CANP--RESPONSE TO TOXIC INSULT AND ROLE IN HOMEOSTASIS

Ca2+激活的中性蛋白酶（CANP）响应于 增加细胞内Ca2+，并已显示下调蛋白质 激酶C（PKC）并降解立即早期基因产物Fos和Jun， 这表明CANP在维持 细胞内稳态 本研究的总体目标是 表征CANP的生化和分子机制 对有毒物质有反应，并根据有毒物质的反应进行调节。 初步 数据显示，对大鼠施用CCl4或叔丁基氢醌（tBHQ 导致肝脏mRNA水平快速（0.5 - 2小时）增加（0.20倍） 即早基因c-fos和c-jun的表达，相应地， 治疗后6至24小时，水平增加5至10倍。 fos和jun 蛋白质形成转录因子复合物AP-1，AP-1结合到 几个活跃基因5端的特定位点（AP-1） 分化和恶性转化。 CANP基因有一个AP-1- 在其5 '区域中的类似位点和初步凝胶阻滞（移位） 分析，结合竞争者和超偏移分析， 证明了在治疗后形成了特异性AP-1复合物 CCI4的 因此，本研究的假设是，Fos，Jun和 PKC在化学损伤后被mCANP降解，并且mCANP基因 表达受Fos/Jun AP-1转录因子复合物调节， 其中一些会逃逸或难以降解，因此 能够进入细胞核，激活mCANP基因，并补充 mCANP的蜂窝商店。 以这种方式，CANP功能返回到 通过去除Fos/Jun AP-1的化学损伤后的细胞稳态 转录因子复合物，其中一些逃逸或难治性， 降解，因此能够进入细胞核，激活细胞核。 mCANP基因和补充mCANP的细胞储存。 以这种方式 CANP的功能是使细胞在化学作用后恢复稳态。 通过去除Fos/Jun和降低磷酸化活性进行损伤 与PKC同工酶有关。 因此，本报告的具体目标 研究内容有：（1）考察已知化学品（CCl_4、tBHQ）对土壤中有机磷的影响 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HepG2细胞中的表达; (2)为了证实CCl4或tBHQ刺激Fos/Jun的产生， 肝组织和HepG2中异源二聚体AP-1转录因子复合物 并评估其他转录因子结合的作用 存在于mCANP基因的5 '侧翼区的位点;（3）存在于mCANP基因的5'侧翼区的位点; 检查CANP是否降解AP-1转录因子复合物;（4） 研究PKC同工酶在mCANP激活中的作用， 体内CCl4或tBHQ处理或CCl4、tBHQ或Ca2+离子载体处理 培养的HepG2细胞和（5）检查在何种程度上，立即 早期基因和mCANP基因的表达依赖于氧化 代谢（CCl 4）或氧化还原循环（tBHQ）活性。 这项研究将 提供关于控制激活的因素的有价值的信息， CANP是一种蛋白酶，它在调节细胞凋亡中起关键作用。 c-Fos和c-Jun水平和信号转导系统响应 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.