Glucocorticoid Enhances Surfactant Proteolipid Phe and pVal Synthesis and RNA in Fetal Lung*

Two newly described surfactant proteolipids (SPL), Phe and pVal, are produced by proteolytic processing of distinct precursors of M, = 40,000 and 22,000, respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVa1) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthe- sis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked im- munosorbent assay and by [3SS]methionine incorporation into the glycosylated M, = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone oc- curred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein- (SAP) 35, a distinct surfactant glycoprotein. 8-Br-CAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined homogenization and 1 phenylmethylsulfonyl fluoride. Sample and anti-bovine proteo- lipid antiserum (1:30,000) were preincubated overnight at 37 prior to addition to the well. The reaction was then developed with im- munoperoxidase conjugated goat anti-rabbit IgG (1:lOOO) and 0- phenylenediamine.

respectively. These proteins are structurally related and intimately associated with surfactant phospholipids. We now demonstrate the expression of both SPL(Phe) and SPL(pVa1) in explants of human fetal lung from 16-24 weeks of gestation. Content, synthesis, and mRNA for the proteolipids were low prior to organ culture of fetal lung. Induction of synthesis of the proteolipids occurred rapidly in explant culture in the absence of exogenous hormones and was enhanced by addition of dexamethasone. Increased synthesis of the proteolipids was detected by enzyme-linked immunosorbent assay and by [3SS]methionine incorporation into the glycosylated M, = 40,000-43,000 SPL (Phe) precursor. The response to dexamethasone occurred rapidly and contrasted with effects of dexamethasone on the expression of surfactant-associated protein-(SAP) 35, a distinct surfactant glycoprotein. 8-Br-CAMP did not significantly increase proteolipid content but markedly increased synthesis of SAP-35 in identical cultures. Increased proteolipid content was associated with increased mRNA for each protein as determined by the Northern blot analysis. Proteolipid RNA was also increased by 8-Br-cAMP, however, not to the extent observed with the glucocorticoid. Immunohistochemical analysis of fetal lung with anti-proteolipid antiserum confirmed that the dexamethasoneenhanced synthesis of the proteins by Type I1 epithelial cells. The time and hormone dependence of the regulation of expression of both SPL(Phe) and SPL(pVa1) precursors were distinct from that of SAP-35. Expression of the surfactant proteolipids increased during explant culture of human fetal lung and was further enhanced by glucocorticoid. Developmental and hormonal regulation of the surfactant proteolipids may be important factors in surfactant function at birth. Surfactant-associated hydrophobic proteins of M , =  membrane disease (1)(2)(3)(4). These human proteins consist of small molecular weight hydrophobic peptides, one of which has been identified as SPL(Phe)' on the basis of the NH,terminal phenylalanine and which arises from a 40,000-dalton precursor protein (5); a second proteolipid SPL(pVa1) has been identified on the basis of its unique polyvaline domain and is encoded by distinct cDNA which encodes a 22,000precursor protein.' Synthesis of proteolipid precursors has been identified in human lung, and antiserum generated against the surfactant proteolipid recognizes both the M, = 40,000 and 22,000 precursors (5).'*3 Recent studies from this laboratory demonstrated the synthesis and processing of pulmonary surfactant protein 9) and proteolipid SPL(PheI3 in fetal lung tissue. Increased expression of SAP-35 and SPL(Phe) was demonstrated during explant culture of human fetal lung (5,8,9 ) . ' s 3 Developmental expression of surfactant and of surfactantassociated proteins has been recently subjected to intense study. Synthesis of surfactant phospholipids is enhanced by corticosteroid, thyroid hormones, cAMP phosphodiesterase inhibitors, and epidermal growth factor (10, for review). In recent studies, expression of SAP-35 was enhanced by cAMP (8,ll) and epidermal growth factor (12); however, contrasting effects of dexamethasone have been recently reported (8, [11][12][13]. The present study was designed to determine whether glucocorticoids alter the expression of SPL(Phe), SPL(pVal), or SAP-35 precursors during explant culture of human fetal lung.

