Differentiation of murine embryonic stem cells induces progesterone receptor gene expression

The role of steroid hormone receptors in very early embryonic development remains unknown. Clearly, expression during organogenesis is important for tissue-specific development. However, progesterone receptor (PR) and estrogen receptors (ERalpha, ERbeta) are expressed during early development through the blastocyst stage in mice and other species, and yet are not essential for embryonic viability. We have utilized the mouse embryonic stem (mES) cell model to investigate the regulated expression of these receptors during differentiation. Surprisingly, one of the earliest changes in gene expression in response to a differentiation signal observed is PR gene induction. It parallels the time course of expression for the patterning genes Hoxb1 and Hoxa5. Unexpectedly, PR gene expression is not regulated in an estrogen-dependent manner by endogenous ERs or by transiently overexpressed ERalpha. Our results suggest a potentially novel mechanism of PR gene regulation within mES cells compared to adult tissues and the possibility of unique targets of PR action during early mES cell differentiation.     

Estrogen-induced genes,WISP-2 and pS2, respond divergently to protein kinase pathway

Recently, we identified WISP-2 (Wnt-1 inducible signaling pathway protein 2) as a novel estrogen-inducible gene in the MCF-7 human breast cancer cell line. In this study, we examined whether WISP-2 expression is modulated by PK activators. Treatment with protein kinase A (PKA) activators [cholera toxin plus 3-isobutyl-1-methylxanthine (CT/IBMX)] induced WISP-2 expression. CT/IBMX induced expression of the other estrogen-responsive gene, pS2, more dramatically than maximum stimulation by 17beta-estradiol (E2). Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), which directly stimulates protein kinase C (PKC) activity, completely prevented WISP-2 mRNA induction by E2, whereas it increased pS2 mRNA expression more dramatically than maximum stimulation by E2. Results of treatments with the protein synthesis inhibitor cycloheximide and the pure antiestrogen ICI182,780 suggest that these PK pathways modulate WISP-2 gene expression via different molecular mechanisms than those for pS2. Because TPA inhibits cell proliferation, we investigated whether WISP-2 induction was dependent on cell growth. Cells were treated with insulin-like growth factor-1 (IGF-1) or interleukin-1alpha (IL-1alpha) to stimulate or inhibit cell growth, respectively. These treatments had no effect on WISP-2 mRNA expression either alone or in combination with E2, suggesting that WISP-2 induction is independent of cell growth.     

Negative regulation of parathyroid hormone-related protein expression by steroid hormones

Elevated parathyroid hormone-related protein (PTHrP) is responsible for humoral hypercalcemia of malignancy (HHM), which is of clinical significance in treatment of terminal patients with malignancies. Steroid hormones were known to cause suppression of PTHrP expression. However, detailed studies linking multiple steroid hormones to PTHrP expression are lacking. Here we studied PTHrP expression in response to steroid hormones in four cell lines with excessive PTHrP production. Our study established that steroid hormones negatively regulate PTHrP expression. Vitamin D receptor, estrogen receptor α, glucocorticoid receptor, and progesterone receptor, were required for repression of PTHrP expression by the cognate ligands. A notable exception was the androgen receptor, which was dispensable for suppression of PTHrP expression in androgen-treated cells. We propose a pathway(s) involving nuclear receptors to suppress PTHrP expression.     

Estradiol restores diabetes-induced reductions in sex steroid receptor expression and distribution in the vagina of db/db mouse model

Sex steroid hormones and receptors play an important role in maintaining vaginal physiology. Disruptions in steroid receptor signaling adversely impact vaginal function. Limited studies are available investigating the effects of diabetic complications on steroid receptor expression and distribution in the vagina. The goals of this study were to investigate type 2 diabetes-induced changes in expression, localization and distribution of estrogen (ER), progesterone (PR) and androgen receptors (AR) in the vagina and to determine if estradiol treatment ameliorates these changes. Eight-week-old female diabetic (db/db) mice (strain BKS.Cg-m+/+ Lepr^db/J) were divided into two subgroups: untreated diabetic and diabetic animals treated with pellets containing estradiol. Control normoglycemic littermates were subcutaneously implanted with pellets devoid of estradiol. At 16 weeks of age, animals were sacrificed, vaginal tissues excised and analyzed by Western blot and immunohistochemical methods. Diabetes produced marked reductions in protein expression of ER, PR, and AR. Diabetes also resulted in marked differences in the distribution, staining intensity and proportion of immunoreactive cells containing these steroid receptors in the epithelium, lamina propria and muscularis. Treatment of diabetic animals with estradiol restored receptor protein expression and distribution similar to those levels observed in control animals. This study demonstrates that type 2 diabetes markedly reduces steroid receptor protein expression and distribution in the vagina. Estradiol treatment of diabetic animals ameliorates these diabetes-induced changes.     

