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Am J Physiol Renal Physiol 293: F904-F913, 2007. First published July 3, 2007; doi:10.1152/ajprenal.00365.2006
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Melanophilin, a novel aldosterone-induced gene in mouse cortical collecting duct cells

Jessica A. Martel, Donna Michael, Géza Fejes-Tóth, and Anikó Náray-Fejes-Tóth

Dartmouth Medical School, Lebanon, New Hampshire

Submitted 11 September 2006 ; accepted in final form 28 June 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The molecular mechanisms of aldosterone-regulated Na+ transport are not entirely clear. The goal of this study was to identify aldosterone-induced genes potentially involved in the trafficking of the epithelial Na+ channel (ENaC). We report that the transcript levels of melanophilin (MLPH), a protein involved in vesicular trafficking in melanocytes, are rapidly increased by aldosterone in cortical collecting duct (CCD) cells. This effect was near maximal at physiological aldosterone concentrations, indicating that it is mediated by the mineralocorticoid receptor. De novo protein synthesis is not required for the induction of MLPH mRNA by aldosterone. To determine whether this induction has functional consequences on transepithelial Na+ current, we generated clonal CCD cell lines that express a tetracycline-inducible MLPH. Induction of MLPH in these cells led to a relatively modest, but statistically significant, increase in amiloride-sensitive Na+ current, suggesting the MLPH may be involved in ENaC trafficking. MyosinVc, the epithelial-specific class V myosin that is highly homologous to MyosinVa, another component of the melanosome trafficking complex, has putative consensus sites for serum and glucocorticoid-induced kinase 1 (SGK1), an early aldosterone-induced kinase that mediates some of aldosterone's effects on Na+ transport. Our results indicate that MyosinVc is phosphorylated by endogenous SGK1, suggesting that this complex may be involved in the aldosterone-regulated trafficking of ENaC in the CCD. These results suggest potential mechanisms by which aldosterone may regulate Na+ transport both directly, by increasing the abundance of MLPH, and indirectly by increasing the transcription of SGK1, which in turn regulates the activity of MyosinVc.

epithelial Na+ channel; myosin; serum and glucocorticoid-induced kinase

ALDOSTERONE IS THE PRIMARY hormone that regulates Na+ reabsorption in the kidney and is therefore a major determinant of long-term regulation of blood pressure. The main target of aldosterone is the epithelial Na+ channel (ENaC) which resides in the apical membrane of mineralocorticoid receptor (MR) target cells, most importantly the cortical collecting duct (CCD) of the kidney. There are two phases of aldosterone-induced Na+ transport (2, 4, 38, 48). The early phase, occurring within 20 to 60 min, is most likely due to activation of preexisting ENaC molecules in the cell membrane, increased ENaC trafficking or recycling to the membrane, and/or decreased degradation of ENaC (77). The delayed phase of aldosterone action (>3 h) is characterized by a change in the anabolic state of the cell, which allows for greater Na+ transport (77). This is due to an increase in abundance of functional ENaC and Na-K-ATPase (basolateral Na+ pump) in the plasma membrane (5, 11, 12, 24, 63). Both the early and delayed phases of aldosterone action are dependent on transcriptional regulation, resulting from the hormone-activating MRs and/or glucocorticoid receptors (GR).

The molecular mechanisms of aldosterone's effects on Na+ transport are still not entirely clear. One of the most promising candidates in mediating these effects is the serum and glucocorticoid-induced kinase 1 (SGK1), which has been identified as an early aldosterone-induced gene (19, 48). SGK1 has been implicated in regulating cell proliferation (31), protecting cells from apoptosis (45), and regulating ion transport (19, 48, 49). Its role in mediating some of the effects of aldosterone on sodium transport is the only process that has been clearly established both in cell culture and in vivo (3, 19, 32, 49, 83). SGK1 increases the abundance of ENaC at the cell surface in oocytes (3, 48) and A6 cells (2); however, it is unclear what the contribution of increased ENaC mRNA levels, increased trafficking or recycling of ENaC subunits to the membrane, or decreased degradation of ENaC is to this effect. In cell culture, SGK1 regulates the transcription of ENaC subunits (13), which is a delayed phase mechanism and does not explain the earlier actions of aldosterone.

The exact mechanism responsible for ENaC trafficking still needs to be established. The channel is made up of three homologous subunits, {alpha}, beta, and {gamma} (15, 17, 37, 40, 41, 79), each consisting of a large extracellular loop, two membrane-spanning domains, and cytoplasmic COOH and NH2 termini (16, 62, 70). Although it appears inefficient, most of the unassembled and some of the assembled ENaC subunits are ubiquitinated and degraded by proteosomes; only 1% of ENaC reaches the plasma membrane after processing in the Golgi (76). Similar to other trafficking systems, in recent years a number of studies have shown a role for syntaxins and other SNARE proteins in the exocytosis of ENaC (9, 20, 58, 66); however, which SNAREs and what additional proteins are involved in ENaC trafficking is still not entirely clear. Furthermore, it is still not known whether exocytosis of ENaC occurs through a constitutively active or a regulated pathway.

