Receptor tyrosine kinases mediate epithelial Na+ channel inhibition by epidermal growth factor
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《美国生理学杂志》
Department of Physiology, University of Texas Health Science Center, San Antonio, Texas
ABSTRACT
Epidermal growth factor (EGF) decreases Na+ reabsorption across distal nephron epithelia. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ transport in this portion of the nephron. Abnormal ENaC activity and EGF signaling are both associated with polycystic kidney disease localized to the distal nephron. We tested here whether EGF and other ligands for receptor tyrosine kinases (RTK) decrease ENaC activity. EGF markedly and quickly decreased ENaC activity. The RTK inhibitor erbstatin blocked EGF actions on ENaC and when added alone increased channel activity, uncovering basal suppression by endogenous RTK. The protein tyrosine phosphatase inhibitor vanadate, similar to EGF, decreased ENaC activity. Growth factors and vanadate decreased ENaC activity by decreasing open probability. ENaC was not phosphorylated in response to EGF, indicating that intermediary proteins transduce the inhibitory signal from the EGF receptor (EGFR) to ENaC. We find that neither MAPK 1/2 nor c-Src is signaling intermediaries between EGFR and ENaC. Inhibition of ENaC paralleled decreases in plasma membrane phosphatidylinositol 4,5-bisphosphate levels [PtdIns(4,5)P2] and was abolished by clamping PtdIns(4,5)P2. We conclude that EGF and other ligands for RTK decrease ENaC open probability by decreasing membrane PtdIns(4,5)P2 levels.
sodium reabsorption; protein tyrosine phosphatase; distal renal tubule
POLYCYSTIC KIDNEY disease, particularly the autosomal recessive form ARPKD, is marked by progressive cyst formation in the distal renal tubule and collecting duct system with cytogenesis, ultimately leading to renal insufficiency and end-stage renal disease (9, 28, 38). Hallmarks common to polycystic kidney disease (PKD) include disruption of the epidermal growth factor receptor (EGFR) axis and abnormal Na+ transport (7, 9, 16, 24, 36, 38).
The epithelial sodium channel (ENaC) is an amiloride-sensitive Na+ channel localized to the luminal plasma membrane of normal distal nephron principal cells where its activity is limiting for trans-epithelial Na+ reabsorption (6, 13). Several studies have demonstrated that EGF inhibits Na+ reabsorption in the distal nephron by decreasing activity of an amiloride-sensitive apical conductive pathway most likely representing ENaC (25, 34, 35, 37). Tyrosine kinase inhibitors attenuate this action of EGF on Na+ transport (21). However, the actions of EGF and receptor tyrosine kinase (RTK) on ENaC activity have not been directly studied. Endothelin-1 via the nonreceptor c-Src tyrosine kinase decreases ENaC activity by reducing channel open probability (8), and chronic blockade of tyrosine kinases with genistein decreases the number of ENaC in the luminal plasma membrane in renal A6 epithelia (19). Understanding the actions of EGF and RTK on ENaC activity becomes particularly important when the recent and apparently conflicting studies reporting that distal nephron Na+ reabsorption is decreased (36) and increased (24) in ARPKD are considered.
The goals of the present study were to determine if EGF directly influences ENaC activity and to glean insight into the cellular mechanism whereby this growth factor impinges on channel activity. We demonstrate here that EGF decreases ENaC activity via RTK signaling by reducing channel open probability. ENaC was not phosphorylated in response to RTK signaling, suggesting that intermediary proteins transduce the inhibitory signal from the EGFR to the channel. We exclude nonreceptor c-Src family tyrosine kinases and constituents in the MAPK 1/2 cascade as potential intermediates and provide evidence that tyrosine kinase signaling decreases ENaC activity by decreasing membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] levels.
EXPERIMENTAL DESIGN AND METHODS
Materials. All chemicals were purchased from Calbiochem (San Diego, CA), BioMol (Plymouth Meeting, PA), or Sigma (St. Louis, MO) unless noted otherwise and were of reagent grade. The plasmids encoding human ENaC subunits and Myc-tagged ENaC subunits have been described previously (2, 31, 32). The plasmid encoding EGFR (pRK5-HER1) was a kind gift from A. Ulrich (Munich, Germany) and has been described previously (15). The plasmids encoding the M1 muscarinic receptor and the fusion of EGFP with the plekstrin homology domain of PLC (PH-PLC-EGFP) were kind gifts from M. Shapiro and T. Meyer, respectively, and have been described previously (5, 10, 32). All material used in Western blot analysis was from Bio-Rad (Hercules, CA). Sulfo-NHS-LC-biotin and streptavidin-agarose were from Pierce (Rockford, IL). The mouse monoclonal anti-myc antibody was from Clontech (Palo Atlo, CA), and the rabbit polyclonal anti-phospho-tyrosine antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-mouse HRP-conjugated 2° antibody was from Kirkegaard-Perry Laboratories (Gaithersburg, MD). ECL reagents were from PerkinElmer Life Sciences (Boston, MA). Chinese hamster ovary (CHO) cells were maintained with standard culture conditions (DMEM + 10% FBS, 37°C, 5% CO2) and transfected using the Polyfect reagent (Qiagen, Valencia, CA) as described previously (2, 31, 32).
Electrophysiology. Whole cell macroscopic current recordings of ENaC reconstituted in CHO cells were made under voltage-clamp conditions using standard methods (2, 31, 32). In brief, CHO cells were transiently transfected using Polyfect with -, -, -hENaC cDNA (0.5 μg ea/35-mm dish). Current through ENaC in whole cell and excised, outside-out patches was the inward, amiloride-sensitive Na+ current with an internal pipette and extracellular bath solutions of (in mM) 120 CsCl, 5 NaCl, 5 EGTA, 2 MgCl2, 2 ATP, 0.1 GTP, 10 HEPES (pH 7.4), and 160 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4), respectively. All electrophysiological experiments were performed under constant bath perfusion (1 ml/min) with a dead time of 10–20 s. Current recordings were acquired with a PC505B patch-clamp amplifier (Warner Instruments) interfaced via a Digidata 1322A (Axon Instruments) to a PC running the pClamp 9 suite of software (Axon Instruments). For excised patches, gap-free current recordings were made at 0 mV. For whole cell experiments, macroscopic currents were elicited by voltage ramping from a holding potential of 40 to –100 mV with 1-s ramps delivered every 4 s. Whole cell capacitance was routinely compensated and was approximately 9 pF for CHO cells. Series resistances, on average 2–5 M, were also compensated.
Biochemistry. Western blot analysis was performed using standard procedures described previously (1, 2, 5, 11, 27). In brief, cells were lysed in gentle lysis buffer (1.0% NP-40), cleared, normalized for total protein concentration, suspended in Laemmeli sample buffer and 20 mM DTT, heated at 85°C for 10 min, run on 7.5% polyacrylamide gels (80 μg total protein/well) in the presence of SDS, transferred to nitrocellulose, and probed with antibody in Tris-buffered saline supplemented with 5% dry milk (Nestle, Solon, OH) and 0.1% Tween 20.
For immunoprecipitation experiments, CHO cells overexpressing all three myc-tagged ENaC subunits were extracted in gentle lysis buffer in the presence of phosphatase inhibitors. Whole cell lysates (400 μl at 1 μg/μl total protein) were treated with anti-myc antibody plus protein A/G PLUS agarose overnight at 4°C. Precipitants were washed three times with 400 μl gentle lysis buffer and resuspended in sample buffer. Blots were probed with anti-phospho-tyrosine antibody as described above.
