Characterization of rat vertebrae cortical bone microstructures using confocal Raman microscopy combined to tomography and electron microscopy.

BACKGROUND: The rat vertebrae is a good model to study bone regeneration after implantation of biomaterials used to treat bone loss, a major problem in oral and dental surgery. However, the precise characterization of bone microstructures in the rat vertebrae has not been reported. Therefore, the aim of this study was to achieve the complete analysis of such bone, at different scales, in order to have a clear model of healthy bone for comparison with regenerated bone. METHODS: In order to image the cortical bone of rat caudal vertebra, confocal Raman microscopy was combined with high resolution X-ray micro computed tomography (micro-CT), with scanning electron microscopy (SEM) using backscatter electron imaging and with more conventional histology coloration techniques. SEM and Raman microscopy were done in various regions of the cortical bone corresponding to external, middle and internal areas. The spongy bone was imaged in parallel. Micro-CT was performed on the whole vertebra to monitor the network of haversian canals in the cortical bone. Osteonic canals characteristics, and relative chemical composition were analysed in several regions of interest, in cortical and spongy bone. Five rats were included in this study. RESULTS: On micro-CT images, differences in intensity were observed in the cortical bone, substantiated by SEM. Chemical analysis with Raman spectra confirmed the difference in composition between the different regions of the cortical and spongy bone. PCA and k-mean cluster analysis separated these groups, except for the external and middle cortical bone. Peak intensity ratio confirmed these results with a CO3 to ν2 PO4 ratio significantly different for the internal cortical bone. Grayscale images stack extracted from micro-CT showed that global architecture of cortical bone was characterized by a dense and complex network of haversian osteonic canals, starting from the surface towards the vertebrae center. The mean diameter of the canals was 18.4 µm (SD 8.6 µm) and the mean length was 450 µm (SD 152 µm). Finally, Raman reconstructed images of the lamellar bone showed an enlargement of the lamellar layer width, both in circumferential lamellar bone and around haversian canals. CONCLUSIONS: Micro-CT and confocal Raman microscopy are good tools to complete classical analysis using optical and electron microscopy. The results and measurements presented in a rat model known for its small inter-individual differences provide the main characteristics of a mature bone. This study will allow the community working on this rat vertebrate model to have a set of characteristics, in particular on the structure of the haversian canals.

Complex regulation of LCoR signaling in breast cancer cells.

Ligand-dependent corepressor (LCoR) is a transcriptional repressor of ligand-activated estrogen receptors (ERs) and other transcription factors that acts both by recruiting histone deacetylases and C-terminal binding proteins. Here, we first studied LCOR gene expression in breast cancer cell lines and tissues. We detected two mRNAs variants, LCoR and LCoR2 (which encodes a truncated LCoR protein). Their expression was highly correlated and localized in discrete nuclear foci. LCoR and LCoR2 strongly repressed transcription, inhibited estrogen-induced target gene expression and decreased breast cancer cell proliferation. By mutagenesis analysis, we showed that the helix-turn-helix domain of LCoR is required for these effects. Using in vitro interaction, coimmunoprecipitation, proximity ligation assay and confocal microscopy experiments, we found that receptor-interacting protein of 140 kDa (RIP140) is a LCoR and LCoR2 partner and that this interaction requires the HTH domain of LCoR and RIP140 N- and C-terminal regions. By increasing or silencing LCoR and RIP140 expression in human breast cancer cells, we then showed that RIP140 is necessary for LCoR inhibition of gene expression and cell proliferation. Moreover, LCoR and RIP140 mRNA levels were strongly correlated in breast cancer cell lines and biopsies. In addition, RIP140 positively regulated LCoR expression in human breast cancer cells and in transgenic mouse models. Finally, their expression correlated with overall survival of patients with breast cancer. Taken together, our results provide new insights into the mechanism of action of LCoR and RIP140 and highlight their strong interplay for the control of gene expression and cell proliferation in breast cancer cells.

Cognitive impairments in adult mice with constitutive inactivation of RIP140 gene expression.

