TTF-1 response element is critical for temporal and spatial regulation and necessary for hormonal regulation of human surfactant protein-A2 promoter activity

Dongyuan Liu, Ming Yi, Margaret Smith, Carole R. Mendelson

Abstract

Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara cells, and is developmentally and hormonally regulated in fetal lung. Using transfected human fetal type II cells, we previously observed that ∼300 bp of 5′-flanking DNA mediated cAMP and interleukin-1 (IL-1) stimulation and dexamethasone (Dex) inhibition of hSP-A2 promoter activity. This region contains response elements for estrogen-related receptor α element (ERRE, −241 bp), thyroid transcription factor (TTF)-1/Nkx2.1 (TTF-binding protein, −171 bp), upstream stimulatory factor 1/2 (E-box, −80 bp), and stimulatory protein (Sp) 1 (G/T-box, −62 bp), which are essential for basal and cAMP induction of hSP-A2 expression. To define genomic regions necessary for developmental, hormonal, and tissue-specific regulation of hSP-A2 expression in vivo, we analyzed transgenic mice carrying hGH reporter genes comprised of 313 bp of hSP-A2 gene 5′-flanking DNA ± mutation in the TBE or 175 bp of 5′-flanking DNA, containing TBE, E-box and G/T-box, but lacking ERRE. Transgenes containing 313 or 175 bp of hSP-A2 5′-flanking DNA were expressed in a lung cell-specific manner and developmentally regulated in concert with the endogenous mouse SP-A gene. In cultured lung explants from hSP-A313:hGH transgenic fetal mice, cAMP and IL-1 induced and Dex inhibited transgene expression. However, the 175-bp hSP-A2 genomic region was insufficient to mediate hormonal regulation of hSP-A2 promoter activity. The finding that expression of the hSP-A313TBEmut:hGH transgene was essentially undetectable in fetal lung and was not hormonally regulated in transgenic fetal lung explants underscores the critical importance of the TBE in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression.

  • fetal lung
  • tissue specific
  • type II cell
  • adenosine 3′,5′-cyclic monophosphate
  • glucocorticoid

pulmonary surfactant, a developmentally regulated, phospholipid-rich lipoprotein required for air breathing, is synthesized exclusively by lung alveolar type II cells. To define the basic mechanisms involved in type II cell-specific, developmental, and hormonal regulation of surfactant synthesis, we have focused on surfactant protein (SP)-A, the major surfactant protein, that is an excellent marker of fetal lung maturity. Transcription of the SP-A gene is initiated in fetal lung after ∼80% of gestation is completed and reaches maximal levels just before birth (44). Developmental regulation of SP-A gene expression in fetal lung is more closely associated with the induction of surfactant glycerophospholipid synthesis and appearance of identifiable type II cells than is the temporal regulation of genes encoding surfactant proteins SP-B, SP-C, or SP-D. SP-A and the related surfactant protein, SP-D, are C-type lectins (members of the collectin family) that play important roles in immune defense within the lung alveolus by binding to a variety of bacterial and viral pathogens and facilitating their uptake by alveolar macrophages (see Ref. 60 for review). Recently, we obtained compelling evidence that augmented SP-A secretion by the fetal lung in amniotic fluid near term stimulates migration of fetal macrophages to the maternal uterus where they release cytokines, resulting in activation of nuclear factor-κB (NF-κB), increased expression of cyclooxygenase-2, enhanced prostaglandin production, and the initiation of labor (14, 22, 23).

SP-A gene expression is essentially lung-specific (9) and occurs primarily in type II cells and to a lesser extent in bronchioalveolar epithelial (Clara) cells of the proximal and distal airways (6, 53, 58). The human (29, 41) and baboon (20) genomes contain two highly similar SP-A genes, SP-A1 and SP-A2. By contrast, SP-A is encoded by a single copy gene in rats, mice, rabbits, and dogs. Although cAMP increases SP-A gene expression in human (25, 42, 51), rabbit (8, 44), and baboon (38, 55) fetal lung in culture, the rat (3, 49) and mouse (J. L. Alcorn, M. E. Smith, and C. R. Mendelson, unpublished observations) SP-A genes are unresponsive to cAMP. Because we observed that cAMP has a pronounced effect to stimulate promoter activity of the human, rabbit, and baboon SP-A genes (3, 46, 62) in transfected rat type II cells, it is likely that the transcription factors that promote cAMP responsiveness are conserved across species while the genetic elements that mediate these effects may differ. In studies using midgestation human fetal lung explants, hSP-A2 was found to be far more responsive to the inductive effects of cAMP analogs than human SP-A1 (hSP-A1; see Refs. 33 and 42). Therefore, we have focused our studies on the gene encoding hSP-A2.

