Interleukin-1beta decreases expression of the epithelial sodium channel alpha-subunit in alveolar epithelial cells via a p38 MAPK-dependent signaling pathway

Acute lung injury (ALI) is a devastating syndrome characterized by diffuse alveolar damage, elevated airspace levels of pro-inflammatory cytokines, and flooding of the alveolar spaces with protein-rich edema fluid. Interleukin-1beta (IL-1beta) is one of the most biologically active cytokines in the distal airspaces of patients with ALI. IL-1beta has been shown to increase lung epithelial and endothelial permeability. In this study, we hypothesized that IL-1beta would decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we measured the effects of IL-1beta on transepithelial current, resistance, and sodium transport in primary cultures of alveolar epithelial type II (ATII) cells. IL-1beta significantly reduced the amiloride-sensitive fraction of the transepithelial current and sodium transport across rat ATII cell monolayers. Moreover, IL-1beta decreased basal and dexamethasone-induced epithelial sodium channel alpha-subunit (alpha ENaC) mRNA levels and total and cell-surface protein expression. The inhibitory effect of IL-1beta on alpha ENaC expression was mediated by the activation of p38 MAPK in both rat and human ATII cells and was independent of the activation of alpha v beta6 integrin and transforming growth factor-beta. These results indicate that IL-1beta may contribute to alveolar edema in ALI by reducing distal lung epithelial sodium absorption. This reduction in ion and water transport across the lung epithelium is in large part due to a decrease in alpha ENaC expression through p38 MAPK-dependent inhibition of alpha ENaC promoter activity and to an alteration in ENaC trafficking to the apical membrane of ATII cells.     

Anionic phospholipids regulate native and expressed epithelial sodium channel (ENaC).

Using patch clamp techniques, we found that the epithelial sodium channel (ENaC) activity in the apical membrane of A6 distal nephron cells showed a sudden rundown beginning at 4 min after forming the inside-out configuration. This sudden rundown was prevented by addition of anionic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP(2)), phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), and phosphatidylserine (PS) to the "cytoplasmic" bath. Conversely, chelation of endogenous PIP(2) with anti-PIP(2) antibody, hydrolysis of PIP(2) with either exogenous phospholipase C (PLC) or activation of endogenous PLC by extracellular ATP, or application of the positively charged molecule, poly-L-lysine, accelerated channel rundown. However, neutral phosphatidylcholine had no effect on ENaC activity. By two-electrode voltage clamp recordings, we demonstrated that PIP(2) and PIP(3) significantly increased amiloride-sensitive current in Xenopus oocytes injected with cRNAs of rat alpha-, beta-, and gamma-ENaC. However, PIP(2) and PIP(3) did not affect surface expression of ENaC, indicating that PIP(2) and PIP(3) regulate ENaC at the level of the inner plasma membrane through a mechanism that is independent of ENaC trafficking. These data suggest that anionic phospholipids may mediate the regulation of ENaC by PLC- or phosphoinositide 3-kinase-coupled receptors.     

Rab proteins regulate epithelial sodium channel activity in colonic epithelial HT-29 cells

ENaC, the sodium-selective amiloride-sensitive epithelial channel, mediates electrogenic sodium re-absorption in tight epithelia and is deeply associated with human hypertension. The ENaC expression at plasma membrane requires the regulated transport, processing, and macromolecular assembly in a defined and highly compartmentalized manner. Ras-related Rab GTPases regulate intracellular trafficking during endocytosis, regulated exocytosis, and secretion. To evaluate the role of these proteins in regulating amiloride-sensitive sodium channel activity, multiple Rab isoforms 3, 5, 6, and Rab27a were expressed in HT-29 cells. Rab3 and Rab27a inhibited ENaC currents, while the expression of other Rab isoforms failed to elicit any statistically significant effect on amiloride-sensitive currents. The immunoprecipitation experiments suggest protein-protein interaction of Rab3 and Rab27a with epithelial sodium channel. Biotinylation studies revealed that modulation of ENaC function is due to the reduced apical expression of channel proteins. Study also indicates that Rabs do not appear to affect the steady-state level of total cellular ENaC. Alternatively, introduction of isoform-specific small inhibitory RNA (SiRNA) reversed the Rab-dependent inhibition of amiloride-sensitive currents. These observations point to the involvement of multiple Rab proteins in ENaC transport through intracellular routes like exocytosis, recycling from ER to plasma membrane or degradation and thus serve as potential target for human hypertension.     

