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Am J Physiol Lung Cell Mol Physiol 284: L738-L748, 2003. First published January 10, 2003; doi:10.1152/ajplung.00375.2002
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Vol. 284, Issue 5, L738-L748, May 2003

Human SP-A 3'-UTR variants mediate differential gene expression in basal levels and in response to dexamethasone

Guirong Wang1, Xiaoxuan Guo1, and Joanna Floros1,2

Departments of 1 Cellular and Molecular Physiology and 2 Pediatrics, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Human surfactant protein A (SP-A) is encoded by two genes (SP-A1, SP-A2), and each is identified with several alleles. SP-A is involved in normal lung function, innate immunity, inflammatory processes, and is regulated by glucocorticoids. We investigated the role of 3'-untranslated region (UTR) of 10 SP-A variants on gene expression using transient transfection of 3'-UTR constructs in the human lung adenocarcinoma cell line NCI-H441. We found: 1) both basal mRNA and protein levels of the reporter gene of SP-A 3'-UTR constructs are significantly (P < 0.01) reduced compared with controls (vector pGL3 and surfactant protein B pGL3) and that differences exist among alleles; and 2) after dexamethasone (Dex) treatment (100 nM for 16 h), mRNA was reduced (31-51%). Seven alleles showed a significant decrease (P < 0.05) in mRNA, and three did not. Reporter activity was also decreased, from 17% (1A1) to 38% (1A), with six alleles showing a significant decrease. The data indicate that the 3'-UTR of SP-As play a differential role in SP-A basal expression and in response to Dex. Therefore, a careful consideration of individual use of steroid treatment may be considered.

allele; gene regulation; surfactant protein A; 3'-untranslated region


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PULMONARY SURFACTANT, a lipoprotein complex, is essential for normal lung function because deficiency of surfactant can lead to various diseases, including respiratory distress syndrome (RDS), in the prematurely born infant. Surfactant protein A (SP-A) is involved in surfactant physiology (19) and plays a major role in innate host defense and the regulation of inflammatory processes in the lung (38, 51).

The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2, in opposite transcriptional orientation, and a nonfunctional gene (25). A number of alleles have been characterized for each SP-A gene (17). On the basis of coding sequence differences to date, four alleles of SP-A1 (6A, 6A2, 6A3, 6A4) and six alleles of SP-A2 (1A, 1A0, 1A1, 1A2, 1A3, 1A5) are found frequently (>1%) in the general population (17). In addition, sequence variability within the 3'-untranslated region (3'-UTR) has been observed between the human SP-A genes/alleles (26, 34, 53). Therefore, sequence differences among human SP-A genes/alleles within both the coding and noncoding regions hold the potential for functional and regulatory differences, respectively. Our preliminary published information indicates that both the coding region and the noncoding region may result in functional (58, 59) and regulatory (26) differences between the two SP-A genes and among the alleles.

Human SP-A is regulated by a variety of molecules, including several hormones, some cytokines, growth factors, and oxygen (43). For example, insulin, TNF-alpha , and glucocorticoids at high concentrations decrease human SP-A expression (8, 15, 18, 28, 31, 57). In clinical practice, steroid therapy is widely used for mothers who are in danger of delivering prematurely and is also used commonly in the management of preterm infants with chronic lung disease [bronchopulmonary dysplasia (BPD)] to improve pulmonary compliance and to wean infants from ventilators (6, 12, 33, 44). However, concerns have been raised about side effects of steroid therapy, such as increased blood pressure, increased blood sugar, transient adrenal suppression in infants, and impaired bone growth in children (30, 48, 63). Recent findings from an animal study indicate that dexamethasone (Dex; a synthetic glucocorticoid) administration impairs normal postnatal lung growth in rats (49). Therefore, a careful consideration of balancing short- and long-term effects of steroid therapy may be warranted. In this regard, weighing genetic and environmental factors together with such untoward effects of steroid therapy may allow for a better assessment of the optimal dose and duration of Dex treatment for a given individual's therapy.

Glucocorticoids (GCs) play a role in the modulation of gene transcription, posttranscription, and translation events by several different mechanisms. The glucocorticoid effect on human SP-A gene expression is complex because it may involve transcriptional and posttranscriptional events (8, 27, 28, 43, 52). In human fetal lung explants, GCs reduce SP-A gene expression in a time- and dose-dependent manner. At low concentrations (<10 nM), GCs increase SP-A mRNA levels (5, 14, 28, 39, 43), and at high concentrations (100 nM), GCs decrease SP-A expression (26-28, 31, 43). In contrast, Dex at 1-100 nM has only an inhibitory effect on SP-A gene expression in the lung adenocarcinoma cell line NCI-H441 (50, 52). The decay rate of SP-A mRNA in the presence of Dex was shown to be biphasic in fetal lung explants, with an initial rapid decrease followed later by a slower decay rate (28). It was suggested that this biphasic decay rate was a result of differential effects of Dex on SP-A1 and SP-A2 mRNA stabilities. In fact, the two SP-A genes have been shown to be differentially regulated by certain agents, including GCs (27, 31, 35, 42, 55), and certain SP-A alleles may be differentially regulated by Dex (26).

