Abstract
The Karyopherin α (KPNA) genes encode a family of soluble proteins that mediate the transport of macromolecules into the nucleus, a central function of eukaryotic cell survival. Due to this important function, dysregulation of these proteins has been implicated in a number of human diseases, including schizophrenia and cancer, as well as human cytomegalovirus, influenza A and HIV nucleoprotein infection. To date, little information is available on the regulation of these proteins, especially at the transcriptional level. The proximal promoter region of human KPNA3 gene was identified by evolutionary conservation, and putative nuclear receptor binding sites identified by in silico analysis. These included one putative binding site for the peroxisome proliferator activated receptor (PPAR), plus four putative binding sites for the vitamin D receptor (VDR). Subsequent cloning of the KPNA3 proximal promoter, plus reporter gene assay, demonstrated a dose-dependent inhibition of KPNA3 expression by several PPAR agonists (fibrates and Wy-14643), while site directed mutagenesis and electromobility shift assay demonstrated the functionality of the identified putative binding site. In contrast, the classical VDR agonist lα, 25(OH)2D3 elicited both negative and positive regulatory functions on human KPNA3 expression in the human hepatoma cell line Huh-7, in a dose-dependent manner. Subsequent mutagenesis experiments demonstrated the input of each of the four putative VDR binding sites in this response. Finally, the impact of over-expression of KPNA3 on VDR sub-cellular localization was studied. KPNA3 was demonstrated to mediate ligand-independent nuclear accumulation of VDR in Huh-7 cells. Taken together, this work is the first direct demonstration of a role for PPARa and VDR in regulation of any nuclear import gene, suggesting that functionality of these transcription factors may be regulated through a regulatory loop involving the mediators of nuclear import, the karyopherin proteins.