Stromal growth and epithelial cell proliferation in ventral prostates of liver X receptor knockout mice.

With specific liver X receptor alpha and beta (LXRalpha and LXRbeta) antibodies, we found that LXRalpha is strongly expressed in the luminal and basal cells of prostatic epithelium. The ventral prostates (VP) of LXRalpha(-/-) mice are characterized by the presence of smooth-muscle actin-positive stromal overgrowth around the prostatic ducts and by numerous fibrous nodules pushing into the ducts and causing obstruction, so that most of the ducts were extremely dilated. BrdU labeling and Ki67 staining revealed epithelial and stromal proliferation in the fibrous nodules. However, the dense stroma surrounding the ducts was not positive for proliferation markers. There was no detectable difference between WT and LXRalpha(-/-) mice VP in the expression of the androgen receptor, but there was an increase in nuclear expression of Snail and Smad 2/3, indicating enhanced TGF-beta signaling. Upon treatment of WT mice for 3 months with the LXR agonist T2320 or for 3 weeks with beta-sitosterol, LXRalpha was downregulated, and a VP phenotype similar to that of LXRalpha(-/-) mice resulted. We conclude that in rodents, LXRalpha seems to control VP stromal growth and that LXRalpha(-/-) mice may be a useful model to study prostatic stromal hyperplasia. Because LXRalpha is expressed in the epithelium, the excessive stromal growth in LXRalpha(-/-) mice indicates that LXRalpha is essential for epithelial stromal communication.

Expression of liver X receptor beta is essential for formation of superficial cortical layers and migration of later-born neurons.

Liver X receptor (LXR) beta regulates cholesterol levels in the brain and is essential for maintenance of motor neurons in the spinal cord and dopaminergic neurons in the substantia nigra. Here, we have examined the expression pattern of LXRbeta protein in the cerebral cortex and looked for defects in cortical development in LXRbeta knockout (LXRbeta(-/-)) mice. LXRbeta protein was widely expressed in the mouse brain at later embryonic stages, and the expression pattern in the cerebral cortex was developmentally regulated. In normal postnatal mice, LXRbeta was localized mainly in the upper layers of the cerebral cortex. In LXRbeta(-/-) mice layers II and III were thinner with fewer neurons. Layer I was slightly thicker, whereas layers IV-VI were essentially normal. Consistent with this finding, Brn2 and NeuN expression were decreased in the upper layers in the LXRbeta(-/-) neonatal cortex. The number of S-phase progenitor cells in the cortex between embryonic day (E) 12.5 to E16.5, was similar in WT and LXRbeta(-/-) littermates but BrdU birth dating revealed that late-generated neurons labeled by BrdU injections administered at E14.5 or E16.5, and destined to cortical layers II/III, were disorganized and failed to migrate. The defect in migration appears to be caused by a reduction in the number of vertical processes emanating from the radial glia. These processes are the architectural guides for later-born migrating neurons. Taken together, these findings suggest that LXRbeta expression in the cerebral cortex is involved in cortex lamination and is essential for the migration of late-generated neocortical neurons.

Pancreatic exocrine insufficiency in LXRbeta-/- mice is associated with a reduction in aquaporin-1 expression.

Liver X receptors (LXRs) alpha and beta are nuclear oxysterol receptors with a key role in cholesterol, triglyceride, and glucose metabolism. In LXRbeta(-/-) mice on a normal diet, there is a reduction in size of perigonadal fat pad and, on high-fat diet there is resistance to obesity. In the present study, we investigated the reason for the resistance of LXRbeta(-/-) mice to weight gain. In LXRbeta(-/-) mice we found pancreatic exocrine insufficiency with reduced serum levels of amylase and lipase, reduced proteolytic activity in feces, chronic inflammatory infiltration, and, in the ductal epithelium, an increased apoptosis without compensatory proliferation. Electron microscopy revealed ductal dilatation with intraductal laminar structures characteristic of cystic fibrosis. To investigate the relationship between LXRbeta and pancreatic secretion, we studied the expression of LXRbeta and the water channel, aquaporin-1 (AQP1), in the ductal epithelium of the pancreas. In WT mice, ductal epithelial cells expressed LXRbeta in the nuclei and AQP1 on the plasma membrane. In LXRbeta(-/-) mice neither LXRbeta nor AQP1 was detectable. Moreover, in WT mice the LXR agonist (T2320) increased AQP1 gene expression. These data demonstrate that in LXRbeta(-/-) mice dietary resistance to weight gain is caused by pancreatic insufficiency and that LXRbeta regulates pancreatic exocrine secretion through the control of AQP1 expression. Pancreatic exocrine insufficiency is the main cause of malabsorption syndrome responsible for weight loss in adults and growth failure in children. Several genes are known to be involved in the pathogenesis and susceptibility to pancreatic insufficiency. LXRbeta should be included in that list.

Phase I-II Open-Label Multicenter Study of Palbociclib + Vemurafenib in BRAF V600MUT Metastatic Melanoma Patients: Uncovering CHEK2 as a Major Response Mechanism.

