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, 306 (5), C471-84

Cholera Toxin Enhances Na(+) Absorption Across MCF10A Human Mammary Epithelia

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Cholera Toxin Enhances Na(+) Absorption Across MCF10A Human Mammary Epithelia

Qian Wang et al. Am J Physiol Cell Physiol.

Abstract

Cellular mechanisms to account for the low Na(+) concentration in human milk are poorly defined. MCF10A cells, which were derived from human mammary epithelium and grown on permeable supports, exhibit amiloride- and benzamil-sensitive short-circuit current (Isc; a sensitive indicator of net ion transport), suggesting activity of the epithelial Na(+) channel ENaC. When cultured in the presence of cholera toxin (Ctx), MCF10A cells exhibit greater amiloride-sensitive Isc at all time points tested (2 h to 7 days), an effect that is not reduced with Ctx washout for 12 h. Amiloride-sensitive Isc remains elevated by Ctx in the presence of inhibitors for PKA (H-89, Rp-cAMP), PI3K (LY294002), and protein trafficking (brefeldin A). Additionally, the Ctx B subunit, alone, does not replicate these effects. RT-PCR and Western blot analyses indicate no significant increase in either the mRNA or protein expression for α-, β-, or, γ-ENaC subunits. Ctx increases the abundance of both β- and γ-ENaC in the apical membrane. Additionally, Ctx increases both phosphorylated and nonphosphorylated Nedd4-2 expression. These results demonstrate that human mammary epithelia express ENaC, which can account for the low Na(+) concentration in milk. Importantly, the results suggest that Ctx increases the expression but reduces the activity of the E3 ubiquitin ligase Nedd4-2, which would tend to reduce the ENaC retrieval and increase steady-state membrane residency. The results reveal a novel mechanism in human mammary gland epithelia by which Ctx regulates ENaC-mediated Na(+) transport, which may have inferences for epithelial ion transport regulation in other tissues throughout the body.

Keywords: ENaC; Isc; amiloride; cholera toxin; epithelial Na+ channel; mammary gland; short-circuit current.

