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Am J Physiol Lung Cell Mol Physiol 285: L569-L577, 2003. First published May 16, 2003; doi:10.1152/ajplung.00426.2002
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Glucocorticoid effects on vitamin K-dependent carboxylase activity and matrix Gla protein expression in rat lung

Kirk A. Gilbert and Stephen R. Rannels

Department of Cellular and Molecular Physiology, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033

Submitted 12 December 2002 ; accepted in final form 14 May 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The role of glucocorticoids in the regulation of vitamin K-dependent carboxylase activity was investigated in fetal and adult lung. Glucocorticoid deficiency induced by adrenalectomy (ADX) stimulated adult lung growth and reduced carboxylation in a tissue-specific manner. Type II epithelial cells were enriched in carboxylase activity, where ADX-induced downregulation was retained in freshly isolated cells. Carboxylase activity in fetal type II cells was one-half that found in fetal fibroblasts isolated from the same lungs, and both populations increased activity with time in culture. Both carboxylase activity and formation of {gamma}-carboxyglutamate (Gla)-containing proteins were stimulated by dexamethasone (Dex) in fetal type II cells. Matrix Gla protein (MGP), a vitamin K-dependent protein known to be synthesized in type II cells, was also found in fetal fibroblasts, where its expression was stimulated by Dex. These combined results suggested an important role for glucocorticoids and MGP in the developing lung, where both epithelial and mesenchymal cells coordinate precise control of branching morphogenesis. We investigated MGP expression and its regulation by Dex in the fetal lung explant model. MGP mRNA and protein were increased in parallel with the formation of highly branched lungs, and this increase was stimulated twofold by Dex at each day of culture. Dex-treated explants were characterized by large, dilated, conducting airways and a peripheral rim of highly branched saccules compared with uniformly branched controls. We propose that glucocorticoids are important regulators of vitamin K function in the developing and adult lung.

branching morphogenesis; type II cells; dexamethasone; matrix {gamma}-carboxyglutamate protein; lung development

THE AMINO ACID {gamma}-carboxyglutamate (Gla) is the product of postribosomal carboxylation of glutamic acid residues in a vitamin K-dependent microsomal reaction. This modification, which requires reduced vitamin K, O2, and CO2 (14), provides calcium-binding sites in a number of proteins, including the clotting factors (prothrombin, VII, IX, and X) and proteins C, S, and Z. Although this system was first identified in liver microsomes, it has since been described in numerous tissues and many cell types where nonhepatic substrates such as bone Gla protein and matrix Gla protein (MGP) have been identified (13, 30). Bell (1) first studied carboxylation in lung after observing that this tissue demonstrated active uptake of vitamin K, and subsequent studies have indicated that lung activity may be as high as 60% of that found in liver (41, 42). Indeed, a high specific activity of the carboxylase is present in primary cultures of adult rat pulmonary type II epithelial cells (36), suggesting that the lung plays an important role in vitamin K regulation and function.

Developmental regulation of the vitamin K-dependent carboxylase enzyme system is not well understood. Administration of dexamethasone (Dex) to newborn rats resulted in delayed enhancement of liver carboxylase activity 6 days after drug administration, with a similar delayed enhancement of carboxylase activity in fetal hepatocytes cultured in the presence of the hormone (43). Maternal hormonal stimulation with Dex and triiodothyronine has been shown to stimulate the maturation of multiple enzyme systems in developing lung, including the vitamin K-dependent carboxylase system (15). Thus glucocorticoids appear to influence the activity of the vitamin K-dependent carboxylase system and, in turn, the regulation of Gla-containing proteins. Interestingly, hormonal induction of these pathways is also associated with decreased lung size, likely because of the pluripotent effects of steroid hormones.

Adrenal glucocorticoids are essential to normal development. In addition to their effects on lung carboxylase activity, these important hormones regulate changes in fetal rat lung growth and development that occur before birth, including the production of both phospholipid and protein components of surfactant (17), tropoelastin synthesis (29), and the synthesis of other proteins involved in control of development (6, 7). Less is known about the role of glucocorticoids in the regulation of postnatal or adult lung function, although these steroids are important in surfactant homeostasis (11, 12, 44). During the postnatal period, glucocorticoids slow growth and reduce alveolization, a process that is partially rescued by retinoids (26). In adult rats, removal of endogenous glucocorticoids by bilateral adrenalectomy (ADX) before left lobe pneumonectomy results in a much faster replacement of lung mass, an effect that can be reversed by administration of hydrocortisone (33, 34); however, neither growth nor lung morphology was affected by ADX alone in these studies (34). These combined results suggest that glucocorticoids may actually function as growth suppressors during postnatal growth and the rapid compensatory replacement of lung mass after pneumonectomy.

