Direct interaction between estrogen receptor alpha and NF-kappaB in the nucleus of living cells.
Inhibition of NF-kappaB transcriptional activity by steroid receptors is the basis for the antiinflammatory actions of steroid hormones and the molecular mechanism underlying this cross-talk is thought to involve direct protein-protein interactions. In this study, we show that estrogen receptor (ER)alpha and NF-kappaB interact in vivo by using fluorescence resonance energy transfer (FRET) and co-immunoprecipitation. U2-OS cells were used to study direct interactions between fluorescent fusion proteins of ERalpha and the NF-kappaB subunits p50 and p65. Interactions were observed only in the nucleus and maximal FRET signal was detected when ERalpha is co-expressed with both NF-kappaB subunits and cells were stimulated with estrogen. This is in agreement with the induction of nuclear co-localization of the proteins under this condition. Moreover, in a U2-OS clone stably expressing ERalpha, interaction with NF-kappaB was confirmed. A p65 deletion mutant lacking the Rel homology domain was strongly impaired in its interaction with ERalpha showing the importance of this domain. Taken together, these findings provide a strong basis for the direct protein-protein interaction model for cross-talk between ERalpha and NF-kappaB.
Quaedackers
Monique E
ME
Hubrecht Laboratory, Netherlands Institute for Developmental Biology, Uppsalalaan 8, 3584 CT Utrecht, The Netherlands. m.quaedackers@erasmusmc.nl
van den Brink
Christina E
CE
van der Saag
Paul T
PT
Tertoolen
Leon G J
LG
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D002470:Cell Survival; D002999:Clone Cells; D047628:Estrogen Receptor alpha / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D047468:Immunoprecipitation; D008164:Luminescent Proteins / Q000378:metabolism; D052002:NF-kappa B p50 Subunit / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051996:Transcription Factor RelA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D047628:Estrogen Receptor alpha; D008164:Luminescent Proteins; D052002:NF-kappa B p50 Subunit; D011993:Recombinant Fusion Proteins; D051996:Transcription Factor RelA