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.
Highlights
Pulmonary surfactant is a mixture of phospholipids and proteins, which functions to reduce surface tension at the air/ liquid interface preventing alveolar collapse during respiration [1]
The Surfactant protein C (SP-C) Promoter Is Transactivated by transcription factor-1 (TTF-1)—In order to determine whether SP-C promoter sequences were responsive to activation by transcription factors that stimulate genes expressed in airway epithelial cells, MLE-15 cells were cotransfected with a 4.8-kb SP-C-chloramphenicol acetyltransferase (CAT) construct and a second plasmid that constitutively expressed one of the recombinant transcription factors TTF-1, HNF-3␣, or HFH-8
CAT assays of cell extracts demonstrated that the SP-C promoter was transactivated by coexpression of TTF-1 but was not transactivated by HNF-3␣ or HFH-8 [24] (Fig. 1A)
Summary
Pulmonary surfactant is a mixture of phospholipids and proteins, which functions to reduce surface tension at the air/ liquid interface preventing alveolar collapse during respiration [1]. SP-C transcriptional activity is detected in primordial respiratory epithelial cells at the earliest stages of lung development (fetal day 11 in the mouse), is restricted to the distal most portions of the developing fetal lung, and is maintained in alveolar Type II epithelial cells in the postnatal lung [6, 7]. As a first step in identifying the cis-active regulatory elements that confer the Type II cell-specific, developmental, and humoral regulation of SP-C gene expression, we have cloned and sequenced both the human and the murine SP-C genes and their flanking sequences [10, 11]. Two recent studies demonstrate that TTF-1 activates the transcription of surfactant proteins A and B [18, 19] We extend those observations to SP-C and identify specific cisacting sequences involved in transactivation of the SP-C promoter by TTF-1
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