Membrane-labeling experiments closely followed those described previously (2). In brief, CHO cells overexpressing all three myc-tagged ENaC subunits were washed 3x with ice-cold PBS (pH 8.0) and subsequently incubated with 1 mM sulfo-NHS-LC-biotin (in PBS, pH 8.0) for 30 min at 4°C in the dark. Cells were washed 3x with ice-cold PBS and extracted in gentle lysis buffer. Preequilibrated streptavidin agarose beads were agitated overnight at 4°C with 100 μg of total protein. Agarose beads were then washed 6x with gentle lysis buffer and subsequently resuspended in sample buffer. Proteins were visualized using standard Western blotting with anti-myc antibody as described above.
Evanescent field fluorescence microscopy. As described previously (32), we used evanescent field (EF) fluorescence [also called total internal reflection fluorescence (TIRF)] microscopy to selectively illuminate the plasma membrane to quantify membrane phospholipid levels. A fusion protein (EGFP-PH-PLC) containing the plekstrin-homology (PH) domain of PLC and EGFP was used as a specific PtdIns(4,5)P2 reporter. Cells were cotransfected with the PtdIns(4,5)P2 reporter and HER1. Methods for EF fluorescence microscopy followed closely those described previously by Almers and colleagues (26, 30). In brief, EF illumination was generated with through-the-lens TIRF. Samples were viewed through a Plan Apo TIRF 60x oil-immersion, high-resolution (1.45 numerical aperture) objective on a Nikon Eclipse TE2000 (Nikon Instruments, Melville, NY) inverted microscope. EGFP was excited with an argon laser with appropriate dichroic mirror and emission filter. Images were collected and processed with a Cascade Photometric CCD camera (Roper Scientific, Tucson, AZ) interfaced with a PC running MetaMorph software. In some instances, to better visualize translocation of the PtdIns(4,5)P2 reporter out of the plasma membrane in response to EGF, we pretreated cells for 30 min with the PI 4-kinase inhibitor wortmannin (50 μM) to prevent recycling/regeneration of PtdIns(4,5)P2.
Statistics. All patch-clamp data are presented as means ± SE. Paired and unpaired data were compared using appropriate t-tests with P 0.05 considered significant.
RESULTS
EGF decreases ENaC activity via RTK. We reconstituted human ENaC in CHO cells to directly test the effects of growth factors on channel activity. The channel was reconstituted by coexpressing -, -, -hENaC subunits as previously described (2, 31, 32). Channel activity was assessed as the amiloride-sensitive inward Na+ current at –80 mV under voltage-clamp conditions. All currents were corrected for cell surface area yielding current density. Figure 1A shows representative macroscopic current traces from a CHO cell expressing ENaC plus the EGF receptor HER1 before (con) and after bath application of 100 ng/ml (17 nM) EGF. As reported previously, CHO cells contain little endogenous leak current with none being sensitive to amiloride (2, 31, 32). Shown in Fig. 1B is the current-voltage (I-V) relationship for the amiloride-sensitive inward Na+ current before and after EGF. As summarized in Fig. 1C, EGF significantly decreased ENaC activity from 130 ± 21 to 31 ± 7 pA/pF. EGF had no effect on cell capacitance (not shown). The time course of EGF action on ENaC is shown in Fig. 1D with this growth factor rapidly decreasing channel activity. The effect of EGF on ENaC was abolished with pretreatment by the EGFR tyrosine kinase inhibitor erbstatin analog (10 μM; 30 min) as shown in Fig. 1E. ENaC activity as summarized in Fig. 1F in the absence and presence of EGF in cells pretreated with erbstatin was not different at 130 ± 53 and 140 ± 20 pA/pF, respectively. These results demonstrate that EGF through its RTK acutely decreases ENaC activity.
We next tested whether inhibition of endogenous tyrosine kinase activity affected ENaC. Figure 2A shows an overlay of typical macroscopic currents from a CHO cell expressing ENaC before (con) and after the erbstatin analog (10 μM). Shown in Fig. 2B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after erbstatin. As summarized in Fig. 2C, erbstatin significantly increased ENaC activity from 83 ± 24 to 158 ± 42 pA/pF. Erbstatin had no effect on cell capacitance (not shown). The time course of erbstatin action on ENaC is shown in Fig. 2D with inhibition of endogenous tyrosine kinases quickly augmenting channel activity.
Because tyrosine kinase signaling decreased ENaC activity, we tested whether phosphotyrosine phosphatases (PTP) also impinged on channel activity. Figure 3A shows typical macroscopic currents from a CHO cell expressing ENaC before (con) and after application of the broad range PTP inhibitor sodium vanadate (100 μM). Shown in Fig. 3B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after vanadate. As summarized in Fig. 3C, vanadate significantly decreased ENaC activity from 77 ± 5 to 15 ± 2 pA/pF. Vanadate had no effect on cell capacitance (not shown). The time course of vanadate action on ENaC is shown in Fig. 3D with inhibition of endogenous PTP decreasing channel activity.
Similar to EGF, IGF-I is a ligand for a RTK. We previously reported that IGF-I increases ENaC activity via PI3-K signaling with the phospholipid products of this lipid kinase directly increasing channel activity (32). This stimulatory action of IGF-I on ENaC is fully apparent only in the presence of repressed tyrosine kinase signaling. Figure 4A shows representative current traces from a CHO cell expressing ENaC before (con) and after IGF-I (100 ng/ml). Shown in Fig. 4B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after IGF-I with the effects of this growth factor on channel activity summarized in Fig. 4C (153 ± 27 to 26 ± 4 pA/pF). Figure 4D shows typical current traces from a CHO cell expressing ENaC and pretreated with erbstatin (10 μM; 30 min), before (con) and after treatment with IGF-I (100 ng/ml) and subsequently amiloride. When IGF-I was added to cells expressing ENaC and pretreated with erbstatin, channel activity significantly increased about twofold (summary graph not shown; see Ref. 32). The broad-range tyrosine kinase inhibitor genistein, similar to erbstatin, attenuated decreases in ENaC activity in response to IGF-I (not shown). The time courses of IGF-I actions on ENaC in the absence and presence of pretreatment with erbstatin are shown in Fig. 4, E and F, respectively. Figure 4G shows the time course of IGF-I actions on ENaC in a cell pretreated with both erbstatin and the PI3-K inhibitor wortmannin (0.2 μM). Pretreatment with both the RTK and PI3-K inhibitor abolished all actions of IGF-I on ENaC with activity being 100 ± 56 and 92 ± 50 pA/pF before and after addition of growth factor (summary graph not shown).
Results in Figs. 1–4 demonstrate that ENaC activity is reciprocally influenced by RTK and PTP. These results demonstrate, moreover, that ENaC is an end-effector for growth factor signaling, such as that initiated by EGF through RTK, with growth factors decreasing channel activity.
Growth factors via RTK decrease ENaC open probability. Gilmore and colleagues (8) reported that endothelin-1, which is a ligand for ETA and ETB heterotrimeric G protein-coupled receptors, decreases the activity of rat ENaC reconstituted in NIH 3T3 cells through the actions of nonreceptor c-Src family tyrosine kinases. Because Src family tyrosine kinases decrease ENaC activity by decreasing channel open probability (8), we wondered whether RTK signaling had a similar effect. Figure 5A shows several ENaC in a typical excised outside-out patch (holding potential = 0 mV; inward current down) made from a CHO cell coexpressing all three channel subunits and HER1 before (inset I control) and after (inset II) addition of EGF (100 ng/ml). As reported previously, ENaC reconstituted in CHO cells has a unitary conductance of 5 pS, is amiloride sensitive, is never observed in untransfected cells, and does not "run-down" in outside-out patches over time (2, 32). As shown by the all point histograms in Fig. 5, B and C, addition of EGF decreased ENaC open probability. The time course of EGF action on ENaC in excised outside-out patches and whole cell experiments was similar.
