Selective activation of estrogen receptor-beta transcriptional pathways by an herbal extract.

Novel estrogenic therapies are needed that ameliorate menopausal symptoms and have the bone-sparing effects of endogenous estrogens but do not promote breast or uterine cancer. Recent evidence suggests that selective activation of the estrogen receptor (ER)-beta subtype inhibits breast cancer cell proliferation. To establish whether ERbeta-selective ligands represent a viable approach to improve hormone therapy, we investigated whether the estrogenic activities present in an herbal extract, MF101, used to treat hot flashes, are ERbeta selective. MF101 promoted ERbeta, but not ERalpha, activation of an estrogen response element upstream of the luciferase reporter gene. MF101 also selectively regulates transcription of endogenous genes through ERbeta. The ERbeta selectivity was not due to differential binding because MF101 binds equally to ERalpha and ERbeta. Fluorescence resonance energy transfer and protease digestion studies showed that MF101 produces a different conformation in ERalpha from ERbeta when compared with the conformations produced by estradiol. The specific conformational change induced by MF101 allows ERbeta to bind to an estrogen response element and recruit coregulatory proteins that are required for gene activation. MF101 did not activate the ERalpha-regulated proliferative genes, c-myc and cyclin D1, or stimulate MCF-7 breast cancer cell proliferation or tumor formation in a mouse xenograft model. Our results demonstrate that herbal ERbeta-selective estrogens may be a safer alternative for hormone therapy than estrogens that nonselectively activate both ER subtypes.

Estradiol and selective estrogen receptor modulators differentially regulate target genes with estrogen receptors alpha and beta.

Estrogens and selective estrogen receptor modulators (SERMs) interact with estrogen receptor (ER) alpha and beta to activate or repress gene transcription. To understand how estrogens and SERMs exert tissue-specific effects, we performed microarray analysis to determine whether ERalpha or ERbeta regulate different target genes in response to estrogens and SERMs. We prepared human U2OS osteosarcoma cells that are stably transfected with a tetracycline-inducible vector to express ERalpha or ERbeta. Western blotting, immunohistochemistry, and immunoprecipitation studies confirmed that U2OS-ERalpha cells synthesized only ERalpha and that U2OS-ERbeta cells expressed exclusively ERbeta. U2OS-ERalpha and U2OS-ERbeta cells were treated either with 17beta-estradiol (E2), raloxifene, and tamoxifen for 18 h. Labeled cRNAs were hybridized with U95Av2 GeneChips (Affymetrix). A total of 228, 190, and 236 genes were significantly activated or repressed at least 1.74-fold in U2OS-ERalpha and U2OS-ERbeta cells by E2, raloxifene, and tamoxifen, respectively. Most genes regulated in ERalpha cells in response to E2, raloxifene, and tamoxifen were distinct from those regulated in ERbeta cells. Only 38 of the 228 (17%) genes were regulated by E2 in both U2OS-ERalpha and U2OS-ERbeta cells. Raloxifene and tamoxifen regulated only 27% of the same genes in both the ERalpha and ERbeta cells. A subset of genes involved in bone-related activities regulated by E2, raloxifene, and tamoxifen were also distinct. Our results demonstrate that most genes regulated by ERalpha are distinct from those regulated by ERbeta in response to E2 and SERMs. These results indicate that estrogens and SERMs exert tissue-specific effects by regulating unique sets of targets genes through ERalpha and ERbeta

Distinct roles of unliganded and liganded estrogen receptors in transcriptional repression.

The decline in estrogen levels during menopause is associated with increased cytokine production and inflammatory diseases. Estrogens exert anti-inflammatory effects by repressing cytokine genes, such as tumor necrosis factor-alpha (TNFalpha). The mechanisms involved in transcriptional repression by estrogens are virtually unknown. Here, we used chromatin immunoprecipitation to investigate how estrogens repress the autoinduction of the TNFalpha gene. TNFalpha assembled a transcriptional activation complex at the TNFalpha promoter that includes c-jun, p50-NFkappaB, p65-NFkappaB, CBP, Hsp90, and unliganded estrogen receptor (ER). Estradiol repressed TNFalpha gene expression by reversing the ligand-independent activation by ERalpha and the stimulatory actions of c-jun, NFkappaB, and CBP on transcription. Silencing of GRIP1 reversed the repression of TNFalpha and other cytokine genes by estradiol, demonstrating that GRIP1 is required for transcriptional repression and can act as a corepressor. Our study demonstrates that ERalpha is a TNFalpha-induced coactivator that becomes a repressor in the presence of estradiol by recruiting GRIP1.

Estradiol represses human T-cell leukemia virus type 1 Tax activation of tumor necrosis factor-alpha gene transcription.

Adult T-cell leukemia is caused by human T-cell leukemia virus type I (HTLV-I). The HTLV-I Tax protein is essential for clinical manifestations because it activates viral and cellular gene transcription. Tax enhances production of tumor necrosis factor-alpha (TNF-alpha), which may lead to bone and joint destruction. Because estrogens might prevent osteoporosis by repressing TNF-alpha gene transcription, we investigated whether estrogens inhibit the transcriptional effects of Tax on the TNF-alpha promoter. Tax activated the -1044, -163, and -125 TNF-alpha promoters by 9-25-fold but not the -82 promoter, demonstrating that Tax activation requires the -125 to -82 region, known as the TNF response element (TNF-RE). Three copies of the TNF-RE upstream of the minimal thymidine kinase promoter conferred a similar magnitude of activation by Tax. We demonstrated that c-Jun, NFkappaB, p50, and p65 interact with and activate the TNF-RE by using mutational analysis of the TNF-RE, Tax mutants that selectively activate NFkappaB or the cAMP-response element binding protein/activating transcription factor pathway, and gel shift assays with nuclear extracts. Estradiol markedly repressed Tax-activated transcription of the TNF-alpha gene with estrogen receptor (ER) alpha or beta. Nuclear extracts from U2OS cells stably transfected with ER(alpha) demonstrated that ERs interact with the TNF-RE. Our studies provide evidence that ERs repress Tax-activated TNF-alpha transcription by interacting with a c-Jun and NFkappaB platform on the TNF-RE. Estrogens may ameliorate bone and inflammatory joint diseases in patients infected with HTLV-I by repressing transcription of the TNF-alpha gene.