Harnessing the reverse cholesterol transport pathway to favor differentiation of monocyte-derived APCs and antitumor responses.

Lipid and cholesterol metabolism play a crucial role in tumor cell behavior and in shaping the tumor microenvironment. In particular, enzymatic and non-enzymatic cholesterol metabolism, and derived metabolites control dendritic cell (DC) functions, ultimately impacting tumor antigen presentation within and outside the tumor mass, dampening tumor immunity and immunotherapeutic attempts. The mechanisms accounting for such events remain largely to be defined. Here we perturbed (oxy)sterol metabolism genetically and pharmacologically and analyzed the tumor lipidome landscape in relation to the tumor-infiltrating immune cells. We report that perturbing the lipidome of tumor microenvironment by the expression of sulfotransferase 2B1b crucial in cholesterol and oxysterol sulfate synthesis, favored intratumoral representation of monocyte-derived antigen-presenting cells, including monocyte-DCs. We also found that treating mice with a newly developed antagonist of the oxysterol receptors Liver X Receptors (LXRs), promoted intratumoral monocyte-DC differentiation, delayed tumor growth and synergized with anti-PD-1 immunotherapy and adoptive T cell therapy. Of note, looking at LXR/cholesterol gene signature in melanoma patients treated with anti-PD-1-based immunotherapy predicted diverse clinical outcomes. Indeed, patients whose tumors were poorly infiltrated by monocytes/macrophages expressing LXR target genes showed improved survival over the course of therapy. Thus, our data support a role for (oxy)sterol metabolism in shaping monocyte-to-DC differentiation, and in tumor antigen presentation critical for responsiveness to immunotherapy. The identification of a new LXR antagonist opens new treatment avenues for cancer patients.

The estrogen receptor α-selective agonist propyl pyrazole triol improves glucose tolerance in ob/ob mice: potential molecular mechanisms

The authors and journal apologise for an error in the above paper, which appeared in volume 199 part 2, pages 275­286. The error relates to Fig. 10, given on page 283.

ERα expression in T lymphocytes is dispensable for estrogenic effects in bone.

Estrogen treatment has positive effects on the skeleton, and we have shown that estrogen receptor alpha (ERα) expression in cells of hematopoietic origin contributes to a normal estrogen treatment response in bone tissue. T lymphocytes are implicated in the estrogenic regulation of bone mass, but it is not known whether T lymphocytes are direct estrogen target cells. Therefore, the aim of this study was to determine the importance of ERα expression in T lymphocytes for the estrogenic regulation of the skeleton using female mice lacking ERα expression specifically in T lymphocytes (Lck-ERα-/-) and ERαflox/flox littermate (control) mice. Deletion of ERα expression in T lymphocytes did not affect bone mineral density (BMD) in sham-operated Lck-ERα-/- compared to control mice, and ovariectomy (ovx) resulted in a similar decrease in BMD in control and Lck-ERα-/- mice compared to sham-operated mice. Furthermore, estrogen treatment of ovx Lck-ERα-/- led to an increased BMD that was indistinguishable from the increase seen after estrogen treatment of ovx control mice. Detailed analysis of both the appendicular (femur) and axial (vertebrae) skeleton showed that both trabecular and cortical bone parameters responded to a similar extent regardless of the presence of ERα in T lymphocytes. In conclusion, ERα expression in T lymphocytes is dispensable for normal estrogenic regulation of bone mass in female mice.

Estrogen receptor ß controls proliferation of enteric glia and differentiation of neurons in the myenteric plexus after damage.

