Surfactant Protein D Gene Regulation: INTERACTIONS AMONG THE CONSERVED CCAAT/ENHANCER-BINDING PROTEIN ELEMENTS

Yanchun He,Erika Crouch

doi:10.1074/jbc.m201126200

Abstract

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury. Given the importance of CCAAT/enhancer-binding protein (C/EBP)-binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs. Five SP-D motifs (-432, -340, -319, -140, and -90) homologous to the C/EBP consensus sequence specifically bound to C/EBPs in gel shift assays, and four of the five sites (-432, -340, -319, and -90) efficiently competed for the binding of C/EBPalpha, C/EBPbeta, or C/EBPdelta to consensus oligomers. Cotransfection of C/EBPalpha, C/EBPbeta, or C/EBPdelta cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transfection of C/EBP also increased the level of endogenous SP-D mRNA in H441 cells. Transactivation of the reporter construct was abrogated by deletion of sequences upstream of -205. Independent site-directed mutagenesis of the sites at -432, -340, and -319 reduced C/EBP-mediated activation by approximately 50%, and mutagenesis of the site at -432 in combination with either of the tandem sites at -340 and -319 blocked activation. The conserved AP-1 element at -109 was required for maximal promoter activity, but not for the transactivation of SS698 by C/EBPs. Thus, interactions among C/EBP elements in the near-distal promoter can modulate the promoter activity of SP-D.

Highlights

Surfactant protein D (SP-D) plays roles in pulmonary host defense and surfactant homeostasis and is increased following acute lung injury
Given the importance of CCAAT/enhancer-binding protein (C/EBP)binding elements in the systemic acute-phase response and lung development and the expression of C/EBP isoforms by lung epithelial cells, we hypothesized that conserved C/EBP motifs in the near-distal and proximal promoters contribute to the regulation of SP-D expression by C/EBPs
The potential importance of the four most upstream motifs is suggested by the spatial conservation of homologous sequences in the rat and mouse SP-D promoters [27, 31] and/or in the promoter sequences of bovine conglutinin and CL-43, hepatic host defense collectins believed to have evolved from a primordial SP-D gene [32,33,34]

Summary

Surfactant Protein D Gene Regulation

Cotransfection of C/EBP␣, C/EBP␤, or C/EBP␦ cDNA in H441 lung adenocarcinoma cells significantly increased the luciferase activity of a wild-type SP-D promoter construct containing 698 bp of upstream sequence (SS698). Transgenic mice deficient in surfactant protein A or SP-D show abnormal microbial clearance or acute inflammatory responses to microbial challenge [9] Based on these and other data, it has been suggested that the lung collectins contribute to a pulmonary acute-phase response, somewhat analogous to the hepatic acute-phase response (APR) to systemic injury [6]. We compared the activity of wildtype constructs and constructs containing mutated consensus sequences in transient transfection assays using luciferase reporter constructs and examined potential functional interactions with the conserved AP-1 element in the proximal promoter

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION