SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression

Russell R. Hoover1 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.

surfactant protein A; 3'-untranslated region; alleles; dexamethasone; messenger ribonucleic acid; transfection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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REFERENCES

PULMONARY SURFACTANT is secreted by alveolar type II cells and lies at the alveolar air-liquid interface where it prevents alveolar collapse by lowering surface tension forces (reviewed in Ref. 12). Surfactant protein (SP) A is the most abundant protein of pulmonary surfactant and is involved in surfactant physiology, structure, and metabolism. In recent years, it has become evident that a major role of SP-A also lies in lung host defense. A number of studies have shown that SP-A is involved in the activation of alveolar macrophages, proliferation of lymphocytes, and the production of inflammatory cytokines (reviewed in Ref. 33). SP-A-deficient ("SP-A knockout") mice do not exhibit respiratory problems in normal situations but have difficulty in clearing bacteria from their lungs (26).

Human SP-A is encoded by two very similar but nonidentical genes, SP-A1 (39) and SP-A2 (21). The two genes are 94% identical at the nucleotide level and encode proteins with ~96% identity. The human SP-A locus has been mapped to 10q22-23 (6, 22) and also contains a nonfunctional pseudogene (23). The SP-A pseudogene lies between the two human SP-A genes, which are ~35-40 kb apart (16).

Based on the available coding sequences for each SP-A gene, a number of alleles have been characterized (9, 10, 18, 19). To date, five alleles for SP-A1 (6A, 6A2, 6A3, 6A4, and 6A5) and six alleles for SP-A2 (1A, 1A0, 1A1, 1A2, 1A3, and 1A4) have been discovered. In addition, splice variability (19) of 5'-untranslated exons and sequence variability within the 3'-untranslated region (UTR) (11) have been described for each human SP-A gene. The physiological significance of different SP-A alleles and/or 5'-untranslated splice variants is currently unknown.

Glucocorticoids have complex effects on human SP-A gene expression. In human fetal lung explants, glucocorticoids at low concentrations (<10 nM) increase SP-A mRNA levels, whereas at higher concentrations (100 nM), glucocorticoids decrease SP-A mRNA and protein levels (2, 8, 17, 27). By contrast, glucocorticoids have only an inhibitory effect on SP-A gene expression in the lung adenocarcinoma cell line NCI-H441 (30, 34). In the NCI-H441 cell line, SP-A mRNA levels are reduced to ~10% of the control level after a 36-h exposure to 10 nM dexamethasone (30). Under the conditions described in the present study, dexamethasone reduces SP-A gene expression in a time- and dose-dependent manner; at a concentration of 100 nM, dexamethasone decreases total SP-A mRNA levels to ~25% of the control level after 18 h of exposure (unpublished data). The effects of glucocorticoids on SP-A gene expression in fetal lung explants are reversible and are glucocorticoid receptor mediated (27).

Glucocorticoids regulate human SP-A gene expression by affecting both SP-A transcription and SP-A mRNA stability (5, 17, 34). Studies using human fetal lung explants have shown either an increase (4) or a decrease (17, 34) in SP-A gene transcription in the presence of the synthetic glucocorticoid dexamethasone.

A number of studies (4, 5, 17) have shown that dexamethasone significantly decreases the half-life of the SP-A message in fetal lung explants. In control conditions, the half-life of SP-A mRNA was shown to be ~9 h, but in the presence of 100 nM dexamethasone, the half-life was reduced to 3 h (17). The decay rate of SP-A mRNA in the presence of dexamethasone was shown to be biphasic in fetal lung explants, with an initial rapid decrease followed by a slower decay rate (17). It was suggested that this biphasic decay rate was a result of differential effects on SP-A1 and SP-A2 mRNA stabilities (17). In this early study, the genotypes of the fetal lung explants used were not known; thus it is not certain whether the biphasic effect seen may have been due to differences in SP-A alleles.

