Differential expression of oestrogen receptors in human secondary lymphoid tissues.

Many autoimmune diseases including rheumatoid arthritis (RA), Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE) occur much more frequently in women than in men. There is much evidence that oestrogen is the major cause of this gender difference. Interestingly, oestrogen relieves the symptoms of RA and SS but it exacerbates SLE. This contradictory effect of oestrogen on autoimmune diseases is not well understood. Most of the effects of oestrogen are mediated by two receptors: oestrogen receptor alpha and beta (ERalpha and ERbeta). To determine whether these contradictory effects of oestrogen relate to the involvement of distinct effects of the two ERs, we investigated expression of ERalpha and ERbeta in human secondary lymphoid tissues. We observed that, in tonsils, ERbeta is expressed in lymphocytes of germinal centres (GC) and the follicular mantle zone as well as in granulocytes, while ERalpha is expressed only in activated germinal centres but not in the follicular zone. ERbeta is the predominant ER in human leucocytes from peripheral blood, spleen and in leucocytes infiltrating cancers in both males and females. In addition, in different human lymphoma cell lines including Hodgkin lymphoma, Burkitt lymphoma, and multiple myeloma, ERbeta is abundant while ERalpha is not detectable. Our results indicate that ERbeta is the predominant type of ER in mature lymphocytes. We suggest that ERalpha and ERbeta have distinct roles in secondary lymphoid tissues and that further studies with ERbeta-specific agonists will help to elucidate the role of ERbeta in these tissues.

Estrogen receptor beta in health and disease.

Estrogens, acting through its two receptors, ESR1 (hereafter designated ER alpha) and ESR2 (hereafter designated ER beta), have diverse physiological effects in the reproductive system, bone, cardiovascular system, hematopoiesis, and central and peripheral nervous systems. Mice with inactivated ER alpha, ER beta, or both show a number of interesting phenotypes, including incompletely differentiated epithelium in tissues under steroidal control (prostate, ovary, mammary, and salivary glands) and defective ovulation reminiscent of polycystic ovarian syndrome in humans (in ER beta-/- mice), and obesity, insulin resistance, and complete infertility (both in male and female ER alpha-/- mice). Estrogen agonists and antagonists are frequently prescribed drugs with indications that include postmenopausal syndrome (agonists) and breast cancer (antagonists). Because the two estrogen receptors (ERs) have different physiological functions and have ligand binding pockets that differ enough to be selective in their ligand binding, opportunities now exist for development of novel ER subtype-specific selective-ER modulators.

Transforming growth factor-beta1 downregulates beating frequency and remodeling of cultured rat adult cardiomyocytes.

We have observed increased levels of transforming growth factor-beta1 (TGF-beta1) in human hibernating myocardium (HM). Impaired ventricular function in HM is known to be restored to normal following revascularization implying that myocardial structure in HM is to a certain degree preserved. We have therefore tested whether TGF-beta1 can imitate features of HM by reducing the number and frequency of beating cells (chronotropism) and structural remodeling of cultured adult rat cardiomyocytes (ARC), thus saving substrate, energy, and oxygen. Parameters measured were cell size, protein synthesis, protein degradation, protein content, myofibrillogenesis, and chronotropism. ARC were stimulated for 6 days with sera from patients with coronary heart disease, as this period led to a maximum response of cells. An increase of 90% in cell surface area following such treatment was reduced to a 20% increase of the original size by TGF-beta1. Concomitantly, the rate of protein synthesis dropped from 3.6-fold to 2.4-fold, and myofibrillogenesis was reduced. TGF-beta1 downregulated both the number of contracting cells from 81% to 10% and the frequency from 52 to nine beats per minute. However, TGF-beta1 treatment did not reduce the augmentation of protein content (1.28-fold versus 1.25-fold) indicating that protein degradation was also inhibited. Similar results were obtained with serum from healthy volunteers. The effects of TGF-beta1 were reversible. We conclude that TGF-beta1 constrains protein turnover and beating activity in underperfused myocardium, thus mediating protection by adapting myocytes to shortages in blood supply.

Aromatase-deficient mice spontaneously develop a lymphoproliferative autoimmune disease resembling Sjogren's syndrome.

Sjögren's syndrome (SS) is an incurable, autoimmune exocrinopathy that predominantly affects females and whose pathogenesis remains unknown. Like rheumatoid arthritis, its severity increases after menopause, and estrogen deficiency has been implicated. We have reported that estrogen receptor-alpha and -beta-knockout mice develop autoimmune nephritis and myeloid leukemia, respectively, but neither develops SS. One model of estrogen deficiency in rodents is the aromatase-knockout (ArKO) mouse. In these animals, there is elevated B lymphopoiesis in bone marrow. We now report that ArKO mice develop severe autoimmune exocrinopathy resembling SS. By 1 year of age, there is B cell hyperplasia in the bone marrow, spleen, and blood of ArKO mice and spontaneous autoimmune manifestations such as proteinuria and severe leukocyte infiltration in the salivary glands and kidney. Also, as is typically found in human SS, there were proteolytic fragments of alpha-fodrin in the salivary glands and anti-alpha-fodrin antibodies in the serum of both female and male ArKO mice. When mice were raised on a phytoestrogen-free diet, there was a mild but significant incidence of infiltration of B lymphocytes in WT mice and severe destructive autoimmune lesions in ArKO mice. In age-matched WT mice fed a diet containing normal levels of phytoestrogen, there were no autoimmune lesions. These results reveal that estrogen deficiency results in a lymphoproliferative autoimmune disease resembling SS and suggest that estrogen might have clinical value in the prevention or treatment of this disease.