SP-A expression in cultured human fetal lung explants and isolated type II cells is stimulated by hormones and factors that increase cAMP (2, 51) and by interleukin-1 (IL-1) (25) and is inhibited by glucocorticoids, such as dexamethasone (Dex) (5, 50, 52). cAMP and IL-1 stimulation of SP-A expression is O2 dependent (1, 25, 26). In previous studies to define cis-acting elements involved in cAMP-regulated hSP-A2 expression in transfected human fetal lung type II cells, we found that as little as ∼300 bp of 5′-flanking sequence were sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types (62, 63). This genomic region contains four response elements that are highly conserved in the SP-A genes of various species (45). These include an element (ERRE) that binds the orphan nuclear receptor estrogen-related receptor α (ERRα) (40, 62); a thyroid transcription factor-1 (TTF-1/Nkx2.1)-binding element (TBE) that binds TTF-1 and NF-κB (25); an E-box that binds the basic helix-loop-helix-zipper transcription factors, upstream stimulatory factors (USF) 1 (18) and USF2 (19) as a heterodimer; and a G/T-box that binds Sp1 and other, as yet, unidentified factors (63). In type II cell transfection studies using reporter constructs containing 5′-flanking sequences from the rabbit (3, 17, 19, 46), human (25, 37, 40, 62, 63), and baboon (38) SP-A genes, we found that the ERRE, TBE, E-box, and G/T-box each serve essential roles in basal and cAMP induction of SP-A promoter activity. Mutation of any one of these elements reduces basal and abrogates cAMP induction of SP-A promoter activity in transfected type II cells (43).

Previous transgenic studies from our laboratory indicated that as little as 378 bp of 5′-flanking DNA from the rabbit SP-A gene was sufficient to direct appropriate lung cell-specific and developmental regulation of transgene expression (3). The same genomic region also was sufficient to mediate cAMP stimulation and glucocorticoid inhibition of transgene expression (3). In the present study, transgenic mice were used to define the genomic sequences upstream of the hSP-A2 gene required for appropriate lung cell-specific, developmental, and hormonal regulation of expression. Transgenic mice were created carrying fusion genes comprised of 313 bp of 5′-flanking region from the hSP-A2 gene linked to human growth hormone (hGH), as reporter, with an intact (hSP-A313:hGH) or mutated (hSP-A313TBEmut:hGH) TBE or with a minimal genomic sequence containing the TBE, E-box, and G/T-box (hSP-A175:hGH), but lacking upstream elements. Fusion genes containing 313 bp of hSP-A2 5′-flanking DNA were expressed in an appropriate lung cell-specific, developmentally- and hormonally-regulated manner in transgenic mice. Interestingly, although as little as 175 bp of hSP-A2 5′-flanking DNA mediated appropriate developmental and lung cell-specific expression, this genomic region was insufficient to mediate cAMP and IL-1 stimulation or glucocorticoid inhibition of hSP-A2 promoter activity. The finding that expression of the hSP-A313TBEmut:hGH transgene was essentially undetectable in fetal lung during late gestation and was not regulated by cAMP, IL-1, or Dex in transgenic fetal lung explants underscores the critical importance of the TBE and of TTF-1 and NF-κB in lung cell-specific, developmental, and hormonal regulation of hSP-A2 gene expression.

MATERIALS AND METHODS

Construction of plasmids containing hSP-A2:hGH fusion genes.

