Surfactant is a mixture of lipids and proteins on the alveolar surface that plays an important role for gas exchange. Surfactant protein B (SP-B), an important protein component of surfactant, is expressed in type II cells and Clara cells in the lung. SP-B expression is mainly controlled by thyroid transcription factor-1 (TTF-1) and hepatocyte nuclear factor-3. Previously, we uncovered a novel polypeptide, BR22, a TTF-1 associated protein (m. wt. 26 kD) (TAP26), which bound to human SP-B promoter sequence at the proximal region containing TTF-1 binding sites and interacted with TTF-1 to activate SF-B promoter. This protein also exhibits a direct protein-protein contact with TTF-1 in vivo and in vitro. In this proposal, we will demonstrate that TAP26 is a co-factor of TTF-1 to regulate the SP-B promoter activity in the lung. In order to understand the mechanism by which TAP26 modulates the promoter activity, the following approaches will be performed: 1) Characterize the contact domain modules of TAP26 and TTF-1 for a better understanding of the detailed protein complex structure. Deletion and mutagenesis analyses will be employed for the characterization. 2) Determine the mechanism by which TAP26 acts with TTF-1 to modulate the SP-B promoter activity. TAP26 in H441 cells will be sequestered to demonstrate its influence on the promoter activity. In addition, the effect of dexamethasone, cAMP, and phorbol ester on SP-B promoter activity will be inspected in the absence of TAP26 to determine if the regulation is mediated through TAP26. 3) Establish the relationship between TAP26 and SPB expression. The amounts of TAP26 message and protein level will be evaluated under conditions in which the SF-B expression is altered in vivo. 4) The DNA-binding activity of TAP26 and TAP-26/TTF-1 complex on the SP-B promoter will be inspected. The association of this protein complex and SP-B promoter in vivo will be examined by chromatin cross-linking and immunoprecipitation (CHIP) analysis. 5) Inspect cellular locations of TAP26 and TTF-1 in lung cells expressing SF-B. Two-color or triple-color staining of endogenous proteins in H441 cells or the lung sections will be performed to detect their expression in SP-B producing cells.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065325-03
Application #
6718368
Study Section
Lung Biology and Pathology Study Section (LBPA)
Program Officer
Denholm, Elizabeth M
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
3
Fiscal Year
2004
Total Cost
$234,000
Indirect Cost
Name
University of Texas Sw Medical Center Dallas
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
800771545
City
Dallas
State
TX
Country
United States
Zip Code
75390
Guo, Yuhong; Yang, Meng-Chun; Weissler, Jonathan C et al. (2007) PLAGL2 translocation and SP-C promoter activity--a cellular response of lung cells to hypoxia. Biochem Biophys Res Commun 360:659-65