As a pathway for Na؉ reabsorption, the epithelial Na ؉ channel ENaC is critical for Na ؉ homeostasis and blood pressure control. Na ؉ transport is regulated by Nedd4-2, an E3 ubiquitin ligase that decreases ENaC expression at the cell surface. To investigate the underlying mechanisms, we proteolytically cleaved/activated ENaC at the cell surface and then quantitated the rate of disappearance of cleaved channels using electrophysiological and biochemical assays. We found that cleaved ENaC channels were rapidly removed from the cell surface. Deletion or mutation of the Nedd4-2 binding motifs in ␣, ␤, and ␥ENaC dramatically reduced endocytosis, whereas a mutation that disrupts a YXXØ endocytosis motif had no effect. ENaC endocytosis was also decreased by silencing of Nedd4-2 and by expression of a dominant negative Nedd4-2 construct. Conversely, Nedd4-2 overexpression increased ENaC endocytosis in human embryonic kidney 293 cells but had no effect in Fischer rat thyroid epithelia. In addition to its effect on endocytosis, Nedd4-2 also increased the rate of degradation of the cell surface pool of cleaved ␣ENaC. Together the data indicate that Nedd4-2 reduces ENaC surface expression by altering its trafficking at two distinct sites in the endocytic pathway, inducing endocytosis of cleaved channels and targeting them for degradation.The epithelial Na ϩ channel ENaC forms a pathway for Na ϩ reabsorption across epithelia, including the kidney, lung, and colon. Therefore, it plays a critical role in Na ϩ homeostasis and blood pressure control (reviewed in Refs. 1 and 2). Defects in ENaC function or regulation cause inherited forms of hypertension and hypotension (3) and may contribute to the pathogenesis of lung disease in cystic fibrosis (4).ENaC is regulated by Nedd4-2, a HECT domain E3 ubiquitin ligase that decreases ENaC expression at the cell surface (5-7). This regulation requires the binding of Nedd4-2 WW domains to PY motifs (PPPXYXXL) located in the C terminus of each of the three subunits that form the channel (␣, ␤, and ␥ENaC). Mutations in the PY motifs of ␤ or ␥ENaC disrupt binding, causing Liddle syndrome (8 -10). In this inherited form of hypertension, increased expression of ENaC at the cell surface results in excessive renal Na ϩ reabsorption (7,8,11). Binding is also modulated by aldosterone and vasopressin via serum and glucocorticoid-regulated kinase and protein kinase A, respectively; both kinases phosphorylate Nedd4-2, which reduces its binding to ENaC (6,12,13). However, the mechanism by which Nedd4-2 reduces ENaC surface expression is uncertain. It is possible that Nedd4-2 regulates ENaC trafficking in the biosynthetic pathway, targeting it for degradation in the proteasome. Consistent with this model, localization of Nedd4-2 at the cell surface is not required for Nedd4-2 to inhibit ENaC (14). Moreover, proteasome inhibitors decrease ENaC degradation (15-17) and increase ENaC surface expression.2 Alternatively, Nedd4-2 could regulate ENaC in the endocytic pathway, altering ENaC endocytosis and/or...

Liddle's syndrome, an inherited form of hypertension, is caused by mutations that delete or disrupt a C-terminal PY motif in the epithelial Na ؉ channel (ENaC). Previous work indicates that these mutations increase expression of ENaC at the cell surface by disrupting its binding to Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. However, it remains uncertain whether this mechanism alone is responsible; increased activity of ENaC channels could also contribute to excessive Na ؉ transport in Liddle's syndrome. ENaC activity is controlled in part by its cleavage state; proteolytic cleavage produces channels with a high openstate probability, whereas uncleaved channels are inactive. Here, we found that Liddle's syndrome mutations have two distinct effects of ENaC surface expression, both of which contribute to increased Na ؉ transport. First, these mutations increased ENaC expression at the cell surface; second, they increased the fraction of ENaC at the cell surface that was cleaved (active). This disproportionate increase in cleavage was reproduced by expression of a dominant-negative Nedd4-2 or mutation of ENaC ubiquitination sites, interventions that disrupt ENaC endocytosis and lysosomal degradation. Conversely, overexpression of Nedd4-2 had the opposite effect, decreasing the fraction of cleaved ENaC at the cell surface. Thus, the data not only suggest that Nedd4-2 regulates epithelial Na ؉ transport in part by controlling the relative expression of cleaved and uncleaved ENaC at the cell surface but also provide a mechanism by which Liddle's syndrome mutations alter ENaC activity.amiloride ͉ hypertension ͉ protease ͉ sodium

