RhoA-ROCK-Dependent Moesin Phosphorylation Regulates Advance Glycation End Products-induced Endothelial Response

Objective:This study was to investigate the involvement of RhoA/ROCK-dependent moesin phosphorylation in endothelial abnormalities induced by advanced glycation end products (AGEs). Our previous studies have reported that AGEs induced endothelial hyper-permeabiliby and F-actin disorganization. And the studies also suggested the involvement of ROCK in AGE-induced endothelial responses. But ROCK is not directly act on F-actin and other cytoskeletal molecules. We think there is a missing link between the activation of ROCK and the cytoskeleton reorganization. Moesin is emerging as the potential candidate that likely to mediate this process. So, RhoA-ROCK is postulated to phosphorylate moesin in AGE-induced endothelial response, but it is still lack of direct evidences for the interaction of ROCK and moesin in this AGE-evoked endothelial alteration. Therefore, we choose dominant negative RhoA mutant (RhoA N19) and constitutively active mutant (RhoA L63) recombinant adenovirus to infect cells. Using G-LISA assay kit to dectect the activity of RhoA and immunoblotting to detect the phosphorylation of ROCK. Combined with our previous results of ROCK inhibitor treated cells, to further clarify the RhoA/ROCK pathway invovle in AGE-HSA-mediated endothelial changes in morphology and function. This present study also aimed to further confirm the interaction of ROCK and its downstream target moesin by using Co-immunoprecipitation (Co-IP). To identify the effect of moesin threonine phosphorylation in AGE/ROCK-induced endothelial response, two mutants pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D were applied in endothelial cells and the AGE-stimulated responses were compared.Methods:AGE-HSA was prepared by incubating human serum albumin (HSA) in PBS with D-glucose for 8 weeks in a sterile environment at 37℃. Choosing the dominant negative RhoA mutant (RhoA N19) and constitutively active mutant (RhoA L63) recombinant adenovirus to infect cells. Applicating the G-LISA assay kit to detect the activity of RhoA and immunoblotting to detect the phosphorylation of ROCK. To further confirm the interaction of ROCK and its downstream target moesin by Co-immunoprecipitation (Co-IP). Using molecular cloning and the site-directed mutagenesis to constructed the eukaryotic expression plasmid pcDNA3/HA-moesin and its mutants pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D. The mutant pcDNA3/HA-moesinT558A was induced by substituting Thr at 558 amino acid residues with Ala as inhibited mutant. And pcDNA3/HA-moesin was substituted by Asp as activated mutant. By Lipofectamine LTX to transfect HUVECs, the endothelial morphological and functional changes were detected by staining F-actin with rhodamine-phalloidin, using immunoblotting to detect the phosphorylation of moesin, and taking a multi-function proteomics imaging system KODAK2000R to image directly, with ImageJ software to analysis of grey value,β-actin as the internal reference calibrated to the gray value of control group area as 100% compared with the experimental group. With TRITC fluorescent marker protein leakage to detect endothelial monolayer permeability coefficient values of Pa. The experimental results are expressed as a percentage change in Pa, Pa%= (Pa value of experimental samples /Pa value of the control sample)×100.Result:1. RhoA activation induced by AGE-HSA in endothelial cells (1) RhoA activity was remarkably increased by AGE-HSA treatment in a time-and concentration-dependent manner in endothelial cells.Treatment with AGE-HSA significantly increased RhoA activity in time and dose dependent. In the time-dependent group, the activation of RhoA was remarkably increased from AGE-HSA stimulated 30 min. Until to 60 min, the RhoA activity reached the peak. In the concentration-dependent group, the activation of RhoA was reached its peak after exposure at 50 mg/L AGE-HSA.