High levels of oestrogen receptor-alpha in tumorigenesis: inhibition of cell growth and angiogenic factors.

We previously found that the stable overexpression of oestrogen receptor-alpha in the human endothelial cell line ECV304* inhibits its growth in vitro, and that this inhibition is possibly mediated through a down-regulation of the vasoactive agents endothelin-1 and vascular endothelial growth factor. Here we show an in vivo growth-inhibitory effect of oestrogen receptor-alpha overexpression in tumours initiated in nude mice from the same clone of ECV304. In addition, we show that this growth inhibition is accompanied by an alphavbeta3-mediated inhibition of cell migration in vitro, and a down-regulation of the integrin alphavbeta3, vascular endothelial growth factor and vascularization in vivo. The levels of vascular endothelial growth factor and integrin alphavbeta3, through their effect on cell growth and migration, contribute to the process of angiogenesis and to the pathogenesis of atherosclerosis and cancer. The results shown here demonstrate that a higher level of oestrogen receptor-alpha in the cell, through its effect on certain angiogenic factors, may play a role in the control of angiogenesis.

Estrogen receptor-alpha in the inhibition of cancer growth and angiogenesis.

A high level of estrogen receptor-alpha (ER-alpha) is believed to be favorable in the prognosis and treatment of certain female cancers. ER-alpha expression in the ER-negative breast cancer cell lines inhibits their proliferation and invasive, metastatic potential in vitro. We stably overexpressed the ER-alpha in the human endometrial cancer cell line Ishikawa and showed that, unlike estradiol, high levels of ER-alpha significantly inhibit the growth of tumors xenografted from the Ishikawa cells. Subsequent to ER-alpha overexpression, in vivo down-regulation of vascular endothelial growth factor was observed in tumor xenografts. In addition, these tumors showed an inhibition of vascularization and of the angiogenic agent, integrin alphavbeta3. Involvement of a switch in the angiogenic pathways during tumorigenesis has been a recent focus of interest. Our results indicate that a high level of ER-alpha may be beneficial in the control of female cancers because of its inhibitory effect on such angiogenic pathways.

Stable over-expression of estrogen receptor-alpha in ECV304 cells inhibits proliferation and levels of secreted endothelin-1 and vascular endothelial growth factor.

Studies with mammalian vascular cells have suggested growth inhibitory effects of estrogen on the vascular wall. To investigate the involvement of estrogen receptor-alpha (ER) in the control of endothelial cell proliferation, we have stably transfected human estrogen receptor-alpha cDNA into the endothelial cell line ECV304. The clone ECV-ER, thus obtained, over-expresses estrogen receptor to a level approximately 10-fold higher than the parent cell line. Effects of this over-expression were studied on the cell growth rate, and on the levels of secreted endothelin-1 and vascular endothelial growth factor (VEGF). Similar to the previously reported data in other cell types, we found the transfection of ER in ECV304 cells to be inhibitory to their growth. Our ER-over-expressing clone of ECV304 also showed an inhibition of secreted endothelin-1 and VEGF levels. Moreover, the growth inhibition of this ER-over-expressing clone was reversed by the addition of endothelin-1 or VEGF to the medium. In view of the growth-stimulatory effect of endothelin-1 and VEGF on vascular cells, our results indicate that estrogen receptor-alpha may bring about its growth inhibition partly by suppressing endothelin-1 and/or VEGF production in ECV304 cells.

Prostaglandin E2 content in herniated lumbar disc disease.

STUDY DESIGN: A prospective series of biochemical assays for prostaglandin E2 content in symptomatic herniated lumbar disc specimens. OBJECTIVES: To help clarify the pathogenesis of lumbar radiculopathy. SUMMARY OF BACKGROUND DATA: Three recent studies have shown elevated levels of prostaglandin E2 in intervertebral disc herniations. None of these studies correlated symptoms with prostaglandin E2 levels. METHODS: Twenty-four disrupted disc samples were purified by a standard solid phase extraction method and analyzed for prostaglandin E2 with an enzyme-linked immunosorbent assay. Clinical and anatomic correlations were sought with analysis of variance and t test. RESULTS: Sequestered discs tended to be associated with a higher prostaglandin E2 content than extruded discs, which in turn, tended to be associated with higher prostaglandin E2 content than protruded ones. A positive straight leg raising test appeared to be associated with a higher prostaglandin E2 content than a negative test. CONCLUSIONS: Prostaglandin E2 appears to mediate some of the inflammatory effects of lumbar disc herniation. An intact anulus may provide some protection against this stimulus.