The current hypothesis for ENaC degradation includes the epithelial cell-specific ubiquitin protein ligase, Nedd4.2. Nedd 4.2 binds to ENaC subunits which causes an increase in endocytosis of the channel resulting in decreased Na+ transport (72). When phosphorylated, Nedd4.2 is unable to bind and ubiquitinate ENaC. Thus it has been suggested that SGK1, which is capable of phosphorylating Nedd4.2, can increase sodium transport by inhibiting ENaC degradation (22, 71). This hypothesis has only been proven in cells overexpressing Nedd4.2 and SGK1 and it is probably not the only mechanism by which aldosterone increases ENaC surface expression. Moreover, although SGK1 knockout mice have a salt-wasting phenotype (83), it is much less severe than that of the MR knockout mice (10). This suggests that other aldosterone-regulated genes are also playing a role in the regulation of Na+ homeostasis.

The goal of this study was to identify additional aldosterone-regulated genes in mouse CCD cells. Our efforts were focused on proteins that could potentially be involved in the trafficking or recycling of ENaC to the plasma membrane. We report for the first time that the transcript levels of melanophilin (MLPH), a protein involved in vesicular trafficking in melanocytes, are significantly and rapidly increased by physiological concentrations of aldosterone in mouse CCD cells. In melanocytes, MLPH has been shown to act as a link between Rab27a and an unconventional myosin, MyosinVa. In this study, we also determined that not only do M1 cells express the epithelial cell-specific MyosinVc (MyoVc), this myosin motor is apparently phosphorylated by endogenous SGK1.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of M1 cell lines stably expressing the tetracycline transactivator, rTA2A-M2. M1 cells, originating from mouse CCDs (73), were cultured in PC-1 medium (PC-1; BioWhittaker, Walkersville, MD; supplemented with 5% FBS, 2 mM L-glutamine, and 200 ng/ml normocin). Once cells had reached 60% confluence, they were transfected with the pBTE construct (59) generously provided by Dr. F. Kern (Southern Research Institute, Birmingham, AL), using Lipofectamine PLUS reagent (Invitrogen, Carlsbad, CA). Clones stably expressing the tet-transactivator, rTA2A-M2, were selected with 5 µg/ml blasticidin. Individual clones were expanded and tested for expression/activity of rTA2A-M2 by luciferase assay. Clonal populations were incubated in DMEM/F12 medium (Cellgro, Herndon, VA) supplemented with 5% tet-free serum, 2 mM L-glutamine, and antibiotics ± 500 ng/ml doxycycline for 24 h, and then transiently transfected with the pREV-TRE-Luc construct (Clontech) and incubated for an additional 24 h before measuring luciferase activity. The clonal cell line that exhibited the greatest luciferase activity and therefore the greatest expression/activity of the rTA2A-M2 was chosen for future experiments and will be referred to as M1-pBTE cells.

Generation of M1 cell lines stably expressing a tet-inducible rat MR. First, we generated the pREV-SG-TRE construct in which an improved tetracycline-regulated promoter, "SG-TRE" (1), was subcloned into the pREV-TRE retroviral construct (Clontech, Mountain View, CA) to replace the original promoter. The "SG-TRE," generously provided by Dr. S. Agha-Mohammadi (University of Pittsburgh, Pittsburgh, PA), contains a shortened cytomegalovirus (CMV) promoter together with eight tetracycline operator sequences, resulting in a greater reduction of basal leakiness than maximal transgene expression (1). Next, the rat MR (rMR) was cloned into the pBBHN vector (78), kindly provided by Dr. V. Ogryzko (Institut André Lwoff, Villejuif, France).

Following the generation of these two constructs, the biotinylatable rMR was subcloned into the pREV-SG-TRE vector and retroviruses were generated by transient transfection into amphotropic Phoenix cells (American Type Culture Collection, Rockville, MD) using Lipofectamine PLUS reagent. Following a 48-h incubation, retroviruses were harvested and used to infect M1-pBTE cells as previously described (32). Individual clones stably expressing the tetracycline-inducible rMR were selected using 200 µg/ml hygromycin. Individual clones were expanded, treated with 500 ng/ml doxycycline for 48 h to induce transcription of the MR, and tested for [3H]aldosterone binding as previously described (51). These cells will be referred to as M1/Tet-ON/MR cells.

Generation of M1 cell lines stably expressing a tet-inducible mouse MLPH. Mouse MLPH (mMLPH) cDNA, generously provided by Dr. M. Fukuda (Tohoku University, Japan), was subcloned into the pREV-SG-TRE retroviral construct, and retroviruses were used to infect M1-pBTE cells as previously described (32). Individual clones stably expressing the tet-inducible mMLPH were selected using 200 µg/ml hygromycin and expanded. These cells will be referred to as M1/Tet-ON/MLPH cells.

Aldosterone treatment, RNA, and protein isolation. M1/Tet-ON/MR cells were grown to confluence in PC-1 medium in 12- or 6-well tissue culture plates. We found that 2x stripped FBS contains residual amounts of corticosteroids that could potentially bind to the MR; therefore, to measure the effects of aldosterone activating the MR, we wanted to decrease the potential of trace amounts of hormones and small peptides to mask the effects of aldosterone. To do this, after reaching confluence, cells were incubated in a steroid-free DMEM/F12 medium containing 2.5%, 3x charcoal-stripped FBS, 2 mM L-glutamine, 200 ng/ml normocin, and 500 ng/ml doxycycline, to induce expression of the rMR, for 48 h. Cells were then treated with 0.01–10 nM aldosterone for 30 min to 24 h. For a separate set of experiments, in addition to aldosterone, M1/Tet-ON/MR cells were treated with 100 nM of the GR antagonist RU486.

