It is now accepted that circulating ROS can cause endothelial barrier dysfunction, which may lead to several pathologies, notably atherosclerosis. However, the molecular mechanisms involved are still poorly understood. We previously suggested that the phosphorylation of Tm1 could contribute to maintain endothelial integrity during oxidative stress. In the present study, we now firmly demonstrate the crucial importance of the Tm1 phosphorylation in the regulation of endothelial permeability and transendothelial migration of cancer cells (TEM).
The major finding of our study is that we show that Tm1 expression is important to maintain the endothelial barrier integrity in response to oxidative stress. Two experiments support these new findings. First, permeability assays confirm that the knockdown of Tm1 by small RNA interference in HUVEC monolayers is tightly associated with a heavy increase of endothelial permeability and TEM of cancer cells in response to oxidative stress. Second, in immunofluorescence assays, HUVEC in which Tm1 was decreased with siRNA7Tm1 under oxidative stress conditions display a marked disappearance of stress fibers, a high level of inter endothelial gaps and membrane blebbing.
Another major accomplishment of the present study is to show that Tm1 phosphorylation at Ser283, downstream of ERK is a crucial mechanism that contributes to maintain the selective transendothelial permeability in response to oxidative stress. This finding is supported by two principal observations. First, HUVEC monolayer expressing human non-phosphorylatable Ser283Ala Tm1 insensitive to siRNA7Tm1 and in which human endogenous Tm1 is knocked-down, are characterized by a strong increase of transendothelial permeability under oxidative stress conditions. In contrast, the ectopic expression of human wild-type Tm1 is rather accompanied by an acute decrease of transendothelial permeability in response to oxidative stress. Second, when the expression of human endogenous Tm1 is knocked-down in cells expressing an exogenous nonphosphorylatable Ser283Ala Tm1 insensitive to the knockdown, the endothelium shows important toxic features such as strong shrinkage, high level of inter-endothelial gaps, presence of membrane blebbing, and a total absence of stress fibers. In contrast, the over-expression of human exogenous wild-type Tm1 in similar condition plays a protective role on the endothelial barrier exposed to oxidative stress.
It has been proposed that actin polymerization and remodelling are important regulators of the endothelial permeability barrier [18, 31]. Along these lines, our results reveal a strong reorganization of F-actin into transcytoplasmic stress fibers in HUVEC monolayers treated with H2O2. Almost all cells were elongated with thick actin stress fibers, which traversed the cells in the direction of cell elongation. Moreover, we distinguished an acute presence of inter endothelial gaps suggesting an H2O2-mediated retraction of the cells. We also noticed the same typical morphological pattern with histamine treatment, a potent inducer of endothelial and vascular permeability . Based on these findings, the H2O2 and histamine-mediated changes in the organization of actin seem to be a fast and strong response that contributes to increase stress resistance. In this context, the actin depolymerising toxin cytochalasin D decreases endothelial barrier function [33, 34]. Hence by its participation to the formation and stabilization of stress fibers, the phosphorylation of Tm1 may regulate the actin-based contraction/retraction of endothelial cells that controls the permeability of the endothelium. Along these lines, other proteins known to modulate actin dynamics and stress fiber formation, such as the small GTPase RhoA, are also known to regulate endothelial cell permeability and TEM [35–37].
The tightness of the vessel walls represents a functional barrier that contributes to limit cancer cell TEM and ultimately metastasis. In that regard, dysfunction of the endothelial integrity is associated with metastasis [16, 38, 39]. Notably, certain breast, bladder, and kidney cancer cells induce alterations of endothelial mechanical properties to ease their TEM . Given the determinant role played by Tm1 phosphorylation at Ser283 in maintaining the integrity of endothelial cell in response to oxidative stress, one could expect that this mechanism may also protect against metastasis. This is, indeed, supported for the first time by our results. First, in response to H2O2, TEM of colon cancer HT-29 cells through HUVEC monolayers in which endogenous Tm1 expression is knocked-down by siRNA7Tm1 shows a higher number of transmigrating cells than through control HUVEC monolayer expressing endogenous Tm1. Second, the ectopic expression of human nonphosphorylatable Ser283Ala Tm1 insensitive to siRNA7Tm1 in HUVEC monolayer in which expression of human endogenous Tm1 is knocked-down is associated with an increased level of transendothelial migrating HT-29 cells in response to H2O2. In contrast, the expression of human exogenous wild-type Tm1 in similar conditions is associated with an acute decreased level of transendothelial migrating HT-29 cells. These results strengthen the point that phosphorylation of Tm1 at Ser283 is crucial to restrict the transendothelial migration of cancer cells under oxidative stress conditions.
In conclusion, we propose that Tm1 is required to maintain the endothelial barrier integrity against molecules as well as against cancer cells both under unstimulated as well as under oxidative stress conditions. We also propose that the phosphorylation of Tm1 on Ser283, downstream of ERK, is a key event for the regulation of endothelial permeability by modulating the contraction/retraction of the endothelial cells via remodelling of the actin cytoskeleton dynamics. These finding strongly support the role for the phosphorylation of Tm1 at Ser283 to prevent endothelial barrier dysfunction associated with oxidative stress.