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NF-[kappa]B signaling is known to induce the expression of antiapoptotic and proinflammatory genes in endothelial cells (ECs). We have shown recently that [Ca.sup.2+] influx through canonical transient receptor potential (TRPC) channels activates NF-[kappa]B in ECs. Here we show that [Ca.sup.2+] influx signal prevents thrombin-induced apoptosis by inducing NF-[kappa]B-dependent A20 expression in ECs. Knockdown of TRPCI expressed in human umbilical vein ECs with small interfering RNA (siRNA) suppressed thrombin-induced [Ca.sup.2+] influx and NF-[kappa]B activation in ECs. Interestingly, we observed that thrombin induced >25% of cell death (apoptosis) in TRPC1-knockdown ECs whereas thrombin had no effect on control or control siRNA-transfected ECs. To understand the basis of EC survival, we performed gene microarray analysis using ECs. Thrombin stimulation increased only a set of NF-[kappa]B-regulated genes 3- to 14-fold over basal levels in ECs. Expression of the antiapoptotic gene A20 was the highest among these upregulated genes. Like TRPC1 knockdown, thrombin induced apoptosis in A20-knockdown ECs. To address the importance of [Ca.sup.2+] influx signal, we measured thrombin-induced A20 expression in control and TRPC1-knockdown ECs. Thrombin-induced p65/RelA binding to A20 promoter-specific NF-[kappa]B sequence and A20 protein expression were suppressed in TRPC1-knockdown ECs compared with control ECs. Furthermore, in TRPC1-knockdown ECs, thrombin induced the expression of proapoptotic proteins caspase-3 and BAX. Importantly, thrombin-induced apoptosis in TRPC1-knockdown ECs was prevented by adenovirus-mediated expression of A20. These results suggest that [Ca.sup.2+] influx via TRPC channels plays a critical role in the mechanism of cell survival signaling through A20 expression in ECs. transient receptor potential canonical channel-1; nuclear factor-[kappa]B doi:10.1152/ajpcell.00456.2009

Caveolin-1 (Cav-1) regulates agonist-induced [Ca.sup.2+] entry in endothelial cells; however, how Cav-1 regulates this process is poorly understood. Here, we describe that Cav-1 scaffold domain (N[H.sub.2]-terminal residues 82-101; CSD) interacts with transient receptor potential canonical channel 1 (TRPC1) and inositol 1,4,5-trisphosphate receptor 3 ([IP.sub.3]R3) to regulate [Ca.sup.2+] entry. We have shown previously that the TRPC1 COOH-terminal residues 781-789 bind to CSD. In the present study, we show that the TRPC1 COOH-terminal residues 781-789 truncated (TRPC1-C[DELTA]781-789) mutant expression abolished [Ca.sup.2+] store release-induced [Ca.sup.2+] influx in human dermal microvascular endothelial cell line (HMEC) and human embryonic kidney (HEK-293) cells. To understand the basis of loss of [Ca.sup.2+] influx, we determined TRPC1 binding to IP3R3. We observed that the wild-type (WT)-TRPC1 but not TRPC1-C[DELTA]781-789 effectively interacted with [IP.sub.3]R3. Similarly, WT-TRPC1 interacted with Cav-1, whereas TRPC1-C[DELTA]781-789 binding to Cav-1 was markedly suppressed. We also assessed the direct binding of Cav-1 with TRPC1 and observed that the WT-Cav-1 but not the Cav-1[DELTA]CSD effectively interacted with TRPC1. Since the interaction between TRPC1 and Cav-1[DELTA]CSD was reduced, we measured [Ca.sup.2+] store release-induced [Ca.sup.2+] influx in Cav-l[DELTA]CSD-transfected cells. Surprisingly, Cav-1[DELTA]CSD expression showed a gain-of-function in [Ca.sup.2+] entry in HMEC and HEK-293 cells. We observed a similar gain-of-function in [Ca.sup.2+] entry when Cav-1[DELTA]CSD was expressed in lung endothelial cells of Cav-1 knockout mice. Immunoprecipitation results revealed that WT-Cav-1 but not Cav-1[DELTA]CSD interacted with [IP.sub.3]R3. Furthermore, we observed using confocal imaging the colocalization of [IP.sub.3]R3 with WT-Cav-1 but not with Cav-1[DELTA]CSD on [Ca.sup.2+] store release in endothelial cells. These findings suggest that CSD interacts with TRPC1 and [IP.sub.3]R3 and thereby regulates [Ca.sup.2+] store release-induced [Ca.sup.2+] entry in endothelial cells. transient receptor potential channel 1; inositol 1,4,5-trisphosphate receptor; caveolin-1 knockout mice