To inhibit protein synthesis, M1/Tet-ON/MR cells were treated with 10 µM cycloheximide (Sigma, Palo Alto, CA), 5 µg/ml anisomycin (Sigma), or vehicle. Thirty minutes after the addition of the protein synthesis inhibitors or vehicle, half of the cells were treated with 1 nM aldosterone for 2 h.

M1/Tet-ON/MLPH cells were also grown to confluence in PC-1 medium; however, after reaching confluence, these cells were incubated for 48 h in a steroid-free DMEM/F12 medium supplemented with 5%, 2x stripped FBS, L-glutamine, and normocin and then treated, or not, with 500 ng/ml doxycycline to induce MLPH expression.

After treatment, both the M1/Tet-ON/MR and the M1/Tet-ON/MLPH cells were lysed in TRI-Reagent (Molecular Research Center, Cincinnati, OH) and RNA was extracted following the manufacturer's protocol. Alternatively, cells were lysed in an SDS solubilization buffer [48.2 mM 2-(N-hexylamino) ethanesulfonic acid, 1% SDS, 10% glycerol, and 1% protease and phosphatase inhibitors; Sigma].

Semiquantitative RT-PCR. To determine the relative amounts of MLPH, we used semiquantitative RT-PCR. Two micrograms of total RNA were reverse transcribed using MMLV Reverse Transcriptase (GIBCO, Gaithersburg, MD). Sense (5'-CGC GTT ACT GAG AAA CTT GAC A-3') and antisense (5'-TCT CCC AGC AGC CAA GGC GTA G-3') primers against the mMLPH sequence were used to amplify a 136-bp product. Four serial dilutions of cDNA were used as templates and PCR was carried out using Taq polymerase (New England Biolabs, Beverly, MA). After a 2-min denaturation at 95°C, the PCR reaction was carried out for 27 cycles (95°C for 30 s, 56°C for 30 s, and 72°C for 45 s). This was followed by a final extension of 8 min at 72°C. beta-Actin mRNA levels were determined from the same cDNA samples using sense (5'-AGA GGG AAA TCG TGC AC-3') and antisense (5'-CAA TAG TGA TGA CCT GGC CGT-3') primers for 23 cycles (95°C, 57°C, and 72°C, 1 min each). After amplification, samples were separated on 5% polyacrylamide gels which were subsequently stained with ethidium bromide and scanned on a FluoroImager 575 (Molecular Dynamics, Sunnyvale, CA). PCR products were quantified using ImageQUANT software (Molecular Dynamics). To determine the relative amount of MLPH mRNA, the slope of the amount of PCR product vs. the amount of template used was derived by linear regression and normalized to the slope of the beta-actin PCR product vs. template used. This method has previously been described in detail (75).

Quantitative real-time RT-PCR. To verify the results obtained by semiquantitative PCR, we used quantitative real-time RT-PCR. The sense and antisense primers mentioned above were used to amplify the 136-bp product using iTaq SYBR Green Supermix with Rox (Bio-Rad, Hercules, CA). The reactions were carried out in triplicate according to the manufacturer's protocol. In brief, 200 nM of both the sense and antisense primers along with 10 ng of cDNA and 12.5 µl of Supermix was brought to a final reaction volume of 25 µl. After an initial 3-min denaturation, PCR reactions were carried out for 40 cycles (95°C for 15 s, 57°C for 30 s). PCR reactions for beta-actin were carried out under the same conditions. The threshold cycle (Ct) of the PCR product generated was determined using the AB 7300 (Applied Biosystems, Foster City, CA) and relative expression (RE) levels were calculated using the following equation: RE = 2–(Ct of MLPH – Ct of beta-actin). At the recommendation of the manufacturer, and to verify that PCR products amplified were not contaminated with nonspecific double-strand DNA, a dissociation stage was carried out by incubating the reactions at 95°C for 15 s, 57°C for 30 s, and 95°C for 15 s.

Transient transfection of HEK293 and M1 cells with MyoVc-GFP. Subconfluent HEK293 cells (American Type Culture Collection), maintained in DMEM/F12 supplemented with 10% FBS, 2 mM L-glutamine, and 200 ng/ml normocin, were transfected with a GFP-tagged human MyosinVc tail construct (MyoVc-GFP, aa 907–1742) (64), generously provided by R. Cheney (University of North Carolina, Chapel Hill, NC), or empty pLEGFP-N1 vector as a control (Clontech) using Lipofectamine PLUS reagent. To avoid phosphorylation of substrates by PKB/AKT, cells were treated with 1–6 µM AKT inhibitor VIII (Calbiochem, San Diego, CA) for 24 h before lysing in immunoprecipitation buffer [20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM ETDA, 1 mM EGTA, 1% Triton X-100, 1% protease and phosphatase inhibitor cocktail (Sigma), 2.5 mM Na-pyrophosphate, 1 mM B-glycerolphosphate, 1 mM Na3VO4]. Similarly, subconfluent M1 cells, maintained in PC-1 complete medium, were transfected with MyoVc-GFP and treated or not with 6 µM AKT inhibitor VIII (Calbiochem) for 24 h before lysing in immunoprecipitation buffer.

