Effects of Transforming Growth Factor-S on Collagen Synthesis by Fetal Rat Lung Epithelial Cells Samuel J. DiMari, Anita M. Howe, and Michael A. Haralson Department of Pathology, Vanderbilt University School of Medicine, Nashville, Tennessee

Studies were performed to characterize the effects of acute and chronic exposure to transforming growth factor-S (TGF-t3) on collagen biosynthesis by fetal rat lung epithelial (FRLE) cells, a cell line established from the fetal rat lung alveolar epithelial cell. Neither condition of exposure to TGF-t3 stimulated cell growth, but both conditions increased total protein synthesis. Quantitative evaluation by carboxymethylTrisacryl chromatography revealed that FRLE cells synthesized types I, III, IV, and V collagen under all circumstances. Acute and chronic exposure to TGF-t3 increased total collagen production approximately 50 % and 300 %, respectively, with the increases in total collagen production exceeding those of total protein synthesis. In addition, these analyses indicated that the production of types I and III molecules was stimulated to a greater extent than was the synthesis of types IV and V molecules. Both experimental conditions increased the ratio of secreted to cell-associated molecules for types I and III molecules, decreased this ratio for type IV collagen, but minimally affected the culture distribution of type V collagen. Additionally, both conditions of exposure to TGF-t3 were found to increase the proportion of the homotrimeric forms of types I and V molecules relative to their heterotrimeric counterparts. Thus, these studies establish that TGF-t3 selectively and type-specifically alters collagen production without affecting growth in an epithelial cell line of fetal rat lung origin.

The type II alveolar epithelial cell plays several major roles in fetal lung development: (1) it is the cell that synthesizes surfactant (1,2); (2) it is the progenitor of other alveolar epithelial cell types (1, 3); and (3) it is, at least in part, the source of the macromolecules that form the extracellular matrix (ECM) that underlies the alveolar epithelium (4-8). Furthermore, immaturity of the alveolar type II cell is clearly linked to the pathogenesis of neonatal respiratory distress syndrome (9, 10). Thus, delineation of the factors that affect the development and state of differentiation of the fetal type II pneumocyte may provide insights into its normal development and into its responses during pathologic conditions. Current evidence indicates that both soluble factors (cytokines and hormones) and the ECM affect the development and function of the type II alveolar epithelial cell (reviewed in reference 11). Studies on the effects of soluble mediators on type II cells have focused primarily on the expression of differentiated functions, i.e., surfactant production. Yet many of the same factors that affect type II pneumocyte function (Received in original form July 16, 1990 and in final form November 5, 1990) Address correspondence to: Dr. Michael A. Haralson, Department of Pathology, Vanderbilt University School of Medicine, Nashville, TN 37232. Abbreviations: carboxymethyl, CM; extracellular matrix, ECM; epidermal growth factor, EGF; fetal bovine serum, PBS; fetal rat lung epithelial cell, FRLE cell; N-ethylmaleimide, NEM; phenylmethylsulfonyl fluoride, PMSF; transforming growth factor-S, TGF-I3. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 455-462, 1991

also affect the biosynthesis and catabolism of the ECM (12, 13). Because the type II pneumocyte produces ECM components (4-8) and because the ECM affects type II cell differentiation (6, 7, 14-20), the possibility exists that the actions of these soluble mediators on the alveolar epithelial cell partially reflect their impact upon matrix production. However, little information exists about the effects of cytokines and hormones on matrix production by cells of fetal alveolar epithelial lineage. One approach to examine this possibility is to determine the effects of cytokines and hormones on ECM biosynthesis using clonal populations of pulmonary epithelial cells in culture. This laboratory has described a new cell line (fetal rat lung epithelial [FRLE] cells), which was established from the fetal rat lung type II pneumocyte (21). The properties of FRLE cells suggest that they are a useful model for investigating ECM metabolism in cells of this lineage. Recently, we have shown that epidermal growth factor (EGF) and retinoic acid, two substances recognized to affect airway epithelial cells, differentially modulate collagen production by FRLE cells (11, 22). In addition to EGF and retinoic acid, transforming growth factor-S (TGF-t3) has been reported also to influence the functional properties of EGF-stimulated fetal type II cells (23). Although the mechanism of action of TGF-tJ on the type II cell remains unresolved, studies in other systems indicate that a major effect of this cytokine is the control of both ECM metabolism and the expression of the integrin family of cell-surface receptors (reviewed in references 24 through 27). Thus, TGF-tJ might also affect fe-

456

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991

tal alveolar epithelial cell development by altering ECM biosynthesis. To explore this issue, we have extended our previous studies to assess the effectsof acute and chronic exposure to TGF-{j on the growth of and collagen biosynthesis by FRLE cells. The results of these investigations are presented in this report.

