CANP--RESPONSE TO TOXIC INSULT AND ROLE IN HOMEOSTASIS

钙激活的中性蛋白水解酶(CANP)被激活以响应 增加细胞内钙离子，并被证明下调蛋白质 激酶C(PKC)和降解即刻早期基因产物Fos和Jun， 提示CANP在维持性脑出血中起重要作用 细胞动态平衡。这项研究的总体目标是 CANP的生化和分子机制特征(S) 对有毒侮辱做出回应，并对其进行监管。初步 数据显示，CCI4或叔丁基对苯二酚(TBHQ)对大鼠 结果肝脏mRNA水平迅速(0.5-2小时)增加(0.20倍) 即早期基因c-fos和c-jun。相应地，CANP mRNA 治疗后6至24小时，血药浓度增加5至10倍。FOS和JUN 蛋白质形成一个转录因子复合体AP-1，它与 几个活跃的基因的5-端的特定位点(AP-1) 分化和恶变。CANP基因有一个AP-1- 在它们的5‘区域有相似的位置和初步的凝胶延迟(移位) 分析，结合竞争对手和超级变换分析， 证实了在治疗后形成了特定的AP-1复合体 使用CCI4。因此，本研究的假设是Fos、Jun和 化学伤害后mCANP和mCANP基因降解PKC 表达受Fos/Jun AP-1转录因子复合体调控， 其中一些逃逸或不易降解，因此 能够进入细胞核，激活mCANP基因，并补充 MCANP的细胞储藏。以这种方式，CANP函数返回到 通过去除Fos/Jun AP-1实现化学损伤后的细胞内稳态 转录因子复合体，其中一些逃逸或对其不敏感 降解，因此能够进入原子核，激活 MCANP基因，并补充mCANP的细胞储存。以这种方式 CANP的作用是使细胞在化学物质作用后恢复内环境平衡 通过去除Fos/Jun和降低磷酸化活性来侮辱 与PKC同工酶相关。因此，这一行动的具体目标是 研究内容有：(1)考察已知化学物质(CCI4、TBHQ)的影响 刺激即刻早期(c-fos、c-jun、c-myc)基因表达 MCANP基因在体内肝组织和体外培养的HepG2细胞中的表达； (2)确认CCI4或TBHQ刺激Fos/Jun的产生 异源二聚体AP-1转录因子复合体在肝组织和HepG2中的表达 细胞和评估其他转录因子结合的作用 存在于mCANP基因5‘侧翼区的位点； 检测CANP是否降解AP-1转录因子复合体；(4) 探讨PKC同工酶在MCANP激活中的作用 体内CCI4或TBHQ处理或CCI4、TBHQ或钙离子载体处理 培养的HepG2细胞和(5)检测即时 早期基因和mCANP基因的表达依赖于氧化 代谢(CCI4)或氧化还原循环(TBHQ)活性。这项研究将 提供有关控制激活的因素的宝贵信息 在调控中起关键作用的蛋白水解酶CANP的表达 C-Fos和c-Jun水平和信号转导系统对 有毒的侮辱。

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.