Expression of genes for estrogen receptors alpha and beta in human articular chondrocytes. (25/1528)

OBJECTIVE: To investigate the gene expression of estrogen receptor (ER) alpha and ERbeta in human articular chondrocytes. METHODS: 16 articular cartilage specimens were obtained from 15 patients during surgery. Three of the specimens were from men and 13 from women; three from hip joints and 13 from knee joints; four were normal and 12 showed osteoarthritic cartilage. Total RNA was extracted from the articular chondrocytes and the expression of both ERalpha and ERbeta genes was investigated by the reverse transcription-polymerase chain reaction (RT-PCR) method. RESULTS: Gene expressions of ERalpha were detected in all specimens and those of ERbeta were found in 15 specimens by the RT-PCR method. There was a significant correlation between the amounts of ERalpha and ERbeta. Expression levels of both genes were significantly higher in men than in women. There were no significant differences in the expression levels of both ER genes between the hip and knee joint sites, nor between normal and osteoarthritic tissues. CONCLUSION: This study is to our knowledge the first to demonstrate the gene expression of both ERalpha and ERbeta in human articular chondrocytes. Since there are some functional differences between the two receptors, the effects of estrogen on cartilage metabolism should be elucidated by two different receptor mechanisms.  (+info)

Dissection of the LXXLL nuclear receptor-coactivator interaction motif using combinatorial peptide libraries: discovery of peptide antagonists of estrogen receptors alpha and beta. (26/1528)

Recruitment of transcriptional coactivators following ligand activation is a critical step in nuclear receptor-mediated target gene expression. Upon binding an agonist, the receptor undergoes a conformational change which facilitates the formation of a specific coactivator binding pocket within the carboxyl terminus of the receptor. This permits the alpha-helical LXXLL motif within some coactivators to interact with the nuclear receptors. Until recently, the LXXLL motif was thought to function solely as a docking module; however, it now appears that sequences flanking the core motif may play a role in determining receptor selectivity. To address this issue, we used a combinatorial phage display approach to evaluate the role of flanking sequences in influencing these interactions. We sampled more than 10(8) variations of the core LXXLL motif with estradiol-activated estrogen receptor alpha (ERalpha) as a target and found three different classes of peptides. All of these peptides interacted with ERalpha in an agonist-dependent manner and disrupted ERalpha-mediated transcriptional activity when introduced into target cells. Using a series of ERalpha-mutants, we found that these three classes of peptides showed different interaction patterns from each other, suggesting that not all LXXLL motifs are the same and that receptor binding selectivity can be achieved by altering sequences flanking the LXXLL core motif. Most notable in this regard was the discovery of a peptide which, when overexpressed in cells, selectively disrupted ERbeta- but not ERalpha-mediated reporter gene expression. This novel ERbeta-specific antagonist may be useful in identifying and characterizing the ERbeta-regulated process in estradiol-responsive cells. In conclusion, using a combinatorial approach to define cofactor-receptor interactions, we have clearly been able to demonstrate that not all LXXLL motifs are functionally equivalent, a finding which suggests that it may be possible to target receptor-LXXLL interactions to develop receptor-specific antagonists.  (+info)

Postnatal sex reversal of the ovaries in mice lacking estrogen receptors alpha and beta. (27/1528)

Mice lacking estrogen receptors alpha and beta were generated to clarify the roles of each receptor in the physiology of estrogen target tissues. Both sexes of alphabeta estrogen receptor knockout (alphabetaERKO) mutants exhibit normal reproductive tract development but are infertile. Ovaries of adult alphabetaERKO females exhibit follicle transdifferentiation to structures resembling seminiferous tubules of the testis, including Sertoli-like cells and expression of Mullerian inhibiting substance, sulfated glycoprotein-2, and Sox9. Therefore, loss of both receptors leads to an ovarian phenotype that is distinct from that of the individual ERKO mutants, which indicates that both receptors are required for the maintenance of germ and somatic cells in the postnatal ovary.  (+info)

Estrogen inhibits the vascular injury response in estrogen receptor beta-deficient female mice. (28/1528)

The protective effects of estrogen in the cardiovascular system result from both systemic effects and direct actions of the hormone on the vasculature. Two estrogen receptors have been identified, ERalpha and ERbeta. We demonstrated previously that estrogen inhibits the response to vascular injury in both wild-type and ERalpha-deficient mice, and that ERbeta is expressed in the blood vessels of each, suggesting a role for ERbeta in the vascular protective effects of estrogen. In the present study, we examined the effect of estrogen administration on mouse carotid arterial injury in ERbeta-deficient mice. Surprisingly, in ovariectomized female wild-type and ERbeta knockout mice, 17beta-estradiol markedly and equally inhibited the increase in vascular medial area and the proliferation of vascular smooth muscle cells after vascular injury. These data demonstrate that ERbeta is not required for estrogen-mediated inhibition of the response to vascular injury, and suggest that either of the two known estrogen receptors is sufficient to protect against vascular injury, or that another unidentified estrogen receptor mediates the vascular protective effects of estrogen.  (+info)

Estradiol repression of tumor necrosis factor-alpha transcription requires estrogen receptor activation function-2 and is enhanced by coactivators. (29/1528)

