Roles of estrogen receptor α and β in the regulation of proliferation in endometrial carcinoma

Endometrial carcinoma (EC), an estrogen-dependent gynecological malignancy, is prevalent worldwide. Estrogen receptor α (ERα) and estrogen receptor β (ERβ) are two main estrogen receptor isoforms, which mediate estrogen-induced proliferation in EC. However, the dynamic changes of ERα and ERβ subtype expression and their functions on proliferation in EC remain elusive. In this study, we aimed to investigate the expression of ERα and ERβ in para-tumor eutopic endometrium, endometrial atypical hyperplasia and EC by immunohistochemistry and then analyse their clinical significance. Subsequently, Ishikawa cells with ERα or ERβ knockdown by lentivirus transfection were used to explore the relationship between ERα/ERβ and cell proliferation, and preliminarily evaluate whether metformin’s inhibitory effect on estrogen-induced cell proliferation was mediated by ERα and ERβ. We found that the expression of ERα and ERβ were markedly changed in endometrial hyperplasia and EC compared with that in para-tumor eutopic endometrium and exhibited different expression trends. Through further analysis, we discovered that ERα presented higher expression in endometrial atypical hyperplasia and early stage of EC than that in para-tumor eutopic endometrium, while the expression of ERβ gradually decreased from para-tumor eutopic endometrium to EC. Additionally, the cell cycle-related protein, CyclinD1 was gradually increased but p21 decreased. Furthermore, knockdown of ERα and ERβ severally in Ishikawa cells either inhibited or promoted estrogen-induced cell proliferation through regulating CyclinD1 and p21 expression. Meanwhile, the inhibitory effect of metformin on estrogen-induced cell proliferation was respectively blunted or partly reversed by knockdown of ERα or ERβ. Altogether, ERα and ERβ have different expression patterns in the progression of EC either facilitating or suppressing cell proliferation through regulating the expression of CyclinD1 and p21 in EC cells, and may also mediate the inhibitory effect of metformin on estrogen-induced EC cells proliferation.     

The role of T-cell receptor α and β genes in MHC-restricted antigen recognition

T cells use α- and β-chain genes, which are organized and diversified by somatic DNA rearrangements much like the immunoglobulin genes, to encode clonally distributed receptor molecules which confer specificity for MHC and antigen. Here Zlatko Dembic and his colleagues summarize recent mutagenesis and gene transfection experiments indicating that α- and β-chain genes are necessary and sufficient for T-cell specificity.     

Cell-specific expression and immunolocalization of nitric oxide synthase isoforms and soluble guanylyl cyclase α and β subunits in postnatal porcine uteri

The aim of the present study was to investigate the cellular expression and immunolocalization of nitric oxide synthase (NOS) isoforms and soluble guanylyl cyclase (sGC) subunits in postnatal porcine uteri. Immunohistochemical experiments showed that three isoforms of NOS were mainly localized in the uterine luminal and glandular epithelium and myometrium, and the intensity of immunostaining for iNOS and eNOS was increased gradually with temporal development of the postnatal uterus. In addition, sGC subunits, sGCα1 and β, were present in the uterine luminal and glandular epithelium, myometrium and stromal cells. The uterine NOS activity data showed that the total NOS and iNOS activities were significantly increased at postnatal days 21 and 35. Although constitutive NOS activity was increased at postnatal day 21, it decreased subsequently at postnatal day 35. Immunoblot analysis revealed that iNOS protein expression was significantly increased at postnatal days 21 and 35. Furthermore, sGCα1 protein expression was not significantly changed throughout days 7 to 35. Collectively, our findings suggest that NO/cGMP signaling is involved in the process of postnatal porcine uterine development.     

Immunolocalization of β-catenin and Lef-1 during postnatal hair follicle development in mice

It is well recognized that the Wnt pathway, in which β-catenin and Lef-1 are important factors, is associated with many physiological processes, including embryogenesis and postnatal development. The Wnt pathway also plays a critical role in the development of skin. It regulates the formation of the dorsal dermis and epidermal appendages in the skin and the activity of epithelial stem cells. In this study, we investigated the presence and localization of β-catenin and Lef-1 in murine hair follicles through the first postnatal month, which encompasses the first hair cycle in mice, using Western blotting and immunohistochemistry. Our results show that β-catenin and Lef-1 are expressed during all stages in a hair cycle, most strongly in the anagen and weakly in the catagen and telogen phases. The results also suggest that the β-catenin-Lef-1 complex may regulate hair follicle cycling. This process will be of considerable interest to future studies.     