Figures

Fig. 1.
Fig. 1.
Pharmacological evidence for Na+ absorption and anion secretion by MCF10A cells, a cell line derived from human mammary epithelium. A: typical tracings of short circuit current (Isc; net ion transport) across MCF10A cells that were cultured for the final 24 h in the absence or presence of cholera toxin (Ctx). Solid lines represent Isc values over time, and the dashed lines represent zero current. Ctx-treated monolayers show elevated baseline Isc and greater amiloride-sensitive Isc. Bumet, bumetanide. B: summary of amiloride-sensitive Isc at all time points tested. All cells were assessed 14 days postseeding and were exposed to Ctx for the indicated duration before assessment. A significant effect of Ctx was observed at all time points compared with vehicle (Veh). Bars represent 5–11 observations at each duration.
Fig. 2.
Fig. 2.
Amiloride and benzamil exhibit concentration-dependent inhibition profiles that are consistent with the block of epithelial Na+ channel (ENaC). A: typical tracings in which MCF10A monolayers were cultured with or without Ctx and were exposed to escalating concentrations of amiloride or benzamil, as indicated. Results are representative from 4 observations in each condition. B: summarized data and derived concentration-response curves for the block of Ctx-induced Isc by amiloride and benzamil. Amiloride- or benzamil-sensitive Isc for each concentration was converted to the percentage of maximal inhibited current for that monolayer. Solid lines represent the best fit of a Michaelis-Menton equation to data sets exposed only to vehicle. Dashed lines represent fits for the data from Ctx-treated cells. Data were summarized from 4 sets of observations in each condition. Parameters of each fit are reported in the text.
Fig. 3.
Fig. 3.
Ctx B subunit alone failed to mimic Ctx effect. A: typical tracings from MCF10A cells that were cultured in the absence or presence of Ctx or Ctx B subunit for 1 day. B: data summarized from A and 4 additional experiments. Amiloride-sensitive Isc was significantly greater only when cells were exposed to the holotoxin. Ctx B subunit, alone, had no detectable effect on amiloride-sensitive Isc. C: Western blot shows Ctx B subunit immunoreactivity was detected in samples derived from cells exposed either to Ctx (holotoxin) or to Ctx B subunit alone. Results from 3 experiments are shown.
Fig. 4.
Fig. 4.
Ctx-enhanced amiloride-sensitive Isc was not reduced with washing out. A: typical data from MCF10A cells that were exposed to vehicle or Ctx for 12 h before assay or followed by culturing for 12 h in typical medium. B: data summarized from A and 6 similar experiments that included each of the 4 conditions. Ctx-enhanced amiloride-sensitive Isc was not reduced with washing out.
Fig. 5.
Fig. 5.
Forskolin (Forsk) failed to mimic Ctx effect on amiloride-sensitive Isc across MCF10A cells. A: MCF10A cells were cultured in the absence or presence of forskolin for 24 h or 2 days. Typical results showing that, in paired monolayers, there was no increment in baseline or amiloride (Amil)-sensitive Isc associated with forskolin exposure. B: typical results showing a clear and sustained increment in basal and amiloride-sensitive Isc was associated with Ctx exposure. C: typical results showing that acute (30 min) exposure to either forskolin or Ctx was without detectable effect. D: data are summarized from 4 experiments in all conditions.
Fig. 6.
Fig. 6.
Isoproterenol failed to mimic and Rp-cAMP and H-89 failed to block the Ctx effect. A: typical outcomes when MCF10A cells were cultured in the presence or absence of Ctx or isoproterenol (Iso; 100 nM) in combination with Rp-cAMP (100 μM) for 24 h. Cells were exposed acutely to amiloride and isoproterenol (1 μM) as indicated. B: data summarized from 8 experiments showing that amiloride-sensitive Isc was significantly greater only when cells were cultured in the presence of Ctx. C: Rp-cAMP did not decrease Ctx-induced amiloride-sensitive Isc, but abolished the acute isoproterenol-induced current. D: MCF10A cells were cultured in the absence or presence of Ctx and/or H-89 (10 μM), a protein kinase A (PKA) inhibitor for 24 h before assessment in Ussing chambers. The effect of Ctx on Isc was not inhibited significantly by H-89. Data are summarized from 7 paired observations.
Fig. 7.
Fig. 7.
Ctx elevated amiloride-sensitive Isc only in the presence of cortisol (Cort) and had no effect on mRNA expression of ENaC subunits in MCF10A cells. A: typical tracings from MCF10A cells that were cultured in the absence or presence of cortisol and/or Ctx for 1 day as indicated. Amiloride-sensitive Isc was detected only in cells that had been exposed to the corticosteroid and was enhanced by Ctx exposure. B: results showing the effect of amiloride are summarized from A and 5 similar experiments. C: RNA was isolated in each culture condition. Semi-quantitative RT-PCR shows that mRNA expressions for α-, β-, or γ-ENaC subunits are detectable in all cells and exhibit cortisol-induced expression. Threshold cycle (CT) value for each ENaC subunit was normalized to 18S, and the ΔΔCT method was used to determine mRNA expression level relative to cells cultured in typical cortisol-containing medium. Withdrawal of cortisol was associated with significant reduction in mRNA coding for all ENaC subunits. The effect of Ctx was not statistically significant for any ENaC subunit. Data are summarized from 4 experiments.