Addition of IGF-I, similar to EGF, decreased ENaC open probability. Figure 6A shows a representative excised outside-out patch (holding potential = 0 mV) containing several ENaC from a CHO cell coexpressing all three channel subunits before (inset I control) and after (inset II) IGF-I (100 ng/ml). Figure 6, B and C, shows all-points histograms before and after IGF-I.
Also similar to EGF, the PTP inhibitor vanadate decreased ENaC open probability as shown in Fig. 7. A representative excised outside-out patch (holding potential = 0 mV) containing a single ENaC from a CHO cell coexpressing all three channel subunits before and after vanadate (100 μM) is shown in Fig. 7A. Figure 7, B and C, shows all-points histograms before and after vanadate. Figure 7, D and E, summarizes ENaC activity (NPo) and open probability (Po) before and after treatment with vanadate. We rarely observed single ENaC in a membrane patch. However, in such an instance, as was the case for Fig. 7A, activity and open probability are equivalent with the effects of activating tyrosine kinase signaling conclusively decreasing ENaC open probability.
Results in Fig. 8 agree with those in Figs. 5–7 with both supporting the idea that RTK and PTP do not influence the number of ENaC in the plasma membrane but rather affect channel open probability. Shown here are two representative Western blots with the left blot containing whole cell lysates (80 μg/lane) from CHO cells not transfected (control) and those coexpressing all three channel subunits engineered with an NH2-terminal myc-tag in the absence and presence of erbstatin (10 μM, 15 min) and vanadate (100 μM, 15 min) treatment. Membrane proteins in these cells were labeled with sulfo-NHS-LC-biotin prior to extraction, with the blot on the right showing the membrane (streptavidin precipitant from 100 μg total protein) levels of ENaC in the respective cells. Both blots were probed with monoclonal anti-myc antibody. As reported previously, - and -ENaC expressed in CHO cells appear as overlying bands of 90 kDa with -ENaC migrating slightly faster (2, 32). These results demonstrate that inhibition of RTK and PTP has no acute effect on the membrane levels of ENaC.
ENaC is not tyrosine phosphorylated in response to growth factors and RTK signaling. Because RTK signaling leads to substrate phosphorylation and often leads to activation of downstream nonreceptor tyrosine kinases, as well as decreases in ENaC activity (see Figs. 1–7), we tested whether EGF and other growth factor ligands for RTK promote tyrosine phosphorylation of the channel. The top Western blot in Fig. 9 contains whole cell lysate from untransfected CHO cells and cells expressing myc-tagged ENaC treated with vanadate, EGF (in cells also coexpressing HER1), erbstatin, IGF-I and IGF-I plus erbstatin. This representative blot was probed with anti-myc antibody. The middle blot was probed with anti-phospho-tyrosine antibody and contains the anti-myc immunoprecipitant from the respective lysates. This blot clearly shows that ENaC subunits are not tyrosine phosphorylated in response to growth factor signaling via RTK. The bottom blot, which is the top blot stripped and reprobed with the anti-phospho-tyrosine antibody used with the middle blot, is a positive control demonstrating that as expected, EGF promotes tyrosine phosphorylation of EGFR (HER1), which runs as an 170-kDa protein.
Tyrosine kinase signaling decreases membrane PtdIns(4,5)P2 levels coincidently with ENaC activity. Because RTK signaling, as mentioned above, often impinges on the activity of nonreceptor tyrosine kinases, such as c-Src, and c-Src decreases ENaC activity (8), we asked whether Src family tyrosine kinases were involved in growth factor-mediated inhibition of ENaC. Figure 10 summarizes the effects of EGF and IGF-I on ENaC activity (amiloride-sensitive current density in CHO cells expressing ENaC) in the presence of the c-Src inhibitor PP2 (0.1 μM). Similar to their effects in the absence of PP2 (see Figs. 1 and 4), both growth factors decreased ENaC activity in the presence of PP2. Although these results do not definitively exclude c-Src from playing a role in RTK-mediated inhibition of ENaC in response to EGF, they do strongly suggest this to be the case. This is further supported by our earlier findings demonstrating that PP2, when employed in a manner identical to the way it was used in the current study, did indeed counter inhibition by c-Src of KCNQ K+ channels reconstituted in CHO cells (5).
ERK/MAPK also are often downstream effectors of RTK. Similar to inhibition of c-Src, inhibition of MEK1/2 with either PD-98059 (10 μM) or U-1026 (0.5 μM) did not affect EGF actions on ENaC (not shown). These results demonstrate that EGF can decrease ENaC activity via RTK signaling independent of c-Src and MAPK 1/2 signaling, suggesting that these latter two constituents do not lie between EGFR and the channel.
Depletion of membrane PtdIns(4,5)P2 levels in response to RTK signaling and subsequent activation of phospholipase C- affects the activity of phospholipid-sensitive ion channels (4, 22, 39). Because ENaC is sensitive to PtdIns(4,5)P2 levels with this phospholipid being permissive for channel activity (17, 32, 40), we asked whether EGF and the balance between RTK and PTP activities in CHO cells impacted membrane PtdIns(4,5)P2 levels in a manner consistent with changes in ENaC activity. Figure 11A shows the amount of a PtdIns(4,5)P2 reporter (23) in the plasma membrane before (top) and after (bottom) treatment with EGF (100 ng/ml, 12 min), vanadate (100 μM, 12 min, column 3), and vehicle (column 4). For these experiments, TIRF microscopy was used to isolate fluorescence signals from the PtdIns(4,5)P2 reporter in the plasma membrane (see EXPERIMENTAL DESIGN AND METHODS). For EGF, we show both an epiflourescence (column 1) and TIRF (column 2) image of the same cell before and after treatment. Both images show translocation of the PtdIns(4,5)P2 reporter out of the membrane in response to EGF, which is consistent with depletion of this phospholipid from the membrane (4, 14, 22, 39). Addition of EGF and vanadate significantly decreased plasma membrane PtdIns(4,5)P2 levels within 12 min to 38 ± 0.5 (n = 16) and 41 ± 4% (n = 5), respectively. Figure 11B shows the time course of PtdIns(4,5)P2 loss from the plasma membrane in response to EGF and vanadate with this time course being similar to that for inhibition of the channel. Although these results do not conclusively demonstrate that PtdIns(4,5)P2 depletion is causative for decreases in ENaC activity in response to EGF-RTK-PLC signaling, they are consistent with such a cellular mechanism of action, and together with the observations of others showing that depletion of PtdIns(4,5)P2 in response to RTK signaling impinges on ion channel activity (see below) provide a rationale for further future study of this possible mechanism.