Receptor-interacting protein 140 (RIP140) is a negative transcriptional coregulator of nuclear receptors such as estrogen, retinoic acid or glucocorticoid receptors. Recruitment of RIP140 results in an inhibition of target gene expression through different repressive domains interacting with histone deacetylases or C-terminal binding proteins. In this study, we analyzed the role of RIP140 activity in memory processes using RIP140-deficient transgenic mice. Although the RIP140 protein was clearly expressed in the brain (cortical and hippocampus areas), the morphological examination of RIP140(-/-) mouse brain failed to show grossly observable alterations. Using male 2-month-old RIP140(-/-) , RIP140(+/-) or RIP140(+/+) mice, we did not observe any significant differences in the open-field test, rotarod test and in terms of spontaneous alternation in the Y-maze. By contrast, RIP140(-/-) mice showed long-term memory deficits, with an absence of decrease in escape latencies when animals were tested using a fixed platform position procedure in the water maze and in the passive avoidance test. Noteworthy, RIP140(-/-) mice showed decreased swimming speed, suggesting swimming alterations that may in part account for the marked alterations measured in the water maze. Moreover, RIP140(+/-) and RIP140(-/-) mice showed a significant increase in immobility time in the forced swimming test as compared with wild-type animals. These observations showed that RIP140 gene depletion results in learning and memory deficits as well as stress response, bringing to light a major role for this transcriptional coregulator in the neurophysiological developmental mechanisms underlying cognitive functions.

ERalpha and ERbeta expression and transcriptional activity are differentially regulated by HDAC inhibitors.

The proliferative action of ERalpha largely accounts for the carcinogenic activity of estrogens. By contrast, recent data show that ERbeta displays tumor-suppressor properties, thus supporting the interest to identify compounds that could increase its activity. Here, we show that histone deacetylase inhibitors (HDI) upregulated ERbeta protein levels, whereas it decreased ERalpha expression. Part of this regulation took place at the mRNA level through a mechanism independent of de novo protein synthesis. In addition, we found that, in various cancer cells, the treatment with different HDI enhanced the ligand-dependent activity of ERbeta more strongly than that of ERalpha. On the other hand, in MDA-MB231 and HeLa cells, the expression of ERs modified the transcriptional response to HDI. The use of deletion mutants of both receptors demonstrated that AF1 domain of the receptors was required. Finally, we show that ERbeta expression led to a dramatic increased in the antiproliferative activity of HDI, which correlated with a modification of the transcription of genes involved in cell cycle control by HDI. Altogether, these data demonstrate that the interference of ERbeta and HDAC on the control of transcription and cell proliferation constitute a promising approach for cancer therapy.

Transcriptional and posttranscriptional regulation of fibulin-1 by estrogens leads to differential induction of messenger ribonucleic acid variants in ovarian and breast cancer cells.

Fibulin-1 is an extracellular matrix protein overexpressed in epithelial ovarian and breast cancers. In estrogen receptor (ER)-positive ovarian and breast cancer cell lines, fibulin-1 mRNA levels are markedly increased by estrogens. Transfection experiments using fibulin-1 promoter constructs indicate that 17beta-estradiol (E2) increases fibulin-1 gene transcription and that ERalpha is more potent than ERbeta to mediate E2 regulation of the transfected fibulin-1 promoter. Using SL2 cells devoid of specificity protein 1 (Sp1) and site-directed mutagenesis of GC boxes, we evidenced that the E2 regulation occurs through a proximal specificity protein 1 binding site. In addition, we show that fibulin-1C and -1D mRNAs, the two major fibulin-1 splicing variants, are differentially induced by E2. The induction of both mRNAs variants is direct and independent of a newly synthesized protein intermediate. Interestingly, actinomycin D chase experiments demonstrate that E2 treatment selectively shortens the fibulin-1D mRNA half-life. This indicates that estrogens affect differentially the stability of fibulin-1 variants and may explain the lower accumulation of fibulin-1D mRNA on E2 treatment. In conclusion, our data show that estrogens, via ERalpha, are key regulators of fibulin-1 expression at both the transcriptional and posttranscriptional levels. The preferential induction of the fibulin-1C variant, which is overexpressed in ovarian and breast cancer, might play an important role in estrogen-promoted carcinogenesis.

Dimerization is required for transactivation by estrogen-receptor-related (ERR) orphan receptors: evidence from amphioxus ERR.

The estrogen-receptor-related (ERR) receptors are orphan members of the nuclear receptor superfamily that bind to their specific DNA target sites as homodimers. However, it has not been shown whether this mode of binding is required for the transcriptional activation they drive. We here show that heterodimerization can also occur between these receptors. Furthermore, we demonstrate that the unique amphioxus ortholog of ERR genes (AmphiERR) is expressed as two isoforms differing by an in-frame insertion. While the short isoform behaves like its mammalian counterparts, the long isoform (AmphiERR(L)) displays divergent transcriptional properties according to the target site to which it binds. Indeed, AmphiERR(L) binds as a monomer but does not activate transcription through the SF1 response element (SFRE). On the contrary, this isoform binds as a homodimer and activates transcription through the classical estrogen-response element. Our results strongly suggest that dimerization is required for transactivation exerted by the ERR receptors.

