Regulation of Thrombomodulin in Endothelial Cells by Stretch
碩士 === 國立臺灣大學 === 化學工程學研究所 === 96 === 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. Vascular Endothelial...
Main Authors: | , |
---|---|
Other Authors: | |
Format: | Others |
Language: | zh-TW |
Published: |
2008
|
Online Access: | http://ndltd.ncl.edu.tw/handle/23806202951025796698 |
id |
ndltd-TW-096NTU05063089 |
---|---|
record_format |
oai_dc |
spelling |
ndltd-TW-096NTU050630892015-11-25T04:04:37Z http://ndltd.ncl.edu.tw/handle/23806202951025796698 Regulation of Thrombomodulin in Endothelial Cells by Stretch 拉伸應力對內皮細胞中凝血酶調節素之調控 Hung-Hsiun Chang 張弘勳 碩士 國立臺灣大學 化學工程學研究所 96 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. Vascular Endothelial cells (ECs) are exposed to stretch caused by the blood pressure. Therefore, stretch is an important regulator of EC functions. In this study, human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were utilized to investigate the signaling pathway of stretch-induced TM and the regulatory mechanism of TM protein stability. When ECs were exposed to stretch (16% strain) for 1 hour, the mRNA level of TM was increased more than 3.2 folds in comparison with static control, but decreased to the same level as that of static control after 6 hours. These results suggest that stretch transiently induces the mRNA level of TM. As for TM protein level, stretch also induced the protein level of TM up to 2.2 folds, and this effect sustained at least for 6 hours. On the other hand, Double immunofluorescence staining for TM and actin showed that the colocalization between TM and actin was induced by stretch, which led to the special distribution of TM in ECs. We further transfected ECs with TM plasmid containing C-terminal FLAG tag to explore the regulatory mechanism of TM stability. To simulate the condition of stretch-induced nitric oxide (NO) release, ECs were treated with short term and long term NO donor (SNAP and NOC18, respectively), and we found that only short term NO donor protected TM protein from degradation.. To explore the effect of phosphorylation/dephosphorylation on the stability of TM, VEGF and protein tyrosine phosphatase (PTP) inhibitor (Na3VO4) were used. We found that inhibition of PTP activity by Na3VO4 increased the degradation of TM, whereas the use of VEGF to stimulate kinase activity had no effect on the TM stability. These results suggest that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. To verify this assumption, Y534A mutant of TM plasmid was transfected into BAECs. We found that in cells transfected with Y534A there was no degradation fragment of TM. Therefore, it is likely that the C-terminal Y534 residue of TM is constantly dephosphorylated by PTP and the dephosphorylation of Y534 residue may stabilize TM protein. Futhermore, our data showed that ubiquitination may play a role in the degradation of TM. Then we used immunoprecipitation to find out that PTEN was associated with TM under normal condition. NO and H2O2 had slight effect on the association between TM and PTEN. In summary, our data suggest that stretch transiently up-regulates the mRNA level of TM. Stretch also upregulates the protein level of TM, and the effect sustains at least for 6 hours. Besides, stretch increases the colocalization between TM and actin and leads to the special distribution of TM in ECs. Further studies indicate that NO may protect TM from degradation. PTEN may dephosphorylate TM Y534 residue and thus stabilize TM protein. On the contrary, the increase in Y534 phosphorylation may lead to the degradation of TM through ubiquitin proteasome system. Hsyue-Jen Hsieh 謝學真 2008 學位論文 ; thesis 99 zh-TW |
collection |
NDLTD |
language |
zh-TW |
format |
Others
|
sources |
NDLTD |
description |
碩士 === 國立臺灣大學 === 化學工程學研究所 === 96 === 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. Vascular Endothelial cells (ECs) are exposed to stretch caused by the blood pressure. Therefore, stretch is an important regulator of EC functions. In this study, human umbilical vein endothelial cells (HUVECs) and bovine aortic endothelial cells (BAECs) were utilized to investigate the signaling pathway of stretch-induced TM and the regulatory mechanism of TM protein stability.
