DEVELOPMENTAL
BIOLOGY
1%
35-41
(1990)
Fetal Rat Lung Fibroblasts Produce a TGFP Homolog That Blocks Alveolar Type II Cell Maturation JOHN S. TORDAY Joint
Program
in Neonatology,
AND
Brigham Accepted
STELLA Women’s
December
KOUREMBANAS Hospital,
Boston,
Massachusetts
02115
20, 1989
Normal growth and differentiation of the lung depends upon mesenchymal-epithelial interactions during development. Recombination experiments using immature (Day 17) and mature (Day 21) fetal rat lung fibroblasts (FRLF) revealed that the stimulatory effect of mature fibroblasts on fetal type II epithelial cells is blocked by immature fibroblasts. Similarly, conditioned medium from Day 17 FRLFs blocks the stimulatory effect (fibroblast-pneumonocyte factor) of Day 21 conditioned medium on type II epithelial cells. This blocking activity is nondialyzable, trypsin sensitive, and heat stable. Its activity is neutralized by an antibody to TGFP, in both conditioned media and recombined cell studies, and its activity is mimicked by TGFP. Developmentally, TGF@-like activity is present in conditioned medium from 15- to Wday FRLF, decreasing precipitously between 19 and 21 days gestation. Northern blot analysis of mRNAs from fetal rat lung fibroblasts on Days 17, 19, and 21 revealed expression of TGFp at all three stages of 0 1990 Academic Press, Inc. development.
three to five dams were used per preparation. The dams were killed with chloroform and the pups were removed from the uterus by laparotomy and kept on ice. The lungs were removed en bloc in a laminar flow hood using sterile technique and put into sterile Hanks’ balanced salt solution without calcium or magnesium on ice. The Hanks’ balanced salt solution was decanted and 5 vol of 0.05% trypsin (Worthington) was added to the lung preparation. The lungs were dissociated in a 37°C water bath using a Teflon stirring bar to disrupt the tissue physically. When the tissue had been completely dispersed into a unicellular suspension (approximately 20 min) the cells were spun down at 500g for 10 min at room temperature in a 50-ml polystyrene centrifuge tube. The supernatant was decanted and the pellet was resuspended in enough minimal essential medium (GIBCO) containing 10% fetal bovine serum (MEM/ fbs) to yield a mixed cell suspension of approximately 3 X lOa cells as determined by a Coulter particle counter. The cell suspension was added to 75-cm2 culture flasks (Corning Glass Works, Corning, NY) for 30-60 min to allow for differential adherence of lung fibroblasts (Smith and Giroud, 1975). Preparation of alveolar type II cell cultures. The unattached cells from the above preparation were then pooled, filtered through a 50-pm nitex filter, spun down at 500g for 10 min in 50-ml polyethylene centrifuge tubes, and allowed to incubate at 37°C for 1 hr (Douglas and Teel, 1976). At the end of this incubation period the cell pellet was resuspended in MEM/fbs to yield a cell suspension of 2 X 10’ cells/ml. Cells (1 X 10’) were then
INTRODUCTION
The normal growth and differentiation of the lung is dependent upon mesenchymal-epithelial interactions during development (Rutter, 1978). Early studies describing this phenomenon were based on observations of recombined mesenchyme and epithelium (Taderera, 1967). The critical experiments by Grobstein (1967) demonstrating that this tissue interaction is mediated by soluble factors has allowed the transition from description to mechanism. One such factor is produced in response to cortisol by lung fibroblasts and stimulates alveolar epithelium to synthesize surfactant phospholipids. Conversely, androgens delay the maturation of fibroblasts (Torday, 1985) and the subsequent increase in surfactant phospholipid synthesis by alveolar type II cells (Torday, 1985). Since the fetal lung fibroblast is able to enzymatitally convert both glucocorticoids (Smith et al, 1973) and androgens (Sultan, 1980) to their bioactive forms and possesses specific receptors for both classes of steroid hormones (Sultan, 1980; Ballard et aZ., 1978) it was hypothesized that the immature fetal lung fibroblast produces a soluble factor which antagonizes the maturational factor produced by the mature fibroblast (FPF). METHODS
AND
MATERIALS
Preparation of rat lung jibroblast mated Sprague-Dawley rats were Charles River Breeders (Wilmington,
cultures. Timeobtained from MA). Usually 35
0012-1606/90 Copyright All rights
$3.00
0 1990 by Academic Press, Inc. of reproduction in any form reserved.
