Steroid-Mediated Regulation of the Epithelial Sodium Channel ...

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Endocrinology 148(8):3958 –3967 Copyright © 2007 by The Endocrine Society doi: 10.1210/en.2006-1741

Steroid-Mediated Regulation of the Epithelial Sodium Channel Subunits in Mammary Epithelial Cells Cary Boyd and Aniko´ Na´ray-Fejes-To´th Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756 The epithelial sodium channel (ENaC) is a key mediator of sodium transport in epithelia; however, little is known about ENaC expression in mammary epithelia. Using realtime PCR, we demonstrated the expression of the ENaC subunit mRNAs in mouse and human mammary cell lines and in vivo mouse mammary tissue. We determined the effects of glucocorticoids, progesterone, and prolactin on ENaC expression in four mammary cell lines. Dexamethasone induced all detectable ENaC subunits in noncancerous cell lines, HC11 and MCF10A. Interestingly, in cancerous cell lines (T-47D and MCF-7), both ␤- and ␥- but not ␣ENaC mRNAs were induced by dexamethasone. Progesterone induced ENaC mRNA only in T-47D cells, and prolactin had no effects. ␥ENaC was rapidly induced by steroids, whereas induction of ␣- and ␤ENaC was slower; moreover, the in-

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HE EPITHELIAL SODIUM channel (ENaC) is located in the apical membrane of epithelial cells and consists of three homologous subunits (␣, ␤, ␥) that share 35% identity in amino acid sequence (1, 2). The mature channel is highly selective for sodium, exhibits low conductance, and is amiloride sensitive (3). ENaC subunits have been identified in a diverse range of tissues including the colon, respiratory tract, kidney, urinary bladder, brain, reproductive organs, and sweat glands (3). Surprisingly, although both the sweat gland and mammary gland arise from the same embryological origin (the germinal epithelium), there are few data regarding ENaC expression in mammary epithelium; furthermore, the role of sodium transport during milk production is not well understood. It is possible that sodium is partially responsible for the secretion of water into milk. The traditional view is that lactose is responsible for this action; however, because species that secrete little or no lactose, such as pinnipeds (walrus, sea lion, seal) still secrete water, other osmolytes such as sodium may also be playing a role (4, 5). The transport of sodium during lactation is further supported by the identification of Na⫹-K⫹ ATPases in the basolateral membrane of mammary secretory cells (6). In the 1970s Peaker and colleagues (4, 7) created a model that predicted First Published Online May 17, 2007 Abbreviations: Act-D, Actinomycin-D; Ct, cycle threshold; EnaC, epithelial sodium channel; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GRE, glucocorticoid response element; HRP, horseradish peroxidase; 11-␤HSD, 11 ␤-hydroxysteroid dehydrogenase type 2; KRT-14, keratin 1–14; MR, mineralocorticoid receptor; RE, relative expression. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

duction of the ␤-subunit required de novo protein synthesis. Dexamethasone treatment did not affect ENaC mRNA stability. Western blot analysis revealed immunoreactive bands corresponding to different forms of ␣-, ␤-, and ␥ENaC; dexamethasone significantly increased the intensity of ␣ENaC (85 kDa) and ␤ENaC (90 kDa). We also showed an in vivo reduction in ␣ENaC levels in the mammary tissue of lactating mice as compared with controls, whereas ␤- and ␥ENaC mRNA levels were significantly increased. Furthermore, dexamethasone in vivo significantly increased ␣-, ␤-, and ␥ENaC mRNA expression. Our data indicate that both mouse and human mammary cells express all ENaC subunits, and they are regulated by steroid hormones in a temporal and cell-specific manner both in culture and in vivo. (Endocrinology 148: 3958 –3967, 2007)

an apical sodium channel in mammary epithelia; perhaps ENaC is the unidentified channel. Prior studies using Ussing chamber and whole-cell patch clamping demonstrated amiloride-sensitive currents in primary cultures of mouse mammary cells and mouse and bovine mammary cell lines (8 –11). In the bovine mammary cell line, BME-UV, chronic exposure to corticosteroids induced an amiloride-sensitive current and reduced transepithelial resistance; moreover, these cells expressed mRNA for all three ENaC subunits (9, 11). In mouse epithelia, immunohistochemistry localized ␤ENaC protein to the apical membrane (8). These data emphasize the importance of determining whether mouse and human mammary cells express ENaC subunits; if so, are both mRNA and protein expression regulated by steroid hormones? Steroid-regulated ENaC expression in the mammary gland may play a role during lactation in establishing and maintaining the low-sodium concentration of milk (12, 13). At the onset of lactation, human breast milk sodium is 65 mEq/liter, after 4 d it is 17 mEq/liter, and after 15 d it is maintained at 7 mEq/liter (13). Some of this reduction is hypothesized to be due to tight junction formation and a decrease in paracellular sodium movement in response to glucocorticoids (14 –16). We hypothesize that glucocorticoids may also stimulate the expression of ENaC in the mammary gland because they do in other epithelial cells such as the colon and lung (17, 18). Similarly to glucocorticoids, prolactin has also been shown to increase both tight junction formation and the absorption of sodium in primary cultures of mouse mammary epithelial cells (10); prolactin is also negatively correlated with the concentration of sodium in human breast milk (19). Another hormone that may regulate ENaC expression is progesterone, its presence is essential for alveolar proliferation in mammary tissue and its withdrawal is