Immunoprecipitation of MyoVc-GFP. Protein content of MyoVc-GFP-transfected HEK293 and M1 cell lysate was measured using the BCA protein assay kit (Pierce, Rockford, IL). GFP-tagged proteins were immunoprecipitated from 0.5–1.0 mg total protein using the mouse monoclonal 3E6 anti-GFP antibody (Molecular Probes, Eugene, OR) according to the company's protocol. Following the last wash step, beads were resuspended in 20 µl of SDS solubilization buffer and incubated for 3 min at 100°C. Insoluble material was pelleted and the supernatant was analyzed by Western blotting.

Western blot analysis. Twenty-five micrograms of total protein from M1/Tet-ON/MLPH cells were separated on 6–10% SDS-PAGE gel and transferred to an Immobilon-P membrane (Millipore, Bedford, MA). The membrane was incubated for 1 h at room temperature in blocking buffer (5% dry milk, 150 mM NaCl, 0.05% Tween, 124 µM Thimerosal, and 10 mM Tris, pH 7.5) and then overnight with a 1:1,000 dilution of the rabbit polyclonal anti-MLPH antibody, MelPep2 (74), kindly provided by Dr. M. Seabra (Imperial College, London, UK). After sufficient washing, the membrane was incubated with 1:2,000 dilution of horseradish peroxidase (HRP)-linked goat anti-rabbit antibody (Cell Signaling, Beverly, MA) for 45 min at room temperature.

Immunoprecipitated GFP-tagged protein from HEK293 cells was separated on 7.5% SDS-PAGE gel in duplicate, transferred to Immobilon-P membrane, and blocked for 1 h at room temperature. One-half of the membrane was probed with an anti-MyoVc antibody (64), generously provided by Dr. R. Cheney, diluted 1:100 in blocking buffer. The duplicate half of the membrane was probed with a 1:1,000 dilution of an antibody against proteins containing a phosphorylated serine/threonine preceded by Lys/Arg at positions –5 and –3, named the Phospho-Ser/Thr AKT/SGK substrate antibody (anti-P.AKT/SGK1s, Cell Signaling). After sufficient washing, both membranes were incubated for 45 min at room temperature with HRP-linked anti-rabbit antibody (Cell Signaling) at a 1:5,000 dilution (MyoVc blot) or 1:1,000 dilution (P.AKT/SGK1s blot). Immunoprecipitated GFP-tagged protein from M1 cells was also separated on a 7.5% SDS-PAGE gel and transferred to an Immobilon-P membrane. The membrane was then probed with a 1:1,000 dilution of the P.AKT/SKG1s antibody and a 1:1,000 HRP-linked anti-rabbit antibody as described above.

Signals on all immunoblots were visualized using SuperSignal West Dura substrate (Pierce) and scanned using the FluoroChem 8900 imaging system (Alpha Innotech, San Leandro, CA) in conjunction with the ChemiImager 5500 software (Alpha Innotech).

Determination of transepithelial voltage, resistance, and Na+ current in M1 cells stably expressing rMR or mMLPH. M1/Tet-ON/MR and M1/Tet-ON/MLPH cells were seeded onto Millicell-permeable membranes (Millipore) and grown to confluence in PC-1 medium. M1/Tet-ON/MR cells were maintained in steroid-free, 5% 3x stripped FBS-containing medium for 3 days before being treated with doxycycline (500 ng/ml) and/or aldosterone (10 nM). M1/Tet-ON/MLPH cells were maintained in steroid-free, 5% 2x stripped FBS-containing medium for 3 days and then treated with doxycycline (500 ng/ml) for 4 days. Transepithelial voltage (VTE) and resistance (RTE) values were measured with an epithelial voltohmeter (World Precision Instruments, Sarasota, FL) and equivalent short-circuit currents (ISC) were calculated. Our laboratory previously determined that in M1 cells, ISC represents transepithelial Na+ current, as it is completely eliminated by 1 µM apical amiloride (23).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Functional characterization of M1/Tet-ON/MR cells. The M1 cell line originates from mouse CCDs (73), but it does not express functional MRs. To study the physiological effects of aldosterone, mediated through the MR, we developed M1 cells that stably express a tet-inducible MR. We determined the number of high-affinity (Kd in the nanomolar range) MRs/cell in several clonal populations of M1/Tet-ON/MR cells. After induction with doxycycline, clone 6 (C6) expressed {approx}860 MRs/cell and clone 4 (C4) expressed {approx}6,600 MRs/cell, whereas the untreated and parent cells had no detectable specific binding of [3H]aldosterone. We chose C4 for future experiments as the number of MRs is more representative of the in vivo state, {approx}10,000 MRs/cell (50, 68). To determine whether the stably expressed, inducible MRs are functional, we examined the effects of aldosterone on the transepithelial Na+ current of M1/Tet-On/MR C$ cells (Fig. 1). When pretreated with doxycycline, aldosterone greatly increased amiloride-sensitive Na+ current within 24 h and these cells continued to have higher ISC values than the control cells through 72 h (Fig. 1A). Figure 1B shows that ISC values were increased as early at 4 h after aldosterone treatment. For all experiments, RTE values remained steady during the experiment. It should also be noted that ISC in M1 monolayers decreases in the absence of steroids, as illustrated in Fig. 1A. These data indicate that not only are the M1/Tet-On/MR C4 cells expressing doxycycline-inducible MRs, these receptors are functional at physiological concentrations of aldosterone.


