Ikappab kinase-beta (ikkbeta) modulation of epithelial sodium channel activity.
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.
Renal-Electrolyte Division, Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15261, USA. Lebowitzj@msx.dept-med.pitt.edu
Jonathan
J
Lebowitz
Robert S
RS
Edinger
Bing
B
An
Clint J
CJ
Perry
Sergio
S
Onate
Thomas R
TR
Kleyman
John P
JP
Johnson
D000450:Aldosterone / Q000494:pharmacology; D000818:Animals; D002461:Cell Line, Transformed; D004847:Epithelial Cells / Q000378:metabolism*; D053503:Epithelial Sodium Channels; D051550:I-kappa B Kinase; D008565:Membrane Proteins; D051379:Mice; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015222:Sodium Channels / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D014981:Xenopus
D053503:Epithelial Sodium Channels; D008565:Membrane Proteins; D015222:Sodium Channels; D000450:Aldosterone; D017346:Protein Serine-Threonine Kinases; C496564:Chuk protein, mouse; D051550:I-kappa B Kinase; C496562:Ikbkb protein, mouse; C496566:Ikbke protein, mouse