DNA purification, restriction endonuclease digestion, ligation, agarose gel electrophoresis, transformation, and maintenance of Escherichia coli were carried out as previously described (4). The plasmid pACsk20-hGH (56), which contains the promoterless hGH structural gene subcloned into pUC12, was used to construct the hSP-A2:hGH fusion genes. Schematic diagrams of the fusion genes used to create transgenic mice are shown in Fig. 1. To construct hSP-A313:hGH, a HindIII-BamHI fragment of human genomic DNA containing ∼313 bp of DNA flanking the 5′-end of the hSP-A2 gene transcription initiation site and the first 25 bp of exon I was fused to the first exon of the hGH structural gene in pACsk20-hGH by ligation to a BamHI site. The hSP-A175:hGH fusion gene was constructed by ligating an EcoRI-Sau3AI fragment of human genomic DNA that included ∼175 bp of 5′-flanking DNA and 25 bp of exon I of the hSP-A2 gene to the hGH structural gene by subcloning into the BamHI site of pACsk20-hGH. The hSP-A313TBEmut:hGH fusion gene, in which the TBE was mutated by scrambling the sequence from CTCAAG (wild-type) to GAATTC (TBEmut), was accomplished by site-directed mutagenesis of the element within hSP-A313:hGH.

Fig. 1.

Schematic diagram of human (h) surfactant protein-A (SP-A):human growth hormone (hGH) fusion genes and their expression in tissues of transgenic mice. A: schematic of hSP-A313:hGH, hSP-A175:hGH, and hSP-A313TBEmut:hGH fusion genes used to create transgenic mice. Gray bars, regions derived from the hSP-A2 gene (+25 bp of 1st exon and 5′-flanking DNA); hatched bars, regions encoding the hGH gene; black, striped, gray, and white bars represent the estrogen-related receptor α element (ERRE; −241 bp), thyroid transcription factor-1-binding element (TBE; −171 bp), E-box (−80 bp), and GT-box (−61 bp) response elements, respectively; arrow, position of hSP-A2 transcription initiation and direction of transcription. Sequences of wild-type and mutated TBE also are indicated. B: Northern blot of total RNA (30 μg) isolated from various tissues of adult transgenic mice carrying hSP-A313:hGH, hSP-A175:hGH, and hSP-A313TBEmut:hGH fusion genes. Blots were analyzed for hGH and cyclophilin mRNA expression. Cyclophilin was analyzed to assess efficiency of RNA loading and transfer.

Production and identification of transgenic mouse lines.

Transgenic mouse lines were created in the University of Texas Southwestern Transgenic Core Facility, as described previously (4). Briefly, after digestion of the recombinant plasmids with appropriate restriction endonucleases to release the fusion genes, the linearized transgenes were isolated on agarose gels and purified using a GENEClean purification kit (MP Biomedicals) before microinjection in the male pronucleus of fertilized F2 hybrid mouse eggs (obtained by mating C57Bl/6 × SJL hybrid adults). After culture to the two-cell stage and reimplantation in pseudopregnant recipients, the embryos were allowed to develop to term. Transgenic progeny were identified, and transgene copy number was analyzed by dot-blot analysis of tail genomic DNA using hGH DNA as a probe. Copy number was determined by comparison of the intensity of the hGH signal in the tail DNA with that of a known amount of hGH structural DNA added to the tail DNA of a nontransgenic mouse.

Northern analysis of hGH mRNA in transgenic mouse tissues.

Total RNA was extracted from lung and other tissues of transgenic mice using the guanidinium isothiocyanate method as described previously (4, 9). Total RNA (∼15 μg) was separated by electrophoresis and transferred to nitrocellulose. The blot was hybridized with 32P-labeled probes corresponding to mouse (m) SP-A cDNA, hGH cDNA, or cyclophilin as a control for loading and transfer of RNA.

Quantitative RT-PCR.

Total RNA from lung tissues of transgenic mice was extracted by the one-step method (11) (TRIzol; Invitrogen). RNA was treated with deoxyribonuclease to remove any contaminating DNA, and four micrograms were reverse transcribed using random primers and Superscript II RNase H-reverse transcriptase (Invitrogen). Primer sets directed against hGH [forward 5′-GGAAACACAACAGAAATCCAACCTA-3′ (335–359 bp); reverse 5′-GCGAAGACACTCCTGAGGAACT-3′ (412–433 bp) and mSP-A mRNA forward 5′-TCCAGGGTTTCCAGCTTACCT-3′ (390–410 bp); reverse 5′-GACAGCATGGATCCTTGCAAG-3′ (480–500 bp)] together with mouse TATA box binding protein [mTBP; forward 5′-GTATCTGCTGGCGGTTTGG-3′ (56–74 bp); reverse 5′-GGCACTGCGGAGAAAATGA-3′ (94–112 bp)], as standard, were generated utilizing Primer Express software (PE Applied Biosystems) based on published sequences.