Histone deacetylase inhibition and estrogen receptor alpha levels modulate the transcriptional activity of partial antiestrogens.

In this study, we have analysed the effects of histone deacetylase (HDAC) inhibition on estrogen receptor (ER) expression and on its transcriptional activity in response to antiestrogens. In several breast cancer cell lines, trichostatin A (TSA), a potent HDAC inhibitor, strongly decreases ERalpha expression in a dose-dependent manner. This repression is observed independently of the presence of ligand and also occurs in ovarian and endometrial cell lines. In addition, we show that in MCF7 cells bearing a stably transfected reporter plasmid (MELN cells), partial antiestrogens such as 4-OH-tamoxifen (OHTam), raloxifen or LY117018, switch to an agonist activity upon HDAC inhibition. This effect is blocked by the pure antiestrogen ICI182780 and exhibits a half-maximal concentration of OHTam equivalent to its affinity for ERalpha. The TSA-dependent decrease of ERalpha expression is required to induce the agonist switch of OHTam properties as it is lost in cells constitutively expressing exogenous receptors (MELN-ERalpha or ERbeta). By contrast, the transrepression activity of OHTam is abolished by TSA independently of the decrease of ERalpha expression. Interestingly, in MELN-ERalpha, ICI182780 remains inhibitory suggesting the involvement of HDAC-independent mechanisms. Finally, in the absence of TSA, transcriptional activity in response to OHTam is significantly raised in MELN cells expressing low levels of ERalpha after transfection of antisense oligonucleotides. In conclusion, inhibition of HDAC enzymatic activity and modulation of ERalpha levels tightly control the relative agonist activity of partial antiestrogens on a stably integrated reporter transgene.

Oestrogen receptor alpha increases p21(WAF1/CIP1) gene expression and the antiproliferative activity of histone deacetylase inhibitors in human breast cancer cells.

We analysed the antiproliferative activity of various histone deacetylase (HDAC) inhibitors such as trichostatin A (TSA) on human breast cancer cells. We observed a lower sensitivity to HDAC inhibition for oestrogen receptor negative (ER-) versus positive (ER+) cell lines. This differential response was associated neither with a modification of drug efflux via the multidrug resistance system nor with a global modification of histone acetyltransferase (HAT)/HDAC activities. In contrast, we demonstrated that in ER+ breast cancer cells the p21(WAF1/CIP1) gene was more sensitive to TSA regulation and was expressed at higher levels. These differences were observed both in transient transfection experiments and on the endogenous p21(WAF1/CIP1) gene. The Sp1 transcription factor, which was shown to interact in vitro with both class I and class II HDACs, is sufficient to confer the differential sensitivity to TSA and participated in the control of p21(WAF1/CIP1) basal expression. Finally, re-expression of ERalpha following adenoviral infection of ER- breast cancer cells increased both p21(WAF1/CIP1) protein accumulation and the growth inhibitory activity of TSA. Altogether, our results highlight the key role of ERalpha and p21(WAF1/CIP1) gene expression in the sensitivity of breast cancer cells to hyperacetylating agents.

Estrogens and receptors: an evolving concept.

Estrogens elicit most of their physiological effects through specific nuclear receptors (ERalpha and ERbeta), which belong to a superfamily of hormone-activated transcription factors. They regulate gene expression either through direct binding to DNA (as homo- or heterodimers) or via protein-protein interactions with other transcription factors. The hormone induces a conformational change that creates a new interface for molecules acting as transcription intermediary factors. These cofactors bind directly to estrogen receptors and mediate transcription activation or repression, usually as part of multimolecular complexes, acting either by stabilization of the basal transcription machinery or by chromatin remodelling; these involve the activities of various enzymes such as histone acetyltransferases and deacetylases. Moreover, different types of post-translational modifications (phosphorylation, acetylation, ubiquitination) also regulate estrogen receptor stability and/or transcriptional activation, and these effects, together with the non-genomic effects of estrogens, are important parameters of this signalling pathway. Finally, all these processes have their own dynamics, which may lead, depending on whether the hormone is provided continuously or as a pulse, to the same or different effects according to the end-point and the cell considered.