Depletion of PtdIns(4,5)P2 decreases ENaC activity. We previously demonstrated that activation of muscarinic signaling in CHO cells by stimulating recombinant M1 receptors (M1-R) with oxotremorine decreases membrane PtdIns(4,5)P2 levels (32). Muscarinic M1 receptors are G protein-coupled receptors linked to PLC--mediated hydrolysis of PtdIns(4,5)P2 (14). With this in mind, we tested whether depletion of PtdIns(4,5)P2 via activation of M1-R coexpressed with ENaC would, like EGFR signaling, lead to decreases in ENaC activity. ENaC and M1-R are never found in the same cell. Thus, while M1-R signaling as used here is an artificial means of depleting PtdIns(4,5)P2 with respect to regulation of ENaC, it does serve as an important proof of principal for this possible mechanism of action. Figure 12A shows typical macroscopic currents from a CHO cell expressing ENaC plus M1-R before (con) and after oxotremorine (3 μM). Shown in Fig. 12B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after oxotremorine. As summarized in Fig. 12C, oxotremorine via M1-R significantly decreased ENaC activity. Oxotremorine signaling via M1-R had no effect on cell capacitance (not shown). The time course of oxotremorine action on ENaC is shown in Fig. 12D with depletion of PtdIns(4,5)P2 through G protein-linked PLC- likely affecting the channel in a manner similar to depletion of this phospholipid through RTK linked PLC-. Figure 12E, which shows a representative excised outside-out patch (holding potential = 0 mV) containing several ENaC from a CHO cell coexpressing all three channel subunits plus M1-R before and after addition of oxotremorine, is also consistent with the idea that depletion of PtdIns(4,5)P2 mediates both the EGF via RTK and oxotremorine via M1 inhibition of ENaC.
Figure 13 shows results from additional experiments probing the role of PtdIns(4,5)P2 depletion in EGF signaling to ENaC. In these experiments, EGFP-PH-PLC was used to buffer PtdIns(4,5)P2. In the presence of buffered PtdIns(4,5)P2, as shown in Fig. 13A, EGF had little effect on ENaC activity. This is in direct contrast to the affect this growth factor has on ENaC when PtdIns(4,5)P2 levels are not buffered and able to change (Fig. 1D). Figure 13B summarizes relative (to start values) ENaC activity in response to EGF in the absence and presence of buffered PtdIns(4,5)P2. Although in the absence of the PtdIns(4,5)P2 buffer relative activity after EGF was markedly decreased to 0.29 ± 0.06 (n = 10), in the presence of EGFP-PH-PLC relative activity decreased substantially less to 0.81 ± 0.10 (n = 4). Interestingly, in every experiment performed in cells expressing EGFP-PH-PLC, we observed a brief transient decrease in channel activity directly following EGF application (relative activity at 2.5 min, peak was 0.63 ± 0.08). We believe that this transient decrease in activity results from a sudden decrease in PtdIns(4,5)P2 levels near the channel in response to growth factor signaling with additional PtdIns(4,5)P2 losses and further decreases in channel activity countered by buffering. The results in Fig. 13 directly demonstrate that when PtdIns(4,5)P2 levels are clamped with a buffer, EGF is less able to markedly decrease ENaC activity allowing us to conclude that PtdIns(4,5)P2 depletion plays a central role in regulation of ENaC by this growth factor.
DISCUSSION
The current finding that EGF potently decreases activity of ENaC reconstituted in CHO cells is consistent with EGF decreasing Na+ reabsorption in cortical collecting duct epithelia (25, 34, 37). Importantly, the current study demonstrates for the first time the direct effects of EGF on ENaC. We find that EGF quickly and markedly decreases ENaC activity for a sustained period. Such a fast time course and sustained inhibition suggest a close signal transduction link between EGFR and ENaC.
Our results position a RTK, such as HER1, between EGF and ENaC with the receptor and channel closely linked for we could reconstitute regulation of ENaC by EGF and RTK in excised, outside-out patches. The present finding that EGF decreases ENaC activity via RTK is consistent with EGF decreasing Na+ reabsorption in isolated rabbit cortical collecting ducts in a herbimycin A-sensitive manner (21), with both suggesting that in native epithelia, ENaC is the final target for EGF-RTK signaling during decreases in Na+ reabsorption.
Bowlby and colleagues (3) demonstrated in a related study that EGF via RTK acutely decreases activity of Kv1.3 K+ channels reconstituted in HEK 293 cells. In this study, Kv1.3 was identified as a substrate for tyrosine phosphorylation with phosphorylation decreasing channel activity. In contrast, we find that ENaC is not a substrate for tyrosine phosphorylation, leading us to propose that signaling intermediaries must transduce the inhibitory signaling form RTK to ENaC. Three candidates were tested. We first asked whether EGF signaling via RTK impinged on ENaC activity via c-Src kinase. Our rationale was that RTK signaling is well known to activate cytosolic tyrosine kinases, such as c-Src, and c-Src decreases ENaC activity (8). We find no evidence that c-Src plays a role in EGF regulation of ENaC.
We next tested whether MAPK 1/2 signaling was involved in EGF regulation of ENaC. The rationale for testing this hypothesis is that others have demonstrated that MAPK 1/2 signaling is necessary for EGF to decrease Na+ reabsorption across an immortalized mouse collecting duct epithelial cell line (25), and MAPK 1/2 signaling decreases ENaC activity (1, 20). We find that MAPK 1/2 signaling is not necessary for the acute actions of EGF and RTK on ENaC.
The third signaling pathway tested that may possibly couple EGFR to ENaC is RTK-mediated depletion of membrane PtdIns(4,5)P2 levels. This signaling pathway has drawn much attention lately with the finding that nerve growth factor via the receptor tyrosine kinase TrkA diminishes membrane PtdIns(4,5)P2 levels via PLC- to regulate TRP channels (4, 22). EGF signaling via RTK also depleted PtdIns(4,5)P2 to modulate TRP channel activity (22) and EGF-mediated hydrolysis of PtdIns(4,5)P2 suppresses activation of GIRK channels (14). Depletion of PtdIns(4,5)P2 in response to RTK signaling, in addition, suppresses P/Q- and N-type Ca2+ channel activity (39). The current findings confirm that EGF and RTK signaling depletes PtdIns(4,5)P2 and demonstrates that depletion of PtdIns(4,5)P2 via M1 signaling independent of RTK also decreases ENaC activity. Moreover, we demonstrate that EGF is unable to affect ENaC activity when PtdIns(4,5)P2 levels are clamped. These findings are consistent with a cellular mechanism where EGF signaling via RTK leads to depletion of membrane PtdIns(4,5)P2 levels to decrease ENaC activity. Such a mechanism fits nicely with the recent findings that ENaC activity is modulated by membrane phospholipid levels to include PtdIns(4,5)P2 (17, 32, 40).
Open probability of ENaC is directly related to membrane PtdIns(4,5)P2 levels with this phospholipid being permissive for normal channel activity (17, 40). It is believed that as ENaC unbinds PtdIns(4,5)P2 open probability decreases. The current findings demonstrating that EGF and RTK signaling decrease ENaC open probability then also are consistent with a mechanism of action dependent on PtdIns(4,5)P2 depletion. Moreover, the quick time course of EGF actions on ENaC and the fact that this signaling pathway is membrane delimited also are consistent with depletion of PtdIns(4,5)P2 as the mechanism of EGF action on ENaC activity.
The EGFR is mislocalized to the luminal plasma membrane in ARPKD epithelial cells (9, 38). Moreover, ARPKD epithelial cells change phenotype, converting from a highly differentiated Na+ absorbing cell type to a less differentiated Cl–-secreting cell type (28, 38). We wonder whether these two hallmarks of ARPKD are related. We speculate that mislocalization of EGFR to the luminal membrane in cystic ARPKD epithelia leads to hyperactivation of this receptor by cystic EGF-like peptides with consequent diminution of membrane PtdIns(4,5)P2 levels, leading to decreases in ENaC activity. Such a transduction cascade may contribute to the abnormal transport properties observed in cystic epithelia.
GRANTS
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-59594, American Heart Association Texas Affiliate Grant 0355012Y, and the American Society of Nephrology Carl W. Gottschalk Research Scholar Grant (to J. D. Stockand).
ACKNOWLEDGMENTS
J. Medina and P. Patel are recognized for excellent technical assistance. We thank Drs. M. Gekle and A. Ulrich, and P. Snyder, respectively, for the HER1 and human ENaC cDNAs used in the current study.