The relative abundance of each RNA transcript was determined by quantitative RT-PCR (qRT-PCR) using the ABI Prism 7700 Detection System (Applied Biosystems) and the DNA binding dye SYBR Green (PE Applied Biosystems) for the detection of PCR products as described in detail previously (13, 22). Relative fold changes were calculated using the comparative cycle times (Ct) method with mTBP as the reference guide. Over a wide range of known cDNA concentrations, all primer sets were demonstrated to have good linear correlation (slope = −3.4) and equal priming efficiency for the different dilutions compared with their Ct values (data not shown). The relative abundance of each primer set compared with calibrator was determined by the formula, 2{μΔΔCt}, whereby ΔΔCt is the calibrated Ct value (primer − internal control).

In situ hybridization analysis of hGH mRNA in transgenic mouse lung tissues.

The procedure for in situ hybridization was described previously (4). Briefly, transgenic mouse lungs were fixed in 4% paraformaldehyde and embedded in paraffin before sectioning and adherence to charged slides. After deparaffinization, 35S-labeled sense and antisense hGH cRNAs were hybridized with the fixed sections. After autoradiography in Kodak NTB2 emulsion (Eastman Kodak, Rochester, NY), the sections were counterstained with hematoxylin and eosin. The images were visualized and photographed under bright- and dark-field microscopy.

Culture of fetal mouse lung tissues.

Lung tissues from 16 days postcoitum (dpc) transgenic and nontransgenic fetal mice were cut into several pieces and placed in organ culture in serum-free Waymouth's MB752/1 medium (GIBCO-BRL, Life Technologies, Grand Island, NY) in the absence and presence of dibutyryl-cAMP (DBcAMP, 10−3 M), Dex (10−7 M), or IL-1β (10 ng/ml), added alone or in various combinations (4, 57). The explants were cultured for up to 2 days in a humidified atmosphere of 95% air-5% CO2.

hGH assays.

Media from the cultured mouse fetal lung explants were collected at 24-h intervals. The concentration of hGH that accumulated in the media between 24 and 48 h of culture was quantified using a hGH Enzyme Immunoassay Test Kit (Diagnostic Automation, Calabasas, CA).

Experimental animals.

Mice used in this research were treated in accordance with the guidelines established by the Animal Welfare Information Center and approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center at Dallas.

RESULTS

hSP-A2 (175 bp) 5′-flanking DNA is sufficient for lung cell-specific and appropriate developmental regulation of transgene expression in mice.

To delineate the genomic sequences required for lung cell-specific and developmental regulation of expression of the hSP-A2 gene in vivo, a number of hSP-A:hGH fusion genes containing various amounts of hSP-A2 5′-flanking DNA were constructed (Fig. 1A) and used to create transgenic mice. Previously, we observed that 378 bp of 5′-flanking DNA from the rabbit SP-A gene were sufficient to direct appropriate lung cell-specific and developmental regulation of expression in transgenic mice (4). In human fetal type II cell transfection studies using reporter constructs containing 5′-flanking sequences from the hSP-A2 gene, we observed that the TBE at −171 to −166 bp plays a critical role in cAMP, IL-1, and glucocorticoid regulation of hSP-A2 promoter activity (5, 25, 27, 38). In the present study, we created transgenic mouse lines carrying hSP-A:hGH fusion genes comprised of 313 bp of hSP-A2 5′-flanking DNA, without and with a scramble mutation in the TBE (hSP-A313TBEmut:hGH) and a fusion gene comprised of 175 bp of 5′-flanking sequence containing the core TBE, E-box, and G/T-box sequences, but lacking the ERRE. The transgenic founders were bred to establish germline transmission, and the offspring were analyzed by Northern blotting and qRT-PCR to assess tissue-specific and developmental regulation of transgene expression, as well as levels of transgene expression in lung between and within each transgenic line (Table 1). Hormonal responsiveness of the transgenes was analyzed using lung explants from 16-day fetal transgenic mice cultured in serum-free medium in the absence or presence of DBcAMP, IL-1β, or Dex added alone or in various combinations.

