Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells

Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.     

A novel ligand-independent function of the estrogen receptor is essential for osteocyte and osteoblast mechanotransduction

Bone senses and adapts to meet mechanical needs by means of an extensive mechanotransduction network comprising osteocytes (former osteoblasts entrapped in mineral) and their cytoplasmic projections through which osteocytes communicate with osteoblasts and osteoclasts on the bone surface. Mechanical stimulation promotes osteocyte (and osteoblast) survival by activating the extracellular signal-regulated kinases, ERKs. Estrogens have similar effects and, intriguingly, the adaptive response of bone to mechanical forces is defective in mice lacking estrogen receptor (ER) alpha or ERbeta. We report that ERKs are not activated by stretching in osteocytic and osteoblastic cells in which both ERalpha and ERbeta have been knocked out or knocked down and this is reversed partially by transfection of either one of the two human ERs and fully by transfection of both receptors. ERK activation in response to stretching is also recovered by transfecting the ligand-binding domain (E) of either receptor or an ERalpha mutant that does not bind estrogens. Furthermore, mechano-responsiveness is restored by transfecting the Ealpha targeted to the plasma membrane, but not to the nucleus, whereas ERalpha mutants with impaired plasma membrane localization or binding to caveolin-1 fail to confer ERK activation in response to stretching. Lastly, the ER antagonist ICI 182,780 abrogates ERK activation and the anti-apoptotic effect of mechanical stimulation. We conclude that in addition to their role as ligand-dependent mediators of the effects of estrogens, the ERs participate in the transduction of mechanical forces into pro-survival signaling in bone cells, albeit in a ligand-independent manner.     

Dynamic regulation of estrogen receptor-alpha isoform expression in the mouse fallopian tube: mechanistic insight into estrogen-dependent production and secretion of insulin-like growth factors

Estrogen receptors (ERs) are members of the nuclear receptor superfamily and are involved in regulation of fallopian tube functions (i.e., enhancement of protein secretion, formation of tubal fluid, and regulation of gamete transport). However, the ER subtype-mediated mechanisms underlying these processes have not been completely clarified. Recently, we identified ERbeta expression and localization in rat fallopian tubes, suggesting a potential biological function of ERbeta related to calcium-dependent ciliated beating. Here we provide for the first time insight into the less studied ERalpha isoforms, which mediate estrogen-dependent production and secretion of IGFs in vivo. First, Western blot studies revealed that three ERalpha isoforms were expressed in mouse fallopian tubes. Subsequent immunohistochemical analysis showed that ERalpha was detected in all cell types, whereas ERbeta was mainly localized in ciliated epithelial cells. Second, ERalpha isoform levels were dramatically downregulated in mouse fallopian tubes by treatment with E(2) or PPT, an ERalpha agonist, in a time-dependent manner. Third, the presence of ICI 182,780, an ER antagonist, blocked the E(2)- or PPT-induced downregulation of tubal ERalpha isoform expression in mice. However, alteration of ERalpha immunoreactivity following ICI 182,780 treatment was only detected in epithelial cells of the ampullary region. Fourth, changes in ERalpha isoform expression were found to be coupled to multiple E(2) effects on tubal growth, protein synthesis, and secretion in mouse fallopian tube tissues and fluid. In particular, E(2) exhibited positive regulation of IGF-I and IGF-II protein levels. Finally, using growth hormone receptor (GHR) gene-disrupted mice, we showed that regulation by E(2) of IGF production was independent of GH-induced GHR signaling in mouse fallopian tubes in vivo. These data, together with previous studies from our laboratory, suggest that the long-term effects of estrogen agonist promote IGF synthesis and secretion in mouse tubal epithelial cells and fallopian tube fluid via stimulation of ERalpha.     

Estrogens inhibit l-glutamate uptake activity of astrocytes via membrane estrogen receptor alpha

We investigated the effects of estrogen-related compounds including xenoestrogens [17beta-estradiol (E2), 17alpha-ethynylestradiol (EE), diethylstilbestrol (DES), p-nonylphenol (PNP), bisphenol A (BPA) and 17alpha-estradiol (17alpha)] on l-glu uptake by cultured astrocytes via glutamate-aspartate transporter (GLAST). After 24 h treatment, E2 inhibited the l-glu uptake at 1 micro m and higher concentrations. EE and DES also inhibited the l-glu uptake at 1 nm and higher concentrations. The other four compounds had no effect. The effects of E2, EE and DES were completely blocked by 10 nm of ICI182 780 (ICI). beta-Estradiol 17-hemisuccinate : bovine serum albumin (E2-BSA), a membrane-impermeable conjugate of E2, also elicited the inhibition of l-glu uptake at 1 nm and higher concentrations, and the effect was blocked by ICI. 16alpha-Iodo-17beta-estradiol (16alphaIE2), an estrogen receptor alpha (ERalpha) selective ligand, revealed an inhibitory effect at 10 nm, while genistein, an ERbeta selective ligand, failed to reveal such an effect at this concentration. Western blot analysis showed that the predominant ER of cultured astrocytes was ERalpha. The colocalization of ERalpha with GLAST on plasma membranes was immunohistochemically detected in these cells. From these results, we concluded that estrogens down-regulate l-glu uptake activity of astrocytes via membrane ERalpha.     

Calmodulin is a selective modulator of estrogen receptors

In the search for differences between ERalpha and ERbeta, we analyzed the interaction of both receptors with calmodulin (CaM) and demonstrated that ERalpha but not ERbeta directly interacts with CaM. Using transiently transfected HeLa cells, we examined the effect of the CaM antagonist N-(6-aminohexyl)-5-chloro-naphthalene sulfonilamide hydrochloride (W7) on the transactivation properties of ERalpha and ERbeta in promoters containing either estrogen response elements or activator protein 1 elements. Transactivation by ERalpha was dose-dependently inhibited by W7, whereas that of ERbeta was not inhibited or even activated at low W7 concentrations. In agreement with these results, transactivation of an estrogen response element containing promoter in MCF-7 cells (which express a high ERalpha/ERbeta ratio) was also inhibited by W7. In contrast, transactivation in T47D cells (which express a low ERalpha/ERbeta ratio) was not affected by this CaM antagonist. The sensitivity of MCF-7 cells to W7 was abolished when cells were transfected with increasing amounts of ERbeta, indicating that the sensitivity to CaM antagonists of estrogen-responsive tissues correlates with a high ERalpha/ERbeta ratio. Finally, substitution of lysine residues 302 and 303 of ERalpha for glycine rendered a mutant ERalpha unable to interact with CaM whose transactivation activity became insensitive to W7. Our results indicate that CaM antagonists are selective modulators of ER able to inhibit ERalpha-mediated activity, whereas ERbeta actions were not affected or even potentiated by W7.