Differential regulation of SP-A1 and SP-A2 gene expression by glucocorticoids has been demonstrated in both fetal lung explants (18, 24, 28) and the NCI-H441 cell line (24). With regard to the inhibitory effect of dexamethasone, studies using fetal lung explants have showed that either SP-A2 (28) was more responsive, SP-A1 was more responsive (18), or the two SP-A genes were equally responsive to dexamethasone (24). In contrast, SP-A1 was found to be more responsive to dexamethasone than SP-A2 in the NCI-H441 cell line (24).

Toward a better understanding of mechanisms involved in the posttranscriptional regulation of human SP-A gene expression, NCI-H441 cells were transfected with chloramphenicol acetyltransferase (CAT) reporter constructs in which the native CAT 3'-UTR was replaced with various regions of the SP-A1 or SP-A2 cDNA. Exposure to dexamethasone reduced CAT activity for only the constructs that contained the SP-A 3'-UTR, indicating a role for this region in the glucocorticoid inhibition of human SP-A gene expression.


    METHODS
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Cell culture. The lung adenocarcinoma cell line NCI-H441 is presumably of Clara cell origin, grows as an attached monolayer with epithelial cell characteristics, and expresses SP-A and SP-B in a regulated manner (30). NCI-H441 cells were purchased from the American Type Culture Collection (Manassas, VA) and were grown in RPMI 1640 medium plus 10% heat-inactivated fetal bovine serum (FBS; Summit Biotechnology, Ft. Collins, CO) and 1× antimycotic-antibiotic solution (Sigma, St. Louis, MO). For all experiments, FBS-containing medium was replaced with serum-free RPMI 1640 medium ~12 h before hormone treatments. Dexamethasone was purchased from Sigma and stored in ethanol at -20°C as a 10 mM stock.

Generation of CAT reporter constructs. The generation of CAT reporter constructs is outlined in Fig. 1. PCR fragments representing the SP-A1, SP-A2, or SP-B 3'-UTRs were cloned into the Sma I site of psvlCAT (29). To generate 3'-UTRs specific for either SP-A1 or SP-A2, long-range PCR was initially performed with oligos specific for SP-A1 (primer pair 326/13) or SP-A2 (primer pair 327/13; see Fig. 1) with genomic DNA from individuals (20) with certain homozygous genotypes (6A36A3/1A11A1 and 6A26A2/1A01A0) as a template.


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Fig. 1.   Construction of chimeric psvlCAT/SP-A 3'-untranslated region (UTR). Human genomic DNA of known surfactant protein (SP) A genotypes served as template in gene-specific PCR. Top: graphic representation of genomic structural organization of SP-A1 or SP-A2 gene. Hatched boxes, UTRs; horizontal arrows, relative position and orientation of primers used; nos. on top and bottom, primers. SP-A1- or SP-A2-specific PCR product then served as template for amplification of SP-A 3'-UTR (or portions thereof; middle). These PCR products were then cloned into vector psvlCAT in place of native chloramphenicol acetyltransferase (CAT) 3'-UTR (bottom). In some cases, recombinant clones were digested with Xho I (X) or Acc I (A). CAT/SP-A 3'-UTR gene was then subcloned into Xho I or Acc I site of pCI-neo vector (data not shown). S, Sma I.

The protocol for long-range PCR was modified from that previously described (3). Briefly, the reaction mixture contained 20 mM Tris · HCl, pH 8.6, 150 mg/ml of BSA, 16 mM (NH4)2SO4, 3.5 mM MgCl2, 250 mM each deoxynucleotide 5'-triphosphate, 30 mM Tris base, 8% glycerol, 100-200 ng of template DNA, and 100 ng of each primer. After an initial denaturation of 1 min, 2.5 U of AmpliTaq (Applied Biosystems, Foster City, CA) combined with 0.15 U of Pfu DNA polymerase (Stratagene, La Jolla, CA) were added. All long-range PCRs were performed with thin-walled tubes (USA/Scientific Plastics, Ocala, FL) in a total volume of 100 ml. Reactions typically consisted of 30-35 cycles of an 8-s denaturation at 94°C, a 30-s annealing at 58-62°C, and a 4-min extension at 68°C.