The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. Here we show that Cblb also has an unexpected function in acute lung inflammation. Cblb attenuates the sequestration of inflammatory cells in the lungs after administration of lipopolysaccharide (LPS). In a model of polymicrobial sepsis in which acute lung inflammation depends on the LPS receptor (Toll-like receptor 4, TLR-4), the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Loss of Cblb significantly increases sepsis-induced release of inflammatory cytokines and chemokines. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of wild-type Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for the transcription factor nuclear factor-[kappa]B (NF-[kappa]B) in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of neutrophils is impaired in the absence of Cblb. Our data reveal that Cblb regulates the TLR4-mediated acute inflammatory response that is induced by sepsis., Author(s): Kurt Bachmaier (corresponding author) [1, 3]; Sophie Toya [1, 2, 3]; Xiaopei Gao [1]; Thomas Triantafillou [1]; Sean Garrean [1]; Gye Young Park [2]; Randall S Frey [1]; Stephen [...]

We determined the effects of TNF-[alpha] on the expression of transient receptor potential channel (TRPC) homologues in human vascular endothelial cells and the consequences of TRPC expression on the endothelial permeability response. We observed that TNF-[alpha] exposure increased TRPC1 expression without significantly altering expression of other TRPC isoforms in human pulmonary artery endothelial cells (HPAEC). Because TRPC1 belongs to the store-operated cation channel family, we measured the [Ca.sup.2+] store depletion-mediated [Ca.sup.2+] influx in response to thrombin exposure. We observed that thrombininduced [Ca.sup.2+] influx in TNF-[alpha]-stimulated HPAEC was twofold greater than in control cells. To address the relationship between store-operated [Ca.sup.2+] influx and TRPC1 expression, we overexpressed TRPC1 by three- to fourfold in the human dermal microvascular endothelial cell line (HMEC) using the TRPC1 cDNA. Thrombininduced store [Ca.sup.2+] depletion in these cells caused approximately twofold greater increase in [Ca.sup.2+] influx than in control cells. Furthermore, the inositol 1,4,5-trisphosphate-sensitive store-operated cationic current was increased greater than twofold in TRPCl-transfected cells compared with control. To address the role of [Ca.sup.2+] influx via TRPC1 in signaling endothelial permeability, we measured actinstress fiber formation and transendothelial monolayer electrical resistance (TER) in the TRPC1 cDNA-transfected HMEC and TNF[alpha]-challenged HPAEC. Both thrombin-induced actin-stress fiber formation and a decrease in TER were augmented in TRPCl-overexpressing HMEC compared with control cells. TNF-[alpha]-induced increased TRPC1 expression in HPAEC also resulted in marked endothelial barrier dysfunction in response to thrombin. These findings indicate the expression level of TRPC1 in endothelial cells is a critical determinant of [Ca.sup.2+] influx and signaling of the increase in endothelial permeability. tumor necrosis factor-[alpha]; store-operated calcium ion influx; transient receptor potential channel 1; endothelial barrier dysfunction

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of [sup.125]I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol * [min.sup.-1] * [10.sup.6] [cells.sup.-1]. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-[beta]-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased [sup.125]I-albumin uptake 2.3-fold. At 37[degrees]C, [sup.125]I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (I[C.sub.50] = 1 [micro]M). Using a two-site model, we estimated by Scatchard analysis the affinity ([K.sub.D]) and maximal capacity ([B.sub.max]) of albumin uptake to be 0.87 [micro]M ([K.sub.D1]) and 0.47 pmol/[10.sup.6] cells ([B.sub.max1]) and 93.3 [micro]M ([K.sub.D2]) and 20.2 pmol/[10.sup.6] cells ([B.sub.max2]). At 4[degrees]C, we also observed two populations of specific binding sites, with high ([K.sub.D1] = 13.5 nM, 1% of the total) and low ([K.sub.D2] = 1.6 [micro]M) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer. capillary permeability; vesicular transport; caveolae; albumin-binding glycoprotein gp60

