growth inhibition in these cells. In contrast, expression of c-jun and c-fos was unaffected. Other factors that have been reported to stimulate growth of airway epithelial cells in other species (17, 21), including EGF, hydrocortisone, epi-

Takizawa, Beckmann, Yoshida et al.: TGF-,B and c-oncogene Expression in Bronchial Epithelial Cells

nephrine, triiodothyronine (T3) , and transferrin, were without effect on either cell proliferation or on oncogene expression in this cell culture system.

Materials and Methods Reagents LHC basal medium was purchased from Biofluids (Rockville, MD). Hanks' balanced salt solution (HBSS), RPMI 1640 medium, Dulbecco's modified Eagle's medium (DMEM), minimal essential medium (MEM), and HBSS containing 0.05% trypsin and 0.53 mM EDTA, streptomycin-penicillin, and fungizone were purchased from GIBCO (Chagrin Falls, OH). FCS was obtained from Biofluids and was heat-inactivated before use. Protease (bacterial type XIV), transferrin, insulin, EGF, and hydrocortisone were purchased from Sigma Chemical Co. (St. Louis, MO). BPE was prepared as previously reported from frozen bovine pituitaries (Pel Freez, Rockdale, AR) (21). 1GF-,BI (from porcine platelets) was obtained from R & D Systems (Minneapolis, MN) and dissolved in sterile, 4 mM HCI containing 1 mg/ml bovine serum albumin. Isolation of Bovine Bronchial Epithelial Cells Bovine bronchial epithelial cells were obtained by the method of Wu as reported previously (22, 23). Briefly, bovine lungs were obtained from a slaughterhouse just after killing. Bronchi were removed, cut into pieces, and incubated in sterile MEM supplemented with 0.1% protease, penicillin-streptomycin, and fungizone at 4° C overnight. The bronchi were removed, and the bronchial lumens were gently rinsed several times with MEM containing 10% FCS to detach the epithelial cells. The collected cells were washed once with 10% FCS-MEM, filtered through 250-j.tm sterile mesh (Tetko, Elmsford, NY), and washed twice with MEM. The total cell count was calculated by a standard hemacytometer, and viability of the cells was examined by trypan blue dye exclusion. Approximately 500 to 1,000 X 106 cells (> 80 to 98 % viability) were obtained from each lung. The cells were then washed with serum-free medium and resuspended in DMEM for use. Culture of Bovine Bronchial Epithelial Cells In Vitro In the present experiments, primary cells and the second passaged cells (see below) were used to assess the growth kinetics and mRNA expression. The primary bovine bronchial epithelial cells were plated at I x 106 cells/dish on 35-mm tissue culture plates (Coming, Corning, NY) and incubated at 37° C, 5 % CO 2 overnight. The plates were then rinsed with DMEM, and the medium was changed for the experimental purposes. The standard medium used in this report was LHC8e medium (21). This is LHC basal medium supplemented with bovine insulin (5 j.tg/ml), EGF (5 ng/ml), transferrin (10 j.tg/ml), epinephrine (5 j.tg/ml), T 3 (10 nM), hydrocortisone (0.2 j.tM), phosphoethanolamine/ethanolamine (5 j.tM), trace elements, calcium (0.11 mM), BPE (0.5 %), penicillin-streptomycin, and fungizone. The medium was changed every 3 days unless otherwise mentioned. To passage the bovine bronchial epithelial cells, the primary cells were plated on lOO-mm tissue culture plates

