Jonathan Lebowitz

Beth Israel Deaconess Medical Center, Boston, MA, United States

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Publications (6)25.36 Total impact

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    ABSTRACT: We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.
    Journal of Biological Chemistry 01/2008; 282(52):37402-11. · 4.65 Impact Factor
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    ABSTRACT: We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.
    Journal of Biological Chemistry 01/2008; 284(1):150-157. · 4.65 Impact Factor
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    ABSTRACT: No definitive treatment exists to restore lost brain function following a stroke. Transplantation of cultured neuronal cells has been shown to be safe and effective in animal models of stroke and safe in a Phase 1 human trial. In the present study the authors tested the usefulness of human neuron transplantation followed by participation in a 2-month stroke rehabilitation program compared with rehabilitation alone in patients with substantial fixed motor deficits associated with a basal ganglia stroke. Human neuronal cells (LBS-Neurons; Layton BioScience, Inc.) were delivered frozen and then thawed and formulated on the morning of surgery. The entry criteria in this randomized, observer-blinded trial of 18 patients included age between 18 and 75 years, completed stroke duration of 1 to 6 years, presence of a fixed motor deficit that was stable for at least 2 months, and no contraindications to stereotactic surgery. Patients were randomized at two centers to receive either 5 or 10 million implanted cells in 25 sites (seven patients per group) followed by participation in a stroke rehabilitation program, or to serve as a nonsurgical control group (rehabilitation only; four patients). The surgical techniques used were the same at both centers. All patients underwent extensive pre- and postoperative motor testing and imaging. Patients received cyclosporine A for 1 week before and 6 months after surgery. The primary efficacy measure was a change in the European Stroke Scale (ESS) motor score at 6 months. Secondary outcomes included Fugl-Meyer, Action Research Arm Test, and Stroke Impact Scale scores, as well as the results of other motor tests. Nine strokes were ischemic in origin and nine were hemorrhagic. All 14 patients who underwent surgery (ages 40-70 years) underwent uncomplicated surgeries. Serial evaluations (maximum duration 24 months) demonstrated no cell-related adverse serological or imaging-defined effects. One patient suffered a single seizure, another had a syncopal event, and in another there was burr-hole drainage of an asymptomatic chronic subdural hematoma. Four of seven patients who received 5 million cells (mean improvement 6.9 points) and two of seven who received 10 million cells had improved ESS scores at 6 months; however, there was no significant change in the ESS motor score in patients who received cell implants (p = 0.756) compared with control or baseline values (p = 0.06). Compared with baseline, wrist movement and hand movement scores recorded on the Fugl-Meyer Stroke Assessment instrument were not improved (p = 0.06). The Action Research Arm Test gross hand-movement scores improved compared with control (p = 0.017) and baseline (p = 0.001) values. On the Stroke Impact Scale, the 6-month daily activities score changed compared with baseline (p = 0.045) but not control (p = 0.056) scores, and the Everyday Memory test score improved in comparison with baseline (p = 0.004) values. Human neuronal cells can be produced in culture and implanted stereotactically into the brains of patients with motor deficits due to stroke. Although a measurable improvement was noted in some patients and this translated into improved activities of daily living in some patients as well, this study did not find evidence of a significant benefit in motor function as determined by the primary outcome measure. This experimental trial indicates the safety and feasibility of neuron transplantation for patients with motor stroke.
    Journal of Neurosurgery 08/2005; 103(1):38-45. · 3.15 Impact Factor
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    ABSTRACT: Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of beta-ENaC and IkappaB kinase-beta (IKKbeta), the kinase that phosphorylates Ikappabeta and results in nuclear targeting of NF-kappaB. A true two-hybrid reaction employing full-length IKKbeta and the carboxyl termini of all three subunits confirmed a strong interaction with beta-ENaC, a weak interaction with gamma-ENaC, and no interaction with alpha-ENaC. Co-immunoprecipitation studies for IKKbeta were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with beta-ENaC, but not gamma-ENaC, resulted in co-immunoprecipitation of IKKbeta. To examine the direct effects of IKKbeta on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for alphabetagamma-ENaC with or without cRNA for IKKbeta. Co-injection of IKKbeta significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKbeta (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCD(c14) cells with aldosterone increased whole cell amounts of IKKbeta. Because this result suggested that aldosterone might activate NF-kappaB, mpkCCD(c14) cells were transiently transfected with a luciferase reporter gene responsive to NF-kappaB activation. Both aldosterone and tumor necrosis factor-alpha (TNFalpha) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKbeta interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKbeta is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-kappaB transcription pathway and steroid regulatory pathways in epithelial cells.
