The role of total and cartilage-specific estrogen receptor alpha expression for the ameliorating effect of estrogen treatment on arthritis

Introduction

Estrogen (E2) delays onset and decreases severity of experimental arthritis. The aim of this study was to investigate the importance of total estrogen receptor alpha (ERα) expression and cartilage-specific ERα expression in genetically modified mice for the ameliorating effect of estrogen treatment in experimental arthritis.

Methods

Mice with total (total ERα^-/-) or cartilage-specific (Col2α1-ERα^-/-) inactivation of ERα and wild-type (WT) littermates were ovariectomized, treated with E2 or placebo, and induced with antigen-induced arthritis (AIA). At termination, knees were collected for histology, synovial and splenic cells were investigated by using flow cytometry, and splenic cells were subjected to a T-cell proliferation assay.

Results

E2 decreased synovitis and joint destruction in WT mice. Amelioration of arthritis was associated with decreased frequencies of inflammatory cells in synovial tissue and decreased splenic T-cell proliferation. E2 did not affect synovitis or joint destruction in total ERα^-/- mice. In Col2α1-ERα^-/- mice, E2 protected against joint destruction to a similar extent as in WT mice. In contrast, E2 did not significantly ameliorate synovitis in Col2α1-ERα^-/- mice.

Conclusions

Treatment with E2 ameliorates both synovitis and joint destruction in ovariectomized mice with AIA via ERα. This decreased severity in arthritis is associated with decreased synovial inflammatory cell frequencies and reduced splenic T-cell proliferation. ERα expression in cartilage is not required for estrogenic amelioration of joint destruction. However, our data indicate that ERα expression in cartilage is involved in estrogenic effects on synovitis, suggesting different mechanisms for the amelioration of joint destruction and synovitis by E2.     

Mapping ERβ Genomic Binding Sites Reveals Unique Genomic Features and Identifies EBF1 as an ERβ Interactor

Considerable effort by numerous laboratories has resulted in an improved understanding of estrogen and SERM action mediated by the two estrogen receptors, ERα and ERβ. However, many of the targets for ERβ in cell physiology remain elusive. Here, the C4-12/Flag.ERβ cell line which stably expressed Flag.ERβ is used to study ERβ genomic functions without ERα interference. Mapping ERβ binding sites in these cells reveals ERβ unique distribution and motif enrichment patterns. Accompanying our mapping results, nascent RNA profiling is performed on cells at the same treatment time. The combined results allow the identification of ERβ target genes. Gene ontology analysis reveals that ERβ targets are enriched in differentiation, development and apoptosis. Concurrently, E2 treatment suppresses proliferation in these cells. Within ERβ binding sites, while the most prevalent binding motif is the canonical ERE, motifs of known ER interactors are also enriched in ERβ binding sites. Moreover, among enriched binding motifs are those of GFI, REST and EBF1, which are unique to ERβ binding sites in these cells. Further characterization confirms the association between EBF1 and the estrogen receptors, which favors the N-terminal region of the receptor. Furthermore, EBF1 negatively regulates ERs at the protein level. In summary, by studying ERβ genomic functions in our cell model, we confirm the anti-proliferative role of ERβ and discover the novel cross talk of ERβ with EBF1 which has various implications in normal physiology.     

Estrogen Receptor β-Selective Agonists Stimulate Calcium Oscillations in Human and Mouse Embryonic Stem Cell-Derived Neurons

Estrogens are used extensively to treat hot flashes in menopausal women. Some of the beneficial effects of estrogens in hormone therapy on the brain might be due to nongenomic effects in neurons such as the rapid stimulation of calcium oscillations. Most studies have examined the nongenomic effects of estrogen receptors (ER) in primary neurons or brain slices from the rodent brain. However, these cells can not be maintained continuously in culture because neurons are post-mitotic. Neurons derived from embryonic stem cells could be a potential continuous, cell-based model to study nongenomic actions of estrogens in neurons if they are responsive to estrogens after differentiation. In this study ER-subtype specific estrogens were used to examine the role of ERα and ERβ on calcium oscillations in neurons derived from human (hES) and mouse embryonic stem cells. Unlike the undifferentiated hES cells the differentiated cells expressed neuronal markers, ERβ, but not ERα. The non-selective ER agonist 17β-estradiol (E₂) rapidly increased [Ca2+]i oscillations and synchronizations within a few minutes. No change in calcium oscillations was observed with the selective ERα agonist 4,4′,4″-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (PPT). In contrast, the selective ERβ agonists, 2,3-bis(4-Hydroxyphenyl)-propionitrile (DPN), MF101, and 2-(3-fluoro-4-hydroxyphenyl)-7-vinyl-1,3 benzoxazol-5-ol (ERB-041; WAY-202041) stimulated calcium oscillations similar to E₂. The ERβ agonists also increased calcium oscillations and phosphorylated PKC, AKT and ERK1/2 in neurons derived from mouse ES cells, which was inhibited by nifedipine demonstrating that ERβ activates L-type voltage gated calcium channels to regulate neuronal activity. Our results demonstrate that ERβ signaling regulates nongenomic pathways in neurons derived from ES cells, and suggest that these cells might be useful to study the nongenomic mechanisms of estrogenic compounds.     

Classical Estrogen Receptors and ERα Splice Variants in the Mouse

Estrogens exert a variety of effects in both reproductive and non-reproductive tissues. With the discovery of ERα splice variants, prior assumptions concerning tissue-specific estrogen signaling need to be re-evaluated. Accordingly, we sought to determine the expression of the classical estrogen receptors and ERα splice variants across reproductive and non-reproductive tissues of male and female mice. Western blotting revealed that the full-length ERα66 was mainly present in female reproductive tissues but was also found in non-reproductive tissues at lower levels. ERα46 was most highly expressed in the heart of both sexes. ERα36 was highly expressed in the kidneys and liver of female mice but not in the kidneys of males. ERβ was most abundant in non-reproductive tissues and in the ovaries. Because the kidney has been reported to be the most estrogenic non-reproductive organ, we sought to elucidate ER renal expression and localization. Immunofluorescence studies revealed ERα66 in the vasculature and the glomerulus. It was also found in the brush border of the proximal tubule and in the cortical collecting duct of female mice. ERα36 was evident in mesangial cells and tubular epithelial cells of both sexes, as well as podocytes of females but not males. ERβ was found primarily in the podocytes in female mice but was also present in the mesangial cells in both sexes. Within the renal cortex, ERα46 and ERα36 were mainly located in the membrane fraction although they were also present in the cytosolic fraction. Given the variability of expression patterns demonstrated herein, identification of the specific estrogen receptors expressed in a tissue is necessary for interpreting estrogenic effects. As this study revealed expression of the ERα splice variants at multiple sites within the kidney, further studies are warranted in order to elucidate the contribution of these receptors to renal estrogen responsiveness.