of biological vol. of 15, pp. 1987 of printed in u. a ...vol. 262, no. 11, issue of april 15, pp....

Vol. 262, No. 11, Issue of April 15, pp. 5256-5261, 1987 Printed in U. S. A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY (0 1987 by The American Society of Biological Chemists, Inc.

Induction of Surfactant Protein in Fetal Lung EFFECTS OF cAMP AND DEXAMETHASONE ON SAP-35 RNA AND SYNTHESIS*

(Received for publication, July 22, 1986)

Jeffrey A. Whitsettz, Tami Pilot§, Jean C. Clark, and Timothy E. Weaver From the University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0541 and SAbbott Laboratories, North Chicago, ~. Illinois 60064

Synthesis of surfactant-associated glycoprotein of M, = 30,000-35,000 (SAP-35) was induced in explant culture of human fetal lung otained from 8 to 24 weeks of gestation. SAP-35 synthesis and content increased markedly during 1-5 days in organ culture in association with the morphologic maturation of Type 11 epithelial cells and the appearance of lamellar bodies. [T3] Methionine labeling of the explants and subsequent immunoprecipitation of SAP-35 demonstrated distinct high-mannose precursors and sialylated SAP-35 forms as early in culture as SAP-35 synthesis was detectable. The increase in SAP-35 synthesis was associated with increased SAP-35 RNA of 2.1 kilobases as assessed by hybridization assay with [32P]cDNA specific for human SAP-35. Specific SAP-35 RNA increased during organ culture and both SAP-35 content and SAP-35 RNA increased in the absence of exogenous hormones in 2% carbon-stripped fetal calf serum. SAP-35 content and synthesis was stimulated by 8-Br-CAMP. Addition of 100 p~ 8-Br-cAMP, enhanced both the concentration of SAP-35 protein and the SAP-35 RNA as assessed by hybridization assay. In contrast, treatment of the explants with dexamethasone was associated with decreased SAP-35 protein synthesis, SAP-35 content, and decreased SAP-35 RNA levels compared to untreated explants. Inhibition by dexamethasone occurred at all gestational ages tested, was dose-dependent, and detectable within 24-48 h during organ culture. Dexamethasone significantly inhibited both basal and CAMP-induced SAP-35 synthesis.

Induction of pulmonary surfactant protein (SAP-35) synthesis during organ culture of human fetal lung was associated with increased SAP-35 RNA. SAP-35 synthesis and SAP-35 RNA were inhibited by dexamethasone and enhanced CAMP.

Protein of M , = 30,000-35,000, herein termed surfactant- associated protein (SAP-35),’ is the predominant glycoprotein

* This work was supported in part by Grants HL 28623, HD 11725, Research Career Development Award HL 10124 (J. W.) and Training Grant HD 07200 (to T. W.) from the National Institutes of Health and a grant from the Children’s Hospital Research Foundation, Cincinnati, OH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 T o whom correspondence should be addressed: University of Cincinnati, College of Medicine, Pediatrics/Neonatology Division, 231 Bethesda Ave., Cincinnati, OH 45267-0541.

The abbreviations used are: SAP-35: surfactant-associated protein M , = 30-35,000; 8-Br-CAMP: 8-bromo-3’,5’-cyclic adenosine monophosphate; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; ELISA, enzyme-linked immunosorbent assay.

associated with surfactant phospholipids and has been isolated from the lungs of numerous mammalian species (1, 2). SAP-35 is synthesized from primary translation products of M , approximately 30,000 (3, 4) and is encoded by a DNA sequence predicting 228 amino acids (5). Size and charge heterogeneity of SAP-35 is related in part to both acetylation and the presence of asparagine-linked oligosaccharides containing sialic acid (6-10). In late gestation SAP-35 content increases in amniotic fluid (11-14) and lung tissue (13). Re- cent work from our laboratory has also demonstrated the induction of SAP-35 synthesis during organ culture of human fetal lung occurring in the absence of serum or exogenous hormones. Such induction of SAP-35 synthesis was not observed in dissociated pulmonary cells during monolayer culture. In those studies, SAP-35 RNA was abundant in adult lung and was undetectable in fetal human lung as determined by in vitro translation assay (15).