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Boyd and Na´ray-Fejes-To´th • Steroid Regulation of ENaC in MEC

necessary to initiate lactation (20). Prior studies have shown that progesterone increases the transcript level of ␥ENaC and suppresses the estrogen induction of the ␣-subunit in the female rat kidney (21). Taken together, these data raise the possibility that glucocorticoids, prolactin, and/or progesterone may regulate ENaC expression in mammary epithelium and thereby regulate the sodium concentration of breast milk. It is also important to understand the regulation of sodium transport during breast cancer. The sodium content of mammary adenocarcinomas has been shown to be significantly higher than that of normal lactating mammary epithelium (22), and recent work has shown a correlation between expression of sodium symporters, increased sodium current, and increased invasion capacity of breast cancer (23–25). Interestingly, tumor proliferation can be prevented by treating cells with the ENaC inhibitor, amiloride (26). However, the relationship between intracellular sodium concentration and cancer remains elusive, although it may involve changes in cell volume and/or membrane potential (22). The goal of this study was to compare the expression levels and hormonal regulation of ENaC in various mammary epithelia. We hypothesized that: 1) human and mouse mammary epithelial cells express all three ENaC subunits; 2) in the lactating mammary gland, ENaC expression is up-regulated in response to lactogenic hormones to facilitate the reabsorption of sodium from breast milk; 3) in cancerous mammary epithelia, the hormonal regulation of ENaC expression is altered. Here we report for the first time that all three ENaC subunits are expressed in mouse mammary tissue and human cell lines and that lactation affects the expression pattern of ENaC mRNA levels. In addition, in human mammary cell lines mRNA levels of the ␤- and ␥ENaC subunits are significantly increased by glucocorticoids, whereas the steroid induction of ␣ENaC occurs exclusively in the noncancerous mammary cell lines. Materials and Methods Cell culture of mammary cell lines MCF10A cells (from American Type Culture Collection, Manassas, VA) were maintained in DMEM/F12 (Cellgro, Herndon, VA), supplemented with 10% fetal bovine serum (FBS), 2 mm l-glutamine, 2 ␮g/ml bovine insulin, 2 ng/ml epithelial growth factor, and 100 ␮g/ml Normocin (InvivoGen, San Diego, CA). HC11 cells (provided by Bernd Groner, University of Frankfurt, Frankfurt, Germany) were maintained in RPMI 1640 medium (Cellgro), 2 mm l-glutamine, 10% FBS, 5 ␮g/ml bovine insulin, 10 ng/ml epithelial growth factor, and 100 ␮g/ml Normocin. Both T-47D cells (American Type Culture Collection) and MCF-7 cells (American Type Culture Collection) were maintained in DMEM/F12 medium containing 10% FBS, 2 mm l-glutamine, and 100 ␮g/ml Normocin. Cells were grown to confluence in complete medium. Before the addition of hormones cells were incubated for 24 h in steroid-free medium containing 5% twice charcoalstripped FBS. The cells were treated for 24 h with dexamethasone (1 nm to 1 ␮m), progesterone (10 nm), prolactin (5 ␮g/ml), or vehicle. The time course of mRNA induction with these treatments was determined at 0, 2, 8, and 24 h. Cells were lysed in 500 ␮l of Tri-Reagent (Molecular Research Center Inc., Cincinnati, OH) and RNA was isolated.

Protein synthesis inhibition with anisomycin and cycloheximide MCF10A and T-47D cells were grown in complete medium until reaching confluence. Cells were then steroid starved for 24 h before an 8-h treatment with: 1) steroid-free medium alone; 2) dexamethasone (1 ␮m); 3) anisomycin (1 ␮g/␮l); 4) cycloheximide (10 ␮m); 5) anisomycin

Endocrinology, August 2007, 148(8):3958 –3967

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⫹ dexamethasone; and 6) cycloheximide ⫹ dexamethasone. After treatment, RNA was extracted from the cells using Tri-Reagent.

RNA polymerase inhibition with actinomycin-D MCF10A cells were grown in complete medium until reaching confluence, steroid starved for 24 h and then treated with either dexamethasone (1 ␮m) or vehicle for 24 h, after which actinomycin-D (5 ␮g/ml) was added to the medium and incubated for 8 h. Cells were then lysed with Tri-Reagent and RNA was isolated.

Real-time quantitative RT-PCR To compare the relative amounts of ENaC subunit mRNA levels in untreated and hormone-treated cells, we used real-time quantitative RT-PCR. Reverse transcription was performed with 2 ␮g of total RNA, using Moloney murine leukemia virus reverse transcriptase (Life Technologies, Inc., Gaithersburg, MD). For human cell lines, a 159-bp product of ␣ENaC was amplified using the following the primers: sense (5⬘-GCC ACA GCA CTG CCC AGA A-3⬘); antisense (5⬘-AGC AGC CCA CGG CGG AGG A-3⬘). An 84-bp product of ␤ENaC was amplified with the primers: sense (5⬘-CAG TTG CCA TAG TGA GGG TAG AAG A-3⬘); antisense (5⬘-GAT GAT CCT GGC CTG CCT ATT CGG A-3⬘). A 174-bp product of ␥ENaC was amplified with the primers: sense (5⬘-TCC GAA ACC ACA GAT GGC CAT T-3⬘); antisense (5⬘-AGC CAA CTA CTG CAA CTA CCA G-3⬘). The expression of ␤-actin was determined using primers sense (5⬘-CAA TAG TGA TGA CCT GGC CGT-3⬘) and antisense (5⬘-AGA GGG AAA TCG TCG GTG AC-3⬘) that amplified a 138-bp product. ␤-Actin mRNA levels were similar in both hormone-treated and control groups, indicating that hormone treatment did not affect mRNA levels or stability. For mouse cell lines, a 159-bp product of ␣ENaC was amplified with the primers: sense (5⬘-GGC AGC CCA CCG AGG AGG A-3⬘); antisense (5⬘-GCC ACA GCA CCG CCC AGA A-3⬘). An 84-bp product of ␤ENaC was amplified with the primers: sense (5⬘-CAG TTG CCA TAA TCA GGG TAG AAG A-3⬘); antisense (5⬘-CAT AAT CCT AGC TTG CCT GTT TGG A-3⬘). A 146-bp product of ␥ENaC was amplified with the primers: sense (5⬘-TCA GAA GCC TCA GAT GGC CAC T-3⬘); antisense (5⬘-CCC AAC TGG ATG TAT TGC TAC T-3⬘). Because the mouse mammary tissue was a heterogeneous mixture of cells, keratin 1–14 (KRT-14) was used as a marker for epithelial cells. An 83-bp product of KRT-14 was amplified with the primers; sense (5⬘-GTC CAC GGT GGC TGC CAG GAT-3⬘); antisense (5⬘-GGC CCA CTG AGA TCA AAG AC-3⬘). Real-time PCR was performed using iTaq SYBR Green Supermix with Rox (Bio-Rad, Hercules, CA). The reactions were performed in triplicate according the manufacturers’ protocol with 200 nm of each sense and antisense primer, and 30 – 40 ng of cDNA, for a final reaction volume of 25 ␮l/well. The thermal cycling parameters were: initial denaturation at 95 C for 3 min, followed by 40 cycles at 95 C for 15 sec and 57 C for 30 sec. The PCR products and threshold cycle were determined and analyzed using AB 7300 (Applied Biosystems, Foster City, CA). Changes between the untreated and hormone-treated ENaC mRNA levels were based on the number of cycles at which the amplified product reached cycle threshold (Ct). Relative expression (RE) of the mRNA was quantified using the equation: RE ⫽ 2-(Ct gene of interest ⫺ Ct ␤-actin). To apply the equation RE ⫽ 2-(Ct gene of interest ⫺ Ct ␤-actin), we calculated the PCR efficiency of each amplicon from standard curves based on a dilution series. The results were graphed as the log of nanograms cDNA vs. cycle threshold: efficiency ⫽ 10(⫺1/slope). The calculated PCR efficiency for each amplicon was 2.0. Because the real-time PCR products were not of equal length (␣, ␤, ␥), there may be unintended variations in fluorescence values between subunits, although the results from realtime PCR were verified with traditional PCR. Reactions were performed under standard conditions with AmpliTaq DNA polymerase (Roche, Indianapolis, IN). Furthermore, to verify that the real-time PCR products were not contaminated from SYBR green binding to nonspecific doublestranded DNA, a dissociation stage was performed: 95 C for 15 sec, 57 C for 30 sec, and 95 C for 15 sec.