36
DEVELOPMENTALBIOLOGY
injected into l-cm’ Gelfoam collagen sponges (The Upjohn Co., Kalamazoo, MI) hydrated with MEM/fbs using a 200-~1 Pipetman (Rainin Instruments, Waltham, MA) with a cutoff tip to allow for better distribution of the cells within the sponges. The injected sponges were allowed to stand in lOO-cm2 petri dishes overnight in a CO2 incubator (Forma Scientific, Marietta, OH) at 37°C with a gas phase of 5% COB, 95% air. Alveolar type II cells were then isolated from the Gelfoam sponges and propagated in monolayer culture by the method of Post et al. (1984) in 24-well multiwell plates. These cells have been characterized by electron microscopy and have been found to be at least 85% pure by histochemical criteria. Heterochronic jibroblust: Type II cell mixing experiment. Day 17 and Day 21 fetal rat lung fibroblasts were
prepared as described above and recombined in organotypic culture with Day 19 fetal rat lung type II cells as follows: fibroblasts were harvested from 75-cm2 tissue culture flasks using 0.25% trypsin/5 mM ethylenediaminetetracetic acid 24 hr after they were initially isolated. The cells were spun down at 500g for 10 min and resuspended in MEM/fbs. Day 19 type II cells were isolated from Gelfoam sponges (as described above) and resuspended in MEM/fbs. Cell numbers for all preparations were determined using a Coulter particle counter and fibroblasts were mixed with type II cells in a 1:2 ratio, spun down at 500s for 10 minutes, and allowed to incubate in a pellet at 37°C for 1 hr (Douglas and Teel, 1976). At the end of the incubation period, the cell pellets were resuspended in MEM/fbs to yield a cell suspension equivalent to 2 X 10’ fibroblasts and 4 X lo7 type II cells/ml. One-twentieth of this suspension was then injected into sponges (see above) and the sponges were maintained in MEM without serum for 24 hr at 37°C in 5% C02, 95% air. For the final 6 hr, the sponges were incubated with [‘HIcholine chloride (1 &i/ml) and the type II cells were then reisolated and analyzed for rH]SPC content. Fibroblust-pneumonocyte factor bioassay. Conditioned media for fibroblast-pneumonocyte factor were prepared using confluent monolayer cultures of fibroblasts which were washed free of serum-containing medium using 2 X 10 ml MEM with 1 X lo-* Mcortisol for 24 hr in the CO2 incubator at 37°C. The media were decanted and frozen at -70°C until they were assayed. On the day of the assay the media were defrosted, mixed 1:l with fresh minimal essential medium containing 2 &i/ml rH]choline chloride (DuPont-New England Nuclear), and filter-sterilized through a OBB-pm-pore-size Nalgene filter (Nalge Co., Rochester, NY). The medium was aspirated from the type II cell cultures and each well was rinsed twice with 2 ml of MEM. The test medium was then added to the type II cell cultures and incubated for 6 hr in a CO2 incubator. To stop the incuba-
VOLUME 139,199O
tion, the incubation medium was aspirated, and 1 ml of 0.05% trypsin solution was added to each well. The cells were trypsinized at 37°C in the CO2 incubator, transferred to 12 X 75-mm test tubes, and vortexed, and loo-p1 aliquots were taken for cell count and determination of tritium-labeled saturated phosphatidylcholine content. The time course for this assay is linear for 20 hr (Post, 1984). Bioassay of jbroblast-pneumonocyte factor blocking activity. This assay was identical to the assay for fibroblast-pneumonocyte factor except that 21-day fetal rat lung fibroblast conditioned medium was used to maximally stimulate type II cell maturation and test media were then added to assay for blocking activity. Determination
of ‘H-saturated
phosphatidylcholine.
