Lung Biology Laboratory, Departments of 1Medicine and 2Pediatrics, Georgetown University School of Medicine, Washington, District of Columbia 20057-1481; 3AVANT Immunotherapeutics, Incorporated, Needham, Massachusetts 02194-2725; and 4Roswell Park Cancer Institute, Buffalo, New York 14263
Submitted 6 May 2003 ; accepted in final form 24 June 2003
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ABSTRACT |
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cell physiology; corticosteroids; pulmonary microvascular cells; retinoids; receptors; hormone
The ability of exogenous all-trans retinoic acid (ATRA) to induce alveolus formation (5, 28-30, 47) and the regulatory role retinoic acid receptors (RARs) and retinoid X receptors (RXRs) (32-34) play in the endogenous formation of alveoli suggest the lung's vitamin A (retinol)-storing cell may be a (the) proximate endogenous source of retinoid signals for alveolus formation. These cells, designated lipid interstitial cells (LICs) (21, 24, 25, 46), are located in the alveolar wall between alveolar capillary endothelial cells and alveolar epithelial cells. During the early postnatal weeks, LICs, although present in alveolar walls throughout the lung's gas-exchange region (9, 20, 23, 24, 45), are especially concentrated at "septal junctions," sites from which several septa emanate (10). After the early postnatal period, LICs are found mainly in the subpleural region (28, 48), the likely site of continued alveolus formation (28). Thus the presence of these retinol storing cells (41, 47) corresponds in time and place with sites of alveolus formation.
These considerations lead us to propose LICs release retinoids synthesized from retinol, but that they are more biologically active than the parent molecule. The released retinoids induce, among other alveolar wall cells, changes in gene expression that initiate the formation of alveoli. This hypothesis would be supported by demonstrating 1) LICs release retinoids, 2) the release is impaired by a potent inhibitor of alveolus formation, i.e., dexamethasone (Dex) (7, 27, 28, 44), a synthetic glucocorticosteroid hormone, and 3) a second alveolar wall cell type responds to a retinoid released by LICs in a molecular manner relevant to alveolus formation, e.g., increasing the expression of a gene important for the synthesis of ATRA from retinol. To pursue this hypothesis, cellular retinol binding protein-I (CRBP-I) mRNA was used as an example of a gene product important to the synthesis of ATRA (36, 37) and whose concentration in lung peaks at a time of rapid alveolus formation (43, 50). Pulmonary microvascular endothelial cells (PMVCs) were used as a second cell type of the alveolar wall. We now show: 1) LICs release ATRA into serum-free, exogenous ATRA-free medium, 2) the release of ATRA is diminished in the presence of Dex, 3) incubation of PMVCs in serum-free, exogenous ATRA-free medium conditioned by LICs rich in retinol storage granules markedly increases CRBP-I mRNA in PMVCs, and 4) this increased expression of CRBP-I mRNA is impaired by an RAR and an RXR pan-antagonist, consistent with the requirement of both classes of receptors for the postnatal formation of alveoli (34).
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MATERIALS AND METHODS |
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Materials. ATRA, retinol, thymidine, trypsin, collagenase, and deoxyribonuclease were purchased from Sigma Chemical (St. Louis, MO). Tissue culture supplies were from BioFluids (Rockville, MD), Percoll was from Amersham Pharmacia Biotech (Piscataway, NJ), and radioactive retinol was from NEN Science Products (Boston, MA). An RXR pan-antagonist (RO-26-5491) was a gift from Dr. Louise H. Foley, Hoffman-La Roche, Nutley, NJ. BMS-189-453, an RAR pan-antagonist, was obtained from Bristol-Myers-Squibb (Princeton, NJ). Dex was purchased from Organon Diagnostics (West Orange, NJ).
Isolation of LICs. LICs were isolated from lungs of 8-day-old rat pups by a procedure generously provided by Dr. Stephen McGowan (Univ. of Iowa School of Medicine), which is a modification of a previously published method of isolating these cells (41). We perfused lungs via the pulmonary artery with PBS, removed major conducting structures, placed the lungs in Hanks' balanced salt solution containing trypsin (500 U/ml), collagenase (0.7 U/ml), and DNase (110 U/ml), minced the lungs, and incubated them in the enzyme solution for 90 min at 37°C. The incubated material was pipetted into and out of a Pasteur pipette several times to dislodge cells. After 2 h of incubation, fetal calf serum was added to inactivate trypsin, and the incubated material was again pipetted into and out of a Pasteur pipette and filtered (150-µm pores). The filtrate was centrifuged at 300 g for 10 min at 22°C. The pelleted cells were dispersed in HEPES-buffered medium, placed on a discontinuous density (1.04 g/ml, 1.06 g/ml, 1.07 g/ml) gradient of Percoll, and centrifuged at 400 g for 30 min at 22°C. The fluffy layer of cells at the top of the least dense layer was aspirated, and residual Percoll was removed by centrifuging the cells and washing them with RPMI 1640 (hereafter designated RPMI) medium containing 10% FBS. LICs were cultured as indicated in the figure legends; the gas phase was always 15% O2-5% CO2-80% N2, which is the concentration of these gases in the alveolus.
PMVCs. PMVCs were isolated from lungs of adult rats as previously described (17). Except where indicated, they were grown in medium 199 E supplemented with 10% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM glutamine, and 10 µM thymidine in Corning 75-mm flasks. The gas phase was 15% O2-5% CO2-80% N2.
Northern analysis and RNase protection assay. Total RNA was isolated from LICs and from PMVCs using TRI Reagent according to the manufacturer's instructions (Molecular Research Center, Cincinnati, OH) and as described by Chomczynski (13). RNA was quantitated by absorbance at 260 nm with an extinction coefficient of 0.025 (µg/ml)-1 cm-1 and analyzed by Northern blot (LICs) or RNase protection assay (RPA; PMVCs).
LIC RNA was separated by electrophoresis through a 1.5% agarose-660 mM formaldehyde gel. RNA ladders (0.16-1.77 kb and 0.24-9.5 kb; Life Technologies, Rockville, MD) were run as size standards. After electrophoresis, RNA was transferred to S & S Nytran Plus nylon membranes (Schleicher & Schuell, Keene, NH) and covalently cross-linked to the membrane by ultraviolet irradiation using a stratalinker (Stratagene, La Jolla, CA). To determine CRBP-I mRNA relative concentration, RNA was hybridized with a double-stranded radiolabeled CRBP-I cDNA probe generated using the random-primer DNA labeling system (Life Technologies), [-32P]dCTP, and a 500-bp ClaI-XbaI fragment of rat liver CRBP-I cDNA generously provided by Dr. Frank Chytil (Vanderbilt Univ. School of Medicine, Nashville, TN). Hybridization proceeded for at least 16 h at 60°C in 600 mM sodium chloride, 65 mM sodium phosphate, 5 mM EDTA, 0.02% Ficoll, 0.02 polyvinylpyrrolidone, 0.02% bovine serum albumin, 50 µg/ml tRNA, and 50% formamide. After hybridization, the blots were washed and subjected to radioautography.
RNA isolated from PMVCs was subjected to RPA using the RPA II kit (Ambion, Austin, TX) following the manufacturer's instructions. To detect CRBP-I mRNA, we prepared a single-stranded antisense species-specific probe as previously described (50). Radiolabeled 18s RNA probe was prepared by in vitro transcription according to the protocol and reagents provided by Ambion (Austin, TX) using pT7 RNA 18s antisense control template, which contains 80 bp of a highly conserved region of the ribosomal RNA gene. RNA from PMVCs was hybridized with antisense CRBP-I RNA and 18s RNA probes for at least 16 h at 45°C. After digestion by RNase A/T1, the protected fragments were resolved on a 6% nondenaturing polyacrylamide gel and detected by radioautography. The intensity of the protected band was measured using a Molecular Dynamics laser densitometer and ImageQuant Software program (Sunnyvale, CA). The concentration of CRBP-I mRNA was expressed as relative densitometry units per 18s RNA.
Assay for retinoids and release of retinoids from LICs. We separated medium from cells in culture, placed the medium in 0.1 M KOH in ethanol, added hexane, agitated the mixture, centrifuged it, aspirated the organic phase (4, 38), dried it under a stream of N2, and stored it at -20°C under N2 in the dark. The cells were washed with ice-cold medium, lifted by scraping, collected by centrifugation, and sonicated. Samples of the disrupted cells were assayed for protein. The rest of the sonicated cellular material was extracted (4, 38) and stored in the same manner as the medium. All procedures involving retinoids, including the handling of cell cultures, were performed under dimmed or yellow light.