Western blot analysis Confluent MCF10A cells were steroid starved for 24 h before a 24-h treatment with dexamethasone (1 ␮m) or vehicle. Cells were then rinsed

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with cold PBS and lysed in a buffer containing 48.2 mm 2-(N-hexylamino) ethanesulfonic acid, 1% sodium dodecyl sulfate, 10% glycerol, and 1% protease and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO). Bicinchoninic acid protein assay reagent (Pierce, Rockford, IL) was used to determine the protein concentration of the samples. Twenty to 40 ␮g of protein were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gel and then transferred to Immobilon-P membrane (Millipore, Billerica, MA). The membranes were blocked for 1 h with 5% dry milk dissolved in Tris-buffered saline-Tween 20 containing 10 mm Tris (pH 7.5), 150 mm NaCl, 124 ␮m thimerosal, and 0.05% Tween 20 (Sigma-Aldrich). Membranes were incubated overnight at 4 C in the 5% dry milk/ Tris-buffered saline-Tween 20 buffer containing the primary antibodies against the ENaC subunits (␣: 1:500, ␤: 1:1000, and ␥: 1:500). The ␣- and ␥ENaC antibodies were a generous gift of Dr. L. Palmer (Cornell University, Ithaca, NY) and were previously characterized in detail (27, 28). ␤ENaC antibody was purchased from Chemicon International (Temecula, CA). Antirabbit IgG conjugated with horseradish peroxidase (HRP) was used as a secondary antibody (Cell Signaling, Danvers, MA). The HRP signal was detected using SuperSignal West Dura substrate (Pierce), and visualized with a Flurochem 8900 in conjunction with the ChemiImager 5500 (Alpha Innotech, San Leandro, CA). The values were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels determined using a GAPDH antibody (1:5000) (American Research Products, Belmont, MA), and antimouse IgG conjugated with HRP (1:5000; Zymed, San Francisco, CA).

Isolation of mouse mammary tissue Mammary tissue was extracted from both nonlactating and lactating (21 d postpartum) female littermates (C57BL/6) to determine the effects of lactation on ENaC mRNA levels. To examine the effects of dexamethasone on ENaC mRNA levels, we used adrenalectomized C57BL/6 female mice (3 months) obtained from Charles River Labs (Wilmington, MA). Mice were treated with dexamethasone (100 ␮g/kg, ip) or vehicle at 4 and 24 h before removing the mammary tissue. All procedures involving animals were approved by the Institutional Animal Care and Use Committee of Dartmouth Medical School. RNA was extracted using Tri-Reagent (1 ml per 100 mg tissue) according to the manufacturers’ protocol.

Statistics All results are presented as means ⫾ sem. Data were analyzed by either unpaired t test or one-way ANOVA followed by Tukey-Kramer posttest. P ⬍ 0.05 was regarded as statistically significant.

Results ENaC mRNA expression and steroid regulation in noncancerous mammary epithelial cells

Using real-time PCR, we initially established that all three ENaC subunit mRNAs (␣, ␤, ␥) are expressed in several human mammary cell lines. Our experiments revealed that in the MCF10A cell line, which originates from normal human mammary gland epithelial cells derived from fibrocystic disease (29), ␣ENaC mRNA levels are more abundant than those of ␤- or ␥ENaC. The ratio of the basal ENaC mRNA levels were 358:1 for ␣- to ␤ ENaC and 240:1 for ␣- to ␥ENaC. Next we determined whether ENaC mRNA levels are regulated by hormones involved in lactation: glucocorticoids, progesterone, or prolactin. Our results revealed that 24 h dexamethasone treatment significantly increased transcript levels of all three ENaC subunits at concentrations between 10 nm and 1 ␮m. After 24 h of 1 ␮m dexamethasone treatment, ␣ENaC mRNA levels increased 5-fold, whereas the levels of ␤- and ␥ENaC increased 19- and 17-fold, respectively (Fig. 1A). The time course of induction varied between subunits, with ␣- and ␤ENaC increasing at a slower rate than ␥ENaC (Fig. 2). Both the ␣- and ␤ENaC subunit

FIG. 1. The effect of dexamethasone on relative expression of ENaC mRNA levels in noncancerous mammary epithelial cells. A, In MCF10A cells, 24 h of dexamethasone (dex; 1 ␮M) significantly increases the mRNA levels of all three ENaC subunits (␣, ␤, ␥) (n ⫽ 8), compared with control (ctrl) cells grown in steroid-free medium. B, In HC11 cells, dexamethasone significantly increases both ␣- and ␥ENaC mRNA (n ⫽ 8). ␤ENaC mRNA levels were below the level of detection. Note the different scales for ␣- and ␤-, ␥ENaC mRNA levels, and values were normalized for ␤-actin. For A and B, results are presented as means ⫾ SEM and data were analyzed by unpaired two-tailed t test. *, P ⬍ 0.05; **, P ⬍ 0.01.