The cell suspension was extracted with chloroform and methanol by the method of Bligh and Dyer (1959) and the organic phase was evaporated under nitrogen and processed for saturated phosphatidylcholine by a modification of the method of Mason et al. (1976). The dried extract was resuspended in 0.5 ml of carbon tetrachloride (spectral grade, Fisher) containing 3.5 mg of osmium tetroxide (Sigma, St. Louis, MO). After 15 min the reaction mixture was evaporated under a stream of nitrogen and the residue was resuspended in 70 ~1 of chloroform:methanol, 9:l (v/v). The extract was transferred to a Silica Gel H thin layer chromatogram (Eastman Kodak, Rochester, NY) using a Pasteur pipet and developed in a chloroform:methanol:water (65:25:4) solution. The developed chromatogram was dipped in bromothymol blue, stained, blotted, and dried at 90°C for 5 min. The area on the chromatogram to which saturated phosphatidylcholine migrated was scraped into vials and counted in a liquid scintillation spectrometer. TGF/3 mRNA analysis. Total cellular RNA was extracted from Day 17,19, and 21 fetal rat lung fibroblasts using a modification of the 6 M guanidine-HCl lysis procedure (Chirgwin et aZ., 1979). Twenty micrograms of RNA were electrophoresed on a 1.2% agarose gel containing formaldehyde and transferred to nitrocellulose paper (BA-85, Schleicher & Schuell, Keene, New Hampshire) by blotting. Filters were probed with a human 32P-labeled TGFB cDNA probe (Derynck et a& 1985). Hybridization was performed for 16 hr at 42°C in 45% formamide, 5X SSPE (3 M NaCl, 0.2 M NaH2P04, 0.012 M EDTA Na2, pH 7.4), 5X Denhardt’s solution, and 0.1% sodium dodecyl sulfate. Final washing-of filters was in 0.2% SSC (3 M NaCl, 0.3 M Na3 citrate 2H20, pH 7.0) solution at 55°C. Autoradiographs were obtained by 24-hr exposure of Kodak XAR film at -70°C using intensifying screens. Other reagents. Transforming growth factor & and transforming growth factor p neutralizing antibody were purchased from R & D Systems (Minneapolis, MN).
TORDAY
Statistics. ance.
AND KOUREMBANAS
Data were compared
by analysis
TGFP
Hmolog
of vari-
3z 0 “0
RESULTS
Heterochronic
Cell Mixing
m’ &
Experiment
To directly test the hypothesis that the immature fetal lung fibroblast blocks the FPF-type II cell interaction, fibroblasts were isolated from Day 17 and Day 21 fetal rat lung; type II cells were prepared from Day 19 fetal rat lung. Day 19 type II cells were then combined with (a) Day 17 fibroblasts, (b) Day 21 fibroblasts, or (c) Day 17 and Day 21 fibroblasts. As can be seen in Fig. 1, the Day 1’7 fibroblasts had no effect on [3H]SPC synthesis, while the Day 21 fibroblasts enhanced rH]SPC synthesis 100%. However, when the Day 17 and Day 21 fibroblasts were combined, the Day 21 fibroblast stimulatory effect was lost. Production of FPF Blocking by Day 17 Fibroblasts
Activity
To determine whether the effect of the Day 17 fibroblasts on Day 21 fibroblast stimulating activity was due to a soluble factor, conditioned medium was collected from monolayer cultures of Day 17 and Day 21 fibroblasts (Fig. 2). When Day 19 fetal rat lung type II cells in monolayer culture were exposed to Day 17 fibroblast conditioned medium in the presence of rH]choline there was no effect on [3H]SPC synthesis; Day 21 fibroblast conditioned medium stimulated [3H]SPC synthesis 40%. However, when both Day 17 and Day 21 conditioned
T 1
control
dl7
I21
117
+
d21
d17
+
d2l
TGF;
Ab
FIG. 1. Heterochronic cell mixing experiment. Day 1’7 and Day 21 fetal rat lung fibroblasts were combined with Day 19 fetal rat lung type II cells in organotypic culture for 24 hr in minimal essential medium without serum. The bar on the far right represents data for cells cultured in the presence of an antibody to TGFb (100 pg/ml). For the last 6 hr the cultured cells were incubated with [3H]choline (1 j&i/ml) and the type II cells were reisolated and analyzed for [3H]SPC content. Each bar represents the mean + SD of five values from two exneriments. *P < 0.01 bv analvsis of variance (n, = 10).
