Biomedical subjects

Dale C Leitman

Publications and source records attributed to Dale C Leitman.

9 recordsLinked to original sources

PubMed2006

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.

Anemarrhena↗

PubMed2006

Tumor necrosis factor-alpha down-regulates human Cu/Zn superoxide dismutase 1 promoter via JNK/AP-1 signaling pathway.

Overexpression of Cu/Zn superoxide dismutase 1 (SOD1) in monocytes blocks reactive oxygen species-induced inhibition of cell growth and apoptosis and renders cells resistant to the toxic effect of tumor necrosis factor (TNF)-alpha, suggesting that TNF-alpha represses the SOD1 gene in these cells. We herein show that TNF-alpha decreases SOD1 mRNA, protein, and promoter activity in U937 cells. Electrophoretic mobility-shift assays (EMSA) show that TNF-alpha decreased binding of three different complexes. Ectopic Sp1 overexpression markedly increased SOD1-basal promoter activity and partially antagonized the TNF-alpha inhibitory effect. In contrast, ectopic c-Jun overexpression mimics TNF-alpha inhibitory effects and antagonizes Sp1 stimulatory effects. In agreement with these findings, EMSA shows a TNF-alpha-induced increase in AP-1 and a decrease in Sp1 DNA binding. Disruption of the C/EBP site decreases, whereas mutation in the Sp1/Egr-1 site completely abolishes DNA-binding and promoter activity. A JNK inhibitor antagonized the negative effects of TNF-alpha on SOD1 promoter activity, suggesting that JNK signaling through c-Jun protein activation is critical for the TNF-alpha-dependent SOD1 repression. A greater understanding of the mechanisms of TNF-alpha-induced SOD1 repression could facilitate the design and development of novel therapeutic drugs for inflammatory conditions.

Base Sequence↗

PubMed2006

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.

CREB-Binding Protein↗

PubMed2005

Differential response of estrogen receptor subtypes to 1,3-diarylindene and 2,3-diarylindene ligands.

Estrogen receptors (ERs) control transcription of genes important for normal human development and reproduction. The signaling networks are complex, and there is a need for a molecular level understanding of the roles of receptor subtypes ERalpha and ERbeta in normal physiology and as therapeutic targets. We synthesized two series of ER ligands, based on a common indene scaffold, in an attempt to develop compounds that can selectively modulate ER-mediated transcription. The 3-ethyl-1,2-diarylindenes, utilizing an amide linker for the 1-aryl extension, bind weakly to the ERs. The 2,3-diarylindenes bind with high affinity to the ER subtypes and demonstrate a range of different biological activities, both in transcriptional reporter gene assays and inhibition of estradiol-stimulated proliferation of MCF-7 cells. Several ligands differentiate between ERalpha and ERbeta subtypes at an estrogen response element (ERE), displaying various levels of partial to full agonist activity at ERalpha, while antagonizing estradiol action at ERbeta.

Antineoplastic Agents↗

PubMed2004

Estrogen receptor beta inhibits human breast cancer cell proliferation and tumor formation by causing a G2 cell cycle arrest.

Studies indicate that estrogen receptor (ER) alpha mediates breast cancer-promoting effects of estrogens. The role of ERbeta in breast cancer is unknown. Elucidating the role of ERbeta in the pathogenesis of breast cancer is important because many human breast tumors express both ERalpha and ERbeta. We show that adenovirus-mediated expression of ERbeta changes the phenotype of ERalpha-positive MCF-7 cells. Estradiol increases cell proliferation and causes tumor formation of MCF-7 cells expressing only ERalpha. In contrast, introducing ERbeta into MCF-7 cells causes an inhibition of proliferation in vitro and prevents tumor formation in a mouse xenograft model in response to estradiol. ERbeta inhibits proliferation by repressing c-myc, cyclin D1, and cyclin A gene transcription, and increasing the expression of p21(Cip1) and p27(Kip1), which leads to a G(2) cell cycle arrest. These results demonstrate that ERalpha and ERbeta produce opposite effects in MCF-7 cells on cell proliferation and tumor formation. Natural or synthetic ERbeta-selective estrogens may lack breast cancer promoting properties exhibited by estrogens in hormone replacement regimens and may be useful for chemoprevention of breast cancer.

