| Myeloid-related protein8(MRP8, S100A8) and MRP14(S100A9) belong to a large family of calcium-binding proteins of the S100family and exit mainly as a MRP8/MRP14heterodimer in the extracellular milieu. MRP8/14can form heterodimers in the absence of calcium and heterotetramer in the presence of calcium. MRP8/14complexes account for up to40%of total cytosolic protein in neutrophils. In quiescent cells, MRP8, MRP14and MRP8/14are retained in the cytoplasm. They are secreted upon the activation of neutrophils and circulate in plasma. MRP8, MRP14and MRP8/14play important roles in myeloid differentiation, in inflammatory response, and also exert antimicrobial activity during infection.The vascular endothelium is a layer of a selective permeability barrier which can regulate the fluid, solute and plasma proteins moving from blood to interstitial spaces and vise versa. Studies show that increase of vascular permeability is involved in a series of pathological processes such as inflammation, burn, shock, immunity and tumor metastasis. It is well known as one of the major causes of death in patients with advanced shock. MRP8and MRP14are known to promote the adhesion of neutrophils to endothelium and MRP8/14induces a proinflammatory and thrombogenic response in human microvascular endothelial cells (HMECs). A recent study found that MRP8/14predominantly down-regulated the expression of genes that contribute to endothelial monolayer integrity. Other evidences showed that MRP8/14complex served as an endogenous activator of TLR4and promoted lethality during septic shock. Therefore, we hypothesize that MRP8and MRP14may induce increase of endothelial permeability in a short time.The receptors responsible for MRP8and MRP14signaling in HUVECs are still under debate. It is now increasingly recognized that extracellular MRP8and MRP14mediate pathological functions through interaction with cell surface pattern recognition receptors such as TLR4and RAGE. The expression of TLR4and RAGE in endothelial cells are pronounced and inflammatory stimulation will enhance the expression of both receptors. So in this study, we will investigate possible receptors for MRP8-and MRP14-induced HUVEC permeability increase.Mitogen-activated protein kinases (MAPKs) are a group of well-described serine/threonine-specific protein kinases generally expressed in all cell types. MAPKs are essential components of signal transduction machinery and occupy a central position in regulation of gene expression, mitosis, metabolism, survival, motility, apoptosis, proliferation and differentiation. It has been well established that MRP8/14activates JNK, p38and ERK1/2signaling pathways in tumor cells. In this study, we aim to clarify the involvement of the three subfamilies of MAPKs in the process of MRP8-and MRP14-indcued increase of endothelial permeability.Objective:In the current study, we will investigate whether MRP8, MRP14and MRP8/14can increase HUVECs permeability and which receptors and signaling pathways are implicated in.Methods:Human umbilical vein endothelial cells (HUVECs) were stimulated with MRP8, MRP14and MRP8/14for120min. Transenothelial electrical resistance (TER) was measured to investigate the change of HUVEC permeability. Immunofluorescence was used to monitor F-actin and ZO-1disorganization in HUVECs. The phosphorylation of MAPKs was determined by immunoblotting. The calcium-dependency was detected in the absent of Ca2+or in the presence of gradient-dose Ca2+. The involvement of TLR4and RAGE were observed by using specific inhibitor for TLR4and neutralizing antibody of RAGE. For signaling pathways investigation, specific inhibitors for p38, ERK1/2and JNK were used.Results:1. MRP8, MRP14and MRP8/14induced a concentration-and time-dependent increase in HUVEC permeability(1) MRP8, MRP14or MRP8/14with concentrations of0,0.5,1.0,2.0and4.0μg/ml were applied, respectively, to HUVEC monolayer. The TER was measured for120min. The results showed when HUVECs were stimulated by MRP8, the TER was significantly different among different treatment factors (F=79.604, P=0.000) and different treatment times (F=114.348, P=0.000). There were crossover effects between the treatment factors and treatment times (F=4.266, P=0.000). MRP8evoked the decreases of TER in a