SAP-35 is synthesized primarily by pulmonary Type I1 epithelial cells; however, recent work also demonstrates its presence in the Clara cell (16, 17). Pulmonary epithelial cells undergo dramatic morphologic maturation in late gestation in association with appearance of Type I1 epithelial cells and increased surfactant phospholipid synthesis. Morphologic and biochemical maturation of the fetal lung epithelium is altered by a variety of hormonal influences including corticosteroids, thyroid hormones, 3’,5’-cAMP, or cyclic nucleotide phosphodiesterase inhibitors, which enhance surfactant phospholipid synthesis both i n vitro and i n uiuo, see Ref. 18 for review. Although SAP-35 levels increased in temporal association with increased phospholipid synthesis during late gestation in the rat (13), factors regulating the synthesis of surfactant proteins have not been elucidated. The present work describes contrasting effects of cAMP analogues and dexamethasone on the induction of both SAP-35 synthesis and specific SAP- 35 RNA levels during explant cultures of second trimester human fetal lung.

MATERIALS AND METHODS

Purification of Human SAP-35“Surfactant was purified from lung lavage fluid from human volunteers and patients with alveolar proteinosis, from amniotic fluid obtained at term Cesarean section, and from lung lavage of human cadavers a t autopsy. All protocols were approved by the Human Research Committee, University of Cincin- nati College of Medicine. Human surfactant was purified by differ- ential centrifugation and SAP-35 purified to homogeneity by prepar- ative isoelectric focusing and Cibacron blue affinity chromatography, as previously described for canine SAP-35 (19).

Explant Culture-Tissue was obtained from consenting donors from pathologic specimens at this institution and from the National Diabetes Research Interchange (Philadelphia, PA), in accordance with a protocol approved by the Human Research Committee, Uni- versity of Cincinnati College of Medicine. Tissue was immediately placed in iced minimum essential medium (GIBCO, Grand Island, NY) at 4 “C and transported in ice to this laboratory within 24 h.

5256

Induction of Surfactant Protein in Fetal Lung 5257

Lung tissues were minced to l-mm3 pieces using a McIlwain tissue chopper and placed on scratched surfaces of 60-mm plastic dishes to which was added Dulbecco's minimum essential medium or Wey- mouth's media with 2% carbon-stripped fetal calf serum (20) containing 100 units/ml penicillin and 0.1 pg/ml gentamicin. Hormones were added to the culture media and maintained throughout the incubation. Explant cultures were placed on a rocker platform at 3 cpm to immerse and expose the explants during the culture period, as described by Gross and Wilson (21). Media was changed on the second or third day of 4-5 days culture. Metabolic labeling of tissues and cells was performed in methionine-deficient medium (I mg/l unla- beled methionine) in which carbon-stripped fetal calf serum was replaced with 0.1% bovine serum albumin. Following a 30-min equil- ibration period, [35S]methionine (New England Nuclear, Boston, MA), specific activity = 1000 Ci/mmol, was added to a final concentration of approximately 150 pCi/ml and the tissues were incubated for 3-6 h. [35S]Methionine-labeled tissues were prepared for electrophoretic analysis after homogenization in 150 mM NaCl, 6 mM EDTA, 0.5% SDS, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 30 mM Tris-HCI, pH 7.5. Cellular debris was removed by centrifugation at 9000 X g (4 "C) for 5 min in an Eppendorf microcentrifuge. An aliquot of the supernatant was precipitated with trichloroacetic acid for determination of [35S]methionine incorporation. [35S]Methionine incorporation was normalized among the samples prior to specific immunoprecipitation of SAP-35 (15). Results represent findings obtained from 15 separate tissue samples from lung obtained from 8 to 24 weeks of gestation. After weighing, the protein content of the explants was determined by the method of Lowry (22) using bovine serum albumin as standard. DNA content was determined by the method of Zamenhof et al. (23) using calf thymus DNA as standard. Morphologic analysis by light and electronmicroscopy was performed by standard techniques as previously described (15).

Analysis of Protein-Antisera were generated against SAP-35 purified from human alveolar proteinosis lavage as previously described (15). Purified protein (100-200 pg) was suspended in Freund's com- plete adjuvant and injected into New Zealand albino rabbits. The resultant monospecific antiserum recognized only SAP-35 in immunoblots of crude human alveolar lavage and term amniotic fluid (not shown) and, after adsorption with a human serum-Sepharose affinity column, did not react with immunoblots of human serum.