549

(Corning) that were precoated with type I collagen (Vitrogen 100; Collagen Corp., Palo Alto, CA). Before confluence, the cells were treated with trypsin-EGTA solution followed by the addition of soybean trypsin inhibitor to harvest the cells (23). After two washes with HBSS, the cells were plated onto Vitrogen-coated dishes with 1 vol LHC8e plus 1 vol RPMI 1640 medium. The second passaged cells were harvested and plated at 5 X 104 cells/Vitrogen-coated 35-mm dishes. To confirm that the cultured cells were of epithelial origin, immunohistochemical studies using the avidin-biotin complex (ABC) method were performed with anti-keratin antibody (MAK-6; Triton, Alameda, CA). These studies demonstrated that the cells were keratin-positive, indicating they were epithelial cells as reported previously (23). Furthermore, staining with the fibroblast specific anti-vimentin antibody (DAKO-VIMENTIN; DAKO, Santa Barbara, CA) did not reveal fibroblast contamination as reported previously (22). Quantification of the Cell Growth in Response to Growth Factors Assessed by Coulter Counter To assess the importance of each growth factor contained in LHC8e medium, the cells were cultured in LHC8e medium minus one of the following factors: BPE, insulin, EGF, transferrin, epinephrine, hydrocortisone, and T3 , as well as in LHC8e medium. After different periods of culture, the cell number was counted by Coulter Counter (Coulter Electronics, Hialeah, FL) as reported previously (23). Briefly, each plate was rinsed once with HBSS, HBSS containing 0.05% trypsin and EDTA was added, and the cells were detached from the plates. Each time after this procedure, it was confirmed by phase-contrast microscopy that all of the cells were detached from the plates. The cell count was determined twice for each of triplicate dishes. As a further step to assess the effect of each growth factor on proliferation and proto-oncogene induction, we used the following system. The bovine bronchial epithelial cells were cultured for 7 days in LHC8e medium, then the media were replaced with LHC8e medium omitting the growth factors as follows: transferrin, insulin, epinephrine, EGF, hydrocortisone, T3 , and BPE. After 48 h, the cells were pulsed with one of the growth factors included in LHC8e, or FCS. Preliminary studies of radiolabeled thymidine uptake into nuclei in this growth factor-free medium demonstrated that the percentage of the DNA-synthesizing cells decreased from 75.4 ± 2.5 % to 7.0 ± 1.2% by 48 h of incubation (23). This result indicated that the cells were extensively quiescent, thus allowing us to assess the effect of growth factors on proliferation and early induction of proto-oncogene expression. Cell number was determined at different times of culture by Coulter Counter (Coulter Electronics) as described. RNA Extraction from Cultured Bovine Bronchial Epithelial Cells The bovine bronchial epithelial cells were cultured in LHC8e medium for 7 days as described above, then the media were replaced by LHC8e omitting the growth factors. After 48 h, the cells were pulsed with a growth factor. After different time periods of incubation, total RNA was extracted

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

by the method of Chomczynski and Sacchi (24) and quantified by a spectrophotometer (DU-62 spectrophotometer; Beckman Instruments, Fullerton, CA) at 260 and 280 nm. RNA isolated from HFL-l fibroblasts (American Type Culture Collection, Rockville, MD) in culture was used as positive control for fos, myc, and jun expression. HFL-l fibroblasts were grown on the tissue culture plates with 10% FCS-DMEM until approximately 70% confluence, replaced with serum-free DMEM for 48 h, and then stimulated with the addition of 10% FCS. After different time periods of incubation, the total RNA was extracted by the method described above.