Injury to the enteric nervous system (ENS) can cause several gastrointestinal (GI) disorders including achalasia, irritable bowel syndrome, and gastroparesis. Recently, a subpopulation of enteric glial cells with neuronal stem/progenitor properties (ENSCs) has been identified in the adult ENS. ENSCs have the ability of reconstituting the enteric neuronal pool after damage of the myenteric plexus. Since the estrogen receptor ß (ERß) is expressed in enteric glial cells and neurons, we investigated whether a selective ERß agonist, LY3201, can influence neuronal and glial cell differentiation. Myenteric ganglia from the murine muscularis externa were isolated and cultured in either glial cell medium or neuronal medium. In glial cell medium, the number of glial progenitor cells (Sox10+) was increased by fourfold in the presence of LY3201. In the neuronal medium supplemented with an antimitotic agent to block glial cell proliferation, LY3201 elicited a 2.7-fold increase in the number of neurons (neurofilament+ or HuC/D+). In addition, the effect of LY3201 was evaluated in vivo in two murine models of enteric neuronal damage and loss, namely, high-fat diet and topical application of the cationic detergent benzalkonium chloride (BAC) on the intestinal serosa, respectively. In both models, treatment with LY3201 significantly increased the recovery of neurons after damage. Thus, LY3201 was able to stimulate glial-to-neuron cell differentiation in vitro and promoted neurogenesis in the damaged myenteric plexus in vivo. Overall, our study suggests that selective ERß agonists may represent a therapeutic tool to treat patients suffering from GI disorders, caused by excessive neuronal/glial cell damage.

The role of membrane ERα signaling in bone and other major estrogen responsive tissues.

Estrogen receptor α (ERα) signaling leads to cellular responses in several tissues and in addition to nuclear ERα-mediated effects, membrane ERα (mERα) signaling may be of importance. To elucidate the significance, in vivo, of mERα signaling in multiple estrogen-responsive tissues, we have used female mice lacking the ability to localize ERα to the membrane due to a point mutation in the palmitoylation site (C451A), so called Nuclear-Only-ER (NOER) mice. Interestingly, the role of mERα signaling for the estrogen response was highly tissue-dependent, with trabecular bone in the axial skeleton being strongly dependent (>80% reduction in estrogen response in NOER mice), cortical and trabecular bone in long bones, as well as uterus and thymus being partly dependent (40-70% reduction in estrogen response in NOER mice) and effects on liver weight and total body fat mass being essentially independent of mERα (<35% reduction in estrogen response in NOER mice). In conclusion, mERα signaling is important for the estrogenic response in female mice in a tissue-dependent manner. Increased knowledge regarding membrane initiated ERα actions may provide means to develop new selective estrogen receptor modulators with improved profiles.

Liver X receptors regulate cerebrospinal fluid production.

Of the two isoforms of Liver X receptor (LXR), LXRß has been shown to have major effects in the central nervous system (CNS) and on the regulation of aquaporins while LXRα has its most marked effects on cholesterol homeostasis. Both receptors have immunomodulatory functions. In LXRαß knockout (ko) mice, the CNS phenotype is much more severe than in the LXRß ko mice, suggesting a contribution of LXRα in CNS functions. One of the most striking abnormalities in the brains of LXRαß ko mice is the occlusion of the lateral ventricles with age. In the present study, we have found by immunohistochemical staining that both LXRα and LXRß are expressed in the cell nuclei of the epithelium of the choroid plexus and in the ependymal cells surrounding the lateral ventricles. The two receptors regulate several genes and can compensate for each other on expression of genes involved in structural integrity (E-cadherin, P-cadherin and ß-catenin) and function (aquaporin 1 and carbonic anhydrase IX). Aquaporin 4 (AQ4) is not expressed in the choroid plexus but is expressed in the astrocytic end feet and ependymal cells. AQP4 expression was increased in white matter around lateral ventricles but not in neurons of LXRαß ko mice. The data show that LXR is a regulator of cerebrospinal fluid (CSF) both at the choroid plexus and at the astrocytic end feet and defects in the synthesis of cerebrospinal fluid may be targeted by LXR agonists to facilitate CSF production, turnover and clearance in CNS diseases.

ERß decreases breast cancer cell survival by regulating the IRE1/XBP-1 pathway.