View this table:
Table 1.

Expression of various fusion genes in transgenic mice

In two of the three lines of hSP-A313:hGH transgenic mice created, transgene expression (measured as hGH mRNA) was essentially lung specific, with very low levels of expression observed in other tissues (Fig. 1B and Table 1). In line no. 0623, by far the highest levels of hGH expression were present in lung; however, elevated expression was observed in mammary gland (Table 1). Of interest was the finding that, in the seven lines of transgenic mice created carrying SP-A:hGH fusion genes with only 175 bp of hSP-A2 5′-flanking DNA, hGH also was expressed in a lung-specific manner. Northern blot analysis revealed predominant expression in lung, with relatively low levels of expression in other tissues. In line no. 3682, which manifested extremely high levels of expression in lung, expression also was evident in heart (Table 1). The 175-bp region contains the core TBE, E-box, and G/T-box, but lacks the ERRE.

On the other hand, in three of the five lines of transgenic mice carrying the hSP-A313TBEmut:hGH fusion gene, in which the TBE was specifically mutated, transgene expression was either extremely low or undetectable in all tissues (Table 1). Although hGH was detected in lung in the two other lines (0252 and 0270), it was not lung specific. In one line (0252), hGH was expressed in brain at levels higher than those of lung, whereas in the other line (0270), which manifested elevated levels of transgene expression in lung, hGH also was highly expressed in heart, kidney, and mammary gland (Table 1). Therefore, it is likely that in the 0270 line tandem copies of the transgene were inserted in the enhancer region of another gene. Shown in Fig. 1B are representative Northern blots of hGH mRNA in tissues from transgenic mice carrying the three fusion gene constructs. As can be seen at the exposure times shown, hGH expression was lung specific in transgenic mice carrying the hSP-A313:hGH and hSP-A175:hGH fusion genes and undetectable in any tissues of transgenic mice with the hSP-A313TBEmut:hGH fusion gene construct.

SP-A (175 bp) 5′-flanking DNA is sufficient for appropriate lung cell-specific expression.

We next performed in situ hybridization analyses of lung tissues of transgenic mice carrying the hSP-A175:hGH fusion gene using a 35S-labeled hGH antisense cRNA probe. As can be seen in the darkfield images shown in Fig. 2, it is apparent that transgene expression in lung was restricted to type II cells and to bronchioalveolar epithelial cells as previously reported for the endogenous SP-A gene in mouse (32, 59), rabbit (58), and pig (24) lung.

Fig. 2.

SP-A (175 bp) 5′-flanking DNA is sufficient for appropriate lung cell-specific expression. Lung tissue sections from adult transgenic mice carrying hSP-A175:hGH fusion genes were analyzed by in situ hybridization using an 35S-labeled antisense hGH cRNA probe and examined by brightfield (A) and darkfield (B) microscopy. Transgene (hGH) expression (shown as white silver grains on darkfield image) is evident in bronchioalveolar epithelial cells (arrowheads) and in type II cells (arrows).

SP-A (175 bp) 5′-flanking DNA contains all the required response elements for appropriate developmental regulation of SP-A expression.

To analyze the hSP-A2 5′-flanking sequences and the role of the TBE in the appropriate developmental regulation of SP-A, expression of hSP-A313:hGH (line 2857), hSP-A−175:hGH (line 3682), and hSP-A313TBEmut:hGH (line 0237) fusion genes and the endogenous mouse SP-A gene was analyzed in lung tissues of fetal and neonatal transgenic mice using qRT-PCR (Fig. 3). At 15.5 dpc, expression of the endogenous mSP-A gene and of each of the transgenes was extremely low or undetectable. Endogenous mSP-A mRNA transcripts were modestly increased on 16.5 dpc and increased markedly on 17.5 dpc, reaching maximal levels on 18.5 dpc. Expression of the hSP-A313:hGH and hSP-A−175:hGH transgenes followed a highly similar pattern (Fig. 3, A and B). By contrast, expression of the hSP-A313TBEmut:hGH transgene remained undetectable throughout gestation.