FOOTNOTES
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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ABSTRACT
Epidermal growth factor (EGF) decreases Na+ reabsorption across distal nephron epithelia. Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ transport in this portion of the nephron. Abnormal ENaC activity and EGF signaling are both associated with polycystic kidney disease localized to the distal nephron. We tested here whether EGF and other ligands for receptor tyrosine kinases (RTK) decrease ENaC activity. EGF markedly and quickly decreased ENaC activity. The RTK inhibitor erbstatin blocked EGF actions on ENaC and when added alone increased channel activity, uncovering basal suppression by endogenous RTK. The protein tyrosine phosphatase inhibitor vanadate, similar to EGF, decreased ENaC activity. Growth factors and vanadate decreased ENaC activity by decreasing open probability. ENaC was not phosphorylated in response to EGF, indicating that intermediary proteins transduce the inhibitory signal from the EGF receptor (EGFR) to ENaC. We find that neither MAPK 1/2 nor c-Src is signaling intermediaries between EGFR and ENaC. Inhibition of ENaC paralleled decreases in plasma membrane phosphatidylinositol 4,5-bisphosphate levels [PtdIns(4,5)P2] and was abolished by clamping PtdIns(4,5)P2. We conclude that EGF and other ligands for RTK decrease ENaC open probability by decreasing membrane PtdIns(4,5)P2 levels.
sodium reabsorption; protein tyrosine phosphatase; distal renal tubule
POLYCYSTIC KIDNEY disease, particularly the autosomal recessive form ARPKD, is marked by progressive cyst formation in the distal renal tubule and collecting duct system with cytogenesis, ultimately leading to renal insufficiency and end-stage renal disease (9, 28, 38). Hallmarks common to polycystic kidney disease (PKD) include disruption of the epidermal growth factor receptor (EGFR) axis and abnormal Na+ transport (7, 9, 16, 24, 36, 38).
The epithelial sodium channel (ENaC) is an amiloride-sensitive Na+ channel localized to the luminal plasma membrane of normal distal nephron principal cells where its activity is limiting for trans-epithelial Na+ reabsorption (6, 13). Several studies have demonstrated that EGF inhibits Na+ reabsorption in the distal nephron by decreasing activity of an amiloride-sensitive apical conductive pathway most likely representing ENaC (25, 34, 35, 37). Tyrosine kinase inhibitors attenuate this action of EGF on Na+ transport (21). However, the actions of EGF and receptor tyrosine kinase (RTK) on ENaC activity have not been directly studied. Endothelin-1 via the nonreceptor c-Src tyrosine kinase decreases ENaC activity by reducing channel open probability (8), and chronic blockade of tyrosine kinases with genistein decreases the number of ENaC in the luminal plasma membrane in renal A6 epithelia (19). Understanding the actions of EGF and RTK on ENaC activity becomes particularly important when the recent and apparently conflicting studies reporting that distal nephron Na+ reabsorption is decreased (36) and increased (24) in ARPKD are considered.
The goals of the present study were to determine if EGF directly influences ENaC activity and to glean insight into the cellular mechanism whereby this growth factor impinges on channel activity. We demonstrate here that EGF decreases ENaC activity via RTK signaling by reducing channel open probability. ENaC was not phosphorylated in response to RTK signaling, suggesting that intermediary proteins transduce the inhibitory signal from the EGFR to the channel. We exclude nonreceptor c-Src family tyrosine kinases and constituents in the MAPK 1/2 cascade as potential intermediates and provide evidence that tyrosine kinase signaling decreases ENaC activity by decreasing membrane phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] levels.
EXPERIMENTAL DESIGN AND METHODS
Materials. All chemicals were purchased from Calbiochem (San Diego, CA), BioMol (Plymouth Meeting, PA), or Sigma (St. Louis, MO) unless noted otherwise and were of reagent grade. The plasmids encoding human ENaC subunits and Myc-tagged ENaC subunits have been described previously (2, 31, 32). The plasmid encoding EGFR (pRK5-HER1) was a kind gift from A. Ulrich (Munich, Germany) and has been described previously (15). The plasmids encoding the M1 muscarinic receptor and the fusion of EGFP with the plekstrin homology domain of PLC (PH-PLC-EGFP) were kind gifts from M. Shapiro and T. Meyer, respectively, and have been described previously (5, 10, 32). All material used in Western blot analysis was from Bio-Rad (Hercules, CA). Sulfo-NHS-LC-biotin and streptavidin-agarose were from Pierce (Rockford, IL). The mouse monoclonal anti-myc antibody was from Clontech (Palo Atlo, CA), and the rabbit polyclonal anti-phospho-tyrosine antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-mouse HRP-conjugated 2° antibody was from Kirkegaard-Perry Laboratories (Gaithersburg, MD). ECL reagents were from PerkinElmer Life Sciences (Boston, MA). Chinese hamster ovary (CHO) cells were maintained with standard culture conditions (DMEM + 10% FBS, 37°C, 5% CO2) and transfected using the Polyfect reagent (Qiagen, Valencia, CA) as described previously (2, 31, 32).
Electrophysiology. Whole cell macroscopic current recordings of ENaC reconstituted in CHO cells were made under voltage-clamp conditions using standard methods (2, 31, 32). In brief, CHO cells were transiently transfected using Polyfect with -, -, -hENaC cDNA (0.5 μg ea/35-mm dish). Current through ENaC in whole cell and excised, outside-out patches was the inward, amiloride-sensitive Na+ current with an internal pipette and extracellular bath solutions of (in mM) 120 CsCl, 5 NaCl, 5 EGTA, 2 MgCl2, 2 ATP, 0.1 GTP, 10 HEPES (pH 7.4), and 160 NaCl, 2 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4), respectively. All electrophysiological experiments were performed under constant bath perfusion (1 ml/min) with a dead time of 10–20 s. Current recordings were acquired with a PC505B patch-clamp amplifier (Warner Instruments) interfaced via a Digidata 1322A (Axon Instruments) to a PC running the pClamp 9 suite of software (Axon Instruments). For excised patches, gap-free current recordings were made at 0 mV. For whole cell experiments, macroscopic currents were elicited by voltage ramping from a holding potential of 40 to –100 mV with 1-s ramps delivered every 4 s. Whole cell capacitance was routinely compensated and was approximately 9 pF for CHO cells. Series resistances, on average 2–5 M, were also compensated.
Biochemistry. Western blot analysis was performed using standard procedures described previously (1, 2, 5, 11, 27). In brief, cells were lysed in gentle lysis buffer (1.0% NP-40), cleared, normalized for total protein concentration, suspended in Laemmeli sample buffer and 20 mM DTT, heated at 85°C for 10 min, run on 7.5% polyacrylamide gels (80 μg total protein/well) in the presence of SDS, transferred to nitrocellulose, and probed with antibody in Tris-buffered saline supplemented with 5% dry milk (Nestle, Solon, OH) and 0.1% Tween 20.
For immunoprecipitation experiments, CHO cells overexpressing all three myc-tagged ENaC subunits were extracted in gentle lysis buffer in the presence of phosphatase inhibitors. Whole cell lysates (400 μl at 1 μg/μl total protein) were treated with anti-myc antibody plus protein A/G PLUS agarose overnight at 4°C. Precipitants were washed three times with 400 μl gentle lysis buffer and resuspended in sample buffer. Blots were probed with anti-phospho-tyrosine antibody as described above.
