Characterization of the rat pulmonary surfactant protein A promoter

Abstract

The expression of the pulmonary surfactant protein A (SP‐A) is developmentally regulated and controlled by several hormones. In an attempt to characterize cis‐acting elements involved in the regulation of SP‐A expression, we have cloned the 5′ flanking sequence of the rat SP‐A gene. The promoter region contains a TATA box but no CAAT box. The transcription start site has been identified by anchored polymerase chain reaction and S1 nuclease mapping of the mature and precursor transcripts. S1 mapping of precursor transcripts has confirmed the stimulating effect of glucocorticoids on SP‐A rat gene transcription in vivo. This hormonal effect may be mediated by a putative glucocorticoid responsive element located 140 bp upstream from the initiation site and protected against DNase 1 digestion in footprinting experiments. In vitro transcription of a G‐free reporter cassette linked to the 212‐bp 5′ flanking DNA fragment has established that this putative promoter region is functional. Efficient transcription of the G‐free reporter cassette was obtained with cell‐free fetal lung extracts, whereas no transcript was detectable with cell‐free liver extracts. Comparative analysis of the human and rat 5′ flanking sequences shows the presence of strongly conserved motifs, unrelated to previously known consensus sequences. Some of these motifs, specifically protected in DNase 1 footprinting studies, could therefore be involved in the regulation of SP‐A gene expression.

Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP‐mediated transcription*

“…Using a CAT-reporter construct in which the CAT expression is under the control of the SP-D 5 H promoter segment (SP-D698), we examined the effect of RB on the SP-D698 reporter activity in the lung epithelial MLE-15 cells [Wikenheiser et al, 1993]. As shown in Figure 2A, when cotransfected to MLE-15 cells, RB stimulated SP-D698/CAT reporter activity more than three fold, whereas RB had only marginal effect on SP-A236/CAT reporter activity despite the presence of a consensus NF-IL6 binding site (TTCTGCAAT) n the SP-A promoter region (À184/À176) [Lacaze-Masmonteil et al, 1992]. The positive effect of RB on the SP-D698/CAT reporter was also observed in HeLa cells (data not shown).…”

Ambroxol suppresses influenza-virus proliferation in the mouse airway by increasing antiviral factor levels

“…Since there is 95% identity between the amino acid sequence of the SP-As of mouse and rat [23,24], and isolated polyclonal antibodies against rat SP-A [6,25] react with mouse SP-A, the levels of SP-A in BALF of mice were analysed by enzyme-linked immunosorbent assay (ELISA) using monospecific biotinylated and nonbiotinylated antibodies against rat SP-A and mouse SP-A as standards. The levels of cytokines (TNF-a, IL-12, IFN-c, IL-6 and IL-4) in BALF were determined using ELISA kits (BioSource International, Camarillo, CA, USA) according to the manufacturer9s protocol.…”

The lung-specific CC10 gene is regulated by transcription factors from the AP-1, octamer, and hepatocyte nuclear factor 3 families.

“…Recently, sequences for several lung-specific promoters have been obtained, such as those for the lung surfactant proteins A, B, and C and for CC10, a secretory product of airway cells (for a review, see reference 40). Genetic studies using these sequences have begun to provide information on transcriptional regulation in the lung (12,22,25). For example, in situ hybridization in mice and rats has shown that the surfactant protein C gene is expressed exclusively in the alveolar epithelium and the CC10 gene is expressed primarily in the airway, while the surfactant A and B genes are expressed at high levels in both of these locations (12,20,45).…”