Accumulating evidence indicates that the 3'-UTR of mRNA may play an important role in regulating gene expression and may be essential for the appropriate expression of many genes (10). Recent findings show that the 3'-UTR of mRNA may affect several processes of gene expression, such as mRNA stability (7, 37, 45, 46), increased efficiency of mRNA formation (21), transport and localization, posttranscriptional modification and degradation, and translation (11, 16, 21, 22). GCs have been shown to influence gene expression through the modulation of mRNA-protein interactions in the 3'-UTR of mRNA (20) and of the 3'-UTR poly(A) of mRNA (23, 24). Moreover, studies of SP-A indicate that certain regulatory regions, including the 3'-UTR of the human SP-A gene, play a role in the GC-induced inhibition of gene expression and may mediate differential allelic expression in response to Dex (26). Allelic differences within regulatory regions may account for differences in drug response among individuals, and these differences may help us to better understand some of the side effects that may occur in the course of steroid therapy of prematurely born infants with RDS, BPD, and/or other pulmonary diseases. Therefore, a more in-depth understanding of the factors affecting the expression of SP-A may lead to better strategies in the treatment and/or prevention of pulmonary diseases of the prematurely born infant.

In the present study, we investigated the role of 3'-UTR on gene expression of 10 hSP-A alleles 1) at the basal level and 2) in response to Dex treatment. We observed that the SP-A 3'-UTR leads to a decrease in basal gene expression compared with controls and reduces expression levels after Dex treatment. Differences in basal level and in response to Dex exist among the 10 hSP-A alleles studied. Nucleotide differences within the 3'-UTR region among SP-A alleles and potential factors that may account for these observations are discussed.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell line and cell culture conditions. The lung adenocarcinoma cell line NCI-H441 from American Type Culture Collection (Manassas, VA) was used. The cells were grown in RPMI 1640 medium (Invitrogen, Carlsbad, CA) containing 10% heat-inactivated fetal bovine serum (FBS), 1× antimycotic-antibiotic solution (Sigma, St. Louis, MO), and 1% L-glutamine (Sigma) at 37°C in 5% CO2 atmosphere. The cells were fed and passaged weekly.

SP-A 3'-UTR plasmid constructs. Luciferase reporter constructs were generated by ligation of the pGL3 luciferase reporter vector (Promega, Madison, WI) with PCR products spanning the entire 3'-UTR of SP-A, including the poly(A) addition recognition signal (Fig. 1). First, SP-A1- and SP-A2-specific segments were amplified using oligonucleotide-specific primers for SP-A1 (primer pair 326/1,074) and SP-A2 (primer pair 327/1,075; all primer information shown in Table 1) together with genomic DNA from individuals of certain homozygous genotypes (e.g., 6A26A2/1A01A0, etc.). PCR of gene-specific genomic segments was performed in duplicate and treated as independent samples throughout to better control for PCR error. PCR products were cloned and sequenced. Lack of PCR errors would most likely result in clones with identical sequence from each independent PCR.


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  		Fig. 1.   Firefly luciferase (Luci) vector pGL3/surfactant protein A (SP-A) 3'-untranslated region (UTR) construct. The firefly luciferase gene was driven by the simian virus 40 (SV40) promoter. SP-A 3'-UTR of ~1.3-kb fragments of alleles (6A, 6A2, 6A3, 6A4, 1A, 1A0, 1A1, 1A2, 1A3, 1A5) was inserted into XbaI site of vector pGL3. A total of 10 constructs, each containing a different 3'-UTR, were generated. In addition, a 1.3-kb fragment from the human surfactant protein B (SP-B) gene cDNA was cloned into this site to generate a control SP-B construct.


                              
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  		Table 1.   Primers used in this study

The SP-A1 and SP-A2 gene-specific PCR products were then used as templates for a second round of amplification with the primer pairs 1073/1074 and 1073/1075, thus amplifying the entire 3'-UTR. Primer location and sequences are shown in Table 1. The nucleotide locations are according to White et al. (60) for SP-A1 and Katyal et al. (32) for SP-A2. All primers at the 3'-UTR contain the specific enzyme recognition site of XbaI. These XbaI PCR products were then cloned into the XbaI site of the pGL3 vector.

A total of 10 constructs, four SP-A1 alleles (6A, 6A2, 6A3, 6A4) and six SP-A2 alleles (1A, 1A0, 1A1, 1A2, 1A3, 1A5), were generated. In addition, a positive reporter control plasmid was prepared by cloning a 1.3-kb fragment of surfactant protein B (SP-B) cDNA consisting of the entire coding region and ~0.3 kb of the 3'-UTR (29). Recombinant DNA was performed according to standard methods (54). Plasmid DNA for transfection was prepared using the Qiagen plasmid Maxi kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions.

