Differentiation (1 989) 41 : 34-41 Differentiat ion Ontogcny and Neoplasia 0 Springer-Verlag 1989

Inhibitory effects of transforming growth factor# on laminin production and growth exhibited by endoderm-like cells derived from embryonal carcinoma cells David Kelly and Angie Rizzino * Eppley Institute for Research in Cancer and Allied Diseases, University of Ncbraska Medical Center, 42nd and Dewey Avenue, Omaha, NE 68105, USA

Abstract. Previous studies have shown that two mouse em- bryonal carcinoma (EC) cell lines do not express cell surface receptors for transforming growth factor type-/3 (TGF-B) until they are induced to differentiate. To understand the effects of TGF-/3 in this model system, we have examined the effects of TGF-/3 on parietal endoderm-like cells derived from EC cells. We have determined that TGF-/3 exerts three effects on these cells. TGF-/3 inhibits proliferation of the parietal endoderm-like cells, and this occurs even in the presence of growth factors that stimulate their proliferation. TGF-/3 also alters the morphology of the parietal endo- derm-like cells by increasing their spreading. Moreover, the morphological effect of TGF-/3 is observed in the presence of dibutyryl cyclic AMP (dbcAMP), which reduces the spreading of these cells. Lastly, TGF-/3, but not other growth factors, decreases the production of laminin by the parietal endoderm-like cells. This was unexpected since TGF-/3 has been shown to increase the production of extra- cellular matrices in other systems. Thus, our findings indi- cate that parietal endoderm-like cells provide a useful sys- tem for broadening the study of TGF-/3. Furthermore, our findings provide additional support for the possibility that TGF-/3 plays important roles during the early stages of mammalian development.

Introduction It is now well established that TGF-j? is a multifunctional regulator of both cell growth and differentiation (reviewed in [21]). Two different forms of TGF-/3, TGF-PI and TGF- pZ, have been purified to homogeneity [2, 8, 26, 321 and recent evidence argues that two additional forms exist [ l l , 12,381. TGF-Bl and TGF-/32 can stimulate or inhibit prolif- eration [28, 391 and they have been shown to influence the differentiation of many different cell types. In general, TGF-/3 can stimulate the differentiation of some cells and inhibit the differentiation of others (reviewed in [21]). Al- though information is limited, it is evident that TGF-/3 can exert important effects in vivo. Implants of TGF-/3 have been shown to inhibit the growth and morphogenesis of mammary glands [33], and injection of TGF-/3 into newborn mice induces fibrosis and angiogenesis [29]. Furthermore, the distribution of TGF-/3 in mid- and late-gestation mouse embryos is consistent with TGF-/3 playing important roles

* To whom offprint requests should be sent

during mammalian development [7], and recent studies indi- cate that mouse embryos begin to produce TGF-/3 as early as the eight-cell stage [18].

Work with EC cells also suggests that TGF-/3 may influ- ence development prior to midgestation. EC cells are widely used as a model system for the study of early mammalian development. These cells, which can be induced by retinoic acid (RA) to differentiate into cells that exhibit the proper- ties of early embryonic cells [36, 371, have been shown to regulate the expression of TGF-/3 receptors when they dif- ferentiate. Whereas nearly all cells express TGF-/3 receptors [41], F9 and PC-13 EC cells lack detectable TGF-/3 recep- tors [20]. When these EC cells are induced to differentiate by RA, expression of TGF-/3 receptors is observed within 2 days and growth of the differentiated cells is inhibited

Attempts to understand the effects of TGF-/3 on the differentiation of other cells led to the finding that TGF-/3 exerts multiple effects on the extracellular matrix of cells. TGF-/3 has been shown to increase the production of var- ious components of the extracellular matrix [l, 6, 10, 17, 34,401 and to decrease the levels of transcripts for metallo- proteases collagenase and stromelysin while increasing the level of transcripts for the specific metalloprotease inhibitor TIMP [5]. Interestingly, several lines of evidence suggest that TGF-/3 may influence cell differentiation, at least in part, by influencing the production of fibronectin, a major component of extracellular matrices. TGF-/3 has been shown to increase the production of fibronectin by myo- blasts, preadipocytes and chondrocytes [9,10,30], and addi- tion of fibronectin to these cells inhibits their differentiation [16, 30, 351.

