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.