Resiliency in lung and arteries is provided by an extracellular matrix that is rich in elastic fibers. Elastin, the principal component of elastic fibers, is the product of crosslinked tropoelastin monomers. The production of elastin is unique among connective tissue proteins in that expression is limited to a brief period of development. By maturity, assembly of elastic fibers is complete, and synthesis of tropoelastin has ceased. However, certain diseases, such as pulmonary hypertension and emphysema, are associated with an abnormal or continued accumulation of elastin. To understand the mechanism of such aberrant production, the normal regulation of tropoelastin expression needs to be delineated; however, only minimal information is currently available on the molecular control of elastogenesis. The preliminary data for this grant indicate that downregulation of tropoelastin expression is primarily controlled posttranscriptionally, and the work proposed here focuses on a detailed, molecular characterization of this unique mechanism. This grant will test the hypotheses that the normal cessation of elastogenesis is controlled by an accelerated decay of tropoelastin mRNA and that specific sequences in the transcript are involved in this regulatory mechanism. For these studies, the generality of this mechanism will be determined by assessing the regulation of tropoelastin production under different conditions, such as hormone treatment, time in culture and age of the cell donor, and with models of in vivo elastin production during development. The effect on tropoelastin transcription, which is expected to be minimal, will be assessed by nuclear run-off assay, by reverse transcription/polymerase chain amplification of tropoelastin pre-mRNA and by transfection with tropoelastin promoter-plasmid constructs. Since transcript turn over can be affected by diverse pathways, a detailed characterization of the accelerated degradation of tropoelastin mRNA will be performed by different methods. Specific enzymatic decay of tropoelastin mRNA will be examined to determine if such mechanisms are activated in response to inhibitors of elastin production. A nuclease protection assay will be used to determine if the status of polyadenylation correlates with the turn over of tropoelastin mRNA, and regulatory sequences in the mRNA will be identified by monitoring the response of reporter constructs containing defined sequences coding for tropoelastin mRNA to downregulation of tropoelastin expression. The interaction of these and other sequences with cellular factors will be determined by gel retardation assay using synthetic fragments of tropoelastin mRNA. Information from these studies will provide new and valuable information on the control of elastogenesis and will lead to the eventual characterization of specific cellular factors that are involved in the regulation of elastin production in development and disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
7R01HL048762-08
Application #
6056245
Study Section
Lung Biology and Pathology Study Section (LBPA)
Project Start
1992-09-30
Project End
2002-08-31
Budget Start
1999-09-01
Budget End
2000-08-31
Support Year
8
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Washington University
Department
Pediatrics
Type
Schools of Medicine
DUNS #
062761671
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
Leidenfrost, Jeremy E; Khan, M Faisal; Boc, Kenneth P et al. (2003) A model of primary atherosclerosis and post-angioplasty restenosis in mice. Am J Pathol 163:773-8
Zhang, M; Pierce, R A; Wachi, H et al. (1999) An open reading frame element mediates posttranscriptional regulation of tropoelastin and responsiveness to transforming growth factor beta1. Mol Cell Biol 19:7314-26
Doyle, G A; Saarialho-Kere, U K; Parks, W C (1997) Distinct mechanisms regulate TIMP-1 expression at different stages of phorbol ester-mediated differentiation of U937 cells. Biochemistry 36:2492-500
Doyle, G A; Pierce, R A; Parks, W C (1997) Transcriptional induction of collagenase-1 in differentiated monocyte-like (U937) cells is regulated by AP-1 and an upstream C/EBP-beta site. J Biol Chem 272:11840-9
Parks, W C (1997) Posttranscriptional regulation of lung elastin production. Am J Respir Cell Mol Biol 17:1-2
Thompson, R W; Parks, W C (1996) Role of matrix metalloproteinases in abdominal aortic aneurysms. Ann N Y Acad Sci 800:157-74
Halpert, I; Sires, U I; Roby, J D et al. (1996) Matrilysin is expressed by lipid-laden macrophages at sites of potential rupture in atherosclerotic lesions and localizes to areas of versican deposition, a proteoglycan substrate for the enzyme. Proc Natl Acad Sci U S A 93:9748-53
Mariani, T J; Roby, J D; Mecham, R P et al. (1996) Localization of type I procollagen gene expression in silica-induced granulomatous lung disease and implication of transforming growth factor-beta as a mediator of fibrosis. Am J Pathol 148:151-64
Pierce, R A; Mariencheck, W I; Sandefur, S et al. (1995) Glucocorticoids upregulate tropoelastin expression during late stages of fetal lung development. Am J Physiol 268:L491-500
Swee, M H; Parks, W C; Pierce, R A (1995) Developmental regulation of elastin production. Expression of tropoelastin pre-mRNA persists after down-regulation of steady-state mRNA levels. J Biol Chem 270:14899-906

Showing the most recent 10 out of 20 publications