Understanding the dynamics of Toll-like Receptor 5 response to flagellin and its regulation by estradiol

© 2017 The Author(s). Toll-like receptors (TLRs) are major players of the innate immune system. Once activated, they trigger a signalling cascade that leads to NF-ΰ B translocation from the cytoplasm to the nucleus. Single cell analysis shows that NF-ΰ B signalling dynamics are a critical determinant of transcriptional regulation. Moreover, the outcome of innate immune response is also affected by the cross-talk between TLRs and estrogen signalling. Here, we characterized the dynamics of TLR5 signalling, responsible for the recognition of flagellated bacteria, and those changes induced by estradiol in its signalling at the single cell level. TLR5 activation in MCF7 cells induced a single and sustained NF-k B translocation into the nucleus that resulted in high NF-k B transcription activity. The overall magnitude of NF-k B transcription activity was not influenced by the duration of the stimulus. No significant changes are observed in the dynamics of NF-k B translocation to the nucleus when MCF7 cells are incubated with estradiol. However, estradiol significantly decreased NF-k B transcriptional activity while increasing TLR5-mediated AP-1 transcription. The effect of estradiol on transcriptional activity was dependent on the estrogen receptor activated. This fine tuning seems to occur mainly in the nucleus at the transcription level rather than affecting the translocation of the NF-k B transcription factor

DAX-1 expression in human breast cancer: comparison with estrogen receptors ER-α, ER-β and androgen receptor status

BACKGROUND: So far there have been no reports on the expression pattern of DAX-1 (dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1) in human breast cells and its relationship to the estrogen receptors, ER-α and ER-β, and the androgen receptor (AR). METHODS: In this study we evaluated, by immunohistochemistry and Western blot analysis, the presence and distribution of DAX-1 in benign breast disease (BBD), in situ carcinoma (CIS), and ductal and lobular breast carcinomas. RESULTS: In BBD and breast carcinomas, DAX-1 was present in both the nuclei and the cytoplasm of epithelial cells, although in infiltrative carcinomas the percentage of nuclear immunoreaction was higher than in CIS. An important relation was observed between DAX-1 and AR expression and between this orphan receptor and nodal status. CONCLUSION: DAX-1 might modify the AR and ER-β intracellular location, and because a direct positive relation between the expression of these three receptors was found it could be assumed that the presence of DAX-1 in neoplastic cells might indicate a possible failure of endocrine therapies

Tamoxifen-elicited uterotrophy: cross-species and cross-ligand analysis of the gene expression program

<p>Abstract</p> <p>Background</p> <p>Tamoxifen (TAM) is a well characterized breast cancer drug and selective estrogen receptor modulator (SERM) which also has been associated with a small increase in risk for uterine cancers. TAM's partial agonist activation of estrogen receptor has been characterized for specific gene promoters but not at the genomic level <it>in vivo</it>.Furthermore, reducing uncertainties associated with cross-species extrapolations of pharmaco- and toxicogenomic data remains a formidable challenge.</p> <p>Results</p> <p>A comparative ligand and species analysis approach was conducted to systematically assess the physiological, morphological and uterine gene expression alterations elicited across time by TAM and ethynylestradiol (EE) in immature ovariectomized Sprague-Dawley rats and C57BL/6 mice. Differential gene expression was evaluated using custom cDNA microarrays, and the data was compared to identify conserved and divergent responses. 902 genes were differentially regulated in all four studies, 398 of which exhibit identical temporal expression patterns.</p> <p>Conclusion</p> <p>Comparative analysis of EE and TAM differentially expressed gene lists suggest TAM regulates no unique uterine genes that are conserved in the rat and mouse. This demonstrates that the partial agonist activities of TAM extend to molecular targets in regulating only a subset of EE-responsive genes. Ligand-conserved, species-divergent expression of carbonic anhydrase 2 was observed in the microarray data and confirmed by real time PCR. The identification of comparable temporal phenotypic responses linked to related gene expression profiles demonstrates that systematic comparative genomic assessments can elucidate important conserved and divergent mechanisms in rodent estrogen signalling during uterine proliferation.</p

Coexpression of Nuclear Receptors and Histone Methylation Modifying Genes in the Testis: Implications for Endocrine Disruptor Modes of Action

BACKGROUND: Endocrine disruptor chemicals elicit adverse health effects by perturbing nuclear receptor signalling systems. It has been speculated that these compounds may also perturb epigenetic mechanisms and thus contribute to the early origin of adult onset disease. We hypothesised that histone methylation may be a component of the epigenome that is susceptible to perturbation. We used coexpression analysis of publicly available data to investigate the combinatorial actions of nuclear receptors and genes involved in histone methylation in normal testis and when faced with endocrine disruptor compounds. METHODOLOGY/PRINCIPAL FINDINGS: The expression patterns of a set of genes were profiled across testis tissue in human, rat and mouse, plus control and exposed samples from four toxicity experiments in the rat. Our results indicate that histone methylation events are a more general component of nuclear receptor mediated transcriptional regulation in the testis than previously appreciated. Coexpression patterns support the role of a gatekeeper mechanism involving the histone methylation modifiers Kdm1, Prdm2, and Ehmt1 and indicate that this mechanism is a common determinant of transcriptional integrity for genes critical to diverse physiological endpoints relevant to endocrine disruption. Coexpression patterns following exposure to vinclozolin and dibutyl phthalate suggest that coactivity of the demethylase Kdm1 in particular warrants further investigation in relation to endocrine disruptor mode of action. CONCLUSIONS/SIGNIFICANCE: This study provides proof of concept that a bioinformatics approach that profiles genes related to a specific hypothesis across multiple biological settings can provide powerful insight into coregulatory activity that would be difficult to discern at an individual experiment level or by traditional differential expression analysis methods