Materials and Methods Materials The sources for all reagents for cell culture and biochemical procedures have been previously detailed (11, 21, 22, 2S, 29). PH]Proline (sp act, lOS Ci/mmol) was purchased from Amersham (Arlington Heights, IL). Transforming growth factor-S, (porcine) was purchased from R&D Systems (Minneapolis, MN). The origin, characteristics, and routine culture maintenance of FRLE cells have previously been detailed (11, 21, 22). Growth of Cells and Metabolic Labeling To assess the effects of TGF-{j on collagen production by FRLE cells, three culture conditions were employed: (1) control cultures; (2) cultures acutely exposed to TGF-,6; and (3) cultures chronically exposed to ffiF-{j. A schematic representation of the protocol used for cell growth is presented in Figure 1. In the experiments detailed herein, all cultures were seeded with the same passage of FRLE cells and were grown simultaneously in medium supplemented with the same lot of fetal bovine serum (FBS). Control cultures were grown in 100-mm-diameter plastic dishes containing 25 ml of standard medium (RPMI 1640 supplemented with 16 % FBS, 2 mM glutamine, and 50 j.tg/ml gentamycin sulfate). The cultures were fed each third day after subculture. Eight days after subculture, the medium was replaced with 10 mll dish of labeling medium (RPMI 1640 containing 1 mg/ml bovine serum albumin, 50 j.tg/ml gentamycin, 50 ug/ml ascorbic acid, 100 j.tg/ml ,6-aminopropionitrile, and 20 j.tCil ml (3H]proline). After incubation for "-'24 h, the culture medium was removed, clarified by centrifugation (2,000 rpm for 15 min at 4 0 C), and the supernatant fraction adjusted

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Figure 1. Schematic representation of the protocol used for cell culture. Fetal rat lung epithelial (FRLE) cells were grown in the absence oftransforming growth factor-S (TGF-fj) (control cultures) or acutely and chronically exposed to TGF-fj using the culture protocol presented. Details of the procedure are given in MATERIALS AND METHODS.

to 10 mM N-ethylmaleimide (NEM) and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). For the preparation of cellassociated collagens, the cell layers were scraped into 0.5 M HOAc ("-'S ml/dish), and the suspension was adjusted to the NEM and PMSF concentrations specified above. After stirring for 24 h at 4 0 C, the suspension was clarified by centrifugation at 5,000 rpm for 30 min. Either the clarified culture medium or the acid-soluble fraction derived from the cell layer was then used as the starting material for all subsequent procedures. Acutely exposed cultures were grown for the same length of time in an identical manner to the control cultures except the labeling medium was additionally supplemented with 3 ng/ml TGF-,6. Chronically exposed cultures were likewise grown for the same length of time, but from the initial day of subculture the standard medium contained 3 ng/ml TGF-{j, and the labeling medium was also supplemented with the growth factor. The secreted and cellassociated fractions from both the acutely and chronically exposed cultures were prepared identically to those of the control cultures. DNA levels (21) and protein contents (30) were assessed for control and acutely and chronically expose~ FRLE cell cultures grown and manipulated in the identical manners described above except that radioactive proline was omitted during the serum-free incubation procedures. DNA values were determined in triplicate, and protein values in duplicate. In each instance, the SEM was less than 5 %. The mean values for the DNA levels were used in all calculations. Isolation and Quantification of FRLE Cell Culture Medium and Cell Layer Collagens The secrete~ and cell-associated collagens synthesized by FRLE cells III the absence or presence of TGF-,6 were isolated using limited pepsin digestion as previously detailed (11, 21, 22, 28, 29). The pepsin-resistant molecules were fractionated by dialysis against 0.5 M HOAc/0.9 M NaCI into preparations containing either types I and ill or types IV and V collagen molecules as described (22,28,29). Collagen ex chains were isolated after reduction and alkylation by molecular sieve chromatography on agarose A-5m. The amount of radioactivity recovered in each genetic type of collagen chain was determined by chromatography of the collagen ex chains recovered in each fraction on carboxymethyl (CM)-Trisacryl as previously detailed (11, 21, 22, 2S, 29). All values were normalized to the equivalent amount of DNA in the original cultures. The values presented therefore reflect the amount of radioactivity recovered in a specific genetic type of collagen chain produced by the number of cells in the original cultures. The molecular compositions of types I and V molecules were calculated using the procedures previously detailed (11, 21, 22, 28).

