Past studies of the applicant have established that oxidative metabolism predominates in chondrocytes in the zones of proliferation and maturation with sharp reductions of oxidative metabolism in the hypertrophic cell zone. These studies have been published for over a decade and involve a combination of microbiochemical, novel biophysical, and cell biological technology. The central question involved in this application is the precise role of energy metabolism in chondrocyte cell differentiation.
In Specific Aim 1, studies will be conducted in a mammalian system that are similar to prior studies in the chick system. In mammalian systems, more blood vessels are found in cartilage and, thus, the oxygen tension in mammalian cartilage may be quite different than in chick cartilage. The goal of this phase of the application is to determine the amount of oxygen available indifferent regions of mammalian cartilage. 3H-MISO, a drug that binds to cells in the presence of low oxygen conditions, will be used to determine oxygen levels at the cellular level and a fluorescent dye will be used to localize blood vessels in sections. In a parallel study, chondrocytes will be grown in vitro and the effects of oxygen tension on gene expression will be monitored.
In Specific Aim 2, chondrocytes will be induced to differentiate by treatment with retinoic acid and grown under different oxygen tensions. The effects of various oxygen tensions on mineralization will be monitored.
In Specific Aim 3, the secretion of phosphagens (ATP, creatine phosphate, and other phosphorylated compounds) will be studied during chondrocyte cell differentiation. The major phosphorylated compounds secreted by cells will be determined by labeling cells with 32P and identifying metabolically labeled compounds by HPLC. The rate of secretion of these compounds and whether they are secreted in matrix vesicles will be determined. Finally, it will be determined whether phosphagen secretion is influenced by oxygen tension or retinoic acid treatment.
In Specific Aim 4, the hypothesis that oxygen tension alters transcription factor activity will be tested. Cells will be transcription factor activity will be tested. Cells will be alkaline phosphatase, and types II and X collagens. The effects of oxygen tension of promoter activity in the systems above will be followed by CAT assays. Gel retardation assays will be carried out to determine whether oxygen tension changes transcription factor binding to either the AP-1 or HIE sites. Since the erythropoietin gene has been shown to be under the control of oxygen tension via an HIE element in its promoter, an alkaline phosphatase-CAT construct will be transfected into a hepatoma cell line to determine if the alkaline phosphatase promoter also responds to oxygen tension changes.
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