    Journal of Biological Chemistry 11/2004; 279(40):41985-90. · 4.65 Impact Factor
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    ABSTRACT: Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of β-ENaC and IκB kinase-β (IKKβ), the kinase that phosphorylates Iκβ and results in nuclear targeting of NF-κB. A true two-hybrid reaction employing full-length IKKβ and the carboxyl termini of all three subunits confirmed a strong interaction with β-ENaC, a weak interaction with γ-ENaC, and no interaction with α-ENaC. Co-immunoprecipitation studies for IKKβ were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with β-ENaC, but not γ-ENaC, resulted in co-immunoprecipitation of IKKβ. To examine the direct effects of IKKβ on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for αβγ-ENaC with or without cRNA for IKKβ. Co-injection of IKKβ significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKβ (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCDc14 cells with aldosterone increased whole cell amounts of IKKβ. Because this result suggested that aldosterone might activate NF-κB, mpkCCDc14 cells were transiently transfected with a luciferase reporter gene responsive to NF-κB activation. Both aldosterone and tumor necrosis factor-α (TNFα) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKβ interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKβ is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-κB transcription pathway and steroid regulatory pathways in epithelial cells.
    Journal of Biological Chemistry 09/2004; 279(40):41985-41990. · 4.65 Impact Factor
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    ABSTRACT: In several in vivo settings, prolonged alterations in the rate of apical Na+ entry into epithelial cells alter the ability of these cells to reabsorb Na+. We previously modeled this load dependence of transport in A6 cells by either decreasing Na+ entry via apical Na+ removal or amiloride or enhancing Na+ entry by chronic short-circuiting (Rokaw MD, Sarac E, Lechman E, West M, Angeski J, Johnson JP, and Zeidel ML. Am J Physiol Cell Physiol 270: C600-C607, 1996). Inhibition of Na+ entry by either method was associated with striking downregulation of transport rate as measured by short-circuit current (Isc), which recovered to basal levels of transport over a period of hours. Conversely, upregulation of Na+ entry by short-circuiting resulted in a sustained increase in transport rate that also returned to basal levels over a period of hours. The current studies were undertaken to determine whether these conditions were associated with alterations in either the whole cell content or apical membrane distribution of sodium channel (ENaC) subunits or on basolateral expression of either of the subunits of the Na+-K+-ATPase. We compared these effects to those achieved by chronic upregulation of Na+ transport by aldosterone. Whole cell levels of ENaC subunits were measured by immunoblot following 18-h inhibition of Na+ entry achieved by either tetramethylammonium replacement of Na+ or apical amiloride or after an 18-h increase in Na+ entry achieved by chronic short-circuiting. None of these maneuvers significantly altered the whole cell content of any of the ENaC subunits compared with control cells. We then examined the effects of these maneuvers on apical membrane ENaC expression using domain-specific biotinylation and immunoblot. Inhibition of Na+ entry by either method was associated with a profound decrease in apical membrane beta-ENaC without significant changes in apical membrane alpha-or gamma-ENaC amounts. Restoration of apical Na+ and/or removal of amiloride resulted in return of Isc to control levels over 2 h and coincided with return of apical beta-ENaC to control levels without change in apical alpha- or gamma-ENaC. Stimulation of Na+ transport by short-circuiting, in contrast, did not significantly alter apical membrane composition of any of the ENaC subunits. Basolateral expression of Na+-K+-ATPase was also measured by biotinylation and immunoblot and was unchanged under all conditions. Aldosterone increased basolateral expression of the alpha-subunit of Na+-K+-ATPase. These results suggest that chronic downregulation of transport is mediated, in part, by a selective decrease in apical membrane ENaC expression, consistent with our previous observations of noncoordinate regulation of ENaC expression under varying transport conditions in A6 cells. The chronic increase in the rate of Na+ entry is not associated with any of the changes in transporter density at either apical or basolateral membrane seen with aldosterone, suggesting that these two mechanisms of augmenting transport are completely distinct.
    American journal of physiology. Renal physiology 10/2003; 285(3):F524-31. · 3.61 Impact Factor