Preparation of DNA Probes DNA probes were as follows: v-fos probe isolated from FBI murine osteosarcoma virus inserted into Pst I site of pBR322 (No. 41040; American Type Culture Collection) originally reported by Curran and associates (25); human c-myc-l probe, an 8.2-kbp HindlII-EcoRI genomic human c-myc fragment originally reported by Land and eo-workers (26) was a kind gift from Dr. Mario Stevenson; v-jun probe inserted into EcoRI-BamHI site ofpBR322 originally reported by Vogt and colleagues (14, 15) was a kind gift from Dr. Timothy Bos; mouse (35 tubulin probe was a kind gift from Dr. Don Cleveland. DNA probes were 32P-labeled by the oligolabeling method reported by Feinberg and Vogelstein (27). Northern Blot Analysis RNA was electrophoresed under denaturing conditions in 0.8% agarose gels containing 1 M formaldehyde (28). Transfer of RNA to nitrocellulose membranes (pore size BA85 0.45 j.tm, S & S NC; Schleicher & Schuell, Keene, NH) was accomplished by capillary blotting (28). Membranes were then air-dried and baked in a vacuum oven for 2 h at 80 0 C. Blots were prehybridized with 5 X SSPE, 1% sodium dodecyl sulfate (SDS), and 5x Denhardt's solution (50x = 1 g/ 100 ml each of Ficoll, polyvinylpyrrolidone, and bovine serum albumin) for more than 20 min at 42 0 C, and then placed in plastic bags (Seal-a-meal). A total of 10 ml of hybridization fluid (5 % dextran sulfate, 5 x SSPE, 50% deionized formamide, 2 x Denhardt's solution, and 0.5 % SDS) containing radiolabeled DNA probe was added and incubated at 42 ° C overnight. Blots were then washed under appropriate conditions, which allowed enough stringency to have specific signals. In most cases, the final wash was at 42 0 C in O.1x SSPE, 0.1% SDS. Autoradiography was at -80 0 C using one intensifying screen and X-Omat AR film (Eastman Kodak, Rochester, NY). Quantification of hybridization signals was performed by densitometric scanning with a OS 300 transmitter/reflectance scanning densitometer (Hoefer Scientific Instruments, San Francisco, CA). RNA integrity and equivalency of loading were routinely assessed by visualization of the 18S and 28S ribosomal subunits in the gels by ethidium bromide fluorescence. As a constitutive expression marker, we also studied tubulin mRNA transcripts in the same blots that had been hybridized with other DNA probes. Treatment of the Bronchial Epithelial Cells by TGF-,6 To evaluate the effect of TGF-,61 on the proliferation of bronchial epithelial cells, TGF-(31 was added to the cell cul-

ture dishes as described below. Because preliminary doseresponse studies demonstrated that 200 pM of TGF-(31 inhibited extensively the growth of the cells in LHC8e medium (after 7 days in culture: % inhibition = [cell count in LHC8e medium - cell count in LHC8e with TGF-(3d/cell count in LHC8e medium = 69 ± 3.6% in three different experiments), we used this dose in further experiments. After the cells were cultured in LHC8e medium for 7 days, the medium was replaced by LHC8e omitting growth factors; then one of the growth factors listed above was added as described. A total of 200 pM of TGF-(3 was added to the dishes either simultaneously or 24 h before the addition of the growth factors. For evaluation of the effect on cell growth, the cell number was counted after different periods of culture as described above. For the evaluation of the induction of protooncogenes, the total RNA was extracted as described after different times of incubation. Statistics Student's t test was used to compare the data of growth and densitometric measurement of mRNA levels.

Results Effect of the Growth Factors on the Proliferation of Bovine Bronchial Epithelial Cells When the cells were cultured in LHC8e medium minus one of the supplemented growth factors, a significant decrease in cell number was observed only if either BPE or insulin were excluded (data not shown). Therefore, we focused subsequent experiments on these two media additives. The bovine bronchial epithelial cells in LHC8e medium showed exponential growth as assessed by counting the cell number at different periods of culture (Figure 1, days 1 through 7) (doubling time: mean, 2.76 days). After replacement of the medium with LHC8e omitting all seven of the growth factor supplements, the increase in cell number be-

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Figure 1. Effect of serum and growth factors on the proliferation of bronchial epithelial cells after 48 h of incubation in growth factor-free medium. The bovine bronchial epithelial cells were grown in LHC8e medium for 7 days and then incubated in LHC8e omitting the growth factors for 48 h (days 7 through 9). The cells stimulated with fetal calf serum (FCS), bovine pituitary extract (BPE) , or insulin (Ins) on day 9 (arrow) showed a significant increase in cell number after 4 days (days 9 through 13). The data were shown in mean ± SEM from three different experiments.