Transient transfection and Dex treatment. Various constructs and reporter control plasmids were transiently cotransfected into NCI-H441 cells, and expression of the reporter gene was analyzed (Fig. 2). The reporter control plasmid used as standardization in this study was Renilla luciferase reporter plasmid pRL-SV40 (Promega) or pCMV-SPORT-beta -gal (Invitrogen). Our preliminary findings unexpectedly indicated that Renilla luciferase reporter plasmid pRL-SV40 responds to Dex treatment, making it an unsuitable control for the Dex experiments. Thus Renilla was used for assessment of basal levels, and beta -galactosidase (beta -gal) was used where assessing levels in response to Dex.


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  		Fig. 2.   Outline of the cotransfection protocol. The experimental construct and transfection control vector were cotransfected in the H441 cells to control for the efficiency of transfection. The results are normalized to the transfection control. beta -gal, beta -galactosidase; Dex, dexamethasone.

NCI-H441 cells were grown to 80-90% confluency in 10-cm dishes and subcultured into six-well culture plates with 1 × 106 cells/well 24 h before transfection. Four hours before transfection, the RPMI 1640 medium plus 10% FBS was replaced with DMEM (Invitrogen) containing neither FBS nor antibiotics.

The transfection procedure was performed using the Lipofectamine Plus reagent kit (Invitrogen). In brief, 1 µg of DNA of experimental construct plus 0.05 µg of DNA of control pRL-SV40 plasmid or 0.3 µg of DNA of pCMV-SPORT-beta -gal plasmid were diluted into 100 µl of DMEM without serum. Six microliters of Plus reagent were added, and the mixture was incubated for 15 min at room temperature. In another tube, 4 µl of Lipofectamine reagent were diluted into 100 µl of DMEM without serum. The components of the two tubes were then combined, mixed, and incubated for 15 min at room temperature. The above DNA-Lipofectamine complex was added to each well containing H441 cells with 2.5 ml of fresh medium. Four hours after transfection, the DMEM with 10% FBS was added to normal culture volume (5 ml/well). Transfection was carried out for 36 h or for the indicated time point at 37°C in 5% CO2 atmosphere for basal gene expression assay. For Dex-treated experiments, the medium was changed to RPMI 1640 plus 1% L-glutamine 24 h after transfection, and then cells were treated with Dex (100 nM) 36 h after transfection.

Enzyme activity assay. Both dual luciferase assay (firefly/Renilla luciferase) and firefly luciferase/beta -gal activity were used in this study. The pRL-SV40 (Renilla luciferase) in dual luciferase assay or pCMV-SPORT-beta -gal was cotransfected with pGL3 reporter vector (firefly luciferase) to H441 cells. Thus all values are expressed as the ratio firefly-Renilla luciferase activity or firefly luciferase activity-beta -gal activity. For all experiments, transfection and luciferase assay were performed in triplicate.

Dual luciferase assay. Dual luciferase assay was performed with the Dual luciferase reporter assay system (kit) (Promega). Transfected cells were harvested at 36 h after transfection or at other time points according to statement in the experiments. The cells were washed with 1× PBS and were dissolved in 500 µl of 1× passive lysis buffer. The culture plates were rocked at room temperature for 15 min, and the lysate was transferred to a tube and centrifuged for 1 min at 4°C to clear the lysate. Twenty microliters of cell extract were transferred into a luminometer tube containing 100 µl of luciferase Assay Reagent II (LAR II). The tube was placed in an FB12 luminometer (Zylux, Maryville, TN) to initiate firefly luciferase activity reading. Stop & Glo Reagent (100 µl) was placed into the tube to initiate Renilla luciferase activity reading. The ratio of firefly luciferase activity to Renilla luciferase activity was calculated.

Luciferase assay and beta -gal enzyme assay. Luciferase assay and beta -gal enzyme assay were performed with the luciferase reporter assay system and the beta -gal enzyme assay system (Promega). Briefly, transfected cells were harvested at various time points after transfection with or without Dex treatment. The cells were washed with 1× PBS and were dissolved in 500 µl of 1× reporter lysis buffer. The culture plates were rocked at room temperature for 15 min, and the lysate was transferred into a tube. The lysate was frozen and thawed for several cycles and centrifuged for 1 min at 4°C to remove cell fragments. Firefly luciferase was analyzed according to the above statement. For the beta -gal activity assay, 150 µl of diluted (3:1) cell extract and 150 µl of assay buffer (2×) were put together and mixed, and the mixture was incubated at 37°C for 30 min. The reaction was stopped by the addition of 500 µl of 1 M sodium carbonate. The absorbance at 420 nm was read with a spectrophotometer (Ultrospec 4050, Cambridge, UK). The ratio of firefly luciferase activity to beta -gal activity was calculated.

