Transcription of the Lung-specific Surfactant Protein C Gene Is Mediated by Thyroid Transcription Factor 1

doi:10.1074/jbc.271.12.6881

Transcription of the Lung-specific Surfactant Protein C Gene Is Mediated by Thyroid Transcription Factor 1 (*)

From the Division of Pulmonary Biology, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

^§ To whom correspondence should be addressed:
Children's Hospital Medical Center, Div. of Pulmonary Biology, TCHRF, 3333 Burnet Ave., Cincinnati, OH 45229-3039
. Tel.: 513-559-7850; Fax: 513-559-7868.

Abstract

Surfactant protein C (SP-C) is expressed in alveolar Type II epithelial cells of the lung. In order to determine the mechanism(s) that regulate gene transcription, we have analyzed the activation of the murine SP-C promoter in mouse lung epithelial cells (MLE cells) and in HeLa cells after co-transfection with a vector expressing rat thyroid transcription factor-1 (TTF-1). TTF-1 transactivated SP-C-chloramphenicol acetyltransferase constructs containing −13 kilobase pairs to −320 base pairs (bp) of the 5′ flanking region of the SP-C gene. Essential cis-acting elements were functionally localized to between −320 and −180 bp from the start of transcription by transfection analysis. Five DNase-protected regions, indicating multiple protein-DNA interactions within the −320 bp TTF-1-responsive region of the SP-C gene, were identified by DNase footprint analysis. A 40-bp segment of SP-C DNA from −197 to −158 linked to a heterologous promoter-chloramphenicol acetyltransferase construct activated expression after co-transfection with CMV-TTF-1 in HeLa and MLE cells. The −197 to −158 segment contained two consensus TTF-1 sites, which were specifically identified as TTF-1 binding sites by gel retardation and antibody supershift with MLE cell nuclear extracts and purified TTF-1 homeodomain protein. Site-specific mutagenesis of either of the TTF-1 binding sites completely blocked activation by TTF-1, indicating both sites are required for TTF stimulation of SP-C transcription.

Footnotes

↵* This work was supported by Grant HL50046 from NHLBI, National Institutes of Health (to S. W. G.) and Training Grant in Pulmonary and Cardiovascular Development HL07752 from the National Institutes of Health (to S. E. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(™)/EMBL Data Bank with accession number(s) M38314.
↵¹ The abbreviations used are:

SP-A-SP-D

surfactant proteins A-D

kb

kilobase pair(s)

bp

base pair(s)

CAT

chloramphenicol acetyltransferase

PCR

polymerase chain reaction

BSA

bovine serum albumin.
↵² S. E. Kelly and S. W. Glasser, unpublished observations.
- Received October 6, 1995.
- Revision received January 10, 1996.