PCRs were performed in duplicate. Gene specificity was ensured by including SP-A1 and SP-A2 genomic clones (16) as template controls. 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 10/13, 10/68, and 10/174, which would amplify differing lengths of the SP-A 3'-UTR (Fig. 1). The SP-B 3'-UTR was generated by PCR with primer pair 101/121 and human genomic DNA as a template. Primer sequences are shown in Table 1.

                              
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Table 1.   Primers used in PCR amplifications

The PCR products were then cloned into the Sma I site of the vector psvlCAT with standard DNA ligation procedures. Ligation products were transformed into competent Escherichia coli (strain XL1-Blue). Recombinant constructs were then identified by Southern hybridization with standard procedures. The inserts were sequenced, and the recombinant plasmids that contained the full-length SP-A or SP-B 3'-UTR in place of the native CAT 3'-UTR are referred to as psvlCAT/SP-A 3'-UTR or psvlCAT/SP-B 3'-UTR, respectively. Expression of CAT in these recombinant plasmids is under the direction of the SV40 promoter.

In some instances, the entire CAT/SP-A 3'-UTR gene was excised from the psvlCAT recombinant plasmids by digestion with Xho I and Acc I. This resulting fragment was then subcloned into the Xho I or Acc I site of the mammalian expression vector pCI-neo (Promega, Madison, WI). This was done so that the chimeric CAT/SP-A 3'-UTR gene would be under the direction of the human cytomegalovirus (CMV) promoter, which is much stronger than the SV40 promoter of psvlCAT. The plasmids in which the CAT/SP-A 3'-UTR was subcloned into the pCI-neo vector are refered to as pCI-neoCAT/SP-A 3'-UTR.

For the generation of CAT constructs that contained the SP-A1 5'-UTR and coding region, the SP-A 3'-UTR was excised from pCI-neoCAT/SP-A 3'-UTR by digestion with Sma I, resulting in a construct designated pCI-neo/CAT. PCR fragments representing the SP-A1 5'-UTR and coding region or the entire SP-A1 cDNA (5'-UTR plus coding plus 3'-UTR) were then subcloned into the Sma I site of pCI-neo/CAT. PCR with primer pair 32/494 and a 6A2 cDNA clone (19) as a template was used to generate a fragment representing the SP-A1 5'-UTR and coding region. The SP-A cDNA clones available in our laboratory lack a significant portion of the 3'-UTR (19); thus to generate the full-length SP-A cDNA, recombinant PCR was performed. A recombinant PCR product that spanned the entire 6A2 2.2-kb cDNA (5'-UTR plus coding plus 3'-UTR) was generated by long-range PCR with the primer pair 32/13. The template for the recombinant PCR consisted of a 1:1 mixture of two overlapping PCR products: one generated by using the primer pair 32/68 with the 6A2 cDNA as a template and the other generated by using the primer pair 10/13 with the SP-A1-specific primer pair 326/13 PCR product as a template.

Transient transfections. DNA suitable for transfection was prepared with the Qiagen Plasmid Maxiprep kit (Qiagen, Hilden, Germany) according to manufacturer's specifications. NCI-H441 cells were transfected with CAT reporter constructs when at ~80% confluency with the calcium phosphate method. Four hours before transfection, the RPMI 1640 medium plus 10% FBS was replaced with Dulbecco's modified Eagle's medium (DMEM) plus 10% FBS. Ten milligrams of CAT reporter construct were combined with 10 mg of the beta -galactosidase expression vector pCMVSPORT-beta -Gal (GIBCO BRL, Life Technologies, Gaithersburg, MD). To this mixture, 50 µl of 2.5 M CaCl2 and H2O to a final volume of 0.5 ml were added. The CaCl2-DNA solution was added dropwise to 0.5 ml of 2× HEPES-buffered saline (0.28 M NaCl, 0.05 M HEPES, and 1.5 mM Na2HPO4) with constant agitation. The resulting 1 ml of calcium phosphate-DNA precipitate was overlaid onto the NCI-H441 cells. Transfections were carried out overnight (16 h) in DMEM plus 10% FBS, after which time the DMEM was removed and the cells were washed twice with cold PBS. Transfected cells were incubated in serum-free RPMI 1640 medium for 8-10 h before drug treatments.

