4-hydroxytamoxifen trans-represses nuclear factor-kappa B activity in human osteoblastic U2-OS cells through estrogen receptor (ER)alpha, and not through ER beta

Estrogens are important mediators of bone homeostasis, and postmenopausal e strogen replacement therapy is extensively used to prevent osteoporosis. Th e biological effects of estrogen are mediated by receptors belonging to the superfamily of steroid/thyroid nuclear receptors, estrogen receptor (ER)al pha and ER beta. ER alpha, not only trans-activates target genes in a hormo ne-specific fashion, but it can also neutralize other transcriptional activ ators, such as nuclear factor (NF)-kappaB, causing repression of their targ et genes. A major mechanism by which estrogens prevent osteoporosis seems t o be repression of transcription of NF-kappaB target genes, such as the ost eoclast-activating cytokines interleukin-6 and interleukin-1. To study the capacity of both ER alpha in repression of NF-kappaB signaling in bone cell s, we first carried out transient transfections with ER alpha or ER beta of the human osteoblastic U2-OS cell line, in which endogenous NF-kappaB was stimulated by tumor necrosis factor alpha. Repression by ER alpha was alrea dy observed without 17 beta -estradiol, whereas addition of the ligand in-c reased repression to 90%. ER beta, however, was able to repress NF-kappaB a ctivity only in the presence of ligand. Because it is known that some antie strogens can also display tissue-specific agonistic properties, 4-hydroxyta moxifen was tested for its capacity in repressing NF-kappaB activity and wa s found to be active (albeit less efficient than 17 beta -estradiol) and, i nterestingly. only with ER alpha. The pure antagonist ICI 164,384 was incap able of repressing through any ER subtypes. Deletion analysis and the use o f receptor ER alpha /ER beta -chimeras showed that the A/B domain, containi ng activation function-1, is essential for this suppressive action. Next, w e developed stable transfectants of the human osteoblastic U2-OS cell line containing ER alpha or ER beta in combination with an NF-kappaB luciferase reporter construct. In these cell lines, repression of NF-kappaB activity w as only mediated through ER alpha and not through ER beta. These findings o ffer new insights into the specific role of both ER subtypes in bone homeos tasis and could eventually help in developing more specific medical interve ntion strategies for osteoporosis.