Differential epigenetic regulation of BDNF and NT-3 genes by trichostatin A and 5-aza-2′-deoxycytidine in Neuro-2a cells

To understand epigenetic regulation of neurotrophins in Neuro-2a mouse neuroblastoma cells, we investigated the alteration of CpG methylation of brain-derived neurotrophic factor (BDNF) promoter I and neurotrophin-3 (NT-3) promoter IB and that of histone modification in Neuro-2a cells. Bisulfite genomic sequencing showed that the CpG sites of BDNF promoter I were methylated in non-treated Neuro-2a cells and demethylated following 5-aza-2′-deoxycytidine (5-aza-dC) treatment. In contrast, methylation status of the NT-3 promoter IB did not change by 5-aza-dC treatment in Neuro-2a cells. Furthermore, we demonstrated that BDNF exon I-IX mRNA was induced by trichostatin A (TSA) treatment. However, NT-3 exon IB-II mRNA was not induced by TSA treatment. Chromatin immunoprecipitation assays showed that the levels of acetylated histones H3 and H4 on BDNF promoter I were increased by TSA. These results demonstrate that DNA methylation and/or histone modification regulate BDNF gene expression, but do not regulate NT-3 gene expression in Neuro-2a cells.     

Differential regulation of CDX1 and CDX2 gene expression by deficiency in methyl group donors

The CDX2 and CDX1 homeobox genes have respectively a tumour suppressor and proliferative role in the intestinal epithelium. We analyzed DNA methylation and histones modifications associated with CDX2 and CDX1 promoters in two human colon cancer cell lines expressing differentially these genes, Caco2/TC7 [CDX2 positive-CDX1 negative] and HT29 [CDX2 negative-CDX1 negative] cells. Chromatin immunoprecipitation experiments indicated that CDX2 and CDX1 gene expression correlated with a histone modifications pattern characterizing active chromatin (H3K4 trimethylated and H3 acetylated). Bisulfite DNA sequencing and methylation-specific PCR showed that CDX2 and CDX1 promoters display no methylation in HT29 cells even though both genes are not expressed. In contrast, the CDX1 promoter is methylated in Caco2/TC7. DNA demethylation by 5aza-dC or the combination of 5aza-dC plus SAHA, an inhibitor of histone deacetylases, restored CDX1 expression in Caco2/TC7 cells but these treatments were inefficient on both CDX2 and CDX1 in HT29 cells. Thus, in colon cancer cells the changes in chromatin conformation are heterogeneous and repression of CDX2 and CDX1 in HT29 cells is not due to epigenetic mechanisms. In vivo, dietary deprivation of methyl groups in rats upregulated CDX1 mRNA and downregulated to a lesser extent CDX2 mRNA expression. Moreover, methyl group deprivation downregulated CDX2 protein by changing its phosphorylation pattern. The changes in CDX2 and CDX1 expression determined by methyl group deprivation may constitute one of the mechanisms sustaining the protective role attributed to folate in colon cancer.     

Sexually dimorphic androgen and estrogen receptor mRNA expression in the brain of túngara frogs

Sex steroid hormones are potent regulators of behavior and they exert their effects through influences on sensory, motor, and motivational systems. To elucidate where androgens and estrogens can act to regulate sex-typical behaviors in the túngara frog (Physalaemus pustulosus), we quantified expression of the androgen receptor (AR), estrogen receptor alpha (ERα), and estrogen receptor beta (ERβ) genes in the brains of male and females. To do so, we cloned túngara-specific sequences for AR, ERα, and ERβ, determined their distribution in the brain, and then quantified their expression in areas that are important in sexual communication. We found that AR, ERα, and ERβ were expressed in the pallium, limbic forebrain (preoptic area, hypothalamus, nucleus accumbens, amygdala, septum, striatum), parts of the thalamus, and the auditory midbrain (torus semicircularis). Males and females had a similar distribution of AR and ER expression, but expression levels differed in some brain regions. In the auditory midbrain, females had higher ERα and ERβ expression than males, whereas males had higher AR expression than females. In the forebrain, females had higher AR expression than males in the ventral hypothalamus and medial pallium (homolog to hippocampus), whereas males had higher ERα expression in the medial pallium. In the preoptic area, striatum, and septum, males and females had similar levels of AR and ER expression. Our results suggest that sex steroid hormones have sexually dimorphic effects on auditory processing, sexual motivation, and possibly memory and, therefore, have important implications for sexual communication in this system.     