CAT and beta -galactosidase assays. Transfected cells were harvested ~36 h after the start of transfection. NCI-H441 cells were washed with and scraped into cold PBS and then centrifuged at 1,000 g for 1 min. The cell pellets were generally resuspended in ~200 µl of 0.25M Tris · HCl, pH 8.0. The cells were lysed by three cycles of freeze-thaw. Cell debris was removed by centrifugation, and the cell lysates were then assayed for CAT and beta -galactosidase activities.

CAT activity was measured with the phase-extraction method (1). Fifty milliliters of cell extract were heated at 65°C for 10 min, and then 20 µl of 0.25 M Tris · HCl, pH 8.0, 0.5 µl of 50 mCi/mmol [3H]chloramphenicol (NEN, Boston, MA), and 5 µl of 5 mg/ml n-butyryl coenzyme A (Sigma) were added. Reactions were incubated at 37°C for 6-16 h (depending on the efficiency of transfection) and then terminated by the addition of 300 µl of mixed xylenes (Sigma). Reactions were vortexed for 1 min and centrifuged at 12,000 g for 1 min. The upper (xylene) phase was reextracted with 100 µl of 0.25 M Tris · HCl, pH 8.0. Two hundred microliters of the upper (xylene) phase were removed and placed in 10 ml of scintillation fluid (ICN Pharmaceuticals, Costa Mesa, CA). CAT activity was determined with a scintillation counter.

To correct for transfection efficiency, CAT activity is expressed as a ratio of CAT to beta -galactosidase activities. beta -Galactosidase activity was determined with a colorimetric assay (15). Fifty to one hundred microliters of cellular extract (not heated) were added to 0.5 ml of reagent buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2, and 50 mM beta -mercaptoethanol) and 0.1 ml of o-nitrophenyl-beta -D-galactopyranoside that had been dissolved in 60 mM Na2HPO4 and 40 mM NaH2PO4. Reactions were incubated at 37°C until the development of a yellow color was apparent and were terminated by the addition of 0.5 ml of 1 M Na2CO3. beta -Galactosidase activity was quantitated by spectrophotometry at a wavelength of 420 nm.

For all experiments, transfections were performed in duplicate as were the CAT and beta -galactosidase assays.


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ABSTRACT
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SP-A 3'-UTR mediates an inhibition of CAT activity by dexamethasone. A number of studies (4, 5, 17) have shown that dexamethasone reduces SP-A mRNA levels in part by affecting the half-life of the SP-A mRNA (i.e., SP-A mRNA stability). As a first step in identifying cis-acting elements within the SP-A mRNA that may mediate the posttranscriptional effect of dexamethasone on SP-A gene expression, chimeric CAT cDNAs that contain various portions of SP-A1 cDNA were introduced into NCI-H441 cells. The transfected NCI-H441 cells were treated with either 0.01% ethanol (control) or 100 nM dexamethasone for 16 h in serum-free medium.

Preliminary results with the pCI-neoCAT/SP-A 3'-UTR constructs that contained different portions of the 6A2 cDNA (the most common SP-A1 allele) in place of the native CAT 3'-UTR indicated that an element(s) within the SP-A 3'-UTR could mediate the inhibitory effects of 100 nM dexamethasone. Figure 2 shows that a modest but significant decrease in CAT activity occurs in the presence of dexamethasone with the constructs that contain either the entire 6A2 cDNA (construct 3) or only the 6A2 3'-UTR (construct 1; P < 0.05 by ANOVA) but not with the construct that contains the 6A2 5'-UTR plus coding (construct 2).