required 24 h of dexamethasone treatment to have significant increases in mRNA levels, whereas the expression of ␥ENaC mRNA increased rapidly, reaching a 22-fold induction at 2 h, and then slightly decreasing at 24 h to a level 17-fold higher than at 0 h (Fig. 2). After establishing that glucocorticoids do affect ENaC mRNA levels, the effects of other lactogenic hormones were measured. MCF10A cells were treated with prolactin (these cells express prolactin receptors, but do not express progesterone receptors) (10, 19, 30, 31). Our results show that in MCF10A cells prolactin did not affect ENaC mRNA levels (not shown), even though prolactin has been associated with increases in sodium transport by mammary epithelial cells (10). Next we determined the levels of ENaC mRNA in HC11 cells, which originate from normal mouse mammary epithelial cells derived from a lactating mouse and tight junctions when grown on a permeable membrane (32, 33). As shown in Fig. 1B, the ratio of the basal ENaC mRNA levels for ␣ to ␥ was: 23:1; however, mRNA expression of the ␤-subunit was below the level of detection, even after dexamethasone treatment. Twenty-four hours of dexamethasone treatment significantly increased the mRNA levels of ␣- (3-fold) and ␥ENaC (16-fold) subunits. The time course of steroid induction of the ␣-subunit occurred at a slower rate than that of ␥ENaC; the ␣-subunit was significantly induced by dexamethasone at 24 h, whereas the levels of the ␥-subunit mRNA were significantly elevated as early as 2 h (5-fold) after dexamethasone treatment (not shown). These temporal differ-



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FIG. 2. Time course of dexamethasone induction on ENaC mRNA in MCF10A cells. ␣ENaC (left panel, n ⫽ 8) and ␤ENaC (right panel, n ⫽ 8) mRNA levels were significantly increased after 24 h dexamethasone (1 ␮M) treatment. The largest induction of ␥ENaC mRNA by dexamethasone occurs within 2 h (n ⫽ 4), with levels slightly decreasing by 8 h (n ⫽ 4) and 24 h (right panel, n ⫽ 8). The level of ␥ENaC mRNA at 24 h was not significantly different from the expression at 2 or 8 h. For all subunits, 0 h time point n ⫽ 8. Results are presented as means ⫾ SEM and data were analyzed by one-way ANOVA followed by Tukey-Kramer posttest. *, P ⬍ 0.05; **, P ⬍ 0.01.

ences in the steroid induction of ␣- and ␥ENaC were similar to the pattern we observed in MCF10A cells. Neither progesterone nor prolactin had a significant effect on ENaC mRNA expression, even though HC11 cells have previously been shown to respond to both hormones (not shown) (34).

breast cancer cell lines. We tested the effects of dexamethasone and progesterone on MCF-7 cells, which are derived from a human adenocarcinoma (36). MCF-7 cells grown in

ENaC mRNA expression in response to hormone treatment in breast cancer cells

Next we asked the question, is steroid regulation of ENaC expression maintained in mammary carcinoma cells? T-47D cells are human mammary cells derived from a ductal carcinoma (35). Unlike the noncancerous cell lines, in T-47D cells, a 24-h treatment with dexamethasone failed to increase the transcript levels of the ␣-subunit, whereas the ␤- and ␥-subunit levels were increased after treatment with dexamethasone or progesterone (Fig. 3A). The ratios of ENaC subunit mRNA levels in untreated T-47D cells were: ␣- to ␤EnaC, 23:1 and ␣- to ␥EnaC, 398:1. Twenty-four hours of dexamethasone treatment increased the levels of ␤ENaC mRNA 2-fold and that of ␥ENaC mRNA 10-fold, whereas progesterone increased ␤ENaC and ␥ENaC mRNA levels by 2.5- and 20-fold, respectively (Fig. 3A). Interestingly, T-47D cells were the only mammary cell line we tested in which progesterone affected ENaC mRNA levels. To determine whether the lack of steroid induction of ␣ENaC was due to an early increase that declined to basal levels by 24 h, we examined the level of ENaC mRNA at 2 and 8 h. ␣ENaC mRNA levels were not increased by steroid treatment at any time point (Fig. 4). Induction of the ␤-subunit by dexamethasone was slow and reached statistical significance only after 24 h. The ␥-subunit was induced 19-fold by dexamethasone as soon as 2 h, further increased to 48-fold by 8 h, and declined to a 10-fold induction by 24 h (Fig. 4). The time course of induction by progesterone was similar to that of dexamethasone for both ␤- and ␥ENaC mRNA. With progesterone treatment ␤ENaC mRNA was significantly induced (2.5-fold) only at 24 h. Progesterone increased the levels of the ␥-subunit 16- and 47-fold at 2 and 8 h, respectively, and mRNA levels declined to a 20-fold induction by 24 h (not shown). Next, we determined whether the lack of ␣ENaC induction was specific to T-47D cells or if it was associated with other

FIG. 3. ENaC mRNA levels in cancerous mammary epithelial cells after dexamethasone or progesterone treatment. A, In T-47D cells, 24 h dexamethasone (dex; 1 ␮M) had no significant effect on ␣ENaC mRNA levels, whereas ␤- and ␥ENaC mRNA levels were significantly increased, compared with control (ctrl) cells (n ⫽ 8). Progesterone (prog; 10 nM) had similar effects to dexamethasone on both ␤- and ␥ENaC expression (n ⫽ 8). B, In MCF-7 cells, neither dexamethasone nor progesterone had an affect on ␣ENaC mRNA levels (n ⫽ 4), whereas dexamethasone increased ␤- and ␥ENaC mRNA levels (n ⫽ 4), and progesterone had no effect on ␤- or ␥ENaC mRNA levels (n ⫽ 4). For A and B, results are presented as means ⫾ SEM and data were analyzed by one-way ANOVA followed by Tukey-Kramer posttest. *, P ⬍ 0.05; **, P ⬍ 0.01.