Vogel, Stephen M., Richard D. Minshall, Milena Pilipovic, Chinnaswamy Tiruppathi, and Asrar B. Malik. Albumin uptake and transcytosis in endothelial cells in vivo induced by albumin-binding protein. Am J Physiol Lung Cell Mol Physiol 281: L1512-L1522, 2001.--The 60-kDa endothelial cell surface albumin-binding glycoprotein (gp60) is postulated to be a docking site for albumin that mediates the uptake of albumin and its transport in cultured microvessel endothelial cells. In the present study, we used an isolated Krebs-perfused rat lung preparation to address the in vivo role of gp60 in mediating albumin uptake and transport. Addition of primary anti-gp60 antibody to the perfusate followed by the secondary antibody to cross-link gp60 increased the vessel wall [sup.125.I]-albumin permeability-surface area (PS) product 2.5-fold without affecting the capillary filtration coefficient ([K.sub.f,c]; a measure of liquid permeability). In contrast, EDTA (5 mM), which induces interendothelial gap formation, produced parallel increases in both [K.sub.f,c] and [sup.125.I]-albumin PS product. Increasing perfusate albumin concentration to > 1 g/100 ml (E[C.sub.50] 1.2 g/100 ml) was sufficient to block [sup.125.I]-albumin PS product, indicating that the perfusate albumin competed with tracer albumin for transendothelial albumin transport. Cross-linking of gp60 in lungs perfused with saturating concentration of albumin resulted in a greater increase in [sup.125.I]-albumin PS product, indicating that gp60 function was capable of being modulated. These results show that activation of gp60 in pulmonary microvessels induces albumin uptake and its transport through a nonhydraulic pathway that fits with a model of albumin permeability via the transcellular pathway. rat lung; filipin; gp60; antibody cross-linking

Changes in cytosolic calcium activation and in human pulmonary artery endothelial cells (HPAEC) retraction response was measured to examine the kinetics of endothelial cell surface expression of proteolytically activated thrombin receptor (PAR-1) in HPAEC. The results indicated that endothelial cell surface PAR-1 can be quickly restored even when it is continuously exposed to a low concentration of thrombin. However, cellular PAR-1 pools are depleted by high thrombin concentrations and longer exposure times.

Immunofluorescence studies and cytosolic calcium determination were undertaken to determine the effects of protein kinase C (PKC) activation on the synthesis and activity of the proteolytically activated thrombin receptor 1 (PAR-1). Results indicate that PKC-catalyzed phosphorylation of PAR-1 leads to receptor inactivation. Further, PKC contributes to the loss of PAR-1 on the cell surface and to the heterologous desensitization of the receptor as well as to the inhibition of cellular signal transduction through calcium.

The thrombin receptor domains responsible for receptor activation in endothelial cells were studied using site-specific antibodies. Results of experiments on polyclonal antibodies directed against peptide sequences corresponding to postulated binding sites of thrombin suggested a critical role for the thrombin receptor's NH2-terminal extension and loop 2 sites. These binding sites influenced receptor activation in endothelial cells, which in turn lead to increased intracellular calcium and transendothelial permeability to albumin.

A decline in endothelial hydraulic conductivity (Lp), a measure of liquid permeability across endothelial and matrix barrier, is affected by albumin and lectin Ricinus communis agglutinin (RCA) in bovine pulmonary microvascular endothelial cell monolayers. This effect of albumin and RCA is consistent with the response associated with the binding of albumin and RCA to extracellular matrix (ECM). The permeability-reducing effect of these molecules is hindered by the administration of protamine sulfate, a polycation, thus suggesting the mechanism whereby albumin and RCA interact with ECM and render the endothelial layer to function as a barricade to liquid infiltration.

Japan F344 rats, deficient in dipeptidyl peptidase IV (DPP IV) were employed in examining DPP IV influence on intestinal hydrolysis and prolyl peptide uptake. Brush-border membrane hydrolysis was lesser in the Japan specimens. Control rats possessed greater resistance to weight loss than Japan F344 rats. In Japan rat's intestinal lumen, hydrolysis-resistant morphiceptin impact was higher. These Japan rat-specific reactions are attributed to the lack of DPP IV in the Japan rat-system.