The Ca2+ activated neutral protease (CANP) is activated in response to increased intracellular Ca2+ and has been shown to down regulated protein kinase C (PKC) and degrade the immediate early gene products Fos and Jun, suggesting that CANP plays an important role in the maintenance of cellular homeostasis. The overall objective of this research is to characterize the biochemical and molecular mechanism(s) by which CANP responds to, and is regulated in response to, toxic insult. Preliminary data show that CCI4 or t-butylhdroquinone (tBHQ) administration to rats results in a rapid (0.5-2 hr) increase (.20-fold) in hepatic mRNA levels of the immediate early genes c-fos and c-jun. Correspondingly, CANP mRNA levels increase 5 to 10-fold from 6 to 24 hr post-treatment. Fos and Jun proteins form a transcription factor complex, AP-1, which binds to specific sites (AP-1) in the 5- end of several genes active in differentiation and malignant transformation. CANP genes have an AP-1- like site in their 5' region and preliminary gel retardation (shift) assays, in conjunction with competitor and super-shift assays, demonstrated the formation of a specific AP-1 complex following treatment with CCI4. Thus, the hypothesis of this research is that Fos, Jun and PKC are degraded by mCANP following chemical insult and that mCANP gene expression is regulated by the Fos/Jun AP-1 transcription factor complex, some of which escapes or is refractory to degradation, and is therefore able to enter the nucleus, activate the mCANP gene and replenish the cellular stores of mCANP. In this manner CANP functions to return to the cell homeostasis following chemical insult by removing Fos/Jun AP-1 transcription factor complex, some of which escapes or is refractory to degradation, and is therefore able to enter the nucleus, activate the mCANP gene and replenish the cellular stores of mCANP. In this manner CANP functions to return the cell to homeostasis following chemical insult by removing Fos/Jun, and decreasing phosphorylation activity associated with PKC isozymes. Thus, the specific objectives of this research are: (1) to examine the effects of chemical (CCI4, tBHQ) known to stimulate immediate early (c-fos, c-jun, c-myc) gene expression on mCANP gene expression in hepatic tissue in vivo and HepG2 cells in vitro; (2) to confirm that CCI4 or tBHQ stimulate production of the Fos/Jun heterodimer AP-1 transcription factor complex in hepatic tissue and HepG2 cells and to evaluate the role of other transcription factor binding sites present in the 5' flanking region of the mCANP gene; (3) t o examine whether CANP degrades the AP-1 transcription factor complex; (4) to investigate the role of PKC isozymes in mCANP activation following CCI4 or tBHQ treatment in vivo or CCI4, tBHQ or Ca2+ ionophore treatment of cultured HepG2 cells and (5) to examine the extent to which immediate early gene and mCANP gene expression are dependent on oxidative metabolism (CCI4) or redox-cycling (tBHQ) activity. This research will provide valuable information on factors which control the activation and expression of CANP, a protease which plays a critical role in modulating c-Fos and c-Jun levels and signal transduction systems in response to toxic insult.
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