Regulation of vimentin expression and protease-mediated vimentin degradation during differentiation of human monocytic leukemia cells.
Terminal differentiation of human monocytic leukemia THP-1 cells is induced in vitro by 12-O-tetradecanoylphorbol-13-acetate (TPA). We investigated the effects of TPA on the expression of vimentin during the differentiation of THP-1 cells at both the mRNA and the protein level. On northern blotting analysis, a 2.1 kb vimentin mRNA was up-regulated by TPA. On western blotting, small vimentin molecules with a molecular mass of approximately 40 kDa were observed in the soluble fraction and increased with TPA-induction of cellular differentiation. Since larger, including intact, vimentin molecules were detectable at a high TPA dose, we assessed the possible existence of protease activity directed against vimentin in THP-1 cells. With incubation of the cellular lysates of THP-1 cells, the endogenous vimentin became increasingly smaller over time, suggesting the presence of a vimentin-degrading protease. Phenylmethylsulfonyl fluoride inhibited this apparent protease activity against vimentin, suggesting the enzyme involved to be a serine protease. Interestingly, the protease activity was down-regulated by TPA treatment. TPA-treated THP-1 cells were found to express a vimentin-filament network based on immunocytochemical analysis using an anti-vimentin monoclonal antibody, V9. Taken together, these observations suggest that post-translational mechanisms work in cooperation with transcriptional regulation to maintain the vimentin-intermediate filament structure in differentiated THP-1 cells.
Honke
K
K
Department of Molecular Medicine, Osaka Medical Center for Maternal and Child Health.
Wada
Y
Y
D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D010450:Endopeptidases / Q000378:metabolism*; D015972:Gene Expression Regulation, Neoplastic; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D007948:Leukemia, Monocytic, Acute / Q000378:metabolism / Q000473:pathology*; D008856:Microscopy, Fluorescence; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014746:Vimentin / Q000235:genetics* / Q000378:metabolism*
D012333:RNA, Messenger; D014746:Vimentin; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010450:Endopeptidases; D013755:Tetradecanoylphorbol Acetate