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FIG. 4. Time course of dexamethasone induction on ENaC mRNA in T-47D cells. Dexamethasone (1 ␮M) had no significant effect on ␣ENaC mRNA levels (left panel, n ⫽ 8) but increased ␤ENaC mRNA levels by 24 h (n ⫽ 8) and ␥ENaC mRNA levels by 2 h (right panel, n ⫽ 4). The levels of ␥ENaC mRNA further increased at 8 h (n ⫽ 4) and then significantly decreased at 24 h but remained 10-fold higher, compared with 0 h. For all subunits, 0 h time point, n ⫽ 8. Results are presented as means ⫾ SEM and data were analyzed by one-way ANOVA followed by Tukey-Kramer posttest. *, P ⬍ 0.05; **, P ⬍ 0.01.

steroid-free media had a ratio of basal ENaC mRNA levels: 133:1 for ␣- to ␤ENaC and 85:1 for ␣- to ␥ENaC. Similarly to T-47D cells, steroid hormones failed to induce the expression of ␣ENaC mRNA, whereas both ␤- and ␥ENaC mRNAs were induced by dexamethasone (24 h), by 10- and 6-fold, respectively (Fig. 3B). Progesterone had no effect on the mRNA levels of any subunit. These data indicate that in both cancer cell lines, ␣ENaC mRNA was constitutively expressed and was not induced by dexamethasone treatment; this response differed from the noncancerous mammary cells. Protein synthesis is not necessary for the rapid induction of ␥ENaC but is required for the induction of ␤ENaC

The rapid induction of ␥ENaC mRNA suggests that steroid hormones directly affect ␥ENaC transcription without requiring de novo protein synthesis. To verify this, we treated MCF10A cells for 8 h with dexamethasone plus the protein synthesis inhibitor anisomycin (1 ␮g/ml) or cycloheximide (10 ␮m), at concentrations previously reported to block protein synthesis (37). These experiments revealed that the steroid induction of both the ␣- and ␥-subunit were maintained in the presence of protein synthesis inhibitors, implying that de novo synthesis of dexamethasone-induced proteins is not required for ␣- or ␥ENaC induction (Fig. 5). The protein synthesis inhibitors caused a decrease in the absolute amount of ␣ENaC mRNA levels, although the fold induction remained constant. The basal levels of ␥ENaC mRNA were 2to 3-fold higher in the presence of protein synthesis inhibitors, compared with control cells. Similarly, dexamethasone combined with cycloheximide caused a 25-fold induction in ␥ENaC mRNA levels, compared with a 16-fold induction without cycloheximide. Therefore, both the absolute amount and the fold induction of ␥ENaC mRNA levels were increased in the presence of cycloheximide. In contrast, steroid induction of ␤ENaC mRNA was completely prevented in the presence of the protein synthesis inhibitors, indicating an indirect effect of dexamethasone (Fig. 5). We also determined the effects of protein synthesis inhibitors on ENaC mRNA levels in T-47D breast cancer cells. The expression of the ␣-subunit was not significantly affected by dexamethasone alone or dexamethasone with protein synthesis inhibitors (not shown), whereas the level of ␤ENaC

tended to increase with dexamethasone alone, and this induction required protein synthesis. The induction of ␥ENaC was similar to MCF10A cells; in T-47D cells, basal levels of ␥ENaC mRNA were 10-fold higher in the presence of protein synthesis inhibitors than in control cells, and dexamethasone resulted in a further 4- to 5-fold induction. Effect of dexamethasone on mRNA stability of ␤- and ␥ENaC

To determine whether the steroid effect on ENaC mRNA levels was due to an increase in transcription or in mRNA stability, MCF10A cells were treated with the RNA polymerase inhibitor, actinomycin-D (Act-D) (8 h), at a concentration previously reported to inhibit 95% of RNA synthesis (5 ␮g/ml) (17). The decrease in ␤- and ␥ENaC mRNA levels after Act-D treatment were similar in dexamethasone-treated (24 h) and untreated cells, indicating that dexamethasone does not affect the mRNA stability of ␤- or ␥ENaC (Fig. 6). In the presence of Act-D, we observed a greater decrease in ␥ENaC than ␤ENaC, suggesting that ␥ENaC mRNA may have a shorter half-life than ␤ENaC mRNA. Similar findings for ENaC mRNA half-lives were previously reported in cells derived from the distal colon (38). Steroid induction of ENaC protein in mammary epithelial cells

Next, using Western Blot analysis, we investigated whether dexamethasone causes an increase in ENaC protein, paralleling the increase in ENaC mRNA in MCF10A cells. As illustrated in Fig. 7, 24 h treatment with dexamethasone caused a significant (1.8-fold) increase in the intensity of the 85-kDa band of ␣ENaC, whereas the intensity of the 30-kDa band of ␣ENaC remained unchanged. The 85-kDa band has been characterized as the immature, full-length form of ␣ENaC; proteolytic cleavage at the NH2 terminus produces mature 60- and 30-kDa fragments (39). It should be noted that the faint 60-kDa immunoreactive band is probably due to a nonspecific reaction and does not represent the mature fragment of ␣ENaC because the ␣ENaC antibody was raised against amino acids 24 – 68, and the 60-kDa subunit begins at amino acid 231 (28, 40). In MCF10A cells, the immature form of the ␤-subunit (90



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␤ENaC was 85–90 kDa (doublet), and ␥ENaC was 85–90 kDa. Proteolytic cleavage of these subunits resulted in bands at 60 and 30 kDa for ␣ENaC and 70 kDa for ␥ENaC. ␤ENaC was processed by removing N-linked sugars, which produced a band at 70 kDa (27). ENaC mRNA expression in mouse mammary tissue in vivo

