SFTPB is critical in maintaining lung function, because SFTPB-deficient mice die of respiratory failure shortly after birth (3
). One of the key events in acute lung injury is the loss of functional surfactant. Thus, surfactant has long been investigated as a treatment for acute lung injury. Surfactant replacement, however, has yet to be demonstrated as an effective therapy for acute lung injury because of immense hurdles in its administration (44
). An alternative strategy may involve the development of treatments that maintain or restore endogenous surfactant production. We previously reported that in a murine model of acute lung injury, the induction of JUN was associated with a diminution of SFTPB expression (7
). SFTPB expression was maintained in resistant, compared with sensitive, murine strains, and inducible SFTPB expression increased the survival of mice (7
). The present work focused on analyses of the core Sftpb
promoter, to identify transcription factor recognition sites that contribute to the JUN-mediated inhibition of SFTPB.
The murine Sftpb
promoter, spanning nucleotides −653 to +42, contains two functionally distinguishable proximal (−132 to −1) and distal (−382 to −283) promoter regions (8
). The distal promoter region can be transcriptionally active in the absence of the proximal promoter region. The distal promoter region contains a putative initiator sequence (CATTCTG) at nucleotides −286 to −280. This initiator element, which was originally identified in the TATA-less murine terminal deoxynucleotidyl transferase gene, encompasses the transcription start site and can direct basal transcription (45
). As determined by primer extension (8
) and expressed-sequence Tag (EST) database analyses, the SFTPB mRNA 5′ untranslated region contains ≤14–16 nucleotides. However, basal promoter activity was lost with the deletion of promoter sequences from −415 to −353 (34
), demonstrating the critical role of the distal promoter region. Although the occurrence of an alternative SFTPB transcriptional initiation site is unknown, the functionality of the distal region as a promoter provided a useful tool for analyzing the role of its putative transcription factor recognition sites, independent of those in the proximal region.
promoter contains AP-1, NKX2–1, trans-acting transcription factors 1 and 3 (SP1, SP3), hepatocyte nuclear factor 3 (HNF3), retinoic acid receptor, and other recognition elements (8
). Although JUN can activate numerous genes by binding to AP-1 recognition elements, it can also inhibit the induction of other genes by a different mechanism. JUN inhibits the induction of the bone γ-carboxyglutamate protein (osteocalcin) gene by retinoic acid and vitamin D3 (35
) and the induction of the kallikrein-related peptidase 3 (prostate-specific antigen) gene by androgen (47
). Direct interactions between JUN and the receptors for vitamin D3 or androgen inhibit the induction of target genes.
The Sftpb proximal and distal promoter regions contain putative AP-1 sites. The proximal AP-1 site is identical to the optimal consensus AP-1 site (TGACTCA), whereas the distal AP-1 site (TGCGTCA) differs by two nucleotides. In addition, the distal CREB site differs from the consensus CREB site (TGACGTCA). However, these nucleotide sequence differences could not explain JUN's ability to inhibit the distal, but not the proximal, promoter region. The patterns of JUN inhibition of the distal AP-1 wild-type and point mutant reporters were comparable. To rule out the possibility that the introduced mutations may not have sufficiently altered the mode of protein–DNA interactions, we investigated the effects of JUN co-expression on reporter constructs that lacked the distal putative AP-1 site. Despite the absence of the putative AP-1 site, JUN inhibited reporter activity. These results suggest that the presence of the putative AP-1 site in the distal promoter region is not required for the inhibition by JUN of Sftpb promoter activity, raising the possibility of protein–protein interactions as a mediator of JUN's inhibitory effect.
