Retinoic acid and dexamethasone affect RAR-beta and surfactant protein C mRNA in the MLE lung cell line

Mary A. Grummer1 and Richard D. Zachman1,2

Departments of 1 Pediatrics and 2 Nutritional Sciences, Meriter Hospital Perinatal Center, University of Wisconsin, Madison, Wisconsin 53715

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Lung development and surfactant biosynthesis are affected by retinoic acid (RA) and dexamethasone (Dex). Using a mouse lung epithelial cell line, we are exploring RA-Dex interactions through the study of RA and Dex effects on RA receptor (RAR) and surfactant protein (SP) C mRNA expression. RA increased expression of RAR-beta (5.5 times) and SP-C (2 times) mRNA, with maximal effects at 24 h and at 10-6 M. The RA induction was not inhibited by cycloheximide, suggesting RA affects transcription. With added actinomycin D, RA did not affect the disappearance rate of RAR-beta mRNA, but SP-C mRNA degradation was slowed, indicating an effect on SP-C mRNA stability. Dex decreased RAR-beta and SP-C expression to 75 and 70% of control values, respectively, with greatest effects at 48 h and at 10-7 M. There was no effect of Dex on either RAR-beta or SP-C mRNA disappearance with actinomycin D. However, cycloheximide prevented the effect of Dex. Despite Dex, RA increased both RAR-beta and SP-C mRNA. This work suggests that RA and Dex affect RAR-beta and SP-C genes by different mechanisms.

gene expression; type II cells; steroid hormone superfamily; lung development; vitamin A; messenger ribonucleic acid

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

VITAMIN A HAS LONG BEEN recognized as essential for lung epithelial cell differentiation, growth, and health. Vitamin A deficiency results in loss of mucus-secreting epithelium because cells fail to differentiate normally and to develop as layers of squamous keratinizing epithelium (7). In the human premature infant, a role for vitamin A in prevention and/or repair of lung injury is speculated (34). The actions of vitamin A in the lung may involve its effects on gene regulation. Retinoic acid (RA) and 9-cis-RA, which are metabolites of retinol, bind to nuclear RA receptor (RAR) and 9-cis-RAR (RXR) proteins, which are ligand-activated DNA-binding proteins that belong to the steroid receptor superfamily. Three distinct types of RARs and RXRs (alpha , beta , and gamma ) interact in dimer and heterodimer forms with response elements to affect target genes (22). The characteristic expression patterns of the different receptors suggest that they have distinct roles during development. Specifically, in the developing lung, in situ hybridization has demonstrated variable expression of receptors in branching fetal mouse lung at midgestation (8, 9). Inactivation of some specific RAR isoforms in compound null mutant mice results in abnormal lung growth and early respiratory deaths (21).

The role of RA in lung development and surfactant production is being explored in various experimental models. Regulation of the phospholipid component of surfactant by RA has been examined in in vivo studies in which maternal administration of RA increases fetal lung surfactant phospholipids and choline incorporation into phosphatidylcholine (PC; see Ref. 13). In isolated fetal rat type II cells, which produce surfactant, RA stimulates choline incorporation into PC despite its ability to inhibit type II cell proliferation (12). Type II cells isolated from vitamin A-deficient adult rats incorporate less choline into PC and disaturated PC (DSPC) compared with controls, whereas adding RA stimulates choline incorporation into both PC and DSPC in control and deficient cells (35), similar to the finding in fetal type II cells.

Fetal lung explants and lung adenocarcinoma cells have been utilized to determine the effect of RA on surfactant protein (SP). In 13.5-day fetal rat lung cultured for up to 9 days, RA added to the media augments the pattern of lung development toward more growth of proximal airways and also suppresses the expression of the SP genes SP-A, SP-B, and SP-C, which usually occur in the peripheral portions of lung during alveolar development (5). In 17- or 19-day fetal rat lung explants, exposure to RA results in increases in SP-A, SP-B, and SP-C mRNA, although each shows different dose-response characteristics (4). At 10-10 M RA, SP-A mRNA maximally increases, but at 10-5 M RA, it decreases. In contrast, 10-5 M RA maximally stimulates SP-C and SP-B mRNA. Human fetal lung explants treated with RA for 6 days show dose-dependent decreases in SP-A protein and mRNA levels, a decrease in SP-C mRNA, and an increase in SP-B mRNA (26). When H441 human lung adenocarcinoma cells are treated with RA, SP-A mRNA levels are unaffected, but SP-B mRNA levels increase in a dose-dependent manner (15). Together, these results are suggestive that RA generally decreases SP-A mRNA expression, increases SP-B mRNA expression, and has a more variable effect on SP-C mRNA in the systems studied thus far. Cell source and the concentration and length of exposure time to RA likely account for the variable responses noted.