MGP is one of the nonhepatic vitamin K-dependent Gla proteins originally discovered in bone extracts (32). MGP is a small 10-kDa secreted protein that contains five residues of Gla and functions as a key regulator of tissue calcification (4, 5, 27, 39). Among the known Gla proteins, MGP is the most widespread in its tissue distribution, with the highest mRNA expression found in adult lung, heart, and kidney (13). These soft tissues, however, contain little of the protein, consistent with the idea that newly synthesized MGP from these tissues is rapidly secreted in the circulation and targeted elsewhere.

During embryogenesis, MGP expression is much more restricted, with localization limited to the epithelial/mesenchymal border of developing lung and limb buds (22). In addition to its known role in regulation of tissue calcification, the embryonic pattern of MGP expression suggests that it may play a regulatory role in development and differentiation of both lung and limb. Additional evidence in support of a growth-regulatory role for MGP is observed in cell culture studies where MGP expression is altered in the presence of various compounds known to influence growth and differentiation. Cancela et al. (3) have carefully described the regulation of MGP expression in normal rat kidney (NRK) cells, where both mRNA concentration and protein secretion were found to be suppressed during active cellular proliferation; when cell proliferation slowed upon confluence, both MGP message and protein levels rapidly increased (3). MGP mRNA and protein expression were also dramatically upregulated by transforming growth factor-{beta}1 (TGF-{beta}1) and inhibited by basic fibroblast growth factor and epidermal growth factor (3). Similarly, exogenous TGF-{beta}1 stimulated MGP mRNA expression in mouse lung explant cultures (45). These studies suggest that the growth and differentiation effects induced by these gene products may be mediated in part by MGP. We have demonstrated that confluent primary cultures of pulmonary type II cells actively synthesize and secrete high levels of MGP (36). Thus MGP may play an essential role in growth, development, and/or differentiation of tissues that express the gene at high levels, the lung in particular.

Because of their important influence in the fetus, we sought to determine whether adrenal glucocorticoids influence vitamin K function in the fetal and adult lung by examining regulation of the vitamin K-dependent carboxylase. The present studies were designed to investigate the regulation of lung carboxylase activity by endogenous glucocorticoids. In addition, cell-specific regulation of carboxylase activity and Gla content in response to Dex treatment was studied in both adult and fetal lung cell cultures. Finally, a well-characterized fetal growth model was used to study the effects of Dex treatment on fetal lung explant growth and MGP expression. Some of these results have been presented in abstract form (16, 37).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
ADX surgical procedure. All procedures involving animals were reviewed and approved by the institution's Animal Care and Use Committee. The institution has an Animal Welfare Assurance on file (A3045-01) with the Office for Protection from Research Risks, National Institutes of Health. Bilateral ADX was accomplished through two 1-cm dorsolateral incisions in rats anesthetized by an intraperitoneal injection of chloral hydrate solution (7.2%; 0.5 ml/100 g body wt). ADX rats had free access to 0.15 M NaCl and water for 6 days before microsome preparation of whole lung or type II cell isolation. For hormone replacement therapy after ADX, hydrocortisone (5 mg/kg in saline) was injected intraperitoneally each day; all sham-operated controls were given a similar volume of 0.9% NaCl.

Isolation and treatment of adult type II cells. Type II cells were isolated from lungs of anesthetized (50 mg pentobarbital sodium/kg body wt) rats by intratracheal installation of 375 units porcine pancreatic elastase (1 unit solubilizes 1 mg elastin in 20 min at 37°C, pH 8.8; ICN) in Joklik's modified Eagle's medium (JMEM) containing 0.05% BaSO4 as previously described (36). Dispersed cells were treated with DNase (80 mg/ml), trypsin inhibitor (0.08%), and 50% newborn calf serum to inhibit proteolysis. After filtration, Percoll gradient centrifugation, and differential adherence on plastic surfaces, type II cells (day 0) were obtained. Cell viability and purity exceeded 90% and were >95% after 24 h in primary culture as determined by acridine orange or osmium tetroxide staining of lamellar inclusions. All cells were plated at 15 x 106 cells/100-mm tissue culture dish (2.0 x 105 cells/cm2; Falcon) in DMEM containing 10% charcoal-stripped FCS unless noted otherwise. The medium for all cultures was changed at 24 and 72 h. Cell viability in all cultures was evaluated routinely using trypan blue exclusion or lactate dehydrogenase release, where no changes were observed relative to control cultures at equivalent intervals.