dose-dependent manner. MRP8-induced barrier disruption demonstrated a sharper decline and reached the bottom in30min, then sustained for over2h. When HUVECs were stimulated by MRP14or MRP8/14, the TER was also significantly different among different treatment factors (F values were47.864and52.972respectively. Both of the P values were0.000) and different treatment times (F values were112.496and158.498respectively. Both of the P values were0.000). There were crossover effects between the treatment factors and treatment times (F values were12.890and12.554respectively. Both of the P values were0.000). MRP14and MRP8/14also induced a concentration-and time-dependent increase in HUVEC permeability. But it is different from MRP8, MRP14and MRP8/14induced a more gradual decrease in TER in120min.(2) Smooth and continuous staining for both F-actin and ZO-1along with the intercellular borders of adjacent endothelial cells was seen in normal untreated HUVECs. The exposure of MRP8, MRP14or MRP8/14in2.0μg/ml to HUVECs caused a noticeable alteration of ZO-1spreading at cellular border, displaying discontinuity and serration in the location of per se sharp lining without stimulation. This change was accompanied by the polymerization of F-actin and the formation of stress fiber, resulting in the appearance of intercellular gaps. These changes became more obvious as the exposure time extended from10to120min.2. MRP8, MRP14and MRP8/14induced increase in HUVEC permeability by a p38and ERK1/2dependent mechanism(1) HUVECs were stimulated with MRP8, MRP14and MRP8/14for0,10,30,60and120min respectively. Phosphorylation of p38, ERK1/2and JNK was assessed by western blotting. The results displayed that the application of MRP8remarkably induced the increase of phosphorylation of p38, ERK1/2and JNK (The values of F were4.930,8.252and6.357respectively; the values of P were0.019,0.003and0.008respectively). As early as in10min, MRP8sparked the significant increase of phosphorylation of p38, ERK1/2and JNK compared to the control group. Phosphorylation of p38, ERK1/2and JNK was significantly different among different groups when HUVECs were treatmented by MRP14(The values of F were7.882,12.974and7.506respectively; the values of P were0.004,0.001and0.005respectively). MRP8/14also significantly increased phosphorylation of p38, ERK1/2and JNK (The values of F were9.186,5.957and4.290respectively; the values of P were0.002,0.010and0.028respectively). MRP14and MRP8/14increased the phosphorylation of p38, ERK1/2and JNK gradually. The different time-point of MAPK phosphorylation induced by MRP8and MRP14matched the pattern of time-dependent decrease of TER in HUVECs respectively.(2) HUVECs were stimulated with MRP8, MRP14or MRP8/14for120min, with or without60min pre-incubated with SB203580, PD98059and SP600125which were used as standard inhibitor for p38, ERK1/2and JNK, respectively. Then the TER was measured. Whether HUVECs were stimulated by MRP8, MRP14or MRP8/14, the TER was all significantly different among different treatment factors (F values were83.647,85.645and137.528respectively. All of the P values were0.000) and different treatment times (F values were399.476,260.949and987.621respectively. All of the P values were0.000). There were crossover effects between the treatment factors and treatment times (F values were31.689,18.633and80.156respectively. All of the P values were0.000). And we found that both SB203580and PD98059significantly attenuated the increase of endothelial monolayer permeability induced by MPR8, MRP14or MRP8/14, while SP600125showed no influence on MRPs-induced endothelial barrier dysfunction.(3) The morphological changes of endothelial cytoskeleton and tight junction protein induced by MRP8, MRP14and MRP8/14were also abolished by inhibiting the activation of p38and EKR1/2while the formation of stress fiber and the serrating discontinuity of ZO-1were attenuated in SB203580or PD98059pretreated HUVECs. The inhibition of JNK showed no effect on the disorganization of F-actin and ZO-1induced by MRPs.3. MRP8induced increase in HUVEC permeability via TLR4and MRP14increased HUVEC permeability via RAGE(1) HUVECs were stimulated with MRP8, MRP14or MRP8/14for120min, with or without60min pre-incubated with specific blockers (TAK242for