Takizawa, Beckmann, Yoshida et al.: TGF-13 and c-oncogene Expression in Bronchial Epithelial Cells

Figure 2. Induction of c-oncoc gene mRNA levels in the bronchial epithelial cells after the C) stimulation of insulin (5 p.g/ml). Lone::> e::>~ e::> ...... ("i")e.ccn...... A total of 10 p.g RNA from each sample was electrophoresed and immobilized on nitrocellulose 28S~ 28S~ membrane as described. Northern blot hybridization analyses were performed using DNA probes for c-fos (A), c-jun (B), and c-myc (C). The blots were exposed for 3 days (A), 5 days (B), and 7 days (C). Small arrow indicates specific signals for c-fos (2.2 kb), c-jun (2.7 kb), and c-myc (2.4 kb). RNA loads were con- tubu1.in tubulin trolled by subsequent hybridization with a tubulin cDNA probe.

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came minimal (Figure 1, days 7 through 9) in accordance with the decrease of the cells positive for DNA synthesis (data not shown). After this, the cultures were pulsed with one of the growth factors. Statistically significant increases of cell counts occurred after stimulation by FCS (20 %), BPE (0.5 %), or insulin (5 JLg/ml) (P < 0.001), but not with medium alone (Figure 1) or by the other growth factors tested. Expressed as percentage of medium only, the change in cell number after 4 days was EGF (5 ng/ml) , 101 ± 2.3%; hydrocortisone (0.2 JLM), 108 ± 2.1%; epinephrine (5 JLg/ml) , 110 ± 3.5%; T 3 (10 nM), 103 ± 1.9%; transferrin (10 JLg/ml) , 105 ± 1.8%, P > 0.1 as compared with the cells with no additives. Thus, the feeding procedure itself had no significant effect on the cell proliferation. For further experiments with FCS, BPE, and insulin, dosages of 20%, 0.5 %, and 5 JLg/ml, respectively, were used because earlier studies suggested these dosages were optimal for the cell proliferation when assessed by cell counting. Effect of the Growth Factors on the mRNA Levels of Proto-oncogenes fos, jun, and mye After the bovine bronchial epithelial cells were cultured for 7 days in LHC8e medium, the medium was replaced with LHC8e omitting growth factors for 48 h. The cells were then stimulated by one of the additives, and the total RNA was isolated and examined for c-fos, jun, and mye expression by Northern blot analysis. Because the proto-oncogene expression is known to be observed rapidly but transiently after the stimulation, studies were performed at different time intervals. Stimulation of the cells with 20 % FCS, 0.5 % BPE, and insulin (5 JLg/ml) resulted in a rapid but transient increase of c-fos expression (Figures 2A and 3A and Table 1). Maximal level of gene expression was observed after 30 min and had returned to baseline by 2 h. FCS, BPE, and insulin also induced a transient increase in the 2.7-kb c-jun mRNA levels (15) (maximal level was after 30 min) and c-mye mRNA levels (maximal level was after 2 h) (Figures 2 and 3 and Table 1). The other growth factors (EGF, hydrocortisone, epinephrine, T3 , and transferrin) and medium alone did not induce a statistically significant increase in these proto-oncogene mRNA levels (Table 1). Thus, the feeding procedure itself did not appear to alter the proto-oncogene mRNA levels.

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Figure 3. Time course of the proto-oncogene mRNA levels after the stimulation of FCS (20%) (e), BPE (0.5%) (.A.), and insulin (5 p.g/ml) (T). PanelA: c-fosipanel B: c-jun;panel C: c-myc. Magnitude of induction was expressed as percentage of induction when the densitometric value without stimulation was calculated as 100% (vertical axis). Horizontal axis shows time in minutes. Representative results of each group were shown. The films were exposed for 5 days (A and B) and 7 days (C).