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Fig. 2.   3'-UTR of SP-A1 allele 6A2 mediates a decrease in CAT activity in presence of 100 nM dexamethasone (Dex). NCI-H441 cells were transiently transfected with various chimeric CAT reporter constructs and exposed to Dex for 16 h in serum-free medium. CAT activity was corrected for transfection efficiency by normalization to beta -galactosidase activity. Con, control. Values are means ± SE; n, no. of experiments (nos. in parentheses). A decrease in CAT activity is seen only with constructs containing 3'-UTR of SP-A1 allele 6A2 (constructs 1, 3, and 4) but not with constructs that contain 6A2 5'-UTR and coding region (construct 2), SP-B 3'-UTR (construct 5), or a minimal 3'-UTR derived from vector sequence (construct 6). Decrease in CAT activity in presence of Dex is independent of promoter used to drive CAT expression (compare constructs 1 and 4). * P < 0.05 compared with construct 6 by ANOVA.

The ability of the 6A2 3'-UTR to mediate inhibition of CAT activity by dexamethasone is independent of the promoter used to drive CAT expression (Fig. 2, compare constructs 1 and 4). The constructs under the direction of the SV40 promoter yielded much lower (~10-fold) CAT activity than those under the direction of the human CMV promoter (data not shown). There was concern that high basal CAT levels may have an impact on subsequent results because trans-acting factors that mediate the effect of dexamethasone may be limiting (in which case extremely high CAT activity may mask any effect that dexamethasone would have). Thus for all subsequent experiments, psvlCAT/SP-A 3'-UTR constructs (where CAT expression is driven by the relatively weaker SV40 promoter) were used.

Figure 2 also shows that the decrease in CAT activity is specific for the SP-A 3'-UTR and is not mediated by vector sequence because no decrease was seen when constructs that contained the SP-B 3'-UTR (construct 5) or a minimal 3'-UTR that came from a vector sequence (construct 6) were tested. The results shown in Fig. 2 suggest that the 6A2 3'-UTR is both necessary and sufficient to mediate a decrease in CAT activity in the presence of 100 nM dexamethasone, possibly at a posttranscriptional level.

SP-A gene and/or allele differences in the SP-A 3'-UTR response to dexamethasone. To determine whether the 3'-UTRs of other SP-A genes and/or alleles respond differently from the 6A2 3'-UTR to dexamethasone treatment, transfections were also performed with CAT reporter constructs that contained the 3'-UTR of the 6A3 and 1A0 alleles. Figure 3 shows that for every allele tested, a decrease in CAT activity was seen in the presence of dexamethasone, indicating that the inhibitory effect is not specific for the 6A2 3'-UTR. However, a slightly but significantly greater response was seen with the 3'-UTR of the 6A3 allele (Fig. 3, construct 2) compared with the 6A2 (Fig. 3, construct 1) and 1A0 (Fig. 3, construct 3) alleles (P < 0.05 by ANOVA). These results indicate that the different SP-A genes and/or alleles may respond differentially to dexamethasone treatment.


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Fig. 3.   Differences in response to Dex among 3'-UTRs of SP-A genes and/or alleles. Values are means ± SE; n, no. of experiments (nos. in parentheses). Decrease in CAT activity in presence of 100 nM Dex is significantly greater with constructs containing 3'-UTR of SP-A1 allele 6A3 (construct 2) compared with constructs with 3'-UTR of 6A2 (construct 1) or SP-A2 allele 1A0 (construct 3). Construct 4, vector sequence. * P < 0.05 compared with constructs 1 and 4 by ANOVA.

As a way to determine which region of the SP-A 3'-UTR might be involved in the dexamethasone inhibition of CAT activity, a DNA sequence comparison among the SP-A alleles was performed. A sequence comparison between the 3'-UTR of the allele with a greater response to dexamethasone (6A3) and the 3'-UTRs of the alleles with a lower response (6A2 and 1A0) may give insight into which 3'-UTR sequences mediate (or modify) the dexamethasone regulation of SP-A gene expression. Figure 4 shows that one area of the 3'-UTR may be of particular interest. Approximately 400 bp past the translation stop site, there are an additional 11 bp in the 3'-UTR of 6A2 and 1A0 that is not found in the 3'-UTR of 6A3. It is possible that this 11-bp insertion/deletion (Indel) modifies the response of the SP-A 3'-UTR to dexamethasone so that the alleles with the 11-bp insertion (6A2 and 1A0) have less of a response to dexamethasone than those without the 11-bp insertion (6A3).