Results Effects of TGF-fj on Growth and Protein Synthesis by FRLE Cells FRLE cells exhibit under standard culture conditions a biphasic growth pattern (21) characterized by the develop~~~t of an organized ~econdary growth pattern on top of the InItial. monolayer (FIgure 2A). As shown in Figure 2B, chronic exposure to TGF-{j did not affect the culture mor-

DiMari, Howe, and Haralson: TGF-J3 Effects on FRLE Cell Collagen Synthesis

457

exposure to TGF-J3. Evaluation of total protein indicated, however, that both experimental conditions increased the total protein content of the cultures relative to control values (Table 1). Additionally, the amounts of radioactivity incorporated per microgram of DNA into total protein before pepsin digestion were increased 8 % and 38 % in acutely and chronically exposed cultures, respectively (data not shown). Thus, these results indicate that TGF-J3 stimulates total protein synthesis without affecting the growth rate of FRLE cells.

Figure 2. Phase-contrast microscopy of FRLE cells grown in the absence or presence of TGF-{l Panel A: FRLE cells grown under standard culture conditions (control cultures). PanelB: FRLE cells grown in medium supplemented with 3 ng/ml TGF-,B (chronically exposed cultures). Both sets were grown for 8 d as detailed in MATERIALS AND METHODS. The cells were photographed using a Nikon diaphot inverted phase-contrast microscope and a green interference filter. (Magnification in each panel: X125.)

phology. A similar culture morphology was observed under conditions of acute exposure to the cytokine (data not shown). These findings suggested that TGF-J3 minimally affected the growth of FRLE cells. To further assess the effects ofTGF-J3on growth, the total DNA content of control and experimental cultures was determined. As shown in Table 1, the DNA contents of the cultures were unchanged by

Quantitative Evaluation of the Effects of lGF-,B on the Genetic Types and Culture Distribution of the Collagens Synthesized by FRLE Cells To assess the effects of TGF-J3 on collagen production, the collagen a chains in each fraction were separately chromatographed on CM-Trisacryl as previously detailed (21, 28, 29). The amounts of radioactivity eluting in an area corresponding to a specific genetic type of collagen chain were determined and then normalized to the equivalent amount of DNA in the original cultures. CM-Trisacryl chromatography of the chains in the secreted types I + III fractions from control (Figure 3, panel A), acutely (Figure 3, panel B), and chronically (Figure 3, panel C) exposed cultures indicated components corresponding to types I and III collagen chains in each instance. Summation of the amounts of radioactivity corresponding to each collagen chain followed by data reduction (Table 2, part A) indicated that acute exposure to TGF-J3 increased the amounts of radioactivity recovered in each component rv50%. Likewise, chronic exposure to the cytokine resulted in increased amounts of radioactivity incorporated into each chain. However, the magnitudes of the increases were significantly greater than those observed under conditions of acute exposure. Analysis of the secreted types IV + V fractions from control (Figure 4, panel A), acutely (Figure 4, panel B), and chronically (Figure 4, panel C) exposed FRLE cells revealed the presence of chains corresponding in chromatographic properties to those present in types IV and V molecules. Additionally, a small amount of radioactivity corresponding to the al(l) chains, which arises from type I-homotrimer molecules (21), was observed. Summation of the radioactivities corresponding to each genetic type of collagen chain followed by data reduction (Table 2, part A) indicated that neither acute nor chronic exposure to TGF-J3 significantly altered the level of al(IV) components. In contrast, both experimental conditions increased the amount of radioactivity recovered in type V components. However, the magni-

TABLE 1

Effects of acute and chronic exposure to TGF-(3 on DNA and protein synthesis by cultured FRLE cells* JLg/IOO-mm Dish Culture Condition

Control Acute exposure Chronic exposure

DNA

Protein

Protein/DNA

360 ± 12 390 ± 17 (108%) 360 ± 10 (100%)

7,760 ± 359 9,290 ± 210 (120%) 10,290 ± 423 (133%)

21.6 23.8 (110%) 28.5 (132%)

Definition of abbreviations: TGF-,B = transforming growth factor-S; FRLE cell = fetal rat lung epithelial cell. * DNA and protein levels of FRLE cell cultures grown in the absence of TGF-,B or acutely and chronically exposed to 3 ng/rnl TGF-,B were determined as described in MATERIALS AND METHODS. The values in parentheses represent the percentage of the DNA or protein content or the ratio of protein to DNA determined for FRLE cell cultures exposed to TGF-,B relative to the corresponding value in the control cultures.