Our conclusion about the role of the Sftpb
AP-1 sites contrasts with those of previous studies (34
). Sever-Chroneos and colleagues (34
) observed that mutation in the distal AP-1 binding site increased basal promoter activity fivefold in MLE-15 cells. In addition, they concluded that the distal AP-1 element is involved in, but is not sufficient for, the inhibition by JUN of promoter activity. In our analysis, although deletion of the distal element containing the overlapping AP1/NKX2–1 recognition sites reduced promoter activity, mutation of the AP-1 site did not induce basal promoter activity or reverse JUN inhibition. It is unclear whether the use of the luciferase reporter (half-life of approximately 0.84 hours) (48
) versus the CAT reporter (half-life of approximately 16 hours) (49
) and other experimental variations (e.g., the use of different promoter fragments) contributed to the discrepancies observed. In support of our observations, a genome-wide analysis of the frequency and distribution of AP-1 sites indicated that the number of AP-1–regulated genes identified is far smaller than the number of genes containing potential AP-1 sites, and that not all AP-1 sites are activated in a given cell under a given condition (50
In addition, the interaction of JUN with NKX2–1 appears unlikely to inhibit Sftpb promoter activity. Point mutations at the NKX2–1 sites in the distal promoter reduced reporter activity, but the mutants remained sensitive to inhibition by JUN, suggesting that other recognition sites or regulatory factors mediated the JUN-mediated inhibition of Sftpb.
Mobility shift assays indicated that JUN may target the Sftpb promoter by binding to a site within the −339/−316 region. Nucleotide sequence analysis for potential transcription factor binding sites in the −339/−316 Sftpb promoter region predicted recognition sites for DBP and C/EBP. This was a pertinent finding because DBP and the C/EBPs, like JUN, belong to the bZIP protein family. The contribution of the identified site to the regulation of Sftpb promoter activity was demonstrated in MLE-15 cells transfected with pGL4−653/+42 wild-type and mutant reporters. Mutagenesis of the putative recognition site reduced Sftpb promoter activity and sensitivity to JUN-mediated inhibition.
Endogenous DBP or C/EBP binding to the Sftpb promoter was investigated by chromatin immunoprecipitation. Immunoprecipitation of MLE-15 chromatin sonicate with anti-DBP antibodies did not enrich the Sftpb promoter PCR amplification product, suggesting that DBP does not associate with Sftpb promoter in vivo, or that the abundance of DBP–Sftpb promoter complexes formed in vivo does not permit detection by this method. In contrast, C/EBP-α and C/EBP-β binding to the Sftpb promoter was evidenced by the enrichment of chromatin immunoprecipitation.
JUN modulates gene transcription by forming homodimeric or heterodimeric complexes (52
). Our results indicate that JUN may not inhibit Sftpb
promoter activity by forming homodimers. The recombinant JUN protein formed a complex with the consensus AP-1 oligonucleotide, but with none of the Sftpb
-derived oligonucleotides spanning the Sftpb
promoter region −375 to −275. However, in the presence of MLE-15 nuclear protein extract, the addition of JUN increased the intensity of DNA–protein complexes formed with the −339/−316 oligonucleotide probe. Further analysis, using point mutant oligonucleotides, suggested that multiple transcription factors bound to the −339/−316 probe.
Previous studies demonstrated that C/EBP-β and JUN interacted through their bZIP region in the absence of DNA. Such interactions altered the specificity of DNA binding (54
). In our EMSA analyses, C/EBP binding to the Sftpb
−339/−316 region, to which JUN by itself cannot bind, was shifted in the presence of purified recombinant JUN protein. These results suggest that the Sftpb
−339/−316 region is recognized by C/EBP-α and/or C/EBP-β dimers or C/EBP-α and/or C/EBP-β/JUN heterodimers, and that the formation of heteromeric complexes is key to Sftpb
promoter targeting by JUN. The PAR-bZIP DBP also bound to the −339/−316 probe, raising the possibility that other transcription factors or bZIP proteins could bind to the same site.
In conclusion, we analyzed the murine Sftpb promoter encompassing nucleotides −653 to +42. Deletion and site-directed Sftpb promoter reporter mutants and EMSAs indicate that the distal Sftpb promoter region −339 to −316 is a critical regulatory element. Mutagenesis in this region, which contains a C/EBP recognition site, reduced Sftpb promoter activity and sensitivity to JUN-mediated inhibition. Endogenous C/EBP-α and C/EBP-β bind to the Sftpb promoter. The transcription factor JUN can partner with C/EBP-α or c/EBP-β, bind to the identified cis-acting regulatory DNA site, and inhibit Sftpb promoter activity. Thus, the inhibition by JUN of the Sftpb promoter is likely indirect and dependent on heteromeric complex formation.