Lung development is also influenced by glucocorticoids, which regulate SP expression and affect morphological differentiation (18). In vivo administration of glucocorticoids to pregnant rats results in stimulation of SP genes, with SP-C mRNA increasing with the lowest dose (11). Studies in fetal lung explants show that SP-A mRNA levels increase at low concentrations of dexamethasone (Dex; 10-10 to 10-9 M; see Ref. 3), which is due to a stimulation of transcription. However, at higher concentrations (10-8 M), Dex has an inhibitory effect resulting from reduced SP-A stability. Induction of SP-B mRNA appears to result from both an increase in gene transcription and mRNA stability (23, 31). Dex also induces an increase in SP-C mRNA in fetal lung explants, although reports on changes in gene transcription and mRNA stability are mixed (2, 31).

Although the roles of Dex and RA in the lung have mostly been examined independently, a limited amount of research has suggested that the actions of Dex and vitamin A are interrelated. The lungs of infants with bronchopulmonary dysplasia (BPD) display alterations in function and morphology similar to those resulting from vitamin A deficiency (28); postnatal Dex is a frequent treatment for these infants. Serum retinol increased in fetuses and infants with BPD in response to glucocorticoids (16, 36). In rats, Dex induced an increase in plasma vitamin A simultaneously with a decrease in liver and lung vitamin A storage (17). Specifically in the lung, the expression of RAR-beta mRNA (20) and RAR binding (24) is decreased by Dex.

Although such interactions between Dex, vitamin A, and lung development exist, the mechanisms involved are presently unknown. The objective of the present study was to begin exploring this relationship by examining the effects of RA and Dex on RAR and SP-C gene expression. To determine any relationship between RA and Dex, this study evaluates their effects together in the same system, utilizing a distal respiratory cell line that can maintain a differentiated phenotype in culture. The effects of retinoids have not previously been investigated in this cell line.

    METHODS
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Methods
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References

Cell culture. Murine lung epithelial cell line MLE-12 (a gift of J. A. Whitsett, Children's Hospital Medical Center, Cincinnati, OH) is a clonal line representing distal bronchiolar and alveolar epithelium. It is derived from pulmonary tumors in mice transgenic for the chimeric gene composed of the simian virus 40 (SV40) large tumor antigen under transcriptional control of the promoter region of the human SP-C gene (33). The MLE-12 cell line possesses many functional and morphological characteristics of pulmonary type II cells, including typical epithelial cell morphology, presence of microvilli and multivesicular bodies, synthesis and secretion of phospholipids, and production of SP-B and SP-C. Cells were maintained in an humidified atmosphere at 37°C and 5% CO2 in modified hydrocortisone-insulin-transferrin-estrogen-selenium medium (6) containing 10 mg/ml transferrin, 4 mM L-glutamine, and 10 mM N-2-hydroxyethylpiperazine-N '-2-ethanesulfonic acid supplemented with 2% fetal bovine serum, 100 U/ml penicillin G, and 100 mg/ml streptomycin. Cells were used at passages 23-27 in culture.

Treatments. Cells were treated with Dex (water soluble) and/or all trans-RA that were dissolved in dimethyl sulfoxide (DMSO). Controls with similar levels of DMSO as RA treatment groups showed no difference in mRNA levels. RA solutions were used under subdued lights, and culture flasks were kept dark during treatment. For most experiments, two or three replicate flasks were used for each experimental condition, and, to further increase sample size, experiments were repeated various times. For determining optimum concentration levels, cells were treated with 10-8 to 10-6 M RA or 10-10 to 10-6 M Dex for 48 h. From these experiments, it was determined that 10-6 M RA and 10-7 M Dex produced maximal effects, and therefore these concentrations were used in subsequent experiments. To determine the effects of RA and Dex together, RA and Dex were added simultaneously to culture media followed by incubation for 48 h. For time-course studies, RA or Dex was added to the media, and cells were cultured for varying lengths of time from 2 to 48 h. The effect of the protein synthesis inhibitor cycloheximide (CHX) was determined by incubation of cells with 0.1 mg/ml CHX for 1 h followed by treatment with or without RA or Dex for 24 h. Preliminary work showed no effect of 0.1 or 0.5 mg/ml CHX at 6 h. Measurement of RNA degradation was evaluated by the use of actinomycin D (Act D). Cells were treated with RA or Dex for 24 h, after which 0.4 mg/ml Act D was added to the media. Cells were then incubated for various lengths of time from 2 to 12 h. The amount of mRNA present after time was expressed relative to its 0-h control. Half-lives were calculated from the equation: t1/2 = ln 2/k [t1/2 is half-life, and k is the degradation rate constant that equals -2.303 (slope)].