FIG. 5. The effect of protein synthesis inhibitors on the steroid induction of ENaC mRNA in MCF10A cells. MCF10A cells were treated for 8 h with dexamethasone (dex; 1 ␮M) or vehicle in the presence of anisomycin (aniso; 1 ␮g/ml) or cycloheximide (cyclo; 10 ␮M). Both protein synthesis inhibitors decreased the absolute amount of ␣ENaC mRNA but had no affect on the fold induction by dexamethasone (n ⫽ 4). In contrast, both anisomycin and cycloheximide completely prevented the induction of ␤ENaC (n ⫽ 4). The induction of ␥ENaC mRNA was augmented in the presence of protein synthesis inhibitors (with or without dexamethasone), compared with cells grown without protein synthesis inhibitors (n ⫽ 4). Note the values on the y-axis are normalized to their respective steroid-free (S.F.) control (ctrl) values, and RE is relative expression. Results are presented as means ⫾ SEM and data were analyzed by unpaired one-tailed t test. *, P ⬍ 0.05; **, P ⬍ 0.01.

kDa) appeared as a doublet and was significantly induced (1.6-fold) by 24 h dexamethasone treatment (Fig. 7). We also detected a band at approximately 70 kDa that may correspond to the ␤ENaC with the complex N-glycans removed (39); this band was not induced by dexamethasone in MCF10A cells. For ␥ENaC, in most experiments we detected only one band at 70 kDa, representing the cleaved form, although in two of five experiments, we also observed a faint band around 85 kDa that may correspond to the uncleaved form (27, 39). The intensity of the 70-kDa band tended to increase after a 24-h dexamethasone treatment without reaching statistical significance (Fig. 7). Our results, revealing several distinct bands for ENaC protein in mammary epithelial cells, are similar to results reported for other tissues (27, 39). Ergonul et al. (27) reported that in rat kidney the nonprocessed form of ␣ENaC was around 85 kDa,

To determine the in vivo relevance of our results obtained with mammary cell lines, we used real-time PCR to measure ENaC mRNA levels from both nonlactating and lactating mice. Our results establish that lactation significantly affects the mRNA level of all three ENaC subunits (Fig. 8). In the lactating mice, we observed a 4-fold decrease in ␣ENaC mRNA, compared with nonlactating mice, whereas ␤- and ␥ENaC mRNA levels were significantly increased (10- and 9-fold, respectively). The ratios of the ␣to ␤- and ␣- to ␥ENaC mRNA levels in the nonlactating mice were 156:1 and 874:1, respectively. During lactation, due to a decrease in ␣ENaC and an increase in ␤- and ␥ENaC, the ratio of ␣- to ␤ENaC decreased to 3:1, whereas ␣- to ␥ENaC decreased to 22:1. To avoid artifacts due to the heterogeneous composition of cells in the mammary tissue, all ENaC mRNA values from mouse mammary tissue were normalized to the mRNA levels of KRT-14, in addition to ␤-actin. KRT-14 is a myoepithelial cell marker and is consistently expressed throughout mammary development (41– 43). The fold change in ENaC mRNA between nonlactating and lactating were similar when normalized to either ␤-actin or KRT-14 (not shown). To determine whether ENaC is regulated by glucocorticoids at the transcriptional level in mammary epithelia in vivo, we examined the effect of dexamethasone in adrenalectomized mice after 4 and 24 h of treatment. A 4-h treatment with dexamethasone significantly increased mRNA levels of the ␥-subunit (35-fold), whereas expression of the ␣- and ␤-subunit tended to increase at 4 h but did not reach statistical significance until 24 h. At 24 h, dexamethasone significantly increased both ␣- and ␤ENaC mRNA levels by 9- and 6-fold, respectively. On the other hand, ␥-subunit mRNA levels declined at 24 h of dexamethasone treatment (Fig. 9). Again, as in the nonlactating mice, the levels of ␣ENaC were significantly higher than those of ␤- or ␥ENaC mRNA, with control adrenalectomized mice at 24 h having ␣- to ␤ENaC 33:1 and ␣- to ␥ENaC 272:1.

FIG. 6. Dexamethasone does not affect the stability of ␤- and ␥ENaC mRNA in MCF10A cells. Cells were cultured for 24 h with dexamethasone (1 ␮M) or vehicle, and then Act-D (5 ␮g/ml) was added for 8 h. Act-D treatment caused a similar decrease for both ␤- and ␥ENaC mRNA levels for cells grown in either steroid-free or dexamethasonetreated medium (n ⫽ 3). Note the values on the y-axis are normalized to their respective control (ctrl) values.

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FIG. 7. Dexamethasone induction of ENaC protein in MCF10A cells. Twenty to 40 ␮g of protein from steroid-free control (ctrl) or dexamethasone (dex; 1 ␮M, 24 h)-treated cells were separated on an SDSPAGE gel and probed with anti-ENaC (␣, ␤, ␥) antibodies. Dexamethasone increased the intensity of the 85-kDa ␣ENaC protein 1.8-fold. In the presence of dexamethasone, the intensity of the 90kDa band of ␤ENaC increased 1.6-fold, whereas that of the 70-kDa band of ␥ENaC tended to increase but did not reach statistical significance. All values were normalized to GAPDH protein levels. Results are presented as means ⫾ SEM, and data were analyzed by unpaired one-tailed t test. *, P ⬍ 0.05, n ⫽ 5.

Discussion ENaC expression and regulation in noncancerous mammary epithelia in culture and in vivo

The main findings of this study are that all three ENaC subunits are expressed and regulated by steroid hormones in both mammary cell lines and in mammary tissue and that lactation affects the level of all three ENaC subunits in vivo. We chose to study the effects of steroid hormones on ENaC expression because steroid hormones are essential for pregnancy and lactation. Progesterone is important for mammary growth and development during pregnancy, and both progesterone and glucocorticoids affect lactogenesis (44). Less is known about the effects of aldosterone on the mammary gland during pregnancy and lactation, although the mammary gland is similar to other aldosterone-sensitive organs in that it expresses the mineralocorticoid receptor (MR) and 11 ␤-hydroxysteroid dehydrogenase type 2 (11-␤HSD) (45, 46). 11-␤HSD prevents inappropriate activation of the MR by converting cortisol to cortisone. During lactation the function of 11-␤HSD in the mammary gland is questionable because its expression is significantly decreased allowing glucocorticoids to remain active (45). Thus, the MRs are likely occu-