Isolation of fetal cells. For the isolation of mesenchymal cells, lungs from two to three litters of embryonic day 19-20 fetuses were placed in sterile 1x Hanks' balanced salts (without calcium or magnesium) and dissected free of other tissues. Lungs were drained and suspended in Hanks' containing 2 mg/ml collagenase and 0.15 mg/ml DNase (larger lungs were minced into approximately 1-mm fragments) and incubated for 45 min at 37°C with shaking (2,000 rpm) and repipetting of the suspension every 10 min to aid in tissue disruption. Cells were then filtered through 160-mm mesh, washed with JMEM, collected by centrifugation at 200 g for 1 min to remove contaminating red blood cells, and then resuspended in JMEM containing 10% FCS. Mixed cells were plated on tissue culture plates and incubated for 2 h for differential adherence of mesenchymal cells, which are the cells of interest in these protocols. Cells are plated in tissue culture dishes using carbon-stripped DMEM containing 5% carbon-stripped FCS.

Type II cells were collected by panning and gentle rinsing of the cell surface. Isolated fetal type II cells were suspended in JMEM and loaded on Percoll gradients with density steps of 1.095 and 1.045; viable intact cells are collected from the interface after centrifugation at 1,200 rpm (400 g) for 20 min and diluted free of Percoll. Type II cells were used as fresh isolates or plated in DMEM or Waymouth's medium; carbon-stripped serum was included during the first 4-12 h to promote adherence. Identification and purity of cell cultures were as described previously (35).

Fetal lung explants. Time-dated pregnant Wistar rats were obtained from Charles River Laboratories, housed in approved facilities, and fed a standard rat chow ad libitum. Pregnant rats were killed 14 days postcoitum, and embryonic fetal rat pups were removed surgically from the uterine decidua into ice-cold, sterile PBS. With the use of a stereomicroscope, fetal lungs were dissected free from the embryos and cultured under standard conditions on cell culture inserts (0.4 µm size; Falcon) in six-well tissue culture plates (Falcon) containing BGJb medium (Fitton-Jackson Modification, Life Technologies) supplemented with 5% carbon-stripped FCS. The medium was changed at 24-h intervals, unless otherwise noted, and lung explants were photographed daily. After 3-4 days in culture, explants were removed from the inserts and flash-frozen in liquid nitrogen, with subsequent storage at -80°C until RNA or protein isolation was performed.

Preparation of microsomes. To prepare cellular microsomes for assay of carboxylase, cultures were washed two times with 250 mM sucrose/25 mM imidazole, pH 7.2, containing 1 mM PMSF, resuspended or scraped in the same buffer, and sonicated. Whole lungs were homogenized (Polytron) in the same buffer and sonicated; both sonicates were centrifuged at 14,000 g for 10 min, and the resulting supernatant was centrifuged at 100,000 g for 60 min. This established method (40) isolates a crude microsomal, or postmitochondrial, pellet, which is subsequently stored at -70°C and resuspended in 25 mM imidazole (pH 7.2) containing 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Protein was determined in each microsomal preparation using bicinchoninic acid (BCA) reagent (Pierce). For assays using a peptide substrate, microsomal pellets were resuspended by gentle homogenization in a Dounce using 10 strokes of the B pestle to 0.5-1.0 mg/ml; for endogenous substrate carboxylation, 2.0-3.0 mg/ml protein was used.

