The overall goal of this proposal is to understand the role of specific rat retinoid metabolizing enzymes (especially retinyl ester hydrolases) in the regulation of retinoid metabolism in hepatic stellate cells. Hepatic stellate cells are the main reservoir of Vitamin A in the liver where it is stored mainly as retinyl palmitate in highly visible intracellular lipid droplets. When animals are exposed to hepatotoxins or humans chronically abuse alcohol, the stellate cells become activated and transform into myofibroblast-like cells. These transformed cells are the sites of collagen and extracellular matrix protein formation in alcohol-induced hepatic fibrosis. One of the earliest events in stellate cell activation is the hydrolysis of retinyl esters and the depletion of the intracellular lipid droplets. The most likely retinyl ester hydrolase candidates are the members of the families of broad substrate specificity lipases and carboxylesterases. The specific retinyl ester hydrolases expressed in hepatic stellate cells and the mechanisms for regulation of their activity during stellate cell activation is not known. In preliminary data, we show that the five most common rat liver carboxylesterases with retinyl palmitate activity are not highly expressed in stellate cells. However, the hormone-sensitive lipase gene is expressed in rat hepatic stellate cells and it has retinyl palmitate hydrolase activity in vitro. The goals of the grant are to identify the specific retinyl ester hydrolases (lipases and carboxylesterases) that are expressed in isolated rat liver cells (stellate cells, Kupffer cells, hepatocytes) by real-time, quantitative PCR of RNA and immunofluorescence microscopy of stellate cells with enzyme-specific antibodies. The expression of retinyl ester hydrolases and retinol dehydrogenases will be correlated with retinoid autofluorescence in lipid droplets and expression of cell-specific markers in cultured rat stellate cells as they undergo activation to myofibroblast-like cells. When feasible, we will measure retinyl palmitate hydrolase activity and perform protein gel electrophoresis of cellular extracts with esterase activity staining. We will examine the kinetics of purified rat stellate cell retinyl ester hydrolases and examine the effects of enzyme activators and inhibitors. Inhibitors of retinyl ester hydrolysis in stellate cells may be an effective therapeutic strategy for arresting stellate cell activation early in hepatic fibrosis. ? ? ?
| Mello, Tommaso; Nakatsuka, Alice; Fears, Sharry et al. (2008) Expression of carboxylesterase and lipase genes in rat liver cell-types. Biochem Biophys Res Commun 374:460-4 |