Isolation of RNA and Northern and data analyses. After MLE cells were treated as above, total RNA was extracted using Trizol Reagent (GIBCO BRL, Grand Island, NY) as specified by the manufacturer. Preparation of gels, transfer to nylon membranes, labeling of probes, and hybridization procedures have been previously described (20). The cDNA probes for RAR-alpha , -beta , and -gamma , RXR-beta , and SP-C were the generous gifts of P. Chambon, Strasbourg, France, D. J. Mangesdorf, Dallas, TX, and J. A. Whitsett, Cincinnati, OH, respectively. To correct for RNA levels, filters were also probed with 28S rRNA (Clontech, Palo Alto, CA). Filters were quantitated using a phosphorimager (Bio-Rad, Hercules, CA). All data were normalized to corresponding 28S rRNA levels. Treatment values were expressed relative to mean control values.

Statistical significance of the difference between control and treatment groups was assessed by nonpaired Student's t-test or by analysis of variance in CHX experiments. Data determining half-lives of mRNA were fitted by linear regression analysis. For all analyses, P < 0.05 was considered significant.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

The MLE-12 cells contain mRNA of all three types of RARs (alpha , beta , gamma ), RXR-beta , and SP-C (Fig. 1). All transcripts are of sizes and patterns previously reported (19, 37). RAR-alpha , RAR-gamma , and RXR-beta mRNA were not changed by RA in these experiments (data not shown) and, therefore, were not studied further. RA increases RAR-beta and SP-C mRNA levels, with the greatest effect occurring at 10-6 M RA (Fig. 2). RA affected the expression of both RAR-beta and SP-C mRNA in a time-dependent manner (Fig. 3). RAR-beta mRNA was higher by 2 h, and SP-C mRNA was higher by 24 h. RA at 10-6 M had the maximal effect by 24 h on both RAR-beta and SP-C mRNA, with no further stimulation noted up to 48 h of incubation.


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Fig. 1.   Expression of retinoic acid (RA) receptors (RAR)-alpha , -beta , and -gamma , 9-cis-RAR (RXR)-beta , and surfactant protein (SP) C mRNAs. Extraction, fractionation on gel, transfer to membrane, and hybridization to cDNA probes are described in text. Sizes of the mRNA species as well as the position of the 28S and 18S mRNA bands are indicated.


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Fig. 2.   Effect of RA dose on expression of RAR-beta and SP-C mRNA. MLE-12 cells were treated with concentrations of RA ranging from 10-8 to 10-6 M for 48 h. Values are expressed relative to untreated controls and are means ± SE of 2-3 replicates from 2 experiments (RA-8 and RA-7) and 4 experiments (RA-6 and control). Significant difference from control: * P < 0.01; ** P < 0.001. [Retinoic acid], RA concentration.


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Fig. 3.   Time course of RA action on RAR-beta and SP-C mRNA. MLE-12 cells were treated with 10-6 M RA for various time intervals from 2 to 48 h. Values are expressed relative to same time untreated controls and are means ± SE of 2-3 replicates from 2 experiments (2, 4, and 8 h) and 8 experiments (24 and 48 h). Significant difference from time 0: * P < 0.05; ** P < 0.01.

Dex decreased RAR-beta and SP-C mRNA in MLE cells over a range of concentrations (Fig. 4). This effect did not occur until after 12 h of Dex treatment (Fig. 5). Dex had no effect on RAR-alpha , RAR-gamma , or RXR-beta mRNA (datanot shown). In cells treated with RA, Dex did not significantly affect the increase in RAR-beta or SP-C caused by RA (Fig. 6).


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Fig. 4.   Effect of dexamethasone (Dex) dose on expression of RAR-beta and SP-C mRNA. MLE-12 cells were treated with concentrations of Dex ranging from 10-10 to 10-6 M Dex for 48 h. Values are expressed relative to untreated controls and are means ± SE of 2-3 replicates from 1 experiment (10-10 M Dex), 3 experiments (10-9, 10-8, and 10-6 M Dex), and 10 experiments (10-7 M Dex and control). Significant difference from control: * P < 0.05; ** P < 0.01. [DEX], Dex concentration.


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Fig. 5.   Time course of Dex action on RAR-beta and SP-C mRNA. MLE-12 cells were treated with 10-7 M Dex for various time intervals from 4 to 48 h. Values are expressed relative to same time untreated controls and are means ± SE of 2-3 replicates from 2 experiments (4 and 12 h) and 7 experiments (24 and 48 h). Significant difference from time 0: * P < 0.05; ** P < 0.01.