151413-
s
11
H f 0
10
** ** * nII1 37
Cell Maturation
i l2
L MEM
dl7CM
d21CM
dl$CM
Ipg
d21CM
,
lOccI3 1owJ + TGFB Ab , dl7CM+ d2lCM
FIG. 2. Production of FPF blocking activity by Day 17 fibroblasts. Day 19 fetal rat lung type II cells in monolayer culture were exposed to conditioned media from Day 17 and Day 21 fibroblasts in the presence of [3H]choline (1 &i/ml) and subsequently analyzed for [3H]SPC content. Where indicated, the fibroblast conditioned medium was preincubated with TGFb antibody (l-100 rg/ml) and then added to the bioassay. The height of the bars represents the mean + of at least five values from two experiments (n = 10).
media were added to the test system in equal amounts, there was no detectable stimulatory activity. Subsequently, a dose-response curve was generated for the effect of Day 17 conditioned medium on Day 21 conditioned medium (Fig. 3)-there was a linear inhibitory response between 1:lO and 1200 dilutions. On further preliminary biochemical characterization of the blocking activity in Day 17 conditioned medium,
* 1IL T*
Blocks
2
15
z w
14 t
0
3 b :
13 I
I
1 10-4
1 10-3 CM/ml
1 10-2 ( -1
1 10-l
,r, r10-1
I
I
1
I
I
0
1
2
3
4
TGFB (r&ml)
,
76
(*-a)
FIG. 3. Dose-response curves for bioactivity of Day 17 fibroblast conditioned medium and TGF@. Day 19 fetal rat lung type II cell monolayers were incubated with rH]choline in the presence of Day 21 fetal rat lung fibroblast conditioned medium and either Day 17 fetal rat lung fibroblast conditioned medium (CM) or TGF@ at the indicated concentrations. Each data point represents the mean f SD of four values from three experiments (n = 12).
38
DEVELOPMENTAL BIOLOGY
it was found that it is nondialyzable trypsin sensitive, and heat resistant
(cutoff, 3500 MW), (56’C for 3 min).
Neutralization of Day 17 CM Blocking with TGFfl Antibody
Activity
Preincubation of the mixture of Day 17 and Day 21 conditioned media with an antibody to TGFP (TGFP, Ab, 100 pg/ml) for 1 hr at 37°C neutralized the blocking activity found in the Day 1’7 conditioned medium (Fig. 2); incubation of this mixture with rabbit IgG had no effect on the blocking activity, nor was there any direct inhibitory effect of TGFP Ab on Day 21 conditioned medium (data not shown). Furthermore, there was a dose-response relationship when decreasing amounts of TGF/3 Ab (100-l pg/ml) were used in the bioassay (Fig.,2). Antibody to EGF, PDGF, and bFGF had no effect on Day 1’7 conditioned medium (data not shown). Efect of TGFP on FPF Stimulatim of [‘H]SPC Synthesis by Day 19 Type II Cells Since the blocking activity in Day 17 conditioned medium was neutralized by TGF/? Ab, we tested the effect of TGFP itself on FPF stimulation of rH]SPC synthesis. TGFP blocked Day 21 conditioned medium stimulatory activity in a dose-response fashion between 0.5 and 4.0 rig/ml (Fig. 3). Comparison of the half-maximal inhibitory activities of TGFP and Day 17 conditioned medium (Fig. 3) leads to the conclusion that there is the equivalent of approximately 200 ng of TGFP/ml of Day 17 conditioned medium. Since 10 fig of TGFP antibody was found to be half-maximally effective in neutralizing the endogenous TGFB-like activity in conditioned medium (Fig. 2) we tested the neutralizing activity of the antibody on a 200 rig/ml solution of TGFP and found that at 10 pg/ml the TGFP antibody inhibited approximately half of the TGF@ activity, further supporting the identification of the endogenous inhibitor as TGFP. As a check that the TGF/3 was specifically affecting the assay system, TGFP Ab was coincubated with TGFP for 1 hr at 37°C and the incubation product was tested in the bioassay and found to be inactivated; moreover, TGF/3 itself had no effect on baseline C3H]SPC synthesis, again indicating that its mechanism of action is as an antagonist. In other experiments, it was found that once stimulated by FPF, the TGF@ had no effect, again indicating that the TGFP is acting as an antagonist to FPF rather than as an inhibitor of rH]SPC synthesis. Eflects of TGFfl and TGFP Antibody on FibroblastType II Cell Interactions in Organotypic Culture To determine whether the TGFP-like activity in Day 17 conditioned medium is related to the originally described effect of Day 17 fibroblasts on the Day 21 fibro-