Unfolded protein response (UPR) is an adaptive reaction that allows cancer cells to survive endoplasmic reticulum (EnR) stress that is often induced in the tumor microenvironment because of inadequate vascularization. Previous studies report an association between activation of the UPR and reduced sensitivity to antiestrogens and chemotherapeutics in estrogen receptor α (ERα)-positive and triple-negative breast cancers, respectively. ERα has been shown to regulate the expression of a key mediator of the EnR stress response, the X-box-binding protein-1 (XBP-1). Although network prediction models have associated ERß with the EnR stress response, its role as regulator of the UPR has not been experimentally tested. Here, upregulation of wild-type ERß (ERß1) or treatment with ERß agonists enhanced apoptosis in breast cancer cells in the presence of pharmacological inducers of EnR stress. Targeting the BCL-2 to the EnR of the ERß1-expressing cells prevented the apoptosis induced by EnR stress but not by non-EnR stress apoptotic stimuli indicating that ERß1 promotes EnR stress-regulated apoptosis. Downregulation of inositol-requiring kinase 1α (IRE1α) and decreased splicing of XBP-1 were associated with the decreased survival of the EnR-stressed ERß1-expressing cells. ERß1 was found to repress the IRE1 pathway of the UPR by inducing degradation of IRE1α. These results suggest that the ability of ERß1 to target the UPR may offer alternative treatment strategies for breast cancer.

Estrogen receptor ß and Liver X receptor ß: biology and therapeutic potential in CNS diseases.

In the last decade of the twentieth century, two nuclear receptors were discovered in our laboratory and, very surprisingly, were found to have key roles in the central nervous system. These receptors have provided some novel insights into the etiology and progression of neurodegenerative diseases and anxiety disorders. The two receptors are estrogen receptor beta (ERß) and liver X receptor beta (LXRß). Both ERß and LXRß have potent anti-inflammatory activities and, in addition, LXRß is involved in the genesis of dopaminergic neurons during development and protection of these neurons against neurodegeneration in adult life. ERß is involved in migration of cortical neurons and calretinin-positive GABAergic interneurons during development and maintenance of serotonergic neurons in adults. Both receptors are present in magnocellular neurons of the hypothalamic preoptic area including those expressing vasopressin and oxytocin. As both ERß and LXRß are ligand-activated transcription factors, their ligands hold great potential in the treatment of diseases of the CNS.

Liver X receptor ß is essential for the differentiation of radial glial cells to oligodendrocytes in the dorsal cortex.

Several psychiatric disorders are associated with aberrant white matter development, suggesting oligodendrocyte and myelin dysfunction in these diseases. There are indications that radial glial cells (RGCs) are involved in initiating myelination, and may contribute to the production of oligodendrocyte progenitor cells (OPCs) in the dorsal cortex. Liver X receptors (LXRs) are involved in maintaining normal myelin in the central nervous system (CNS), however, their function in oligodendrogenesis and myelination is not well understood. Here, we demonstrate that loss of LXRß function leads to abnormality in locomotor activity and exploratory behavior, signs of anxiety and hypomyelination in the corpus callosum and optic nerve, providing in vivo evidence that LXRß deletion delays both oligodendrocyte differentiation and maturation. Remarkably, along the germinal ventricular zone-subventricular zone and corpus callosum there is reduced OPC production from RGCs in LXRß(-/-) mice. Conversely, in cultured RGC an LXR agonist led to increased differentiation into OPCs. Collectively, these results suggest that LXRß, by driving RGCs to become OPCs in the dorsal cortex, is critical for white matter development and CNS myelination, and point to the involvement of LXRß in psychiatric disorders.

The oxysterol receptor LXRß protects against DSS- and TNBS-induced colitis in mice.