(2) Suppression of RhoA with a dominant negative adenovirus prevented activation of RhoA and phosphorylation of ROCK and moesin.To further specify the role of the RhoA/ROCK pathway in AGE-HSA-related endothelial changes, RhoA activity and ROCK, moesin phosphorylation were assessed in HDMEC treated with AGE-HSA at 24 h after infection with dominant negative RhoA N19 recombinant adenovirus. Transfection of RhoA N19 down-regulated RhoA activity in HDMEC, with relative RhoA activity being decreased from 123.561±2.003% in the AGE-HSA group to 96.862±1.750% in the AGE-HSA group with RhoA N19 adenovirus. It was also found that phosphorylation of ROCK and moesin were significantly suppressed by transfection of RhoA N19. Transfection of the constitutively activated recombinant adenovirus (RhoA L63) mimicked the effects of AGE-HSA on RhoA, ROCK and moesin in cultured HDMEC. Relative activity of RhoA increased by 32.454±2.215% after active RhoA L63 transfection, along with markedly enhanced phosphorylation of ROCK and moesin.2. Endogenous ROCK interacted directly with moesin in endothelial cellsTo further verifying the relationship of ROCK and moesin, the direct interaction of endogenous ROCK and moesin after AGE-HSA stimulation was assessed using Co-Immunoprecipitation (Co-IP)-Immunoblotting (IB) analysis of HUVEC extracts after stimulation with 50 mg/L AGE-HSA for 1 h. Physical interaction between ROCK and moesin was confirmed in HUVECs with or without AGE-HSA application. The binding of moesin with phospho-ROCK was decreased after AGE-HSA administration. We speculated that the phosphorylation of ROCK activated moesin and released it to act as the linker protein, resulting in the redistribution of cytoskeleton. These data demonstrated that RhoA-ROCK pathway might work through moesin activation in AGE-HSA induced endothelial responses.3. The eukaryotic expression plasmid of moesin and two mutants were successfully constructed and expressed in endothelial cells.The mutants of moesin were induced by substituting Thr at 558 amino acid residues with Ala (pcDNA3/HA-moesinT558A) as inhibited mutant, or Asp (pcDNA3/HA-moesinT558D) as activated mutant, respectively. The eukaryotic expression of plasmids of two mutants and the eukaryotic expression plasmid pcDNA3/HA-moesin were all identified by nucleotide sequencing. Realtime PCR showed that the expression of moesin mRNA in pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D transfection groups were higher than that in non-transfected, while no difference existed between the two transfection groups. Immunoblotting detected the protein expression of HA-moesin in transfected pcDNA3/HA-moesinT558A and pcDNA3/HA-moesinT558D groups, but not detected in non-transfected group.4. The inhibiting mutation of moesin attenuated AGE-HSA induced phosphorylation of moesinThe transient transfection of inhibiting mutant pcDNA3/HA-moesinT558A into HUVECs decreased the level of phosphorylated moesin after stimulation of 50 mg/L AGE-HSA for 1h, while the transient transfection of activating mutant pcDNA3/HA-moesinT558D itself educed phosphorylation of moesin without AGE-HSA application.5. The inhibiting mutation of moesin attenuated AGE-HSA induced endothelial hyper-permeabiliby response and F-actin disorganizationOur previous report has shown that AGE-HSA increased the monolayer permeability and caused the formation of F-actin stress fiber in HUVECs and HMVECs. The transient transfection of plasmid pcDNA3/HA-moesinT558A into HUVECs decreased the hyper-permeability response induced by AGE-HSA administration (50 mg/L, 1h) from 158.342±4.166% to 116.670±3.213%. It also preserved the distribution of F-actin in endothelial cortex area after AGE-HSA stimulation. The transient transfection of plasmid pcDNA3/HA-moesinT558D itself increased endothelial monolayer permeability for 152.619±7.137%, and it also triggered the formation of F-actin stress fiber. These results provides direct evidence for the involvement of phosphorylation of moesin in Thr 558 residues in AGE-induced endothelial dysfunction.Conclusion:1. RhoA activity was remarkably increased by AGE-HSA treatment in a time-and concentration-dependent manner in endothelial cells.2. The downstream target molecule ROCK was evoked by AGE-HSA mediated RhoA activation, and ROCK interacted directly with moesin, resulting in morphological and functional change in endothelial cell.3. The phosphorylation of moesinT558 induced by AGE/ROCK played a important role in the pathological process of endothelial cell morphology and functional changes, and inhibition of phosphorylation of moesinT558 could abolish the endothelial response induced by AGE-HSA.