Vitamin K-dependent carboxylation. Carboxylase activity was measured in microsomal preparations after the addition of chemically reduced vitamin K1H2 (100 µg/ml) to reaction mixtures containing 5.2 mM dithiothreitol, 5 mM MnCl2, 20 µCi/ml NaH14CO3 (4 mM), and the pentapeptide Phe-Leu-Glu-Glu-Leu (FLEEL) at 4-6 mM (total reaction volume, 245 µl). Endogenous protein precursors were labeled in the absence of peptide. Although the specific activity of the bicarbonate substrate varied somewhat, within a single experimental protocol it was equal for all assays. Peptide reactions remained linear for as long as 180 min at 25°C and were terminated at 60 min by the addition of 1 ml cold TCA, followed by removal of unbound 14CO2 by bubbling of CO2 through the TCA supernatant (which contained all peptide). For endogenous substrate carboxylation, reactions (120 min) were stopped as above and TCA-insoluble pellets were washed three times with NaCO3, dissolved in NaOH, and counted by liquid scintillation. Activity is expressed as disintegrations per minute per milligram microsomal protein per hour or per 2 hours as indicated in the legends for Figs. 1, 2, 3, 4.



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  		Fig. 1. Effect of adrenalectomy (ADX) on vitamin K-dependent activity in lung and liver. Microsomes were isolated from the lungs and livers of 5-7 rats from each group 6 days after ADX or ADX plus hydrocortisone (ADX-H) treatment. Vitamin K-dependent carboxylase activity was measured using Phe-Leu-Glu-Glu-Leu (FLEEL) as a substrate as described in EXPERIMENTAL PROCEDURES. Values representing means ± SE are shown as a percentage of the control activity. **P < 0.02 vs. control lung. *P < 0.05 vs. ADX-H liver. Actual activities (dpm · mg microsomal protein-1 · h-1) for lung and liver, respectively, were as follows: control, 2,817 ± 165 and 21,400 ± 4,163; ADX, 1,498 ± 91 and 24,100 ± 4,744; ADX-H, 3,532 ± 287 and 18,800 ± 4,590.

 


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  		Fig. 2. Carboxylase activity in adult rat lung and isolated adult type II cells. Microsomes were prepared from whole rat lung and freshly isolated type II cells as described in EXPERIMENTAL PROCEDURES. Carboxylase activity was measured as dpm 14CO2 incorporated into FLEEL · mg microsomal protein-1 · h-1 (lung, 12,527 ± 1,318; type II cells, 27,198 ± 824) and shown as a percentage of lung activity. Values represent means ± SE of 6 determinations representative of 3 experiments. *P < 0.02 vs. lung.

 


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  		Fig. 3. Carboxylase activity in type II cells isolated from lungs of ADX rats. After (6 days) ADX, type II cells were isolated, and microsomes were prepared as described. Carboxylase activity using FLEEL (dpm · mg-1 · h-1) is shown in A; endogenous activity (dpm · mg-1 · 2 h-1) is shown in B, where values represent means ± SE of 6 assays. Similar results were obtained using 4 different bicarbonate concentrations and 2 different microsomal protein concentrations. *P < 0.05 vs. control.

 


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  		Fig. 4. Dexamethasone (Dex) stimulates fetal type II cell carboxylation. Fetal type II cells were isolated from lungs at embryonic day 19-20 as described. Parallel cultures of eight 100-mm plates for each condition were cultured for 2 days in the presence of 50 µM warfarin (Warf) or 100 nM Dex and compared with controls. The cells from 2 plates of each condition were combined for isolation of microsomes; 1 plate was used for the determination of DNA, and the remaining 5 plates were combined for {gamma}-carboxylglutamate (Gla) analysis as described in EXPERIMENTAL PROCEDURES. Carboxylase activity was determined in 6 separate assays and expressed as 14CO2 incorporated into FLEEL · mg microsomal protein-1 · h-1; Gla is expressed as pmol/mg DNA and is the mean of 3 separate HPLC analyses. The entire experiment was performed 2 times with similar results.

 

Determination of Gla. To determine total cellular Gla content, cells from five 100-mm plates were scraped and lysed in water and hydrolyzed under vacuum in 2.5 M KOH at 100°C for 16-24 h. Hydrolysates were centrifuged, adjusted to pH 4-5 with perchloric acid, and recentrifuged before HPLC analysis of Gla. Gla was quantitated by detecting the fluorescence of opthalaldehyde/2-mercaptoethanol amino acid derivatives separated on a C18-Ultrasphere-ODS column (Beckman) and a guard column of C18-ODS-bonded Corasil (Waters). The internal standard was {beta}-carboxyaspartic acid; DNA was quantitated from parallel cultures. Parallel samples were acid hydrolyzed to convert all Gla residues to glutamic acid residues, where Gla peaks were completely absent and their identity could be confirmed.