To understand the possible effects of TGF-/3 during early development, we examined the effects of TGF-/3 on the parietal endoderm-like cell line PYS-2 and on the pari- etal endoderm-like cells derived from F9 EC cells by RA- induced differentiation. These cells were selected because parietal endoderm (parietal extraembryonic endoderm) is one of the first cell types to form during early development and because these cells produce laminin, which is a major component of Reichert’s basement membrane. In this re- port, we demonstrate that TGF-p inhibits the growth of the parietal endoderm-like cells and dramatically alters their morphology by increasing cell spreading. In addition, we determined that TGF-/3 decreases laminin production by the parietal endoderm-like cells. Although further studies are needed, our findings provide additional support for the

by TGF-/3 [20].

35

possibility that TGF-P plays one or more roles during early mammalian development and provides a basis for predict- ing how TGF-P may influence the growth and behavior of extraembryonic endoderm in vivo.

Methods

Cells and culture medium. Stock cultures of F9 EC cells and PYS-2 cells were maintained in Dulbecco's Modified Eagle's Medium (DME, Gibco # 430-2100, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Irvine Scientific, Santa Ana, Calif., USA) as reported previously [22, 231. Differentiation of F9 EC cells was in- duced by 48 h treatment with 5 pM RA. Stock cultures and all experimental cultures were maintained at 37" C in a moist atmosphere of 95% air and 5% CO,.

Monolayer growth and sizing experiments were per- formed in DME supplemented with FBS at the concentra- tions indicated or in serum-free medium supplemented with newborn calf plasma at the concentrations indicated. Se- rum-free medium (DME/F-12) consisted of a 1 : 1 mixture of DME and Ham's F-12 (Gibco #43&1700) supple- mented with 15 mM 4-(2-hydroxyethyl)-I -piperazineethane sulfonic acid (HEPES) and 25 nM selenous acid. DME/F- 12 and plasma (Irvine Scientific, Santa Ana, Calif.) were prepared as described previously [25]. Bovine basic fibro- blast growth factor (FGFb), TGF-PI , human platelet-de- rived growth factor (PDGF; all obtained from R & D Sys- tems, Inc., Minneapolis, Minn., USA), epidermal growth factor (EGF; Collaborative Research, Waltham, Mass., USA) and dbcAMP (Boehringer Mannheim GmbH, Feder- al Republic of Germany) were used at the concentrations indicated. Average cell number for each treatment was de- termined by trypsinizing the attached cells from triplicate dishes, and counting the cells from each dish electronically with a Coulter counter. Cell counts varied by less than 10% within each condition. To determine the average cell size for each treatment, an inverted phase-contrast micro- scope equipped with a calibrated grid in the eyepiece was used at magnification x 200 to make direct visual measure- ments of the vertical and horizontal diameters of 75 ran- domly selected cells. The measured diameters for each treat- ment were then averaged and cell volumes were calculated from the formula 4/37rr3.

Metabolic labeling. At 18-24 h prior to metabolic labeling, PYS-2 cells (2.5 x lo5 per 35-mm culture dish) were plated in DME supplemented with 10% FBS plus the factors indi- cated in the text, and the F9-differentiated cells ( 5 x lo4 per 35-mm culture dish), which were derived from F9 EC cells as described above, were refed with DME supple- mented with 10% FBS plus the factors indicated in the text. The cells were labeled in 1 ml medium consisting of methionine-free DME, 0.3 mg/ml glutamine, 10% dialyzed FBS, and 200 pCi [35S]-methionine (New England Nuclear, Boston, Mass.). Incubation was for 3 h at 37" C (similar results were obtained when an incubation time of 18 h was used - data not shown). After labeling, the cells were washed twice with DME/F-12 at 4" C and solubilized at 4" C with radioimmunoprecipitation assay (RIPA) buffer, pH 7.4 (50 mM Tris-HC1, 0.15 M NaC1, 1% Triton, 1% deoxycholate and 0.1% sodium dodecyl sulfate, SDS) for 10-1 5 min. Each extract was clarified by centrifugation at 11 400 g for 15 min and total [35S]-methionine trichloro-

acetic acid precipitable counts were determined. The ob- served values of incorporated 35S-methionine for each ex- tract were normalized to the value observed for the un- treated cells. This value is shown in Tables 3 and 4 as rela- tive methionine incorporation.