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Fig. 6.   Effect of Dex and RA, alone or in combination, on RAR-beta and SP-C mRNA. MLE-12 cells were treated with 10-7 M Dex and/or 10-6 M RA for 48 h. Values are expressed relative to untreated controls and are means ± SE of 2-3 replicates of 2 experiments. Significant difference from control: * P < 0.05; ** P < 0.01.

To determine the need for protein synthesis in the response of RAR-beta and SP-C mRNA levels to RA and Dex treatment, the protein synthesis inhibitor CHX was utilized. Treatment with CHX alone resulted in an increase in abundance of RAR-beta mRNA (Fig. 7), which has been shown previously (29, 32). CHX did not affect the stimulation by RA of RAR-beta mRNA, but it prevented the inhibition by Dex.


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Fig. 7.   Effect of cycloheximide (CHX) on response of RAR-beta and SP-C mRNA to RA or Dex. Before the 24-h incubation with 10-7 M Dex or 10-6 M RA, cells receiving CHX treatment were preincubated with 0.1 mg/ml CHX for 1 h. Values are expressed relative to untreated controls and are means ± SE of 2-3 replicates from 3 experiments. Significant difference from control: * P < 0.05; ** P < 0.01. x P < 0.05, CHX vs. CHX + Dex; z P < 0.005, CHX vs. CHX + RA.

In contrast to its effect on RAR-beta , CHX alone caused a marked decrease in the level of SP-C transcripts (Fig. 7). A similar effect has been reported on SP-A mRNA (3). The same level of decrease occurred using a greater concentration of CHX (0.5 mg/ml; data not shown). Addition of RA and Dex resulted in an increase in SP-C transcript abundance above the level found in the presence of CHX alone.

To determine whether changes in RAR-beta and SP-C mRNA levels with RA and Dex may be due to alterations in mRNA stability, cells were treated with the RNA synthesis inhibitor Act D. Act D reduced the level of RAR-beta mRNA to 50% after 2.5 h and of SP-C mRNA after 12.3 h (Fig. 8). These half-lives are similar to previously reported values (2, 30). The decline in RAR-beta and SP-C mRNA induced by Act D was similar in control and Dex-treated cells. However, RA slightly slowed the decline 1.5-fold (t1/2 = 18.9 h) in SP-C mRNA; RA did not affect the RAR-beta half-life.


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Fig. 8.   Effect of actinomycin D (Act D) on response of RAR-beta (A) and SP-C (B) mRNA to RA or Dex. After 24 h of treatment with 10-7 M Dex or 10-6 M RA, Act D (4 mg/ml) was added to media, and cells were incubated for various time intervals from 2 to 12 h. Values are expressed relative to untreated controls at 0 h. Three experiments with 2-3 replicates were conducted; this graph is data from 1 representative experiment.

All of the above experiments were conducted in media containing 2% fetal bovine serum as well as other supplements. Analysis of the amount of retinol in one batch of serum determined that medium concentrations of retinol were 10-9 M; when this level of RA was added to media, no effect was detected on RAR-beta or SP-C mRNA. Because other factors present in the media, such as albumin and hormones, could affect results, several experiments were conducted that compared the effects of RA and Dex on RAR-beta and SP-C mRNA in control media and in media without added serum and hydrocortisone. There were no differences in RAR-beta or SP-C mRNA levels and the magnitude of response to RA or Dex between control and serum-free groups.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

Lung differentiation, growth, and health are affected by vitamin A, as is apparent from several lines of evidence. In vivo and organ culture studies demonstrate that vitamin A deficiency causes bronchiolar and upper airway changes characterized by loss of ciliated and mucus-secreting cells and replacement of normal columnar epithelium by stratified squamous keratinizing epithelium (7). The need for retinol in cell differentiation and proliferation has been confirmed using cultures of various pulmonary cells (7). Recent genetic evidence demonstrates abnormal lung growth and early respiratory deaths in compound null RAR mice (21). Clinical findings also associate vitamin A deficiency with pulmonary morbidity (10), and some speculate that vitamin A might have a role in the prevention and/or repair of lung injury in human premature newborns (7, 36). These observations have led to recent active investigations regarding the effect of RA on lung development and surfactant in several systems.

With the increasing use of Dex both antenatally and in neonates with BPD, a clear understanding of the mechanism of the interaction between Dex and vitamin A is necessary because Dex has dramatic effects on vitamin A. Dex transiently raises neonatal plasma vitamin A and decreases liver stores (16, 35). Dex decreases RAR binding (24) and decreases specifically the RAR-beta mRNA expression in rat whole lung and fetal lung explants (20). Fetal lung development may be affected by Dex through its ability to change fetal retinoid levels and distribution (14), with subsequent morphological and biochemical alterations.