Total mRNA preparation and measurement by real-time PCR method. Total mRNA was prepared from cells according to the method of RNA-Bee kit (Tel-Test, Friendswood, TX). Briefly, the culture medium in the wells was removed, and the cells were washed with PBS buffer. One milliliter of RNA-Bee solution was added, and the cells were homogenized after addition of 0.2 ml of chloroform. The aqueous phase was transferred to a clean tube, and total RNA was precipitated by adding 0.5 ml of isopropanol for 5-10 min at room temperature and centrifuging at 12,000 g for 5 min at 4°C. The pellet was washed with 75% ethanol. The total RNA was dissolved in RNase-free ddH2O. Any contaminating DNA was removed from RNA preparations by using a DNA-free kit (Ambion, Austin, TX). Real-time PCR was performed using the ABI Prism 7700 sequence detection system and a kit of TaqMan one-step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA). In brief, 100 ng of RNA were added into 50 µl of real-time PCR mix buffer. The buffer contained forward/reverse primer pairs (each 50 nM), such as primers 1152/1153 targeting the reporter gene (luciferase gene) of pGL3, and a probe primer, such as probe-pGL (see Table 1), and other enzyme and mixing reagents provided by the manufacturer (Applied Biosystems). The real-time PCR reaction was carried out through two steps: 1) one cycle of 48°C for 30 min and 95°C for 10 min, and 2) 43 cycles of 95°C for 15 s and 60°C for 1 min. The mRNA of luciferase from RNA preparation was standardized with control vector and expressed as copies per nanogram of total RNA.

Statistical analysis. In the present study, at least three independent experiments and triplicate culture dishes for each experiment were performed. The data were analyzed using the ANOVA test. Statistical significant differences were considered when P < 0.05, and the results are expressed as means ± SD.

Alignment of SP-A 3'-UTR DNA sequences. The alignment was performed using a megAlign program of DNASTAR (version 5.0) by clustal V method and included entire 3'-UTR sequences of all 10 alleles after the translation termination codon.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Comparison of 3'-UTR DNA sequences of 10 hSP-A alleles. The entire 3'-UTR of 10 hSP-A alleles from human genomic DNA was cloned using PCR amplification. To eliminate clones with PCR errors, two independent PCR amplifications were performed for each SP-A allele, and then three independent clones from each PCR reaction were sequenced. The sequence identity and divergence of 10 SP-A 3'-UTR DNA sequences are summarized in Table 2. Specific sequence differences between the SP-A genes and/or alleles include the following: 1) an 11-bp insertion/deletion (indel) that is present at nucleotide positions 405-416 (26) in all six SP-A2 alleles and the SP-A1, 6A2 allele; this 11-bp sequence is absent from the other three commonly found alleles of SP-A1; 2) all SP-A2 alleles lack 3 bp starting at the nucleotide position 713 after the stop codon compared with SP-A1; 3) all SP-A1 alleles lack 5 bp starting at the nucleotide position 934 after the stop codon compared with SP-A2; 4) other single nucleotide transitions and transversions among the alleles were identified. The nucleotide length of the 3'-UTR for all six SP-A2 alleles is 1,308, for 6A2 is 1,304, and for the other three SP-A1 alleles (6A, 6A3, 6A4) is 1,293. Alignment results of the 10 3'-UTR DNA sequences indicate that the identities between SP-A1 and SP-A2 ranged from 87.8 to 89.2%; identities among SP-A1 alleles ranged from 98.4 to 99.8%, and among SP-A2 alleles are from 99.2 to 99.8% (Table 2).

                              
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  		Table 2.   Identity and divergence percentages of human SP-A 3'-UTR based on DNA sequence comparison

Time course of mRNA and protein mediated by SP-A 3'-UTR. The mRNA and protein (luciferase activity) contents were assessed from 6 to 52 h after transfection. The SP-A2 (1A allele) 3'-UTR construct was used for this experiment. As shown in Fig. 3, the results indicate that the mRNA level increases starting at 10 h after transfection and reaches the highest level at 18 h. From 18 to 24 h, the mRNA level slowly deceases and then reaches a plateau that is maintained at least up to 52 h from the start of the experiment. The time course of luciferase activity follows the mRNA time course as it may be expected. Therefore, a significant increase of luciferase activity starts at 24 h after transfection and peaks at 38 h (Fig. 3). On the basis of these results, the 36-h time point was used for subsequent experimentation.


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  		Fig. 3.   Time course of SP-A (1A) 3'-UTR-mediated expression. Time courses of mRNA and protein (luciferase activity) were analyzed from 6 to 52 h after transfection using the SP-A (1A) 3'-UTR construct. The mRNA level increases starting 10 h after transfection and reaches the highest level at the 18-h time point. From 18 to 24 h, the mRNA level slowly deceases and then stays at a plateau for up to 52 h of experimentation. The time course of luciferase activity is similar to that of mRNA, but a significant increase of luciferase activity starts at 24 h after transfection, reaching the highest level at the 38-h time point. The experiments were repeated 3 times.

