Kringle 1-5 of Plasminogen Downregulated Thrombomodulin Through Proteosome-Lysosome Degradation Pathway

• Main Authors: Chia-Fong Cho, 卓家楓
• Other Authors: Guey-Yueh Shi
• Format: Others
• Language: zh-TW
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/95587374389221918345

碩士 === 國立成功大學 === 生物化學研究所 === 93 === 　Angiogenesis plays an important role in many physiological and pathological processes, including embryonic vascular system development, wound healing, and reproductive cycle in adult female and tumorigenesis. Many reports indicate that cancer cells can secrete angiogenic factors to stimulate the growth of new blood vessels during the formation of tumor. Angiostatin, a proteolytic fragment of plasminogen, is a potent antagonist of angiogenesis and inhibitor of endothelial cell proliferation and migration. Thrombomodulin (TM) is a glycoprotein receptor and cofactor for thrombin’s activity in a physiologically important natural anticoagulant system. 　In this study, we used Pichia pastoris expression system to prepare kringle 1-5 (K15) protein, a kind of angiostatin. First, we found that K15 downregulated TM expression in bovine aortic endothelial cells (BAECs) at 15 hours after treatment. The effect of K15 was endothelial cells-specific since other cell lines, such as A549 cells, HaCat cells, and A2058 cells transfected with full-length TM, had no such effect. To determine the mechanism of TM downregulation by K15, the protease inhibitors were used to inhibit this effect. The result showed that the downregulation of TM by K15 was caused by neither matrix metalloproteinases (MMPs) nor serine proteases. However, pretreatment with the internalization, proteasome or lysosome inhibitors, the expression of TM was not declined in BAECs by treatment with K15. Furthermore, the result of RT-PCR showed that the downregulation of TM expression was not due to decrease RNA expression. Additionally, pretreatment of protein kinase A (PKA) inhibitors, KT5720 and H89, could inhibit the TM downregulation by K15. 　Based on these results, we suggested that K15 induced downregulation of TM expression through proteasome-lysosome degradation pathway, not by induction of protease activity or downregulation of its transcription level. Moreover PKA was shown to be involved in this pathway.