Methods of immunoprecipitation of labeled proteins from cell lysates and media have been described elsewhere (3, 6, 9). Protein samples were analyzed under reducing conditions by SDS-polyacrylamide gel electrophoresis (13%) or by two-dimensional isoelectric focusing, pH 3.5-5.8, polyacrylamide gel electrophoresis (13%), as described by Garrison and Wagner (24). Proteins were electrophoret-

The [35S]methionine-labeled SAP-35 was then exposed to Kodak ically transferred to nitrocellulose and analyzed by autoradiography.

XAR-2 film at -70 "C. ELISA Assay-Tissue was homogenized in approximately 10 vol-

umes of buffer containing 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, 0.1% Nonidet P-40, 50 mM Tris-HCI, pH 7.4. Protein was assessed after a further 100-fold dilution by the method of Lowry in 0.001% Nonidet P-40 using bovine serum albumin diluted in the same buffer as the standards. A two-antibody capture ELISA was utilized to measure SAP-35 content following methods described by Katyal and Singh (13). Goat anti-SAP-35 immunoglobulin was prepared by repeated NH,SO, precipitation and used as the primary capture antibody added to the plastic ELISA plate at 1:lOO dilution. Rabbit anti-SAP-35 was used as the second antibody (1:500) and the assay developed using horseradish-conjugated goat-anti-rabbit (Miles Inc.) using @phenylenediamine as substrate. Standard SAP-35 and tissue samples were diluted in the homogenizing buffer. Standard curves were generated between 1 and 100 ng of purified SAP-35 and were entirely linear (regression coefficient = 0.90-0.99) over the range used for the assay. Duplicate samples generally varied less than 10%. SAP-35 content was determined at 3-4 dilutions of each tissue homogenate and each dilution was assayed in duplicate within the linear portion of the assay curve. Statistical analysis of differences in SAP-35 content and SAP-35 RNA were analyzed using an Apple IIE computer using ANOVA with correction for multiple group comparisons, ANOVA for nonparametric comparisons, or Student's paired t test where appropriate.

RNA Extraction for Hybridization Experiments-Approximately 30 mg of the explant tissues was homogenized in buffer containing 4 M guanidine thiocyanate, 0.5% N-lauroyl sarcosine, 20 mM sodium citrate, 0.1 M @-mercaptoethanol, and 0.1% Antifoam A. RNA was

extracted either by precipitation in lithium chloride (25) or by centrifugation through a cushion of 5.7 M cesium chloride (26). The RNA pellet was dissolved in solubilizing buffer containing 0.1% SDS, 1 mM EDTA, 10 mM Tris-HC1, pH 7.5, or in water, extracted with phenol and chloroform, and precipitated with ethanol. The amount of RNA in an aqueous solution was determined by optical density at 260 nm. In order to minimize variability among samples, control and treated explants were processed and analyzed in parallel using the same lung specimens for SAP-35 content, synthesis, and RNA.

For Northern blot analysis, approximately 15 pg of total RNA prepared from 100 mg of tissue was separated on a 1.2% agarose formaldehyde gel (27) and transferred to nitrocellulose. The filter was baked at 80 "C for 2 h. Nitrocellulose blots were made by applying 500 or 250 ng of formaldehyde-denatured RNA to nitrocellulose using a "Slot" blot manifold (Schleicher and Schuell).

SAP-35 cDNA-Nitrocellulose filters containing RNA were hybridized with an 0.8-kilobase cDNA clone isolated from a Xgtll expression library generated from poly(A+) RNA purified from adult human lung. The cDNA consists of nearly the entire coding sequence of SAP-35. The entire sequence analysis of this cDNA demonstrated close identity to that published by White et al. (5) and will be described in detail elsewhere. The SAP-35 cDNA prohe was labeled with [cI-~'P]~CTP usinga nick translation reagent kit purchased from Bethesda Research Laboratories. In some experiments, filters were washed free of SAP-35 probe and reprobed in an identical fashion with a cDNA from the 3' untranslated region of human fetal liver B- actin, the kind gift of Dr. L. H. Kedes, Stanford University.