VOLUME 139,199O
blast-Day 19 type II cell interaction in organotypic culture, TGF/3 was added to the Day 21 fibroblast/Day 19 type II cell combination as described above and the rate of [3H]choline incorporation into [3H]SPC was measured (Fig. 1). TGFP at the half-maximal dose used in the conditioned medium experiments completely blocked the stimulatory effect of Day 21 fibroblasts; when TGFP Ab was added it neutralized the effect of the TGF& while having no significant effect by itself. Conversely, when Day 17 fibroblasts were cocultured with Day 21 fibroblasts and Day 19 type II cells in organotypic culture in the presence of TGFP Ab, the blocking effect of the Day 17 fibroblasts was neutralized, while TGF/3 Ab had no effect on [3H]SPC synthesis by Day 19 type II cells in the presence of Day 17 fibroblasts alone. Ontogeny of TGF@-like Activity Rat Lung Fibroblasts
by Fetal
When conditioned medium from Day 15 to Day 21 fetal rat lung fibroblasts was assayed for FPF blocking activity, it was found that there was activity from Day 15 to Day 19, but that the activity decreased significantly between Days 19 and 21, being undetectable by Day 21 (Fig. 4, top). The ontogeny of FPF activity is shown for the sake of comparison (Fig. 4, bottom). TGF@ Expression
by Fetal Rat Lung Filmblasts
Total RNAs from 17-, 19-, and 21-day-old fetal rat lung fibroblasts were subjected to Northern blot analysis and probed for TGFP. As can be seen in Fig. 5, TGFP is expressed by these cells on Day 17. Transcript levels for Days 19 and 21 (not shown) are similar to those seen on Day 17, suggesting that regulation of TGF/3 occurs at a post-translational site. DISCUSSION
Normal development of the lung and other organs is regulated by mesenchymal-epithelial interactions and the timing of these events can be accelerated or delayed by various hormones (Van Golde et aL, 1988). Glucocorticoids are well-recognized accelerators of lung maturation, enhancing fetal lung surfactant phospholipid synthesis via a mechanism involving mesenchymal-epithelial interactions (Smith, 1979). The fetal lung fibroblast produces a low molecular weight polypeptide (FPF) in response to glucocorticoids which accelerates surfactant phospholipid synthesis (Floros et aL, 1985). Androgens, on the other hand, delay surfactant synthesis in vivo (Nielsen et aL, 1982) and in vitro (Torday, 1985), by delaying FPF expression (Floros et aL, 1987) and production by fetal lung fibroblasts (Floros et al, 1987). At the molecular level, the fetal lung fibroblast qualifies as a target for circulating fetal adrenal hormone for several reasons: 11-oxidoreductase, which is neces-
TORDAY
AND
KOUREMBANAS
39
TGF/3 Homolog Blocks Cell Maturation
TGF/3 was first identified by its ability to cause phenotypic transformation of rat fibroblasts (Roberts, 1981) and is now recognized to have numerous regulatory actions in a wide variety of both normal and neoplastic cells (Sporn et aZ., 1986). Using immunolocalization techniques, Heine et al., (1987) have shown that TGFP is expressed in mesenchymal cells during mouse development, usually during critical phases of morphogenesis, particularly at times when cell-cell interactions between mesenchyme and epithelium are critically important for normal epithelial cell differentiation. In support of the physiological role of TGFP during morphogenesis, McLachlan et al, (1988) have established that during limb formation TGFP is present
day
I
I
I
I
1
I6
I7
I8
I9
20 DAYS
I
21
17 Fibs
1
22
GESTATION
FIG. 4. Ontogeny of TGF@-like activity (upper panel). Day 19 fetal rat lung type II monolayer cultures were incubated with [3H]eholine in the presence of Day 21 fetal rat lung fibroblast conditioned medium and conditioned medium from Day 15-21 fetal rat lung fibroblasts. Each data point represents the mean + SD of four values from three to five experiments. Ontogeny of FPF activity (lower panel). Same protocol as upper panel without addition of Day 21 fetal rat lung fibroblast conditioned medium. Each data point represents the mean + SD of four values from three to five experiments (n = 12-20).
