Epoxyeicosatrienoic acids (EETs) regulate epithelial sodium channel activity by extracellular signal-regulated kinase 1/2 (ERK1/2)-mediated phosphorylation

Abstract

The epithelial sodium channel (ENaC) participates in the regulation of plasma sodium and volume, and gain of function mutations in the human channel cause salt-sensitive hypertension. Roles for the arachidonic acid epoxygenase metabolites, the epoxyeicosatrienoic acids (EETs), in ENaC activity have been identified; however, their mechanisms of action remain unknown. In polarized M1 cells, 14,15-EET inhibited amiloride-sensitive apical to basolateral sodium transport as effectively as epidermal growth factor (EGF). The EET effects were associated with increased threonine phosphorylation of the ENaC β and γ subunits and abolished by inhibitors of (a) mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal regulated kinases 1 and 2 (MEK/ERK1/2) and (b) EGF receptor signaling. CYP2C44 epoxygenase knockdown blunted the sodium transport effects of EGF, and its 14,15-EET metabolite rescued the knockdown phenotype. The relevance of these findings is indicated by (a) the hypertension that results in mice administered cetuximab, an inhibitor of EGF receptor binding, and (b) immunological data showing an association between the pressure effects of cetuximab and reductions in ENaCγ phosphorylation. These studies (a) identify an ERK1/2-dependent mechanism for ENaC inhibition by 14,15-EET, (b) point to ENaC as a proximal target for EET-activated ERK1/2 mitogenic kinases, (c) characterize a mechanistic commonality between EGF and epoxygenase metabolites as ENaC inhibitors, and (d) suggest a CYP2C epoxygenase-mediated pathway for the regulation of distal sodium transport.

FIGURE 1.

The transport effects of 14,15-EET are amiloride-insensitive and, as with EGF, ERK1/2-mediated. A, changes in J_Na+ induced by control, vehicle (black bars) or amiloride (10 μm) (white bars), or by 14,15-EET (5 μm) added in the presence of vehicle (black bars) or amiloride (10 μm) (white bars). Values (in pmol/h/cm²) are averages ± S.E. (error bars) calculated from five different cell samples. Differences from vehicle are indicated as follows: *, p < 0.0008; +, p < 0.0001; ≠, p < 0.002. The differences between amiloride and 14,15-EET or between amiloride added in the absence or presence of 14,15-EET were not significant (p > 0.05). The amiloride-sensitive components of the J_Na+ responses to the agonists were 34, 26, and 37%, respectively, for amiloride, 14,15-EET, and a combination of both. B, cells incubated without (control) or with U0126 (apical; 10 μm) were exposed to vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (black, gray, and white bars, respectively), and J_Na+ was determined 2 h later. Values (in pmol/h/cm²) are averages ± S.E. (error bars) calculated from five different cell samples. Differences from controls are indicated as follows: *, p < 0.05; +, p < 0.0001; ≠, p < 0.01. The amiloride-sensitive components of the total cell J_Na+ effects corresponded to 39, 30, and 25% for control untreated and to 46, 54, and 40% for U0126-treated cells for vehicle, 14,15-EET, and EGF, respectively. C and D, Western blots of cell lysates probed with anti-phospho-ERK1/2 (p-ERK) (upper panels) or -ERK1/2 (lower panels) antibodies. Shown are immunoreactive proteins with the mobilities of phospho-ERK1/2 and ERK1/2. C, lysates (50 μg of protein/lane) isolated from cells incubated for 10, 30, or 60 min with vehicle (lane 1) or 14,15-EET (5 μm) (lane 2). D, lysates from cells incubated without (control) or with U0126 (10 μm) for 1 h prior to a 30-min exposure to vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (lanes 1, 2, and 3, respectively; 50–60 μg of protein/lane) probed with peptide antibodies.

FIGURE 2.