Hybridization Assay-Optimal conditions for hybridization were determined in preliminary experiments with varying methods of RNA preparation, hybridization, and washing conditions. The filters were prehybridized in 40% deionized formamide, 4 X SSC (1 X SSC is 0.15 M NaCI, 0.015 M sodium citrate), 40 mM NaH'PO,, pH 7.0, 1.4 X Denhardt's solution (1 X Denhardt's is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinyl pyrollidine), 0.1% SDS, and 100 pg/ml denatured salmon sperm DNA at 41 "C overnight. RNA was hybridized in the same solution at 41 "C overnight with approximately 5 X lo6 cpm ml" 32P-labeled SAP-35 cDNA. Following hybridization, filters were washed four times at room temperature with 2 X SSC, 0.2% SDS, once at 41 "C with the same solution and once with 0.2 X SSC, 0.2% SDS at 50 "C. Filters were wrapped between two sheets of Saran Wrap and exposed to Kodak XAR-2 film. After exposure to 32P-labeled SAP-35 probe, filters were washed in 0.05 X SSC, 0.05% SDS at 65 "C for 3 h, and reprobed with 32P-labeled @-actin cDNA. This cDNA is a 404-base pair fragment of the 3' untranslated region of human cytoplasmic &actin and is constitutively expressed in most human tissues (28).

RESULTS

Morphological analysis of the lung tissue (8-24 weeks gestation) prior to explant culture demonstrated the undifferen- tiated appearance of the respiratory epithelium as previously described (15). In samples from second trimester gestation, epithelial cells contained large glycogen stores and were lacking lamellar bodies. During explant culture, characteristic Type I1 cells were observed in association with appearance of lamellar bodies. Morphologic maturation was observed in serum-free or 2% or carbon-stripped fetal calf serum containing media. In preliminary experiments, induction of SAP-35 content was consistently higher in Weymouth's medium than in Dulbecco's modified Eagle's medium. Cellular outgrowth on the edges of the explants observed by phase-contrast microscopy and morphologic integrity of the interstitium were better preserved in the presence of 2% carbon-stripped serum than in serum-free medium. Thus, Weymouth's media with 2% carbon-stripped fetal calf serum was chosen for the experiments reported. Total protein and DNA content did not change significantly during organ culture 1-4 days and were not significantly altered by the addition of dexamethasone or 8-Br-CAMP (Table I).

SAP-35 Content-Prior to culture, SAP-35 was undetectable or barely detectable in lung tissue from 15-24 weeks gestation as determined by the ELISA assay. SAP-35 did not vary significantly with gestational age (15-24 weeks) of the

5258 Induction of Surfactant Protein in Fetal Lung TABLE I

Tissue protein to DNA ratio (pg/pg) Protein and DNA content were determined in triplicate in three

separate samples a t 18-22 weeks of gestation and sampled prior to culture. Lung tissue was also assessed during explant culture in Weymouth’s media with 2% carbon-stripped fetal calf serum on day 1 and day 4 cultured in the presence and absence of 100 ~ L M 8-Br- cAMP or 10 p~ dexamethasone. The n represents the number of separate determinations from distinct explants obtained from three separate tissue samples. Values represent mean f S.D. There were no significant differences related to the time in culture or addition of dexamethasone or 8-Br-CAMP.

Control 8-Br-CAMP Dexamethasone

24 h 7.7 f 2.7 6.4 f 1.9 7.7 f 3.0

96 h 7.8 f 2.4 6.3 f 2.9 6.8 -C 2.6 n = 11 n = 9 n = 6

n = 9 n = 6 n = 6

Prior to culture 7.47 f 0.31, mean f S.D.

TABLE I1 SAP-35 content in adult and fetal lung tissue

Adult lung tissue was obtained from a young adult with no history of lung disease at the time of death and immediately frozen in liquid nitrogen. SAP-35 content was determined in three separate samples obtained from the lung parenchyma. Fetal lungs were sampled prior to explant culture. When SAP-35 content was less than the lowest standard utilized in the assav. the value is listed as <0.1 udme.

TABLE I11 Effect of dexamethasone and 8-Br-CAMP on SAP-35 content

Tissue from lungs a t 16-24 weeks gestation were placed iE culture in Weymouth’s media with or without 10 p~ dexamethasone or 100 p~ 8-Br-CAMP from 4 days or 5 days (n = separate fetal lungs). SAP-35 was determined by ELISA assay as described under “Mate- rials and Methods.” Significance of the differences among the groups was determined by ANOVA for rank sum as described by Kruskal- Wallis (48). Content of SAP-35 prior to culture was less than 0.1 pg/ mg in these tissues.