Retinoids were separated by reverse phase high-pressure liquid chromatography (Waters, Milford, MA) with an Analytical C18 column (Waters) and a Nova-Pak C18 as a guard column at a flow rate of 1.0 ml/min. The elution profile was monitored at 340 nm; 0.5-ml fractions of the eluate were collected, and radioactivity was measured in a liquid scintillation system. Known nonradioactive retinoids added to the samples served to identify the composition of the radioactive peaks.
To study the release of retinoids, LICs were incubated in RPMI medium containing 5% fetal calf serum and retinol (3.0 µM) under 15% O2-5% CO2-80% N2 for 48 h. Then, 5 µCi of [3H]retinol was added, and the incubation was continued. After 24 h, the medium was removed, and the cells were washed three times in cold PBS. The cells were reincubated in RPMI with 3.0 µM nonradioactive retinol but without fetal calf serum. After 4 h, the medium was separated from the cells, and both were separately extracted and assayed for radioactive retinol and ATRA. The percentages of radioactive ATRA and radioactive retinol released into the medium were calculated by dividing the total medium radioactivity of the retinoid by the total (cells + medium) radioactivity of the same retinoid and multiplying the quotient by 100.
Identification of retinyl esters in LICs. Gold chloride selectively stains retinyl esters (49). LICs were cultured on coverslips in the absence or presence of ATRA, fixed in 4% formaldehyde for 10 min at room temperature, and then incubated in the dark for 5 min with 0.01% gold chloride solution in 0.01 N HCl at 22°C. The LICs were examined in a light microscope.
Protein analysis and staining with oil red O. Protein concentration was measured using Coomassie Plus assay reagent (Pierce Chemical, Rockford, IL) with bovine serum albumin as the standard. LICs were tested for the presence of lipid granules using oil red O (23).
Electron microscopy. Cells present in the least dense layer of the Percoll gradient were removed and placed in cold 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 30 min at 4°C.
Fixation was continued in 2% osmium tetroxide in 0.1 M sodium cacodylate buffer for 30 min at 4°C. The cells were dehydrated and embedded in PolyBed 812 in BEEM capsules, and thin sections were cut and examined in a JEOL 1200EX electron microscope.
Statistical analysis. The mean ± SE of each group of measurements was calculated, and a two-tailed unpaired t-test analysis was used to assess the statistical significance of the difference between means of two groups (StatMost Statistical Analysis and Graphics version 2.5 8, DataMost). The Bonferroni adjustment was used when more than one comparison was made.
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RESULTS |
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Retinoids synthesized and secreted by LICs. Retinol is the precursor of other cellular retinoids. In the extract of cells incubated with radioactive retinol in a pulse-chase manner, radioactive 4-oxo-retinoic acid, ATRA, retinol, retinyl acetate, and retinyl palmitate were identified (Fig. 2A). 4-Oxo-retinoic acid, retinol, and ATRA were secreted by LICs, but their respective storage forms, retinyl acetate and retinyl palmitate, remained in the cells (Fig. 2B). However, we have not excluded the presence of isomers of ATRA that may elute with known ATRA.
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Dex diminished the release by LICs of ATRA, but not retinol. In pulse-chase experiments with radioactive retinol, the presence of Dex (2 x 10-8 M) did not influence the radioactivity (dpm/mg of protein) of radioactive cellular ATRA or retinol (Fig. 3A). However, Dex, a potent inhibitor of alveolus formation (4, 11, 28, 29), caused a threefold lower radioactivity per milligram of cellular protein of ATRA in the medium (Fig. 3A) and halved the percentage of radioactive ATRA released into the medium (Fig. 3B). Dex did not alter the percentage of radioactive retinol released into the medium (Fig. 3B). Thus Dex differentially affected release of ATRA and retinol. The identical cellular radioactive ATRA per milligram of cell protein in the absence and presence of Dex, in light of the much greater percentage release of radioactive ATRA in the absence of Dex, suggests Dex alters intracellular metabolism of ATRA, which may, in fact, affect its release from the cell.
