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.
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