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Fig. 4.   Sequence comparison of 3'-UTRs of different SP-A alleles shows that SP-A2 allele 1A0 and SP-A1 allele 6A2 have an 11-bp insertion (boldface uppercase letters) ~400 bp after translational stop site. Nos. on top, nucleotide positions from transcriptional start site of SP-A1 genomic sequence (39). Boldface lowercase letters, single nucleotide differences. Only ~200 bp surrounding 11-bp insertion are shown.

Deletional analysis of the 6A3 3'-UTR. A deletional analysis was performed on the 6A3 3'-UTR to identify sequences involved in the dexamethasone inhibition of CAT activity. PCR fragments representing three deletions of the 6A3 3'-UTR were subcloned into the Sma I site of the psvlCAT vector. Figure 5 shows that a decrease in CAT activity in the presence of dexamethasone was seen with each of the constructs tested, even for the construct that contains only ~350 bp of the 6A3 3'-UTR (construct 3). These results indicate that a cis-acting element that can mediate the dexamethasone response lies within the first 350 bp of the SP-A 3'-UTR. This region does not include the portion of the 3'-UTR that would contain the aforementioned 11-bp Indel, indicating that the 11-bp Indel may have, if any, an indirect or modifying role in the response to dexamethasone.


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Fig. 5.   Deletional analysis of 6A3 3'-UTR (constructs 1-3) indicates that 1st 350 bp are sufficient to mediate a decrease in CAT activity in presence of 100 nM Dex. First 350 bp of 3'-UTR (construct 3) do not include region that contains the 11-bp insertion (arrow), indicating that the 11-bp insertion may not be directly involved in response to Dex but may modify it. Nos. above solid line, positions of 3'-ends of constructs relative to transcriptional start site of SP-A1 genomic sequence (5). Data are means ± SE; n, no. of experiments (nos. in parentheses).

Effect of hydrocortisone, dihydrotestosterone, and 12-O-tetradecanoylphorbol-13-acetate on the 6A3 psvlCAT/SP-A 3'-UTR construct. The inhibitory response of the 6A3 3'-UTR is not dexamethasone specific but is glucocorticoid specific because both dexamethasone and hydrocortisone decrease CAT activity. By contrast, dihydrotestosterone, which does not affect human SP-A mRNA levels (27), has no affect on CAT activity (Fig. 6). The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which affects SP-A gene expression at a transcriptional level (34), also does not decrease the CAT activity of the 6A3 psvlCAT/SP-A 3'-UTR construct (Fig. 6).


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Fig. 6.   Inhibition of CAT activity mediated by 6A3 3'-UTR is not Dex specific but is glucocorticoid specific. Chimeric CAT construct that contains 6A3 3'-UTR was transiently transfected into NCI-H441 cells; transfected cells were then exposed to various agents for 16 h in serum-free medium. Data are means ± SE. Decrease in CAT activity is seen with 100 nM Dex and 1 µM naturally occurring glucocorticoid hydrocortisone (CORT) but not with 1 µM sex steroid dihydrotestosterone (DHT), which has no affect on SP-A mRNA levels (27). There is also no decrease in CAT activity when transfected cells were exposed to 50 nM phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), which is a transcriptional inhibitor of human SP-A (34).


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Glucocorticoids have both anti-inflammatory and lung-maturational properties. Their effects on human SP-A1 and SP-A2 gene expression are of particular interest because of the roles that SP-A has in lung host defense and surfactant physiology. The synthetic glucocorticoid dexamethasone has complex effects on human SP-A gene expression that occur at both the transcriptional and posttranscriptional levels. In the human adenocarcinoma cell line NCI-H441, dexamethasone reduces total SP-A mRNA levels in a time- and dose-dependent manner (30, 34). In the present study, we present evidence that the human SP-A 3'-UTR is involved in the dexamethasone-mediated reduction of human SP-A mRNA levels, possibly at a posttranscriptional level. Our results also suggest that there are differences in the degree of response to dexamethasone treatment among alleles.

