In airway and renal epithelia, the glucocorticoid-mediated stimulation of amiloride-sensitive Na ؉ transport is associated with increased expression of the epithelial Na ؉ channel ␣ subunit (␣ENaC). In H441 lung cells, 100 nM dexamethasone increases amiloride-sensitive shortcircuit current (3.3 A/cm 2 to 7.5 A/cm 2 ), correlating with a 5-fold increase in ␣ENaC mRNA expression that could be blocked by actinomycin D. To explore transcriptional regulation of ␣ENaC, the human ␣ENaC 5-flanking region was cloned and tested in H441 cells. By deletion analysis, a ϳ150-base pair region 5 to the upstream promoter was identified that, when stimulated with 100 nM dexamethasone, increased luciferase expression 15-fold. This region, which contains two imperfect GREs, also functioned when coupled to a heterologous promoter. When individually tested, only the downstream GRE functioned in cis and bound GR in a gel mobility shift assay. In the M-1 collecting duct line Na ؉ transport, m␣ENaC expression and luciferase expression from ␣ENaC genomic fragments were also increased by 100 nM dexamethasone. In a colonic cell line, HT29, trans-activation via a heterologously expressed glucocorticoid receptor restored glucocorticoid-stimulated ␣ENaC gene transcription. We conclude that glucocorticoids stimulate ␣ENaC expression in kidney and lung via activation of a hormone response element in the 5-flanking region of h␣ENaC and this response, in part, is the likely basis for the up-regulation of Na ؉ transport in these sites.

H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na(+) transport pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li, R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas. J Biol Chem 274: 12431-12437, 1999). To understand its molecular basis, we examined the effect of glucocorticoids (GC) on epithelial Na(+) channel (ENaC)-alpha, -beta, and -gamma and sgk1 expression and determined the biophysical properties of Na(+) channels in these cells. GC stimulated the expression of ENac-alpha, -beta, and -gamma and sgk1 mRNA, with the first effect seen by 1 h. These effects were abolished by actinomycin D, but not by cycloheximide, indicating a direct stimulatory effect on ENaC and sgk1 mRNA synthesis. The GC effect on transcription of ENaC-alpha mRNA was accompanied by a significant increase in ENaC-alpha protein levels. GC also stimulated ENaC-alpha, -beta, and -gamma and sgk1 mRNA expression in A549 cells, an alveolar type II cell line. To determine the biophysical properties of the Na(+) channel, single-channel currents were recorded from cell-attached H441 membranes. An Na(+)-selective channel with slow kinetics and a slope conductance of 10.8 pS was noted, properties similar to ENaC-alpha, -beta, and -gamma expressed in Xenopus laevis oocytes. These experiments indicate that amiloride-sensitive Na(+) transport is mediated through classic ENaC channels in human lung epithelia and that GC-regulated Na(+) transport is accompanied by increased transcription of each of the component subunits and sgk1.

Vascular endothelial-cadherin (VE-cad) is localized to adherens junctions at endo-thelial cell borders and forms a complex with- ,- ,-, and p120-catenins (p120). We previously showed that the VE-cad complex disassociates to form short-lived "gaps" during leukocyte transendo-thelial migration (TEM); however, whether these gaps are required for leukocyte TEM is not clear. Recently p120 has been shown to control VE-cad surface expression through endocytosis. We hypothesized that p120 regulates VE-cad surface expression, which would in turn have functional consequences for leukocyte transmigration. Here we show that endo-thelial cells transduced with an adenovi-rus expressing p120GFP fusion protein significantly increase VE-cad expression. Moreover, endothelial junctions with high p120GFP expression largely prevent VE-cad gap formation and neutrophil leuko-cyte TEM; if TEM occurs, the length of time required is prolonged. We find no evidence that VE-cad endocytosis plays a role in VE-cad gap formation and instead show that this process is regulated by changes in VE-cad phosphorylation. In fact, a nonphosphorylatable VE-cad mutant prevented TEM. In summary, our studies provide compelling evidence that VE-cad gap formation is required for leukocyte transmigration and identify p120 as a critical intracellular mediator of this process through its regulation of VE-cad expression at junctions. (Blood. 2008;112:2770-2779) Introduction Vascular endothelial-cadherin (VE-cad) is a transmembrane protein expressed in the vascular endothelium 1 that participates in endothelial barrier function, angiogenesis, signaling, and endothelial cell survival (reviewed in Dejana et al 2). Surface-expressed VE-cad localizes to cell-cell junctions and associates with-catenin,-catenin, plakoglobin (-catenin), and p120-catenin (p120) through its cytoplasmic tail, and with the actin cytoskeleton 3,4 in combination with vinculin and-actinin, which is thought to be critical for VE-cad adhesive interactions (reviewed in Vestweber 5). p120 is a substrate for Src family kinases and other receptor tyrosine kinases 6,7 and regulates cadherin-dependent adhesion positively and negatively, depending on the cell system under study (reviewed in Alemà and Salvatore 8). p120 associates with the juxtamembrane cytoplasmic region of VE-cad, 9 and this is crucial to maintain cadherin surface expression. 10 Overexpres-sion of VE-cad mutants that competed for p120 binding, or siRNA knockdown of p120 in endothelium, resulted in dramatically decreased surface-expressed VE-cad and concomitant increased VE-cad degradation by an endocytic pathway. In contrast, overexpression of wild-type p120 augments VE-cad surface expression and diminishes its endocytosis. 11 The precise mechanism(s) by which p120 controls the turnover and endocy-tosis of junctional VE-cad is not completely understood, 8,11 but it is conclusive that cytosolic levels of p120 regulate VE-cad surface expression in endothelial cells, and the level of E-cadherin in epithelial cells. 12 The idea that VE-ca...

Leukocyte recruitment to tissues and organs is an essential component of host defense. The molecular mechanisms controlling this process are complex and remain under active investigation. The combination of biochemical techniques and live cell imaging using in vivo and in vitro flow model approaches have shed light on several aspects of neutrophil transmigration through the vascular endothelial lining of blood vessels. Here we focus on the role of adhesion molecule signaling in endothelial cells and their downstream targets during the process of transendothelial migration at cell-cell borders (paracellular transmigration). An emerging model involves leukocyte β2 integrin engagement of endothelial cell ICAM-1, which triggers integrin-ICAM-1 clustering (rings) and stabilizes leukocyte adhesion at cell-cell junctions. This step recruits nonreceptor tyrosine kinases that phosphorylate key tyrosine residues in the cytoplasmic tail of VE-cadherin, which destabilizes its linkage to catenins and the actin cytoskeleton, triggering the transient opening of VE-cadherin homodimers to form a gap in the cell junction, through which the neutrophil transmigrates. Interestingly, the signaling events that lead to neutrophil transmigration occur independent of shear flow in vitro.