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

TABLE 1

Induction of c-fos, c-jun, and c-myc mRNA in bovine bronchial epithelial cells by the stimulation with growth factors Relative Signal Intensity (%)* c-fos

c-jun

c-myc

100 625 ± 44.7t 334 ± 11It 230 ± 16.9t 139 ± 38.2:1: 92.9 ± 19.1:1: 93.3 ± 10.0:1: 110.5 ± 10.9:1: 139.4 ± 40.3:1:

100 502 ± 31.2t 382 ± 4.35t 338 ± 11.5t 109 ± 22.0:1: 99.1 ± 11.2:1: 121.2 ± 10.0:1: 100.5 ± 2.3:1: 99.8 ± 3.7:1:

100 272 ± 48.3t 241 ± 19.2t 221 ± 11.3t 91.9 ± 33.1:1: 99.4 ± 1.83:1: 112 ± 10.9:1: 112 ± 10.8:1: 107 ± 9.33:1:

Growth Factor

None Fetal calf serum (20%) Bovine pituitary extract (0.5 %) Insulin (5 tLg/ml) . Epidermal growth factor (5 ng/ml) Hydrocortisone Epinephrine Triiodothyronine Transferrin

* Ratio of densitometric intensity to the baseline value x 100 was listed at the peak level for each proto-oncogene expression: c-fos 30 min, c-jun 30 min, and c-myc 2 h after the stimulation. The data: are mean ± SEM from three representative results. < 0.01 as compared with controls. Not significant compared with controls.

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Inhibitory Effect of lGF-{31 on the Growth Factor-induced Proliferation of Bovine Bronchial Epithelial Cells

200

As shown in Figure 4, TGF-t3 inhibited the FCS-, BPE-, and insulin-induced proliferation by 89 ± 2.1%,84 ± 1.3%, and 86 ± 3.1%, respectively, when added simultaneously with as well as 24 h before the addition of the stimulation. Similar results were obtained when the second passaged cells were used (data not shown).

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Effect of TGF-131 on Proto-oncogene e-fos, c-jun, and c-myc Expression TGF-t3 itself (200 pM) showed no statistically significant induction of the expression of c-fos, c-jun, or c-myc (Table 2). As shown in Figure 5 and Table 2, lGF-13 did not alter the kinetics of c-fos expression. This finding suggests that TGF-t3 does not interfere with the signal pathway of c-fos gene activation after stimulation by growth factor. Similar results were obtained when the second passaged cells were used (data not shown). Similarly, TGF-t3 did not change the magnitude of c-jun mRNA levels as shown in Figure 6 and Table 2. In contrast, when TGF-t3J was added simultaneously

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Figure 4. Growth-inhibitory effect of transforming growth factor{3\ (TGF-{31) on the growth factor-induced proliferation of bovine bronchial epithelial cells. The cells were grown in LHC8e medium for 7 days, then incubated in LHC8e omitting growth factors. After 48 h (days 7 to 9), the cells were stimulated with FCS, BPE, or insulin (Ins). TGF-{31 (200 pM) was added to the medium simultaneously (A) or 24 h before addition of the growth factor (B). After 96 h (days 9 to 13), the cell numbers were counted in triplicate from each group and expressed in percentage when the count of day 9 was calculated as 100% (vertical axis). The data are shown as mean ± SEM. TGF-{31 significantly inhibited the FCS-, BPE-, and insulin-induced cell proliferation (P < 0.01).

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Figure 5. Effect of TGF-{31 on the induction of c-fos after stimulation by growth factors. TGF-{31 did not significantly alter the c-fos mRNA levels in bovine bronchial epithelial cells when added to the medium 24 h before the insulin stimulation. The results were similar when TGF-{3J was used simultaneously with growth stimulation (see Table 2). The blot was exposed for 5 days. Large arrows show 288 (top) and 18S (bottom) ribosomal RNA locations. Small arrow indicates c-fos signal. Subsequent hybridization with a tubulin cDNA indicates RNA load equivalency.

Takizawa, Beckmann, Yoshida et al.: TGF-11 and c-oncogene Expression in Bronchial Epithelial Cells

553

TABLE 2

Effect of transforming growth factor-S, (TGF-fiJ) on the induction of c-fos, c-jun, and c-myc mRNA levels in response to the growth factors Relative Signal Intensity (%)* Treatment

c-fos

None TGF-{31 alone Fetal calf serum (FCS) FCS + TGF-{3}, simultaneously FCS + TGF-{3}, 24 h before Insulin Insulin + TGF-{3}, simultaneously Insulin + TGF-{3}, 24 h before

105 542 556 601 267 233 258

* Ratio of the densitometric intensity to the baseline value c-myc 2 h after the stimulation. Results are mean t p < 0.05 (Student's t test).

c-jun

l*

100 ± 22.3 + 110 ] ± 53.5 ± 52.0 ± 11.9] ± 32.8 ± 39.5

]1 ]1

100 111 ± 3.55 512 ± 31.2 Not tested 541 ± 33.3 338 ± 41.2 Not tested 305 ± 51.2

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]*

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100 123 ± 31.5 291 ± 13.5] 153 ± 32.5 134 ± 51.5 218 ± 33.3] 108 ± 10.1 99.2 ± 18.3

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with 20% FCS and 5 ILg/ml insulin, the increase in c-myc mRNA levels was reduced as shown in Figure 7 and Table 2 (47 % and 54 %, respectively). This effect was also observed when TGF-111 was added 24 h before the stimulation by FCS and insulin (Table 2). Similar results were also obtained when the second passaged cells were used (data not shown). As a control, the effect of TGF-111 on tubulin mRNA was also studied. Tubulin mRNA levels were unchanged after the addition of each growth factor tested including TGF-I1. This result suggested that tubulin mRNA was constitutively transcribed and that this signal can be used as an internal control for densitometric normalization.

Discussion In the present report, we have demonstrated that complex and defined growth factors including serum, BPE, and insulin induce the rapid but transient increase in c-fos, jun, and myc mRNA levels in bovine bronchial epithelial cells. These three factors consistently induced growth when added to growth factor-free medium, whereas other growth factors tested in the current system showed no apparent stimulation

o 30 min

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Figure 6. Effect of TGF-.81 on the induction of c-jun after stimulation by growth factors. TGF-.81 did not change the c-jun mRNA levels when added to the medium 24 h before the insulin stimulation. The blot was exposed for 7 days. Large arrows show 28S (top) and 18S (bottom) ribosomal RNA locations. Small arrow in" dicates c-jun signal. Equivalent RNA loads are indicated by the tubulin transcript signals.

TGFb +-

tUbulin

of growth nor induction of proto-oncogenes. Therefore, it is likely that these proto-oncogene inductions were associated with their growth-stimulatory activities. Moreover, we also found that TGF-{1l, which inhibited the growth of these cells stimulated with serum, BPE, or insulin, reduced the induction of c-myc mRNA levels but did not affect c-jun or c-fos. TGF-{11 had no effect on any of the proto-oncogene mRNA levels tested when added alone. These findings might suggest that c-myc expression may play a crucial role in the regulation of growth of bovine bronchial epithelial cells, and that TGF-{11 possibly exerts its growth inhibitory effect through the pathway that involves the processes of c-myc regulation within the nuclei. In comparison with human bronchial epithelial cells prepared from explant outgrowths, bovine bronchial epithelial cells prepared by enzymatic digestion differed in several aspects of their proliferation and differentiation. EGF had no effect in our present system, but had a growth-stimulatory effect on human bronchial epithelial cells (17). More interestingly, FCS stimulated the proliferation of the bovine cells, whereas it inhibited the growth of human cells and induced squamous differentiation (29). Purified TGF-{11 does, however, inhibit growth and promote a squamous phenotype (1. D. Beckmann et al., in press). Although the reasons for these differences remains unknown, one possibility is that

