XBP-1 Enhances The Transcriptional Activity Of Estrogen Receptor α

Estrogen receptor (ERa) is a member of a large superfamily of nuclear receptors that regulate the transcription of estrogen-responsive genes. ERa is an effective treatment target as well as a prognostic factor of breast cancer. ERa can be subdivided into six regions (A-F), which contain two transactivation function regions, called AFI and AF2, respectively. AF1 locates in the A/B region, which has ligand-independent transactivation function. AF2 locates in the E/F region, with ligand-dependent transactivation function. The effective transactivation of AF1 and AF2 depends on their interaction with other proteins. Recently, endocrine therapy of breast cancer is acquired mainly by decreasing ERa transcriptional activity. Therefore, it is of great significance to identify and characterize the proteins regulating ERa transcriptional activity, in order to develop drugs for breast cancer.Several recent studies have demonstrated that expression of human X-box binding protein 1 (XBP-1) is associated with ERa status in breast tumors and over-expressed in a subset of breast tumors. These studies suggest that XBP-1 may interact with ERa. More recently, two forms of XBP-1 were identified due to their unconventional splicing. The two splicing variants of XBP-1 were respectively designated XBP-1S and XBP-1U, which were different only at the carboxyl-terminus. Since ERa can bind to ERE(estrogen responsive element), ERE-containing luciferase reporter assay was used to determine the effects of XBP-1 S and XBP-1U on the transcription activity of ERa in this study. I found that XBP-1S andXBP-IU enhanced ERa-dependent transcriptional activity in a ligand-independent manner, and XBP-IS had stronger activity than XBP-IU. XBP-IS and XBP-IU enhanced ERa transcriptional activity in a dose-dependent manner. The anti-estrogen 4-OHT and ICI182,780 decreased the effects of XBP-IS and XBP-IU on ERa. XBP-1 specifically enhanced ERa-mediated transactivation since it could not enhance the transcriptional activity of AR, another steroid receptor. XBP-IS and XBP-IU enhanced both ERa API and AF2 transcriptional activities, and the maximal effects of XBP-IS and XBP-IU were observed when they were coexpressed with full-length ERa. SRC-1, one of the p160 steroid receptor coactivator family members, synergized with XBP-IS and XBP-IU. Co-immunoprecipitation showed that XBP-IS and XBP-IU also bound to ERa in vivo in a ligand-independent manner. The binding of XBP-IS to ERa was stronger than that of XBP-IU to ERa. GST pull-down assay also showed that XBP-IS and XBP-IU bound to ERa. XBP-IS and XBP-IU interacted with the ERa region containing DNA binding domain. The ERa-interacting regions on XBP-IS and XBP-IU were mapped to two regions, including the amino-terminal basic region-leucine zipper domain and the carboxyl-terminal activation domain. The deletion mutants of XBP-IS and XBP-IU that disrupted the interaction of XBP-IS and XBP-IU with ERa completely abolished the ERa transactivation by XBP-IS and XBP-IU. RT-PCR analysis showed that both XBP-IS and XBP-IU mRNAs were constitutively expressed in many breast cancer cell lines. Gel shift assay showed that neither XBP-IS nor XBP-IU bound to ERE.These findings suggest that XBP-1S and XBP-1U may directly modulate ERa signaling in both the absence and presence of estrogen. Therefore, XBP-1, especially XBP-1S, may play important roles in the development of breast cancer, and represent a new target for therapeutic intervention.