EXPERIMENTAL PROCEDURES
Purification of Hydrophobic Surfactant Proteins-Surfactant was purified by differential centrifugation and the hydrophobic proteins extracted by chloroform/methanol or ether/ethanol extraction as previously reported (1, 2). Protocols for use of human tissue were approved by the Human Research Committee, University of Cincinnati College of Medicine. Human preparations contain two surfactant peptides, SPL(Phe) and SPL(pVal), identified by their distinct NHZterminal amino acid sequences (5).' The N-terminal sequence of SPL(Phe) peptide Phe-Pro-Ile-Pro, etc., represented approximately 2/3 of the sequence obtained in the human preparation as previously reported (5). The remainder consisted of SPL(pVa1): Ile-Pro-Cys-Cys, etc. Antiserum was generated in rabbits by repeated injection of the bovine surfactant proteolipid prepared by chloroform/methanol extraction of surfactant (1,2). This antiserum (bovine surfactant proteolipid antiserum) immunoprecipitates both the 40,000-43,000- dalton SPL(Phe) and the 22,000-dalton SPL(pVa1) precursors after [35S]methionine labeling (5)? The antiserum reacts with protein of M, = 6,000-14,000 in immunoblots of organic extracts of bovine, human, and canine surfactants (1,2).
Explant Culture-Tissue was obtained from consenting donors from pathologic specimens from the National Diabetes Research Interchange (Philadelphia, PA), in accordance with a protocol approved by the Human Research Committee, University of Cincinnati College of Medicine. Tissue was immediately placed in iced minimum essential medium, (GIBCO) at 4 "C and transported on ice to this laboratory within 24 h. Lung tissues were minced to 1-mm3 pieces using a McIlwain tissue chopper and placed on scratched surfaces of 60-mm plastic dishes to which was added Weymouth's media with 2% carbon-stripped fetal calf serum (14) containing 100 units/ml penicillin and 0.1 pg/ml gentamicin. Explant cultures were placed on a rocker platform at 3 cycles per minute, to immerse and expose the explants during the culture period, as described by Gross,et al. (15).  (16). Labeling with ["Clphenylalanine was performed as above except that the media was deficient in phenylalanine; ["C] phenylalanine (450 mCi/mmol) was present at 150 pCi/ml.
Immunohistochemistry-Fetal lung explant tissue, fixed in 2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.3, was dehydrated in a graded series of acetones and embedded in polymerized polymethylmethacrylate (Aldrich) which was dissolved in dichloromethane as described by Grobsky and Borisy (18). Embedded tissue was sectioned at a thickness of 1-2 pm using a Reichert Om U3 Ultramicrotome (Optische Werke A.G., Austria). Plastic was extracted from tissue sections by submerging sections (which were on a film of water on glue-coated glass slides) in a bath of 75% chloroform and 25% acetone for 45 min. Immunoperoxidase staining of the sections was then performed using the specific antiserum at a dilution of 1:500 in a three-step biotin-avidin system (19).
RNA Extraction for Hybridization Experiments-Approximately 100 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 P-mercaptoethanol, and 0.1% antifoam A. RNA was extracted by centrifugation through a cushion of 5.7 M cesium chloride (20). The RNA pellet was dissolved 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.
