ABSTRACT
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.
Subject(s)
JNK Mitogen-Activated Protein Kinases/metabolism , Liver/metabolism , Lymphoid Enhancer-Binding Factor 1/metabolism , PAX5 Transcription Factor/metabolism , Proto-Oncogene Protein c-ets-1/metabolism , Receptors, Steroid/biosynthesis , Binding Sites/genetics , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Hep G2 Cells , Humans , Pregnane X Receptor , Promoter Regions, Genetic , Protein Binding/genetics , Proto-Oncogene Protein c-ets-1/genetics , Proto-Oncogene Proteins/metabolism , RNA Interference , RNA, Small Interfering , Receptors, Steroid/genetics , Trans-Activators/metabolism , Transcription, Genetic , Transcriptional Activation/geneticsABSTRACT
Microbial enzymatic reduction of a toxic form of chromium [Cr(VI)] has been considered as an effective method for bioremediation of this metal. This study reports on the in vitro reduction of Cr(VI) using cell-free extracts from a Cr(VI) reducing Bacillus firmus KUCr1 strain. Chromium reductase was found to be constitutive and its activity was observed both in soluble cell fractions (S12 and S150 and membrane cell fraction (P150). The reductase activity of S12 fraction was found to be optimal at 40 microM Cr(VI) with enzyme concentration equivalent to 0.493 mg protein/ml. Enzyme activity was dependent on NADH or NADPH as electron donor; optimal temperature and pH for better enzyme activity were 70 degrees C and 5.6, respectively. The Km value of the reductase was 58.33 microM chromate having a V(max) of 11.42 microM/min/mg protein. The metabolic inhibitor like sodium azide inhibited reductase activity of membrane fraction of the cell-free extract. Metal ions like Cu2+, Co2+, Ni2+ and As3+ stimulated the enzyme but others, such as Ag+, Hg2+, Zn2+, Mn2+, Cd2+ and Pb2+, inhibited Cr(VI) reductase activity.
Subject(s)
Bacillus/metabolism , Bacterial Proteins/metabolism , Cell Extracts/chemistry , Chromium/metabolism , Oxidoreductases/metabolism , Bacillus/chemistry , Cell Fractionation , Chromium/chemistry , Hydrogen-Ion Concentration , NAD , NADP , Oxidoreductases/chemistry , TemperatureABSTRACT
A chromium resistant bacterial strain KUCr1 exhibiting potential Cr(VI) reducing ability under in vitro aerobic condition is reported. The bacterial strain showed varied degree of resistance to different heavy metals. The MIC of chromium to this strain was found to be 950 mM under aerobic culture condition in complex medium. The factors affecting Cr(VI) reduction by this strain under culture condition were evaluated. Maximal Cr(VI) reduction was observed at the pH 8 to 10 and at a temperature of 35 degrees C. Higher concentration of Cr(VI) slowed down the reduction, eventually all the metal could be reduced with longer incubation time. Different toxic metals showed differential effect on reduction. Cadmium and zinc were found to inhibit reduction. Cr(VI) reduction and bioremediation were found to be related to the growth supportive condition in terms of carbon, phosphorous and nitrogen supply in wastewater fed with tannery effluent indicating cell mass dependency of Cr(VI) reduction. Through biochemical characterization and 16S rDNA sequence analysis, the strain KUCr1, as the name given to it, was identified as a strain of Bacillus firmus.