Fig. 3.

hSP-A2 (175 bp) 5′-flanking DNA contains the required response elements for appropriate developmental regulation of transgene expression. RNA isolated from lung tissues of 15.5–18.5 days postcoitum (dpc) fetal and newborn (NB) transgenic mice carrying hSP-A313:hGH, hSP-A313TBEmut:hGH, and hSP-A175:hGH fusion genes was analyzed for hGH and mSP-A mRNA transcripts using quantitative RT-PCR (qRT-PCR). Data are expressed as means ± SE of values from 4 tissue samples.

The TBE, as well as upstream sequences between −175 and −313 bp are required for stimulation of hSP-A2 promoter activity by cAMP and IL-1 and inhibition by glucocorticoids in fetal mouse lung explants.

As mentioned above, unlike the endogenous mSP-A gene (unpublished observations), expression of the hSP-A2 gene is stimulated by cAMP and by IL-1 (25) and is inhibited by glucocorticoids (5, 27, 50). To analyze the sequences required for appropriate hormonal regulation of transgene expression, lung explants from 16 dpc fetal transgenic mice carrying hSP-A313:hGH, hSP-A175:hGH, and hSP-A313TBEmut:hGH fusion genes were placed in organ culture and incubated for 48 h in the absence or presence of DBcAMP (1 mM), Dex (10−7 M), or IL-1β (10 ng/ml), added alone or in various combinations. Transgene expression was analyzed by accumulation of hGH in the medium between 24 and 48 h of culture. hGH accumulation in the medium of each culture dish was normalized to the total amount of RNA in the explants of that dish and compared with those in the untreated control group. As can been seen in Fig. 4, treatment of fetal lung explants from hSP-A313:hGH transgenic mice (line 2857) with DBcAMP (1 mM) or with IL-1β caused an approximately threefold increase in hGH production. In fetal lung explants treated with DBcAMP + IL-1β, hGH expression was stimulated approximately fivefold. Furthermore, Dex treatment markedly inhibited basal levels of hSP-A313:hGH transgene expression and abrogated the stimulatory effects of DBcAMP and IL-1β (Fig. 4). Although in lung explants from hSP-A175:hGH transgenic mice basal expression of hGH was readily detectable, no stimulatory effects of DBcAMP or IL-1β on transgene expression were evident. On the other hand, a modest inhibitory effect of Dex on hSP-A175:hGH expression was observed. This is of interest, since we previously reported that Dex/glucocorticoid receptor inhibition of hSP-A2 promoter activity is mediated via the TBE (5, 27). Finally, hGH production by lung explants of fetal transgenic mice carrying the hSP-A313TBEmut:hGH was essentially undetectable and unaffected by any hormonal treatment (Fig. 4). These findings further emphasize the crucial role of the TBE in hormonal regulation of hSP-A2 gene expression.

Fig. 4.

TBE and upstream elements are required for cAMP and interleukin (IL)-1 induction and dexamethasone (Dex) repression of hSP-A2 promoter activity in lung explants from transgenic mice. Lung explants from 16 dpc fetal transgenic mice carrying hSP-A313:hGH, hSP-A175:hGH, and hSP-A313TBEmut:hGH fusion genes were placed in organ culture and incubated for 48 h in the absence or presence of dibutyryl-cAMP (DBcAMP/Bt2cAMP, 1 mM), Dex (10−7 M), or IL-1β (10 ng/ml), added alone or in various combinations. Transgene expression was analyzed by accumulation of hGH in the medium between 24 and 48 h of culture. hGH in the medium from each culture dish was normalized to the total amount of RNA in the explants of that dish and compared with that in the untreated control group, which was assigned a value of 1. Data are means ± SE of values from 4 culture dishes of fetal lung explants. **P < 0.01 and *P < 0.05, significantly different from untreated lung explants.