For Northern blot analysis, 15 pg of total RNA was prepared from 100 mg of tissue and was separated on a 1.2% agarose-formaldehyde gel (21) and transferred to nitrocellulose. The filter was baked at 80 "C for 2 h. Nitrocellulose "slot" blots were made by applying 500 or 250 ng of formaldehyde-denatured RNA to nitrocellulose using a slot blot manifold (Schleicher & Schuell, Inc.). Optimal conditions for hybridization were determined in preliminary experiments varying method of RNA preparation, hybridization, and washing conditions. The filters were prehybridized in 40% deionized formamide, 5 X SSC (1 X SSC is 0.15 M NaCl, 0.015 M sodium citrate) 50 mM NaH2P04, pH 7.0, 5 X Denhardt's solution (1 X Denhardt's is 0.02% bovine serum albumin, 0.02% Ficoll, 0.02% polyvinyl pyrollidine), 0.2% SDS, and 100 pg/ml denatured salmon sperm DNA at 41 "C overnight. Filters were hybridized in the same solution containing 10% dextran sulfate and cDNA probes at approximately 5 X lo6 cpm/ml at 41 "C overnight. 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 placed between two sheets of Saran wrap and exposed to Kodak XAR-2 film.
ELISA Assay-ELISA of the surfactant proteolipid was performed using the anti-proteolipid antibody which recognizes both SPL(Phe) and SPL(pVa1). Competition curves were generated with bovine SPL. The standard completely inhibited immunoreactivity of the antibody under assay conditions. Standard SPL protein was estimated after silver staining and in relationship to previous amino acid analysis after HC1 hydrolysis (5). This antiserum is reactive with both proteolipid percursors in immunoprecipitation assays. ELISA was performed by competition assay using purified, delipidated SPL which was adsorbed to each of the plastic wells. Purified SPL inhibited the reactivity of the antiserum in a dose-dependent manner (10-10,000 ng) and duplicates generally varied less than 10%. Assays of the tissue homogenates were performed at two or three different dilutions of the sample in 10% propanol in water to assure parallel dilution in the competition curve. Explant samples were homogenized immediately or frozen at -70 "C for 1-2 weeks prior to homogenization in phosphate-buffered saline, pH 7.2, containing 10 mM EDTA and 1 mM phenylmethylsulfonyl fluoride. Sample and anti-bovine proteolipid antiserum (1:30,000) were preincubated overnight at 37 "C prior to addition to the well. The reaction was then developed with immunoperoxidase conjugated goat anti-rabbit IgG (1:lOOO) and 0phenylenediamine.

RESULTS
Competition ELISA assay of the surfactant proteolipid in fetal lung of 16-21 weeks gestation demonstrated low or undetectable levels of activity prior to organ culture. Proteolipid content increased during organ culture in the absence of exogenous hormones, Table I. Continuous exposure to dexamethasone enhanced content of the proteolipids (Table I)  surfactant proteolipids by the ELISA assay, Table I.
Culture-dependent increases in SPL(Phe) were confirmed by ["Slmethionine-labeling and immunoprecipitation of 40,000-43,000 glycosylated peptides, Fig. 1. After 2-4 days in culture, synthesis of the proteolipid precursors was high under all conditions and not further enhanced by addition of corticosteroid. Similar to the findings in the ELISA studies, there were no detectable effects of 8-Br-CAMP on synthesis of the SPL precursor protein at 3-5 days (not shown); however, synthesis of SAP-35 was markedly enhanced by 8-Br-CAMP in these explants as previously reported (8).
Smaller molecular weight proteins, M , = 6,000-14,000, which co-migrated in SDS-PAGE with the proteolipids from surfactant, were immunoprecipitated from the explants but were present in relatively low abundance, Fig. 2 Identification of SPL(Phe) and SPL(pVa1) precursors was confirmed by hybrid arrested translation assay which identified the preproteins as M , = 40,000 and 22,000, respectively, Fig. 3, a and b. The presence of N-linked carbohydrate on SPL(Phe) was demonstrated by treatment of the immunoprecipitates with endoglycosidase F which decreased the size and size heterogeneity of the 42,000-43,000 proteolipid precursors, Fig. 4. Endoglycosidase-F also trimmed immunoprecipitable protein of M , = 25,000 which may represent a proteolytic fragment of SPL(Phe).4 These findings confirm previous demonstration of a censensus sequence for N-linked carbohydrate on the SPL(Phe) precursor (5).