Immunoprecipitation of labeled extracts. Equal numbers of counts (incorporated 35S-methionine) from each extract were diluted to the same volume (1 ml) with RIPA buffer at 4" C. To remove proteins that nonspecifically bind to Protein A-agarose, 30 p1 Protein A-agarose (CalBiochem, La Jolla, Calif.): RIPA buffer mixture (1 : 1 v/v) was added to each extract, incubated for 30 min at 4" C with mixing, and the Protein A-agarose was pelleted by centrifugation at 11 400 g for 2 min. The supernatants were removed and mouse polyclonal anti-laminin (Collaborative Research, Waltham, Mass.) was added to each supernatant at a final dilution of 1/200. These antibody-extracts were incubated at 4" C with mixing. After 30 min, 30 p1 1 : 1 Protein A- agarose: RIPA buffer mixture were added to the antibody extracts and incubated for an additional 30 rnin at 4" C with mixing. Each sample was pelleted by centrifugation at 4200 g for 2 min and washed three times with RIPA buffer at 4" C. In preparation for gel electrophoresis, the final pellet was boiled in 100 pl 2.25% SDS, 12.5% 0.5 M Tris (pH 6 4 , 10% glycerol, 5% P-mercaptoethanol, and 0.05% bromophenol blue for 3-5 rnin and then recentri- fuged at 11 400 g for 2 min.

Gel electrophoresis. SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli [15]. Equal volumes of the immunoprecipitates (20 pl) were sepa- rated on vertical 7.5% polyacrylamide minigels (0.75 mm thick). Following separation, the gels were incubated in EN- 3HANCE (New England Nuclear, Boston, Mass.) for 30min, dried, and exposed to Kodak X-OMAT film for 1 4 days. Band intensities on the fluorographs were deter- mined with an LKB 2202 Ultroscan Laser Densitometer (Bromma, Sweden). For the purpose of comparison, these values were normalized with respect to the intensities of the laminin bands observed in the extracts prepared from the untreated cells. Since the immunoprecipitation step was performed on the same number of counts (35S-methionine incorporated into protein) from each extract, these values are also corrected with respect to total protein, i.e., the levels of 35S-methionine incorporated into total protein during the 3-h incubation period.

Results

Effects of TGF-P on cell proliferation

Previous studies from this laboratory have shown that TGF-P inhibits the proliferation of RA-induced differen- tiated cells derived from F9 and PC-13 EC cells [20]. Since F9- and PC-13-differentiated cells exhibit properties of ex- traembryonic endoderm, the parietal endoderm-like cell line PYS-2 was examined for its responses to TGF-D. This cell line was examined for its monolayer growth response to TGF-P in both serum-containing media (SCM) and plasma- supplemented media (PSM). Under both conditions, TGF-P decreased the monolayer growth by 4245% (Table 1). The inhibition induced by TGF-P was due primarily, if not ex- clusively, to a reduction in the growth rate and was not

36

Table 1. Effects of growth factors on the proliferation of PYS-2 cells*

Factors added Cell number' Percent ( ~ 1 0 - 5 ) of control

None (control) 3.35 100 TGF-/3 1.84 55 EGF 3.32 99 PDGF 6.62 187 FGFb 4.85 145 TGF-B+ FGFb 3.11 93 TGF-B+PDGF 3.83 114 TGF-/3+ EGF 2.01 60 TGF-B+ EGF+FGFb+PDGF 3.43 102 5% FBS 7.91 236 5% FBS+TGF-B 4.31 128

FBS, bovine fetal serum a PYS-2 cells (1 x lo5 per 35-mm culture dish) were plated in Dul- becco's modified Eagle's medium (DME)/F-12 supplemented with 2% plasma

Growth factor concentrations were: epidermal growth factor (EGF; 10 ng/ml); transforming growth factor$ (TGF-B; 5 ng/ml); platelet-derived growth factor (PDGF; 10 ng/ml); and fibroblast growth factor b (FGFb; 5 ng/ml). All growth factors were added at the time of plating

At 72 h, the average cell number for each condition was deter- mined from triplicate dishes as described in Methods. Cell numbers varied by less than 10% within each condition

1 0 4 1 , 1 2 3 4

Time (days) Fig. 1. Effect of TGF-/I on the growth of PYS-2 cells. PYS-2 cells (5 x lo4 cells per 35-mm tissue culture dish) were plated in medium containing 5% FBS (+) and in medium containing 5% FBS plus 2 ng/ml TGF-B (-A-). At the times indicated, the no. of cells was determincd. The numbers shown are the averages of triplicates that varied by less than 10% in all cases and by less than 5% in most cases

due to an effect on the plating efficiency of the cells. Plating efficiency of PYS-2 cells was 75%-80% in the presence and absence of TGF-/?; whereas the doubling time of the cells increased from 12 h to 16 h when TGF-/l was added to SCM (Fig. 1) and increased from 20 h to 26 h when TGF-B was added to PSM (data not shown).

