ERp29 regulates epithelial sodium channel functional expression by promoting channel cleavage.

The epithelial Na(+) channel (ENaC) plays a key role in the regulation of blood pressure and airway surface liquid volume. ERp29 is a 29-kDa thioredoxin-homologous endoplasmic reticulum (ER) protein that has only a single cysteine instead of the usual thioredoxin CXXC motif. Our group previously demonstrated that ERp29 promotes biogenesis of the cystic fibrosis transmembrane conductance regulator (CFTR). On the basis of similarities of CFTR and ENaC trafficking, we hypothesized that ERp29 would also regulate ENaC biogenesis and functional expression. In epithelial cells, overexpression of wild-type (wt) ERp29 increased ENaC functional expression [amiloride-sensitive short-circuit current (Isc)] in Ussing chamber experiments, as well as the abundance of the cleaved form of γ-ENaC in whole cell lysates. In contrast, siRNA-mediated depletion of ERp29 or overexpression of a mutant ERp29 lacking its single cysteine (C157S ERp29) decreased ENaC functional expression. Cells in which wt ERp29 was overexpressed had a smaller fractional increase in amiloride-sensitive Isc when trypsin was applied to the apical surface to activate uncleaved ENaC, while cells in which C157S ERp29 was overexpressed or ERp29 was depleted had a significantly greater fractional increase in amiloride-sensitive Isc in response to trypsin. Interestingly, these observations were not associated with altered expression of ß-ENaC at the apical surface. Instead, ERp29 appeared to promote the interaction of ß-ENaC with the Sec24D cargo recognition component of the coat complex II ER exit machinery. Together, these data support the hypothesis that ERp29 directs ENaC toward the Golgi, where it undergoes cleavage during its biogenesis and trafficking to the apical membrane.

IL-13-induced changes in endogenous glucocorticoid metabolism in the lung regulate the proasthmatic response.

Endogenous glucocorticoid (GC) activation is regulated by the intracellular GC-activating and -inactivating enzymes 11ß-hydroxysteroid dehydrogenase (11ß-HSD)1 and 11ß-HSD2, respectively, that catalyze interconversion of inert cortisone and its bioactive metabolite cortisol. Because endogenous GCs are critically implicated in suppressing the asthmatic state, this study examined the roles of the 11ß-HSD enzymes in regulating GC activation and bronchoprotection during proasthmatic stimulation. Airway hyperresponsiveness to methacholine and inflammation were assessed in rabbits following inhalation of the proasthmatic/proinflammatory cytokine IL-13 with and without pretreatment with the 11ß-HSD inhibitor carbenoxolone (CBX). Additionally, IL-13-induced changes in 11ß-HSD isozyme expression and GC metabolism were examined in epithelium-intact and -denuded tracheal segments and peripheral lung tissues. Finally, the effects of pretreatment with CBX or 11ß-HSD2-targeted siRNAs were investigated with respect to cortisol prevention of IL-13-induced airway constrictor hyperresponsiveness and eotaxin-3 production by airway epithelial cells. IL-13-exposed rabbits exhibited airway hyperresponsiveness, inflammation, and elevated bronchoalveolar lung fluid levels of eotaxin-3. These responses were inhibited by pretreatment with CBX, suggesting a permissive proasthmatic role for 11ß-HSD2. Supporting this concept, extended studies demonstrated that 1) IL-13-treated tracheal epithelium and peripheral lung tissues exhibit upregulated 11ß-HSD2 activity, 2) the latter impairs cortisone-induced cortisol accumulation and the ability of administered cortisol to prevent both IL-13-induced heightened airway contractility and eotaxin-3 release from epithelial cells, and 3) these proasthmatic responses are prevented by cortisol administration in the presence of 11ß-HSD2 inhibition. Collectively, these data demonstrate that the proasthmatic effects of IL-13 are enabled by impaired endogenous GC activation in the lung that is attributed to upregulation of 11ß-HSD2 in the pulmonary epithelium.

Regulation of endogenous ENaC functional expression by CFTR and ΔF508-CFTR in airway epithelial cells.