Estrogen receptor beta regulates epithelial cellular differentiation in the mouse ventral prostate.

We have previously reported epithelial cellular hyperplasia in ventral prostates (VP) of mice lacking estrogen receptor beta (ER beta). To investigate the causes of this phenomenon, we measured cellular proliferation and apoptosis in VP of ER beta(-/-) and WT mice. With BrdUrd labeling, the number of proliferating cells was 3.6-fold higher in ER beta(-/-) mice. There was also a decrease in apoptosis as measured by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay and an increase in expression of the anti-apoptotic bcl-2. The state of differentiation of the epithelial cells of the VP was studied by immunohistochemical staining, Western blotting, and fluorescence-activated cell sorting (FACS). In ER beta(-/-) mouse VP, the number of p63-positive cells (basal phenotype) was 2.6-fold higher, and expression level of cytokeratin (CK) 8, a luminal cell marker, was lower. FACS analysis with p63 showed that in WT mice the ratio of basal to intermediate/luminal cell populations expressing p63 was 1:2.5, whereas in ER beta(-/-) mice it was 1:9. The expression of basal/intermediate marker CK 19 in three FACS areas, g1, g2, and g3, gated according to cellular size and granularity, was 1:0.6:2 in WT and 1:4:6.7 in ER beta(-/-) mice, showing a shift of CK 19-positive cells toward a cell population of intermediate size and granularity. We conclude that, in ER beta(-/-) mouse VP, there is increased epithelial proliferation, decreased apoptosis, and accumulation of incompletely differentiated cells in an intermediate pool. The continued proliferation of intermediate cells leads to the prostatic epithelial hyperplasia observed in the absence of ER beta signaling.

Autoimmune glomerulonephritis with spontaneous formation of splenic germinal centers in mice lacking the estrogen receptor alpha gene.

In mice, ovariectomy accelerates the progression of the end-stage renal disease glomerulosclerosis. In women, the incidence of this disease increases after menopause, and estrogen alters its progression. Polymorphisms in the human estrogen receptor alpha (ERalpha) gene have been suggested to constitute a genetic predisposition for lupus nephritis. Here we show that by 1 year of age, mice lacking ERalpha (ERalpha(-/-)) but not those lacking ERbeta (ERbeta(-/-)) exhibit immune complex-type glomerulonephritis, proteinuria, and destruction of tubular cells with severe infiltration of B lymphocytes in the kidney and the presence of anti-DNA antibodies in serum. No gender difference occurred in the incidence or severity of these symptoms. However, in female but not in male ERalpha(-/-) mice there were elevated serum levels of IgG3. Other prominent features of these mice were (i) spontaneous formation of germinal centers in the spleen in the absence of antigen challenge and (ii) infiltration of plasma cells in the kidney and plasmacytosis in the spleen. Immunohistochemistry indicated a selective expression of ERalpha protein in the germinal centers but not in the follicular mantle zone of murine spleens and human tonsils. Our results indicate that ERalpha has indispensable functions in the kidney and in germinal centers, and that defective ERalpha signaling results in glomerulonephritis.

Disruption of the estrogen receptor beta gene in mice causes myeloproliferative disease resembling chronic myeloid leukemia with lymphoid blast crisis.

Proliferation of pluripotent, bone marrow stem cells, which develop to lymphoid and myeloid progenitors, is negatively regulated by estrogen. Although in estrogen deficiency and in estrogen receptor knockout mice there is significant alteration in bone marrow hematopoiesis, the effects of aging on estrogen receptor deficiencies in mice have not been investigated yet. In this study we show that by 1.5 years of age, estrogen receptor beta knockout (ERbeta-/-) mice develop pronounced splenomegaly that is much more severe in females than in males. Further characterization of these mice revealed myelogenous hyperplasia in bone marrow, an increase in the number of granulocytes and B lymphocytes in blood, lymphadenopathy, and infiltration of leukocytes in the liver and lung. Analysis by flow cytometry of the bone marrow cells revealed that the percentage and total number of Gr-1hi/Mac-1hi-positive granulocytes were increased by 15-30% and 100%, respectively. The numbers of B cells in the bone marrow and spleen were significantly higher in ERbeta-/- mice than in WT littermates. Some of the ERbeta-/- mice also had a severe lymphoproliferative phenotype. Thus the absence of ERbeta results in a myeloproliferative disease resembling human chronic myeloid leukemia with lymphoid blast crisis. Our results indicate a previously unknown role for ERbeta in regulating the differentiation of pluripotent hematopoietic progenitor cells and suggest that the ERbeta-/- mouse is a potential model for myeloid and lymphoid leukemia. Furthermore, we suggest that ERbeta agonists might have clinical value in the treatment of leukemia.