r--,

-+-+

Figure 7. Effect of TGF-{31 on the induction of c-myc after stimulation by growth factors. TGF-.81 reduced the magnitude of c-myc mRNA levels when added 24 h before the stimulation. The results were similar when TGF-.81 was used simultaneously with growth stimulation (Table 2). The blot was exposed for 7 days. Large arrows show 28S (top) and 18S (bottom) ribosomal RNA locations. Small arrow indicates c-myc signal. RNA loads were tested by hybridization with a tubulin cDNA.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 5 1991

the "net" effect of serum can be dependent on the potency and receptor-binding affinity of each growth factor included in serum. Because the regulation of cell growth depends on the balance of a variety of growth and hormones, and because . these factors can interact with each other, species differences or differences in experimental conditions might cause different reactions to certain growth factors (30). There have been accumulating data that suggest that certain proto-oncogenes play important roles in the regulation of growth and differentiation in various cell types (1-16). Among these proto-oncogenes, c-fos and c-myc are both induced rapidly but transiently after the exposure of the cells to growth factors. These proto-oncogenes, therefore, are considered to be closely related to the mechanisms of growth regulation (12). Recent studies (31-33) have also shown that transcription of c-jun is induced by growth factors, suggesting a role in the regulation of cell growth. Our present studies showing that some growth factors (insulin, FCS, and BPE) induced growth and c-fos, c-jun, and c-myc expression, but others showed neither growth nor proto-oncogene induction, suggest that the induction of these proto-oncogenes may play a role in the regulation of growth in bovine bronchial epithelial cells. Airway epithelial cells are known to respond to a number of identified growth factors as well. as uncharacterized growth-stimulatory activities (17, 18, 22, 23). Although the mechanisms that lead from growth factor binding to proliferation are not fully elucidated, activities of protein kinase C and increase in intracellular cyclic adenosine monophosphate (cAMP) are thought to play a role. In this regard, activation of protein kinase C and increased levels of cAMP (34-36) have been reported to lead to increases in c-myc and c-fos expression (37, 38). Interestingly, other agents that alter bronchial epithelial cell protein kinase C or cAMP levels such as 12-0-tetradecanoylphorbol-13-acetate (TPA) (39) or dibutyryl cAMP (40) have also been shown to affect growth and differentiation of these cells. Taken together, these results suggest that a variety of growth stimuli may lead to similar activation of proto-oncogenes in bronchial epithelial cells.. Among the growth factors that affect the proliferation of airway epithelial cells, TGF-~ has attracted considerable attention because it is known to be a potent growth inhibitor for epithelial cells (19). In human bronchial and rabbit tracheal epithelial cells, it also induces squamous differentiation (19, 20). In our present system, TGF-~I consistently inhibited the growth-stimulatory effect of FCS, insulin, and BPE on bovine bronchial epithelial cells in vitro. Moreover, TGF-~l also induced squamous differentiation based on the morphologic changes of the cells (data not shown). Interestingly, TGF-~l reduced the increase in c-myc mRNA levels when added either simultaneously or 24 h before growth stimulation. Its effect was selective because it showed no effect on the transcription of c-fos, c-jun, or tubulin mRNA levels, and resulted in an increase of fibronectin mRNA level (41). Coffeyand eo-workers (42) recently reported that TGF-~ selectively inhibited expression of certain of the EGF-induced genes (c-myc and KC) in a nontumorigenic keratinocyte cell line BALB/MK, which is EGF dependent. They also found that c-fos induction was not affected by TGF-~. Pfeifer and associates (29) referred to their unpublished data

