Abstract
Surfactant protein B (SP-B) is selectively expressed in bronchiolar and alveolar epithelial cells of the lung. We identified an upstream enhancer located in the 5'-flanking region of the human SP-B gene (-439 to -331 base pair, hSP-B(-439/-331)) by deletion analysis of SP-B-luciferase constructs assessed in transfection assays in vitro. The element cis-activated the expression of an SV40 promoter-luciferase reporter gene in a human pulmonary adenocarcinoma cell line (H441-4). Three distinct binding sites for the nuclear transcription protein, thyroid transcription factor 1 (TTF-1), were identified, and the purified TTF-1 homeodomain was bound to bhe region of hSP-B(-439/-331). Co-transfection of H441-4 cells with the expression vector pCMV-TTF-1 trans-activated the native human SP-B promoter and the SV40 promoter fused with the SP-B enhancer. Mutations of the TTF-1 binding sites in the upstream enhancer blocked TTF-1 binding and transactivation activity. In summary, TTF-1 interacts with distinct proximal (-80 to -110) and distal (-439 to -331) cis-acting elements than regulate lung epithelial cell-specific transcription of the human SP-B gene.
Highlights
Surfactant protein B is a small, hydrophobic protein that interacts with phospholipids to reduce surface tension at the air-liquid interface of the alveoli in the lung
In order to determine the biological function of the stimulatory element in the human gene, the hSP-B(Ϫ439/Ϫ331) sequence was subcloned into the polymerase chain reaction (PCR) II
Genetic ablation of the SPB gene in transgenic mice caused perinatal respiratory failure associated with atelectasis and the lack of both lamellar bodies and tubular myelin in the lungs of newborn Surfactant protein B (SP-B) deficient mice [2]
Summary
Surfactant protein B is a small, hydrophobic protein that interacts with phospholipids to reduce surface tension at the air-liquid interface of the alveoli in the lung. The lung epithelial cell specificity of surfactant protein gene expression is mediated at the level of gene transcription [4]. Analysis of the 5Ј regions of several genes expressed in a lung-specific manner (SP-A, -B, -C, and Clara cell secretory protein) supports an important role for thyroid transcription factor 1 (TTF-1) in the control of surfactant protein gene expression. Analysis of the SP-B gene promoter demonstrated that both TTF-1 and HNF-3 were activators of SP-B gene transcription mediated by cis-acting elements located between Ϫ218 to ϩ41 bp in the SP-B gene [4]. Point mutation, and transfection assays showed that TTF-1 is the critical nuclear transcription protein activating this SP-B enhancer element in the human gene
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