Variation of 3'-UTR-mediated mRNA and protein among SP-A alleles. Basal mRNA levels of the 10 experimental SP-A 3'-UTR constructs, SP-B pGL3 (control), and vector pGL3 (control) were analyzed 36 h after transfection (Fig. 4). The basal mRNA levels of all 10 alleles (6A, 6A2, 6A3, 6A4, 1A, 1A0, 1A1, 1A2, 1A3, 1A5) are significantly reduced compared with the SP-B pGL3 and pGL3 vector control (P < 0.01). Variation in the basal mRNA level of various SP-A alleles is also observed. The 6A3 and 6A2 alleles have higher levels than the other (6A and 6A4) SP-A1 alleles; the 1A and 1A2 alleles of SP-A2 have higher levels than the other four SP-A2 alleles.


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  		Fig. 4.   Comparison of SP-A 3'-UTR-mediated expression at the mRNA level. This figure depicts basal mRNA levels of the experimental SP-A 3'-UTR constructs, SP-B pGL3 (control), and vector (pGL3) (control). The basal levels of all 10 alleles (6A, 6A2, 6A3, 6A4, 1A, 1A0, 1A1, 1A2, 1A3, 1A5) are significantly (P < 0.01) and differentially reduced compared with the SP-B pGL3 and pGL3 vector. Of the 4 SP-A1 alleles, both 6A3 and 6A2 show higher (P < 0.05) mRNA content than the 6A4 allele; of the 6 SP-A2 alleles, 1A and 1A2 show higher (P < 0.05) mRNA content than the other SP-A2 alleles (n = 3). The symbols a, b, c, and ab depict the statistical results (P < 0.05) from ANOVA test. Significant difference exists between any 2 alleles when they do not have the same symbols, for example, between 6A3 (ab) and 6A4 (c ) or between 6A (b) and 6A4 (c). In turn, if 2 alleles have any of the symbol(s) in common, no significant difference exists between them, for example, between 6A2 (ab) and 6A (b) or between 6A2 (ab) and 6A3 (ab). Bar shows ± SD.

The relative activities of firefly/Renilla luciferase (indicating protein level) were assessed 36 h after transfection (Fig. 5). The relative activities of firefly/Renilla luciferase of all 10 alleles are significantly reduced compared with SP-B pGL3 (control) and vector alone (P < 0.01). As with mRNA levels, variation in reporter gene activity among the alleles is observed (Fig. 5). Of the SP-A1 alleles, the 6A2 and 6A3 show higher levels (P < 0.05) than the 6A and 6A4 alleles. The 1A allele shows a higher level than all other SP-A2 alleles (P < 0.05), and the 1A1 and 1A5 alleles show lower levels than all other SP-A2 alleles (P < 0.05; Fig. 5). In addition, the SP-B pGL3 control was not significantly different from the pGL3 vector. These results indicate that the SP-A 3'-UTR may play a role in determining differentially, among alleles, both basal mRNA levels and basal protein levels.


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  		Fig. 5.   Comparison of SP-A 3'-UTR-mediated expression at the protein (luciferase activity) level. This figure depicts basal protein (luciferase activity) levels of the experimental SP-A 3'-UTR constructs, SP-B pGL3 (control), and vector (pGL3) (control). These experiments were repeated 4 times. The luciferase activities of all 10 alleles (6A, 6A2, 6A3, 6A4, 1A, 1A0, 1A1, 1A2, 1A3, 1A5) are significantly (P < 0.01) reduced compared with the SP-B pGL3 and pGL3 vector. Variation in basal levels among alleles is also observed. From the 4 SP-A1 alleles, 6A2 and 6A3 exhibit higher (P < 0.05) activity than 6A and 6A4 alleles; from the 6 SP-A2 alleles, 1A allele shows higher (P < 0.05) activity than the others; 1A0, 1A2, and 1A3 have higher activity (P < 0.05) than 1A1 and 1A5. The symbols a, b, and c depict the statistical results (P < 0.05) from ANOVA test (see legend for Fig. 4). Bar shows ± SD.

Comparison of luciferase activities with different transfection controls. Because the dual luciferase assay system with Renilla luciferase as control is a simple, accurate, and easy system, all basal level experiments were performed using this system. In our preliminary experiments, we found that the Renilla control vector pRL responded to Dex, i.e., Renilla luciferase activity of the Renilla control vector pRL was strongly inhibited by Dex. Therefore, we used the beta -gal expression vector pCMV-SPORT-beta -gal as the control vector because this vector is not sensitive to Dex treatment. To assess equity between the two cotransfection control vectors (pRL and pCMV-SPORT-beta -gal), we studied five representative alleles with regards to basal level. The relative basal level of the five alleles studied is similar with both control vectors pRL or pCMV-SPORT-beta -gal (Fig. 6). Therefore, pCMV-SPORT-beta -gal is an appropriate control vector in studies where the response to Dex is investigated.