Sexually dimorphic gene expression in the heart of mice and men

The prevalence and clinical manifestation of several cardiovascular diseases vary considerably with sex and age. Thus, a better understanding of the molecular basis of these differences may represent a starting point for an improved gender-specific medicine. Despite the fact that sex-specific differences have been observed in the cardiovascular system of humans and animal models, systematic analyses of sexual dimorphisms at the transcriptional level in the healthy heart are missing. Therefore we performed gene expression profiling on mouse and human cardiac samples of both sexes and young as well as aged individuals and verified our results for a subset of genes using real-time polymerase chain reaction in independent left ventricular samples. To tackle the question whether sex differences are evolutionarily conserved, we also compared sexually dimorphic genes between both species. We found that genes located on sex chromosomes were the most abundant ones among the sexually dimorphic genes. Male-specific expression of Y-linked genes was observed in mouse hearts as well as in the human myocardium (e.g. Ddx3y, Eif2s3y and Jarid1d). Higher expression levels of X-linked genes were detected in female mice for Xist, Timp1 and Car5b and XIST, EIF2S3X and GPM6B in women. Furthermore, genes on autosomal chromosomes encoding cytochromes of the monoxygenase family (e.g. Cyp2b10), carbonic anhydrases (e.g. Car2 and Car3) and natriuretic peptides (e.g. Nppb) were identified with sex- and/or age-specific expression levels. This study underlines the relevance of sex and age as modifiers of cardiac gene expression

Effects of estrogens and bladder inflammation on mitogen-activated protein kinases in lumbosacral dorsal root ganglia from adult female rats

BACKGROUND: Interstitial cystitis is a chronic condition associated with bladder inflammation and, like a number of other chronic pain states, symptoms associated with interstitial cystitis are more common in females and fluctuate during the menstrual cycle. The aim of this study was to determine if estrogens could directly modulate signalling pathways within bladder sensory neurons, such as extracellular signal-related kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. These signalling pathways have been implicated in neuronal plasticity underlying development of inflammatory somatic pain but have not been as extensively investigated in visceral nociceptors. We have focused on lumbosacral dorsal root ganglion (DRG) neurons projecting to pelvic viscera (L1, L2, L6, S1) of adult female Sprague-Dawley rats and performed both in vitro and in vivo manipulations to compare the effects of short- and long-term changes in estrogen levels on MAPK expression and activation. We have also investigated if prolonged estrogen deprivation influences the effects of lower urinary tract inflammation on MAPK signalling. RESULTS: In studies of isolated DRG neurons in short-term (overnight) culture, we found that estradiol and estrogen receptor (ER) agonists rapidly stimulated ER-dependent p38 phosphorylation relative to total p38. Examination of DRGs following chronic estrogen deprivation in vivo (ovariectomy) showed a parallel increase in total and phosphorylated p38 (relative to beta-tubulin). We also observed an increase in ERK1 phosphorylation (relative to total ERK1), but no change in ERK1 expression (relative to beta-tubulin). We observed no change in ERK2 expression or phosphorylation. Although ovariectomy increased the level of phosphorylated ERK1 (vs. total ERK1), cyclophosphamide-induced lower urinary tract inflammation did not cause a net increase of either ERK1 or ERK2, or their phosphorylation. Inflammation did, however, cause an increase in p38 protein levels, relative to beta-tubulin. Prior ovariectomy did not alter the response to inflammation. CONCLUSIONS: These results provide new insights into the complex effects of estrogens on bladder nociceptor signalling. The diversity of estrogen actions in these ganglia raises the possibility of developing new ways to modulate their function in pelvic hyperactivity or pain states

Propofol-Induced Changes in Neurotrophic Signaling in the Developing Nervous System In Vivo

Several studies have revealed a role for neurotrophins in anesthesia-induced neurotoxicity in the developing brain. In this study we monitored the spatial and temporal expression of neurotrophic signaling molecules in the brain of 14-day-old (PND14) Wistar rats after the application of a single propofol dose (25 mg/kg i.p). The structures of interest were the cortex and thalamus as the primary areas of anesthetic actions. Changes of the protein levels of the brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), their activated receptors tropomyosin-related kinase (TrkA and TrkB) and downstream kinases Akt and the extracellular signal regulated kinase (ERK) were assessed by Western immunoblot analysis at different time points during the first 24 h after the treatment, as well as the expression of cleaved caspase-3 fragment. Fluoro-Jade B staining was used to follow the appearance of degenerating neurons. The obtained results show that the treatment caused marked alterations in levels of the examined neurotrophins, their receptors and downstream effector kinases. However, these changes were not associated with increased neurodegeneration in either the cortex or the thalamus. These results indicate that in the brain of PND14 rats, the interaction between Akt/ERK signaling might be one of important part of endogenous defense mechanisms, which the developing brain utilizes to protect itself from potential anesthesia-induced damage. Elucidation of the underlying molecular mechanisms will improve our understanding of the age-dependent component of anesthesia-induced neurotoxicity