Organochlorine pesticide dieldrin upregulate proximal promoter (PII) driven CYP19A1 gene expression and increases estrogen production in granulosa cells.

Organochlorine pesticides are highly persistent environmental pollutants, generally shown to act through estrogen receptor alpha and alter estrogen biosynthesis. However, the molecular mechanism of regulation of estrogen biosynthesis by these pesticides is not clear. Estrogen is main female fertility hormone regulated by rate-limiting enzyme aromatase. It is encoded by the CYP19A1 gene, which is expressed using specific promoters. In the present study, the attempt has been made to elucidate the effect of dieldrin on the promoter-specific CYP19A1 gene expression and estrogen hormone production in buffalo granulosa cells. The buffalo granulosa cells were cultured and treated with dieldrin in a dose (100,150 and 200 ng/mL) and time (6, 12, and 24 h) dependent manner, followed by analysis of CYP19A1, promoter-specific CYP19A1 transcript expression, and estrogen production. Results showed that dieldrin significantly increased the expression of the CYP19A1 gene after 6 and 12 h while its expression was decreased after 24 h. To understand the upregulation of CYP19A1 gene, promoters' specific CYP19A1 transcript analysis was done. The finding showed that dieldrin significantly increased the proximal promoter specific CYP19A1 transcript while there was no effect on distal promoter specific CYP19A1 transcripts. This specific-promoter activity was quantified by chromatin immunoprecipitation assay (ChIP). Results confirmed the involvement of the proximal promoter in the overexpression of CYP19A1 gene. Furthermore, a significant increase in estradiol-17ß level was also observed. Overall, the present study demonstrated the stimulatory effect of dieldrin on the CYP19A1 gene and will help to understand the toxicological role of dieldrin on the reproductive system.

Reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for detection of AhR receptor responsive xenobiotics.

Dioxins are a group of highly toxic environmental persistent organic pollutants, which are lipophilic in nature. 2, 3, 7, 8- tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic representative of this class. TCDD causes several human health effects like endocrine disruption, carcinogenesis and reproductive toxicity mediated by aryl-hydrocarbon receptor. Current detection methods of dioxins like gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry etc. are costly and time consuming. Therefore, the present study aims to develop a relatively faster and cheaper technique called reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay to detect dioxins. Cultured granulosa cells used as a model system were treated with different doses (5, 10 and 15 pg/mL) of aryl hydrocarbon receptor (AhR)responsive xenobiotic, TCDD, in accordance with maximum residue limit values. Cells were treated for 6, 12 and 24 h, respectively to study the gene expression of TCDD receptor called AhR and AhR responsive genes, CYP1A1 and CYP1B1, in a dose and time dependent manner. All targeted genes expression significantly increased after 6 and 12 h by 1.3-8 folds. For the development of RT-LAMP assay, CYP1A1 gene was used with 6 h TCDD treatment. RT-LAMP assay was standardized with optimal color change at 30 min using 50 ng of cellular RNA. In all the cases, we could distinguish RT-LAMP-positive condition from one sample to another sample due to intensity of color. The method was also validated by spectrometric method. In conclusion, the developed method will be used to screen AhR receptor responsive xenobiotics by observing the color change in RT-LAMP assay like dioxin used in the present study.

Activation of GPR56, a novel adhesion GPCR, is necessary for nuclear androgen receptor signaling in prostate cells.

The androgen receptor (AR) is activated in patients with castration resistant prostate cancer (CRPC) despite low circulating levels of androgen, suggesting that intracellular signaling pathways and non-androgenic factors may contribute to AR activation. Many G-protein coupled receptors (GPCR) and their ligands are also activated in these cells indicating that they may play a role in development of Prostate Cancer (PCa) and CRPC. Although a cross talk has been suggested between the two pathways, yet, the identity of GPCRs which may play a role in androgen signaling, is not established yet. By using blast analysis of 826 GPCRs, we identified a GPCR, GPCR 205, which exhibited maximum similarity with the ligand binding domain of the AR. We demonstrate that adhesion GPCR 205, also known as GPR56, can be activated by androgens to stimulate the Rho signaling pathway, a pathway that plays an important role in prostate tumor cell metastasis. Testosterone stimulation of GPR56 also activates the cAMP/ Protein kinase A (PKA) pathway, that is necessary for AR signaling. Knocking down the expression of GPR56 using siRNA, disrupts nuclear translocation of AR and transcription of prototypic AR target genes such as PSA. GPR56 expression is higher in all twenty-five prostate tumor patient's samples tested and cells expressing GPR56 exhibit increased proliferation. These findings provide new insights about androgen signaling and identify GPR56 as a possible therapeutic target in advanced prostate cancer patients.

Hyaluronan-binding protein 1 (HABP1) overexpression triggers induction of senescence in fibroblasts cells.

