Identification and interplay of sequence specific DNA binding proteins involved in regulation of human Pregnane and Xenobiotic Receptor gene.

Pregnane and Xenobiotic Receptor (PXR), a member of nuclear receptor superfamily, acts as a 'xenosensor' in our body and modulates a network of genes involved in xenobiotic metabolism and elimination. Expression levels of PXR in certain metabolic disorders including cancer are reported to be altered and its induced expression is associated with the development of resistance towards chemotherapy and adverse drug-drug interactions. Though the transcriptional regulation of PXR target genes have been elucidated in significant details, the structure and functional control of PXR promoter itself remains inadequately explored. In this work, we identify a Composite Element (CE) located within the proximal PXR promoter region that consists of multiple overlapping cis-elements and demonstrated that CE interacts specifically with some critical nuclear proteins. Subsequent DNA-protein interaction studies revealed mutually exclusive interactions on CE occurring between Sp1 and two unidentified DNA binding proteins with molecular masses of 50 and 54kDa. Here, we report the identification of 54kDa CE binding protein as a heterogeneous nuclear ribonucleoprotein K (hnRNPK) and demonstrate the effect of hnRNP K and Sp1 on PXR promoter transcriptional activity. Overall, the study indicates that PXR gene is tightly regulated to maintain a low receptor level.

Pregnane and Xenobiotic Receptor gene expression in liver cells is modulated by Ets-1 in synchrony with transcription factors Pax5, LEF-1 and c-Jun.

Nuclear receptor PXR is predominantly expressed in liver and intestine. Expression of PXR is observed to be dysregulated in various metabolic disorders indicating its involvement in disease development. However, information available on mechanisms of PXR self-regulation is fragmentary. The present investigation identifies some of the regulatory elements responsible for its tight regulation and low cellular expression. Here, we report that the PXR-promoter is a target for some key transcription factors like PU.1/Ets-1, Pax5, LEF-1 and c-Jun. Interestingly, we observed that PXR-promoter responsiveness to Pax5, LEF-1 and c-Jun, is considerably enhanced by Ets transcription factors (PU.1 and Ets-1). Co-transfection of cells with Ets-1, LEF-1 and c-Jun increased PXR-promoter activity by 5-fold and also induced expression of endogenous human PXR. Site-directed mutagenesis and transfection studies revealed that two Ets binding sites and two of the three LEF binding sites in the PXR-promoter are functional and have a positive effect on PXR transcription. Results suggest that expression of Ets family members, in conjunction with Pax5, LEF-1 and c-Jun, lead to coordinated up-regulation of PXR gene transcription. Insights obtained on the regulation of PXR gene have relevance in offering important cues towards normal functioning as well as development of several metabolic disorders via PXR signaling.

Overexpression of hyaluronan-binding protein 1 (HABP1/p32/gC1qR) in HepG2 cells leads to increased hyaluronan synthesis and cell proliferation by up-regulation of cyclin D1 in AKT-dependent pathway.

Overexpression of the mature form of hyaluronan-binding protein 1 (HABP1/gC1qR/p32), a ubiquitous multifunctional protein involved in cellular signaling, in normal murine fibroblast cells leads to enhanced generation of reactive oxygen species (ROS), mitochondrial dysfunction, and ultimately apoptosis with the release of cytochrome c. In the present study, human liver cancer cell line HepG2, having high intracellular antioxidant levels was chosen for stable overexpression of HABP1. The stable transformant of HepG2, overexpressing HABP1 does not lead to ROS generation, cellular stress, and apoptosis, rather it induced enhanced cell growth and proliferation over longer periods. Phenotypic changes in the stable transformant were associated with the increased "HA pool," formation of the "HA cable" structure, up-regulation of HA synthase-2, and CD44, a receptor for HA. Enhanced cell survival was further supported by activation of MAP kinase and AKT-mediated cell survival pathways, which leads to an increase in CYCLIN D1 promoter activity. Compared with its parent counterpart HepG2, the stable transformant showed enhanced tumorigenicity as evident by its sustained growth in low serum conditions, formation of the HA cable structure, increased anchorage-independent growth, and cell-cell adhesion. This study suggests that overexpression of HABP1 in HepG2 cells leads to enhanced cell survival and tumorigenicity by activating HA-mediated cell survival pathways.