PURPOSE: In BRAF V600MUT metastatic melanoma, cyclin D-CDK4/6-INK4-Rb pathway alterations are involved in resistance to MAPK inhibitors, suggesting a clinical benefit of cyclin-dependent kinase 4 (CDK4) inhibitors. In this phase I-II study, we aimed to establish the MTD of palbociclib when added to vemurafenib. PATIENTS AND METHODS: Patients with BRAF V600E/KMUT metastatic melanoma harboring CDKN2A loss and RB1 expression were included and stratified into two groups according to previous BRAF inhibitor treatment (no:strata 1; yes:strata 2). Treatment comprised palbociclib once daily for 14 days followed by a 7-day break + continuous dosing of vemurafenib. The primary endpoint was the occurrence of dose-limiting toxicity (DLT), and the secondary endpoints included the best response, survival, pharmacokinetics, and tumor molecular profiling. RESULTS: Eighteen patients were enrolled, with 15 in strata 2. Characteristics at inclusion were American Joint Committee on Cancer stage IVM1c (N = 16; 88.9%), high lactate dehydrogenase (N = 9; 50.0%), and median number of previous treatments of 2. One and 5 patients experienced DLT in strata 1 and 2, respectively, defining the MTD at palbociclib 25 mg and vemurafenib 960 mg in strata 2. No significant evidence for drug-drug interactions was highlighted. The median progression-free survival was 2.8 months, and 5 (27.8%) patients showed a clinical response. The baseline differential mRNA expression analysis and in vitro data revealed the role of CHEK2 in the response to palbociclib. CONCLUSIONS: Although the combination of palbociclib + fixed-dose vemurafenib did not allow an increased palbociclib dosage above 25 mg, a significant clinical benefit was achieved in pretreated patients with melanoma. An association between the transcriptomic data and clinical response was highlighted.

Deoxyribonucleic acid response element-dependent regulation of transcription by orphan nuclear receptor estrogen receptor-related receptor gamma.

The estrogen receptor-related receptor gamma (ERR gamma/ERR3/NR3B3) is the newest member of the ERR subfamily that also includes ERR alpha and ERR beta. All three isoforms share a high degree of amino acid identity especially in the DNA binding domain. ERR gamma is a constitutively active transcriptional activator that regulates reporter elements driven by steroidogenic factor 1 response element (SF-1RE) and estrogen response element. However, it has the highest potency on a derivative of SF-1RE present in the small heterodimer partner gene promoter called sft4 and unlike ERR alpha and -beta, it fails to activate a palindromic thyroid hormone response element. To investigate the mechanism behind this response element-specific differential transcriptional activity of ERR gamma, the interactions of ERR gamma and the aforementioned response elements was monitored. EMSA and chromatin immunoprecipitation assays demonstrated that ERR gamma binds to sft4, SF-1RE, and palindromic thyroid hormone response element albeit with different degrees of affinity, but causes hyperacetylation of sft4 and SF-1RE templates only. Limited proteolysis assays showed that ERR gamma, bound to different elements, shows differential trypsin sensitivity. A search for novel coregulators of ERR gamma led to the identification of receptor interacting protein 140 as a potent corepressor and peroxisome proliferator-activated receptor gamma coactivator 1 as a potent coactivator of ERR gamma. DNA-dependent pull-down and transient transfection assays demonstrated that, on different DNA elements, ERR gamma exhibits differential cofactor interactions, which in turn dictate its transcriptional activity. Because ERR gamma shows a similar tissue distribution as peroxisome proliferator-activated receptor gamma coactivator 1 and receptor interacting protein 140, these two coregulators may act as key components of ERR gamma-mediated transcription.

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.

A functionally conserved member of the FTZ-F1 nuclear receptor family from Schistosoma mansoni.

The fushi tarazu factor 1 (FTZ-F1) nuclear receptor subfamily comprises orphan receptors with crucial roles in development and sexual differentiation in vertebrates and invertebrates. We describe the structure and functional properties of an FTZ-F1 from the platyhelminth parasite of humans, Schistosoma mansoni, the first receptor from this family to be characterized in a Lophotrochozoan. It contains a well conserved DNA-binding domain (55-63% identity to other family members) and a poorly conserved ligand-binding domain (20% identity to that of zebrafish FF1a). However, both the ligand domain signature sequence and the activation function 2-activation domain (AF2-AD) are perfectly conserved. Phylogenetic analysis confirmed that SmFTZ-F1 is a member of nuclear receptor subfamily 5, but that it clustered with the Drosophila receptor DHR39 and has consequently been named NR5B1. The gene showed a complex structure with 10 exons and an overall size of 18.4 kb. Two major transcripts were detected, involving alternative promoter usage and splicing of the two 5' exons, but which encoded identical proteins. SmFTZ-F1 mRNA is expressed at all life-cycle stages with the highest amounts in the larval forms (miracidia, sporocysts and cercariae). However, expression of the protein showed a different pattern; low in miracidia and higher in adult male worms. The protein bound the same monomeric response element as mammalian SF-1 (SF-1 response element, SFRE) and competition experiments with mutant SFREs showed that its specificity was identical. Moreover, SmFTZ-F1 transactivated reporter gene transcription from SFRE similarly to SF-1. This functional conservation argues for a conserved biological role of the FTZ-F1 nuclear receptor family throughout the metazoa.