Comparative activity of pulsed or continuous estradiol exposure on gene expression and proliferation of normal and tumoral human breast cells.

Intranasal administration of hormone replacement therapy presents an original plasma kinetic profile with transient estrogen levels giving rise to the concept of pulsed therapy. To further understand the molecular effects of this new therapy, we have compared the effects of pulsed and continuous estradiol treatments on two critical aspects of estradiol action: gene expression and cell proliferation. Cells were stimulated with estradiol as 1-h pulsed or 24-h continuous treatments at concentrations such that the 24-h exposure (concentration x time) was identical in both conditions. In MCF7 cells, the transcriptional activity of estrogen receptors (ER) on a transiently transfected responsive estrogen response element-luciferase reporter construct was shown to be drastically (approximately 10-fold) and similarly stimulated after both treatments. Moreover, the increased mRNA expression of three representative estradiol-sensitive genes (pS2, cathepsin D, progesterone receptor), evaluated by Northern blot, was identical after 1-h pulse with 7 nM estradiol or continuous treatment with 0.29 nM estradiol with the same kinetic profile over 48 h. Proliferation was quantified by a histomorphometric method on primary cultures of human normal breast cells from reduction mammoplasties and using a fluorescence DNA assay in six human breast cancer cell lines which were ER positive or negative. After a 7-day treatment period, estradiol had no effect on the proliferation of the three ER negative cell lines (BT20, MDA MB231, SK BR3) but significantly stimulated the proliferation of the normal cells and of the three tumoral hormone-sensitive cell lines (MCF7, T47D, ZR 75-1); both hormone treatments producing the same increases in cell growth. In conclusion, we have shown that the genomic or proliferative effects of estradiol were identical with pulsed or continuous treatments, thus indicating that estrogenic effects are not strictly related to concentrations but rather to total hormone exposure.

The human estrogen receptor alpha dimer binds a single SRC-1 coactivator molecule with an affinity dictated by agonist structure.

Nuclear receptors act as ligand-inducible transcription factors. Agonist binding leads to interaction with coactivator proteins, and to the assembly of the general transcription machinery. In addition to structural information, a thorough understanding of transcriptional activation by the nuclear receptors requires the characterization of the thermodynamic parameters governing these protein/protein interactions. In this study we have quantitatively characterized the interactions of full-length baculovirus expressed human estrogen receptor alpha (ERalpha), as well as ERalpha hormone binding domain (ERHBD) with a fragment of the coactivator protein SRC-1 (amino acid residues 570 to 780). Fluorescence anisotropy and fluorescence correlation spectroscopy of fluorescently labeled SRC-1(570-780) demonstrate unambiguously that the stoichiometry of the SRC-1/ERalpha/estradiol complex is one coactivator molecule per ERalpha dimer. The affinity of the estradiol or estriol bound ERalpha/SRC-1 complexes was found to be significantly higher than that observed in the presence of estrone. No binding was observed in the absence of ligand or in the presence of antagonists. Distinct anisotropy values for the ERalpha-SRC-1 complexes with different agonists suggest distinct conformations of the complexes depending upon agonist structure.

Semiquantitative reverse transcription-polymerase chain reaction to evaluate the expression patterns of genes involved in the oestrogen pathway.

The increasing number of factors to be taken into account in the oestrogen transcriptional process has created a need to develop a rapid screening method to evaluate their role in physiology and pathology. Molecular biology techniques enable gene expression studies at the mRNA level with small amounts of tissues. We therefore developed a semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique using fluorescent oligonucleotides to analyse simultaneously a large panel of interrelated genes involved in the oestrogen transcriptional pathway using a moderately expressed housekeeping gene, the hypoxanthine phosphoribosyltransferase gene (HPRT), as the reference gene. Expression levels of oestrogen receptors (ERalpha, ERbeta), cofactors AIB1, RIP140, SMRT and the Fas-associated protein-tyrosine phophatase-1 (FAP-1) genes were evaluated in breast, endometrial and ovarian cancer cell lines and in three ERalpha-positive and three ERalpha-negative breast cancer tumours. This technique provides a rapid and reliable way to quantitate simultaneously numerous mRNAs of genes involved in the oestrogen pathway from small amounts of tissues.