pied by endogenous glucocorticoids during lactation. Furthermore, whether the mammary gland is responsive to aldosterone is still controversial. The effects of both glucocorticoids and aldosterone were found to be blocked by RU486 but not spironolactone, suggesting that they were mediated via the glucocorticoid receptor and not the MR (47). These factors prompted us to study the effects of glucocorticoids on ENaC expression, rather than aldosterone. Glucocorticoids are important for inducing and maintaining lactation (14, 20, 44). It has been previously shown that receptor binding of glucocorticoids increases with the onset of lactation (44, 48), and glucocorticoids play an important role in tight junction formation in mouse mammary epithelial cells (10). In addition, glucocorticoids are important for increasing milk protein secretion and differentiation of the alveolar system (44). During the first few days postpartum, plasma glucocorticoid concentrations increase, whereas the sodium content of milk decreases from 65 to 7 mEq/liter (13). This relationship raises the question of whether there is a direct connection between glucocorticoid concentrations and milk sodium content. This question may have clinical implications because high concentrations of sodium in breast milk have been correlated with impaired lactogenesis and failure to nurse and are associated with malnutrition, dehydration, and hypernatremia in infants (49 –52). Despite this, there is limited information regarding the transport of sodium in mammary epithelia, compared with other epithelia. We hypothesize that glucocorticoids regulate the sodium concentration of breast milk by regulating the expression of ENaC in the mammary gland. To our knowledge, this is the first report of hormonal regulation of ENaC mRNA and protein expression in mouse and human mammary epithelial cell lines and in vivo expression during lactation. In the human mammary cell line, MCF10A, the mRNA levels of all three ENaC subunits were increased by glucocorticoids in a time-dependent manner. This hormone response was similar to the glucocorticoid-induction of ENaC mRNA in the fetal lung (17) but different from the regulation observed in the kidney and colon in which only one or two subunits are induced by steroids (18, 28). The reason for the

FIG. 8. The effect of lactation on the relative expression of ENaC mRNA. Mammary tissue was obtained from nonlactating (ctrl) and lactating (lact) mice (21 d postpartum). Lactation significantly decreased the level of ␣ENaC mRNA and increased both ␤- and ␥ENaC mRNA levels, compared with nonlactating littermates. ␣ENaC mRNA levels were significantly higher than ␤- and ␥ENaC mRNA levels in both nonlactating and lactating mice (note the different scales between ␣ vs. ␤, ␥). Results are presented as means ⫾ SEM and data were analyzed by unpaired two-tailed t test. *, P ⬍ 0.05, n ⫽ 5.


FIG. 9. The in vivo effect of dexamethasone treatment on the relative expression of ENaC mRNA. Mammary tissue was obtained from adrenalectomized mice treated with dexamethasone (100 ␮g/kg, ip) or vehicle (0.9% saline) for 4 and 24 h. At 4 h dexamethasone significantly increased the RE of ␥ENaC mRNA levels, whereas ␣- and ␤ENaC mRNA levels tend to increase but did not reach significance. At 24 h, dexamethasone treatment significantly increased both ␣- and ␤ENaC mRNA levels, whereas ␥ENaC mRNA levels significantly declined from the 4-h mRNA levels. Note the values on the y-axis are normalized to the respective control (ctrl). Results are presented as means ⫾ SEM and data were analyzed by one-way ANOVA followed by Tukey-Kramer posttest. *, P ⬍ 0.05, **, P ⬍ 0.01 (4 h, n ⫽ 4; 24 h, n ⫽ 5).

noncoordinate regulation of ENaC transcription in various tissues such as the lung, kidney, and colon is unknown, although it may play a role in the biophysical and pharmacological properties of the channel (53). We found a slow steroid induction of ␣ENaC and a rapid induction of ␥ENaC in both human and mouse noncancerous cell lines. In the human cells, ␤ENaC is seemingly regulated indirectly by glucocorticoids because the induction is slow and requires de novo protein synthesis. It is possible that the undetectably low ␤ENaC mRNA levels in the mouse cell line are due to immortalization. This is supported by results obtained in the porcine vas deferens cell line, PVD9902, in which ␤ENaC mRNA expression could not be detected and the cells failed to develop an amiloride-sensitive current in response to glucocorticoids (54). Using an RNA polymerase inhibitor, we determined that steroid-induced increases in mRNA were due to increases in transcription and were not a result of increasing mRNA stability. Our results correspond to data in the human fetal lung, which demonstrated that glucocorticoids do not affect ENaC mRNA stability (17). In both MCF10A and HC11 cells, ␥ENaC mRNA had the largest and most rapid response to glucocorticoid treatment. The induction did not require protein synthesis, rather cycloheximide caused a super-induction of ␥ENaC mRNA levels. A superinduction has previously been reported for ␣ENaC but not for ␥ENaC in Madin-Darby canine kidney cells (37). The rapid hormone induction of ␥ENaC mRNA is intriguing because a functional glucocorticoid response element (GRE) could not previously be identified in H441 human lung epithelial cells within the 3-kb promoter region upstream from the transcription start site (55). Perhaps activation of the GRE requires transacting factors that were not present in H441 cells or the functional GRE(s) is located further upstream in the promoter. Our analysis of the ␥ENaC promoter using mVISTA (56) and NUBIScan (57) identified a putative GRE located in the first intron that is conserved in human, mouse, and rat. In addition to an induction of ENaC mRNA, we also ob-


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served a corresponding induction of ENaC protein by dexamethasone in noncancerous mammary cells. In MCF10A cells, dexamethasone significantly induced the uncleaved 85-kDa form of ␣ENaC protein. For ␤ENaC, we observed a significant increase in the intensity of the 90-kDa band. For ␥ENaC protein, in a majority of the experiments, only the mature form (70 kDa) could be detected. There was a trend for dexamethasone to increase the 70-kDa form of ␥ENaC; however, the change did not reach statistical significance. This change may still have biological relevance because Volk et al. (58) showed that increases in ␥ENaC mRNA, which resulted in unquantifiable changes in protein levels, were able to augment ENaC channel activity. We also noted that the fold-induction of protein for ␣- and ␤ENaC did not parallel the fold induction of mRNA levels. This suggests that either mRNA is not being translated into protein or protein is being degraded at an increased rate or that the techniques used to determine protein levels are not as sensitive as the techniques used to determine mRNA levels. ENaC expression and regulation in cancerous mammary cell lines

