(taken directly from application) It is now well established that acetaldehyde, an intermediate in alcohol metabolism, may be responsible for inducing the biosynthesis of type I collagen, the major component of fibrotic liver. Hepatic stellate cells, when transformed to myofibroblast-like cells, are the principal source of type I collagen; and its production leads to fibrosis and ultimately cirrhosis. Recently, acetaldehyde was found to increase collagen production in cultured rat hepatic myofibroblast-like cells, and this appears to occur at the level of gene transcription. The purpose of this proposal is to explore further the mechanisms whereby acetaldehyde increases collagen production by cultured rat myofibroblast-like cells. Initial studies will assess whether acetaldehyde increases the rate of transcription of a(I) collagen by examining the effects of actinomycin D on mRNA production. Nuclear run-off experiments will be conducted to affirm nascent mRNA production since nuclear factor I (NF-I) is a key regulatory element in the a2(I) collagen promoter, transient transfection studies using deletion and substitution mutation constructs of the a2(I) collagen promoter will be performed to confirm the importance of NF-I in the gene's activation, and to determine whether additional upstream cis- regulatory elements are important as regulatory elements are essential to confer the effect of acetaldehyde since transforming growth factor beta one (TGFB-1) has been shown to increase production of type I collagen, studies to assess the potential modulation by this cytokine in conferring the effect of acetaldehyde will be conducted; including, studies to demonstrate that acetaldehyde can independently activate the a2(I) collagen promoter. Nuclear extracts will be prepared from cultured myofibroblast-like cells in order to perform DNA-protein binding assays, including DNase I Protection Analysis, and Electrophoretic Mobility Shift Assays (EMSAs). DNA-protein assay binding data, along with prior transfection analyses, will allow further characterization of additional trans-acting factors which are also important in mediating the transcriptional effect of acetaldehyde on the a2(I) collagen promoter. Potentially important transcription factors will also be identified by sequence analysis and computer searches. To establish the mechanism by which acetaldehyde influences collagen gene transcription, studies to demonstrate whether acetaldehyde directly forms nuclear protein adducts are planned. Recent evidence also suggests protein kinase C (PKC) is necessary for collagen gene activation by acetaldehyde. Experiments to confirm that PKC activity is required for the transcriptional effect of acetaldehyde will be performed; and, whether PKC activity is increased by the presence of acetaldehyde. Localization of PKC activity will be elucidated since PKC activation is associated with translocation from the cytosol in some cells.
| Anania, F A; Womack, L; Jiang, M et al. (2001) Aldehydes potentiate alpha(2)(I) collagen gene activity by JNK in hepatic stellate cells. Free Radic Biol Med 30:846-57 |
| Anania, F A; Womack, L; Potter, J J et al. (1999) Acetaldehyde enhances murine alpha2(I) collagen promoter activity by Ca2+-independent protein kinase C activation in cultured rat hepatic stellate cells. Alcohol Clin Exp Res 23:279-84 |
| Potter, J J; Womack, L; Mezey, E et al. (1998) Transdifferentiation of rat hepatic stellate cells results in leptin expression. Biochem Biophys Res Commun 244:178-82 |