Figure 1
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  		Fig. 1. A: short-circuit current (ISC) values of M1/Tet-ON/MR cells treated with doxycycline and/or aldosterone for 3 days. Cells were incubated in steroid-free medium (SF; DMEM/F12 supplemented with 5%, 3x charcoal stripped FBS) + 500 ng/ml doxycycline for 24 h before treatment with aldosterone. On day 0, doxycycline treatment continued and 10 nM aldosterone was added (Aldo), or not (Control), to the medium. On days 1 through 3 doxycycline and aldosterone treatment continued. Transepithelial voltage (VTE) and resistance (RTE) were measured every 24 h and ISC was calculated; n = 3. *P < 0.05, **P < 0.001, 1-tailed t-test, vs. control cells from the corresponding time point. B: early response to aldosterone in M1/Tet-ON/MR cells. Cells were pretreated with 500 ng/ml doxycycline for 24 h before the addition of aldosterone to the culture media. RTE and VTE were measured at 4 h and ISC was calculated; n = 3. **P < 0.001, 1-tailed t-test, vs. control cells.

 
Aldosterone increases MLPH transcript levels. Our recent DNA microarray data with M1 CCD cells (A. Fejes-Tóth, unpublished results) indicate that MLPH, a trafficking protein in melanocytes, is increased by corticosteroids. This observation prompted us to ask two questions: 1) is MLPH directly regulated by aldosterone in CCD cells, and if so 2) is MLPH involved in the regulation of Na+ transport? To answer the first question, we treated M1/Tet-On/MR C4 cells with physiological concentrations of aldosterone and determined the relative amounts of MLPH mRNA. As shown in Fig. 2, as low as 0.1 nM concentration of aldosterone significantly increased MLPH mRNA levels ~3.6-fold after 24 h. It is important to note that 1 nM aldosterone did not further increase the levels of MLPH mRNA. At this concentration, aldosterone occupies only the MR and not the GR, which has a lower affinity for aldosterone than the MR (26). These data indicate that the effect is mediated by the MR. To confirm this, we attempted to block GR-mediated effects using the GR antagonist, RU486. As expected, even in the presence of RU486 both 1 and 10 nM aldosterone significantly increased the levels of MLPH transcript. Some of the effects of 10 nM aldosterone are attenuated by RU486 indicating slight activation of the GR at this concentration. This is not the case with 1 nM aldosterone (Fig. 3) indicating that, at least at this concentration, the effect is mediated by the MR.


Figure 2
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  		Fig. 2. Physiological concentrations of aldosterone increase the expression of melanophilin (MLPH) transcript in M1/Tet-ON/MR cells. Cells were treated with varying concentrations of aldosterone or vehicle for 24 h. mRNA levels were determined by semiquantitative RT-PCR and are normalized for the level of beta-actin mRNA; n = 2 for 0.1 nM, n = 3 for all other concentrations. *P < 0.05; **P < 0.001, 1-tailed t-test, vs. control, untreated cells.

 

Figure 3
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  		Fig. 3. Aldosterone-induction of MLPH is mediated by the mineralocorticoid receptor. Cells were treated with 100 nM of the glucocorticoid receptor antagonist, RU486, and aldosterone (1 or 10 nM) or vehicle solution for 24 h. MLPH mRNA levels were determined by semiquantitative RT-PCR and are normalized to the level of beta-actin mRNA; n = 3. *P < 0.05, **P < 0.001, 1-tailed t-test, vs. cells receiving steroid-free medium only.

 
We then examined the time course MLPH mRNA induction in CCD cells using 1 nM aldosterone. As shown in Fig. 4A, aldosterone increased the levels of MLPH transcript as early as 2 h and MLPH expression remained significantly higher than in control cells through 24 h. These results, obtained by semiquantitative PCR, were verified by quantitative real-time PCR. As shown in Fig. 4, traditional semiquantitative PCR and real-time PCR yielded very similar results. After determining that aldosterone increases the levels of MLPH transcript, we attempted to determine whether this increase is paralleled at the protein level; however, our Western blot analysis revealed that, in M1 cells, endogenous MLPH expression was too low to detect using the MelPep2 antibody (kindly provided by Dr. M. Seabra; Imperial College, London, UK).


Figure 4
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  		Fig. 4. One nanomolar aldosterone increases the level of MLPH transcript in M1/Tet-ON/MR C4 cells by 2 h. A: MLPH mRNA levels were determined by semiquantitative RT-PCR and normalized to the levels of beta-actin mRNA. For 30 min, n = 4, for 2 h n = 7 and for 24 h n = 8. **P < 0.005, 1-tailed t-test, vs. vehicle-treated cells. B: relative MLPH expression was determined using real-time PCR, n = 2. *P < 0.05, 1-tailed t-test, vs. vehicle-treated cells.

 
Effect of aldosterone on MLPH mRNA levels does not require protein synthesis. The rapid time course of MLPH mRNA induction by aldosterone suggests that it is a direct effect. To confirm this, we tested whether de novo protein synthesis is required for the increase of MLPH mRNA levels. We used two structurally different protein synthesis inhibitors, cycloheximide and anisomycin, at concentrations previously shown to inhibit protein synthesis in distal tubular cells (34). As in our previous experiments, in M1/Tet-ON/MR C4 cells aldosterone increased the expression of MLPH mRNA about threefold in cells not receiving protein synthesis inhibitors (Fig. 5). In the presence of either anisomycin or cycloheximide, aldosterone increased the expression of MLPH mRNA about three- to fourfold compared with the vehicle-treated group, and these increases are not significantly different from the induction obtained with aldosterone alone. Treatment with anisomycin or cycloheximide alone did not affect MLPH transcript levels. These findings indicate that de novo protein synthesis is not required for aldosterone induction of MLPH at the transcriptional level.


