The 17␤-estradiol (E2) receptor isoforms [estrogen receptor (ER) ␣ and ER␤] bind E2 and selective ER modulators (SERMs) as homodimers (␣/␣ or ␤/␤) or heterodimers (␣/␤) to regulate gene expression. Although recent studies have shown that ER homodimers regulate unique sets of E2-responsive genes, little information exists regarding the transcriptional actions of the ER␣/␤ heterodimer. This paper describes the development of a U2OS human osteosarcoma (osteoblast) cell line stably expressing both

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... R␣ and ER␤ isoforms at a ratio of 1:4, a ratio reported to exist in normal, mature osteoblast cells derived from cancellous bone. The regulation of endogenous genes by E2 and 4hydroxy-tamoxifen were measured in these cells using gene microarrays and real-time RT-PCR. Both E2 and 4-hydroxy-tamoxifen were shown to regulate unique sets of endogenous genes in the U2OS-ER␣/␤ heterodimer cell line (20% and 27% of total, respectively), compared with all the genes regulated in U2OS-ER homodimer cell lines. Furthermore, two novel E2-regulated genes, retinoblastoma binding protein 1 and 7-dehydrocholesterol reductase, were found to contain estrogen response element-like sequences that directly bind the ER␣/␤ heterodimer. These results suggest that the expression of both ER isoforms, forming functional ER␣/␤ heterodimers, result in unique patterns of gene regulation, many of which are distinct from the genes regulated by the ER homodimers. (Molecular