28s
TGFP 18s
sary for activation of circulating 11-oxidized nonactive glucocorticoid (Bush, 1956), is present in fetal lung fibroblasts (but not type II cells) (Torday, 1985); high affinity glucocorticoid receptors are also present in fetal rat lung fibroblasts (Ballard et aZ., 1978), though they are also present in type II cells (Ballard et aL, 1978); and fetal lung fibroblasts can activate various androgens via 5cu-reductase (Sultan et aZ., 1980) and bind androgen via high affinity receptors (Sultan et aL, 1980). It is because the fetal lung is a target for these steroid hormones that we hypothesized that the immature fibroblast might also produce a soluble factor which would play a role in the regulation of alveolar epithelial cell maturation-in this case acting to block the subsequent maturational step, glucocorticoid-stimulated type II cell maturation via FPF.
FIG. 5. Northern hybridization of mRNA from Day 17 fetal rat lung fibroblasts using TGFP cDNA as a probe. Total cellular RNA was extracted from Day 17 fetal rat lung fibroblasts and 20 pg of RNA were electrophoretically separated on a formaldehyde-1.2% agarose gel and blotted onto nitrocellulose filters. A 32P-labeled cDNA probe was used under high stringency conditions. The positions of 28 S and 18 S ribosomal RNAs are shown for reference.
40
DEVELOPMENTAL BIOLOGY
in conditioned medium from chick limb and tail buds. TGFP has been shown to block the maturation of various cell lines (Keski-Oja, 1988; Sparks and Scott, 1986; Morris et aZ., 1988; Liu et al., 1988) as well as that of normal cells in primary culture (Barnard et ah, 1989); Pfeilschifter et al, 1988; Chenu et ah, 1988; Allen and Boxhorn, 1987). Though little is known about the role of TGFP in normal lung development, Masui et al. (1986) have shown that TGFfl can block the maturation of bronchial epithelial cells, both normal and transformed, and Crystal’s group has shown that TGF@ is present in high levels in the epithelial lining fluid from normal adult lung (Yamauchi et aZ., 1988). As to a role in normal lung development, Whitsett and his co-workers (1987) have shown that exogenous TGFP can block both epidermal growth factor-dependent and basal surfactant-associated protein A synthesis by human fetal lung explants in culture. We have made similar observations with regard to the effect of TGFP on such human fetal lung explants using surfactant-associated phospholipid synthesis as a marker of maturation (Torday, 1989). The present studies further support the role of TGF/3 during normal lung maturation. Although the existence of an endogenous mesenchyma1 “antagonist” to maturation would not necessarily be predicted by prior studies of fetal lung development, in retrospect these studies fit with several key characteristics of this process. Buckingham and Avery (1962) had noted in their early study of mouse lung maturation that the number of lamellar bodies per type II cell increased in a discontinuous fashion, i.e., from none to several, rather than finding type II cells with an arithmetically increasing number per cell, a finding confirmed by Post and Smith (personal communication) in observing isolated fetal rat lung type II cells (Post and Smith, 1988). Another observation consistent with the existence of an endogenous “inhibitor” is the observed centripetal development, both morphologic and biochemical, of the lung (Sorokin, 1965) which would not be expected in a simple diffusional system. Yet another reason to think that endogenous inhibitors of maturation exist is the observation by Rutter (1967) and others (Wessels and Cohen, 1967) of the so-called protodifferentiated state in which epithelium is induced to become what it is destined to be but does not elaborate its differentiated product, suggesting that there is a native inhibitor of maturation. Consistent with this concept is the observation of automaturation of lung (RousseauMerck, 1976), gut (Black and Moog, 1977), and kidney (Skea and Nemeth, 1969) in vitro, which has been speculated to be due to the removal of the organ from its native environment (Rousseau-Merck, 1976), in which a putative (Rousseau-Merck et al, 1976; Black and Moog, 1977; Skea and Nemeth, 1969) inhibitor blocks maturation of these organs.