To understand the growth-inhibitory effects of TGF-8, the growth of PYS-2 cells was examined in the presence

of growth factors previously shown to stimulate the growth of F9-differentiated cells [24]. PDGF and FGF stimulated the growth of the PYS-2 cells, but the growth stimulatory effects of PDGF and FGF were not observed in the pres- ence of TGF-/? (Table 1). It is unclear whether TGF-B blocks the action of PDGF and FGF or whether the growth-stimulatory effects of PDGF and FGF are offset by a corresponding growth inhibition induced by TGF-B. Interestingly, EGF on its own did not stimulate the growth of PYS-2 cells, and it had little or no effect on the growth- inhibitory effects of TGF-/3 pable 1). The lack of an effect by EGF is likely to be due to the very low EGF receptor number exhibited by PYS-2 cells [19]. In contrast, PYS-2 cells have been shown previously to exhibit receptors for PDGF and FGF [22,24].

Treatment of F9differentiated cells with dbcAMP is known to lead to increased TGF-/3 binding [20] and elevated expression of properties characteristic of parietal extraem- bryonic endoderm [36, 371. Therefore, we compared the effect of TGF-B on the monolayer growth of PYS-2 and F9-differentiated cells in the presence and absence of dbcAMP. On its own, dbcAMP increased the monolayer growth of F9-differentiated cells, but decreased the mono- layer growth of PYS-2 cells (Table 2). Nonetheless, TGF-/? inhibited the growth of both PYS-2 cells and F9-differen- tiated cells in the presence and absence of dbcAMP (Ta- ble 2).

Effects of TGF-/? on cell morphology Besides its effect on cell growth, TGF-j? exerted a significant effect on the morphology of the F9-differentiated cells by increasing cell spreading (Fig. 2A, B). To understand this effect, we examined the morphological effects of TGF-B in the presence of dbcAMP, since dbcAMP has been shown to induce a pronounced effect on the morphology of F9- differentiated cells [14]. As shown previously, dbcAMP in- duced a radical change in the morphology of the F9-differ-

Table 2. Effects of TGF-8 and dibutyryl cyclic AMP (dbcAMP) on the growth of F9-differentiated and PYS-2 cells'

~~

Cells Factors Cell no.' Percent added ( x lo+) of control

F9-differentiated None (control) TGF-B

dbcAMP +TGF-B dbcAMP

PYS-2 None (control) TGF-8

dbcAMP + TGF-/I dbcAMP

6.47 4.00 9.53 6.02 8.50 5.16 4.62 3.06

100 62

147 93

100 61 54 36

F9-differentiated cells (1.2 x lo5 per 35-mm culture dish) were derived from F9 EC cells cultured in DME supplemented with 10% FBS and 5 M retinoic acid (RA) for 48 h. After 48 h, the cells were refed with medium lacking RA but containing the factors indicated. PYS-2 cells (5 x lo4 per 35-mm culture dish) were plated in DME supplemented with 10% FBS plus the factors indicated

Concentrations of the added factors were: dbcAMP, 1 mM; TGF-B, 5 ng/ml

After treatment with the indicated factors for 72 h, the average cell number for each condition was determined from triplicate dishes as described in Methods. Cell numbers varied by less than 10% within each condition

37

Fig. 2A-D. Effect of TGF-B on the morphology of F9-differentiated cells. F9-differentiated cells were derived from F9 EC cells cultured for 48 h in DME supplemented with 10% FBS plus 5 pM RA. After 48 h, the F9-differentiated cells were refed with fresh medium lacking RA but containing the factors indicated. A Untreated. B TGF-/I ( 5 ng/ ml). C dbcAMP (1 mM). D dbcAMP (1 mM) and TGF-/? (5 ng/ml). After 72 h, cells from each condition were photographed at the same magnification. Scale bur in D represents 50 pm

