| Endothelial dysfunction is one of the early and decisive event in the development of the atherosclerosis. There is no doubt that the ox-LDL can cause the endothelial dysfunction via the reduced activity of eNOS and the reduced production of NO. LOX-1 was identified from endothelial cells as the main receptor specially recognizing and uptaking the ox-LDL, LOX-1 plays a key role in the endothelial dysfunction triggered by ox-LDL. Phosphorylation of endothelial nitric oxide synthase (eNOS) serine 1179/1177(bovine/human) by protein kinase Akt is a central mechanism of eNOS regulation. Recently, the report showed:ox-LDL can cause ER stress of the endotheliocyte, so cause the endothelial dysfunction. However, the molecular mechanism of the reduced activity of eNOS caused by ox-LDL is not clear, we donot know whether there is a connection with the ER stress caused by ox-LDL, so we need to do research to clarify these questions.To address these issues, we treated the human umbilical venous endothelial cells (HUVEC) with ox-LDL(200μg/ml). The ER stress reaction was estimated by the phosporylation of the PERK(one of the ER stress marker) and the expression level of the glucose-regulated protein 78 (GRP78, ER stress marker) via Western blotting method. The result showed, the ER stress reaction can be caused by ox-LDL just after 0.5hrs treatment(P<0.01, n=3). Further detection of the phosphorylation of eNOS revealed that ox-LDL induced a rapid reduced phosphorylation of eNOS Ser1177 site, and it is time-dependent. This time-dependent action of ER stress reaction was highly specific because neither eNOS Thr 497 phosphorylation nor the total eNOS level in HUVECs was changed. At the same time, we also detected the enzymatic activity of the eNOS, the result showed:Compared with the control group, the enzymatic activity of the eNOS could be markedly reduced by the treatment of ox-LDL(P<0.05, n=3), and it was time-dependent, this result is consistent with the dephosphorylation of the eNOS Ser1177 site caused by ox-LDL. We also found:the phosphorylation of the Thr308 and Ser473 sites of Akt was markedly reduced by the treatment of ox-LDL, this result indicated the activity of Akt was markedly reduced. But the expression of the protein phosphatase 2A(PP2A)and protein phosphatase 1(PP1) in the total proteins of the HUVEC wasn't changed. We further utilized Pull-down technology to prove that in the ox-LDL-treated HUVEC, the de-phosphorylation of eNOS Ser1177 site was caused by the inactivation of Akt, not due to the elevated association of PP2A and PP1 with eNOS.We further utilized the chemical chaperone PBA (which can decrease the ER stress) to pretreat the HUVEC for 14 hours, then incubated cells with ox-LDL for 0-4 hours. The result indicated:PBA treatment group could markedly decrease the expression of the p-PERK and GRP78 caused by the ox-LDL (P<0.01), that is to say the PBA reduced the ER stress reaction of HUVEC. At the same time, PBA could markedly reverse the dephosphorylation of the eNOS Ser1177 site and the Akt Thr308,Akt Ser473 sites caused by ox-LDL. In order to further prove the key role of the ER stress in the process of the reduced activity of eNOS caused by ox-LDL, we used the inducer of ER stress BFA(5μg/ml) to treat the HUVEC, we found that the phosphorylation of Akt and eNOS were markedly reduced, and it was time-dependent. This result proves:The ER stress can cause the dephosphorylation of Akt and eNOS Ser1177 site.We further investigated whether the ER stress reduced the phosphorylation of eNOS through the PI3K/Akt signal transduction pathway, so we used PI3K inhibitor LY294002(0.5nM) or PI3K inducer IGF-1(100ng/ml) to treat the HUVEC, the result indicated:the ER stress inducer BFA can directly influnce the PI3K/Akt signal transduction pathway, so caused the dephosphorylation of eNOS Ser1177 site.We also detected whether the LOX-1 mediated the ER stress reaction caused by ox-LDL. We used LOX-1 blocking antibody JTX20 to treat the cells, the result proved: the JTX20 pretreatment could markedly reverse the dephosphorylation of the eNOS and Akt caused by ox-LDL. We further estimated the ER stress reaction in HUVEC, we found JTX20 could also reverse the expression of the p-PERK and GRP78(P<0.01), that is to say JTX20 can decrease the ER stress reaction.So, our result indicated:ox-LDL can cause the ER stress reaction via LOX-1, and can reduce the phosphorylation of eNOS through the Akt signal transduction pathway, so reduce the function of eNOS.
|
PDF Full Text Request