Estrogen Receptor Isoform-specific Regulation of the Retinoblastoma-binding Protein 1 (RBBP1) Gene

Abstract

Estrogen (E2) is involved in mediating many important functions relevant to osteoblast biology through the actions of the estrogen receptors (ER) ␣ and ␤. To further understand the mechanisms of ER-specific regulation, we used microarray and reverse transcription-PCR analyses of E2-treated U2OS-ER␣ or -ER␤ cells and identified retinoblastoma-binding protein 1 (RBBP1) as a major E2-regulated gene. RBBP1 is a retinoblastoma cofactor involved in the control of osteoblastic proliferation. Although RBBP1 mRNA levels rapidly increased after 2 h of E2 treatment in both U2OS-ER-expressing lines, a sustained induction was only observed in U2OS-ER␣ cells. Examination of the RBBP1 genomic sequence revealed an ER response element and a Sp1 site located within the first intron. Chromatin immunoprecipitation analyses demonstrated that E2-dependent ER␣ binding to the intron 1 enhancer region was constitutive, whereas ER␤ binding was transient, consistent with the mRNA time course. Interestingly, transient transfection and receptor mutational studies revealed that RBBP1 induction by ER␣ only requires the Sp1 site, whereas ER␤ utilizes both the Sp1 and estrogen response elements binding sites for maximal E2-dependent activation. Stable U2OS transfectants containing a deletion of the ER␣ activation function 1 (AF1) resulted in a temporal mRNA induction profile similar to that of wild type ER␤. Further, overexpression and chromatin immunoprecipitation analyses also demonstrated that E2-dependent RBBP1 induction is SRC2-dependent for both ER isoforms. These results describe an E2-dependent, ER isoform-specific transcriptional activation of the RBBP1 gene, which in part, is explained by the differential activity of ER AF1 and enhancer element binding.The transcriptional effects of estrogens (e.g. 17␤-estradiol or E2) 2 are largely mediated by two related, but distinct, estrogen receptor (ER) isoforms ER␣ and ER␤ (1-7). The ERs are ligandinducible transcription factors that bind E2 through a C-terminally located ligand-binding domain (or E/F domain). The ER isoforms contain a centrally located and highly conserved DNA-binding domain (or C domain) that associates directly with estrogen response elements (ERE) or indirectly through protein tethering to AP1 or Sp1 (8, 9). Both ER␣ and ER␤ contain two activation functions (AF); a highly divergent N-terminal A/B (AF1) domain and a more conserved C-terminal E/F (AF2) domain that overlaps with the ligand-binding domain (10 -13). Activation of transcription is achieved through the recruitment of specific transcriptional coregulators resulting in chromatin remodeling and the recruitment of the basal transcriptional machinery (1, 9, 14 -16). Although coregulator recruitment and activation is largely mediated through AF2, the A/B domain (AF1) is known to bind the p160 and p300/CBP (CREB-binding protein) families of coregulators (16 -20) as well as p68/p72 (21, 22), steroid receptor RNA activator (23, 24), and MMS19 (25) proteins. Recent data from our laboratory (26, 27) and others (28, 29) have...

Estrogen receptors alpha and beta in bone

“…Expression arrays show only 21% overlap between E2-regulated genes in U2OS-ERα and U2OS-ERβ cell lines [79], demonstrating that the two receptors have different functions in osteoblast-like cells. When ERα and ERβ are expressed together, a distinct set of E2-regulated genes was observed [80].…”

Estrogen and the selective estrogen receptor modulator (SERM) protection against cell death in estrogen receptor alpha and beta expressing U2OS cells

“…Thus, we chose to use these drug concentrations also on the following experiments. The pre-incubation time of 24 hrs with SERMs has been previously used also by others (Monroe et al, 2006;Kian Tee et al, 2004, Brady et al, 2002. The incubation times for OPG and IL-6 assays were chosen on the basis of cell death analyses.…”

Retinoblastoma binding protein-1 (RBP1) is a Runx2 coactivator and promotes osteoblastic differentiation

“…We have previously identified RBP1 as an estrogen regulated gene in U2OS osteosarcoma cells stably expressing ERα [11-13], however, no information exists on the function of RBP1 during the process of osteoblastic differentiation. As an initial step in understanding the role of RBP1 in this process, we generated a primary osteoblastic cell model in which RBP1 expression was reduced.…”

“…More recently, RBP1 deletion in mice was shown to result in a genomic imprinting defect at the Prader-Willi syndrome and Angelman syndrome imprinting center [9] and has been identified as a leukemia suppressor gene in mice [10]. Our laboratory has recently identified RBP1 as an estrogen-regulated gene in a U2OS osteosarcoma cell model expressing the estrogen receptors (ER) ERα and ERβ [11-13]. …”