Novel Functions for the Pregnane X Receptor include Regulation of mRNA Turnover and Involvement in Colon Cancer Progression
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To understand the mechanisms of transcriptional regulation of PXR, we performed yeast two-hybrid screenings to search for PXR-interacting proteins in a human liver cDNA library using the PXR ligand binding domain as the bait. More than one million independent clones were screened. One positive clone was a partial cDNA of CNOT2 (amino acid 183-540). CNOT2 is a component of CCR4-NOT that is a multi-subunit protein complex highly conserved from yeast to humans. Using a mammalian two-hybrid system in CV-1 cells and GST-pull down assays, we confirmed the direct interaction between PXR and CNOT2 and mapped the specific domains of association. In HepG2 cells, over expression of CNOT2 suppressed the PXR-regulated luciferase reporter gene activity. siRNA knockdown of CNOT2 potentiated PXR-transcriptional activity. These results strongly suggest that the CCR4-NOT complex is significantly involved in transcriptional regulation of PXR. The immuno-precipitated CNOT2 complex contained deadenylase activity as determined by an in vitro RNA decay assay. The presence of transfected PXR inhibited the cNOT2-associated deadenylase activity, as demonstrated by poly(A) tail PCR. Cellular localization of PXR and cNOT2 by immuno-fluorescence microscopy indicates that the interaction might occur within Cajal Bodies. Taken together, these results suggest that PXR regulates the mRNA turnover through direct interaction with the NOT2 component of the CCR4-NOT complex. PXR is also involved in colon cancer progression. Our results indicate that the evolutionarily conserved PXR protects organisms from carcinogenesis by inhibiting tumor growth as well as eliminating carcinogenic substances. Our laboratory proposes that pregnane X receptor has an important role in maintaining the balance of cells progressing through the cell cycle. In vitro and in vivo experiments demonstrate expression of PXR in colon cancer cells slows the progression of tumor formation. Colony growth of the PXR-transfected HT29 cells was suppressed in soft agar assay. In the xenograft assay, the tumor size formed in nude mice was significantly suppressed in HT29 cells stably transfected with PXR (310 mg /- 6.2 vs. 120 mg±6, p<0.01). The number of Ki-67 positive cells were significantly decreased in PXR-transfected HT29 xenograft tumor tissue compared vector-transfected HT29 controls (p<0.01) as determined by immuno-histochemistry suggesting that PXR inhibits proliferation of colon cancer cells. Results of flow cytometry analysis indicated that PXR-transfection in HT29 cells caused G0/G1 arrest. The growth inhibitory effects of PXR are likely mediated through the E2F/Rb-regulated check point since E2F1 nuclear expression was significantly inhibited by PXR over expression.
Eagleton, Navada Lorraine (2010). Novel Functions for the Pregnane X Receptor include Regulation of mRNA Turnover and Involvement in Colon Cancer Progression. Master's thesis, Texas A&M University. Available electronically from