Inhibition of the EGFR tyrosine kinase or EGF binding blunts the effects of 14,15-EET and EGF on ERK1/2 activation and transcellular conductance. The transcellular conductance responses of cells preincubated for 1 h with vehicle or Inhibitor III (apical; 1 μm) (black and white bars, respectively) (A) or with vehicle or cetuximab (basolateral; 20 ng/ml) (black and white bars, respectively) (B) were calculated from TER measurements performed 2 h after the addition of 14,15-EET (5 μm), amiloride (10 μm), or EGF (10 ng/ml). Values are averages ± S.E. (error bars) calculated from three experiments, each performed in triplicates. A, different from controls lacking Inhibitor III: *, p < 10⁻⁵; +, p < 10⁻⁵; ≠, p < 10⁻⁴. B, different from controls lacking cetuximab: *, p < 10⁻³; ≠, p < 10⁻⁵. The differences between amiloride in the presence or absence of cetuximab were not significant (p > 0.05). Western blots of cell lysates probed with anti-EGFR tyrosine 1173 (C) or anti-ERK1/2 and -phospho-ERK1/2 (P-ERK1/2) peptide antibodies (D and E) show immunoreactive proteins with the mobilities of glycosylated EGFR (C) and ERK1/2 and phospho-ERK1/2 (D and E). C, cells (50 μg of protein/lane) were isolated 30 min after the addition of vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml). D, cells were preincubated for 1 h without (lane 1) or with Inhibitor III (apical; 1 μm) (lane 2) and for 30 min in the presence of vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (15–20 and 40–60 μg of protein/well for ERK1/2 and phospho-ERK1/2, respectively). D, cells were preincubated for 1 h without or with cetuximab (basolateral; 20 ng/ml) (lanes 1 and 2, respectively) and for 30 min with vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (30–45 and 70–90 μg of protein/well for ERK1/2 and phospho-ERK1/2, respectively). P-Tyr, phosphotyrosine; S, siemens.

FIGURE 3.

EGF and EETs stimulate threonine phosphorylation of ENaCβ and -γ. A and B, equivalent volumes of lysates from cells (at 80–85% confluence) treated for 15 min with vehicle, 11,12-EET (10 μm), 14,15-EET (5 μm), or EGF (lanes 1, 2, 3, and 4, respectively) (10 ng of protein/lane) were exposed to anti-phosphothreonine (P-Thr) antibodies, and after protein G affinity purification, the anti-phosphothreonine immunoreactive proteins were analyzed by Western blot using anti-ENaCβ (A) or -ENaCγ (B) antibodies. Arrows show mobilities for 75- and 125-kDa proteins. C and D, lysates from ³²P-labeled cells incubated for 15 min with vehicle, 14,15-EET (5 μm), or EGF (10 ng/ml) (black, gray, and white bars, respectively) were exposed to anti-ENaCβ (C) or -ENaCγ (D) antibodies, and the ³²P contents of affinity-purified immunoreactive proteins were determined by β-counting. Values are averages ± S.E. (error bars) calculated from two different experiments, each done in duplicates. C, different from vehicle: *, p < 0.001; ≠, p < 0.0003. D, different from vehicle: *, p < 0.0004; ≠, p < 0.0004.

FIGURE 4.

CYP2C44 knockdown reduces epoxygenase expression and blunts the effects of EGF on sodium transport. A, quantitative real time PCR analysis of mRNAs present in cells expressing non-coding (mock) (black bars) or CYP2C44-coding silencing RNAs (shRNA) (gray bars) using CYP2C44-selective primers (23). Values, normalized to β-actin mRNA levels, are averages ± S.E. (error bars) calculated from three different cell samples, each analyzed in triplicates. Difference from mock cells is indicated as follows: *, p < 0.004. Inset, Western blots of lysates from mock and shRNA cells probed with anti-CYP2C44 antibodies (upper panel) and normalized to the levels of anti-β-actin immunoreactive protein (lower panel). The arrows indicate approximate mobilities for 56- and 65-kDa proteins. B, the sum of EETs and dihydroxyeicosatrienoic acids (DHETs) present in mock and shRNA cells was extracted and quantified using ultrahigh pressure liquid chromatography-tandem mass spectrometric techniques as described under “Experimental Procedures.” Values are -fold change averages calculated from three different experiments. Differences from mock controls are indicated as follows: *, p < 0.02; ≠, p < 0.04. C, amiloride-sensitive J_Na+ responses for mock (black bars) and shRNA cells (gray bars) exposed to vehicle or EGF (10 ng/ml) in the absence or presence of 14,15-EET (5 μm). Values (in pmol/h/cm²) are averages ± S.E. (error bars) calculated from five cell samples. Differences are indicates as follows: from vehicle-treated mock cells: *, p < 0.004; +, p < 0.001; from EGF-treated mock cells: ≠, p < 0.04. The differences between vehicle- and EGF-treated shRNA cells, EGF- and EGF plus 14,15-EET- treated mock cells, and EGF plus 14,15-EET-treated mock and shRNA cells were not significant (p > 0.05).

