Modulation of epithelial sodium channel in human alveolar epithelial cells by lipoxin A 4 through AhR-cAMP-dependent pathway

Purpose: To investigate the effect of lipoxin A4 (LXA4) on the expressions of protein and mRNA of alveolar epithelial sodium channel (ENaC) in normal and lipopolysaccharide (LPS)-stimulated A549 cells. Methods: A549 cell-lines were randomized into 11 groups (N = 8) and treated. EnaC level was evaluated by Western blot. Total RNA was extracted and reverse-transcribed and then levels of ENaC mRNA, cGMP and cAMP in the cells were determined. Results: LXA4 (10mol/L) increased the expressions of α-subunit of ENaC relative to LPS group. In addition, LXA4 significantly up-regulated the expression of mRNAs of α, β and γ subunits of ENaC (p < 0.01). The level of cAMP was increased in LXA4 group, but significantly reduced in LPS group relative to control group (p < 0.05). However, treatment with LXA4 annulled the increased cAMP concentration, compared with LPS group (p < 0.05) Conclusion: These results show that LXA4 influences ENaC up-regulation in normal and LPS stimulated A549 alveolar epithelial cells.


INTRODUCTION
Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) lead to respiratory failure and pulmonary edema [1].Alveolar epithelial injury is a major contributor to alveolar flooding.Injury to alveolar epithelium prevents reabsorption of edema fluid, a key step in the resolution of ALI/ARDS [1,2].Earlier studies showed that alveolar epithelial sodium channel (ENaC) and Na-K-ATPase play critical roles in reducing edema in ALI/ARDS [3,4].
The present study aims to test the hypothesis that LXA 4 increases ENaC protein and mRNA expressions in normal and LPS-stimulated A549 cells.In addition, it investigated LXA 4 receptors, ALX, AhR, cysLRT 1 and cysLRT 2 to determine the key receptors involved in up-regulation of ENaC function in A549 cells, as well as the levels of cAMP and cGMP in A549 cells.

EXPERIMENTAL Materials
Lipopolysaccharide (LPS; E. coil serotype 055: B5) and AhR inhibitor, α-naphthoflavone (ANF) were purchased from Sigma (St. Louis, MO).Lipoxin A 4 was obtained from Cayman Chemical Company (Ann Arbor, MI).CysLT2 receptor inhibitor, BAY-u9773 was from Bimol Company (Farmingdale, NY).RPMI Medium 1640 and FCS were purchased from Gibco (Grand Island, NY).Saline citrate-buffered streptomycin and penicillin were products of Invitrogen (CA).The α, β and γ subunits of anti-ENaC were products of Abcam Company (Cambridge, MA), while anti-β-actin was supplied by Santa Cruz Company (Santa Cruz, CA).ELISA kits for cAMP and cGMP were purchased from R&D systems (Minneapolis, MN).Rp-cAMP (cAMP inhibitor) was obtained from Biomol-Enzo Life Sciences (Farmingdale, NY), while BCA protein assay kits and RT-PCR kits were purchased from Thermo Scientific (Rockford, IL).Real-time PCR Master Mix (SYBR Green) was obtained from Toyobo (Japan).

Western blot for α, β and γ subunits of EnaC
Samples were lysed in standard RIPA lysis solution composed of 50 mM Tris buffer, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 4 mM sodium pyrophosphate, 5 mM sodium fluoride, 1 mM EDTA, 0.02 mM leupeptin and 1mM phenylmethyl sulfonylfluoride (PMSF) on ice for 30 min, and thereafter centrifuged for 10 min at 12,000 rpm.The protein contents of the supernatants were estimated using BCA protein assay kitx.The protein mixture from the supernatants (80 µg) were separately boiled to achieve denaturation, and then subjected to 10 % SDS-PAGE prior to transfer onto PVDF membranes.Following blocking for 2 h with 5% non-fat dry milk in TBS-T buffer, expression of the α subunit of ENaC was determined using primary anti-epithelial sodium channel alpha antibody (1:750).The expression of the β subunit was determined using primary anti-SCNN1B antibody (1:500), while the expression of γ subunit was measured using primary antiepithelial sodium channel gamma antibody (1:1000), and secondary goat anti-rabbit IgG.βactin was used as internal control.Detection of the bound antibody was carried out on X-ray films by enhanced chemiluminescence (ECL).

RNA isolation, reverse transcriptase and PCR
Total RNA was extracted using Trizol reagent according to manufacturer's protocol.One (1) microgram of RNA sample was reversetranscribed into complementary DNA (cDNA) using a RT-PCR kit on the MJ Mini Thermal Cycler (Bio-Rad, Hercules, CA, USA), according to the manufacturer's instructions.The primer sequences used for PCR amplification are in Table 1.
Then semi-quantitative analysis was performed by UVP-gel densitometry.

Quantitative real-time PCR
Levels of ENaC mRNA in A549 cells were determined with SYBR Prime Script Kit on the CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA).Primers used for amplification are in Table 2.
Data was analyzed as previously described [9] using GAPDH as a reference gene.