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ATRA increased the concentration of CRBP-I mRNA in LICs and in PMVCs. ATRA increased CRBP-I mRNA in LICs (Fig. 4A) and in PMVCs (Fig. 4B), demonstrating both cells are responsive to ATRA. The dose response data for PMVCs allow evaluation of CRBP-I mRNA by these cells in the experiments using conditioned media (Figs. 5 and 6).
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The concentration of CRBP-I mRNA in PMVCs was increased by culturing the cells in serum-free, exogenous ATRA-free medium conditioned by LICs. To compare the effect of medium conditioned by LICs rich in retinol granules on expression of CRBP-I mRNA by PMVCs to the effect of medium conditioned by LICs with few retinol storage granules, we prepared four media. Medium 1 was serum-free, exogenous ATRA-free RPMI not preconditioned by cells. We produced medium conditioned by LICs under three different conditions. This was achieved by incubating LICs in RPMI medium plus 10% FBS 1) without any additions; these LICs would eventually generate medium 2; 2) with (0.25%), the diluent for ATRA; these LICs would eventually generate medium 3; and 3) with ATRA (25 µM) in DMSO; these LICs would eventually produce medium 4. After 72 h, we removed the medium from each group of LICs and discarded it. We washed the LICs three times with serum-free, ATRA-free RPMI and incubated each group of LICs in RPMI without serum or other additions. After 24 h, we removed the medium from each group of LICs and centrifuged it at 1,000 g to eliminate cells. The supernatant fluids, conditioned medium 2, 3, and 4, without any additions, were used immediately as media in which we cultured PMVCs for 24 h. CRBP-I mRNA expression by PMVCs incubated in serum-free, exogenous ATRA-free medium 2 and medium 3 was twice as high as in PMVCs incubated in unconditioned medium (medium 1; Fig. 5). However, incubation of PMVCs in serum-free, exogenous ATRA-free medium 4 produced a 10-fold higher CRBP-I mRNA than medium 2 or 3 (Fig. 5).
An RAR pan-antagonist and an RXR pan-antagonist diminished the increase of CRBP-I mRNA produced by incubating PMVCs in medium conditioned by LICs. The results shown in Figs. 2, 3, 4, 5 suggest ATRA, or a retinoid with the same chromatographic mobility as ATRA, is synthesized from retinol and secreted by LICs. It is also clear that PMVCs take up authentic exogenous ATRA with the resultant increase of CRBP-I mRNA (Fig. 4B). These findings support the hypothesis that LICs secrete a retinoid that enters PMVCs where it increases transcription of CRBP-I mRNA. If this is the case, antagonists of the transcriptional activity of ATRA should block the ability of ATRA to increase CRBP-I mRNA in PMVCs.
In the absence of ATRA, a RAR pan-antagonist (BMS-189-453), which is an antagonist of RAR-mediated transcription, did not alter the concentration of CRBP-I in PMVCs (Fig. 6A). However, BMS-189-453 substantially prevented the ATRA-induced increase of CRBP-I mRNA (Fig. 6A). Medium 3, conditioned by LICs with few retinol storage granules, did not increase CRBP-I mRNA in PMVCs (Fig. 6B). By contrast, medium 4, which was conditioned by LICs rich in retinol storage granules, caused a fourfold increase of CRBP-I mRNA; this increase was halved by the RAR pan-antagonist (BMS-189-453; Fig. 6B).
A second hormone receptor, the RXR, plays a major role in retinoid signaling in PMVCs. Retinoid signaling commonly involves the formation of RAR-RXR heterodimers. Therefore, the effect of a RXR pan-antagonist (RO-26-5491) was tested. RO-26-5491 did not alter the concentration of CRBP-I mRNA in PMVCs in the absence of exogenous ATRA (Fig. 6C), but it impaired the ATRA-induced increase of CRBP-I mRNA (Fig. 6C). RO-26-5491 did not alter the concentration of CRBP-I mRNA in PMVCs incubated with medium conditioned by LICs with few retinol storage granules, but it halved the increase of CRBP-I mRNA in PMVCs incubated in medium conditioned by LICs with many retinol storage granules (Fig. 6D).