Effect of ligand and DNA binding on the interaction between human transcription intermediary factor 1alpha and estrogen receptors.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. To achieve efficient regulation of gene expression, these receptors must interact with different type of molecules: 1) the steroid hormone, 2) the DNA response element, and 3) various proteins acting as transcriptional cofactors. In the present study, we have investigated how ligand and DNA binding influence the in vitro interaction between estrogen receptors (ERs) and the transcription intermediary factor hTIF1alpha (human transcriptional intermediary factor 1alpha). We first optimized conditions for the coactivator-dependent receptor ligand assay to lower ED50, and we then analyzed the ability of various natural and synthetic estrogens to allow the binding of the two types of proteins. Results were compared with the respective affinities of these ligands for the receptor. We then developed a protein-protein-DNA assay allowing the quantification of cofactor-ER-estrogen response element (ERE) complex formation in the presence of ligand and used measurements of fluorescence anisotropy to define the equilibrium binding parameters of the interaction. We demonstrated that the leucine-charged domain of hTIF1alpha is sufficient to interact with ERE-bound ERalpha in a ligand-dependent manner and showed that binding of ERalpha onto DNA does not significantly affect its hormone-dependent association with TIF1alpha. Finally, we show that, mainly in the absence of hormone, hTIF1alpha interacts better with ERbeta than with ERalpha independently of the presence of ERE.

Estrogen receptor cofactors expression in breast and endometrial human cancer cells.

In order to approach the molecular basis of the tissue-specific agonistic activity of antioestrogens, we have compared, at the mRNA level, the expression of various transcriptional cofactors (activators or repressors) of estrogen receptors in different breast (MCF7, ZR75-1, T47D, MDA-MB231) and endometrial (Ishikawa, RL-95-2 and HEC1A) human cancer cell lines. We showed that for SRC-1, CBP, TIF1alpha, RIP140, N-CoR, and SMRT, no significant differences in the expression levels were observed between breast and endometrial cells. For TIF1alpha mRNA, both isoforms were also detected at similar levels in all the cells tested. By contrast, over-expression of AIB1 mRNA was observed in MCF7 cells, but not in other breast or endometrial cells, irrespective of their ER-status. We then used protein-protein interaction assay (far-Western blot) to confirm the increased expression of at least one of the p160 proteins in MCF7 cells. Finally, we demonstrated that RIP140 mRNA is directly induced by estrogens in ER-positive MCF7 breast cancer cell lines but not in Ishikawa endometrial cells. Together these results indicate that some differences exist between breast and endometrial cancer cell lines at the level of estrogen receptor transcription cofactor expression.

Transcriptional activities of the orphan nuclear receptor ERR alpha (estrogen receptor-related receptor-alpha).

Estrogen receptor-related receptor alpha (ERR alpha) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR alpha binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR alpha and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR alpha originating from various species. All ERR alpha orthologs act as potent transactivators through the consensus SFRE. ERR alpha activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.

Differential interaction of nuclear receptors with the putative human transcriptional coactivator hTIF1.

Hormonal regulation of gene activity is mediated by nuclear receptors acting as ligand-activated transcription factors. Intermediary factors interacting with their activation functions are required to mediate transcriptional stimulation. In search of such receptor interacting proteins, we have screened a human cDNA expression library and isolated a human protein that interacts in vitro with transcriptionally active estrogen receptors (ER). Sequence analysis reveals that this protein is the human homolog of mouse TIF1 (transcription intermediary factor 1) shown to enhance nuclear receptor ligand-dependent activation function 2 (AF2) in yeast. We have characterized the nuclear receptor binding site on hTIF1 and shown that a region of 26 residues is sufficient for hormone-dependent binding to the estrogen receptor. As shown by point mutagenesis, the AF2 activation domain of ER is required for the binding of hTIF1 but not sufficient, since a short region encompassing the conserved amphipathic alpha-helix corresponding to this domain fails to precipitate hTIF1. We also demonstrate that hTIF1 association with DNA-bound ER requires the presence of estradiol. Finally, we show that the interaction of hTIF1 with receptors is selective since strong in vitro hormone-dependent binding is only observed with some members of the nuclear receptor superfamily.

RIP 140 enhances nuclear receptor-dependent transcription in vivo in yeast.