Immunohistochemical identification of the proteins was performed using both surfactant proteolipid anti'serum and SAP-35 antisera. Staining for the proteolipid was detected in J. Whitsett  in all of the explants (not shown) and differences in treated versus untreated explants were not discernible. The immunoreactive staining was detected primarily in the basalar and apical regions of Type I1 epithelial cells. Staining for the proteolipids was also present in the luminal contents of the explants with increasing time in culture. The immunohistochemical localization and the timing of induction contrasted with the appearance of SAP-35, which was present in larger granular organelles (lamellar bodies) in the apical portion of all of the terminal respiratory epithelial cells and in the luminal contents of the acini.3 RNA Studies-Northern blot analysis demonstrated that the abundance of SPL(Phe) and SPL(pVa1) RNA was low in fetal compared to adult lung tissue, Fig. 6. There was a progressive increase in expression of both SPL(Phe) and SPL(pVa1) from 16-20 weeks gestation, Fig. 6. Lungs from later gestation (22-24 weeks) showed significant variability, some lungs expressing the proteolipid RNAs at relatively high levels, (not shown). Northern blot analysis of the RNA from the explant cultures demonstrated the mRNAs for SPL(Phe) a t 2.0 kilobases, SPL(pVa1) at 1.0 kilobases and SAP-35 at 2.15 kilobases, Figs. 6 and 7. Increased mRNA for both proteolipids was observed in the presence of 10 nM dexamethasone, Fig. 7. 8-Br-CAMP also resulted in a smaller but consistent enhancement of SPL(pVa1) and SPL(Phe) RNA in the Northern and slot blot analysis especially after 2-5 days in culture. Relative effects of the hormones were somewhat variable since rapid and variable induction occurs in the absence of added hormones, especially in the tissue of older gestational age. Induction of the proteolipid RNA in the   16,19, and 20 weeks gestation and adult tissue and separated by agarose gel electrophoresis. RNA was blotted to nitrocellulose and hybridized to 32P-labeled SPL(Phe) and SPL(pVa1) cDNAs. Though weakly detectable at this exposure, SPL(Phe) and SPL(pVa1) RNA in fetal lungs increased with gestational age (16-20 weeks). Scanning densitometry resulted in ratios of the fetal RNAs (week 161920) of 1:4:10 for SPL(Phe) and <1:2:10 for SPL(pVa1). presence of dexamethasone occurred rapidly, reaching maximal levels after 24 h and occurred more rapidly than for the induction of SAP-35 RNA which was maximal by 3-4 days, Figs. 8 and 9. SAP-35 RNA was markedly enhanced by 100 p~ 8-Br-CAMP at 3-4 days as previously reported (8,9). Dexamethasone did not increase SAP-35 RNA and higher concentrations of dexamethasone (10 nM to 10 p~) inhibited SAP-35 RNA as previously reported (8,9). No consistent effects of T3 (5 nM) were observed on SPL(Phe) or SPL(pVa1) RNA, Fig. 8.

DISCUSSION
These studies demonstrate glucocorticoid induction of newly described proteolipids SPL(Phe) and SPL(pVa1) in explant cultures of human fetal lung. Increased proteolipid content during culture was associated with increased [ 9 ] methionine incorporation into glycosylated precursor protein of SPL(Phe) of M , = 40,000-43,000. Increases in proteolipid content and RNA were observed during explant culture in the absence of exogenous hormones and were further enhanced by dexamethasone. The time course of induction and glucocorticoid control of proteolipid expression were distinct from that of SAP-35 expression in these cultures.