DNA and protein quantitation. DNA was quantitated in cell monolayers dissolved in buffered saline containing 0.02% SDS and reacted with 5 µM Hoechst 33258. Fluorescence was determined in a Hoeffer fluorometer using calf thymus DNA standard. Protein was determined using a BCA kit (Boehringer) with BSA as the standard.

RNA isolation. Fetal lung explants were homogenized in 0.75 ml Tri-Reagent (Molecular Research) for isolation of total RNA according to the manufacturer's protocol. Total RNA from lung cell cultures was obtained in a similar fashion by using 1 ml Tri-Reagent for each tissue culture well. Sample RNA was analyzed for quantitation by ultraviolet spectrophotometry and absorption at 260 and 280 nm followed by storage at -80°C for subsequent use. In samples used for RT-PCR, DNase digestion of total RNA samples before spectrophotometric quantitation was performed.

Reverse transcription. Total RNA (0.1-1 µg) samples were reverse-transcribed using poly(dT) primers and Superscript H- RT (Invitrogen) in a total volume of 20 µl for 1 h at 42°C for subsequent use in competitive PCR analysis of MGP mRNA expression.

MGP competitive PCR. Competitive PCR was used to quantify MGP mRNA expression in cultured fetal lung explants. An MGP mimic was constructed by linearizing a nearly full-length MGP/pUC8 cDNA construct with StuI and inserting a 174-bp piece of foreign DNA in the new construct. For PCR, a known amount of mimic was included in each sample, and both mimic and endogenous MGP were coamplified in a 50-µl reaction containing 5 ng of reverse-transcribed total RNA, deoxynucleotides (200 µM), 10x polymerase buffer, forward and reverse primers (0.25 µM each), and Pfu polymerase (Clontech). Primers for amplification of MGP included forward primer 5'-CGGAGAAATGCCAACACCTT-3' and reverse primer 5'-GCAACGAACAATCTGTG-3' to give a 292-bp product corresponding to base pair 120-412 of rat MGP. The MGP mimic amplified with the same set of primers to give a 466-bp product. After an initial denaturation at 94°C for 5 min, samples were amplified in a programmable thermocycler for 30 cycles with denaturation at 94°C (20 s), annealing at 50°C (30 s), and extension at 72°C (1 min), with a final extension at 72°C for 8 min. For analysis, 10 µl of the PCR reaction was run on 1.5% agarose gels with ethidium bromide staining. Gels were scanned on a Fluorimager imaging system (Molecular Dynamics) and quantified with the ImageQuant software package. Comparison of the ratio of target to mimic for a given series of samples provided an accurate measure of experimentally induced changes in MGP mRNA expression.

Northern blot analysis. Total RNA samples from cell culture studies (5-20 µg) were denatured and size-fractionated on 1.2% agarose gels containing 0.4 M formaldehyde. After electrophoresis, fractionated RNA was transferred to nylon membranes using downward capillary blotting. The 495-bp rat MGP cDNA restriction fragments were isolated from a near-full-length MGP/pUC8 construct by restriction endonuclease digestion with EcoRI. Purified cDNA fragments were labeled with [{alpha}-32P]dCTP by random priming with hexanucleotide primers (DECAPrimeII Random Primed DNA Labeling Kit; Ambion). MGP mRNA expression was normalized to glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA expression to account for variations in sample loading.

Membranes were hybridized at 65°C overnight to labeled cDNA fragments using a temperature-controlled rolling incubator and sodium phosphate-based prehybridization and hybridization solutions (8). This solution consisted of 7% SDS, 0.5 M Na2HPO4, 1% nonfat dry milk, and 1 mM EDTA. Washes were performed in 2x 0.3 M NaCl, 20 mM NaH2PO4 (pH 7.4), and 20 mM EDTA (pH 7.4) containing 0.5% SDS at 65°C for 30 min. Northern blots were subsequently quantitated using a Betascope 603 analyzer (Betagen), where the ratios of MGP (dpm) to GAPDH (dpm) were calculated to show relative expression of MGP.