about the correlation between the proto-oncogene expression and TGF-~ effectin their review article and reported that TGF-~ (100 pg/ml) reduced the c-myc mRNA level but increased the c-fos mRNA level in normal human bronchial epithelial cells. In our present system using bovine bronchial epithelial cells, TGF-~ itself did not show any significant effect on proto-oncogene expression. This result is similar to that obtained by Coffey and eo-workers (42). In other nonepithelial cell systems, the effects on c-myc expression by certain inhibitory peptides have been reported. In HL-60 cells, tumor necrosis factor-a treatment reduced c-myc expression at the level of transcription (43). Interferon (IFN)-a or IFN-~I inhibited c-myc expression in Burkitt lymphoma-derived Daudi cells (44, 45). In BALB/3T3 cells, Einat and colleagues (46) have reported that IFN -a and IFN-~I block PDGF-induced c-myc expression. It has also been reported that TGF':"~ reduces c-myc expression in human endothelial cells (47) and in a human breast cancer cell line (48). Thus, it is likely that reduction of c-myc mRNA levels is a common occurrence in response to growth-inhibitory polypeptides. Selective reduction of c-myc mRNA levels by TGF-~ suggests that c-myc transcription plays a role in the regulation of bovine bronchial epithelial cell proliferation. Further studies such as nuclear run-on assays will be necessary to elucidate the molecular mechanism by which TGF-~ exerts this effect. References 1. Greenberg, M. E., and E. B. Ziff. 1984. Stimulation of 3T3 cells induces transcription of the c-fos protooncogene. Nature 311:433-438. 2. Kruijer, W., J. A. Cooper, T. Hunter, and I. M. Verma. 1984. Plateletderived growth factor induces rapid but transient expression of the c-fos gene and protein. Nature 312:711-716. 3. Kelly, K., B. H. Cochran, C. D. Stiles, and P. Leder. 1983. Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35:603-610. 4. Gonda, T. J., and D. Metcalf. 1984. Expression of myb, myc and fos proto-oncogenes during the differentiation of a murine myeloid leukemia. Nature 310:249-251. 5. Makino, R., K. Hayashi, and T. Sugimura. 1984. C-myc transcript is induced in rat liver at a very early stage of regeneration or by cycloheximide treatment. Nature 310:697-698. 6. Fausto, N., and J. E. Mead. 1989. Biology of disease. Regulation of liver growth: protooncogenes and transforming growth factors. Lab. Invest. 60:4-13. 7. Waterfield, M. D., G. T. Scrace, N. Whittle et al. 1983. Platelet-derived growth factor is structurally related to the putative transforming protein p28 sis of simian sarcomic virus. Nature 304:35-39. 8. Downward, J., Y. Yarden, E. Mayes et al. 1984. Close similarity of epidermal growth factor receptor and v-erbB oncogene protein sequences. Nature 307:521-527. 9. Green, S., P. Walter, V. Kumar et al. 1986. Human estrogen receptor cDNA: sequences, expression and homology to v-erb-A. Nature 320: 134-139. 10. Sherr, C. J., C. W. Rettenmier, R. Sacca, M. F. Roussel, A. T. Look, and E. R. Stanley. 1985. The c-fms protooncogene product is related to the receptor for the mononuclear phagocyte growth factor, CFS-1. Cell 41:665-676. 11. Lau, L. F., and D. Nathans. 1987. Expression of a set of growth-related immediate early genes in BALB/c 3T3 cells: coordinate regulation with c-fos or c-myc. Proc. Natl. Acad. Sci. USA 84:1182-1186. 12. King, R. J., M. B. Jones, and P. Minoo. 1989. Regulation of lung cell proliferation by polypeptide growth factors. Am. J. Physiol. 257(Lung Cell. Mol. Physiol. 1):L23-L38. 13. Maki, Y., T. J. Bos, C. Davis, M. Starbuck, and P. K. Vogt. 1987. Avian sarcoma virus 17 carries the jun oncogene. Proc. Natl. Acad. Sci. USA 84:2848-2852. 14. Vogt, P. K., T. J. Bos, and R. F. Doolittle. 1987. Homology between the DNA-binding domain of the GCN4 regulator, protein of yeast and the carboxyl-terminal region of a protein coded for by the oncogene jun. Proc. Natl. Acad. Sci. USA 84:3316-3319. 15. Rauscher, F. J., Ill, D. R. Cohen, T. Curran et al. 1988. Fos-associated

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