ACTIVATION OF KAPPA-B-SPECIFIC PROTEINS BY ESTRADIOL

The kappaB enhancer serves as a recognition site for the nuclear transcription factor NF-kappaB and other kappaB-specific proteins which are activated in many cell types in response to a variety of extracellular signals. But a steroid-dependent activation of NF-kappaB or any other kappaB-specific protein has not previously been reported, to our knowledge. In this report we demonstrate that estrogen can activate kappaB-specific protein in its target tissue, uterus. We have done this by analyzing the interaction of nuclear extracts with kappaB enhancers, using DNA mobility shift assays. The activation by estradiol was time dependent, reaching a maximum at approximately 2 hr after steroid treatment, and was not inhibited by prior cycloheximide treatment. The protein-DNA complexes formed in response to estradiol did not contain NF-kappaB and, when compared with other kappaB enhancer motifs, had a higher affinity to the kappaB enhancer corresponding to the PRDII element present in duplicate motifs. These protein-DNA complexes also did not appear to contain estrogen receptor, since antibodies to estrogen receptor were without any effect on either their formation or their mobility. The protein-DNA complexes formed in response to estradiol, however, exhibited a high affinity for the estrogen-responsive element, suggesting the participation of an estrogen-receptor-like molecule in the DNA binding. In contrast, the protein-DNA complexes formed constitutively contained NF-kappaB, had equivalent affinities to various kappaB enhancers, and did not have a high affinity for the estrogen-responsive element. On the basis of these findings, we propose that estrogen-dependent activation of the as-yet-unidentified kappaB-specific protein involves the association of this protein with an estrogen-receptor-related molecule and binding of the resulting complex to PRDII. The high affinity and specificity of this binding to PRDII suggests that this may serve as a composite regulatory element in mediating estrogen-dependent gene expression. The potential significance of such a mechanism for steroid hormone action is discussed.