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  		Fig. 6.   Comparison of relative luciferase activity with different transfection control vectors (pRL or pCMV-SPORT-beta -gal). Five representative allele constructs [6A, 6A3, 1A, 1A0, and 1A1 were cotransfected with control vector pRL (Renilla luciferase) or pCMV-SPORT-beta -gal (beta -gal)]. These experiments were repeated 3 times. We observed that the relative basal level of the 5 alleles studied is similar with both control vectors, pRL or pCMV-SPORT-beta -gal. The symbols a, b, and c depict the statistical results (P < 0.05) from ANOVA test (see legend for Fig. 4). Bar shows ± SD.

Time course of SP-A 3'-UTR-mediated response to Dex treatment. On the basis of our previous studies (26, 27), we used 100 nM Dex to treat transfected cells and to investigate the time course after Dex treatment with an SP-A2 allele (1A). Cells were treated with 100 nM Dex at 36 h after transfection, and luciferase activity was determined at several time points (0, 0.5, 1, 2, 4, 8, and 16 h) (Fig. 7). The results showed no significant differences in luciferase activity in either the presence or absence of Dex up to ~3 h from the start of the experiment. From 3 to 8 h, the luciferase activity decreased and reached a plateau after Dex treatment, but no decrease was observed in the absence of Dex. On the basis of the time course experiment, we decided to use the 16-h time point after Dex treatment (which corresponds to 52 h after transfection) for further experiments where we compared 3'-UTR-mediated differences among alleles in response to Dex.


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  		Fig. 7.   Time courses of protein (luciferase activity) with and without Dex treatment. The time course of protein (luciferase activity) was analyzed from 0 to 16 h after Dex treatment. The cells at 36 h after transfection were treated with 100 nM Dex, and luciferase activities at several time points, i.e., 0, 0.5, 1, 2, 4, 8, and 16 h, were analyzed. The results shown are from a representative experiment (n = 3). No significant differences in luciferase activity in the presence or absence of Dex up to ~3 h were observed. From ~3 to 8 h, luciferase activity decreased and reached a plateau after Dex treatment, but no decrease was observed in the absence of Dex.

Differential response to Dex among SP-A 3'-UTR alleles. We determined SP-A 3'-UTR-mediated mRNA expression among alleles in the absence or presence of 100 nM Dex at 16 h after Dex treatment (Fig. 8). Luciferase mRNA in Dex-treated cells was reduced 31-51% relative to untreated cells. Alleles 6A**, 6A4*, 1A**, 1A0**, 1A2**, 1A3**, 1A5* showed a significant decrease in luciferase mRNA (*P < 0.05 or **P < 0.01) in the presence of Dex compared with that observed in the absence of Dex. However, three alleles (6A2, 6A3, 1A1) failed to show significant differences in response to Dex (P > 0.05). The vector pGL3 and the control SP-B pGL3 also failed to respond to Dex.


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  		Fig. 8.   SP-A 3'-UTR mediates mRNA expression in the presence or absence of Dex. Total RNAs were extracted from the cells transfected with SP-A 3'-UTR constructs in the absence or presence of 100 nM Dex for 16 h after Dex treatment (see MATERIALS AND METHODS). These experiments were performed 3 times. Dex treatment decreases luciferase mRNA 31-51%. Alleles 6A**, 6A4*, 1A**, 1A0**, 1A2**, 1A3**, and 1A5* show a significant decrease in luciferase mRNA (*P < 0.05 or ** P < 0.01) in the presence of Dex compared with that observed in the absence of Dex. Three other alleles (6A2, 6A3, 1A1) show no significant differences in response to Dex (P > 0.05). The mRNA level of vector pGL3 shows no response to Dex treatment.

The relative luciferase activity in the presence or absence of 100 nM Dex was also analyzed. Firefly luciferase activity was normalized using beta -gal activity of pGL3 vector. We observed that Dex treatment decreased luciferase activity ranging from 17% (1A1) to 38% (1A) in the 10 constructs, each containing a different 3'-UTR. Alleles 6A2, 1A, 1A0, 1A2, 1A3, and 1A5 show a significant decrease in luciferase activity (P < 0.05) in the presence of 100 nM Dex (Fig. 9). Vector pGL3 and control SP-B pGL3 activities showed no response to Dex treatment. These results indicate that the SP-A 3'-UTR may differentially mediate the Dex response among SP-A alleles (Fig. 9).