RIP140 has previously been cloned as a factor that interacts with the estrogen receptor (ER) in vitro. We demonstrate in this study that RIP140 is a co-factor for nuclear receptor in yeast. RIP140 enhances the ER transcriptional activity by increasing 1.5- to 4-fold the induction factor of the reporter gene response at saturating hormone concentrations, this effect being magnified at suboptimal doses of estradiol. Moreover, RIP140 decreases the ED50 of the dose-response curve. These effects are recovered with an N-terminal truncated ER, but impaired by point mutations that abolish AF2-AD activity. We did not observe any modulation of the partial agonist 4-hydroxytamoxifen activity in the presence of RIP140. Thus, RIP140 modulates transcriptional activity of ER through the AF2-AD domain and in a agonist-dependent fashion. RIP140 is also a strong coactivator for the retinoid pathway, as its expression enhances 10-fold the transactivation of a chimeric retinoic acid-alpha receptor at saturant hormone concentration and left shifted 5-fold the ED50 of the dose-response curve. We have investigated whether RIP140 could be involved in cross-talk between estrogenic and retinoid pathways.

A natural transactivation mutation in the thyroid hormone beta receptor: impaired interaction with putative transcriptional mediators.

The syndrome of resistance to thyroid hormone is characterized by elevated serum free thyroid hormones, failure to suppress pituitary thyrotropin secretion, and variable peripheral refractoriness to hormone action. Here we describe a novel leucine to valine mutation in codon 454 (L454V) of the thyroid hormone beta receptor (TR beta) in this disorder, resulting in a mutant receptor with unusual functional properties. Although the mutant protein binds ligand comparably to wild-type receptor and forms homo- and heterodimers on direct repeat, everted repeat, or palindromic thyroid response elements, its ability to activate transcription via these elements is markedly impaired. The hydrophobic leucine residue lies within an amphipathic alpha-helix at the carboxyl terminus of TR beta and the position of the homologous residue in the crystal structure of TR alpha indicates that its side chain is solvent-exposed and might interact with other proteins. We find that two putative transcriptional mediators (RIP140 and SRC-1) exhibit hormone-dependent association with wild-type TR. In comparison, the interaction of this natural mutant (L454V) and artificial mutants (L454A, E457A) with RIP140 and SRC-1 is markedly reduced. Furthermore, coexpression of SRC-1 is able to restore the transcriptional activity of the L454V mutant receptor, indicating that the interaction of this residue with accessory proteins is critical for transcriptional activation. Finally, the occurrence of the L454V mutation in resistance to thyroid hormone, together with impaired negative regulation of the thyroid-stimulating hormone alpha promoter by this mutant, suggests that the amphipathic alpha-helix also mediates hormone-dependent transcriptional inhibition, perhaps via interaction with these or other accessory factors.

RIP-140 interacts with multiple nuclear receptors by means of two distinct sites.

We have characterized two distinct binding sites, called site 1 and site 2, in the nuclear protein RIP-140 which interact with the ligand binding domain of the estrogen receptor both in solution and when the receptor is bound to DNA. Both sites are capable of independently interacting with other nuclear receptors, including the thyroid hormone and retinoic acid receptors, but they are not identical since the interaction with retinoid X receptor is mediated primarily by site 1. The interaction is enhanced by agonists but not by antagonists, and the in vitro binding activities to a number of mutant receptors correlate with their abilities to stimulate transcription in vivo. When RIP-140 is fused to heterologous DNA binding domains, it is able to stimulate the transcription of reporter genes in both yeast and mammalian cells. Thus, RIP-140 is likely to function as a bridging protein between receptors and the basal transcription machinery and thereby stimulate the transcription of target genes.

Biochemical characterization and novel isolation of pure estrogen receptor hormone-binding domain.

Biologically active, mouse estrogen receptor hormone-binding domain (residues 313-599) overexpressed in Escherichia coli was purified to apparent homogeneity as a single component with a molecular mass of 32.831 kDa determined by electrospray ionization mass spectrometry, and was identical to the mass predicted from the amino acid sequence. The intact domain was isolated using a novel, rapid purification scheme without recourse to any chromatographic process. Pure ERhbd maintained both high affinity estradiol binding (at optimum pH 8.0) and specificity for estrogens and anti-estrogens. The steroid-binding domain sedimented as a 4S component in the presence or absence of bound [3H]estradiol and at 2S in the presence of urea. The molecular mass of the 4S steroid unoccupied ERhbd (from dynamic light scattering) was approximately 72 kDa, suggesting that the pure, unlabelled ERhbd formed homodimers. Steroid-labelled ERhbd electrofocussed as a single, acidic component at a pI of 5.6. Binding of ERhbd to [3H]estradiol was unaffected by Ca2+ and Mg2+ ions up to 1 mM but was significantly inhibited by Zn2+ ions at concentrations above 10 microM, an effect reversed by EDTA.