The objective of the present study was to more clearly understand the roles of RA and Dex in the lung by comparing the effects of RA and Dex on RAR-beta and SP-C mRNAs. To do this, we have used the MLE cell line in which morphological and functional characteristics of distal respiratory epithelial cells are maintained (33). MLE-12 cells are derived from pulmonary adenocarcinomas of transgenic mice produced by expression of SV40 large T antigen under transcriptional control of the bronchoalveolar-specific SP-C promoter. No prior studies have been done to determine the effects of RA and Dex in this cell line.

Other lung carcinoma cells express all or a partial component of the alpha , beta , and gamma  types of RAR (27). RAR-beta is usually, but not universally, responsive to RA in these cell lines. All three types of RARs were expressed in the MLE-12 cell line studied here, but only RAR-beta was responsive to RA, which occurred within 2 h of exposure to 10-6 M RA. The rapid response of RAR-beta mRNA to RA can be explained by the presence of a response element for RAR-beta (RARE) within the promoter of the RAR-beta gene (22). SP-C mRNA also increased with 10-6 M RA, the effect beginning after 8 h of exposure and peaking after 24 h of incubation. Previous studies involving the effect of RA on SP-C mRNA have been conducted in fetal lung explants (4, 15, 26) and have shown variable results, apparently due to RA concentration and length of exposure.

The decrease in RAR-beta mRNA in response to Dex concurs with our previous observations in whole neonatal rat lung, in which Dex decreased RAR binding (24) and RAR-beta mRNA accumulation (20). Studies in vivo and in fetal lung explants demonstrate a stimulation of SP-C mRNA in response to Dex (11, 23, 31). The inhibition by Dex of SP-C mRNA in the MLE-12 cell line has been previously observed (J. Whitsett, personal communication). Perhaps this indicates an absence in this particular cell line of a type II cell-specific factor required in the Dex response pathway.

The rapid response of RAR-beta to RA suggests that the regulation by RA of RAR-beta is a primary effect through the RARE. However, the slower response of SP-C to RA and of both RAR-beta and SP-C to Dex could be indicative of a requirement for synthesis of an intermediate protein involved in regulation of the mRNA. The results of experiments utilizing the protein synthesis inhibitor CHX suggest that the stimulation by RA of not only RAR-beta but also SP-C mRNA is independent of protein synthesis. These experiments also indicate that the inhibition by Dex of RAR-beta and SP-C mRNA requires protein synthesis. The effect of CHX alone on RAR-beta and SP-C, however, indicates that caution should be exercised in interpreting the data. RAR-beta transcript levels are increased in the presence of CHX alone. This may be due to inhibition of synthesis of a repressive trans-acting factor or may result from a nonspecific stabilizing effect on polysomal RNA (25). Simultaneously, SP-C mRNA levels are reduced by CHX, which could result from inhibition of an essential labile transcription factor or may involve modification of posttranscriptional events.

Modulation of mRNA stability is an important posttranscriptional mechanism for control of gene expression (1). In this study, mRNA stability was assessed by the use of Act D. No change in the rate of RAR-beta mRNA decline resulted from RA or Dex, but RA did somewhat decrease the degradation rate of SP-C mRNA. Although the increase in SP-C mRNA levels in cells treated with RA in the presence of CHX suggests that the response to RA does not involve synthesis of a stabilizing protein, RA may be involved in stimulating the modification of a protein factor that stabilizes SP-C mRNA.

The different responses of the two genes studied here to RA and Dex suggest that the effect of RA and Dex on each specific mRNA may involve separate promoter regions in the gene with separate factors involved in transcription. However, the lack of inhibition by Dex on both RAR-beta and SP-C mRNA abundance in the presence of RA indicates some level of interaction in the gene regulation mechanisms that is not understood at this point. An examination of the promoter regions of these genes would be helpful in clarifying the mechanism of action of RA and Dex. Also important for future consideration is the role of heterodimer formation that occurs between different steroid receptors.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grant 1-P50-HL-46478 and National Institute of Child Health and Human Development Grant ROI-HD-33916.

    FOOTNOTES

Address for reprint requests: R. D. Zachman, Univ. of Wisconsin, Meriter Perinatal Center, 202 S. Park St., Madison, WI 53715.

Received 19 May 1997; accepted in final form 23 September 1997.

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Abstract
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Discussion
References

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AJP Lung Cell Mol Physiol 274(1):L1-L7
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