| Human organic anion transporter 3 (hOAT3) belongs to a family of organic anion transporters that play critical roles in the body disposition of numerous clinically important drugs. Therefore, understanding the regulation of this transporter has profound clinical significance. In the current study, we investigated the short-term and long-term regulation of hOAT3 by protein kinase C (PKC). We showed that short-term activation of PKC by phobol 12-Myristate 13-Acetate (PMA) inhibited hOAT3 activity through accelerating its internalization from cell surface to intracellular recycling endosomes. The colocalization of hOAT3 with EEA1-positive recycling endosomes was demonstrated by immunolocalization with confocal microscopy. Furthermore, we showed that long-term activation of PKC resulted in the enhanced degradation of cell surface hOAT3. The pathways for hOAT3 degradation were further examined using proteasomal and lysosomal inhibitors. Our results showed that both proteasomal inhibitors and the lysosomal inhibitors significantly blocked hOAT3 degradation. These results demonstrate that PKC plays critical roles in the trafficking and the stability of hOAT3. I established five stable transfected cell lines. Those are COS-7 cells stably expressing hOAT3-c-myc, hOAT4-c-myc and HEK293 cells stably expressing hOAT1-c-myc, hOAT3-c-myc, hOAT4-c-myc. The stable transfected cell lines ensure long term and reproducible specific gene expression because it is accomplished by integration of transfected certain DNA of interest into cell's chromosome. By using HEK293 cells stably expressing hOAT4-c-myc I established, I did anti-cancer drug library screening. The purpose of this experiment is the investigation of anti-cancer drug which has function to inhibit hOAT4. |