Expression of estrogen receptors α and β in the trigeminal mesencephalic nucleus of adult women and men

Temporomandibular disorders are more prevalent in women than in men and phases of pain relate to the estrous cycle. Several studies described the location of estrogen receptors (ER) in the temporomandibular joint (TMJ), the masseteric muscles and cartilage, but it was unknown whether they are also expressed within the pseudounipolar neurons of the trigeminal mesencephalic nucleus, which receives direct sensory inputs from these structures. Therefore, we studied expression of ERα and ERβ protein in the trigeminal mesencephalic nucleus of ten human brains (five female/five male). Both receptors were uniformly expressed on neurons, but not other cell types within the target structure. Thus, sensory inputs from the TMJ and adjacent structures are likely to be modulated by estrogen at the level of the first sensory neuron which may underlie the well-known correlation of pain incidence and phases of the estrous cycle.     

Peroxisome proliferator-activated receptor β activation promotes myonuclear accretion in skeletal muscle of adult and aged mice

We reported recently that peroxisome proliferator-activated receptor β (PPARβ) activation promotes a calcineurin-dependent exercise-like remodelling characterised by increased numbers of oxidative fibres and capillaries. As physical exercise also induces myonuclear accretion, we investigated whether PPARβ activation alters myonuclear density. Transgenic muscle-specific PPARβ over-expression induced 14% increase of myonuclear density. Pharmacological PPARβ activation promoted rapid and massive myonuclear accretion (20% increase after 48 h), which is dependent upon calcineurin/nuclear factor of activated T cells signalling pathway. In vivo bromodeoxyuridine labelling and proliferating cell nuclear antigen immunodetection revealed that PPARβ activation did not promote cell proliferation, suggesting that the PPARβ-promoted myonuclear accretion involves fusion of pre-existing muscle precursor cells to myofibres rather than cell division. Finally, we showed that in skeletal muscle, ageing led to a down-regulation of PPARβ accompanied by decrease of both oxidative fibre number and myonuclear density. PPARβ pharmacological activation counteracts, at least in part, the ageing-driven muscle remodelling.     

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit of the GABAA receptor

A line of mice was recently created in which the gabrb3 gene, which encodes the β₃ subunit of the GABA_A receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA_A receptors in the CNS enabled an investigation of the role of GABA and GABA_A receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA_A and GABA_B receptor agonists. Homozygous null (β₃^−/−) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (β₃^+/+) and heterozygous (β₃^+/−) littermates. The β₃^−/− mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their β₃^+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in β₃^−/− mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA_A receptors in the spinal cord. As expected, subcutaneous administration of the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in β₃^−/− mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, β₃^+/+ and β₃^+/− mice. However, the antinociceptive effect of the GABA_B receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in β₃^−/− mice compared to the progenitor strains, β₃^+/+ or β₃^+/− mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA_A receptors can affect the production of antinociception by other analgesic drugs as well.     

Regulation of cannabinoid receptor gene expression and endocannabinoid levels in lymphocyte subsets by interferon-β: a longitudinal study in multiple sclerosis patients

Evidence suggests the involvement of the cannabinoid system in the pathogenesis of multiple sclerosis (MS). We studied cannabinoid receptor (CB)1 and CB2 receptor gene expression in B, natural killer (NK) and T cells from MS patients before and after 1 year of interferon beta therapy, and compared these levels to those of healthy controls. We also measured the production of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) and the gene expression of the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) in these cells. Prior to interferon therapy, MS patients showed significantly elevated CB₂ expression in B cells, but not in T or NK cells. These levels decreased gradually within 6 months to 1 year of interferon treatment. CB₁ expression was elevated in all cell subsets, but only reached statistical significance in T cells; all levels decreased progressively over time. Before treatment, AEA but not 2-AG levels were significantly elevated in the three cell populations; after 1 year of treatment, all values decreased to control levels. The expression of FAAH was unchanged. The different expression of cannabinoid receptor genes and the increased level of AEA in lymphocytes point to a possible role of the cannabinoid system in MS immune response and its modulation by interferon.     

Expression of oestrogen receptor α in the endometrium of porcine reproductive and respiratory syndrome virus-infected gilts