DISCUSSION

In the present study, we observed that a 313-bp region upstream of the hSP-A2 gene mediated appropriate lung-specific, developmental, and hormonal regulation of expression in transgenic mice. Expression of transgenes containing 313 bp of hSP-A2 5′-flanking DNA was markedly stimulated by cAMP and IL-1 and profoundly inhibited by Dex treatment. This genomic region contains all of the conserved response elements that we previously found to be critical for cAMP and IL-1 induction and for glucocorticoid repression of promoter activity of the human, rabbit, and baboon SP-A genes in transfected type II cells (3, 5, 17, 38, 62, 63). Response elements required for cAMP stimulation of SP-A promoter activity include an element that binds the orphan nuclear receptor ERRα (ERRE) (40), a composite element (TBE) that binds TTF-1 and NF-κB (25), an E-box that binds USFs 1 and 2 (1719), and a G/T-box that binds Sp1 (63). This genomic region functions as an “enhancesosome,” since mutation of any one of these elements abrogates cAMP induction of SP-A promoter activity in transfected type II cells (3, 5, 17, 38, 62, 63).

Interestingly, we found that fusion genes comprised of as little as 175 bp of hSP-A2 5′-flanking DNA, which contain the TBE, E-box, and G/T-box, but lack the ERRE, also were expressed in a lung- and cell-specific manner in the transgenic mice. As we observed in transgenic mice carrying the hSP-A313:hGH fusion gene, hSP-A175:hGH expression was evident in type II cells and in bronchioalveolar epithelial cells, the cell types that express the highest levels of endogenous SP-A (4, 58). Furthermore, this small genomic region also mediated developmental induction of transgene expression in a temporal pattern identical to that of the endogenous mSP-A gene. Thus genomic sequences between −175 and −313 bp, including the ERRE, are not essential for lung cell-specific and developmental regulation of hSP-A2 expression.

On the other hand, the 175-bp region was not sufficient to mediate cAMP and IL-1β stimulation of hSP-A2 promoter activity in transgenic mouse fetal lung explants, although a modest inhibitory effect of Dex on basal transgene expression was observed. This emphasizes the importance of 5′-flanking sequences between −175 and −313 bp, containing the ERRE, for cAMP and IL-1 regulation of expression. Previously, we observed in transfected human fetal type II cells that hSP-A231:hGH fusion genes lacking the ERRE or hSP-A296ERREmut:hGH fusion genes containing an ERRE mutation manifested decreased basal and loss of cAMP stimulation of hSP-A2 promoter activity (62). On the other hand, in transgenic studies to define the regulatory regions of the rabbit SP-A gene, we found that SP-A-378ERREmut:hGH transgenes containing an ERRE mutation were expressed in an appropriate spatial and temporal manner (4). Collectively, these findings indicate that, whereas response elements between −175 and −313 bp containing the ERRE are required for cAMP and IL-1 modulation of SP-A expression, these are not crucial for lung cell-specific and developmental timing of expression. We, therefore, propose that the ERRE and other upstream sequences may serve to fine tune levels of SP-A expression in response to hormonal and environmental signals, such as changes in O2 tension.

In the present study, we observed that, in three out of five transgenic lines carrying an hSP-A313TBEmut:hGH fusion gene with a mutation in the core TBE sequence, lung expression was extremely low or undetectable, and there was a lack of developmental and hormonal regulation of transgene expression. In the other two lines carrying this mutation, transgene expression in lung was readily detectable; however, elevated levels of expression also were observed in other tissues. This underscores the critical importance of the TBE as a “master regulator” of lung-specific and developmental regulation of hSP-A2 promoter activity. Furthermore, in studies using lung explants from 16 dpc fetal transgenic mice carrying the hSP-A313TBEmut:hGH fusion gene, hGH production was essentially undetectable, and inductive effects of cAMP and IL-1 were not observed. This suggests that the TBE is essential for basal and cAMP/IL-1 induction of hSP-A2 expression and support findings of type II cell transfection studies, which indicated that mutation of this TBE site abrogated cAMP induction of hSP-A2 promoter activity (37). Because fetal lung explants from hSP-A175:hGH transgenic mice also were nonresponsive to cAMP and IL-1, these findings collectively suggest that the TBE is necessary, but not sufficient, for cAMP and IL-1 stimulation of hSP-A2 expression.

