This grant was originally developed to assess the metabolic state of chondrocytes at different stages of differentiation and the possibility that ATP dynamics could be a major regulator of mineralization. Along the way, a major change in emphasis has occurred, albeit using many of the same techniques that were originally employed. The proposed studies will now focus on the cells within the hypertrophic chondrocyte pathway that undergo programmed cell death (apoptosis). Once the genetic signal is given (possibly expression from the BCL-2 gene located on the outer membrane of the mitochondrial membrane), a series of metabolic events occur which culminate in the formation of matrix vesicles and cell death. It is the steps distal to the initial signaling event that are examined in great detail and for which important preliminary data is presented. Two models for studying these events are utilized. The first is an intact chick tibial growth plate in which he has shown evidence for DNA fragmentation and FACTS evidence for apoptosis. The second is a cell cultured system, derived from the cephalic region of chick sterna, that is induced to undergo hypertrophic changes with retinoic acid. The model system appears to generate membrane vesicles with morphologic and biochemical hallmarks similar to those extracted from chick cartilage. In the in vitro system, the metabolic events have been shown to consist of a decrease in mitochondrial activity and a fall in intracellular levels of glutathione and cysteine. Three enzymatic systems appear to play an important role in this programmed event. These include activation of the interleukin-1 beta converting enzyme (ICE) which is a mammalian homologue to a nematode cell death gene (CED-3). This enzymatic activity is capable of cleaving poly-[ADPribose]polymerase (PARP), which is important for DNA repair. Loss of its activity may mediate inter nucleosomal DNA cleavage. The third enzyme is a membrane bound transglutaminase which may play a role in maintaining the cell membrane and particularly the matrix vesicles as the cells undergo their programmed cell death. In this competitive renewal, four specific aims will look at the events leading to programmed cell death and the generation of matrix vesicles.
The first aim will examine the intracellular levels of NADH, NAD, glutathione, free oxygen radicals, DNA fragmentation, transglutaminase in intact tibial tissue from the chick. A variety of immunological and fluorescent approaches are used to make these sophisticated measurements. In the second aim, the same type of measurements are applied to the chick culture system, but in this case, direct measurement for many of these metabolic compounds will be made. FACS analysis of the cells in culture will be used to distinguish the proportion of cells undergoing programmed cell death.
The third aim examines the correlation between the metabolic events in the cells and subsequent cleavage of the PARP enzyme, presumably as a consequence to activation of ICE-3 cysteine protease. Direct measurement of the cleavage products is determined by an activity - Western blot technique. The consequences of modulating the activity of the ICE-3 cysteine protease on subsequent events will be explored by the use of various inhibitors of the enzyme. The cDNA for the enzyme will be cloned so that it can be used for RNA analysis. The activity of transglutaminase will be assessed, and the consequences of either inhibition or overexpression will be determined. In the final aim, the formation of matrix particles in their cultured system will be compared to authentic vesicles. The analysis will consist of FACTS flow cytometry based on alkaline phosphatase or annexin content and phospholipid packing.
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