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  		Fig. 9.   SP-A 3'-UTR mediates protein expression (luciferase activity) in the presence or absence of Dex. Luciferase activities were measured using the cell lysate from transfected cells with SP-A 3'-UTR constructs in the absence or presence of 100 nM Dex for 16 h after Dex treatment (see MATERIALS AND METHODS). These experiments were performed 4 times. Dex treatment decreased luciferase activity from 17% (1A1) to 38% (1A) in the 10 SP-A constructs. Alleles 6A2, 1A, 1A0, 1A2, 1A3, and 1A5 show a significant decrease in luciferase activity (P < 0.05) in the presence of Dex compared with that observed in the absence of Dex. Vector pGL3 and a control SP-B pGL3 activity show no response to Dex treatment.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Steroid therapy is widely used in the clinic to enhance lung maturity and surfactant production, to improve pulmonary compliance, and to help wean infants from the ventilator. However, concerns about the side effects of steroid therapy (30, 48, 63) and of possible long-term consequences in lung development (41, 49) have been raised. SP-A, a Dex-responsive molecule, plays important roles in surfactant-related activities, innate host defense, and in the regulation of inflammatory processes of the lung. Pilot studies indicate that Dex may have a differential effect on expression of SP-A variants and that this effect may be mediated via the 3'-UTR (26). In the present study, we tested the effects of 3'-UTR on basal levels of gene expression as well as on expression levels in response to Dex of the 10 most frequently found SP-A1 and SP-A2 alleles. The results of these in vitro transient transfection experiments indicate that the 3'-UTR of SP-A differentially mediates both basal expression and expression in response to Dex. Both basal mRNA and protein (activity) levels are significantly reduced compared with either vector alone or control construct, with some differences observed among alleles. Dex treatment also decreased differentially mRNA and protein levels, with no such decrease observed with the control construct or vector alone. With some of the SP-A1 3'-UTR constructs, an apparent discrepancy in their response to Dex was observed, i.e., some showed a response to Dex at the mRNA level but not at the protein and vice versa. No such discrepancy was observed with the SP-A2 alleles.

In the present study, we observed that the basal mRNA level of 6A4 is significantly lower than those of 6A2 and 6A3, and the basal level of protein of both 6A and 6A4 are significantly lower than those of 6A2 and 6A3. DNA sequence analysis indicated that the 6A and 6A4 alleles differ from the 6A2 and 6A3 in 3'-UTR at positions 213/214 (CA to TC) and 249 (A to C) (see Table 4). The nucleotide sequence changes at 213/214 generate, in 6A and 6A4, potential recognition binding sites for several DNA binding factors, such as GATA-1, -2, -3, Sox-5, and Ik-2, whereas the sequences at these positions for 6A2 and 6A3, although are lacking in recognition binding sites for the aforementioned factors, are the sites for other factors such as p300 and c-Myb. GATA family factors are of the zinc finger protein family, and recently, it was found that the GATA-6 is required for maturation of the lung in late gestation (40). Whether or how these potential recognition binding sites for different DNA binding factors between these two groups of SP-A1 alleles may contribute to the changes observed in the basal level of SP-A 3'-UTR-mediated expression remains to be determined. Recently, Gehring et al. (21) found that only one nucleotide mutation (G to A) at the 3'-UTR of the prothrombin gene causes increased cleavage site recognition, increased 3'-end processing, and increased mRNA accumulation and protein synthesis. Similarly, a nucleotide insertion (1484insG) at the 3'-UTR of the PTP1B gene increases specific gene expression and may be responsible for insulin resistance (16). Together, these findings indicate that the few nucleotide differences between the two groups of SP-A alleles may, as supported by the examples mentioned, cause a significant change in gene expression levels. This change in turn may influence the efficiency of mRNA 3'-UTR formation (21), transport and localization, posttranscriptional modification and degradation, and translation (10, 11, 16, 22).

GCs play a role in several processes of gene expression, including transcription, posttranscription, and translation (1-3). The action of GCs may occur via direct and indirect mechanisms, such as GC-mediated destabilization of mRNA (7, 37, 45, 46) and mRNA-protein interactions in 3'-UTR of mRNA (20). GCs also influence both protein synthetic (translational) and protein degradative pathways and are shown to attenuate mRNA translation at two levels: translational efficiency (i.e., translation initiation) and translational capacity (i.e., ribosome biogenesis) (20, 56). The present results summarized in Table 3 show that differences in response to Dex exist among the 10 SP-A 3'-UTR constructs and that there are some apparent discrepancies between mRNA and protein level among some of the SP-A1 3'-UTR constructs, but not for the SP-A2. Whether these discrepancies are due to a particular nucleotide difference either alone or in the context of other surrounding nucleotide differences and/or as to which these nucleotide differences may be are currently unknown and warrant further investigation. Among the SP-A2 alleles, the 1A1 construct did not respond to Dex as assessed by measurements at either the mRNA or protein level. The 1A1 allele has several unique nucleotide differences in 3'-UTR from the other SP-A2 alleles at positions 29 (A to G), 275 (C to T), and 557 (C to G) (Table 4). These differences may cause a change in recognition binding sites for many factors. For example, potential binding sites for factors Ttk 69 and CF1 exist at the region that includes nucleotide position 29; binding sites for factors GRC1, C-Rel, and Ik-2 exist at the region that includes nucleotide position 275. However, the precise factors and mechanisms responsible for the difference between 1A1 and other SP-A2 alleles remain to be determined.