We examined the function of the oxysterol receptors (LXRs) in inflammatory bowel disease (IBD) through studying dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and by elucidating molecular mechanisms underlying their anti-inflammatory action. We observed that Lxr-deficient mice are more susceptible to colitis. Clinical indicators of colitis including weight loss, diarrhea and blood in feces appeared earlier and were more severe in Lxr-deficient mice and particularly LXRß protected against symptoms of colitis. Addition of an LXR agonist led to faster recovery and increased survival. In contrast, Lxr-deficient mice showed slower recovery and decreased survival. In Lxr-deficient mice, inflammatory cytokines and chemokines were increased together with increased infiltration of immune cells in the colon epithelium. Activation of LXRs strongly suppressed expression of inflammatory mediators including TNFα. While LXRα had anti-inflammatory effects in CD11b(+) immune cell populations, LXRß in addition had anti-inflammatory effects in colon epithelial cells. Lack of LXRß also induced CD4(+)/CD3(+) immune cell recruitment to the inflamed colon. Expression of both LXRA and LXRB was significantly suppressed in inflamed colon from subjects with IBD compared with non-inflamed colon. Taken together, our observations suggest that the LXRs could provide interesting targets to reduce the inflammatory responses in IBD.

Estrogen receptor ß upregulates FOXO3a and causes induction of apoptosis through PUMA in prostate cancer.

Estrogen receptor ß (ERß) is emerging as a critical factor in understanding prostate cancer biology. Although reduced in prostate cancer above Gleason grade 3, ERß is a potential drug target at the initial stage of the disease. In human prostate cancer cells, we found that ERß causes apoptosis by increasing the expression of pro-apoptotic factor p53-upregulated modulator of apoptosis (PUMA), independent of p53, but dependent on the forkhead transcription factor class-O family member, FOXO3a. FOXO3a has previously been shown to induce PUMA after growth factor withdrawal and inhibition of the Akt pathway. Surprisingly, the phosphorylation of FOXO3a remained unchanged, while the mRNA and total protein levels of FOXO3a were increased in response to ERß expression or treatment of PC3, 22Rv1 and LNCaP cells with the ERß-specific ligands 3ß-Adiol (5α-androstane-3ß,17ß-diol), DPN (diarylpropionitrile) or 8ß-VE2 (8-vinylestra-1,3,5 (10)-triene-3,17ß-diol). Knockdown of FOXO3a or ERß expression abolished the increase of PUMA in response to 3ß-Adiol in LNCaP and PC3 cells, suggesting that FOXO3a mediates the apoptotic effect of 3ß-Adiol-activated ERß. Moreover, the ventral prostate of ERß-/- mice had decreased expression of FOXO3a and PUMA compared with the ERß+/+ mice, indicating a relationship between ERß and FOXO3a expression. The regulation of FOXO3a by ERß in normal basal epithelial cells indicates a function of ERß in cell differentiation and maintenance of cells in a quiescent state. In addition, the expression of ERß, FOXO3a and PUMA is comparable and higher in benign prostatic hyperplasia than in prostate cancer Gleason grade 4 or higher, where there is substantial loss of ERß, FOXO3a and PUMA. We conclude that ERß induces apoptosis of prostate cancer cells by increasing transcription of FOXO3a, leading to an increase of PUMA and subsequent triggering of apoptosis via the intrinsic pathway involving caspase-9. Furthermore, we conclude that ligands specifically activating ERß could be useful pharmaceuticals in the treatment of prostate cancer.

Involvement of estrogen receptor ß in maintenance of serotonergic neurons of the dorsal raphe.

The serotonergic neurons of the dorsal raphe (DR) nucleus in the CNS are involved in fear, anxiety and depression. Depression and anxiety occur quite frequently in postmenopausal women, but estrogen replacement to correct these CNS disorders is at present not favored because estrogen carries with it an increased risk for breast cancer. Serotonin synthesis, release and reuptake in the DR are targets of pharmaceuticals in the treatment of depression. In the present study we have examined by immunohistochemistry, the expression of two nuclear receptors, that is, the estrogen receptors ERα and ERß. We found that ERß but not ERα is strongly expressed in the DR and there is no sex difference and no change with ageing in the number of tryptophan hydroxylase (TPH)-positive neurons in the DR of wild-type (WT) mice. However, in ovariectomized (OVX) WT and in ERß(-/-) mice, there was a marked reduction in the number of TPH-positive normal-looking neurons and a marked increase in TPH-positive spindle-shaped cells. These neuronal changes were prevented in mice 1-3 weeks (but not 10 weeks) after OVX by the selective ERß agonist, LY3201, given as continuous release pellets for 3 days. The ERß agonist had no effects on glucose homeostasis. Thus, the onset of action of the ERß agonist is rapid but there is a limited window in time after estrogen loss when the drug is useful. We conclude that, rather than estradiol, ERß agonists could be useful pharmaceuticals in maintaining functional DR neurons to treat postmenopausal depression.

