Regulation of Vimentin Expression and Protease‐mediated Vimentin Degradation during Differentiation of Human Monocytic Leukemia Cells

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Abstract
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‐fUament 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 vimenti intermediate filament structure in differentiated THP‐1 cells.