Retention and transmission of active transcription memory from progenitor to progeny cells via ligand-modulated transcription factors: elucidation of a concept by BIOPIT model.

Observations made in live cells have clearly demonstrated that agonist-activated steroid/nuclear receptors reorganize in the nucleoplasm into hundreds of discrete speckled structures commonly referred to as nuclear foci. Subsequent studies have shown that nuclear foci are formed only with agonist- and not with pure antagonist-bound receptors. Also, the other accessory components of transcriptional machinery co-localize in nuclear foci with the activated receptors, suggesting these to be active gene transcription sites. Recently, it has been observed that during mitosis nuclear foci present in interphase of progenitor cells co-migrate with condensing chromatin and are inherited into the progeny cells. Ensuing events imply that as memory, the cells inherit only a biomolecular blueprint of transcription status over to next generations to express and sustain their characteristic proteome. Thus, cells achieve self-renewal via mitosis but not without ensuring that the characteristic proteome and traits are distinctively preserved during this transcription phase. This mechanism, although somewhat analogous to epigenetic marking, differs in Nature since transcription factors themselves execute this transmission. To uphold the mechanistic distinctions the phenomenon has been termed BIOPIT (biomolecular imprints offered to progeny for inheritance of traits). The BIOPIT model proposed herein attempts to explain how the disruption of BIOPIT markings by therapeutic anti-hormones or endocrine disruptors over prolonged periods may lead to eradication of cellular transcription memory with deleterious cellular consequences. It is anticipated that our model has the potential to explain the concerted actions and consequences of ligand-receptor interactions with the chromatin in the perspective of normal and aberrant physiological situations.

Intracellular localization and nucleocytoplasmic trafficking of steroid receptors: an overview.

Subcellular compartmentalization and dynamic movements of steroid receptors are major steps in executing their transcription regulatory function. Though significant progress has been made in understanding the mechanisms underlying nuclear import of NLS-bearing proteins, our general and mechanistic understanding about the nuclear export processes has begun to emerge only recently. The discovery of most commonly utilized CRM1/exportin1 dependent nuclear export pathway is attributed to a potent nuclear export inhibitor leptomycin B that helped dissecting this and other nuclear export pathways. Simultaneously, utilization of green fluorescent protein (GFP)-tagged intracellular steroid receptors has contributed to not only resolving controversial issue of subcellular localization of unliganded hormone receptors but also provided further insight into finer details of receptor dynamics in living cells. With judicious use of leptomycin B and expression of GFP-tagged receptors in living cells, existence of exportin1/CRM1 independent pathway(s), nuclear export signals and receptors for bi-directional translocation that are unique to steroid receptor trafficking have been specified. Currently, we appear to be arriving at a consensus that steroid/nuclear receptors follow dynamic nucleocytoplasmic processes that deviate from the ones commonly utilized by majority of other proteins.

Pregnane and Xenobiotic Receptor (PXR/SXR) resides predominantly in the nuclear compartment of the interphase cell and associates with the condensed chromosomes during mitosis.

Pregnane and Xenobiotic Receptor (PXR) is a transcription factor that is activated by a diverse range of xenobiotics and endogenous metabolites including steroids, bile acids and about 50% of the prescription drugs. In specific cell types (e.g. liver and intestine) it serves as a 'xenosensor' by regulating expression of a network of genes involved in xenobiotic clearance from the body. PXR expression in several cancerous tissues and its regulated expression of multi-drug resistance proteins highlight its significance in prognosis of malignancies. The view that subcellular localization and ligand induced movements of transcription factors is one of the major phenomena in regulating transcriptional activity, we used a green fluorescent protein tagged PXR chimera to study its dynamic behaviour in living cells. Under all experimental conditions, PXR was observed to be a predominantly nuclear protein maintaining a dynamic equilibrium between the nuclear and cytoplasmic compartments of the interphase cells. Interestingly, for the first time, a member of the nuclear receptor superfamily, PXR, has been observed to be associated with condensed chromosomes during all the mitotic stages of cell division. The significance of PXR association with mitotic chromosomes is discussed.

Purification of full-length human pregnane and xenobiotic receptor: polyclonal antibody preparation for immunological characterization.

Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body's homeostatic mechanisms, we for the first time, report successful prokaryotic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.