When ECs were exposed to stretch (16% strain) for 1 hour, the mRNA level of TM was increased more than 3.2 folds in comparison with static control, but decreased to the same level as that of static control after 6 hours. These results suggest that stretch transiently induces the mRNA level of TM. As for TM protein level, stretch also induced the protein level of TM up to 2.2 folds, and this effect sustained at least for 6 hours. On the other hand, Double immunofluorescence staining for TM and actin showed that the colocalization between TM and actin was induced by stretch, which led to the special distribution of TM in ECs.
We further transfected ECs with TM plasmid containing C-terminal FLAG tag to explore the regulatory mechanism of TM stability. To simulate the condition of stretch-induced nitric oxide (NO) release, ECs were treated with short term and long term NO donor (SNAP and NOC18, respectively), and we found that only short term NO donor protected TM protein from degradation..
To explore the effect of phosphorylation/dephosphorylation on the stability of TM, VEGF and protein tyrosine phosphatase (PTP) inhibitor (Na3VO4) were used. We found that inhibition of PTP activity by Na3VO4 increased the degradation of TM, whereas the use of VEGF to stimulate kinase activity had no effect on the TM stability. These results suggest that the phosphorylation of C-terminal tyrosine residue (Y534) of TM may regulate the TM protein stability. To verify this assumption, Y534A mutant of TM plasmid was transfected into BAECs. We found that in cells transfected with Y534A there was no degradation fragment of TM. Therefore, it is likely that the C-terminal Y534 residue of TM is constantly dephosphorylated by PTP and the dephosphorylation of Y534 residue may stabilize TM protein. Futhermore, our data showed that ubiquitination may play a role in the degradation of TM.
Then we used immunoprecipitation to find out that PTEN was associated with TM under normal condition. NO and H2O2 had slight effect on the association between TM and PTEN.
In summary, our data suggest that stretch transiently up-regulates the mRNA level of TM. Stretch also upregulates the protein level of TM, and the effect sustains at least for 6 hours. Besides, stretch increases the colocalization between TM and actin and leads to the special distribution of TM in ECs. Further studies indicate that NO may protect TM from degradation. PTEN may dephosphorylate TM Y534 residue and thus stabilize TM protein. On the contrary, the increase in Y534 phosphorylation may lead to the degradation of TM through ubiquitin proteasome system.
|
author2 |
Hsyue-Jen Hsieh |
author_facet |
Hsyue-Jen Hsieh Hung-Hsiun Chang 張弘勳 |
author |
Hung-Hsiun Chang 張弘勳 |
spellingShingle |
Hung-Hsiun Chang 張弘勳 Regulation of Thrombomodulin in Endothelial Cells by Stretch |
author_sort |
Hung-Hsiun Chang |
title |
Regulation of Thrombomodulin in Endothelial Cells by Stretch |
title_short |
Regulation of Thrombomodulin in Endothelial Cells by Stretch |
title_full |
Regulation of Thrombomodulin in Endothelial Cells by Stretch |
title_fullStr |
Regulation of Thrombomodulin in Endothelial Cells by Stretch |
title_full_unstemmed |
Regulation of Thrombomodulin in Endothelial Cells by Stretch |
title_sort |
regulation of thrombomodulin in endothelial cells by stretch |
publishDate |
2008 |
url |
http://ndltd.ncl.edu.tw/handle/23806202951025796698 |
work_keys_str_mv |
AT hunghsiunchang regulationofthrombomodulininendothelialcellsbystretch AT zhānghóngxūn regulationofthrombomodulininendothelialcellsbystretch AT hunghsiunchang lāshēnyīnglìduìnèipíxìbāozhōngníngxuèméidiàojiésùzhīdiàokòng AT zhānghóngxūn lāshēnyīnglìduìnèipíxìbāozhōngníngxuèméidiàojiésùzhīdiàokòng |
_version_ |
1718135152622698496 |