Fig. 3A-D. Effect of TGF-B on the morphology of PYS-2 cells. PYS-2 cells were cultured in DME supplemented with 2% plasma plus the factors indicated. A Untreated. B TGF-B (5 ng/ ml). C dbcAMP (1 mM). D dbcAMP (I mM) and TGF-/? (5 ng/ml). After 72 h, cells from each condition were photographed at the same magnification. Scale bar in D represents 50 pm

38

'"1 804

-40 -201 0 20 40 80 80 100 120

ELAPSED TIME (hrs.) Fig. 4. Time-course of the TGF-P effect on the spreading of PYS-2 cells. PYS-2 cells (1 x lo5 per 35-mm culture dish) were plated in DMEIF-12 supplemented with 2% plasma with (-A-) and with- out (-*) TGF-/I (2 ng/ml). At the indicated times, the average cell size of 75randomly selected cells from each condition was determined as described in Methods. Average cell size at initial attachment (5 h) was the Same for both treated and untreated cells and was therefore used as a reference point. Any increase or de- crease in cell area at subsequent time points is plotted relative to the cell size for thc 5-h point (expressed as a percentage)

entiated cells, and this effect was partially reversed by TGF- /? (compare Fig. 2C, D). We also examined the effects of TGF-jl and dbcAMP on the morphology of PYS-2 cells. As in the case of the F9-differentiated cells, TGF-/3 in- creased, while dbcAMP decreased, the spreading of PYS-2 cells (Fig. 3 A-D). Again, TGF-B only partially reversed the morphological effect of dbcAMP on cell spreading (Fig. 3D).

To characterize the morphological effect of TGF-8 in more detail, we examined the effects of TGF-8 over several

T Y

w Y

4

5 d d

W > F

W U

days. PYS-2 cells were used for this study because they are an established cell line and this eliminates some of the variability that occurs when F9 EC cells are induced to differentiate. In most cases, similar observations were made with F9differentiated cells. PYS-2 cells were grown in PSM because the TGF-B-induced morphological effects were maximized in PSM and because serum contains TGF-B. Under these conditions, the TGF-/3-induced cell spreading was evident within 20 h, and the extent of spreading did not change significantly during the next 72 h (Fig. 4). It was also determined that increasing the concentration of TGF-/3 from 50 pg/ml to 5 ng/ml led to increases in cell spreading. Interestingly, the increases in cell spreading were closely paralleled by the decreases in cell growth (Fig. 5). Finally, we determined that removal of TGF-/? reversed the morphological effect exerted on PYS-2 cells. Similarly, a return to the normal morphology was observed when PSY-2 cells that had been treated with TGF-#I were replated in the absence of TGF-/3 (data not shown).

Effects of TGF-B on the properties of EC-derived dij'Jerenriated cells

Recent studies have shown that TGF-/3 influences the differ- entiation of many cell types, and it specifically influences the production of plasminogen activator [31] and extracellu- lar matrices in several different systems [l, 6, 10, 17, 34, 403. Since production of laminin and the release of plasmin- ogen activator are known to rise dramatically when EC cells are induced to differentiate to endoderm-like cells, we examined the effect of TGF-/I on these markers of differen- tiation.

Initially, the possible effect of TGF-/I on plasminogen activator was examined. We did not detect a significant effect of TGF-B on the release of plasminogen activator by PYS-2 cells or F9-differentiated cells (data not shown). Therefore, we focused our efforts on the production of la- minin. Laminin production was monitored in cells that had been metabolically labeled with [35S]-methionine. Fluoro- graphs of immunoprecipitated laminin from treated and untreated PYS-2 cells indicated the presence of two bands (Fig. 6). One band migrated at approximately 200-k Da and

220-

200-

180-

180-

140- w -60

0 50 100 200

TGF-f3 ADDED (w/ml) Fig. 5. Erect of TGF-B on the growth and spreading of PYS-2 cells. PYS-2 cells (1 x lo5 per 35-mm culture dish) were plated in DME/F-12 supplemented with 2% plasma and TGF-/l at the concentrations indicated. At 72 h, the average cell size of 75 randomly selected cells from each condition was determined as described in Methods. Relative cell size (-A-) is the average cell size for each condition relative to the average cell size for the untreated control (expressed as a percentage). Also at 72 h, the average cell number for each condition was determined as described in Methods. Cell numbers from the triplicate samples varied by less than 10% within cach condition. Relative cell number (-t) is the average cell number for each condition relative to the average cell number for the untreated control (expressed as a percentage)