Estrogen receptor beta growth-inhibitory effects are repressed through activation of MAPK and PI3K signalling in mammary epithelial and breast cancer cells.

Two thirds of breast cancers express estrogen receptors (ER). ER alpha (ERα) mediates breast cancer cell proliferation, and expression of ERα is the standard choice to indicate adjuvant endocrine therapy. ERbeta (ERß) inhibits growth in vitro; its effects in vivo have been incompletely investigated and its role in breast cancer and potential as alternative target in endocrine therapy needs further study. In this work, mammary epithelial (EpH4 and HC11) and breast cancer (MC4-L2) cells with endogenous ERα and ERß expression and T47-D human breast cancer cells with recombinant ERß (T47-DERß) were used to explore effects exerted in vitro and in vivo by the ERß agonists 2,3-bis (4-hydroxy-phenyl)-propionitrile (DPN) and 7-bromo-2-(4-hydroxyphenyl)-1,3-benzoxazol-5-ol (WAY). In vivo, ERß agonists induced mammary gland hyperplasia and MC4-L2 tumour growth to a similar extent as the ERα agonist 4,4',4''-(4-propyl-(1H)-pyrazole-1,3,5-triyl) trisphenol (PPT) or 17ß-estradiol (E2) and correlated with higher number of mitotic and lower number of apoptotic features. In vitro, in MC4-L2, EpH4 or HC11 cells incubated under basal conditions, ERß agonists induced apoptosis measured as upregulation of p53 and apoptosis-inducible factor protein levels and increased caspase 3 activity, whereas PPT and E2 stimulated proliferation. However, when extracellular signal-regulated kinase 1 and 2 (ERK ½) were activated by co-incubation with basement membrane extract or epidermal growth factor, induction of apoptosis by ERß agonists was repressed and DPN induced proliferation in a similar way as E2 or PPT. In a context of active ERK ½, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) signalling was necessary to allow proliferation stimulated by ER agonists. Inhibition of MEK ½ with UO126 completely restored ERß growth-inhibitory effects, whereas inhibition of PI3K by LY294002 inhibited ERß-induced proliferation. These results show that the cellular context modulates ERß growth-inhibitory effects and should be taken into consideration upon assessment of ERß as target for endocrine treatment.

Generation of ERα-floxed and knockout mice using the Cre/LoxP system.

Estrogen receptor alpha (ERα) is a nuclear receptor that regulates a range of physiological processes in response to estrogens. In order to study its biological role, we generated a floxed ERα mouse line that can be used to knock out ERα in selected tissues by using the Cre/LoxP system. In this study, we established a new ERα knockout mouse line by crossing the floxed ERα mice with Cre deleter mice. Here we show that genetic disruption of the ERα gene in all tissues results in sterility in both male and female mice. Histological examination of uterus and ovaries revealed a dramatically atrophic uterus and hemorrhagic cysts in the ovary. These results suggest that infertility in female mice is the result of functional defects of the reproductive tract. Moreover, female knockout mice are hyperglycemic, develop obesity and at the age of 4 months the body weight of these mice was more than 20% higher compared to wild type littermates and this difference increased over time. Our results demonstrate that ERα is necessary for reproductive tract development and has important functions as a regulator of metabolism in females.

Soy protein isoflavones differentially regulate liver X receptor isoforms to modulate lipid metabolism and cholesterol transport in the liver and intestine in mice.

