To determine how histamine regulates endothelial barrier function through an integrative cytoskeletal network, we mathematically modeled the resistance across an endothelial cell-covered electrode as a function of cell-cell, cell-matrix, and transcellular resistances. Based on this approach, histamine initiated a rapid decrease in transendothelial resistance predominantly through decreases in cell-cell resistance in confluent cultured human umbilical vein endothelial cells (HUVECs). Restoration of resistance was characterized by initially increasing cell-matrix resistance, with later increases in cell-cell resistance. Thus histamine disrupts barrier function by specifically disrupting cell-cell adhesion and restores barrier function in part through direct effects on cell-matrix adhesion. To validate the precision of our technique, histamine increased the resistance in subconfluent HUVECs in which there was no cell-cell contact. Exposure of confluent monolayers to an antibody against cadherin-5 caused a predominant decrease in cell-cell resistance, whereas the resistance was unaffected by the antibody to cadherin-5 in subconfluent cells. Furthermore, we observed an increase predominantly in cell-cell resistance in ECV304 cells that were transfected with a plasmid containing a glucocorticoid-inducible promoter controlling expression of E-cadherin. Transmission electron microscopy confirmed tens of nanometer displacements between adjacent cells at a time point in which histamine maximally decreased cell-cell resistance.

TRAF2 is an adaptor protein that regulates the activation of the c-Jun N-terminal kinase (JNK) and IB kinase (IKK) signaling cascades in response to tumor necrosis factor alpha (TNF-␣) stimulation. Although the downstream events in TNF-␣ signaling are better understood, the membrane-proximal events are still elusive. Here, we demonstrate that TNF-␣ and cellular stresses induce TRAF2 phosphorylation at serine 11 and that this phosphorylation is required for the expression of a subset of NF-B target genes. Although TRAF2 phosphorylation had a minimal effect on the TNF-␣-induced rapid and transient IKK activation, it was essential for secondary and prolonged IKK activation. Consistent with this, TRAF2 phosphorylation is not required for its recruitment to the TNFR1 complex in response to TNF-␣ stimulation but is required for its association with a cytoplasmic complex containing RIP1 and IKK. In addition, TRAF2 phosphorylation was essential for the full TNF-␣-induced activation of JNK. Notably, TRAF2 phosphorylation increased both basal and inducible c-Jun and NF-B activities and rendered cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some lymphomas. These results unveil a new, finely tuned mechanism for TNF-␣-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation contributes to elevated levels of basal NF-B activity in certain human cancers.Tumor necrosis factor (TNF) receptor (TNFR)-associated factors (TRAFs) are characterized by the presence of a TRAF domain at the C terminus. Currently, six members of this family are known (TRAR1 to TRAR6), and all TRAFs, except TRAF1, contain N-terminal RING finger domains followed by five or seven zinc finger motifs (5, 31). TRAF2 is a prototypical member of the TRAF family and regulates signals from TNFR superfamily members.One of the well-characterized members of the TNFR superfamily is TNFR1. Currently, it is believed that TNFR1 activation by soluble TNF-␣ induces the sequential formation of two complexes with opposing effects on cell fate. In the first step, TNFR1 recruits the TNFR1-associated death domain (TRADD) protein, which in turn recruits TRAF2 and receptor-interacting protein 1 (RIP1) to form the membrane-bound prosurvival complex I. This leads to the sequential activation of mitogen-activated protein kinase (MAPK) kinase kinase (such as MEKK1/3), MAPK kinase (e.g., MKK4/7), and MAPK (e.g., c-Jun N-terminal kinase [JNK]) as well as in activation of transforming growth factor ␤-activated kinase 1 (TAK1), RIP1, and IB kinase (IKK). In the next step, the TRADD/ RIP1/TRAF2 complex dissociates from TNFR1 and associates with the Fas-associated death domain protein and caspase-8 to form the cytoplasmic proapoptotic complex II (23). The balance between these pathways is usually tipped by two events. One is the expression of cFLIP in response to NF-B activation by complex I; cFLIP then binds to complex II and inhibits apoptosis by interfering with caspase-8 activation. T...

We compared temporal changes in isometric tension in cultured human umbilical vein endothelial cells inoculated on a polymerized collagen membrane with changes in cell-cell and cell-matrix adhesion derived by a mathematical model of transendothelial cell resistance. Thrombin and histamine disrupt barrier function by targeting a greater loss in cell-cell adhesion, which preceded losses in overall transendothelial resistance. There were minor losses in cell-matrix adhesion, which was temporally slower than the decline in the overall transendothelial resistance. In contrast, thrombin and histamine restored barrier function by initiating a restoration of cell-matrix adhesion, which occurred before an increase in overall transendothelial resistance. Thrombin mediated a second and slower decline in cell-cell adhesion, which was not observed in histamine-treated cells. This decline in cell-cell adhesion temporally correlated with expressed maximal levels of tension development, suggesting that actin-myosin contraction directly strains cell-cell adhesion sites. Pretreatment of cells with ML-7 mediated more rapid recovery of cell-cell adhesion and had no effect on cell-matrix adhesion. Taken together, expression of actin-myosin contraction affects the restoration of barrier function by straining cell-cell adhesion sites.

TRAF2 regulates JNK and IKK activation in response to TNF-α stimulation. This study found that TNF-α and oxidative stress induce TRAF2 phosphorylation and that this phosphorylation inhibits apoptosis by promoting the prolonged phase of IKK activation while inhibiting the prolonged phase of JNK activation.

Tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2) is an adaptor protein that modulates the activation of the c-Jun NH 2 terminal kinase (JNK)/c-Jun and IKB kinase (IKK)/nuclear factor-KB (NF-KB) signaling cascades in response to TNFA stimulation. Although many serine/threonine kinases have been implicated in TNFA-induced IKK activation and NF-KB-dependent gene expression, most of them do not directly activate IKK. Here, we report that protein kinase CZ phosphorylates TRAF2 at Ser 55 , within the RING domain of the protein, after TNFA stimulation. Although this phosphorylation event has a minimal effect on induction of the immediate/transient phase of IKK and JNK activation by TNFA, it promotes the secondary/prolonged phase of IKK activation and inhibits that of JNK. Importantly, constitutive TRAF2 phosphorylation increased both basal and inducible NF-KB activation and rendered Ha-Ras-V12-transformed cells resistant to stress-induced apoptosis. Moreover, TRAF2 was found to be constitutively phosphorylated in some malignant cancer cell lines and Hodgkin's lymphoma. These results reveal a new level of complexity in TNFA-induced IKK activation modulated by TRAF2 phosphorylation and suggest that TRAF2 phosphorylation is one of the events that are responsible for elevated basal NF-KB activity in certain human cancers.