39

1 2 3 4 5 6 7

Fig. 6. lmmunoprecipitation of laminin from PYS-2 cells. PYS-2 cells (2.5 x lo5 per 35-mm culture dish) were plated in DME supple- mented with 10% FBS plus the factors indicated for each lane. Lme I , untreated; lane 2, TGF-8 (2 ng/ml); lane 3, dbcAMP (1 mM); lane 4, dbcAMP (1 mM) and TGF-8 (2 ng/ml); lane 5, dbcAMP (1 mM) and FGFb (500 pglml); fune 6, dbcAMP (1 mM) and PDGF (20 ng/ml); fane 7, dbcAMP (1 mM) and ECF (20 ng/ ml). In each case, the cells were metabolically labeled with [35S]- methionine and the production of laminin was measured as de- scribed in Methods

Table 3. Effects of TGF-8, dibutyryl CAMP, and various growth factors on the production of laminin by PYS-2 cells"

Factors Relative Relative Relative added incorporation of intensity intensity

["SI-methionine of 200-k Da of 400-k Da band band

None 1 .00 1 .00 1 .00 TGF-8 0.91 0.17 0.50 dbcAMP 1.19 1.39 1.47 dbcAMP + TGF-8 0.96 0.58 1.07 dbcAMP + FGFb 1.1 3 1.38 1.38 dbcAMP + PDGF 1.29 1.42 1.28 dbcAMP+EGF 1.11 1.78 1.48

* PYS-2 cells (2.5 x lo5 per 35-mm culture dish) were plated in DME supplemented with 10% FBS plus the factors indicated ' Concentrations of the added factors were: TGF-8, 2ng/ml; FGFb, 500 pg/ml; PDGF, 20 ng/ml; EGF, 20 ng/ml; dbcAMP, 1 mM ' Intensities of the immunoprecipitated laminin bands from Fig. 6 were determined with a scanning densitometer as described in Methods. These values were normalized to the level of total ["S]- methionine incorporated under each culture condition as described in Methods. The ratio of the absorbances between the 200-k Da band and the 400-k Da band in the untreated control was 1 : 1.6. This experiment was repeated in whole or in part three times with similar results

corresponds to the reported molecular weights of the la- minin B1 and B2 subunits [3, 4, 131. The second band mi- grated at a substantially higher molecular weight, which is characteristic of the 400-k Da laminin A subunit [3, 4, 131. Treatment of PYS-2 cells with TGF-j? for 24 h reduced methionine incorporation by only 9% (Table l), which was similar to the effect of TGF-j? on cell proliferation (Fig. 1). In contrast, TGF-j? significantly reduced the production of both laminin A and B subunits (Fig. 6; compare lanes 1 and 2). In these experiments, laminin production was nor- malized to protein production as described in Methods. Densitometric measurements of the fluorograph indicated that the laminin A subunit was decreased by approximately 50% and the laminin B subunit was decreased by approxi- mately 80% when TGF-fi was present (Table 3). Other

Table 4. Effects of TGF-8, dibutyryl CAMP, and various growth factors on the production of laminin by F9-differentiated cells"

~ ~- Factors Relative Relative Relative added incorporation of intensity intensity

[35S]-methionine of 200-k Da of 400-k Da band band -

Group A None 1 .oo 1 .oo 1 .00 TG F-8 0.93 0.62 k0.10 0.63 k0.16 FGFb 1.11 1.10 & 0.19 0.92 k0.04 PDGF 0.91 1.12 f 0.16 1.18 k 0.10 EGF 0.80 1.17 k0.21 1.05k0.03 dbcAMP 1.07 2.86k0.06 1.50k0.03

Group B None 1 .00 1 .OO 1 .00 TGF-8 0.93 0.63 k0.16 0.56k0.10 dbcAMP 1.07 2.02 & 0.29 1.27 f 0.26 dbcAMP +TGF-B 0.97 1.53k0.42 0.99k0.39 dbcAMP+FGFb 1.11 2.20k 0.63 1.37 f0.48 dbcAMP + PDGF 1.02 2.22 k 0.35 1.35 0.38 dbcAMP + EGF 1.05 2.04k0.49 1.26kO.36

a F9-differentiated cells (5 x lo4 per 35-mm culture dish) were derived from F9 EC cells that were cultured for 48 h in DME supplemented with 10% FBS and 5 M RA. After 48 h, the cells were refed with fresh medium lacking RA but containing the factors indicated