Fig. 8.
Fig. 8.
Ctx has no effect on α-, β-, or γ -ENaC expression. Representative Western blot of MCF10A cells that were exposed to Ctx or vehicle for 24 h. Western blot analysis of whole cell lysates was conducted using primary antibodies raised against human α-, β-, or γ-ENaC, as indicated. Membranes were subsequently stripped and reprobed with antibodies to Na-K-ATPase α1 or occludin, as indicated, to verify equal protein loading. Arrows indicate bands of expected mobility. Results are representative from at least four experiments for each target protein.
Fig. 9.
Fig. 9.
Ctx elevated the abundance of β- and γ-ENaC at the apical cell surface. MCF10A cells were exposed to Ctx for 2 h or 1 day. Biotinylation followed by Western blot analysis was used to detect surface expression of β-ENaC (A) and γ-ENaC (B). Densitometric analysis showed that Ctx exposure was associated with increased intensities of bands that had been biotin labeled. Occludin labeling is used as an internal standard for protein loading. Results are representative from at least 3 observations in each condition. The protein was loaded as the following sequence: vehicle with apical biotinylation (Veh_AP), vehicle whole cell lysates (Veh_WCL), Ctx 2 h with apical biotinylation (Ctx2h_AP), Ctx 2-h whole cell lysates (Ctx2h_WCL), Ctx 1 day with apical biotinylation (Ctx1d_AP), and Ctx 1-day whole cell lysates (Ctx1d_WCL).
Fig. 10.
Fig. 10.
Effects of Ctx were not blocked by cytosolic pathway inhibitors. A: amiloride-sensitive Isc across MCF10A cells was reduced by brefeldin A (BFA; 10 mg/ml), a drug disrupting protein trafficking from endoplasmic reticulum to Golgi but remained sensitive to Ctx exposure. Data are summarized from 6 observations. B: data summarized from 5 observations show that the effect Ctx was not blocked by a phosphatidylinositol-3-kinase (PI3K) inhibitor, LY294002 (LY; 50 μM).
Fig. 11.
Fig. 11.
Ctx increased Nedd4-2 phosphorylation. A: Western blot of lysates from 5 paired MCF10A monolayers exposed to either vehicle or Ctx for 1 day using antibodies raised against Nedd4-2 and β-actin. B: densitometric analysis showed Ctx increased apparent Nedd4-2 expression of both the phosphorylated (130 kDa) and the nonphosphorylated (110 kDa) forms of Nedd4-2 although only the effect of Ctx on 130-kDa phosphorylated form achieved statistical significance. Densities were expressed relative to the intensity of β-actin and are summarized from 5 paired samples.
Fig. 12.
Fig. 12.
Ctx elevated β- and γ-ENaC monoubiquitination. A: ubiquitinated β-ENaC was detected by immunoprecipitation of ubiquitin and followed by Western blot of lysates from MCF10A monolayers exposed to either vehicle or Ctx for 1 day using antibodies raised against β-ENaC. B: ubiquitinated γ-ENaC was detected similarly. C: increasing membrane exposure time from 20 s (B) to 60 s, the Ctx-induced increment of the signal of γ-ENaC pulled down by immunoprecipitation of ubiquitin is more evident. D: densitometric analysis showed Ctx increased β- and γ-ENaC monoubiquitination although only the effect of Ctx on γ-ENaC reached statistical significance. Densities were expressed relative to the intensity of whole cell lysates and are summarized from 4 paired samples.
Fig. 13.
Fig. 13.
Mammary cell model to account for monovalent ion transport and especially for Ctx enhanced Na+ absorption. In this model, cortisol binds to glucocorticoid receptor and induces (β and γ) or enhances (α) the expression of mRNA coding for ENaC subunits, which are transcribed in the endoplasmic reticulum, processed through the Golgi apparatus and trafficked to the apical membrane. Under normal conditions, Nedd4-2 binds to ENaC in the apical membrane to induce its retrieval and, upon ubiquitination, its degradation. Ctx elevates amiloride-sensitive Isc by increasing the number of ENaC channels in the apical membrane. As depicted, Ctx is composed of 1 A subunit and 5 B subunits and binds to GM1 by Ctx B subunits to internalize into cells. After internalization, Ctx is transported in a retrograde fashion from the apical membrane to the Golgi apparatus and then to the endoplasmic reticulum where the A subunit becomes dissociated from the B subunits and translocates to the cytosol. In the cytosol, the Ctx A subunit catalyzes the ADP-ribosylation of Gsα to irreversibly activate the G protein and increase cAMP level that activates PKA. However, the effect of Ctx on increased amiloride-sensitive Isc is independent of the cAMP/PKA pathway. Ctx increases phosphorylation of Nedd4-2, which would decrease its ability to promote retrieval of ENaC from the apical membrane, although the underlying mechanism is not defined. Ctx elevates monoubiquitination of ENaC, which may lead to more surface ENaC expression. Solid lines represent pathways that have been defined, and short dash lines represent predicted pathways. α, β, and γ represent ENaC mRNAs. GR, glucocorticoid receptor; CFTR, a cystic fibrosis transmembrane conductance; AC, of adenylyl cyclase.

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