AIMS/HYPOTHESIS: Liver X receptor (LXR)α regulates the genes involved in cholesterol, fatty acid and glucose metabolism. Soy protein (SP) consumption reduces the hepatic accumulation of cholesterol and triacylglycerol, and improves insulin sensitivity. However, it is not known whether these effects are mediated via LXRα. We therefore investigated whether the consumption of SP regulates metabolic changes in cholesterol metabolism and insulin sensitivity via LXRα. METHODS: Wild-type (WT) and Lxrα(-/-) (Lxrα, also known as Nr1h3) mice were fed an SP diet with or without cholesterol for 28 days. The expression of LXRα target genes was measured in liver and intestine, as were hepatic lipid content and faecal bile acid concentration. Oral glucose and insulin tolerance tests were also performed. Hepatocytes were used to study the effect of isoflavones on LXR activity. RESULTS: The livers of WT and Lxrα(-/-) mice fed an SP high-cholesterol diet showed less steatosis than those fed casein. The SP diet increased the expression of the ATP-binding cassette (ABC) sub-family genes Abca1, Abcg5 and Abcg8 in the liver and intestine, as well as increasing total faecal bile acid excretion and insulin sensitivity in WT mice compared with mice fed a casein diet. However, these effects of SP were not observed in Lxrα(-/-) mice. The SP isoflavone, genistein, repressed the activation of LXRα target genes by T0901317, whereas it stimulated the activation of LXRß target genes. The AMP-activated protein kinase inhibitor, compound C, had the opposite effects to those of genistein. CONCLUSIONS/INTERPRETATION: Our results suggest that SP isoflavones stimulate the phosphorylation of LXRα or LXRß, resulting in different biological effects for each LXR isoform.

LXRß activation increases intestinal cholesterol absorption, leading to an atherogenic lipoprotein profile.

OBJECTIVES: Liver X receptors (LXRs) are essential for the regulation of intestinal cholesterol absorption. Because two isoforms exist, LXRα and LXRß, with overlapping but not identical functions, we investigated whether LXRα and LXRß exert different effects on intestinal cholesterol absorption. DESIGN: Wild-type (WT), LXRα(-/-) and LXRß(-/-) mice were fed control diet, 0.2% cholesterol-enriched diet or 0.2% cholesterol-enriched diet plus the LXR agonist GW3965. RESULTS: When fed a control diet, all three genotypes showed similar levels of cholesterol absorption. Of interest, a significant increase in cholesterol absorption was found in the LXRα(-/-) mice, but not in the WT or LXRß(-/-) animals, when fed a diet enriched with 0.2% cholesterol or 0.2% cholesterol + GW3965. Reduced faecal neutral sterol excretion and a hydrophobic bile acid profile were also observed in LXRα(-/-) mice. Greater increases in the apolipoprotein (apo)B-containing lipoproteins in serum were seen in the LXRα(-/-) mice. A 0.2% cholesterol +GW3965 diet suppressed intestinal Npc1l1 protein expression to the same extent for all genotypes, while Abca1 and Abcg5 were elevated to the same degree. CONCLUSIONS: In the intestine, LXRα and LXRß seem to exert similar effects on expression of cholesterol-transporting proteins such as Npc1l1. Selective activation of LXRß may generate effects such as increased cholesterol absorption and elevated serum levels of apoB-containing lipoproteins, which seem to be counteracted by LXRα. Therefore, an intestinal LXRß-specific pathway might exist in terms of cholesterol transportation in addition to the main pathway.

Estrogen receptors: therapies targeted to receptor subtypes.

Over the past two decades, we have learned that estrogens play important physiological roles not only in women but also in men and that the biological effects of estrogen are mediated by not one but two distinct estrogen receptors (ERs), ERα and ERß. Our appreciation of the physiological importance of estrogen and the mechanisms by which it acts has significantly increased over the years; however, we are only now beginning to decipher the roles of ERα and ERß in different organs and to elucidate how selective ligands, acting through either of the two ERs, can prevent or treat various age- or sex-specific diseases. The specific roles of ERα and ERß and the therapeutic potential of ER subtype-selective agonists in bone and metabolic homeostasis, depression, vasomotor symptoms, neurodegenerative diseases, and cancer are reviewed herein. It must be stated, however, that appropriate clinical studies are necessary to validate these compounds as agents for the prevention and treatment of diseases.