Modulation of Na+ transport and epithelial sodium channel expression by protein kinase C in rat alveolar epithelial cells

Although the amiloride-sensitive epithelial sodium channel (ENaC) plays an important role in the modulation of alveolar liquid clearance, the precise mechanism of its regulation in alveolar epithelial cells is still under investigation. Protein kinase C (PKC) has been shown to alter ENaC expression and activity in renal epithelial cells, but much less is known about its role in alveolar epithelial cells. The objective of this study was to determine whether PKC activation modulates ENaC expression and transepithelial Na+ transport in cultured rat alveolar epithelial cells. Alveolar type II cells were isolated and cultured for 3 to 4 d before they were stimulated with phorbol 12-myristate 13-acetate (PMA 100 nmol/L) for 4 to 24 h. PMA treatment significantly decreased alpha, beta, and gammaENaC expression in a time-dependent manner, whereas an inactive form of phorbol ester had no apparent effect. This inhibitory action was seen with only 5-min exposure to PMA, which suggested that PKC activation was very important for the reduction of alphaENaC expression. The PKC inhibitors bisindolylmaleimide at 2 micromol/L and G?6976 at 2 micromol/L diminished the PMA-induced suppression of alphaENaC expression, while rottlerin at 1 micromol/L had no effect. PMA elicited a decrease in total and amiloride-sensitive current across alveolar epithelial cell monolayers. This decline in amiloride-sensitive current was not blocked by PKC inhibitors except for a partial inhibition with bisindolylmaleimide. PMA induced a decrease in rubidium uptake, indicating potential Na+-K+-ATPase inhibition. However, since ouabain-sensitive current in apically permeabilized epithelial cells was similar in PMA-treated and control cells, the inhibition was most probably related to reduced Na+ entry at the apical surface of the cells. We conclude that PKC activation modulates ENaC expression and probably ENaC activity in alveolar epithelial cells. Ca2+-dependent PKC is potentially involved in this response.     

Phosphatidylinositol 4-phosphate 5-kinase reduces cell surface expression of the epithelial sodium channel (ENaC) in cultured collecting duct cells

Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.     

Chloride and potassium channel function in alveolar epithelial cells

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na(+) transport and, in particular, the role of amiloride-sensitive Na(+) channels in the apical membrane and the Na(+)-K(+)-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl(-) and K(+) channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl(-) and K(+) channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.     

Surfactant protein D regulates the cell surface expression of alveolar macrophage beta(2)-integrins

The beta(2)-integrin receptors (CD11a/CD18, CD11b/CD18, and CD11c/CD18) are expressed on the surface of alveolar macrophages and are important for the phagocytic clearance of pathogens. In the present study, we demonstrate that surfactant protein D (SP-D) modulates surface expression of CD11b and CD11c, but not CD11a or CD18, on alveolar macrophages. While cell surface receptors were reduced, CD11b and CD11c mRNAs were increased by SP-D deficiency. CCSP-rtTA(+)/(tetO)(7)-rSPD(+)/SP-D(-/-) mice, which conditionally express SP-D in the lung, were used to study the kinetics and reversibility of beta(2)-integrin receptors in response to changes in alveolar SP-D. Surface CD11b and CD11c were reduced on the alveolar macrophages within 3 days of SP-D deficiency and were restored with 3 days for CD11b and 7 days for CD11c of repletion of SP-D. SP-D deficiency caused a loss of cellular CD11b and CD11c content, indicating that the decrease in total cell content of the receptors was related to degradation rather than to redistribution of the receptor within the macrophage. CD11b and CD11c staining colocalized with Lamp-1 during SP-D deficiency, supporting the concept that reduced macrophage receptor levels resulted from increased lysosomal trafficking. Hydroxychloroquine, a lysomotropic agent, prevented the reduction of cellular and surface CD11b and CD11c. SP-D regulates surface CD11b and CD11c levels on the alveolar macrophage by modulating receptor trafficking, providing a mechanism by which SP-D mediates phagocytic activity in the alveolar macrophage.     

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.     

Role of intracellular Ca2+ in the expression of the amiloride-sensitive epithelial sodium channel

The amiloride-sensitive epithelial sodium channel (ENaC), a multimeric plasma membrane protein composed of alpha-, beta-, and gamma-ENaC subunits, mediates Na(+) reabsorption in epithelial tissues, including the distal nephron, colon, lung, and secretory glands, and plays a critical role in pathophysiology of essential hypertension and cystic fibrosis (CF). The function of ENaC is tightly regulated by signals elicited by aldosterone, vasopressin, agents that increase intracellular cAMP levels, ions, ion channels, G-protein-coupled mechanisms, and cytoskeletal proteins. In this paper, the effects of Ca(2+) on the expression of the human ENaC subunits expressed in human embryonic kidney cells (HEK-293 cells) were examined. Incubation of cells with increased extracellular Ca(2+) and treatment of cells with A23187 and thapsigargin stimulated the expression of the monomeric ENaC subunits. Treatment of cells with Ca(2+)-chelating agents, EGTA and BAPTA-AM, reduced the levels of ENaC subunit expression. The pulse-chase experiments suggested that a rise in the intracellular Ca(2+) increases the ENaC subunit expression. Immunoblot analysis using the anti-ubiquitin antibody indicated that ENaC undergoes ubiquitination. A correlation between the processes that regulate ENaC function with the intracellular Ca(2+) was discussed.     

