Figure 7

PPAR regulation of cubilin expression. A, upstream flanking sequence of the mouse cubilin gene (Cubn). Putative PPAR/RXR heterodimer binding sites, identified by Genomatix DiAlign, are indicated. Sequence elements are numbered relative to the translation start site (+1). B, representation of the pCub -431 Luc mouse Cubn promoter construct containing mouse Cubn proximal flanking sequence (-431 to -5), including the -97 putative PPAR/RXR binding element (shaded box), linked to a luciferase cassette. C, an established PPAR reporter construct is transactivated by PPARα and PPARγ in transfected BN cells. Reporter plasmid (pPPRE X3-TK-luc) was cotransfected with either a PPARα expression plasmid (pSG5 PPAR alpha), a PPARγ expression plasmid (pcDNA flag PPAR gamma) or a negative control plasmid (Con). Relative luciferase activity in lysates from transfected cells is shown. D, PPARα and PPARγ transactivate the mouse cubilin proximal promoter. Cotransfections were done with the pCub-431 Luc promoter plasmid and either PPARα, PPARγ, or a negative control plasmid (Con) as described for C. Relative luciferase activity in lysates from transfected cells is shown. E and F, qPCR analysis of cubilin (Cubn) and megalin mRNA in NRK cells cultured in the presence or absence of PPARα or PPARγ antagonists, GW6471 and GW9662 (each at 10 μM) for 24 h. Asterisks indicate p<0.05.