SAP-35 pg/mg protein

Control 8-Br-CAMP Dexamethasone

Weeks SAP-35 gestation content

p d w a

Fetal 15 0.04 16 <0.1 18 <o. 1 19 <0.1 20 0.07 (0.05-0.09) 24 0.1

Adult 24 f 2 (n = 3)

Day 4 1.45 f 0.37 6.03 f 1.38” 0.29 f 0.07’ n = 12

Day 5 1.37 f 0.49 7.3 ? 3.4‘ n = 11

0.43 f O.lgd n = 10

n = 9 n = 7 n = 6 ’ 8-Br-CAMP > control p < 0.001. ’ Dexamethasone < control p < 0.001. E 8-Br-CAMP > control p = 0.02.

Dexamethasone < control p = 0.06.

20 week explant

25 I 8-Br-CAMP (100 pM)

SAP-35 ( w m s ) 1

5 ,LLL 0 .-a -./:-:-:

0 1 2 3 4 5 DAYS

FIG. 1. The course of SAP-35 content during explant culture of 20 week fetal lung. SAP-35 content was determined in replicate samples of the explants taken during 1-5 days of organ culture in Weymouth’s media in 2% carbon-stripped fetal calf serum. Explants were cultured in the presence or absence of 100 p~ 8-Br- cAMP and SAP-35 determined by ELISA assay as described under “Materials and Methods.” The experiment is representative of five similar experiments with tissue from 15-24 weeks gestation.

a FIG. 2. Effects of 8-Br-CAMP and dexamethasone on incor-

poration of [35S]methionine into SAP-35 after explant culture. On day 4 of culture, explants were homogenized, normalized to 3 X IO6 cpm, immunoprecipitated, subjected to two-dimensional electrophoresis, and exposed to film for 4 days. Charge heterogeneity, indicative of sialylated SAP-35, is evident in control explants (a) and in explants treated with 100 p~ 8-Br-CAMP ( b ) and (c) 10 p~ dexamethasone. The pH gradient increases from left to right. Panel d demonstrates electrophoretic analysis of the induction of SAP-35 synthesis by 0, 0.1, 10, and 100 p~ and 1 mM 8-Br-CAMP on day 4 of explant culture. The charge trains seen on the two dimensional isoelectric focusing polyacrylamide gel electrophoresis migrate with PI 4.6-5.2, M , approximately 30,000-36,000.

tissue (Table 11). In contrast, SAP-35 was readily detec1.ed in culture and the incorporation of [“S]methionine into SAP- homogenates from adult lung where SAP-35 is a relatively 35 (Figs. 1 and 2). Maximal induction of SAP-35 synthesis

creased during explant culture in all experiments. ~~~~~~~~d with a similar dose-dependent increase in SAP-35 content

culture and increased thereafter during the next 3-5 days were inhibitory. (Fig. 1). Induction of SAP-35 was observed even at the earliest Newly synthesized SAP-35 consisted primarib of high- gestational age tested (one sample of 8 weeks gestation, data mmnOSe Precursors and mature sialylated forms as previously not shown). Addition of 100 PM 8-Br-CAMP further increased described (15) which were detected 3-6 h after [3sS]methio- tissue SAP-35 content (Fig. 1 and Table 111). 8-Br-CAMP nine labeling and two-dimensional isoelectric focusing SDS- significantly increased SAP-35 content at days 4 or 5 of PAGE (Fig. 2). Heterogenous sialylated forms were identical

abundant protein (Table 11). Fetal lung SAP-35 content in- was Observed at loo PM 8-Br-cAMP (Fig. 2) and correlated

SAP-35 was first readily detectable after 24-48 h in organ (not shown). Higher concentrations of 8-Br-cAMP (1 mM)


in migration to those previously detected in normal human surfactant. The pattern of SAP-35 glycosylation was not altered by gestational age, duration of culture, or addition of dexamethasone or 8-Br-CAMP (Fig. 2).

Inhibitory Effects of Dexamethasone-In contrast to the marked stimulation of synthesis of SAP-35 observed in the presence of 8-Br-cAMP, dexamethasone significantly inhibited SAP-35 synthesis and content in all experiments (Table I11 and Fig. 3, a and b). Decreased SAP-35 content observed in the presence of dexamethasone was noted within 24-48 h of incubation and persisted throughout the 5 days of culture. The reduction in SAP-35 levels was dose-dependent and was not accompanied by changes in total protein or RNA re- covered from the explants (Fig. 3). Inhibitory effects of dexamethasone were observed at all gestational ages studied (8- 24 weeks) and occurred in both Dulbecco’s minimum essential medium or Weymouth’s media with 2% carbon-stripped fetal calf serum. Dexamethasone (10 p ~ ) also completely inhibited CAMP-induced SAP-35 synthesis after 5 days of culture; SAP- 35 content was 7.72 * 1.9 pg/mg (mean * S.E. of n = 5 explants) in the presence of 100 p~ 8-Br-CAMP uersus 0.23 k 0.16 ( n = 5) in the presence of 100 p~ 8-Br-CAMP and 10 PM dexamethasone, p < 0.01.