Hyaluronan-binding protein 1 (HABP1), a multi-compartmental, multi-functional protein has a wide range of functions, which can be attributed to its ability to associate with a variety of cellular ligands. Earlier we have reported that HABP1 overexpression in rat normal fibroblasts (F-HABP07) shows chronic generation of reactive oxygen species (ROS), induction of autophagy, and apoptosis. However, a significant proportion of cells remained viable after the majority went through apoptosis from 60 to 72 h. In this study, an attempt has been made to delineate the cellular events in the declined population of surviving cells. It has been elucidated here that, these cells at later time points of growth, that is, 72 and 84 h, not only appeared to shrink but also are devoid of autophagic vacuoles and displayed polyploidy. F-HABP07 cells exhibited an altered cytoskeletal structure from their parental cell line F111, assumed to be caused upon inhibition of actin polymerization and decrease in IQ motif-containing GTPase activating protein 1 (IQGAP1), a key protein associated with maintenance of cytoskeletal integrity. Enhanced expression and nuclear localization of AKT observed in F-HABP07 cells appears to be contributing toward the maintenance of high ROS levels in these cells and also potentially modulating the IQGAP1 activity. These observations, in fact have been considered to result in sustained DNA damage, which then leads to increased expression of p53 and activation of p21 and carry out the cellular events responsible for senescence. Subsequent assessment of the presence of positive ß-gal staining and enhanced expression of p16INK4a in F-HABP07, confirmed that HABP1 overexpressing fibroblasts undergo senescence.

Mapping of functional domains and characterization of the transcription factor Cph1 that mediate morphogenesis in Candida albicans.

Cph1, a transcription factor of the Mitogen Activated Protein (MAP) kinase pathway, regulates morphogenesis in human fungal pathogen Candida albicans. Here, by following a systemic deletion approach, we have identified functional domains and motifs of Cph1 that are involved in transcription factor activity and cellular morphogenesis. We found that the N-terminal homeodomain is essential for the DNA binding activity; however, C-terminal domain and polyglutamine motif (PQ) are indispensable for the transcriptional activation function. Complementation analysis of the cph1Δ null mutant using various deletion derivatives revealed functional significance of the N- and C-terminal domains and PQ motif in filamentation process, chlamydospore formation and sensitivity to the cell wall interfering compounds. Genome-wide identification of the Cph1 binding site and quantitative RT-PCR transcript analysis in cph1Δ null mutant revealed that a number of genes which are associated with the filamentous growth, maintaining cell wall organization and mitochondrial function, and the genes of the pH response pathway are the transcriptional targets of Cph1. The data also suggest that Cph1 may function as a positive or negative regulator depending on the morphological state and physiological conditions. Moreover, differential expression of the upstream MAP kinase pathway genes in wild type and cph1Δ null mutant indicated the existence of a feedback regulation.

Acetylation of pregnane X receptor protein determines selective function independent of ligand activation.

Pregnane X receptor (PXR), like other members of its class of nuclear receptors, undergoes post-translational modification [PTM] (e.g., phosphorylation). However, it is unknown if acetylation (a major and common form of protein PTM) is observed on PXR and, if it is, whether it is of functional consequence. PXR has recently emerged as an important regulatory protein with multiple ligand-dependent functions. In the present work we show that PXR is indeed acetylated in vivo. SIRT1 (Sirtuin 1), a NAD-dependent class III histone deacetylase and a member of the sirtuin family of proteins, partially mediates deacetylation of PXR. Most importantly, the acetylation status of PXR regulates its selective function independent of ligand activation.

Subcellular localization and mechanisms of nucleocytoplasmic trafficking of steroid receptor coactivator-1.

Steroid hormone receptors are ligand-stimulated transcription factors that modulate gene transcription by recruiting coregulators to gene promoters. Subcellular localization and dynamic movements of transcription factors have been shown to be one of the major means of regulating their transcriptional activity. In the present report we describe the subcellular localization and the dynamics of intracellular trafficking of steroid receptor coactivator 1 (SRC-1). After its synthesis in the cytoplasm, SRC-1 is imported into the nucleus, where it activates transcription and is subsequently exported back to the cytoplasm. In both the nucleus and cytoplasm, SRC-1 is localized in speckles. The characterization of SRC-1 nuclear localization sequence reveals that it is a classic bipartite signal localized in the N-terminal region of the protein, between amino acids 18 and 36. This sequence is highly conserved within the other members of the p160 family. Additionally, SRC-1 nuclear export is inhibited by leptomycin B. The region involved in its nuclear export is localized between amino acids 990 and 1038. It is an unusually large domain differing from the classic leucine-rich NES sequences. Thus SRC-1 nuclear export involves either an alternate type of NES or is dependent on the interaction of SRC-1 with a protein, which is exported through the crm1/exportin pathway. Overall, the intracellular trafficking of SRC-1 might be a mechanism to regulate the termination of hormone action, the interaction with other signaling pathways in the cytoplasm and its degradation.

Dynamics of subcellular compartmentalization of steroid receptors in living cells as a strategic screening method to determine the biological impact of suspected endocrine disruptors.

Although a number of screening methods being used for identifying potential endocrine disruptors have generated a wealth of information, a search for alternative combination of methods is still needed to overcome experimental artefacts. There are no generally accepted or validated screening methods for monitoring and studying impact of environmental endocrine disruptors. Also, no single assay can accurately predict all the deleterious effects of endocrine disruptors. For this reason various environmental protection agencies, mainly European and US, have urged that a battery of tests in current use need to be designed to assess their adequacy in detecting the effects of endocrine disruptors. Some details about endocrine disruptors and screening programs can be found at http://www.epa.gov/scipoly/oscpendo/whatis.htm. Several studies in recent years have used fusion proteins between steroid receptors (estrogen, androgen, progesterone, etc.) and green fluorescent protein (GFP) that can serve as an alternative potent screening method to study intracellular dynamics of receptors in living cells. An approach employing nucleocytoplasmic trafficking of steroid receptors as a parameter in response to potential xenobiotic chemicals in living cells may prove to be promising in terms of being direct, fast, reliable, simple and inexpensive.