Introduction of various chimeric CAT/3'-UTR mRNAs into NCI-H441 cells and subsequent exposure of the cells to 100 nM dexamethasone indicated that the SP-A 3'-UTR was sufficient to mediate a decrease in CAT activity. Furthermore, only the first 350 bp of the SP-A 3'-UTR were necessary for this effect. Interestingly, the full-length 3'-UTRs of different SP-A genes and/or alleles appeared to respond differently to dexamethasone treatment, an effect that may be accounted for by sequence differences among alleles. Of particular interest is an 11-bp insertion found ~400 bp past the SP-A translation stop site in alleles that had a lesser response to dexamethasone (6A2 and 1A0; Fig. 3). However, the exact role, if any, of the 11-bp Indel in the regulation of SP-A mRNA stability either in vivo or in vitro is presently unclear.

The in vivo relevance of the differential effect seen among SP-A 3'-UTRs is not known. The 11-bp insertion is found in the 3'-UTR of all known SP-A2 alleles (e.g., 1A0) and in the 3'-UTR of only the SP-A1 allele 6A2 (19; unpublished observations). The genotype of the NCI-H441 cell line is 6A46A4/1A51A5. One would expect that if the 3'-UTR of alleles without the 11-bp insertion had a greater response to dexamethasone than those with the 11-bp insertion, then the 6A4 allele (which does not have the 11-bp insertion) of the NCI-H441 cell line would have a greater response to dexamethasone than the 1A5 allele. Indeed, a recent study (24) has shown that SP-A1 is more responsive to dexamethasone exposure than SP-A2 in NCI-H441 cells. However, the 6A4 allele was not tested in this study.

Nevertheless, it is likely that the final outcome of dexamethasone exposure to NCI-H441 cells (i.e., decrease in SP-A gene expression) is due to a decrease in both SP-A mRNA stability and transcription. Some alleles may have a greater response at the level of mRNA stability, whereas others may have a greater response at the level of transcription. Depending on the relative contribution of the posttranscriptional and transcriptional mechanisms, the overall result could be that of either differential or nondifferential regulation. Thus the response of SP-A1 and SP-A2 to dexamethasone may depend on an individual's SP-A genotype.

In the present study, dexamethasone did not affect the CAT activity from a chimeric CAT/SP-B 3'-UTR mRNA. Pryhuber et al. (36) have shown similar findings when chimeric human growth hormone/SP-B 3'-UTR constructs were stably transfected into NCI-H441 cells. The similarity of the two findings indicates that the results obtained in the present study are not specific to CAT or to the method of transfection used. Dexamethasone treatment also did not affect CAT activity when the psvlCAT vector, which has a minimal 50-bp 3'-UTR, was introduced into NCI-H441 cells. The absence of a dexamethasone response with the CAT/SP-B 3'-UTR and psvlCAT vector controls indicates that the dexamethasone-mediated reduction in CAT activity is specific to the SP-A 3'-UTR and is not due to vector sequences. It may also be of interest to note that the basal CAT activity for the psvlCAT vector was significantly lower (about fivefold) than the CAT activity from the psvlCAT constructs containing the full or partial SP-A or SP-B 3'-UTR (data not shown). This observation indicates that the presence of a longer 3'-UTR increases gene expression, possibly affecting constitutive stability or translation. There was no evidence for differences in basal CAT activity among constructs containing 3'-UTRs from the different SP-A genes and/or alleles.