                              
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  		Table 3.   Summary of SP-A allele response to dexamethasone


                              
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  		Table 4.   SP-A 3'-UTR nucleotide sequence differences among gene-specific (SP-A1 or SP-A2) alleles

Because an element of "pyrimidine-rich domain of 37 nucleotides" and AUUUA (Au-rich) elements have been implicated in GC-mediated mechanisms, we investigated the presence and/or similarity of these elements between SP-A1 and SP-A2 alleles in the context of the present study. At nucleotide positions 787-826, an element of pyrimidine-rich domain of 37 nucleotides was observed in both SP-A1 and SP-A2 alleles, but 7 of the nucleotides in this region differ between SP-A1 and SP-A2. Because this element is involved in RNA-protein interaction of the GC-mediated destabilization of cyclin D3 mRNA in murine T lymphoma cells (20), these seven nucleotide differences may play a differential regulatory role between SP-A1 and SP-A2 alleles, although our present experimental conditions do not support this. However, a combination of factors that may include the pyrimidine tract may provide allele specificity. Moreover, the published literature indicates that AUUUA sequences (AUUUA element) in the 3'-UTR of several mRNAs influence mRNA stability (9, 36, 62, 64), and Dex may affect mRNA stability through the AUUUA element (4). The 3'-UTR of SP-A2 contains two such AUUUA elements at positions 6-10 and 927-931, but the SP-A1 has only one at position 6-10. However, because no discrete differences were observed between SP-A1 and SP-A2 alleles, it is unlikely that the AUUUA element plays, by itself, a role in the processes studied here. But, it is possible that this element, in the presence of other unknown elements/factors, contributes to the observed SP-A allele differences. For example, the 1A1 3'-UTR has a G at position 29, whereas all other SP-A2 alleles have an A. This nucleotide difference is located near the AUUUA element at the nucleotide positions 6-10 after the translation stop codon (Table 5). Whether any interactions exist between this AUUUA element and the region that contains nucleotide 29 that may explain the lack of Dex response of 1A1 compared with other SP-A2 alleles remains to be determined. Further study is necessary to investigate the mechanisms involved in the 3'-UTR-mediated differential allele regulation, both in the basal level and in the response to Dex.

                              
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  		Table 5.   Sequences of SP-A2 3'-UTR potential AUUUA element and adjacent region

Furthermore, in our present study, we observed for the SP-A1 alleles (but not for the SP-A2 alleles) some apparent discrepancy in response to Dex. Some variants were only responsive at the mRNA levels (6A, 6A4) or only at the protein level (6A2). Recent studies indicate that the 3'-UTR and associated proteins such as the poly(A)-binding protein (PABP) and the PABP-interacting protein-1 may be involved in the regulation of translation through interactions with translational factors, such as elF4G, and thus influence mRNA stability or translational efficiency, or both (11, 13, 22, 47). It would be of interest to investigate whether such processes are involved in SP-A1 allele expression.

In summary, we have characterized the 3'-UTR sequences of 10 SP-A alleles and compared the identities and divergence of 3'-UTR of 6 alleles of SP-A2 and 4 alleles of SP-A1. With the use of transient transfection assays at the optimal time point and duration of Dex treatment, we observed that: 1) the basal mRNA and protein levels of all 10 alleles are significantly (P < 0.01) and differentially reduced, compared with those of vector alone or a control SP-B construct; and 2) Dex treatment significantly and differentially decreases luciferase mRNA and luciferase activity in some alleles but not in others compared with controls. Given the allele-dependent differences, it is necessary to carry out a detailed study of the mechanisms involved that may help explain the differential gene expression of SP-A alleles for basal and Dex-responsive regulation. We speculate that such studies may provide the basis for a careful consideration of individual use of steroid treatment.


    ACKNOWLEDGEMENTS

We thank Dr. Myung-Ho Oh for contributions with constructs at the initial stages of this study, Susan DiAngelo for expert technical assistance, and Jeffrey Sandstrom for help and comments with the manuscript.


    FOOTNOTES

This work is supported by National Heart, Lung, and Blood Institute Grant R37 HL-34788.

Address for reprint requests and other correspondence: J. Floros, Dept. of Cellular and Molecular Physiology, H166, The Pennsylvania State Univ. College of Medicine, 500 University Dr., Hershey, PA 17033 (E-mail: jfloros{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.

First published January 10, 2003;10.1152/ajplung.00375.2002

Received 1 November 2002; accepted in final form 2 January 2003.


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