Figure 5
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  		Fig. 5. De novo protein synthesis is not required for aldosterone to increase the levels of MLPH mRNA in M1/Tet-ON/MR C4 cells. Cells were treated with protein synthesis inhibitors anisomycin (5 µg/ml) or cycloheximide (10 µM) 30 min before a 2-h treatment with 1 nM aldosterone. MLPH mRNA levels were determined by semiquantitative RT-PCR and normalized to the levels of beta-actin mRNA, n = 3. *P < 0.05, 1-tailed t-test, vs. untreated cells.

 
Transepithelial Na+ current in M1/Tet-On/MLPH cells overexpressing mMLPH. To determine whether the aldosterone-stimulated MLPH expression has functional consequences on transepithelial Na+ current in CCD cells, we generated several clonal M1 cell lines that stably express a tet-inducible mMLPH. The expression levels of MLPH mRNA in two clonal cell lines of M1/Tet-On/MLPH cells treated or not with doxycycline are shown in Fig. 6A. Compared with the untreated cells, MLPH mRNA levels in clone 5 (C5) were ~7- and 11-fold higher at 24 and 48 h of doxycycline treatment, respectively. Similarly, doxycycline increased mRNA levels in clone 6 (C6) by ~6- and 8-fold after 24 and 48 h, respectively. To determine whether these changes in MLPH mRNA expression are also paralleled by changes in protein levels, we analyzed doxycycline-treated M1/Tet-On/MLPH C6 cells by Western blotting. Figure 6B shows that while endogenous MLPH was undetectable in the vehicle-treated cells, a strong signal at the expected size for MLPH protein was observed in cells treated with doxycycline for 24 h. These data confirm that doxycycline induces expression of MLPH transcript, as well as MLPH protein.


Figure 6
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  		Fig. 6. Doxycycline increases the level of MLPH transcript and protein within 24 h in M1/Tet-ON/MLPH C6 cells. A: cells were maintained in SF medium (DMEM/F-12 supplemented with 5%, 2x stripped FBS) for 48 h before the addition of 500 ng/ml doxycycline or vehicle. MLPH mRNA levels were determined by semiquantitative RT-PCR and are normalized to beta-actin mRNA levels. All measurements were made in duplicate. B: M1/Tet-ON/MLPH C6 cells were incubated in SF medium ± 500 ng/ml doxycycline for 24 h and then lysed in SDS solubilization buffer. Twenty-five micrograms of total protein were separated on an SDS-PAGE gel and probed with the anti-MLPH antibody, MelPep2.

 
Next, we asked the question, does MLPH affect amiloride-sensitive Na+ current in M1 cells? Figure 7 shows the ISC values for M1/Tet-On/MLPH C6 monolayers treated with doxycycline over 4 days. Induction of MLPH with 500 ng/ml doxycycline led to a significantly greater Na+ current within 2 days of treatment (Fig. 7, **P < 0.001) compared with untreated cells. This increase in current continued to be significant through day 4 of the experiment. RTE values were similar in control and doxycycline groups on day 0 (control: 2.0 ± 0.11 k{Omega}·cm2; doxycycline: 2.1 ± 0.08 k{Omega}·cm2) and on day 4 (control: 1.8 ± 0.06 k{Omega}·cm2; doxycycline: 2.0 ± 0.04 k{Omega}·cm2). As expected, ISC of control monolayers decreased over 4 days due to the absence of steroid hormones in the medium.


Figure 7
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  		Fig. 7. MLPH induction increases ISC of M1/Tet-ON/MLPH C6 cells. Cells were incubated with SF medium for 3 days before treatment with 500 ng/ml doxycycline on day 0. VTE and RTE were measured every 24 h and Isc was calculated, n = 8. *P < 0.05, 1-tailed t-test, vs. untreated cells, and P < 0.001, using 2-way ANOVA.

 
MyosinVc tail is phosphorylated by endogenous SGK1. The current model for melanosome trafficking in melanocytes involves at least three trafficking proteins (see Fig. 9): MLPH, Rab27a, and MyosinVa (MyoVa) (7, 25, 33, 39, 57, 80). Class V myosins are broadly distributed and are hypothesized to participate in actin-dependent trafficking complexes (14, 56, 61, 81). MyosinVc (MyoVc) has a similar structure, a conserved globular tail and is expressed specifically in epithelial and glandular tissues (64). It could therefore be involved in epithelial cell-specific transport processes including Na+ transport. It has been shown that phosphorylation of the tail domain of MyoVa regulates its activity (65). Our analysis using MIT Scansite 2.0 revealed that MyoVc has a SGK/AKT phosphorylation consensus sequence (53) with a score of 0.2245 and 0.000% (a score of 0.0000 is considered an optimal match and a % <0.2 falls within the "high-stringency" threshold). The amino acid that is putatively phosphorylated by SGK/AKT is Ser1539, located within the COOH-terminal tail of MyoVc. Because SGK1 is an aldosterone-regulated gene and MyoV is a member of the same trafficking complex as MLPH, we hypothesized that SGK1 phosphorylates, and thereby activates, MyoVc. We tested this hypothesis in cell culture by transiently transfecting both HEK293 cells and M1 cells with a GFP-tagged MyoVc tail and immunoprecipitating the protein with an anti-GFP antibody before Western blot analysis.