   RESULTS
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 DISCLOSURES
 REFERENCES
 
Adult rat lung microsomes isolated 6 days after ADX contained 50% of the vitamin K-dependent carboxylase activity measured in controls. Treatment of adrenalectomized rats with replacement hydrocortisone (ADX-H) restored lung carboxylase activity to control values (Fig. 1). The decrease in ADX lung was not because of a difference in the recovery of microsomal protein, which was 2.8 ± 0.2, 3.0 ± 0.2, and 2.5 ± 0.2 mg/lung for control, ADX, and ADX-H, respectively. The ADX-induced effect on carboxylase activity was paralleled by an overall enhancement of lung growth in the absence of endogenous glucocorticoids; ADX alone led to a 15% increase in lung mass over 6 days (Table 1), but previous studies reported normal lung morphology (34). In contrast, ADX had no significant effect on liver carboxylase activity or liver growth (data not shown), a tissue noted for its synthesis of the coagulation-related Gla proteins. Absolute liver activity was ~10-fold higher than that found in lung, as has been previously described (1). The overshoot in carboxylase activity seen with hydrocortisone replacement also suggests that excess glucocorticoids may further induce Gla protein processing in normal lung.


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  		Table 1. Effect of adrenalectomy on lung weight

 

We extended these studies to examine the cell-specific localization of glucocorticoid regulation and determined that vitamin K-dependent carboxylase activity is also decreased in adult type II epithelial cells isolated from ADX rats. The rationale for this approach was based on the known regulation of type II cell differentiation by glucocorticoids and the assumption that both endogenous substrates and the carboxylase complex would be more homogenous in type II cells. Microsomes were prepared from freshly isolated adult type II cells and were directly compared with microsomes isolated in parallel from whole lung (Fig. 2). Type II cells had a higher specific carboxylase activity than lung, suggesting enrichment in these cells. Carboxylase activity was also determined in freshly isolated type II cells obtained from ADX rats (Fig. 3). Both FLEEL carboxylation and the carboxylation of endogenous precursors were reduced in cellular microsomes isolated from ADX rats. Carboxylation of endogenous lung substrates reflects those Gla protein precursors that remain bound to the carboxylase during microsome isolation. Addition of reduced vitamin K (vitamin K1H2) results in the carboxylation of these proteins within the microsomes. These results mimicked the effects of ADX on lung carboxylase activity observed in Fig. 1; however, treatment with Dex over the culture interval did not further increase carboxylase activity (data not shown).

To further define the role of glucocorticoids in regulating carboxylation in specific cells, we chose to work with fetal type II cells and fibroblasts, both of which are easily obtained from the same tissue and cultured in parallel (35). Furthermore, the important interaction of the epithelium and mesenchyme during development is well known (19, 38), and specific Gla proteins produced by one cell population may influence growth and differentiation in the other cell type. Fetal type II cells isolated from embryonic day 19-20 embryonic lungs also responded to Dex with a 2.5-fold increase in activity (Fig. 4). Total cellular Gla increased nearly threefold from 494 to 1,211 pmol Gla/mg DNA in Dex-treated cells, suggesting that FLEEL carboxylation is an accurate reflection of the degree of processing of endogenous precursors through this pathway. Warfarin, an inhibitor of vitamin K-dependent carboxylation, reduced total Gla content by 37% to 313 pmol/mg DNA, and it paradoxically increased carboxylation of FLEEL. This stimulation is thought to be secondary to a microsomal accumulation of noncarboxylated precursors (reflected by the decrease in Gla), which activate the carboxylase enzyme complex and lower the Km for FLEEL (9, 20, 36).

To investigate the contribution of the mesenchyme to overall fetal {gamma}-carboxylation, the relative carboxylase activities of fetal type II cells and fibroblasts isolated from the same lungs were compared after 24 and 48 h in primary culture (Table 2). Mesenchymal activity was approximately two times that measured in type II cells at both culture intervals. Endogenous substrate carboxylation was also higher in fibroblast cultures, and a time-dependent increase in carboxylation was similar to that previously observed for adult type II cells (36).


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  		Table 2. Vitamin K-dependent carboxylase activity in fetal type II cells and fibroblasts

 

We investigated the possibility that fetal fibroblast vitamin K-dependent carboxylase activity may also be regulated by glucocorticoids and that MGP is an important product of this enzymatic pathway based on its potential role in the regulation of growth and differentiation during development (16, 45). MGP mRNA expression increased from culture days 3-6, and Dex greatly enhanced fibroblast MGP expression at both intervals (Fig. 5). These results, combined with those of Fig. 4, indicate that glucocorticoids influence both epithelial and mesenchymal cell carboxylase and substrate activities in the fetal lung.



