Molecular cloning and characterization of reptilian estrogen receptor cDNAs

cDNAs encoding alligator (caiman), Caiman crocodilus and whiptail lizard, Cnemidophorus uniparens estrogen receptors (ERs) were cloned and sequenced. This is the first report of full-length cDNA sequences for reptilian ERs, to our knowledge. The full-length alligator (caiman) ER cDNA (1764 bp, 587 amino acid residues) shows 68% amino acid homology with the full-length whiptail lizard ER cDNA (1746 bp, 581 amino acid residues). The respective ligand binding E domains have 87 and 83% amino acid homology with human ER while the DNA binding C domains show 100% amino acid homology with the human, rat and chicken forms. When the cDNAs were inserted into the pRc/RSV vector and transfected into HeLa cells with a reporter plasmid, the encoding proteins were confirmed to be functional through the interaction of the receptor-ligand complex with the estrogen responsive element (ERE).     

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.     

Expression of adipokines and estrogen receptors in adipose tissue and placenta of patients with gestational diabetes mellitus

The purpose of this study was to assess the expression profile of genes with potential role in the development of insulin resistance (adipokines, cytokines/chemokines, estrogen receptors) in subcutaneous adipose tissue (SAT), visceral adipose tissue (VAT) and placenta of pregnant women with gestational diabetes mellitus (GDM) and age-matched women with physiological pregnancy at the time of Caesarean section. qRT-PCR was used for expression analysis of the studied genes. Leptin gene expression in VAT of GDM group was significantly higher relative to control group. Gene expressions of interleukin-6 and interleukin-8 were significantly increased, whereas the expressions of genes for estrogen receptors α and β were significantly reduced in SAT of GDM group relative to controls, respectively. We found no significant differences in the expression of any genes of interest (LEP, RETN, ADIPOR1, ADIPOR2, TNF-α, CD68, IL-6, IL-8, ERα, ERβ) in placentas of women with GDM relative to controls. We conclude that increased expression of leptin in visceral adipose depot together with increased expressions of proinflammatory cytokines and reduced expressions of estrogen receptors in subcutaneous fat may play a role in the etiopathogenesis of GDM.     

Immunohistochemical detection of estrogen receptors in fish scales

Calcium mobilization from internal stores, such as scales, induced by 17β-estradiol during sexual maturation in salmonids is well documented. This calcium mobilization from scales is proposed to be mediated by the estrogen receptor (ER). However, the ER subtypes involved and signaling mechanisms responsible for this effect remain to be fully characterized. In the present study, we have localized ERα, ERβa and ERβb proteins in juvenile and adult sea bream (Sparus auratus) and Mozambique tilapia (Oreochromis mossambicus) scales by immunohistochemistry with sea bream ER subtype specific antibodies. The three ERs were detected in isolated or small groups of round cells, in the basal layer of the scales of both juvenile and adult fish and the localization and signal intensity varied with the species and age of the animals. The ERs may be co-localized in cells of the scale posterior region that expressed tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts. These results suggest that the calcium mobilizing action of 17β-estradiol on fish scales is via its direct action on ERs localized in osteoclasts.     

Body size and food size in freshwater zooplankton

We used double-label liquid scintillation techniques to measure the efficiencies with which eight different-sized zooplankton species ingested four cell types relative to a standard cell type (Chlamydomonas). Efficiency ratios (ERs: clearance rate on cell type X ÷ clearance rate on Chlamydomonas) on the three ultraplankton (<5 μm in diameter) cells (a coccoid bacterium and the algae Synechococcus and Nannochloris) varied greatly among zooplankton species but were not correlated with zooplankton body length. Variation in ERs on a much larger (17 × 14 μm) algal cell (Cryptomonas) was only partly explained by zooplankton body length. The eight zooplankton species were classified into three functional groups: (i) species having moderate to high ERs on all ultraplankton (0.4 < ER < 1.6) and ERs on Cryptomonas proportional to their body lengths (Conochilus, Diaphanosoma, and probably Keratella cochlearis and Ceriodaphnia); (ii) species having extremely low ERs on bacteria (mean ER < 0.05), higher but still low ERs on ultraphytoplankton (ER generally < 0.4), and ERs on Cryptomonas proportional to their body lengths (Bosmina, Diaptomus copepodites and adults); (iii) species having extremely low ERs on all ultraplankton (mean ER < 0.05) and ERs on Cryptomonas much higher than expected given their body lengths (Keratella crassa, Polyarthra, and Diaptomus nauplii). These functional groups follow neither taxonomic nor body-length groupings. We conclude that zooplankton body length may influence the maximal particle size a species can ingest but has little influence on the ingestion of smaller particles. Two frequently used models relating zooplankton body size and food size are unrealistic.     