Porcine reproductive and respiratory syndrome (PRRS) virus infection often lead to infertility in gilts and sows. Nevertheless, the impact of PRRS virus on the endometrial function has not been fully elucidated. The present study aimed to determine the effect of PRRS virus infection on the expression of oestrogen receptor (ER) α in the endometrium of gilts. Uterine tissues from 54 gilts were classified into two groups according to PRRS virus detection using immunohistochemistry (26 positive and 28 negative samples). The reproductive status was classified as prepubertal, luteal and follicular phases. The expression of ERα in the epithelium, subepithelium and glandular tissue layers of the endometrium was determined by immunohistochemistry. ERα immunostaining was detected in 22.2, 13.5 and 33.6 % of the cells in the epithelial, subepithelial and glandular tissue layers, respectively. The ERα immunostaining in the glandular layer of the endometrium in follicular-phase gilts was higher than that in prepubertal gilts (46.1 and 15.2 %, respectively, P?=?0.011). The ERα immunostaining in the glandular layer of the endometrium was positively correlated with body weight (r?=?0.138, P?=?0.020) and average daily gain (r?=?0.169, P?=?0.005) of the gilts. The ERα immunostaining in all tissue layers of the endometrium with PRRS virus detection did not differ significantly compared to that without PRRS virus (P?>?0.05). However, in prepubertal gilts, the ERα immunostaining cells in the epithelial layer of PRRS virus negative (9.5 %) tended to be lower than PRRS virus positive (26.3 %, P?=?0.196) uterine tissues. This tendency indicates some impact of PRRS virus infection on the ERα expression in prepubertal gilts.     

Immunolocalisation of 11β-HSD-1 and -2, glucocorticoid receptor, mineralocorticoid receptor and Na+ K+-ATPase during the postnatal development of the rat epididymis

Glucocorticoids have been implicated in male reproductive function and 11β-HSD-1 and -2, the glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), all of which are known to modulate glucocorticoid action, have been localised in the adult rat epididymis, but their developmental expression has not been investigated. Na(+)K(+)-ATPase activity, responsible for sodium transport, is induced by both mineralocorticoids and glucocorticoids in the kidney and colon, and has been localised in epididymal epithelium. This study examined the immunolocalisation of 11β-HSD-1 and -2, GR, MR and Na(+)K(+)-ATPase in rat epididymal epithelium (n = 5) at postnatal days (pnd) 1, 7, 15, 28, 40, 60, 75 and 104, and relative mRNA expression of 11β-HSD-1 and -2, and GR at pre-puberty (pnd 28) and post-puberty (pnd 75). 11β-HSD-1, GR and MR were localised in the epididymal epithelium from pnd 1, and 11β-HSD-2 and Na(+)K(+)-ATPase reactivity from pnd 15. At pnd 28 there was maximal immunoreactivity for both the GR and MR and 11β-HSD-1 and -2. 11β-HSD-1 mRNA expression in the caput increased from pre- to post-puberty, whereas 11β-HSD-2 mRNA expression fell over the same period (P < 0.01). GR mRNA expression was similar at pre- and post-puberty in both caput and cauda. Developmental changes in expression of 11β-HSD-1 and -2 suggest that overall exposure of the epididymis to glucocorticoids increases post-puberty, but cell-specific expression of the 11β-HSD enzymes still provides a capacity for intricate local control of glucocorticoid exposure.     

Neuronal nicotinic acetylcholine receptors expressed in Xenopus oocytes: role of the α subunit in agonist sensitivity and desensitization

Neuronal nicotinic acetylcholine receptors (nAChRs) were expressed in Xenopus laevis oocytes after nuclear injection of complementary deoxyribonucleic acid (cDNA) expression vectors. The two receptor subtypes 4/n1 and 3/n1 were readily distinguishable from one another by ACh sensitivity and desensitization. 3/n1 receptors showed lower ACh sensitivity and stronger desensitization than 4/n1 receptors. Furthermore, although the current/voltage relationship was very similar in both receptor subtypes, the voltage dependence of desensitization was found to be strikingly different. As the n1 subunit was unchanged, the subunits must be responsible for these functional differences. Symmetric hybrid cDNAs, 43 and 34, were constructed and functional receptors were obtained by co-injection with n1. These hybrid receptors displayed an ACh sensitivity that was mainly defined by the extracellular sequence of the subunit. In contrast, no part of the subunit was found fully to determine desensitization.     

Impact of estrogen receptor α on the tamoxifen response and prognosis in luminal-A-like and luminal-B-like breast cancer

Clinical and Experimental Medicine - The luminal-A-like and luminal-B-like breast cancer groups have distinct biological features that lead to differences in the treatment response and clinical...     

Potential of β-galactosidase-expressing Toxoplasma gondii for in situ localization and observation of rare stages of the parasite life cycle

A cyst-forming strain of Toxoplasma gondii was transfected with the Escherichia coli LacZ gene and expressed beta-galactosidase constitutively. This strain has been used to localize and analyze the early stages of development and reactivation of T. gondii in mice. The chromogenic detection of the enzyme allows an easy detection of the parasites after light fixation and therefore allows a submacroscopic analysis of tissue distribution within the organism. Also, it allows further embedding and retrieval of rare stages for electron microscopic observation. that detect the presence of the parasite and initiate the response, and (2) the early stages of reactivation, when the cysts are supposed to break open and release the infectious bradyzo?tes. We have taken advantage of the possibility of detecting the enzymatic activity of beta-galactosidase (beta-gal) in transfected parasites to show that one could perform a semi-macroscopic detection and that this was compatible with further analysis by histological or electron microscopic techniques, being therefore able to detect the rare events and then to analyze them further with more refined morphological techniques.     

