Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: UNIPROT:P41181 (collecting duct)
 5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Ikappab kinase-beta (ikkbeta) modulation of epithelial sodium channel activity. 1529 20

Renal tubulo-interstitial inflammation is frequently associated with polyuria and urine concentration defects. This led us to investigate the effects of the major pro-inflammatory nuclear factor kappaB (NF-kappaB) pathway on aquaporin 2 (AQP2) expression by the collecting duct. Using immortalized collecting duct principal cells (mpkCCDcl4), we found that, acting independently of vasopressin, activation of NF-kappaB by lipopolysaccharide (LPS) decreased AQP2 mRNA and protein levels in a time- and dose-dependent manner but did not decrease AQP2 mRNA stability. Consistently, constitutively active IkappaB kinase beta decreased AQP2 expression. The LPS-induced decrease in AQP2 mRNA levels was confirmed in rat kidney slices and was reproduced both under conditions of elevated cAMP concentration and V(2) receptor antagonism. Computer analysis of the AQP2 promoter revealed two putative kappaB elements. Mutation of either kappaB element abolished the LPS-induced decrease of luciferase activity in cells expressing AQP2 promoter-luciferase plasmid constructs. Chromatin immunoprecipitation revealed that LPS challenge decreased p65, increased p50 and p52, and had no effect on RelB and c-Rel binding to kappaB elements of the AQP2 promoter. RNA-mediated interference silencing of p65, p50, and p52 confirmed controlled AQP2 transcription by these NF-kappaB subunits. We additionally found that hypertonicity activated NF-kappaB in mpkCCDcl4 cells, an effect that may counteract the Tonicity-responsive enhancer binding protein (TonEBP)-dependent increase in AQP2 gene transcription. Taken together, these findings indicate that NF-kappaB is an important physiological regulator of AQP2 transcription.
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PMID:NF-kappaB modulates aquaporin-2 transcription in renal collecting duct principal cells. 1870 15

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.
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PMID:Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2. 1898 Nov 74