Surfactant proteolipids are intimately associated with surfactant phospholipids, and their developmental regulation is likely to be vital to surfactant function during respiratory adaptation to postnatal life. There was detectable immunoreactivity prior to culture of lung explants (between 15-24 weeks of gestation) although abundance of RNA and immunocytochemical abundance was low compared to cultured explants or that in adult lung. Prior to culture, SPL(pVa1) and SPL(Phe) RNA was variable but tended to be higher in lung tissue from older fetal lungs, generally increasing from 16 to 24 weeks of gestation. In the present study, surfactant synthesis of proteolipids increased very rapidly during explant culture. In contrast to the proteolipids, induction of SAP-35 mRNA occurred more slowly (8, 12, 13).3 SPL(Phe) and SPL(pVa1) RNA was rapid (1-2 days) and coordinate, but was distinct from that of SAP-35 expression which reached maximal levels only after 3-5 days of explant culture (8, 12,13).3 Enhancement of SPL(Phe) and SPL(pVa1) expression by dexamethasone was detected at concentrations which were similar to those previously described for the stimulatory effects of corticosteroids on the phospholipid synthesis by fetal lung and consistent with the affinity of dexamethasone for pulmonary corticosteroid receptors (6,22). In contrast to the marked effects of 8-Br-CAMP on SAP-35 synthesis (8, ll), 8-Br-CAMP had no detectable effect on SPL expression or content although some enhancement of proteolipid RNAs by 8-Br-CAMP was detected. Influences of 8-Br-CAMP on surfactant proteolipid RNA occurred in a time frame similar to that observed with the effects of 8-Br-CAMP on SAP-35 synthesis. However, effects of 8-Br-CAMP on proteolipid RNA were less than those observed for dexamethasone and were not associated with increased proteolipid content. SPL(Phe) and SPL(pVa1) arise from larger precursors. The SPL(Phe) precursor was recently shown to be a glycoprotein containing asparagine-linked carbohydrate sensitive to endoglycosidase F (5).3 Both preproteins must undergo further proteolytic processing to generate the smaller molecular weight proteins (proteolipids) detected in surfactant. The site and nature of such proteolytic processing remains to be clarified. Small amounts of [35S]methionine-labeled protein of M , 14,000) migrated in at M , = 18,000 in the absence of pmercaptoethanol, supporting their sulfhydryl-dependent aggregation (1,2). The presence of a surfactant protein of M , = 18,000 was recently reported by Hawgood et al. (7) who identified a canine surfactant protein with high homology to human SPL(Phe) presently described (5).
Immunohistochemistry was used to demonstrate the localization of the proteolipids in Type I1 epithelial cells. The intracellular distribution of the proteolipids was similar but distinguishable from that of SAP-35. SAP-35 was previously shown to be concentrated primarily in lamellar bodies in the apical region of the cells; immunoreactivity of the proteolipids was noted both in the basilar and apical regions of the Type I1 cells.3 Immunostaining of the proteolipid increased rapidly in culture in a time frame distinct from that of 12). The proteolipid antiserum utilized in the present work recognizes both SPL(Phe) and SPL(pVa1). The hydrophobic N-terminal regions of the small molecular weight peptides are structurally similar (5).' It is therefore unclear whether the material detected by immunohistochemistry and the ELISA assay represents both SPL(Phe) and SPL(pVa1). Likewise, it is unclear whether proteolipid precursors and the proteolytically processed peptides are equally reactive. Small peptides ( M , = 6,000-14,000) were detected after [35S]methionineor ['4C]phenylalanine-labeling but were present at low levels in the explant cultures. Thus, it is likely that the immunoreactivity detected in the acinus of distal respiratory structures comprises SPL(Phe) or SPL(pVa1) precursors which are secreted intact into the lumen, suggesting that proteolytic processing occurs after their secretion. It is also possible, however, that some proteolytic processing is not expressed under the explant conditions utilized in this study.
Corticosteroids have dramatic effects on maturation of developing lung cells and are used clinically for the prevention of hyaline membrane disease in premature infants. The present study demonstrates marked effects of dexamethasone on the expression of SPL(Phe) and SPL(pVa1) in vitro and supports the hypothesis that enhancement of surfactant proteolipid synthesis may be important for adaptation to air breathing in postnatal life.