NGF/PI3K signaling-mediated epigenetic regulation of delta opioid receptor gene expression

The G protein-coupled delta opioid receptor gene (dor) has been associated with neuronal survival, differentiation, and neuroprotection. Our previous study identified PI3K/Akt/NF-κB signaling is a main downstream signaling pathway in nerve growth factor (NGF)-induced temporal expression of the dor gene in the PC12 cell model. It is still unknown how NGF/PI3K signaling regulates the expression of the dor gene in the nucleus. In the current study, we investigated how PI3K signaling affected epigenetic modifications of histone H3 Lys⁹ (H3K9) in the 5′-UTR region of the rat dor gene locus. NGF treatment resulted in the global reversal of H3K9 trimethylation in cells. Moreover, the locus-specific reversal of H3K9 trimethylation and acetylation of H3K9 were dependent upon NGF/PI3K signaling and temporally well correlated with NGF-induced gene expression. These results indicate the importance of epigenetic modifications of H3K9, particularly the reversal of trimethylated H3K9, in the regulation of NGF/PI3K-dependent genes during neuronal differentiation.     

Protein Phosphatase 2A Enables Expression of Interleukin 17 (IL-17) through Chromatin Remodeling

Protein phosphatase 2A (PP2A) is a heterotrimeric serine/threonine phosphatase involved in essential cellular functions. T cells from patients with systemic lupus erythematosus (SLE) express high levels of the catalytic subunit of PP2A (PP2Ac). A mouse overexpressing PP2Ac in T cells develops glomerulonephritis in an IL-17-dependent manner. Here, using microarray analyses, we demonstrate that increased expression of PP2Ac grants T cells the capacity to produce an array of proinflammatory effector molecules. Because IL-17 is important in the expression of glomerulonephritis, we studied the mechanism through which PP2Ac dysregulation facilitates its production. We report that PP2Ac is involved in the regulation of the Il17 locus by enhancing histone 3 acetylation through a mechanism that involves activation of interferon regulatory factor 4. Increased histone 3 acetylation of the Il17 locus is shared between T cells of PP2Ac transgenic mice and patients with SLE. We propose that, by promoting the inflammatory capacity of T cells, PP2Ac dysregulation contributes to the pathogenesis of SLE.     

Human estrogen receptor alpha gene is a target of Runx2 transcription factor in osteoblasts

Several studies into the mechanisms involved in control of osteoblast-specific gene expression have identified Runx2 and ERalpha (estrogen receptor alpha) as essential regulators of osteoblast differentiation. Recently, interactions between Runx2 and ERalpha have been described. Here, we investigate the role of Runx2 on the regulation of ERalpha expression by determining its interaction with the F promoter, one of the multiple promoters of the human ERalpha gene and the only one active in bone. We found that, in this promoter, three Runx2-like sites are present. By electrophoretic mobility shift assay in combination with supershift and ChIP experiments, we demonstrated that Runx2 preferentially binds one of the Runx2 motifs of the F promoter. To understand whether or not they are involved in influencing F promoter activity, different promoter-reporter deletion and mutation constructs were transiently transfected into human osteoblastic cells. Comparison of luciferase activities allowed the identification of a prevalent negative role of a sequence context, within the -117,877/-117,426 region, which may be under the control of Runx2 (a) site. Finally, silencing and overexpression of endogenous Runx2 provided evidence that Runx2 has a more complex role than initially expected. In fact, Runx2 (a) and Runx2 (b) sites carried out opposite roles which are conditioned by Runx2 levels in bone cells. Therefore, the resulting F promoter activity may be tightly regulated by a dynamic interplay between these two Runx2 sites, with a predominance of negative effect of the Runx2 (a) site.