Histamine selectively interrupts VE-cadherin adhesion independently of capacitive calcium entry

Histamine is an important agent of innate immunity, transiently increasing the flux of immune-competent molecules from the vascular space to the tissues and then allowing rapid restoration of the integrity of the endothelial barrier. In previous work we found that histamine alters the endothelial barrier by disrupting cell-cell adhesion and identified VE-cadherin as an essential participant in this process. The previous work did not determine whether histamine directly interrupted VE-cadherin adhesion, whether the effects of histamine were selective for cadherin adhesion, or whether capacitive calcium flux across the cell membrane was necessary for the effects of histamine on cell-cell adhesion. In the current work we found that histamine directly interrupts adhesion of L cells expressing the type 1 histamine (H1) receptor and VE-cadherin to a VE-cadherin-Fc fusion protein. In contrast, integrin-mediated adhesion to fibronectin of the same L cells expressing the H1 receptor was not affected by histamine, demonstrating that the effects of histamine are selective for cadherin adhesion. Some of the effects of many edemagenic agonists on endothelium are dependent on the capacitive flux of calcium across the endothelial cell membrane. Blocking capacitive calcium flux with LaCl3 did not prevent histamine from interrupting VE-cadherin adhesion of transfected L cells, nor did it prevent histamine from interrupting cell-cell adhesion of human umbilical vein endothelial cells. These data support the contentions that histamine directly and selectively interrupts cadherin adhesion and this effect on cadherin adhesion is independent of capacitive calcium flux. endothelium; integrin; human histamine receptor H1; vascular endothelial cadherin ACUTE INFLAMMATORY EDEMA is an important component of innate immunity that facilitates the transfer of immune-competent molecules from the vascular space to the tissues. Histamine and serotonin are well-acknowledged physiological agonists of acute inflammatory edema that disrupt cell-cell apposition in postcapillary venules Vascular endothelial (VE)-cadherin is an endothelial unique cadherin that mediates cell-cell adhesion of endothelial cells (17). Function-interrupting antibody to VE-cadherin increases permeability of endothelium in vivo, indicating that integrity of VE-cadherin adhesion is essential to the integrity of the endothelial barrier (9, 16). It was of note that although antibody to VE-cadherin increased in vivo endothelial permeability within 1 h of its administration, no light microscopic changes were evident in the endothelium even at 2 h, demonstrating that no large gaps had formed between the cells. However, similar to the effects of histamine and serotonin, transmission electron micrographs detected very small gaps in lung microvessels exposed to antibody to VE-cadherin (9). ECV304 cells are a transformed bladder epithelial cell line that express the type 1 histamine receptor (H1) and P-and N-cadherin, but not VE-cadherin. When mock-transfected ECV304 cells were exposed to histamine, there was no change in the electrical resistance of the cell-cell barrier they created (24). However, when ECV304 cells transfected with VEcadherin were exposed to histamine, the electrical resistance of the cell-cell barrier fell, identifying an important role for VE-cadherin in the response to histamine of a monolayer of polarized cells (24). ECV304 cells express claudin 2 and develop tight junctions (5). This tight junction is a necessary component of the electrical resistance decreased by histamine. The observation that histamine decreased the integrity of the cell-cell barrier created by VE-cadherin transfected ECV304 cells did not discriminate between effects of histamine on an interaction of VE-cadherin with the tight junction versus a direct effect of histamine on VE-cadherin adhesion. Histamine and other agonists that increase endothelial permeability initiate signaling in endothelial cells that increases cell calcium (6, 7). Although some of the increase in cell calcium caused by these agonists reflects release of calcium from intracellular stores, capacitive flux of calcium across the cell membrane is also activated, and the capacitive calcium flux is necessary for some of the effects of these agonists on endothelial cells In these investigations we used a model of cadherin adhesion that lacks tight junctions and asked whether histamine directly affects VE-cadherin adhesion as opposed to affecting an interaction between VE-cadherin and the tight junction. Our results indicate that histamine directly affects VE-cadherin adhesion. Using a similar model of integrin-based adhesion, we foun

Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function