Na؉ transport across epithelia is mediated in part by the epithelial Na ؉ channel ENaC. Previous work indicates that Na ؉ is an important regulator of ENaC, providing a negative feedback mechanism to maintain Na ؉ homeostasis. ENaC is synthesized as an inactive precursor, which is activated by proteolytic cleavage of the extracellular domains of the ␣ and ␥ subunits. Here we found that Na ؉ regulates ENaC in part by altering proteolytic activation of the channel. When the Na ؉ concentration was low, we found that the majority of ENaC at the cell surface was in the cleaved/active state. As Na ؉ increased, there was a dosedependent decrease in ENaC cleavage and, hence, ENaC activity. This Na ؉ effect was dependent on Na ؉ permeation; cleavage was increased by the ENaC blocker amiloride and by a mutation that decreases ENaC activity (␣ H69A ) and was reduced by a mutation that activates ENaC (␤ S520K ). Moreover, the Na ؉ ionophore monensin reversed the effect of the inactivating mutation (␣ H69A ) on ENaC cleavage, suggesting that intracellular Na ؉ regulates cleavage. Na ؉ did not alter activity of Nedd4-2, an E3 ubiquitin ligase that modulates ENaC cleavage, but Na ؉ reduced ENaC cleavage by exogenous trypsin. Our findings support a model in which intracellular Na ؉ regulates cleavage by altering accessibility of ENaC cleavage sites to proteases and provide a molecular explanation for the earlier observation that intracellular Na ؉ inhibits Na ؉ transport via ENaC (Na ؉ feedback inhibition).Transport of Na ϩ across epithelia is critical to maintain Na ϩ homeostasis. Defects in Na ϩ transport cause inherited forms of hypertension (e.g. Liddle syndrome) and hypotension (pseudohypoaldosteronism type 1) (1). In the distal nephron of the kidney, lung, colon, and sweat duct, transport is mediated by the epithelial Na ϩ channel ENaC, a heterotrimer composed of homologous ␣, ␤, and ␥ subunits (2-5). ENaC is located at the apical membrane, where it functions as a conduit for Na ϩ to enter the cell (reviewed in Refs. 6 and 7). Coupled with Na ϩ exit at the basolateral membrane via the Na ϩ -K ϩ -ATPase, ENaC provides a pathway for Na ϩ reabsorption across these tissues. Although Na ϩ is the permeant ion for this pathway, Na ϩ also regulates its own transport through negative feedback mechanisms (8 -16). Under conditions of Na ϩ /volume excess, the Na ϩ concentration in the distal nephron is high (Ͼ50 mM) (17), which inhibits ENaC in order to minimize Na ϩ reabsorption. Conversely, under conditions of Na ϩ /volume depletion, the Na ϩ concentration is low (ϳ1 mM) (17), which activates ENaC to maximize Na ϩ reabsorption. By countering changes in Na ϩ delivery to the distal nephron, this pathway functions to maintain Na ϩ homeostasis. Previous work indicates that both extracellular Na ϩ ("Na ϩ self-inhibition" (10, 11, 18)) and intracellular Na ϩ ("Na ϩ feedback inhibition" (12-16)) regulate ENaC activity. However, an understanding of the underlying mechanisms has remained elusive.In this work, we tested the hypothesis that N...

Objectives: Targeting tumor necrosis factor (TNF) with biologic agents, such as infliximab and adalimumab, is a widely used and effective therapeutic strategy in inflammatory bowel disease (IBD). Unfortunately, a significant number of patients fail to respond or lose response over time to these agents. Previous studies have defined multiple complex roles for canonical NF-κB signaling in the pathogenesis of IBD. However, preliminary evidence suggests that the lesser defined noncanonical NF-κB signaling pathway also contributes to disease pathogenesis and response to anti-TNF agents. The objective of this study was to evaluate this hypothesis in Crohn’s disease (CD) and ulcerative colitis (UC) patients.Design: A total of 27 subjects with IBD (19 with CD and 8 with UC) and 15 control subjects were tested. Clinical criteria, patient history, and endoscopic disease activity were factors used to categorize patients and define therapeutic response. Biopsy specimens were collected during colonoscopy and expression was determined for 88 target genes known to be associated with noncanonical NF-κB signaling and IBD.Results: Noncanonical NF-κB signaling was significantly upregulated in IBD patients and was associated with increased gastrointestinal inflammation, epithelial cell death, lymphocyte migration, and Nod-like receptor signaling. Furthermore, noncanonical NF-κB signaling was further upregulated in patients unresponsive to anti-TNF agents and was suppressed in responsive patients. MAP3K14, NFKB2, CCL19, CXCL12, and CXCL13 were significantly dysregulated, as were genes that encode pathway regulators, such as CYLD, NLRP12, and BIRC2/3.Conclusion: Our study identifies a previously uncharacterized role for the understudied noncanonical NF-κB signaling pathway in the pathogenesis of IBD and anti-TNF therapy responsiveness. The genes and pathways identified may ultimately prove useful in IBD management and could potentially be used as biomarkers of drug response.