The Basic Helix-Loop-Helix-Zipper Transcription Factor USF1 Regulates Expression of the Surfactant Protein-A Gene

Department of Obstetrics-Gynecology, The University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/1997; 272(37):23398-406. DOI: 10.1074/jbc.272.37.23398
Source: PubMed


Expression of the rabbit pulmonary surfactant protein A (SP-A) gene is lung-specific, occurs primarily in type II cells, and is developmentally regulated. We previously identified two E-box-like enhancers, termed the distal binding element (DBE) and proximal binding element (PBE), in the 5'-flanking region of the rabbit SP-A gene. In the present study, the PBE was used to screen a rabbit fetal lung cDNA expression library; a cDNA insert was isolated which is highly similar in sequence to human upstream stimulatory factor 1 (hUSF1). By use of reverse transcription polymerase chain reaction, two isoforms of rabbit USF1 (rUSF1) mRNAs were identified in fetal rabbit lung and other tissues. The levels of rUSF1 mRNAs reach a peak in fetal rabbit lung at 23 days gestation, in concert with the time of initiation of SP-A gene transcription. Binding complexes of nuclear proteins obtained from fetal rabbit lung tissue and isolated type II cells with the DBE and PBE were supershifted by the addition of anti-rUSF1 IgG. Binding activity was enriched in type II cells compared with lung fibroblasts. Overexpression of rUSF1s in A549 adenocarcinoma cells positively regulated SP-A promoter activity of cotransfected reporter gene constructs. It is suggested that rUSF1s, which bind to two E-box elements in the SP-A gene 5'-flanking region, may serve a key role in the regulation of SP-A gene expression in pulmonary type II cells.

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    • "USF is ubiquitously expressed (Sirito et al. 1994), but contributes to the transcriptional regulation of a considerable number of genes that are expressed in a tissue-speci®c manner. Among these are the gene for cardiac alpha-myosin heavy chain (Xiao & Ojamaa 1998), cardiac myosin light-chain 2 (Navankasattusas et al. 1994), chicken (Cvekl et al. 1994) and mouse aA-crystallin (Sax et al. 1997), surfactant protein A (Gao et al. 1997) and a1(I) collagen (Rippe et al. 1997). In addition, to be required for constitutional expression, there is evidence that in several genes, the "
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    ABSTRACT: Mutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal-dominant juvenile primary open-angle glaucoma (POAG). In patients with non-autosomal-dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. DNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt -106 to -77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA-box was protected, as well as at least three downstream sites, including an AP-1-like sequence. Deletion of the -106 to -77 region caused a substantial loss of functional promotor activity in all cell types. Site-directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the -106 to -77 region. Each of these cis-elements is essential for minimal promoter activity. The 5'-half of the region contains a sequence with similarities to NF-kappaB-related sites, however, binding of NF-kappaB could not be confirmed by EMSA. The 3'-half contains a canonical E-box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E-box sequence and that the binding can be supershifted by specific antibodies. Several DNA-protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.
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    ABSTRACT: Pulmonary surfactant is a developmentally and hormonally regulated lipoprotein synthesized exclusively in alveolar type II cells. Surfactant protein-A (SP-A) gene transcription in human fetal lung in culture is stimulated by glucocorticoids and cAMP; cAMP also enhances the rate of type II cell differentiation. The CCAAT/enhancer-binding protein (C/EBP) family of transcription factors serves an important role in the regulation of genes involved in energy metabolism, lipid biosynthesis, and cellular differentiation. The gene encoding C/EBPdelta, which is induced by glucocorticoids during the early phases of adipocyte differentiation, is expressed at relatively high levels in lung compared with other tissues. In the present study we have analyzed developmental changes in C/EBPdelta messenger RNA levels in fetal rabbit lung as well as changes in the levels of immunoreactive C/EBPdelta in human fetal lung during differentiation in organ culture and after treatment with cAMP and glucocorticoids. We observed that C/EBPdelta messenger RNA is detectable in fetal rabbit lung on day 19 of gestation and is increased approximately 3.7-fold to maximum levels on day 28 of gestation, the time when SP-A gene transcription increases to maximum levels. Immunohistochemical analysis of C/EBPdelta in midgestation human fetal lung before culture revealed trace nuclear staining in epithelial and occasional stromal cells. After 12 h of organ culture in serum-free medium, nuclear staining of C/EBPdelta was markedly increased in epithelial cells lining the prealveolar ducts of the human fetal lung tissue. By immunoblot analysis, it was found that C/EBPdelta levels were induced rapidly during organ culture in control medium and were increased further by treatment with dexamethasone and (Bt)2cAMP. C/EBPdelta levels were maximally induced during the first 24 h of culture and declined thereafter; after 72 h of incubation in control or cAMP-containing medium, C/EBPdelta was reduced markedly. By contrast, in fetal lung tissues incubated in medium containing dexamethasone or dexamethasone plus (Bt)2cAMP, the decline in C/EBPdelta was more modest, so that levels remained elevated throughout the 96-h culture period. Our findings that C/EBPdelta is localized primarily to alveolar epithelial cells, rapidly induced during differentiation of human fetal lung in culture, and increased by cAMP and glucocorticoids suggest a possible role in the regulation of type II cell differentiation and in the synthesis of surfactant phospholipids and proteins.
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