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  		Fig. 5. Effect of Dex on matrix Gla protein (MGP) mRNA expression in fetal fibroblasts. Fibroblasts were isolated from embryonic day 19-20 lungs and cultured for 6 days in the absence or presence of 100 nM Dex. RNA was isolated from each of 6 100-mm plates per condition at the intervals shown, and MGP mRNA was quantitated by Northern analysis and normalized to GAPDH expression as described in EXPERIMENTAL PROCEDURES. *P < 0.01 vs. controls on the same culture day.

 

Based on our results in isolated fetal pulmonary cell cultures, we extended these studies in intact embryonic lungs using a well-established in vitro model of branching morphogenesis. Fetal lung explant cultures were initiated at embryonic day 14, and MGP mRNA was determined through the 3 days of culture by competitive PCR (Fig. 6). In explant cultures, MGP mRNA increased in a linear manner with time, and the addition of 100 nM Dex stimulated MGP mRNA expression approximately twofold at each interval, suggesting that glucocorticoid-induced regulation of MGP expression is present at an early time in lung development (Fig. 7). Dex-induced changes in explant morphology were evident over the entire culture interval, with the most dramatic changes observed at day 3. Normally, explants show an increasingly branched tree-like structure where the proximal trachea and main bronchi lead to finely branched terminal saccules, the precursors of alveoli. With Dex treatment, conducting airways and proximal acinar structures became extremely dilated, accompanied by a thinning mesenchyme. The most distal rim of terminal lung buds, however, retained a relatively normal morphology by the end of the culture interval in Dex-treated explants (Fig. 7). These Dex-induced morphological changes are consistent with those reported by others where it was also determined that distal epithelial cells were more differentiated (28).



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  		Fig. 6. Competitive PCR standard curves. Two standard competitive PCR reactions were performed to determine the effectiveness of an MGP cDNA competitor on quantification of MGP mRNA expression in fetal explants. The ethidium bromide-stained gel on the left contains products from a PCR reaction in which 0-1,600 fg MGP cDNA were amplified in the presence of 50 fg MGP mimic cDNA. The gel on the right contains products from a PCR reaction in which the templates were derived from RT reactions, where 0-320 ng total RNA were reverse transcribed and amplified in the presence of 50 fg MGP mimic cDNA. Competitive PCR standard curves had identical slopes when the log(MGP/mimic) was plotted against log(MGP). Thus MGP mimic cDNA was used to accurately compare MPG mRNA expression in a given series of tissue samples.

 


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  		Fig. 7. MGP mRNA expression and morphology in fetal lung explants. Embryonic day 14 fetal lung explants were cultured for 3 days with and without 100 nM Dex. MGP mRNA was quantitated from 3 individual explants per condition using RT-PCR as described in EXPERIMENTAL PROCEDURES. The results are representative of at least 3 separate experiments. Shown also are control and Dex-treated explant photographs taken at day 3 in culture. *P < 0.05 vs. control.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
DISCLOSURES
 REFERENCES
 
The influence of adrenal glucocorticoids on specific functions of the adult lung is not well defined. They are known to be important developmentally in the process of cellular maturation and differentiation, as exemplified by stimulation of surfactant production. A potential mechanism for increased differentiation in the presence of glucocorticoids is the hormone-induced inhibition of cell proliferation and DNA synthesis indicated by decreased thymidine incorporation (2). In support of this concept, we have found that Dex inhibits type II cell thymidine incorporation, whereas glucocorticoid deficiency leads to an increase in thymidine incorporation and protein synthesis in type II cells isolated from ADX rats (10). Similarly, the rate and extent of tissue accumulation during rapid compensatory growth of the lung are increased in rats that were adrenalectomized before partial pneumonectomy (34). It is thus consistent that, in the present study, ADX alone influenced basal growth rates, where both body and lung mass were accumulating (Table 1). Growth inhibition may be independent of the function of lung vitamin K-dependent proteins; however, it is suggested that MGP may have growth-suppressive or -stabilizing effects on differentiated cells of the tissues where it is located (3). There are numerous vitamin K-dependent proteins in lung that would likely be affected by glucocorticoid-induced changes in carboxylase activity. Therefore, it is possible that the function of MGP and known growth-regulating lung Gla proteins, including protein S and Gas6 (24), may be mediated in part by endogenous glucocorticoids.