Membrane-labeling experiments closely followed those described previously (2). In brief, CHO cells overexpressing all three myc-tagged ENaC subunits were washed 3x with ice-cold PBS (pH 8.0) and subsequently incubated with 1 mM sulfo-NHS-LC-biotin (in PBS, pH 8.0) for 30 min at 4°C in the dark. Cells were washed 3x with ice-cold PBS and extracted in gentle lysis buffer. Preequilibrated streptavidin agarose beads were agitated overnight at 4°C with 100 μg of total protein. Agarose beads were then washed 6x with gentle lysis buffer and subsequently resuspended in sample buffer. Proteins were visualized using standard Western blotting with anti-myc antibody as described above.
Evanescent field fluorescence microscopy. As described previously (32), we used evanescent field (EF) fluorescence [also called total internal reflection fluorescence (TIRF)] microscopy to selectively illuminate the plasma membrane to quantify membrane phospholipid levels. A fusion protein (EGFP-PH-PLC) containing the plekstrin-homology (PH) domain of PLC and EGFP was used as a specific PtdIns(4,5)P2 reporter. Cells were cotransfected with the PtdIns(4,5)P2 reporter and HER1. Methods for EF fluorescence microscopy followed closely those described previously by Almers and colleagues (26, 30). In brief, EF illumination was generated with through-the-lens TIRF. Samples were viewed through a Plan Apo TIRF 60x oil-immersion, high-resolution (1.45 numerical aperture) objective on a Nikon Eclipse TE2000 (Nikon Instruments, Melville, NY) inverted microscope. EGFP was excited with an argon laser with appropriate dichroic mirror and emission filter. Images were collected and processed with a Cascade Photometric CCD camera (Roper Scientific, Tucson, AZ) interfaced with a PC running MetaMorph software. In some instances, to better visualize translocation of the PtdIns(4,5)P2 reporter out of the plasma membrane in response to EGF, we pretreated cells for 30 min with the PI 4-kinase inhibitor wortmannin (50 μM) to prevent recycling/regeneration of PtdIns(4,5)P2.
Statistics. All patch-clamp data are presented as means ± SE. Paired and unpaired data were compared using appropriate t-tests with P 0.05 considered significant.
RESULTS
EGF decreases ENaC activity via RTK. We reconstituted human ENaC in CHO cells to directly test the effects of growth factors on channel activity. The channel was reconstituted by coexpressing -, -, -hENaC subunits as previously described (2, 31, 32). Channel activity was assessed as the amiloride-sensitive inward Na+ current at –80 mV under voltage-clamp conditions. All currents were corrected for cell surface area yielding current density. Figure 1A shows representative macroscopic current traces from a CHO cell expressing ENaC plus the EGF receptor HER1 before (con) and after bath application of 100 ng/ml (17 nM) EGF. As reported previously, CHO cells contain little endogenous leak current with none being sensitive to amiloride (2, 31, 32). Shown in Fig. 1B is the current-voltage (I-V) relationship for the amiloride-sensitive inward Na+ current before and after EGF. As summarized in Fig. 1C, EGF significantly decreased ENaC activity from 130 ± 21 to 31 ± 7 pA/pF. EGF had no effect on cell capacitance (not shown). The time course of EGF action on ENaC is shown in Fig. 1D with this growth factor rapidly decreasing channel activity. The effect of EGF on ENaC was abolished with pretreatment by the EGFR tyrosine kinase inhibitor erbstatin analog (10 μM; 30 min) as shown in Fig. 1E. ENaC activity as summarized in Fig. 1F in the absence and presence of EGF in cells pretreated with erbstatin was not different at 130 ± 53 and 140 ± 20 pA/pF, respectively. These results demonstrate that EGF through its RTK acutely decreases ENaC activity.
We next tested whether inhibition of endogenous tyrosine kinase activity affected ENaC. Figure 2A shows an overlay of typical macroscopic currents from a CHO cell expressing ENaC before (con) and after the erbstatin analog (10 μM). Shown in Fig. 2B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after erbstatin. As summarized in Fig. 2C, erbstatin significantly increased ENaC activity from 83 ± 24 to 158 ± 42 pA/pF. Erbstatin had no effect on cell capacitance (not shown). The time course of erbstatin action on ENaC is shown in Fig. 2D with inhibition of endogenous tyrosine kinases quickly augmenting channel activity.
Because tyrosine kinase signaling decreased ENaC activity, we tested whether phosphotyrosine phosphatases (PTP) also impinged on channel activity. Figure 3A shows typical macroscopic currents from a CHO cell expressing ENaC before (con) and after application of the broad range PTP inhibitor sodium vanadate (100 μM). Shown in Fig. 3B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after vanadate. As summarized in Fig. 3C, vanadate significantly decreased ENaC activity from 77 ± 5 to 15 ± 2 pA/pF. Vanadate had no effect on cell capacitance (not shown). The time course of vanadate action on ENaC is shown in Fig. 3D with inhibition of endogenous PTP decreasing channel activity.
Similar to EGF, IGF-I is a ligand for a RTK. We previously reported that IGF-I increases ENaC activity via PI3-K signaling with the phospholipid products of this lipid kinase directly increasing channel activity (32). This stimulatory action of IGF-I on ENaC is fully apparent only in the presence of repressed tyrosine kinase signaling. Figure 4A shows representative current traces from a CHO cell expressing ENaC before (con) and after IGF-I (100 ng/ml). Shown in Fig. 4B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after IGF-I with the effects of this growth factor on channel activity summarized in Fig. 4C (153 ± 27 to 26 ± 4 pA/pF). Figure 4D shows typical current traces from a CHO cell expressing ENaC and pretreated with erbstatin (10 μM; 30 min), before (con) and after treatment with IGF-I (100 ng/ml) and subsequently amiloride. When IGF-I was added to cells expressing ENaC and pretreated with erbstatin, channel activity significantly increased about twofold (summary graph not shown; see Ref. 32). The broad-range tyrosine kinase inhibitor genistein, similar to erbstatin, attenuated decreases in ENaC activity in response to IGF-I (not shown). The time courses of IGF-I actions on ENaC in the absence and presence of pretreatment with erbstatin are shown in Fig. 4, E and F, respectively. Figure 4G shows the time course of IGF-I actions on ENaC in a cell pretreated with both erbstatin and the PI3-K inhibitor wortmannin (0.2 μM). Pretreatment with both the RTK and PI3-K inhibitor abolished all actions of IGF-I on ENaC with activity being 100 ± 56 and 92 ± 50 pA/pF before and after addition of growth factor (summary graph not shown).
Results in Figs. 1–4 demonstrate that ENaC activity is reciprocally influenced by RTK and PTP. These results demonstrate, moreover, that ENaC is an end-effector for growth factor signaling, such as that initiated by EGF through RTK, with growth factors decreasing channel activity.
Growth factors via RTK decrease ENaC open probability. Gilmore and colleagues (8) reported that endothelin-1, which is a ligand for ETA and ETB heterotrimeric G protein-coupled receptors, decreases the activity of rat ENaC reconstituted in NIH 3T3 cells through the actions of nonreceptor c-Src family tyrosine kinases. Because Src family tyrosine kinases decrease ENaC activity by decreasing channel open probability (8), we wondered whether RTK signaling had a similar effect. Figure 5A shows several ENaC in a typical excised outside-out patch (holding potential = 0 mV; inward current down) made from a CHO cell coexpressing all three channel subunits and HER1 before (inset I control) and after (inset II) addition of EGF (100 ng/ml). As reported previously, ENaC reconstituted in CHO cells has a unitary conductance of 5 pS, is amiloride sensitive, is never observed in untransfected cells, and does not "run-down" in outside-out patches over time (2, 32). As shown by the all point histograms in Fig. 5, B and C, addition of EGF decreased ENaC open probability. The time course of EGF action on ENaC in excised outside-out patches and whole cell experiments was similar.
