Targeting of aminopeptidase N to bile canaliculi correlates with secretoryactivities of the developing canalicular domain

We have used human hepatoma cell lines as an in vitro model to study the de velopment of hepatic bile canaliculi (BC). Well-differentiated hepatoma cel ls cultured for 72 hours could develop characteristic spheroid structures a t sites of cell-cell contact that contained tight junctions and various mem brane protein markers, resembling BC found in vivo. Intact cytoskeleton was essential for this differentiation process. In the coculture experiments i n which cells of different origins were populated together, BC only formed between hepatic cells and preferentially among well-differentiated cells. P oorly differentiated hepatoma cells never formed BC among themselves, but c ould be induced to undergo canalicular differentiation by interacting with well-differentiated cells. During BC morphogenesis, integral canalicular me mbrane proteins were gradually delivered and accumulated at the developing BC. Among them, targeting of aminopeptidase N (APN) seemed to correlate wit h activation of certain secretory functions. Specifically, only APN-positiv e BC supported excretion of fluorescein diacetate (FDA) and 70-kd dextran, but had no relationship with secretion of horseradish peroxidase (HRP), Tar geting of another BC protein, dipeptidyl peptidase IV (DPPIV), on the other hand, bore no association with any secretory activity examined. In additio n, inhibition of enzymatic activity of APN could perturb canalicular differ entiation without affecting cell proliferation. Our results suggest that ta rgeting of APN proteins may reflect or even play an important role in the d evelopment and functional maturation of the canalicular structures.