1.2 - a

Control

0.8 - -

SAP-35 w m g

Dexa-

100 b

80

d 0 L u

60 V

L 0

X 40

20

0 1 2 3 4 5 DAYS

L Log [Dexamethasone. nH1

FIG. 3. Effects of dexamethasone on SAP-35 content 1-5 days in organ culture. Explants were cultured in the presence or absence of dexamethasone and SAP-35 content determined by ELISA assay in samples from day 1-5 of explant culture. a, the time course of SAP-35 content during explant culture. b, the effects of increasing concentrations of dexamethasone from 0.01 to 1000 nM on SAP-35 content. Values are the mean of five separate experiments with tissue from 16 and 24 weeks gestation.

SAP-35 RNA-SAP-35 RNA increased during organ culture as assessed by hybridization assay, using a specific SAP- 35 cDNA which encodes most of the translated region of human SAP-35. SAP-35 RNA was not reproducibly detected in fetal lung tissue prior to culture. Increased SAP-35 RNA was noted after 1-5 days of organ culture and by days 4 and 5 was enhanced approximately 13-fold by 100 p~ 8-Br-CAMP (Fig. 4, a and b). Hybridization of the identical filters with 3’ untranslated human @-actin probe demonstrated no concomitant time or agonist-dependent change in human @-actin

DAYS

a

CONTROL

1 2 3 4 5

500ng

250ng

8-Br-CAMP

DEXAMETHASONE

b 2000

1000

- - e 0 0 c

aQ

? 100 ln

c?

2 a

T

5 0 0 0 9

250”s

500ng

250ng

8-Br-CAMP Dexamethasone

FIG. 4. Effects of 8-Br-CAMP and dexamethasone on SAP- 35 RNA during organ culture. a, total RNA from control, 100 PM 8-Br-cAMP, and 10 PM dexamethasone-treated explants was applied to the nitrocellulose and hybridized to the 32P-labeled SAP-35 cDNA as described under “Materials and Methods” and subjected to autoradiography for approximately 20 h. There was no detectable hybridization to equal amounts of RNA from the tissue prior to explant culture. b, represents summary of changes in SAP-35 RNA in the presence of 10 nM dexamethasone and 100 p~ 8-Br-CAMP after 3-5 days in culture. Slot blot preparations were subjected to autoradiography and optical density determined by scanning densitometry. Effects of agents were determined in relation to an equal number of control (untreated) explants which had been prepared in parallel and subjected to autoradiography for identical exposure times. Statistical differences were determined by ANOVA, comparing control, dexamethasone-, and 8-Br-CAMP-treated cultures. Values are mean S.D. (note that they axis is logarithmic). Dexamethasone was 25 f 6.8% (n = 4) of control; 8-Br-cAMP, 1308 f 449% (n = 6) of separate experiments with tissue varying from 13 to 24 weeks gestation. There were no statistically significant differences in RNA hybridizing to the human &actin cDNA (not shown) from time 0 to 5 days or in response to the agents.

5260 Induction of Surfactant Protein in Fetal Lung

RNA (not shown) during the time in which specific SAP-35 RNA content increased. Although variable, total RNA recov- ery from control, 8-Br-cAMP, or dexamethasone-treated explants did not differ: 372 f 74, 285 f 42, and 373 * 106 ng RNA/mg, wet weight, respectively. SAP-35 RNA was consistently decreased by 10 PM dexamethasone during culture (Fig. 4, a and b ) .

Northern Blot Analysis-Northern blot analysis of SAP-35 mRNA is represented by Fig. 5. The SAP-35 cDNA hybridized primarily with RNA of approximately 2.1 kilobases from adult human lung. Likewise, SAP-35 RNA of 2.1 kilobases was detected after organ culture and was increased in the presence of 100 PM 8-Br-CAMP and decreased in the presence of 10 nM dexamethasone. A weak band of 1.5 kilobases hybridizing to the SAP-35 cDNA was observed in some preparations and its origin is unclear at present. SAP-35 RNA was not detected in RNA isolated from fetal lung under identical conditions.