Addition of IGF-I, similar to EGF, decreased ENaC open probability. Figure 6A shows a representative excised outside-out patch (holding potential = 0 mV) containing several ENaC from a CHO cell coexpressing all three channel subunits before (inset I control) and after (inset II) IGF-I (100 ng/ml). Figure 6, B and C, shows all-points histograms before and after IGF-I.
Also similar to EGF, the PTP inhibitor vanadate decreased ENaC open probability as shown in Fig. 7. A representative excised outside-out patch (holding potential = 0 mV) containing a single ENaC from a CHO cell coexpressing all three channel subunits before and after vanadate (100 μM) is shown in Fig. 7A. Figure 7, B and C, shows all-points histograms before and after vanadate. Figure 7, D and E, summarizes ENaC activity (NPo) and open probability (Po) before and after treatment with vanadate. We rarely observed single ENaC in a membrane patch. However, in such an instance, as was the case for Fig. 7A, activity and open probability are equivalent with the effects of activating tyrosine kinase signaling conclusively decreasing ENaC open probability.
Results in Fig. 8 agree with those in Figs. 5–7 with both supporting the idea that RTK and PTP do not influence the number of ENaC in the plasma membrane but rather affect channel open probability. Shown here are two representative Western blots with the left blot containing whole cell lysates (80 μg/lane) from CHO cells not transfected (control) and those coexpressing all three channel subunits engineered with an NH2-terminal myc-tag in the absence and presence of erbstatin (10 μM, 15 min) and vanadate (100 μM, 15 min) treatment. Membrane proteins in these cells were labeled with sulfo-NHS-LC-biotin prior to extraction, with the blot on the right showing the membrane (streptavidin precipitant from 100 μg total protein) levels of ENaC in the respective cells. Both blots were probed with monoclonal anti-myc antibody. As reported previously, - and -ENaC expressed in CHO cells appear as overlying bands of 90 kDa with -ENaC migrating slightly faster (2, 32). These results demonstrate that inhibition of RTK and PTP has no acute effect on the membrane levels of ENaC.
ENaC is not tyrosine phosphorylated in response to growth factors and RTK signaling. Because RTK signaling leads to substrate phosphorylation and often leads to activation of downstream nonreceptor tyrosine kinases, as well as decreases in ENaC activity (see Figs. 1–7), we tested whether EGF and other growth factor ligands for RTK promote tyrosine phosphorylation of the channel. The top Western blot in Fig. 9 contains whole cell lysate from untransfected CHO cells and cells expressing myc-tagged ENaC treated with vanadate, EGF (in cells also coexpressing HER1), erbstatin, IGF-I and IGF-I plus erbstatin. This representative blot was probed with anti-myc antibody. The middle blot was probed with anti-phospho-tyrosine antibody and contains the anti-myc immunoprecipitant from the respective lysates. This blot clearly shows that ENaC subunits are not tyrosine phosphorylated in response to growth factor signaling via RTK. The bottom blot, which is the top blot stripped and reprobed with the anti-phospho-tyrosine antibody used with the middle blot, is a positive control demonstrating that as expected, EGF promotes tyrosine phosphorylation of EGFR (HER1), which runs as an 170-kDa protein.
Tyrosine kinase signaling decreases membrane PtdIns(4,5)P2 levels coincidently with ENaC activity. Because RTK signaling, as mentioned above, often impinges on the activity of nonreceptor tyrosine kinases, such as c-Src, and c-Src decreases ENaC activity (8), we asked whether Src family tyrosine kinases were involved in growth factor-mediated inhibition of ENaC. Figure 10 summarizes the effects of EGF and IGF-I on ENaC activity (amiloride-sensitive current density in CHO cells expressing ENaC) in the presence of the c-Src inhibitor PP2 (0.1 μM). Similar to their effects in the absence of PP2 (see Figs. 1 and 4), both growth factors decreased ENaC activity in the presence of PP2. Although these results do not definitively exclude c-Src from playing a role in RTK-mediated inhibition of ENaC in response to EGF, they do strongly suggest this to be the case. This is further supported by our earlier findings demonstrating that PP2, when employed in a manner identical to the way it was used in the current study, did indeed counter inhibition by c-Src of KCNQ K+ channels reconstituted in CHO cells (5).
ERK/MAPK also are often downstream effectors of RTK. Similar to inhibition of c-Src, inhibition of MEK1/2 with either PD-98059 (10 μM) or U-1026 (0.5 μM) did not affect EGF actions on ENaC (not shown). These results demonstrate that EGF can decrease ENaC activity via RTK signaling independent of c-Src and MAPK 1/2 signaling, suggesting that these latter two constituents do not lie between EGFR and the channel.
Depletion of membrane PtdIns(4,5)P2 levels in response to RTK signaling and subsequent activation of phospholipase C- affects the activity of phospholipid-sensitive ion channels (4, 22, 39). Because ENaC is sensitive to PtdIns(4,5)P2 levels with this phospholipid being permissive for channel activity (17, 32, 40), we asked whether EGF and the balance between RTK and PTP activities in CHO cells impacted membrane PtdIns(4,5)P2 levels in a manner consistent with changes in ENaC activity. Figure 11A shows the amount of a PtdIns(4,5)P2 reporter (23) in the plasma membrane before (top) and after (bottom) treatment with EGF (100 ng/ml, 12 min), vanadate (100 μM, 12 min, column 3), and vehicle (column 4). For these experiments, TIRF microscopy was used to isolate fluorescence signals from the PtdIns(4,5)P2 reporter in the plasma membrane (see EXPERIMENTAL DESIGN AND METHODS). For EGF, we show both an epiflourescence (column 1) and TIRF (column 2) image of the same cell before and after treatment. Both images show translocation of the PtdIns(4,5)P2 reporter out of the membrane in response to EGF, which is consistent with depletion of this phospholipid from the membrane (4, 14, 22, 39). Addition of EGF and vanadate significantly decreased plasma membrane PtdIns(4,5)P2 levels within 12 min to 38 ± 0.5 (n = 16) and 41 ± 4% (n = 5), respectively. Figure 11B shows the time course of PtdIns(4,5)P2 loss from the plasma membrane in response to EGF and vanadate with this time course being similar to that for inhibition of the channel. Although these results do not conclusively demonstrate that PtdIns(4,5)P2 depletion is causative for decreases in ENaC activity in response to EGF-RTK-PLC signaling, they are consistent with such a cellular mechanism of action, and together with the observations of others showing that depletion of PtdIns(4,5)P2 in response to RTK signaling impinges on ion channel activity (see below) provide a rationale for further future study of this possible mechanism.
Depletion of PtdIns(4,5)P2 decreases ENaC activity. We previously demonstrated that activation of muscarinic signaling in CHO cells by stimulating recombinant M1 receptors (M1-R) with oxotremorine decreases membrane PtdIns(4,5)P2 levels (32). Muscarinic M1 receptors are G protein-coupled receptors linked to PLC--mediated hydrolysis of PtdIns(4,5)P2 (14). With this in mind, we tested whether depletion of PtdIns(4,5)P2 via activation of M1-R coexpressed with ENaC would, like EGFR signaling, lead to decreases in ENaC activity. ENaC and M1-R are never found in the same cell. Thus, while M1-R signaling as used here is an artificial means of depleting PtdIns(4,5)P2 with respect to regulation of ENaC, it does serve as an important proof of principal for this possible mechanism of action. Figure 12A shows typical macroscopic currents from a CHO cell expressing ENaC plus M1-R before (con) and after oxotremorine (3 μM). Shown in Fig. 12B is the I-V relationship for the amiloride-sensitive inward Na+ current before and after oxotremorine. As summarized in Fig. 12C, oxotremorine via M1-R significantly decreased ENaC activity. Oxotremorine signaling via M1-R had no effect on cell capacitance (not shown). The time course of oxotremorine action on ENaC is shown in Fig. 12D with depletion of PtdIns(4,5)P2 through G protein-linked PLC- likely affecting the channel in a manner similar to depletion of this phospholipid through RTK linked PLC-. Figure 12E, which shows a representative excised outside-out patch (holding potential = 0 mV) containing several ENaC from a CHO cell coexpressing all three channel subunits plus M1-R before and after addition of oxotremorine, is also consistent with the idea that depletion of PtdIns(4,5)P2 mediates both the EGF via RTK and oxotremorine via M1 inhibition of ENaC.