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991

458

25

TABLE 2

A. CONTROL

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EFFLUENT VOLUME, MILLILITERS Figure 3. Carboxymethyl-Trisacryl chromatographies of collagen chains recovered in the secreted types I + III fractions synthesized by control and FRLE cells acutely and chronically exposed to TGF13. Panel A: An aliquot (458,700 cpm) of collagen a chains recovered after agarose chromatography of the secreted types I + III fraction from control FRLE cell cultures was chromatographed. Panel B: An aliquot (561,200 cpm) of collagen a chains recovered after agarose chromatography of the secreted types I + III fraction from FRLE cells acutely exposed to TGF-13 was chromatographed. Panel C: An aliquot (502,800 cpm) of collagen a chains recovered after agarose chromatography of the secreted types I + III fraction from FRLE cells chronically exposed to TGF-13 was chromatographed. The conditions of chromatography have been previously described (21, 28). The amounts of radioactivity eluting in the positions corresponding to the a1(1), a1(ID), and a2(1) chains, respectively, were individually determined for each chromatography to establish the fraction of total radioactivity recovered as the genetic type of collagen chain. These values are presented in Table 2, part A.

tudes of the increases in type V components synthesized by the experimental cultures were less than those observed for the types I and III chains. Chromatographic analyses were also performed on the fractions derived from the cell-associated molecules. The results of these evaluations are presented in Table 2, part B. These evaluations indicated that changes in the amounts of radioactivity recovered in types I, III, and V collagen chains

A. Secreted molecules I + III fraction al(l) al(III) a2(1) IV + V fraction al(IV) al(l) a2(V)&a3(V) al(V) B. Cell-associated molecules I + III fraction al(l) al(III) a2(1) IV + V fraction al(IV) al(l) a2(V)&a3(V) al(V) C. Total collagen synthesized al(l) al(III) a2(1) al(IV) a2(V)&a3(V) al(V) Total

542 (369%) 152 (345%) 95 (279%)

(88%) (100%) (140%) (150%)

7 (88%) 1 (50%) 8 (160%) 7 (175%)

176 50 37

265 (151 %) 81 (162 %) 48 (130%)

445 (253%) 145 (290%) 81 (219%)

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10 (250%) 3 (300%) 4 (200%) 5 (166%)

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8 2 5 4

326 94 71 12 7 7 517

7 2 7 6

Chronic Exposure

498 159 97 17 8 9 788

(153%) 991 (304%) (169%) 297 (316%) (137%) 176 (248%) (142%) 17 (142%) (114%) 11 (157%) (129%) 12 (171%) (152%) 1,504 (291%)

Definition of abbreviations: see Table 1. * Values presented were calculated from the amounts of radioactivity recovered from the appropriate areas of the corresponding carboxymethyl- Trisacryl chromatographies (Figures 2 and 3) of the collagen a chains recovered in the indicated fraction. The numbers in parentheses are the percentage of the radioactivity recovered in a fraction from the cells exposed to TGF-~ relative to that in the corresponding control fraction.

essentially paralleled those observed for the secreted molecules. However, whereas the relative amount of secreted type IV chains was unchanged, both conditions of exposure to TGF-13 resulted in a 2.5-fold increase in the amount of radioactivity recovered in cell-associated type IV molecules. Summation of the amounts of radioactivity corresponding to each genetic type of collagen chain (Table 2, part C) indicated that acute exposure to TGF-13 enhanced approximately 50 % the amount of radioactivity recovered in the different types of collagen chains and that chronic exposure resulted in greater increases. To evaluate changes, without regard to molecular composition, in each type of collagen synthesized by FRLE cells acutely or chronically exposed to TGF-I3, the values obtained from the CM-Trisacryl chromatographies (Table 2) were normalized to the corresponding control culture value. This analysis (Figure 5) demonstrated that, although the amount of radioactivity recovered in each genetic type of collagen was increased under both experimental conditions, the degree of the increases differed for each collagen type. Addi-

DiMari, Howe, and Haralson: TGF-{3 Effects on FRLE Cell Collagen Synthesis

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