The functional expression of the epithelial sodium channel (ENaC) appears elevated in cystic fibrosis (CF) airway epithelia, but the mechanism by which this occurs is not clear. We tested the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) alters the trafficking of endogenously expressed human ENaC in the CFBE41o⁻ model of CF bronchial epithelia. Functional expression of ENaC, as defined by amiloride-inhibited short-circuit current (I(sc)) in Ussing chambers, was absent under control conditions but present in CFBE41o⁻ parental and ΔF508-CFTR-overexpressing cells after treatment with 1 µM dexamethasone (Dex) for 24 h. The effect of Dex was mimicked by incubation with the glucocorticoid hydrocortisone but not with the mineralocorticoid aldosterone. Application of trypsin to the apical surface to activate uncleaved, "near-silent" ENaC caused an additional increase in amiloride-sensitive I(sc) in the Dex-treated cells and was without effect in the control cells, suggesting that Dex increased ENaC cell surface expression. In contrast, Dex treatment did not stimulate amiloride-sensitive I(sc) in CFBE41o⁻ cells that stably express wild-type (wt) CFTR. CFBE41o⁻ wt cells also had reduced expression of α- and γ-ENaC compared with parental and ΔF508-CFTR-overexpressing cells. Furthermore, application of trypsin to the apical surface of Dex-treated CFBE41o⁻ wt cells did not stimulate amiloride-sensitive I(sc), suggesting that ENaC remained absent from the surface of these cells even after Dex treatment. We also tested the effect of trafficking-corrected ΔF508-CFTR on ENaC functional expression. Incubation with 1 mM 4-phenylbutyrate synergistically increased Dex-induced ENaC functional expression in ΔF508-CFTR-overexpressing cells. These data support the hypothesis that wt CFTR can regulate the whole cell, functional, and surface expression of endogenous ENaC in airway epithelial cells and that absence of this regulation may foster ENaC hyperactivity in CF airway epithelia.

Th2 cytokine-induced upregulation of 11beta-hydroxysteroid dehydrogenase-1 facilitates glucocorticoid suppression of proasthmatic airway smooth muscle function.

The anti-inflammatory actions of endogenous glucocorticoids (GCs) are regulated by the activities of the GC-activating and -inactivating enzymes, 11beta-hydroxysteroid dehydrogenase (11beta-HSD)-1 and 11beta-HSD2, respectively, that catalyze the interconversion of the inert GC, cortisone, and its bioactive derivative, cortisol. Proinflammatory cytokines regulate 11beta-HSD1 expression in various cell types and thereby modulate the bioavailability of cortisol to the glucocorticoid receptor (GR). Since endogenous GCs reportedly attenuate the airway asthmatic response to allergen exposure, we investigated whether airway smooth muscle (ASM) exhibits cytokine-induced changes in 11beta-HSD1 expression that enable the ASM to regulate its own bioavailability of GC and, accordingly, the protective effect of GR signaling on airway function under proasthmatic conditions. Human ASM cells exposed to the primary proasthmatic T helper type 2 (Th2) cytokine, IL-13, exhibited upregulated expression of 11beta-HSD1, an effect that was attributed to activation of the transcription factor, AP-1, coupled to MAPK signaling via the ERK1/2 and JNK pathways. The induction of 11beta-HSD1 expression and its oxoreductase activity by IL-13 (also IL-4) served to amplify the conversion of cortisone to cortisol by the cytokine-exposed ASM and, hence, heighten GR-mediated transcriptional activation. Extended studies demonstrated that this amplified 11beta-HSD1-dependent GC activation enabled physiologically relevant concentrations of cortisone to exert enhanced protection of ASM tissues from the proasthmatic effects of IL-13 on ASM constrictor and relaxation responsiveness. Collectively, these novel findings identify a Th2 cytokine-driven homeostatic feedback mechanism in ASM that enhances its responsiveness to endogenous GCs by upregulating 11beta-HSD1 activity, thereby curtailing the adverse effects of the proasthmatic cytokine on airway function.

LXA4 stimulates ZO-1 expression and transepithelial electrical resistance in human airway epithelial (16HBE14o-) cells.

Lipoxin A(4) (LXA(4)) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, LXA(4) production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of LXA(4) on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to LXA(4) (100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. LXA(4) (100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 (LXA(4) receptor antagonist). LXA(4) also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that LXA(4) plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the LXA(4) receptor.

RhoA GTPase regulates M-cadherin activity and myoblast fusion.

The Rho family of GTP-binding proteins plays critical roles during myogenesis induction. To elucidate their role later during myogenesis, we have analyzed RhoA function during myoblast fusion into myotubes. We find that RhoA activity is rapidly and transiently increased when cells are shifted into differentiation medium and then is decreased until myoblast fusion. RhoA activity must be down-regulated to allow fusion, because expression of a constitutively active form of RhoA (RhoAV14) inhibits this process. RhoAV14 perturbs the expression and localization of M-cadherin, a member of the Ca2+-dependent cell-cell adhesion molecule family that has an essential role in skeletal muscle cell differentiation. This mutant does not affect N-cadherin and other proteins involved in myoblast fusion, beta1-integrin and ADAM12. Active RhoA induces the entry of M-cadherin into a degradative pathway and thus decreases its stability in correlation with the monoubiquitination of M-cadherin. Moreover, p120 catenin association with M-cadherin is decreased in RhoAV14-expressing cells, which is partially reverted by the inhibition of the RhoA effector Rho-associated kinase ROCK. ROCK inhibition also restores M-cadherin accumulation at the cell-cell contact sites. We propose that the sustained activation of the RhoA pathway inhibits myoblast fusion through the regulation of p120 activity, which controls cadherin internalization and degradation.