Statistical analysis
Data are expressed as mean ± SEM, and groups were compared by one-way ANOVA followed by Newman-Keuls multiple inter-group comparisons, using SPSS version 16.0 software.A probability of p < 0.05 was considered significant.

Dose-dependency of LXA 4 regulated ENaC expression in A549 cells stimulated with LPS
In all responses, only 10 -7 M LXA 4 increased ENaC α subunit expression when compared with LPS group (Figure 1A).However, the expressions of the β and γ subunits of ENaC were increased dose-dependently, peaking at 10 - 7 M LXA 4 (Figure 1B and C).Therefore, in subsequent experiments, the expression of ENaC in A549 cells treated with LPS was assessed using 10 -7 M LXA 4 .

Time-dependency of LXA 4 regulated ENaC expression in A549 cells stimulated with LPS
At 6 h, LPS significantly decreased the expressions of α, β and γ subunits of ENaC when compared with control group (p < 0.01); while LXA 4 significantly increased the expressions of these subunits, compared with control group (p < 0.01; Figure 2).At 12 h, the protein levels of the α, β and γ subunits were decreased in the LPS group, but elevated in the LPS + LXA 4 group relative to the LPS group (p < 0.05; Figure 3).At 24 h, the levels of the α, β and γ subunits of ENaC were significantly decreased in the LPS group, but increased in the LPS + LXA 4 group when compared with the LPS group (p < 0.01; Figure 4).Therefore, in subsequent experiments, the effect of LXA 4 on ENaC expression in LPS-stimulated A549 cells was assessed at 24 h.

Effect of LXA 4 on the ENaC mRNA expression in A549 cells stimulated with LPS
The cells were incubated with LPS (1μg/mL) with or without LXA 4 (10 -7 M) for 6 h.It was found that LXA 4 significantly up-regulated the expressions of mRNAs of α, β and γ subunits of ENaC (p < 0.01), and LPS decreased the mRNA level of ENaC α subunit relative to the control group (p < 0.05; Figure 5A).However, no significant change in mRNA expression of β and γ subunits was observed (p > 0.05; Figure 5B and C).Interestingly, the expressions of mRNAs of the β and γ subunits of ENaC were increased in LPS + LXA 4 group when compared with LPS group (p < 0.05), but not the ENaC α subunit.

Receptors of LXA 4 on A549 cells
As shown in Figure 6, LXA 4 receptor cysLT 2 and AhR mRNA were expressed in A549 cells.However, cysLT 1 and LXR mRNA were not expressed in A549 cells.

Effect of LXA 4 receptors on expression of ENaC protein in A549 cells following LPS exposure
Protein expressions of the α、β and γ subunits of ENaC were up-regulated by LXA 4 , but were blocked by the LXA 4 receptor inhibitor, ANF.However, BAY-u9773 was not inhibitory (Figure 7).

LXA 4 promoted ENaC expression
To test whether LXA 4 (10 -7 M) had an impact on cAMP and cGMP levels in A549 cells, we measured cAMP and cGMP concentration in A549 cells exposed to LPS (1 μg/mL) for 24 h by ELISA kits.It was found that cAMP concentration was increased in the LXA 4 group, but was reduced in the LPS group relative to control group (p< 0.05)., and treatment with LXA 4 reversed the increased cAMP concentration when compared with the LPS group (p < 0.05; Figure 8A).In addition, when BAY-u9773 (cysLT 2 antagonist, 3 μM) and ANF (AhR antagonist, 1nM) were used to treat A549 cells in the presence of LXA 4 for 24 h, cAMP concentration was decreased in the LXA 4 + ANF group relative to the LXA 4 group (p < 0.05), but not in the LXA 4 + BAY-u9773 group (Figure 8C).However, no significant change was seen in cGMP levels in these groups (Figure 8B & Figure 8D).