The 3'-UTR-mediated inhibition of CAT activity by dexamethasone presumably occurs at the posttranscriptional level because it is unlikely that the 3'-UTR itself would have a trans effect on the SV40 (or CMV) promoter; however, this was not proven. In addition, the possibility that the effect of dexamethasone on CAT activity is due to effects on translation has not been ruled out. However, there is presently no evidence that dexamethasone affects human SP-A gene expression at a translational level. Recently, it has been reported (13) that actinomycin D (an inhibitor of transcription) prevents dexamethasone inhibition of SP-A gene expression. This result was interpreted to mean that, in the NCI-H441 cell line, dexamethasone acts solely at a transcriptional level (13). However, it is possible that dexamethasone-regulated SP-A1 or SP-A2 mRNA stability requires RNA synthesis or that actinomycin D and dexamethasone interact in an unknown manner. In the present study, CAT activity, but not CAT/SP-A 3'-UTR mRNA levels or half-life, was measured. The studies herein were undertaken with the assumption that CAT activity reflects the levels of CAT mRNA; however, this may not be entirely accurate considering the relatively long half-life of the CAT enzyme. Thus a reduction in CAT activity by 30% may actually correspond to a reduction in CAT mRNA levels by 60%, which may explain the relatively modest effect seen on CAT activity.

A number of genes appear to be regulated via actions on the 3'-UTR of the mRNA, although the mechanisms involved are poorly understood. The 3'-UTR of SP-B mediates a destabilization of the SP-B message in response to both 12-O-tetradecanoylphorbol-13-acetate and tumor necrosis factor-alpha (36), an effect that may involve stem-loop structures. With the use of chimeric CAT constructs, it was found that the human 70-kDa heat shock protein mRNA is stabilized by heat stress through actions on its 3'-UTR (29). Not every stability determinant resides within the 3'-UTR of an mRNA (reviewed in Ref. 37), as is the case with beta -tubulin, c-fos, and c-myc. In the present report, removal of the SP-A 5'-UTR and coding sequences had no effect on dexamethasone regulation of CAT activity, indicating that these areas are not involved in dexamethasone regulation.

Hormones have been shown to affect the stability of a number of mRNAs. In particular, glucocorticoids destabilize the mRNAs for procollagen (14), interleukin-1beta (25), interferon-beta (32), and 3-hydroxy-3-methylglutaryl CoA reductase (38), whereas they stabilize the mRNAs for fibronectin (7), growth hormone (31), and beta -casein (35). The specific mechanisms by which glucocorticoids produce these effects are still unknown. One of the few reported examples of glucocorticoids affecting mRNA stability via the 3'-UTR is the interferon-beta mRNA where AU-rich sequences within the 3'-UTR mediate glucocorticoid destabilization of the message (32). There are no corresponding sequences within the first 350 bp of the SP-A1 or SP-A2 3'-UTR.

Regulation of mRNA stability is not clearly understood but often occurs through the interaction of trans-acting factors and cis-acting elements residing within the mRNA. This may be the case with human SP-A mRNA, where cis-trans interactions within the 3'-UTR decrease the half-life of the message. It is likely that dexamethasone treatment induces the binding of a destabilizing protein or reduces the binding of a stabilizing protein to the SP-A 3'-UTR. However, to what extent (if any) RNA secondary structure and dexamethasone-regulated protein binding to the SP-A 3'-UTR play a role in the regulation of SP-A1 and SP-A2 mRNA stability is presently unknown. The exact nature and identity of these cis-trans interactions need to be the focus of future investigations.


    ACKNOWLEDGEMENTS

We thank Dr. David S. Phelps and Todd Umstead for invaluable input with cell cultures, Dr. Jeffrey A. Kern for the kind gift of the psvlCAT vector, and Gina Deiter for expert technical assistance.


    FOOTNOTES

This study was funded by National Heart, Lung, and Blood Institute Grant HL-49823 and by an American Heart Association (Pennsylvania Affiliate) Student Research Award (to R. R. Hoover).

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Floros, The Pennsylvania State Univ. College of Medicine, Dept. of Cellular and Molecular Physiology H166, PO Box 850, Hershey, PA 17033 (E-mail: jxf19{at}psu.edu).

Received 4 December 1998; accepted in final form 16 February 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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