Endocrine disruptors targeting ERbeta function.

Endocrine disruptive chemicals (EDCs) circulating in the environment constitute a risk to ecosystems, wildlife and human health. Oestrogen receptor (ER) alpha and beta are targeted by various kinds of EDCs but the molecular mechanisms and long-term consequences of exposure are largely unknown. Some biological effects of EDCs are mediated by the aryl hydrocarbon receptor (AhR), which is a key player in the cellular defence against xenobiotic substances. Adding complexity to the picture, there is also accumulating evidence that AhR-ER pathways have an intricate interplay at multiple levels. In this review, we discuss some EDCs that affect the oestrogen pathway by targeting ERbeta. Furthermore, we describe some effects of AhR activities on the oestrogen system. Mechanisms as well as potential adverse effects on human health are discussed.

Development of osteoarthritic features in estrogen receptor knockout mice.

OBJECTIVE: Estrogens are suggested to play a role in the development of osteoarthritis as indicated by the increased prevalence in women after menopause. We studied whether deletion of the estrogen receptor (ER) alpha, beta, or both in female mice results in cartilage damage, osteophytosis, and changes in subchondral bone of skeletally mature animals. METHODS: We studied knee joints of 6-month-old female ERalpha-/-, ERbeta-/-, and (double) ERalpha-/-beta-/- mice and their wild type (wt) littermates. The presence and size of osteophytes and osteoarthritic changes in cartilage were analyzed using histology. Changes in subchondral plate and trabecular bone were studied using micro-CT. RESULTS: In ERalpha-/-beta-/- mice, we observed an increase in number and/or size of osteophytes and thinning of the lateral subchondral plate. However, cartilage damage was not different from wt. In ERalpha-/- or ERbeta-/- mice, no significant differences in cartilage damage score, osteophyte formation, or subchondral plate thickness were found. The bone volume fraction of the epiphyseal trabecular bone was unchanged in ERalpha-/- mice, increased in ERbeta-/- mice, and decreased in ERalpha-/-beta-/- mice. CONCLUSIONS: We conclude that deletion of both ERs leads to increased osteophytosis, but deletion of one or both ERs does not lead to overt cartilage damage in 6-month-old mice.

Spatiotemporal dynamics of the expression of estrogen receptors in the postnatal mouse brain.

This study reports on the spatiotemporal dynamics of the expression of estrogen receptors (ERs) in the mouse central nervous system (CNS) during the early postnatal and the peripubertal period. At postnatal day 7 (P7), neurons with strong nuclear immunostaining for both ERalpha and ERbeta1 were widely distributed throughout the brain. Sucrose density gradient sedimentation followed by western blotting supported the histochemical evidence for high levels of both ERs at P7. Over the following 2 days, there was a rapid downregulation of ERs. At P9, ERalpha expression was visible only in the hypothalamic area. Decline in ERbeta1 expression was slower than that of ERalpha, and ERalpha-negative, ERbeta1-positive cells were observed in the dentate gyrus and walls of third ventricle. Between P14 and P35, ERs were undetectable except for the hypothalamic area. As before P7, the ovary does not produce estrogen but does produce 5alpha-androstane-3beta, 17beta-diol (3betaAdiol), an estrogenic metabolite of dihydrotestosterone, we examined the effects of high levels of 3betaAdiol in the postnatal period. We used CYP7B1 knockout mice which cannot hydroxylate and inactivate 3betaAdiol. The brains of these mice are abnormally large with reduced apoptosis. In the early postnatal period, there was 1-week delay in the timing of the reduction in ER expression in the brain. These data reveal that the time when ERs might be activated in the brain is limited to the first 8 postnatal days. In addition, the importance of aromatase has to be reconsidered as the alternative estrogen, 3betaAdiol, is important in neuronal function in the postnatal brain.