DISCUSSION

The present work demonstrates the induction of synthesis of surfactant-associated protein, SAP-35 in second trimester human fetal lung tissue during explant culture. SAP-35 RNA synthesis and content increased during 1-5 days in organ culture in the absence of exogenous hormones. 8-Br-CAMP further stimulated, while dexamethasone inhibited SAP-35 synthesis. Changes in SAP-35 content related either to time in culture or in response to CAMP and dexamethasone were associated with similar changes in SAP-35 RNA and the incorporation of [35S]methionine into the protein. These findings are consistent with the hypothesis that the induction of

-0 -0

1)- 215 kb

a b c d e FIG. 5. Northern blot analysis of SAP-35 RNA. Approxi-

mately 15 pg of RNA was purified as described under “Materials and Methods” and separated on a 1.2% agarose formaldehyde gel and transferred to nitrocellulose. After hybridization the nitrocellulose was applied to x-ray film for approximately 6 h. Lane a, adult lung poly(A+) RNA (2 pg); lane b, fetal lung RNA (19 weeks) prior to culture; lane c, fetal lung RNA (19 weeks) after 4 days culture; lane d, fetal lung RNA (19 weeks) with 10 p~ dexamethasone; lane e, fetal lung RNA (19 weeks) with 100 p~ 8-Br-cAMP, kb, kilobase.

surfactant protein synthesis occurs at a transcriptional or translational level.

Dexamethasone specifically inhibited SAP-35 RNA synthesis and content. These findings were surprising and suggest the possibility that SAP-35 synthesis may be regulated inde- pendently of some aspects of pulmonary maturation known to be induced by corticosteroids. The respiratory epithelium undergoes dramatic morphologic and biochemical changes in late gestation. Differentiation of the Type I1 epithelial cells occurs during this time and is associated with synthesis of phospholipid components of surfactant required for respiratory adaptation. Surfactant-associated proteins are integral components of alveolar surfactant and several surfactant- associatedproteins including SAP-35 have been demonstrated to have significant biophysical effects, enhancing surface ten- sion-lowering properties of the surfactant phospholipids (29- 32). In contrast to the inhibitory effects of corticosteroid on SAP-35 synthesis presently observed, glucocorticoids increase lung phospholipid synthesis in fetal lung in numerous mammalian species both in vivo and in vitro, see Ref. 18 for review. Corticosteroids also enhance phospholipid synthesis in explant cultures of fetal rabbit, rat, and human fetal lung under virtually identical conditions to those utilized in the present study (33-35). In previous studies, SAP-35 content increased in the rat lung in vivo in temporal association with the increased phospholipid synthesis in late gestation in that species (13). Whether morphologic and biochemical maturation, as determined by the appearance of lamellar bodies, increased phospholipid synthesis, are intrinsically linked with SAP-35 synthesis in vivo remains to be tested. The observation that morphologic maturation of isolated fetal lung cells occurs in association with increased phospholipid synthesis and the appearance of lamellar bodies but without SAP-35 synthesis, suggests alternative regulatory control in vitro. In previous studies, we were unable to demonstrate significant SAP-35 synthesis in monolayer cultures of fetal lung during the same time period in which SAP-35 content increased dramatically in explant culture (15). This may reflect a re- quirement for specific cell-cell interactions in vivo or in vitro in the explant cultures which are lacking in the isolated cells. In the present study and in previous studies, morphologic maturation of Type I1 cell occurs in vitro in the absence of exogenous hormones. In the present work, SAP-35 increased in the absence of exogenous hormones in carbon-stripped fetal calf serum, a preparation which is depleted of hormones including thyroid hormone, insulin, and corticosteroids (20). We cannot exclude the contribution of other serum factors which may have influenced our findings.

