REGULATION OF LYSOSOMAL AND UBIQUITIN DEGRADATIVE PATHWAYS IN DIFFERENTIATING HUMAN INTESTINAL CACO-2 CELLS

The expression of various components of the lysosomal and ubiquitin-de pendent degradative pathways was characterized in an in vitro model of differentiating enterocytes, the human colon adenocarcinoma Caco-2 ce ll line. The activities of the cell-associated lysosomal enzymes alpha -D-mannosidase, beta-hexosaminidase, beta-glucuronidase, and beta-gala ctosidase increased similar to 2- to 4-fold as differentiation proceed ed. In contrast, the protein levels of the two mannose 6-phosphate rec eptors (MPRs), the insulin like growth factor II/cation-independent MP R (IGF-II/CI-MPR) and the cation-dependent MPR (CD-MPR), did not chang e significantly during Caco-2 differentiation. In addition, quantitati ve Western blot analyses revealed that on a molar basis the CD-MPR is 3.5 times more abundant than the IGF-II/CI-MPR in Caco-2 cells. Since only limited secretion of lysosomal enzymes was observed throughout di fferentiation, the level of expression of the MPRs was sufficient to t arget the increased levels of lysosomal enzymes to the lysosome. Unlik e the expression of lysosomal enzymes, Western blot analysis demonstra ted an similar to 40% and similar to 30% decrease, respectively, in th e steady-state levels of free and conjugated ubiquitin during Caco-2 d ifferentiation. Taken together, these results show that the ubiquitin- dependent proteolytic pathway is regulated differently than the lysoso mal degradative pathway during Caco-2 differentiation.