Calreticulin positively regulates the expression and function of epithelial sodium channel

Epithelial sodium channel (ENaC) is a heteromultimeric Na⁺ channel at the apical membrane in the kidney, colon, and lung. Because ENaC plays a crucial role in regulating Na⁺ absorption and extracellular fluid volume, its dysregulation causes severe phenotypes including hypertension, hypokalemia, and airway obstruction. Despite the importance of ENaC, its protein quality control mechanism remains less established. Here we firstly show the role of calreticulin (CRT), a lectin-like molecular chaperone in the endoplasmic reticulum (ER), on the regulation of ENaC. Overexpression and knockdown analyses clearly indicated that CRT positively affects the expression of each ENaC subunit (α, β and γ). CRT overexpression also up-regulated the cell surface expression of α-, β- and γ-ENaC. Moreover, we found that CRT directly interacts with each ENaC subunit. Although CRT knockdown did not affect the de novo synthesis of ENaC subunits, CRT overexpression decreased α-, β- and γ-ENaC expression in the detergent (RIPA)-insoluble fraction, suggesting that CRT enhanced the solubility of ENaC subunits. Consistent with the increased intracellular and cell surface expression of ENaC subunits, increased channel activity of ENaC was also observed upon overexpression of CRT. Our study thus identifies CRT as an ER chaperone that regulates ENaC expression and function.     

Enhancement of alveolar epithelial sodium channel activity with decreased cystic fibrosis transmembrane conductance regulator expression in mouse lung

We sought to establish whether the cystic fibrosis transmembrane conductance regulator (CFTR) regulates the activity of amiloride-sensitive sodium channels (ENaC) in alveolar epithelial cells of wild-type, heterozygous (Cftr(+/-)), knockout (Cftr(-/-)), and ΔF508-expressing mice in situ. RT-PCR studies confirmed the presence of CFTR message in freshly isolated alveolar type II (ATII) cells from wild-type mice. We patched alveolar type I (ATI) and ATII cells in freshly prepared lung slices from these mice and demonstrated the presence of 4-pS ENaC channels with the following basal open probabilities (P(o)): wild-type=0.21 ± 0.015: Cftr(+/-)=0.4 ± 0.03; ΔF508=0.55 ± 0.01; and Cftr(-/-)=and 0.81 ± 0.016 (means ± SE; n ≥ 9). Forskolin (5 μM) or trypsin (2 μM), applied in the pipette solution, increased the P(o) and number of channels in ATII cells of wild-type, Cftr(+/-), and ΔF508, but not in Cftr(-/-) mice, suggesting that the latter were maximally activated. Western blot analysis showed that lungs of all groups of mice had similar levels of α-ENaC; however, lungs of Cftr(+/-) and Cftr(-/-) mice had significantly higher levels of an α-ENaC proteolytic fragment (65 kDa) that is associated with active ENaC channels. Our results indicate that ENaC activity is inversely correlated to predicted CFTR levels and that CFTR heterozygous and homozygous mice have higher levels of proteolytically processed ENaC fragments in their lungs. This is the first demonstration of functional ENaC-CFTR interactions in alveolar epithelial cells in situ.     

Heterologous expression of a mammalian epithelial sodium channel in yeast

The alpha and beta subunits of the amiloride-sensitive rat epithelial sodium channel (alpha beta ENaC) were expressed in the yeast Saccharomyces cerevisiae. We used a combination of yeast strains, including a mutant in the secretory pathway (sec6), and Western blotting techniques, to show that alpha beta ENaC was synthesized and targeted through the secretory system to the plasma membrane. Yeasts expressing alpha beta ENaC were more sensitive to salt than the parent strain. In addition, amiloride, a specific blocker of ENaC, was found to suppress salt sensitivity in the yeast strain expressing alpha beta ENaC.     