Ligand-dependent synergy of thyroid hormone and retinoid X receptors.

The binding of thyroid hormone receptors to DNA is enhanced by heterodimerization with nuclear proteins. One such heterodimerization partner has recently been characterized as the retinoid X receptor. 9-cis-Retinoic acid has been identified as a natural ligand for retinoid X receptors, suggesting a potential receptor-mediated interaction between thyroid hormone and 9-cis-retinoic acid in the regulation of thyroid hormone-responsive genes. A transient cotransfection assay was used to test for such an interaction. When a complex thyroid hormone response element composed of both direct and inverted repeat hexamers was tested, these two ligands activated gene expression synergistically. In contrast, when the response element consisted only of directly repeated hexamers, unliganded retinoid X receptors enhanced thyroid hormone responsiveness, but 9-cis-retinoic acid induced no additional activation. The results suggest a unique mechanism to achieve differential suggest a unique mechanism to achieve differential thyroid hormone sensitivity of thyroid hormone-responsive genes within a cell. Genes with appropriate response elements will show amplification of the thyroid hormone response by 9-cis-retinoic acid in the presence of retinoid X receptors; other thyroid hormone-responsive genes will be influenced by retinoid X receptors, but not 9-cis-retinoic acid.

Thyroid hormone receptor mutations that interfere with transcriptional activation also interfere with receptor interaction with a nuclear protein.

A 20 amino acid segment within the ligand-binding domain of the rat beta 1-thyroid hormone receptor (T3R; amino acids 286-305) is conserved in all members of the erbA superfamily. Mutations in this region impair T3 induction of reporter gene expression in a transfection system without impairing T3 binding or nuclear localization of the receptors. We postulate that a nuclear protein may need to interact with this domain for full transcriptional activity and provide evidence for the existence of this putative protein. Specifically, a JEG-3 cell protein (denoted TRAP, T3R auxiliary protein) interacts with wild-type T3R-beta 1 to increase binding of the receptor to T3 response elements in vitro. Mutations within amino acids 286-305 impair the ability of TRAP to enhance T3R binding to hormone response elements. Cross-linking studies indicate that TRAP has an apparent mol wt of 63 kDa. Surprisingly, TRAP does not enhance binding of erbA-alpha 2 and v-erbA to DNA, even though these proteins contain highly conserved versions of the 20-amino acid beta 1 T3R sequence under study. TRAP or a similar protein, however, does enhance binding of retinoic acid receptor-beta to a hormone response element. We conclude that the inability of T3Rs with mutations in this domain to fully activate transcription may be due to an inability of these proteins to fully interact with TRAP. We postulate that TRAP or related proteins may be important regulators of ligand-dependent transcriptional activation for other members of the erbA family in addition to the T3R.

Mutational analysis identifies a new functional domain of the thyroid hormone receptor.

We studied the functional significance of a region of the thyroid hormone receptor ligand binding domain which is conserved in all members of the erbA superfamily. The homologous region of the glucocorticoid receptor has been implicated in the binding of heat shock protein 90, and deletions of the glucocorticoid receptor that include this region result in constitutive activity. Both deletion and point mutations were made in this area of the rat beta 1 thyroid hormone receptor (amino acids 286-305), and the functional consequences of these mutations were analyzed in JEG cells by transient cotransfection along with a T3 responsive reporter gene. All mutations studied resulted in significant inhibition of ligand-dependent transcriptional regulation without inducing significant constitutive activity. For some mutations, the lack of transcriptional response correlated with a diminished ability to bind ligand. However, point mutations of amino acids 288, 290, and 300 resulted in impaired transcriptional regulation despite wild type T3 binding affinity. In addition, mutations of these three amino acids failed to impair localization of the receptor to the nucleus or binding to DNA in vitro. Cotransfection of plasmids expressing the wild type and mutant T3 receptor proteins resulted in inhibition of the wild type T3 receptor function. We conclude that this region of the rat beta 1 T3 receptor is essential for full transcriptional activity, but this is not due to a role in T3 binding, DNA binding, or nuclear localization. We postulate that nuclear factors may need to bind to this region for full transcriptional activity.