FIGURE 5.

Cetuximab causes hypertension in mice and reduces ENaCγ threonine phosphorylation. Mice on normal salt (NS) or high salt (HS) diets were left untreated or administered cetuximab every other day for 10 days. A, systolic BPs of untreated or cetuximab-treated mice (white and black bars, respectively). Values are averages ± S.E. (error bars) calculated from ≥30 measurements/mouse performed for groups of untreated mice on normal or high salt diets (five and six animals, respectively) or for cetuximab-treated mice on normal or high salt diets (six and nine mice, respectively). Differences are indicated as follows: *, untreated on high salt diet, p < 10⁻³; cetuximab on normal diet, p < 10⁻⁵; cetuximab on high salt diet, p < 10⁻⁶; ≠, cetuximab on high salt diet, p < 10⁻⁴; untreated on high salt diet, p < 10⁻⁵. B and C, Western blots of kidney membranes from cetuximab-treated and untreated mice fed normal salt or high salt diets and probed with ENaCγ (B) or phosphothreonine (P-Thr) (C) antibodies. Loadings (60–40 and 30–20 μg of protein/lane for B and C, respectively) were normalized by comparisons with Coomassie Blue-stained membranes. Lanes 1 and 3, untreated normal salt and high salt diets, respectively. Lanes 2 and 4, cetuximab-treated normal salt and high salt diets, respectively. D, paraffin-embedded kidney sections from mice fed normal salt diets and exposed to D. biflorus agglutinin followed by anti-phosphothreonine antibodies (p-Thr) were incubated in the absence or presence of a threonine phosphorylated peptide coding for the ENaCγ target threonine (Control and Peptide panels, respectively), and the immunofluorescence analyses proceeded as described under “Experimental Procedures.” Shown are 400× images of green D. biflorus (DB), red (p-Thr), and overlay fluorescence (DB + p-Thr) emissions showing D. biflorus-positive signals in CDs and of anti-phosphothreonine-positive signals in CDs and non-CD tubular segments as well as the presence of anti-phosphothreonine immunoreactive proteins in the CD (white arrows) only in those sections exposed to IgGs incubated in the absence of the ENaCγ peptide. The white scale bars correspond to 0.5 μm.

FIGURE 6.

Regulation of ENaC and sodium transport by up-regulation of the kidney. CYP2C44 epoxygenase and EET biosynthesis (for example, by increased salt intake) activates an EGFR-mediated signaling cascade, leading to increased ERK1/2-mediated ENaCβ and ENaCγ phosphorylation, channel inactivation, and increased sodium excretion. Alterations in EGFR-mediated signaling and/or CYP2C44 activity and/or expression reduce ERK1/2-mediated ENaC regulatory inhibition, leading to sodium retention and increased blood pressure. Upward or downward arrows denote increases or decreases in concentration or enzymatic activity, respectively. Red squares 1, 2, 3, and 4 depict potential sites of EET action: a membrane-bound EET receptor (EET-R) (1), direct actions on EGFR ligand binding or signaling (2), and direct effects on MEK1/2 (3) or ENaC (4) activity.