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DISCUSSION |
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Although no vertebrate has been identified that lacks CRBP, CRBP-I-/- mice demonstrate the protein is not essential for normal development and growth if provided a vitamin A-enriched diet (16). However, CRBP-I-/- mice have a 50% reduction of retinyl ester in liver retinol storing cells, due to decreased synthesis of the ester and a sixfold faster turnover. The latter is not related to alteration of enzymes that metabolize retinyl ester but may be due to impaired delivery of retinol to lecithin-retinol acyltransferase (16). In the presence of a diet sufficient in vitamin A, there is no obvious physical phenotype of CRBP-I-/- mice. However, as shown before, a lung phenotype, not previously found, may be identified if lungs are fixed when inflated and examined using sensitive morphometric procedures (32, 33); such an examination in CRBP-I-/- mice has not been published. The mRNAs encoding the isoenzymes RODH (I) and RODH (II), and CRBP-I, which is upregulated in lung by ATRA (43, 50), are coexpressed in alveolar wall cells (51). Therefore, it is likely, as in other tissues (36, 37), that CRBP-I is involved in the synthesis of ATRA in cells of the alveolar wall.
The actions of ATRA on gene expression are mediated through RARs and RXRs, which are ligand-activated nuclear receptors (12) that have three subclasses (,
,
) (12). Studies with RAR knockout mice indicate that RAR-
contributes to the regulation of alveolus formation after, but not during, the perinatal period (32), whereas RAR-
is an inhibitor of alveolus formation during, but not after, the perinatal period (33). In addition to identifying molecules important to the formation of alveoli, these findings demonstrate there are developmental period-specific regulators of alveolus formation. The absence of RAR-
plus RXR-
markedly depresses the formation of alveoli (34). However, it is not clear if these receptors are developmental period-specific regulators of alveolus formation (34). Thus genetic studies reveal an important role for endogenous retinoids, RARs, and RXRs in the formation of alveoli.
Exogenous ATRA can influence the formation of alveoli in newborn (29, 47) and adult (30, 31) rodents. Exogenous ATRA increases the formation of alveoli in newborn rats (29, 47), prevents the inhibition by Dex of alveolus formation in newborn rats (28) and mice (20), and partially rescues corticosteroid-induced failure of septation (30) and a genetic failure of septation (31). Retinol diminishes the extent to which prolonged mechanical ventilation impairs alveolus formation in premature lambs (1). ATRA treatment of adult rats with preexisting elastase-induced emphysema induces alveolus formation returning the size, number, and surface area of alveoli, and tissue elastic recoil, to values present in same-aged rats not treated with elastase (30). ATRA diminishes the formation of pulmonary emphysema in mice exposed to cigarette smoke (40) and decreases the distance between alveolar walls in mice with emphysema produced by cigarette smoke (40). ATRA does not cause alveolar regeneration in guinea pigs with cigarette-induced emphysema (35) and, inexplicably, diminishes the gain of lung function after unilateral pneumonectomy in dogs (15).
The present work provides support for the inference, based on the high concentration of LICs at sites of alveolus formation (9, 10, 21, 24, 25, 28, 46, 48), that LICs are the source of endogenous ATRA that induces spontaneous alveolus formation. It also indicates retinoid signaling may follow the classic pattern for hormones: synthesis and subsequent storage in granules in a specialized cell type, followed by release and action on a second cell type. The demonstration that exogenous ATRA increases CRBP-I mRNA in both alveolar wall cells studied allows two explanations for the action of exogenous ATRA to increase (29) or induce (30) alveolus formation. The increase of CRBP-I mRNA could reflect a direct response of alveolar wall cells to exogenous ATRA, or ATRA could increase the number of retinol storage granules (Fig. 1, B-E), thereby increasing secretion of ATRA, and, by that means, induce or increase alveolus formation.
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DISCLOSURES |
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ACKNOWLEDGMENTS |
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D. Massaro is Cohen Professor, Georgetown University School of Medicine.
Present address of G. Dirami: Center for Scientific Review, National Institutes of Health, 7601 Rockledge Dr., Rockledge II, MSC 7818, Bethesda, MD 20892.
GRANTS
This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-20366, HL-59432, HL-60115, HL-47413, and HL-37666.
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FOOTNOTES |
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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.
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REFERENCES |
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