VOLUME 139,199O
By Northern blot analysis, TGFP messenger RNA levels in 17-, 19-, and 21-day-old fibroblasts remained constant, unlike the ontogenetic changes seen when the bioactivity in fibroblast conditioned media was tested. These observations suggest that regulations of TGFP occurs at a post-translational level, consistent with reports that TGFP is synthesized as a large precursor molecule which is subsequently cleaved to its bioactive form (Miyazono, 1988). The emerging picture, therefore, is one of a developing lung alveolar environment in which the immature mesenchyme actively produces TGF& or a homolog, and maturation is dependent on activation by such classic maturation stimulators as glucocorticoids or thyroid hormone. We thank Dr. Rik Derynck for his gift of the TGF/3 cDNA probe. This study was supported by a National Heart, Lung, and Blood Institute Grant-HL34616-05 (Respiratory Disorders of Neonates and Children). REFERENCES ALLEN, R. E., and BOXHORN, L. K. (1987). Inhibition of skeletal muscle satellite cell differentiation by transforming growth factor 0. J. Cell Physiol 133,567572. BALLARD, P. L., MASON, R. J., and DOUGLAS, W. H. J. (1978). Glucocorticoid binding by isolated lung cells. Endocrinology 102, 1570-1575. BARNARD, J. A., BEAUCHAMP, R. D., COFFEY, R. J., and MOSES, H. L. (1989). Regulation of intestinal epithelial cell growth by transforming growth factor type p. Proc. Natl. Acad. Sci. USA 86, 15781582. BLACK, B. L., and MOOG, F. (1977). Goblet cells in embryonic intestine: Accelerated differentiation in culture. Science 197,368-370. BLIGH, E. G., and DYER, W. J. (1959). A rapid method of total lipid extraction and purification. Canad J. Biochem. Physiol. 37,911-917. BUCKINGHAM, S., and AVERY, M. E. (1962). Time of appearance of lung surfactant in fetal mouse. Nature (Lwdm) 193,688-689. BUSH, I. E. (1956). The 11-oxygen function in steroid metabolism. Eqerientia 12,325-331. CHENU, C., PFEILSCHIFTER, J., MUNDY, G. R., and ROODMAN, G. D. (1988). Transforming growth factor fl inhibits formation of osteoclast-like cells in long-term human marrow cultures. Proc. Natl. Acad
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CHIRGWIN, J. M., PRYZYBA, A. E., MACDONAL, R. J., and RUTTER, W. J. (1979). Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry l&5294-5299,1979. DERYNCK, R., JARRETT, J. A., CHEN, E. Y., EATON, D. H., BELL, J. R., ASSOIAN, R. K., ROBERTS, A. B., SPORN, M. D., and GOEDDEL, D. V. (1985). Human transforming growth factor-p complementary DNA sequence and expression in normal and transformed cells. Nature (London) 316,701-705. DOUGLAS, W. H. J., and TEEL, R. W. (1976). An organotypic in vitro model system for studying pulmonary surfactant production by type II alveolar pneumonocytes. Amer. Rev. Respir. Dis. 113.1723. FLOROS, J., POST, M., and SMITH, B. T. (1985). Glucocorticoids affect the synthesis of pulmonary fibroblast-pneumonocyte factor at a pretranslational level. J. BioL Chem. 260,2265-2267. FLOROS, J., NIELSEN, H. C., and TORDAY, J. S. (1987). Dihydrotestosterone blocks fetal lung fibroblast-pneumonocyte factor at a pretranslational level. J. BioL Chem 262,13,592-13,598.