Oyster estrogen receptor: cDNA cloning and immunolocalization

A cDNA encoding the Pacific oyster, Crassostrea gigas, estrogen receptor (cgER) was cloned using degenerate PCR primers. The open reading frame predicted 485 amino acid residues. Comparisons of the amino acid sequence of cgER with other mollusk ERs show high similarities of the C domain (95-97%), and the E domain (56-66%). The amino acid sequence of the C domain of cgER shows 86 and 89% identity with the respective sequences of human ER-alpha and ER-beta. The amino acid sequence of the E domain of cgER shows 45% identity with those of human ER-alpha and ER-beta. The phylogenetic analysis indicated that the cgER is an ortholog of the other mollusk ERs. In the C domain, the positions of cysteine residues and other residues around them that constitute the two zinc finger motifs and the P-box are conserved. The cgER mRNA was expressed in various tissues including the ovary. Reporter gene assay revealed that cgER is unresponsive to estrogen. This result is similar to those of other mollusk ERs. ER immunoreactivity was localized mainly in the nuclei of follicle cells, the site of vitellogenin synthesis, and in oocytes. This result suggests that cgER could work as a nuclear receptor.     

Identification of mesotocin and vasotocin nucleotide sequences in two species of urodele amphibian

We amplified and identified, for the first time in urodele amphibians, cDNA sequences that encode preprovasotocin (preproVT) and prepromesotocin (preproMT) from two distinct urodelian species, Taricha granulosa (the rough-skinned newt) and Plethodon shermanii (the spotted salamander). Each of these cDNA sequences encoded proteins that contained the characteristics of known neurohypophysial peptide precursors; each sequence consisting of (1) a signal peptide, (2) VT- or MT-like peptides, (3) neurophysin, and for the preproVTs, (4) copeptin. In T. granulosa, cDNA sequences encoded for the nine amino acids that define VT or MT. In P. shermani, cDNA sequences encoded for the VT peptide and a previously unidentified isoform of MT, ([Val⁴]-MT).     

Autoregulation and crossregulation of nuclear receptor genes.

Whereas crossregulation of nuclear receptors has been known for some time, recently several examples of autoregulation have been described, especially during development and specific gene expression. In this review, I discuss both these phenomena, based on some studies from our laboratory on amphibian metamorphosis and egg protein gene expression. These include autoinduction of estrogen receptor (ER) accompanying egg protein gene expression in adult and larval Xenopus; autoinduction of thyroid hormone receptor (TR) during metamorphosis and in adult Xenopus; crossregulation by triiodothyronine (T(3)) and dexamethasone of autoinduction of ER; and inhibition by PRL of autoinduction and crossinduction of TR and ER genes. A dual receptor threshold model to explain the interplay between T(3), estrogen and PRL is presented and its significance to the general question of nuclear receptor autoregulation and crossregulation during development is also discussed.     

Activation of estrogen receptor-α and of angiotensin-converting enzyme 2 suppresses ischemic brain damage in oophorectomized rats

Like the angiotensin II type 1 receptor blocker, endogenous estrogen (17β-estradiol) is neuroprotective against cerebral ischemia; its effects are thought to be mediated by estrogen receptors (ERs). To verify the role of ERs and the brain renin-angiotensin system in estrogen-deficient rats with ischemia induced by middle cerebral artery occlusion, we compared rats subjected to oophorectomy (OVX(+)) with sham-oophorectomized rats (OVX(-)) and OVX(+) rats treated with 0.3 or 3.0 mg/kg of olmesartan for 2 weeks before middle cerebral artery occlusion. Independent of the blood pressure, the cortical infarct volume was larger in OVX(+) than in OVX(-) rats. It was smaller in olmesartan-pretreated OVX(+) rats. The expression of ERα in the peri-infarct region was correlated with the reduction of cortical infarct but not that of ERβ or G protein-coupled estrogen receptor. Olmesartan prevented ERα downregulation in the cortical peri-infarct area, without affecting ERβ or G protein-coupled estrogen receptor. Olmesartan also increased mRNA expression of angiotensin-converting enzyme 2, Bcl-2, and Bcl-xL and reduced angiotensin II and cleaved caspase 3. These effects were augmented by olmesartan and abolished by the ER inhibitor. In OVX(+) rats treated with the ERα agonist alone, the infarct size was decreased, and the neuroprotective genes were upregulated. These findings suggest that the transactivation of neuroprotective genes and the reduction in brain angiotensin II are ERα dependent and that this may augment neuroprotection together with an angiotensin II type 1 receptor blockade by olmesartan. We present the new insight that the activation of ERα independent of estrogen contributes at least partly to limiting cerebral ischemic damage.     

Neuroprotection and estrogen receptors

This review is intended to assess the state of current knowledge on the role of estrogen receptors (ERs) in the neuroprotective effects of estrogens in models for acute neuronal injury and death. We evaluate the overall evidence that estrogens are neuroprotective in acute injury and critically assess the role of ERα, ERβ, GPR 30, and nonreceptor-mediated mechanisms in these robust neuroprotective effects of this ovarian steroid hormone. We conclude that all three receptors, as well as nonreceptor-mediated mechanisms can be involved in neuroprotection, depending on the model used, the level of estrogen administrated, and the mode of administration of the steroid. Also, the signaling pathways used by both ER-dependent and ER-independent mechanisms to exert neuroprotection are considered. Finally, further studies that are needed to parse out the relative contribution of receptor versus nonreceptor-mediated signaling are discussed.