A correlation between sodium transport and oncogenesis has been reported for decades. In 1980 transformed mouse mammary cells were shown to have a 3-fold higher intracellular sodium content than untransformed cells (22), and increasing the inward sodium current through the voltagegated sodium channel increased the invasive capacity of breast cancer (24, 59). Furthermore, growth and proliferation of mammary adenocarcinomas can be inhibited with amiloride (26), suggesting that ENaC activity is correlated with the proliferation of breast cancer. It was recently published that ␤ENaC is one of the most commonly mutated genes in breast cancer and ion transporter mutations account for approximately 60% of breast cancer mutations (60). Here we report for the first time that ␣ENaC mRNA has dissimilar regulation between cancerous and noncancerous mammary cells. Surprisingly, in the cancerous mammary cell lines, T-47D and MCF-7, ␣ENaC mRNA levels, which are upregulated by steroid hormones in several other tissues (17, 18, 28), were not induced by glucocorticoids or progesterone. On the other hand, the expression of both the ␤- and ␥-subunits were induced by steroid hormones in a manner similar to the noncancerous cell lines. The inability of steroid hormones to induce ␣ENaC mRNA levels is intriguing because ␣ENaC has a GRE(s) in its promoter region, and a reporter-construct containing this GRE can be directly activated by dexamethasone (61). One possible explanation for these findings is that ␣ENaC mRNA levels are not responsive to steroid hormones because of a significant up-regulation of expression limiting further up-regulation. However, we feel this conclusion is unlikely because our results indicate that noncancerous MCF10A cells express 6-fold more basal ␣ENaC mRNA than the cancerous T-47D cells. This suggests that T-47D cells are not expressing maximal ␣ENaC mRNA levels, raising the possibility that the mechanisms regulating ␣ENaC transcription may be constitutively repressed in breast cancer cells, limiting the induction. It is also possible that the developmental stage of the mammary gland determines ␣ENaC’s response to glucocor-

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ticoids. The developed mammary gland contains two types of mature epithelial cells, luminal epithelia and myoepithelia. The principal cells implicated in breast cancer are the luminal epithelial cells, with the resulting breast cancer cells being less differentiated than their mature noncancerous counterparts (62). It is possible that the dexamethasone induction of ␣ENaC is also affected in these less differentiated cells. It was shown in rat and mouse fetal lung tissue that the dexamethasone induction of ␣ENaC was dependent on the stage of lung development (63– 65). Whatever the mechanism behind the lack of steroid induction of ␣ENaC expression, it may also be similar in the colon where the ␣ subunit mRNA levels are not induced by steroids, whereas the ␤ and ␥ subunits are induced (2, 18, 66, 67). In vivo expression of ENaC mRNA during lactation and dexamethasone treatment

Our experiments revealed that in nonlactating mice ␣ENaC mRNA levels were much higher than ␤- or ␥ENaC levels, whereas during lactation a decrease in ␣ENaC mRNA and an increase in ␤- and ␥ENaC mRNA levels led to a decrease in the ratio between the subunits. This distinct expression pattern of ENaC in the mammary gland is similar to that reported for the developing lung; during the prenatal phase ␣ENaC mRNA levels are predominantly high, whereas ␤- and ␥ENaC levels remain low. In the postnatal lung, ␤- and ␥ENaC levels are increased, whereas ␣ENaC levels decrease (63, 68). This developmental-dependent regulation of ␣ENaC may explain the high levels of ␣ENaC in the nonlactating mammary gland vs. the decreased levels of ␣ENaC in the lactating mammary gland. To elucidate the role of glucocorticoids in ENaC regulation in vivo in mammary epithelium, we determined the relative expression of ENaC mRNA in mammary tissue obtained from adrenalectomized mice treated with dexamethasone or vehicle. Interestingly, both ␣- and ␤ENaC mRNA levels were significantly increased in the mice treated with dexamethasone for 24 h, whereas ␥ENaC mRNA levels were increased only at 4 h, compared with control adrenalectomized mice. This rapid effect of dexamethasone on ␥ENaC mRNA, and after decline by 24 h, is very similar to the time course we observed in both MCF10A and T-47D cells. Our current findings advance the understanding of ENaC regulation in mammary epithelial cells and build on the recent observations by Quesnell et al. (11). While our manuscript was pending review, they reported that glucocorticoids regulate ENaC expression in the immortalized bovine mammary epithelial cell line, BME-UV (11). They found a correlation between amiloride-sensitive current and ENaC expression in response to glucocorticoids (dexamethasone, cortisol, or prednisolone) but not aldosterone. In their studies they used a modified Ussing chamber to measure a short circuit current and found that dexamethasone (0.1 ␮m) treatment resulted in a 6-fold increase in short circuit current that was amiloride sensitive. ␣ENaC mRNA levels were not significantly affected by dexamethasone treatment, whereas the ␤- and ␥-subunit mRNA levels were induced 7- and 15-fold, respectively. They also found that ␣ENaC was in great excess relative to ␤- and ␥ENaC mRNA. The data from bovine mammary cells contribute to the global understanding of ENaC and sodium transport in mammary


cells. We have expanded on their observations with our current results using human and mouse mammary epithelial cell lines and in vivo mouse mammary cells. Understanding the steroid regulation of ENaC mRNA and protein expression in mammary epithelial cells is important for deciphering the mechanism of sodium transport during lactation and oncogenesis. During lactation, the steroid induction of all three subunits may facilitate sodium reabsorption from milk and prevent infant hypernatremia. In breast cancer, the steady mRNA expression ␣ENaC may be a result of the developmental stage of the cell and lead to increased sodium content in the cell, which has been linked to oncogenesis. Acknowledgments We thank Dr. Lawrence Palmer (Cornell University, Ithaca, NY) for providing the ␣- and ␥ENaC antibodies. Received December 26, 2006. Accepted May 9, 2007. Address all correspondence and requests for reprints to: Aniko´ Na´ray-Fejes-To´th, Department of Physiology, Dartmouth Medical School, Borwell Building 744W, 1 Medical Center Drive, Lebanon, New Hampshire 03756. E-mail: [email protected]. This work was supported by National Institutes of Health Grants DK41841 and DK07508. Disclosure Statement: The authors have nothing to disclose.

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