Figure 9
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  		Fig. 9. Current model for melanosome transport in melanocytes. MyoV, Myosin V; SHD, Slp homology domain; MBD, myosin binding domain.

 
Figure 8A (lane 1) shows a strong band at 120 kDa in the MyoVc-GFP-transfected HEK293 cells when the membrane was blotted for MyoVc. The size of this band is consistent with the appropriate size of the MyoVc-GFP and is not detected in the control cells (lane 2). To determine whether the transiently expressed MyoVc is phosphorylated by endogenous SGK1, we blotted a duplicate membrane with the anti-P.AKT/SGK1s antibody. The results shown in Fig. 8A (lane 3) indicate that a band with the same size (120 kDa) as MyoVc-GFP is also recognized by the anti-P.AKT/SGK1s antibody in the MyoVc-transfected cells. It is important to note that the anti-P.AKT/SGK1s antibody did not interact with the immunoprecipitated PLEGFP-N1 protein (lane 4). We performed similar experiments in M1 cells. Figure 8B (lanes 1 and 2) shows a strong band at 120 kDa in MyoVc-GFP-transfected M1 cells when the membrane was blotted with the anti-P.AKT/SGK1s antibody. The size of this band is again consistent with the appropriate size of the MyoVc-GFP.


Figure 8
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  		Fig. 8. COOH-terminal tail of MyoVc is a potential SGK1 substrate. A: 500 µg total protein extracted from MyoVc-GFP or pLEGFP-N1 (control)-transfected HEK293 cells was immunoprecipitated with an anti-GFP antibody. Immunoprecipitated protein was separated on a SDS-PAGE gel, transferred to an Immobilon-P membrane, and probed with an anti-MyoVc antibody (lanes 1 and 2) or an anti-P.AKT/SGK1s antibody (lanes 3 and 4). B: 500 µg total protein from MyoVc-GFP-transfected M1 CCD cells treated (lane 1) or not (lane 2) with an AKT inhibitor was immunoprecipitated with an anti-GFP antibody. Immunoprecipitated protein was separated on an SDS-PAGE gel, transferred to an Immobilon-P membrane, and probed with an anti-P.AKT/SGK1s antibody.

 
To determine whether MyoVc is being phosphorylated by endogenous SGK1 or AKT, MyoVc-GFP-transfected HEK293 cells were incubated for 24 h with an AKT inhibitor at a concentration previously shown to inhibit AKT phosphorylation in cell culture (8); therefore, proteins recognized by the anti-P.AKT/SGK1s antibody, such as the 120-kDa MyoVc-GFP in Fig. 8A, are most likely phosphorylated by endogenous SGK1 and not AKT. To verify this, M1 cells were treated with the AKT inhibitor or vehicle before immunoprecipitation and Western blotting (Fig. 8B). Treatment with the AKT inhibitor (lane 1) had no effect on the density of the 120-kDa band, and therefore relative amount of phosphorylation of MyoVc-GFP, compared with vehicle-treated cells (lane 2). Since the anti-P.AKT/SGK1s antibody recognizes phosphorylated residues specifically within the SGK1/AKT consensus sequence, these data together suggest that MyoVc is a potential SGK1 substrate in CCD cells (Fig. 8).


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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The main finding of this study is that MLPH, a gene involved in vesicular trafficking, is rapidly induced by aldosterone in mouse CCD cells. This effect was near maximal at physiological aldosterone concentrations indicating that it is mediated by the MR and could potentially have biological significance. Furthermore, the rapid time course suggests that the induction of MLPH transcript is a direct effect. This is supported by our results showing that induction of MLPH mRNA by aldosterone does not require de novo protein synthesis. Indeed, NUBIScan (55) analysis of the MLPH gene revealed several putative GRE/MREs located within the MLPH promoter in both human and mouse. The time course of MLPH induction by aldosterone is consistent with the stimulatory effect of aldosterone on Na+ transport in M1/Tet-ON/MR cells, which occurs as early as 4 h and persists through 72 h.

MLPH is a Rab effector and a member of the synaptotagamin-like protein (SLP) family. A 21-bp deletion in the MLPH gene is responsible for the "leaden" phenotype (silver coat color) first identified in mice in 1933 (39, 47). MLPH is an essential member of the melanosome trafficking complex in melanocytes, which also includes MyoVa and Rab27a (25, 33, 39, 43, 44, 46, 80, 84) (Fig. 9) . The NH2 terminus of MLPH contains a SLP homolog domain that directly binds to Rab27a, which binds to the melanosome cargo, whereas the middle region of MLPH binds to the tail of MyosinVa. Thus MLPH is the link between the cargo and myosin motor (25). In humans, defects in members of the melanosome trafficking complex cause Griscelli syndrome, a rare, autosomal-recessive disorder that results in pigment dilution of skin and hair, and accumulation of melanosomes in melanocytes (42). Thus far, melanosome trafficking is the only system in which MLPH has been characterized.