The tumor necrosis factor-alpha (TNF-alpha) promoter was used to explore the molecular mechanisms of estradiol (E(2))-dependent repression of gene transcription. E(2) inhibited basal activity and abolished TNF-alpha activation of the TNF-alpha promoter. The E(2)-inhibitory element was mapped to the -125 to -82 region of the TNF-alpha promoter, known as the TNF-responsive element (TNF-RE). An AP-1-like site in the TNF-RE is essential for repression activity. Estrogen receptor (ER) beta is more potent than ERalpha at repressing the -1044 TNF-alpha promoter and the TNF-RE upstream of the herpes simplex virus thymidine kinase promoter, but weaker at activating transcription through an estrogen response element. The activation function-2 (AF-2) surface in the ligand-binding domain is required for repression, because anti-estrogens and AF-2 mutations impair repression. The requirement of the AF-2 surface for repression is probably due to its capacity to recruit p160 coactivators or related coregulators, because overexpressing the coactivator glucocorticoid receptor interacting protein-1 enhances repression, whereas a glucocorticoid receptor interacting protein-1 mutant unable to interact with the AF-2 surface is ineffective. Furthermore, receptor interacting protein 140 prevents repression by ERbeta, probably by interacting with the AF-2 surface and blocking the binding of endogenous coactivators. These studies demonstrate that E(2)-mediated repression requires the AF-2 surface and the participation of coactivators or other coregulatory proteins.  (+info)

Estrogen receptors alpha and beta in the rodent mammary gland. (30/1528)

An obligatory role for estrogen in growth, development, and functions of the mammary gland is well established, but the roles of the two estrogen receptors remain unclear. With the use of specific antibodies, it was found that both estrogen receptors, ERalpha and ERbeta, are expressed in the rat mammary gland but the presence and cellular distribution of the two receptors are distinct. In prepubertal rats, ERalpha was detected in 40% of the epithelial cell nuclei. This decreased to 30% at puberty and continued to decrease throughout pregnancy to a low of 5% at day 14. During lactation there was a large induction of ERalpha with up to 70% of the nuclei positive at day 21. Approximately 60-70% of epithelial cells expressed ERbeta at all stages of breast development. Cells coexpressing ERalpha and ERbeta were rare during pregnancy, a proliferative phase, but they represented up to 60% of the epithelial cells during lactation, a postproliferative phase. Western blot analysis and sucrose gradient centrifugation confirmed this pattern of expression. During pregnancy, the proliferating cell nuclear antigen was not expressed in ERalpha-positive cells but was observed in 3-7% of ERbeta-containing cells. Because more than 90% of ERbeta-bearing cells do not proliferate, and 55-70% of the dividing cells have neither ERalpha nor ERbeta, it is clear that the presence of these receptors in epithelial cells is not a prerequisite for estrogen-mediated proliferation.  (+info)

Estrogen receptor beta is coexpressed with ERalpha and PR and associated with nodal status, grade, and proliferation rate in breast cancer. (31/1528)

The role of estrogen (ER) and progesterone receptors (PR) in breast cancer is well established. Identification of the second human estrogen receptor, the estrogen receptor beta (ERbeta), prompted us to evaluate its role in breast cancer. We studied the expression of ERbeta by immunohistochemistry and mRNA in situ hybridization in 92 primary breast cancers and studied its association with ERalpha, PR, and various other clinicopathological factors. Sixty percent of tumors were defined as ERbeta-positive (nuclear staining in >20% of the cancer cells). Normal ductal epithelium and 5 of 7 intraductal cancers were also found to express ERbeta. Three-fourths of the ERalpha- and PR-positive tumors were positive for ERbeta, whereas ERalpha and PR were positive in 87% and 67% of ERbeta-positive tumors, respectively. ERbeta was associated with negative axillary node status (P < 0.0001), low grade (P = 0.0003), low S-phase fraction (P = 0.0003), and premenopausal status (P = 0.04). In conclusion, the coexpression of ERbeta with ERalpha and PR as well as its association with the other indicators of low biological aggressiveness of breast cancer suggest that ERbeta-positive tumors are likely to respond to hormonal therapy. The independent predictive value of ERbeta remains to be established.  (+info)

Increased expression of estrogen receptor beta in human uterine smooth muscle at term. (32/1528)

Expression of the cx43 gene for the gap junction protein, connexin43 (Cx43), through activator protein (AP)-1 activity has been shown to be inhibited in human primary myometrial cultures pretreated with estrogen. In the present study, the primary myometrial cultures were shown to express predominantly ERbeta, a subtype of estrogen receptor that inhibits AP-1 activity when bound to agonists. ERbeta levels were decreased in the primary myometrial cultures after treatment with the phorbol ester, 12-O-tetradecanolyl-13 acetate, to stimulate AP-1 activity, and this effect is inhibited if cells were pretreated with estrogen. Two isoforms of ERbeta were found in primary myometrial and leiomyoma cultured cells. Immunoblot and RT-PCR analyses indicated that ERbeta expression was increased in human term myometrial tissue compared with non-pregnancy tissue. Immunohistochemistry localized ERbeta to the nucleus in cells of term myometrial tissue samples that had high ERbeta expression. ERbeta was increased in term tissue in which Cx43 protein levels were low. In myometrial tissue in which Cx43 protein levels are greatest (e.g. during active labor), ERbeta was barely detectable. Only low levels of ERbeta were detected in non-pregnancy myometrial and leiomyoma tissues, and the lowest levels were found in tissues from mid cycle. In contrast, ERalpha was highly detectable in the non-pregnancy myometrial and leiomyoma tissues, but not in term myometrial tissue samples. This work indicates there is a dramatic switch from ERalpha to ERbeta expression in the myometrium during pregnancy. The results suggest that, during gestation, myometrial ERbeta may inhibit AP-1 activity and thus block induction of the cx43 gene and other labor-associated genes. Labor may ensue after a loss of myometrial ERbeta expression.  (+info)