Figure 13 shows results from additional experiments probing the role of PtdIns(4,5)P2 depletion in EGF signaling to ENaC. In these experiments, EGFP-PH-PLC was used to buffer PtdIns(4,5)P2. In the presence of buffered PtdIns(4,5)P2, as shown in Fig. 13A, EGF had little effect on ENaC activity. This is in direct contrast to the affect this growth factor has on ENaC when PtdIns(4,5)P2 levels are not buffered and able to change (Fig. 1D). Figure 13B summarizes relative (to start values) ENaC activity in response to EGF in the absence and presence of buffered PtdIns(4,5)P2. Although in the absence of the PtdIns(4,5)P2 buffer relative activity after EGF was markedly decreased to 0.29 ± 0.06 (n = 10), in the presence of EGFP-PH-PLC relative activity decreased substantially less to 0.81 ± 0.10 (n = 4). Interestingly, in every experiment performed in cells expressing EGFP-PH-PLC, we observed a brief transient decrease in channel activity directly following EGF application (relative activity at 2.5 min, peak was 0.63 ± 0.08). We believe that this transient decrease in activity results from a sudden decrease in PtdIns(4,5)P2 levels near the channel in response to growth factor signaling with additional PtdIns(4,5)P2 losses and further decreases in channel activity countered by buffering. The results in Fig. 13 directly demonstrate that when PtdIns(4,5)P2 levels are clamped with a buffer, EGF is less able to markedly decrease ENaC activity allowing us to conclude that PtdIns(4,5)P2 depletion plays a central role in regulation of ENaC by this growth factor.
DISCUSSION
The current finding that EGF potently decreases activity of ENaC reconstituted in CHO cells is consistent with EGF decreasing Na+ reabsorption in cortical collecting duct epithelia (25, 34, 37). Importantly, the current study demonstrates for the first time the direct effects of EGF on ENaC. We find that EGF quickly and markedly decreases ENaC activity for a sustained period. Such a fast time course and sustained inhibition suggest a close signal transduction link between EGFR and ENaC.
Our results position a RTK, such as HER1, between EGF and ENaC with the receptor and channel closely linked for we could reconstitute regulation of ENaC by EGF and RTK in excised, outside-out patches. The present finding that EGF decreases ENaC activity via RTK is consistent with EGF decreasing Na+ reabsorption in isolated rabbit cortical collecting ducts in a herbimycin A-sensitive manner (21), with both suggesting that in native epithelia, ENaC is the final target for EGF-RTK signaling during decreases in Na+ reabsorption.
Bowlby and colleagues (3) demonstrated in a related study that EGF via RTK acutely decreases activity of Kv1.3 K+ channels reconstituted in HEK 293 cells. In this study, Kv1.3 was identified as a substrate for tyrosine phosphorylation with phosphorylation decreasing channel activity. In contrast, we find that ENaC is not a substrate for tyrosine phosphorylation, leading us to propose that signaling intermediaries must transduce the inhibitory signaling form RTK to ENaC. Three candidates were tested. We first asked whether EGF signaling via RTK impinged on ENaC activity via c-Src kinase. Our rationale was that RTK signaling is well known to activate cytosolic tyrosine kinases, such as c-Src, and c-Src decreases ENaC activity (8). We find no evidence that c-Src plays a role in EGF regulation of ENaC.
We next tested whether MAPK 1/2 signaling was involved in EGF regulation of ENaC. The rationale for testing this hypothesis is that others have demonstrated that MAPK 1/2 signaling is necessary for EGF to decrease Na+ reabsorption across an immortalized mouse collecting duct epithelial cell line (25), and MAPK 1/2 signaling decreases ENaC activity (1, 20). We find that MAPK 1/2 signaling is not necessary for the acute actions of EGF and RTK on ENaC.
The third signaling pathway tested that may possibly couple EGFR to ENaC is RTK-mediated depletion of membrane PtdIns(4,5)P2 levels. This signaling pathway has drawn much attention lately with the finding that nerve growth factor via the receptor tyrosine kinase TrkA diminishes membrane PtdIns(4,5)P2 levels via PLC- to regulate TRP channels (4, 22). EGF signaling via RTK also depleted PtdIns(4,5)P2 to modulate TRP channel activity (22) and EGF-mediated hydrolysis of PtdIns(4,5)P2 suppresses activation of GIRK channels (14). Depletion of PtdIns(4,5)P2 in response to RTK signaling, in addition, suppresses P/Q- and N-type Ca2+ channel activity (39). The current findings confirm that EGF and RTK signaling depletes PtdIns(4,5)P2 and demonstrates that depletion of PtdIns(4,5)P2 via M1 signaling independent of RTK also decreases ENaC activity. Moreover, we demonstrate that EGF is unable to affect ENaC activity when PtdIns(4,5)P2 levels are clamped. These findings are consistent with a cellular mechanism where EGF signaling via RTK leads to depletion of membrane PtdIns(4,5)P2 levels to decrease ENaC activity. Such a mechanism fits nicely with the recent findings that ENaC activity is modulated by membrane phospholipid levels to include PtdIns(4,5)P2 (17, 32, 40).
Open probability of ENaC is directly related to membrane PtdIns(4,5)P2 levels with this phospholipid being permissive for normal channel activity (17, 40). It is believed that as ENaC unbinds PtdIns(4,5)P2 open probability decreases. The current findings demonstrating that EGF and RTK signaling decrease ENaC open probability then also are consistent with a mechanism of action dependent on PtdIns(4,5)P2 depletion. Moreover, the quick time course of EGF actions on ENaC and the fact that this signaling pathway is membrane delimited also are consistent with depletion of PtdIns(4,5)P2 as the mechanism of EGF action on ENaC activity.
The EGFR is mislocalized to the luminal plasma membrane in ARPKD epithelial cells (9, 38). Moreover, ARPKD epithelial cells change phenotype, converting from a highly differentiated Na+ absorbing cell type to a less differentiated Cl–-secreting cell type (28, 38). We wonder whether these two hallmarks of ARPKD are related. We speculate that mislocalization of EGFR to the luminal membrane in cystic ARPKD epithelia leads to hyperactivation of this receptor by cystic EGF-like peptides with consequent diminution of membrane PtdIns(4,5)P2 levels, leading to decreases in ENaC activity. Such a transduction cascade may contribute to the abnormal transport properties observed in cystic epithelia.
GRANTS
This research was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-59594, American Heart Association Texas Affiliate Grant 0355012Y, and the American Society of Nephrology Carl W. Gottschalk Research Scholar Grant (to J. D. Stockand).
ACKNOWLEDGMENTS
J. Medina and P. Patel are recognized for excellent technical assistance. We thank Drs. M. Gekle and A. Ulrich, and P. Snyder, respectively, for the HER1 and human ENaC cDNAs used in the current study.
FOOTNOTES
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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