TTF-1 is expressed in lung from the earliest stages of development (21, 31, 36) and serves a critical role in lung morphogenesis (21, 31, 36). TTF-1 also acts later in fetal development to increase transcription of genes encoding SP-A (10, 37), SP-B (60), SP-C (30), and Clara cell-specific protein (CC10) (54), which manifest different cell type and developmental patterns of expression. This raises the interesting question of how TTF-1 exerts these diverse temporal and spatial effects.

Developmental, cell-specific, and gene-specific actions of TTF-1 could possibly be mediated by alterations in its posttranslational modification, which may promote changes in nuclear localization (34, 35, 61), TBE-binding activity (37), and differential interactions with other tissue/cell-specific transcription factors and coregulators (15, 16, 39). As mentioned, the TBE of the hSP-A2 gene is a composite element that binds both TTF-1 and NF-κB proteins, p50 and p65 in vivo (25, 26); these factors act synergistically to increase hSP-A2 transcription in response to cAMP and IL-1 treatment (25).

The role of endogenous NF-κB in hSP-A expression was confirmed by the finding that overexpression in human fetal type II cells of an inhibitory factor κBα phosphorylation site mutant, resistant to proteolytic degradation, reduced TBE-binding, as well as basal, cAMP-, and IL-1α induction for SP-A expression (25). A role for endogenous NF-κB was further supported by the finding that the antioxidant NF-κB inhibitor pyrrolidine dithiocarbamate blocked cAMP- and IL-1-induced binding of nuclear proteins both to the TBE and to a consensus NF-κB response element. Furthermore, this inhibitor abrogated cAMP and IL-1 induction of SP-A expression in the cultured type II cells (25). In previous studies using human fetal type II cells, we observed that cAMP, acting through cAMP-dependent protein kinase (PKA), increased TTF-1 phosphorylation, acetylation (61), TBE binding, and transcriptional activity (37). Cytokines (48), PKA (64, 65), and reactive O2 species (7) also have been reported to increase phosphorylation, DNA binding, and transcriptional activity of NF-κB. Phosphorylation of NF-κB p65 by PKA increases its transcriptional activity by enhancing p65 and p50 association with coactivators cAMP response element binding protein (CBP) (65) and steroid receptor coactivator-1 (SRC-1) (47), respectively.

We have suggested that developmental regulation of SP-A gene expression in fetal lung may be influenced by increased vasculogenesis, resulting in enhanced O2 availability to the developing lung epithelium (1, 26). Previously, we observed that, when human fetal lung type II cells were cultured in a 20% O2 environment, TBE binding of endogenous TTF-1 and NF-κB was stimulated by DBcAMP and IL-1 (26). cAMP and IL-1 also enhanced recruitment to the TBE of inhibitor of κB kinase α and the histone acetyltransferases, CBP and SRC-1, resulting in increased local phosphorylation and acetylation of key residues on histone H3 (2527) that are known marks of “active” chromatin (28). Importantly, these stimulatory effects of cAMP and IL-1 were prevented when type II cells were cultured in a hypoxic environment (26). Thus changes in O2 availability during late fetal development and after birth may enhance formation of a transcriptional activation complex at the TBE stabilized by multivalent interactions with coregulators. This results in posttranslational modifications of histones, resulting in the local opening of chromatin structure and activation of SP-A transcription.

Our present transgenic studies provide further evidence for the critical role of the TBE in lung cell-specific and developmental regulation of hSP-A2 expression. They further suggest that, although the TBE is required for cAMP, cytokine, and glucocorticoid regulation of hSP-A2 promoter activity in fetal lung, it is not sufficient. Based on these and on previous studies (3, 17, 38, 62, 63), it is apparent that cooperative interaction of transcription factors and coregulators bound to the TBE with those bound to other critical response elements upstream of the SP-A promoter likely plays an important role in its hormonal regulation.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grant R37 HL-050022.

Acknowledgments

We thank John Shelton and Dr. James Richardson of the Molecular Pathology Core for in situ hybridization studies and John Ritter of the Transgenic Core for generation of transgenic mice. We also thank Barbara Murry for generating the hSP-A-hGH fusion gene constructs.

Footnotes

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

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View Abstract