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Expression of the human surfactant protein-A2 (hSP-A2) gene is lung specific, occurs in type II and Clara ceils, 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 a 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 [+ or -] 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-A_313: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-A_313TBEmut: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

Alcorn, Joseph L., Kazi N. Islam, Pampee P. Young, and Carole R. Mendelson. Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element. Am J Physiol Lung Cell Mol Physiol 286: L767-L776, 2004. First published November 21, 2003; 10.1152/ajplung.00280.2003.-Induction of surfactant protein-A (SP-A) gene expression in fetal lung type II cells by cAMP and IL-1 is mediated by increased binding of thyroid transcription factor-1 (TTF-1) and NF-[kappa]B proteins p50 and p65 to the TTF-1-binding element (TBE) at--183 bp. In type II cell transfections, dexamethasone (Dex) markedly inhibits cAMP-induced expression of rabbit SP-A:human growth hormone (hGH) fusion genes containing as little as ~300 bp of the SP-A 5'-flanking sequence. Dex inhibifion is blocked by RU-486, suggesting a role of the glucocorticoid receptor (GR). The present study was undertaken to define the mechanisms for GR inhibition of SP-A expression. Cotransfection of primary cultures of type II cells with a GR expression vector abrogated cAMP induction of SP-A promoter activity while, at the same time, causing a 60-fold induction of cotransfected mouse mammary, tumor virus (MMTV) promoter. In lung cells transfected with a fusion gene containing three TBEs fused to the basal SP-A promoter, Dex prevented the stimulatory effect of IL-1 on TTF-1 inducfion of SP-A promoter activity, suggesting that the GR inhibits SP-A promoter activity through the TBE. In gel shift assays using nuclear extracts from human fetal type II cells cultured in the absence or presence of cAMP, Dex markedly reduced binding of nuclear proteins to the TBE and blocked the stimulatory effect of cAMP on TBE-binding activity. Our finding that Dex increased expression of the NF-[kappa]B inhibitory partner I[kappa]B-[alpha] suggests that the decrease in TBE-binding activity may be caused, in part, by GR inhibition of NF-[kappa]B interaction with this site. surfactant protein A; fetal lung; glucocorticoid receptor; cAMP; thyroid transcription factor-1

Expression of the pulmonary surfactant protein A (SP-A) gene is lung specific, developmentally regulated, and enhanced by hormones and factors that increase cAMP. We previously identified two E-box-like enhancers termed distal binding element (DBE) and proximal binding element (PBE) in the 5'-flanking region of the rabbit (r) SP-A gene that are essential for cAMP induction of rSP-A promoter activity (Gao E, Alcorn JL, and Mendelson CR. J Biol Chem 268: 19697-19709, 1993). We also found that DBE and PBE serve as binding sites for the basic helix-loop-helix-leucine zipper transcription factor, upstream stimulatory factor-1 (USF1) (Gao E, Wang Y, Alcorn JL, and Mendelson CR. J Biol Chem 272: 23398-23406, 1997). In the present study, PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert encoding the structurally related rabbit upstream stimulatory factor-2 (rUSF2) was isolated. The levels of rUSF2 mRNA reach peak levels in fetal rabbit lung at 28 days of gestation, in concert with the time of maximal induction of SP-A gene transcription. In yeast two-hybrid analysis, rUSF2 was found to preferentially form heterodimers, compared with homodimers, with rUSF1. Binding complexes of nuclear proteins isolated from fetal rabbit lung type II cells with the DBE and PBE were supershifted by anti-rUSF2 antibodies. Binding activity was enriched in nuclear proteins from type II cells compared with fibroblasts. Overexpression of rUSF2 in transfected lung A549 cells increased rSP-A promoter activity and acted synergistically with rUSF1. We suggest that heterodimers of USF2 and USF1 bound to two E-box elements in the SP-A gene 5'-flanking region serve a key role in developmental and hormonal regulation of SP-A gene expression in pulmonary type II cells. upstream stimulatory factor; E-box; surfactant protein A; gene expression; development; type II cells; regulation

This review discusses the growth and differentiation of the fetal lung at molecular levels during the transition of fetus to an air-breathing environment. Data on the lung development processes and fetal capacity to synthesize the surfactant protein are presented.

In humans, aromatase P450 (product of CYP19 gene), which catalyzes conversion of [C.sub.19] steroids to estrogens, is expressed in a number of tissues, including ovary, adipose, and syncytiotrophoblast of the placenta. The 5[prime] untranslated regions of CYP19 mRNA transcripts in these tissues are encoded by different tissue-specific first exons, which are spliced onto a common site just upstream of the translation initiation site in exon II. In placenta, the 5[prime] untranslated region of CYP19 mRNA transcripts is encoded by exon I.1, which lies [approximately equal to]40 kb upstream of exon II. To map genomic sequences required for placenta-specific CYP19 expression, fusion genes containing 2,400 and 501 bp of placenta-specific exon 1.1 5[prime] flanking DNA linked to the human growth hormone gene (hGH), as reporter, were introduced into transgenic mice. Expression of CYP19(I.1):hGH fusion genes containing as little as 501 bp of 5[prime] flanking DNA was placenta-specific and developmentally regulated. Furthermore, transgene expression occurred specifically in the labyrinthine trophoblast of the mouse placenta, which contains syncytial cells that may be analogous to the human syncytiotrophoblast. We show that a relatively small segment of DNA ([approximately equal to]500 bp) >40 kb upstream of the protein coding region of a human gene is able to direct expression in an appropriate tissue- and cell-specific manner in transgenic mice. These findings suggest that 5[prime] flanking DNA within 501 bp of exon I.1 of the human CYP19 gene contains cis-acting elements that bind placenta-specific transcription factors that are conserved between humans and mice.