DISCUSSION
ALI/ARDS is a common, devastating clinical syndrome that affects large numbers of patients and has up to 40 % mortality [10].The current treatment for ALI/ARDS is aimed at removal of polymorphonuclear neutrophils, and alveolar fluid re-absorption.Cation and anion channels and ion transporters are involved in the clearance of alveolar fluid.Several studies have reported the key role of ENAC not only in the uptake of Na+ from the alveolar fluid but also in the clearance of the fluid itself [2,3,[11][12][13].In the present study, it has been clearly demonstrated that LXA 4 upregulated the expressions of the mRNAs and proteins of the α, β and γ subunits of ENAC in normal and LPS-treated human alveolar epithelial cells.These up-regulations were both dose-and time-dependent.These effects were blocked by the inhibitor of AhR and Rp-cAMP, an  10-7 M) and LPS (1 ug/mL) for 24 h.After incubation, the cells were harvested, and sonicated.ENaC α, β and γ subunits protein expression in the cell lysates were determined by western blotting using a specific antibody indication that LXA 4 enhanced the expression of ENaC through AhR-cAMP signaling pathway.
The expressions of the mRNAs and proteins of the α, β and γ subunits of ENAC were decreased by 1μg/mL LPS, but these decreases were dosedependently reversed by LXA 4, with maximal effect at 10 -7 M.These findings are in disagreement with recent reports which suggest that LXA 4 enhanced the expression of ENaC in A549 alveolar cells [10].Interestingly, it was found that 10 -6 M LXA 4 was less effective than 10 -7 M.This implies that the effect of LXA 4 was not concentration-dependent.It is also likely that at 10 -6 M, LXA 4 actually brought about decreased expression of ENaC.Thus, 10 -6 M LXA 4 was used in subsequent experiments.LXA 4 significantly improved the expressions of the α, β and γ subunits of ENAC in healthy A549 cells and in LPS-treated cells at 6, 12 and 24 h.The expressions in the LPS group at 24 h were much lower than those at 6 h and 12 h, suggesting a greater degree of cellular damage at 24 h than at any of the other periods.It has been reported that LXA 4 increased ENaC concentrations in primary rat alveolar type II epithelial cells exposed to LPS 24 h [21].Therefore, in subsequent studies, the effect of LXA 4 on ENaC expression in LPS-treated A549 cells was evaluated at 24 h.The results obtained in the present study show that LXA 4 regulates the expression of ENaC in healthy and pathological states.This is consistent with previous results which suggest that LXA 4 may be involved in endotoxin-induced ALI/ARDS [14].
The expression of the mRNA of the α subunit of ENAC was decreased following exposure to 1 μg/mL LPS for 6 h, but there were no significant changes in the expressions of mRNAs for the β and γ subunits.This shows that the expression of mRNA of the sodium channel was inhibited by LPS.In a previous study, it was reported that mouse alveolar epithelial cells treated with LPS for 8 h had significantly reduced expressions of mRNAs for the α, β and γ subunits of ENAC [27].The fact that LXA 4 increased mRNA expressions for α, β and γ of ENAC at 6 h indicates that LXA 4 protected the cells by enhancing ENaC gene translation.
LXs mediate their effects by interacting with one or more specific receptors such as ALX, CysLTR1, CysLTR2 and AhR.In this study, CysLTR2 and AhR were the only receptors present on the basolateral membrane of the A549 cells, which is consistent with previous findings showing that A549 cells expressed mRNAs of cysLT 2 and AhR only [16][17][18].The beneficial effects of LXA 4 were reversed by ANF, an AhR antagonist, but not by the cysLT 2 receptor antagonist BAY-u9773.This is clear evidence that the effect of LXA 4 on expression of the α, β and γ subunits of ENAC occurred through binding to AhR. cAMP and cGMP are important second messengers by which cells transduce extracellular signals into intracellular responses.Extracellular signals interact with GPCRs to activate adenylate cyclase (AC) and increase intracellular cAMP levels.A previous study showed that LPS-induced immune responses are associated with decreases in intracellular cAMP levels [7].It has been reported that LPS activates inhibitory G protein, thereby inhibiting formation of cAMP [19].Stimulation by cAMP enhances Na + transport and the activity of Na-K-ATPase in the plasma membrane [20,21].This is in agreement with a model that suggests that at the early stages, cAMP enhances the transport of ENaC to the cell surface, whereas the synthesis of ENaC is necessary for the sustenance of this effect [20].Extracellular signals also interact with GPCR to activate guanylyl cyclases (GC) and increase intracellular cGMP levels.Studies have shown that AhR induces signaling pathways that entail ERK, PKA, MMP9 and cAMP, cGMP and Ca 2+ [22].It has also been shown that AhR is more sensitive to the cAMP signaling pathway [22,23], and that cAMP induces nuclear translocation of the AhR [24].Thus it appears that the two signaling pathways for regulation of ENaC, cAMP, and cGMP are compartmentalized.Levels of intracellular cAMP were decreased after LPS stimulation, and the decreases were reversed by LXA 4 in the LPS group.The reversal of the effects of LXA 4 by ANF and Rp-cAMP, without any significant effects on intracellular cGMP levels indicates that LXA 4 increases the levels of cAMP through AhR.Thus LXA 4 up-regulates the expressions of α, β and γ subunits of ENaC by activating cAMP via AhR.The role of LXA 4 in LPS-treated human alveolar epithelial cells can be depicted as indicated in Figure 10.

CONCLUSION
This study has provided evidence for upregulation of ENAC in normal and LP-stimulated A549 alveolar epithelial cells, most probably through the AhR-cAMP signaling pathways.Thus, it is proposed that LXA 4 can be applied for the induction of ENaC expression in the treatment of ALI/ARDS and related diseases.

Figure 9 :
Figure 9: LXA4 increased ENaC expression dependent on cAMP in A549 cells stimulated with LPS.Cells were treated with RP-cAMP (cAMP inhibitor, 10 uM) in the presence of LXA4 (10-7 M) and LPS (1 ug/mL) for 24 h.After incubation, the cells were harvested, and sonicated.ENaC α, β and γ subunits protein expression in the cell lysates were determined by western blotting using a specific antibody

Figure 10 :
Figure 10: Modulation of sodium channel in human alveolar epithelial cells LXA 4 through AhR-cAMP-dependent pathway