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TGFfl
GROBSTEIN, C. (1967). Mechanisms of organogenetic tissue interaction. NatL Cancer Inst. Monogr. 26,279-299. HEINE, V. I., MUNOZ, E. F., FLANDERS, K. C., ELLINGSWORTH, L. R., LAM, H. Y. P., THOMPSON, N. L., ROBERTS, A. B., and SPORN, M. B. (1987). Role of transforming growth factor-0 in the development of the mouse embryo. J. Cell Biol. 105,2861-2876. KESKI-OJA, J., BLASI, F., LEOF, E. B., and MOSES, H. L. (1988). Regulation of the synthesis and activity of urokinase plasminogen activator in A549 human lung carcinoma cells by transforming growth factor-p. J. Cell BioL 106,451-459. LIU, C., TSAO, M. S., and GRISHAM, J. W. (1988). Transforming growth factors produced by normal and neoplastically transformed rat liver epithelial cells in culture. Cancer Res. 48, 850-855. MASUI, T., LECHNER, J. F., YOAKUM, G. H., WILLEY, J. C., and HARRIS, C. C. (1986). Growth and differentiation of normal and transformed human bronchial epithelial cells. J. CeU Physiol. 4, (Suppl), 73-81. MASON, R. J., NELLENBOGEN, J., and CLEMENTS, J. A. (1976). Isolation of disaturated phosphatidylcholine with osmium tetroxide. J. Lipid Res. 17,281-284. MCLACHLAN, J. C., MACINTYRE, J., HUME, D. D., and SMITH, J. (1988). Direct demonstration of production of transforming growth factor activity by embryonic chick tissue. Experientia 44,351-352. MIYAZONO, K., HELLMAN, V., WERSTEDT, C., and HELDIN, C.-H. (1988). Latent high molecular weight complex of transforming growth factor-/3. J. BioL Chem. 263,6407-6415. MORRIS, J. C., RANGANATHAN, G., HAY, I. D., NELSON, R. E., and JIANG, N-S. (1988). The effects of transforming growth factor-p on growth and differentiation of the continuous rat thyroid follicular cell line, FRTL-5. Endocrinology 123,1385-1394. NIELSEN, H. C., ZINMAN, H. M., and TORDAY, J. S. (1982). Dihydrotestosterone inhibits fetal rabbit pulmonary surfactant production. J. Clin. Invest. PFEILSCHIFTER,
69,611-616. J., SEYEDIN,
S. M., and MUNDY, G. R. (1988). Transforming growth factor fi inhibits bone resorption in fetal rat long bone cultures. J. Clin. Invest. 82,680-685. POST, M., FLOROS,J., and SMITH, B. T. (1984). Inhibition of lung maturation by monoclonal antibodies against fibroblast-pneumonocyte factor. Nature (London) 308,284-286. POST, M., TORDAY, J. S., and SMITH, B. T. (1984). Alveolar type II cells isolated from fetal rat lung organotypic cultures synthesize and secrete surfactant-associated phospholipids and respond to fibroblast-pneumonocyte factor. Exp. Lung Res. 6,53-65. POST, M., and SMITH, B. T. (1988). Histochemical and immunocytochemical identification of alveolar type II epithelial cells isolated from fetal rat lung. Amer. Rev. Respir. Dis. 137,525-530. ROBERTS, A. B., ANZANO, M. A., LAMB, L. C., SMITH, J. M., and SPORN, M. B. (1981). New class of transforming growth factors potentiated by epidermal growth factor: Isolation from non-neoplastic tissues. Proc.
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ROUSSEAU-MERCK, M. F., WYLLIE, L., BASSET, F., and NEZELOF, C. (1976). In vitro behavior of human fetal lung maintained in organ culture. Virchows Arch. A: PathoL Anat. HistoL 371,305-321. RUTTER, W. J., BALL, W. D., BRADSHAW, W. S., CLARK, W. R., and SANDERS, T. G. (1967). Levels of regulation in cytodifferentiation in morphology and biochemical aspects of cytodifferentiation. Exp. Biol. Med 1, 110-124.
Homolog
Blocks
Cell
Maturation
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