TLR4and anti-human RAGE antibody for RAGE). Then the TER was measured. The results showed that whether HUVECs were stimulated by MRP8, MRP14or MRP8/14, the TER was all significantly different among different treatment factors (F values were126.431,74.816and30.440respectively. All of the P values were0.000) and different treatment times (F values were355.094,356.445and186.168respectively. All of the P values were0.000). There were crossover effects between the treatment factors and treatment times (F values were51.834,45.956and17.810respectively. All of the P values were0.000). And we also found that the inhibition of TLR4with TAK242attenuated MRP8-induced increase of monolayer hyper-permeability, and the addition of anti-RAGE did not enhanced the TAK242-evoked decrease of MRP8-induced endothelial barrier dysfunction. While, the blockage of RAGE abolished MRP14-induced but not MRP8-evoked increase of HUVEC permeability.(2) Phosphorylation of p38and ERK1/2was also assessed by western blotting. The results displayed that when HUVECs were stimulated by MRP8, phosphorylation of p38and ERK1/2was significantly different among different groups (The values of F were49.885and86.672respectively. Both of the P values were0.000). And the usage of TLR4inhibitor TAK242, but not antibody of RAGE, abolished MRP8-induced p38and ERK1/2phosphorylation. Phosphorylation of p38and ERK1/2was also significantly different among different groups when HUVECs were treatmented by MRP14(The values of F were11.023and8.402respectively. Both of the P values were0.000). While the utilization of RAGE antibody, but not TAK242, blocked MRP14-induced activation of p38and ERK1/2. Both TAK242and anti-RAGE could abolish MRP8/14-evoked activation of p38and ERK1/2, and compared to TAK242or anti-RAGE alone, the combination of TAK242and anti-RAGE further strengthened the inhibition of p38and ERK1/2phosphorylation in HUVECs exposed to MRP8/14.4. MRP8and MRP14increased HUVEC permeability in a Ca2+-dependent manner(1) HUVECs were incubated with Ca2+-free medium (PBS) with and without MRP8(2.0μg/ml) for90min and in the30th min1.3mM Ca2+were added to the medium. TER was measured every10min. The TER was significantly different among different treatment factors (F=136.026; P=0.000) and different treatment times (F=187.980; P=0.000). There were crossover effects between the treatment factors and treatment times (F=101.373; P=0.000). We also found that the absence of calcium of first caused a decline in TER, while the restore of proper calcium dose resumed the barrier function with a recovering TER. But when the restore of calcium was accompanied with MRP8, the TER was continuously decreased. Then the calcium dependency of MRP8was further confirmed by adding different portion of calcium concentration which could just saturate calcium binding sites of2.0μg/ml MRP8. The results demonstrated that the TER was significantly different among different treatment factors (F=405.592; P=0.000) and different treatment times (F=1647.036; P=0.000). There were crossover effects between the treatment factors and treatment times (F=128.156; P=0.000). And the presence of escalating dose of calcium enhanced the reduction of TER in MRP8-treated endothelial monolayer. These results suggested that MRP8increased the HUVEC permeability in a Ca2+-dependent manner. Using the same methods, we also confirmed that MRP14induced increase of HUVEC permeability in a Ca2+-dependent manner.(2) HUVECs were stimulated with MRP8(2.0μg/ml) and MRP14(2.0μg/ml) with or without Ca2+. Phosphorylation of p38(p-p38) and ERK1/2(p-ERK1/2) were assessed by western blotting. The results showed that the activation of p38and ERK1/2induced by MRP8or MRP14were also property of calcium-dependent. The absence of calcium abolished the phosphorylation of p38and ERK1/2under the treatment of MRP8or MRP14. Conclusions:1. MRP8, MRP14and MRP8/14induced a concentration-and dependent increase in HUVEC permeability;2. MRP8, MRP14and MRP8/14increased HUVEC permeability by a p38and ERK1/2dependent mechanism;3. MRP8induced increase in HUVEC permeability via TLR4and MRP14increased HUVEC permeability via RAGE;4. MRP8and MRP14increased HUVEC permeability in a Ca2+-dependent manner. |
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