Several studies support a role for MGP in the regulation of growth and differentiation. For example, NRK cells secrete very high levels of MGP, and the production of both mRNA and protein are tightly linked and highly dependent on cell density (3). Proliferating, nonconfluent cells expressed very low amounts of MGP, whereas confluent or hyperconfluent nonproliferating cells increased their synthesis and export of MGP in an exponential manner. In both adult and fetal lung, type II pneumocytes follow the same regulation. If maintained in a nonproliferative type II-like phenotype through culture on laminin-rich Matrigel, type II cells maintain high levels of MGP mRNA expression compared with cells plated on tissue culture plastic (unpublished observation). In addition, nonproliferating high-density cultures of type II cells express high levels of MGP mRNA compared with low-density cultures that are moderately proliferating (37). Thus MGP upregulation either is the cause of reduced rates of cellular proliferation or results as a consequence of quiescence. It should be emphasized at this point that all of these studies show correlations rather than direct effects of the protein.

Because Dex dramatically influences branching morphogenesis of fetal lung explants (6, 7, 28), we studied the potential influence of Dex on MGP expression during the pseudoglandular period, an interval characterized by rapid growth and airway branching. In the explant model, MGP shows a mesenchymal distribution pattern (16); however, the present results demonstrate both epithelial and mesenchymal synthesis. It is likely that both cell populations play an important role in producing MGP and other vitamin K-dependent proteins, and, as cellular differentiation proceeds, MGP immunostaining appears in additional cell types, including vascular smooth muscle cells. The morphological changes in embryonic lung explants seen with Dex treatment in the present study are similar to those observed by Oshika et al. (28), who concluded that Dex causes premature differentiation of epithelial function while also thinning the mesenchymal component.

The mechanism whereby dilation of conducting airway structures occurs with Dex treatment is unknown. It is tempting to speculate that some of the effects of Dex on embryonic lung growth may be mediated through MGP and possibly other extracellular matrix (ECM) proteins. For example, the rapid synthesis and secretion of MGP by pulmonary epithelial cells may set up subsequent interactions of this protein with adjacent cells and proteins in the mesenchyme. MGP is known to bind fibronectin, an additional ECM protein localized in the cleft region during branching morphogenesis (18). Interestingly, TGF-{beta}1, which is also involved in cellular differentiation and growth inhibition, is deposited in the branch point cleft (18) and stimulates both MGP (45) and fibronectin (25) expression. It is possible that these proteins work in concert to stabilize the cleft region and promote cellular differentiation during the repetitive process of branching morphogenesis. The Dex effects on explant morphology could also be independent of MGP expression and may be direct effects on additional pathways or other protein mediators of branching. Direct inhibition or elevation of MGP protein is required to resolve this issue. It is intriguing, however, that adrenal glucocorticoids have a dual effect to promote MGP mRNA expression and to stimulate the posttranslational carboxylation of the same protein, a modification that renders it functional.

The mechanism of action of MGP likely involves calcium ion regulation since it has been determined that homozygous MGP-deficient mice die prematurely of arterial calcification (23), a conclusion confirmed using other in vivo approaches (31). If MGP is important for normal branching morphogenesis and perhaps vasculogenesis, it is not clear why the MGP null mice are born with apparently functional lungs. One possibility is that blood-born MGP can cross the placenta and rescue the fetus during development, as occurs with TGF-{beta}1 null mice (21). Newborns would then acquire the described phenotype as they grow in the absence of this protein. The precise role of MGP in the regulation of growth and differentiation is currently an active area of study.

These studies demonstrate that vitamin K-dependent carboxylase activity in both adult and fetal lung is influenced by glucocorticoids. Furthermore, results show that expression of MGP, a specific substrate of the carboxylase pathway, is regulated by glucocorticoids in isolated cell populations and in fetal lung explants. Dex-induced changes in explant morphology are accompanied by increased MGP expression, suggesting that some of the steroid-induced changes in lung growth may be mediated by MGP. Taken together, these results provide evidence for an additional regulatory pathway in the lung influenced by endogenous steroid hormones.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grant HL-62869.


   ACKNOWLEDGMENTS
 
We are grateful for the technical assistance of Jing Zhou.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. R. Rannels, Dept. of Cellular & Molecular Physiology, H-166, The Pennsylvania State Univ. College of Medicine, 500 Univ. Dr., Hershey, PA 17033 (E-mail: srannels{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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