Signaling Mechanism of Shear Stress-regulated Thrombomodulin and the Regulation of Its Protein Stability in Endothelial Cells

• Main Authors: Chung-Yu Hsieh, 謝宗佑
• Other Authors: 謝學真
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/23384278822760349869

碩士 === 國立臺灣大學 === 化學工程學研究所 === 95 === During atherogenesis, inflammation and coagulation play an important role. Thrombomodulin (TM), in vascular endothelial cells, has anti-coagulation and anti-inflammation properties, it can also regulate cell migration and angiogenesis. Transcription factor, KLF2, has been shown to participate in the regulation of the expression of eNOS, TM and other anti-inflammation genes that can regulate the physiological function of endothelial cells (ECs). ECs are constantly under the influence of flow-induced shear stress. Therefore, shear stress is an important regulator of EC functions. In this study, we focused on the signaling pathway of TM and KLF2 under the shear stress stimulation and the regulatory mechanism of TM protein stability. Shear stress elevated TM promoter activity in ECs after 6 hours under both high (25 dyn/cm2) and low (5 dyn/cm2) shear stress conditions, but high shear had more eminent induction (12 folds vs. unsheared control) than low shear (6 folds vs. control). The mRNA level of TM was increased more than 2 folds in comparison with unsheared control and so did the mRNA level of KLF2 (more than 100-fold increase). As for TM protein level, there was a significant difference between high shear (60% increase) and low shear (23% increase) conditions, but no difference in KLF2 protein level was observed under different shear conditions. These results suggest that shear stress increases the protein stability of TM and the effect is proportional to the magnitude of shear stress. We further explored the regulatory mechanism of TM stability. To simulate the condition of shear-induced NO release, ECs were treated with NOC18 (an NO donor), and we found that TM protein level increased 0.8 folds after 3 hours but no significant increase was observed after 5 hours. It is likely that NO may not be a major factor that regulates TM stability or there are too many down-stream signal molecules activated by NO to observe significant effect of NO on TM. Subsequently, ECs were treated with NAC to mimic the effect of antioxidants which were present in ECs exposed to high shear stress. The results showed that the TM protein level was almost totally down-regulated after 5 hours of shear treatment, but the soluble form of TM in the medium was increased. It suggests that NAC (or other antioxidants) may activate specific protease(s) to cleave TM into the solube form that can promote cell migration or angiogenesis. To explore the effect of phosphorylation/dephosphorylation on the stability of TM, tyrosine kinase inhibitor (PP2) and PTP inhibitor (Na3VO4) were used. We found that TM protein level was increased about 2.2 folds in the presence of PP2, and decreased to 0.2 fold in the presence of Na3VO4 in ECs stimulated by VEGF for 5 hours. Based on these findings, we proposed that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. In order to verify this assumption, Y534D (superactive) and Y534A (dominant negative) TM mutants were transfected into HUVECs. We found that Y534D was more unstable in the presence of Na3VO4 but Y534A was not affected by PP2 or Na3VO4. Therefore the phosphorylation of Y534 may destabilize TM protein. Among various PTPs, we found that PTEN was involved in the regulatory mechanism of TM. PTEN silencing down-regulated TM protein level but had no influence on Y534A mutant, and this mechanism was found to be independent of PI3K/Akt pathway. In summary, our data suggest that both high and low shear stresses up-regulate the TM promoter activity and KLF2 mRNA, but only high shear elevates the level of TM mRNA. Besides, shear stress dose-dependently increases the TM protein stability. Further studies indicate that NO may not play a significant role in the regulatory mechanism. As for NAC, it can increase the soluble form of TM, and this phenomenon is due to the cleavage of TM by some unknown protease(s). The key point in the regulation of TM stability seems to be the phosphorylation of Y534. Our results suggest that the phosphorylation of Y534, may be mediated by Src kinase family, decreases the stability of TM, but the activation of PTP such as PTEN enhances the TM protein stability.