K(+) channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na(+) absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K(ATP) K(+) channel activities exerts sustained control in Na(+) transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K(+) channels, and 2) to determine the physiological impact of K(+) channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K(ATP) channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24h) increased α-ENaC expression, similarly to K(ATP) activation by pinacidil. Conversely, pharmacological KvLQT1 and K(ATP) inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K(+) channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K(ATP) activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K(+) channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K(+) channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

K+ channels regulate ENaC expression via changes in promoter activity and control fluid clearance in alveolar epithelial cells

Active Na⁺ absorption by alveolar ENaC is the main driving force of liquid clearance at birth and lung edema resorption in adulthood. We have demonstrated previously that long-term modulation of KvLQT1 and K_ATP K⁺ channel activities exerts sustained control in Na⁺ transport through the regulation of ENaC expression in primary alveolar type II (ATII) cells. The goal of the present study was: 1) to investigate the role of the α-ENaC promoter, transfected in the A549 alveolar cell line, in the regulation of ENaC expression by K⁺ channels, and 2) to determine the physiological impact of K⁺ channels and ENaC modulation on fluid clearance in ATII cells. KvLQT1 and K_ATP channels were first identified in A549 cells by PCR and Western blotting. We showed, for the first time, that KvLQT1 activation by R-L3 (applied for 24 h) increased α-ENaC expression, similarly to K_ATP activation by pinacidil. Conversely, pharmacological KvLQT1 and K_ATP inhibition or silencing with siRNAs down-regulated α-ENaC expression. Furthermore, K⁺ channel blockers significantly decreased α-ENaC promoter activity. Our results indicated that this decrease in promoter activity could be mediated, at least in part, by the repressor activity of ERK1/2. Conversely, KvLQT1 and K_ATP activation dose-dependently enhanced α-ENaC promoter activity. Finally, we noted a physiological impact of changes in K⁺ channel functions on ERK activity, α-, β-, γ-ENaC subunit expression and fluid absorption through polarized ATII cells. In summary, our results disclose that K⁺ channels regulate α-ENaC expression by controlling its promoter activity and thus affect the alveolar function of fluid clearance.     

Modulation of epithelial sodium channel trafficking and function by sodium 4-phenylbutyrate in human nasal epithelial cells

Sodium 4-phenylbutyrate (4-PBA) has been shown to correct the cellular trafficking of several mutant or nonmutant plasma membrane proteins such as cystic fibrosis transmembrane conductance regulator through the expression of 70-kDa heat shock proteins. The objective of the study was to determine whether 4-PBA may influence the functional expression of epithelial sodium channels (ENaC) in human nasal epithelial cells (HNEC). Using primary cultures of HNEC, we demonstrate that 4-PBA (5 mm for 6 h) markedly stimulated amiloride-sensitive sodium channel activity and that this was related to an increased abundance of alpha-, beta-, and gamma-ENaC subunits in the apical membrane. The increase in ENaC cell surface expression (i) was due to insertion of newly ENaC subunits as determined by brefeldin A experiments and (ii) was not associated with cell surface retention of ENaC subunits because endocytosis of ENaC subunits was unchanged. In addition, we find that ENaC co-immunoprecipitated with the heat shock protein constitutively expressed Hsc70, that has been reported to modulate ENaC trafficking, and that 4-PBA decreased Hsc70 protein level. Finally, we report that in cystic fibrosis HNEC obtained from two cystic fibrosis patients, 4-PBA increased functional expression of ENaC as demonstrated by the increase in amiloride-sensitive sodium transport and in alpha-, beta-, and gamma-ENaC subunit expression in the apical membrane. Our results suggest that in HNEC, 4-PBA increases the functional expression of ENaC through the insertion of new alpha-, beta-, and gamma-ENaC subunits into the apical membrane and also suggest that 4-PBA could modify ENaC trafficking by reducing Hsc70 protein expression.     

Expression and functional implications of CCR2 expression on murine alveolar epithelial cells

Acute lung injury results in damage to the alveolar epithelium, leading to leak of proteins into the alveolar space and impaired gas exchange. Lung function can be restored only if the epithelial layer is restored. The process of reepithelialization requires migration of lung epithelial cells to cover denuded basement membranes. The factors that control the migration of lung epithelial cells are incompletely understood. We examined isolated murine type II alveolar epithelial cells (AECs) for expression of CC chemokine receptor 2 (CCR2) and functional consequences of the binding of the main CCR2 ligand monocyte chemoattractant protein-1 (MCP-1). We found that primary AECs bound MCP-1 and expressed CCR2 mRNA. These cells demonstrated functional consequences of CCR2 expression with migration in response to MCP-1 in chemotaxis/haptotaxis assays. Primary AECs cultured from mice lacking CCR2 did not respond to MCP-1. Monolayers of AECs lacking CCR2 demonstrated delayed closure of mechanical wounds compared with AEC monolayers expressing CCR2. Delayed closure of mechanical wounds of wild-type AECs was also demonstrated in the presence of anti-MCP-1 antibody. These data demonstrate for the first time that AECs express CCR2 and are capable of using this receptor for chemotaxis and healing of wounds. CCR2-MCP-1 interactions may be important in the process of reepithelialization after lung injury.