The obvious question in regards to our primary finding is what is the function of MLPH in renal epithelium? Very little is known about the proteins involved in ENaC trafficking and it is our understanding that, at this point, MLPH is the only trafficking protein known to be regulated by aldosterone at the transcriptional level. We also determined that both Rab27a and the epithelial cell-specific MyoVc are expressed in M1 cells (data not shown) suggesting that a trafficking complex similar to that seen in melanocytes might exist in renal epithelia. Therefore, it is logical to assume that aldosterone increases MLPH expression to increase the abundance of ENaC in the cell membrane. Indeed, our results show that stable, tet-inducible expression of MLPH in M1 cells leads to a modest, but statistically significant, increase in amiloride-sensitive Na+ current within 48 h that continues through day 4 of treatment with doxycycline. Interestingly, although we observe an increase in both the levels of MLPH mRNA and protein within 24 h of doxycycline treatment, the increases in current were not significant until 48 h. This could be due to the fact that the actions of aldosterone are pleiotropic; other members of the trafficking complex, including mature ENaC subunits, may need to be upregulated as well. In addition, both subcellular location and the potential need for posttranslational modification of MLPH, or other members of the trafficking complex such as MyoVc, may delay the effect of increasing Na+ transport via elevated MLPH levels alone.

To support the hypothesis that proteins responsible for transporting melanosomes in melanocytes play a role in ENaC trafficking, our studies also revealed that in both HEK293 and M1 cells, the COOH-terminal tail of MyoVc is phosphorylated by an endogenous kinase, possibly SGK1, an early aldosterone-induced gene (19, 48). Although it is possible that AKT could be phosphorylating MyoVc, based on our studies in which cells were treated or not with an AKT inhibitor, the phosphorylation in M1 cells does not seem to be AKT dependent. The tail of myosin V family members is responsible for binding cargo (18, 35, 54, 67, 82) and it is likely that modification of this domain confers specificity for adapter proteins, such as MLPH, and therefore cargo (54). Most importantly, Serine1539, located within the putative SGK1 consensus sequence in MyoVc, is conserved across species and between myosin V family members. This serine is also part of putative consensus sequences for other protein kinases such as protein kinase A, protein kinase C, and Clk2 kinase and has been shown to be phosphorylated, and its activity regulated, by calcium/calmodulin-dependant protein kinase in mitotic Xenopus laevis egg extracts (36). These observations raise the possibility that phosphorylation of Ser1539 of human MyoVc (corresponding to Ser1650 in mouse MyoVa) provides a common mechanism of regulation for vesicular or organelle transport via multiple kinases. Therefore, it seems reasonable to hypothesize that, in the kidney, aldosterone-induced SGK1 is capable of regulating the activity MyoVc by phosphorylation, which could result in increased association of ENaC trafficking proteins, and consequently elevated abundance of ENaC in the plasma membrane.

There are several unanswered questions that still surround our main hypothesis. First, is MyoVc the motor responsible for transporting ENaC? Thus far, we can conclude that MyoVc is expressed in mouse CCD cell lines and the human kidney (64) and is probably phosphorylated by endogenous SGK1 in M1 cells; however, the contribution of this protein to the regulation of Na+ transport remains to be established. Second, which Rab protein associates with ENaC and MLPH in CCD cells? Rab proteins are a ubiquitously expressed family of GTPases that have been implicated in the regulation of vesicular trafficking (reviewed in Ref. 29). Currently, more than 60 Rabs have been identified in mammalian cells with differential expression (69), reflecting the specialized transport processes among various tissues. The kidney expresses numerous Rabs (6, 21, 27, 28, 30, 52, 60), including Rab27a, but it is unclear which, if any, associates with ENaC and MLPH in the CCD.

In conclusion, the major findings of this study raise the possibility of a novel mechanism by which aldosterone could regulate Na+ transport in the CCD. Given the well-established role of MLPH and MyoVa in vesicular trafficking, and our studies indicating that induction of increased MLPH levels in M1 cells leads to an increase in amiloride-sensitive Na+ current, it is reasonable to assume that aldosterone may regulate the trafficking of ENaC directly by regulating the transcript levels of MLPH, and possibly other members of the MLPH-Rab-Myosin trafficking complex. In addition, aldosterone may affect this trafficking pathway indirectly by increasing the transcription of SGK1, which phosphorylates MyoVc, possibly regulating its activity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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REFERENCES
 
This work was supported by National Institutes of Health Grants DK-41841, DK-55845, and DK-58898.


   ACKNOWLEDGMENTS
 
We thank Dr. F. Kern (Southern Research Institute, Birmingham, AL) for providing the pBTE construct, Dr. S. Agha-Mohammadi (University of Pittsburgh, Pittsburgh, PA) for the SG-TRE construct, Dr. V. Ogryzko (Insitut André Lwoff, Villejuif, France) for the pBBHN construct, and Dr. M. Fukuda (Tohoku University, Japan) for the mouse MLPH cDNA. We also thank Dr. M. Seabra (Imperial College, London, UK) for the MelPep2 antibody and Dr. R. Cheney (University of North Carolina, Chapel Hill, NC) for the MyoVc-GFP construct and the anti-MyoVc antibody.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Náray-Fejes-Tóth, Dept. of Physiology, Dartmouth Medical School, 1 Medical Center Drive, Lebanon, NH 03756 (e-mail: Aniko.Fejes-Toth{at}Dartmouth.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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