Effects of tumour necrosis factor α and interleukin 1 β on the proliferation of cultured glomerular epithelial cells

Rat glomerular epithelial cells were cultured with human monocyte supernatant or with recombinant cytokines. A primary glomerular culture and a glomerular epithelial cell culture were made; supernatant from monocyte cultures derived from healthy humans, and recombinant tumour necrosis factor (TNF ) or recombinant interleukin 1 (IL-1 ) were added. Cell proliferation rates were assayed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. In serum-free media, consistent proliferation of glomerular epithelial cells (GEC) was observed throughout the 3 week culture period. Significant growth-stimulatory effects were induced by lipopolysaccharide-treated monocyte conditioned medium and by 1–50 ng/ml of TNF , growth being up to 400% more than in the control culture. The effect of TNF depended mainly on its interaction with epidermal growth factor (EGF). In contrast to TNF , IL-1 inhibited GEC proliferation; this was due to the early appearance and proliferation of mesangial cells, despite the culture being serum-free. This study showed that activated monocytes secrete growth factors for GEC in vitro, and that interaction between both TNF and IL-1 and between TNF and EGF can modulate GEC proliferation. These findings suggest that, under pathological conditions, monocytes or macrophages affect GEC proliferation, probably being involved in crescent formation.     

Postsynaptic and extrasynaptic localization of κ-opioid receptor in selected brain areas of young rat and chick using an anti-receptor monoclonal antibody

Summary -opioid receptors were visualized by light and electron microscopical immunohistochemistry in young rat and chick brains, using a monoclonal antibody KA8 (IgG1, ) raised against a -opioid receptor preparation from frog brain, which recognizes selectively the -type receptor with preference for the -2 subtype. The most pronounced -opioid receptor-like immunoreactivity was observed in the hypothalamic nuclei of the rat brain and in the chick optic tectum, in regions where the functional significance of -opioid receptors is well documented. Both neurons and glia were stained, the former on both somata and dendrites. At the ultrastructural level, the receptor-like immunoreactivity was similar in both species. Immunoprecipitate decorated the inner surface of the plasma membrane of glial cells, neuronal somata and dendrites, in a discontinuous arrangement. In the cytoplasm, labelling was associated with ribosomes, polyribosomes and rough endoplasrnic reticulum membranes but not with Golgi cisternae. In the neuropil, the immunoprecipitate was observed along the dendritic microtubules and was also associated with postsynaptic sites. Nuclei and axons were devoid of label and immunoreactivity was never visible presynaptically. Our findings indicate that the antibody used in the present study marks various forms of the -opioid receptor protein including those synthesised in ribosomes, transported along dendritic microtubules and incorporated into postsynaptic and non-synaptic membranes. The antibody also recognizes glial opioid receptors. The observed subcellular distribution appears to be conserved in phylogenetically distant species.     

Effects of systemic 3-nitropropionic acid-induced lesions of the dorsal striatum on cannabinoid and µ-opioid receptor binding in the basal ganglia

Systemic administration of 3-nitropropionic acid (3NPA) in experimental animals produces bilateral striatal lesions similar to those seen in Huntington’s disease (HD) caudate and putamen. [³H]-CP55,940 binding to cannabinoid receptors in human basal ganglia nuclei has been shown to be highly susceptible to the earliest pathological changes in the HD brain. In this study, to assess further the suitability of 3NPA-induced striatal lesions as a model for HD neuropathology, we examined the effects of striatal lesions induced by the systemic administration of 3NPA on the binding of [³H]-CP55,940 to pre- and postsynaptic cannabinoid receptors in striatum, globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata and also the effect of 3NPA-induced striatal lesions on the binding of [³H]-DAMGO to μ-opioid receptors in striatal striosomes. Systemic administration of 3NPA induced bilateral and symmetrical lesions in dorsolateral striatum. Within the lesion core, [³H]-CP55,940 and [³H]-DAMGO binding density was reduced to background levels. Beyond the immediate borders of the central core of the 3NPA-induced lesion, striatal binding density was not significantly different from that measured in unlesioned rats. [³H]-CP55,940 binding in globus pallidus, entopeduncular nucleus and substantia nigra in 3NPA-lesioned rats was significantly reduced compared to controls, and the individual decreases were similar for each site. However, these reductions were statistically marginal. These data suggest that, while producing striatal lesions which bear some similarity to those seen in HD, the consequences of 3NPA for striatopallidal and striatonigral efferent projections do not reflect the reported neurodegenerative changes seen in the HD brain. Received: 18 November 1998 / Accepted: 12 July 1999