Concentrations of the added factors were: TGF-8, 2 ng/ml; FGFb. 500 pg/ml; PDGF, 20 ng/ml; EGF, 20 ng/ml; dbcAMP, 1 mM

Average relative intensities of the immunoprecipitated la- minin bands from three separate experiments were determined densitometrically. These values were normalized to the level of total ["SI-methionine incorporated under each culture con- dition as described in Methods. In each of the three experi- ments, TGF-8 inhibited laminin production, both in the pres- ence and the absence of dbcAMP. The ratio of absorbances between the 200-k Da band and the 400-k Da band of the un- treated controls for group A and group B are 1 : 1.2 and 1 : 1.1, respectively

growth factors were also tested for their effects on laminin production. FGFb, PDGF, and EGF had no effect on the production of laminin by PYS-2 cells (data not shown). Treatment with dbcAMP, as has been shown for F9differ- entiated cells [37], increased laminin production by PYS-2 cells (Fig. 6; compare lanes 1 and 3), although the effect was not as large (Tables 3, 4). Under the latter conditions, TGF-j? reduced the increase of laminin production by PYS- 2 cells, whereas FGFb, PDGF, and EGF had little or no inhibitory effect (Fig. 6, Table 3).

The effect of TGF-fi on laminin production was also examined in FPdifferentiated cells. As was observed for PYS-2 cells, densitometric measurements of fluorographs of laminin immunoprecipitates from F9differentiated cells indicate that TGF-j? decreased the production of both la- minin A and B subunits. The production of both A- and B-subunits was reduced approximately 40% (Table 4). TGF-fi also reduced the dbcAMP-induced increase in la- minin production by F9-differentiated cells. As in the case of PYS-2 cells, FGFb, PDGF, and EGF did not significant- ly alter the production of laminin by F9differentiated cells, either in the presence or absence of dbcAMP (Table 4).

40

Discussion

It is evident from the work desribed above that TGF-8 exerts several effects on EC-derived endoderm-like differen- tiated cells. TGF-/3 inhibits the growth of the parietal endo- derm-like cells and alters their morphology. Although TGF-8 has been shown t o inhibit the growth and alter the morphology o f other cells [27, 28, 391, TGF-8 has not been shown previously t o decrease the production of com- ponents that make u p extracellular matrices. In previous studies with other cells, TGF-/? has been shown to increase the production of proteoglycans, collagens, fibronectin and laminin [I, 6, 10, 17, 34, 401. Consequently, EC-derived endoderm-like cells provide a useful model system for ex- panding the study of TGF-8 and its effects on the produc- tion of extracellular matrices and their components. A t present, the mechanism by which TGF-8 decreases laminin production is unknown. Although TGF-8 may indirectly reduce laminin production as a consequence o f its inhibition of cell growth, there are two reasons for suspecting that laminin production is not tightly associated with the growth of these cells. First, growth factors such as PDGF and F G F b do not increase laminin production (Tables 3, 4), yet they stimulate the growth o f both F9-differentiated cells [24] and PYS-2 cells (Table 1). Second, dbcAMP increases the production o f laminin by both F9-differentiated cells and PYS-2 cells, yet d b c A M P stimulates the growth of F9- differentiated cells and inhibits the growth o f PYS-2 cells.

Clearly, additional studies will be needed t o understand the mechanisms by which TGF-8 affects EC-derived endo- derm-like differentiated cells in general and laminin produc- tion in particular. Nonetheless, our findings suggest that one of the earliest effects of TGF-8 during mammalian de- velopment is t o regulate the growth of parietal endoderm and its production of Reichert’s membrane.

Acknowledgements. Max Wicha is thanked for helpful advice con- cerning the measurements of laminin production and Heather Riz- zino is thanked for excellent editorial assistance. This work was supported by grants from the National Institute of Child Health and Human Development (HD 21568 and H D 19837) and corc grants from the National Cancer Institute (Laboratory Cancer Re- search Center Support Grant, CA 36727)) and the American Can- cer Society (ACS SIG-16).

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Accepted in revised form March 16, 1989