The observation that the inhibitory effects of dexamethasone on SAP-35 content were associated with concomitant decreased SAP-35 RNA and SAP-35 synthesis suggests control by transcriptional or translational, rather than by post- translational mechanisms. Whether dexamethasone directly inhibits SAP-35 RNA synthesis and stability, or indirectly alters factors which regulate SAP-35 synthesis, remain un- clarified in the present study. The dose-response of the dexamethasone effects on SAP-35 synthesis are similar but some- what higher than the EC,, for stimulatory effects of dexamethasone on phospholipid synthesis in fetal lung and the ECso for binding of dexamethasone to the corticosteroid re- ceptor (36). Inhibition of SAP-35 synthesis and RNA does not appear to be related to nonspecific changes in the tissue since total RNA, DNA, protein, and [35S]methionine incorporation were not altered by dexamethasone. We were unable to detect SAP-35 in the media of the explants under any conditions, therefore it is unlikely that secretion of SAP-35 accounts for the decreased content observed in the presence of dexamethasone. We did not assess the potential role of SAP-35 degradation in the present work, although degradation of SAP-35 is not a significant factor mediating SAP-35


content in cultured Type I1 cells in tissue culture.' In contrast to the inhibitory effects of glucocorticoids, 8-

Br-CAMP markedly enhanced SAP-35 synthesis. As with the findings with dexamethasone, changes in SAP-35 content correlated, in general, with SAP-35 RNA and [35S]methionine incorporation into the protein suggesting that CAMP-dependent regulation occurs by transcriptional or translational mechanisms. cAMP mediates gene expression in a variety of cell systems. For example, marked induction of urokinase synthesis and urokinase RNA was observed in porcine kidney cells in response to CAMP-dependent agonists and 8-Br- cAMP (37). In that study, dexamethasone also inhibited the induction of urokinase synthesis. Terbutaline stimulates synthesis of cAMP in the adult Type I1 epithelial cells, enhancing phospholipid secretion (38, 39) in association with activation of CAMP-dependent protein kinase activity (40). CAMP, cAMP phosphodiesterase inhibitors, and @-adrenergic agonists also increase surfactant phospholipid synthesis in fetal lung (41,42). Treatment of premature labor with P-adrenergic agonists or cAMP phosphodiesterase inhibitors for uterine tocolysis is a common clinical practice and has been associated with decreased hyaline membrane disease in the treated in- fants (43, 44). Whether SAP-35 synthesis is altered in vitro by exposure to @-adrenergic agents or its appearance in surfactant alters the clinical outcomes of hyaline membrane disease are unknown. P-Adrenergic receptors have been identified in fetal lung from numerous mammalian species including humans and their numbers increase dramatically in late gestation (45-47).

SAP-35 content increased in association with increased SAP-35 RNA of 2.15 kilobases. The size of the message is consistent with the recent work of White et al. ( 5 ) predicting the size of the mRNA from this gene. There were no detectable changes in the size of the SAP-35 mRNA in adult, compared with fetal lung (after explant culture) or after exposure to 8- Br-CAMP or dexamethasone. Increased SAP-35 RNA was not associated with changes in @-actin RNA which did not vary significantly before or after explant culture or in response to these agents. White et al. (5) also suggested that the control of SAP-35 synthesis might occur at the transcriptional level since they observed a relative abundance of SAP-35 cDNA clones in adult compared with a fetal lung cDNA library. In addition, they identified 5' regions of the human SAP-35 gene containing consensus sequences associated with glucocorti- coid control of transcription. Whether this region is involved in the presently observed inhibition of synthesis of SAP-35 by dexamethasone in explant culture remains to be clarified.

We have previously demonstrated synthesis of glycosylated SAP-35 by human fetal lung explants during culture (15). In the present study both endoglycosidase H-sensitive forms and sialylated forms were detected under all culture conditions. Inhibition of SAP-35 synthesis by dexamethasone and stimulation by 8-Br-CAMP resulted in decreased synthesis of all forms of the protein. Two-dimensional isoelectric focusing SDS-PAGE of the newly synthesized SAP-35 were identical in charge heterogeneity to those previously observed in surfactant (4, 8). Thus, there is no evidence that regulation of SAP-35 synthesis occurs by post-translational modification of the asparagine-linked oligosaccharides on SAP-35.

In summary, synthesis of SAP-35 by fetal lung explants was inhibited by dexamethasone and stimulated by CAMP. Increased SAP-35 content correlated, in general, with SAP- 35 synthesis and SAP-35 RNA. Whether glucocorticoids or changes in intracellular cAMP mediate the ontogenic expres-

* J. A. Whitsett, T. Pilot, J. C. Clark, and T. E. Weaver, unpub- -.

lished observations.

sion of SAP-35 during normal human development in vivo or its expression in the mature Type I1 epithelial cell in vivo remains to be clarified.

Acknowkdgrnents-We wish to acknowledge the technical assist- ance of William Hull and Georgianne Ciraolo.

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