Animals↗

PubMed2003

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

Estradiol↗

PubMed2003

Estrogens activate bone morphogenetic protein-2 gene transcription in mouse mesenchymal stem cells.

Estrogens exert their physiological effects on target tissues by interacting with the estrogen receptors, ERalpha and ERbeta. Estrogen replacement is one the most common and effective strategies used to prevent osteoporosis in postmenopausal women. Whereas it was thought that estrogens work exclusively by inhibiting bone resorption, our previous results show that 17beta-estradiol (E2) increases mouse bone morphogenetic protein (BMP)-2 mRNA, suggesting that estrogens may also enhance bone formation. In this study, we used quantitative real-time RT-PCR analysis to demonstrate that estrogens increase BMP-2 mRNA in mouse mesenchymal stem cells. The selective ER modulators, tamoxifen, raloxifene, and ICI-182,780 (ICI), failed to enhance BMP-2 mRNA, whereas ICI inhibited E2 stimulation of expression. To investigate if estrogens increase BMP-2 expression by transcriptional mechanisms and if the response is mediated by ERalpha and/or ERbeta, we studied the effects of estrogens on BMP-2 promoter activity in transient transfected C3H10T1/2 cells. E2 produced a dose-dependent induction of the mouse -2712 BMP-2 promoter activity in cells cotransfected with ERalpha and ERbeta. At a dose of 10 nM E2, ERalpha induced mouse BMP-2 promoter activity 9-fold, whereas a 3-fold increase was observed in cells cotransfected with ERbeta. Tamoxifen and raloxifene were weak activators of the mouse BMP-2 promoter via ERalpha, but not via ERbeta. ICI blocked the activation of BMP-2 promoter activity by E2 acting via both ERalpha and ERbeta, indicating that mouse BMP-2 promoter activation is ER dependent. In contrast to E2 and selective ER modulators, the phytoestrogen, genistein was more effective at activating the mouse BMP-2 promoter with ERbeta, compared with ERalpha. Using a deletion series of the BMP-2 promoter, we determined that AP-1 or Sp1 sites are not required for E2 activation. A mutation in a sequence at -415 to -402 (5'-GGGCCActcTGACCC-3') that resembles the classical estrogen-responsive element abolished the activation of the BMP-2 promoter in response to E2. Our studies demonstrate that E2 activation of mouse BMP-2 gene transcription requires ERalpha or ERbeta acting via a variant estrogen-responsive element binding site in the promoter, with ERalpha being the more efficacious regulator. Estrogenic compounds may enhance bone formation by increasing the transcription of the BMP-2 gene.

Animals↗

PubMed2002

Alternate surfaces of transcriptional coregulator GRIP1 function in different glucocorticoid receptor activation and repression contexts.

Members of the mammalian p160 family, such as GRIP1, are known as glucocorticoid receptor (GR) coactivators; at certain glucocorticoid response elements (GREs), however, GRIP1 acts as a GR corepressor. We characterized functional interactions of GR and GRIP1 in a repression complex where GR tethers to DNA-bound activator protein-1 (AP-1), as at the human collagenase-3 gene, and tested whether the identified interactions were similar or different at other response elements. At the AP-1 tethering GRE, we mapped the GRIP1 corepressor activity to a domain distinct from the two known GRIP1 activation domains; it exhibited intrinsic GR-independent repression potential when recruited to DNA via Gal4 DNA-binding domain. Interestingly, neither the domain nor the activity was detected in the other two p160 family members, SRC1 and RAC3. The same GRIP1 corepression domain was required for GR-mediated repression at the nuclear factor-kappaB (NF-kappaB) tethering GRE of the human IL-8 gene. In contrast, at the osteocalcin gene GRE, where GR represses transcription by binding to a DNA site overlapping the TATA box, both GRIP1 and SRC1 corepressed, and the GRIP1-specific repression domain was dispensable. Thus, in a single cell type, GR and GRIP1 conferred one mode of activation and two modes of repression by selectively engaging distinct surfaces of GRIP1 in a response element-specific manner.

Carrier Proteins↗

PubMed2002

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.

Binding Sites↗