Distinct Mechanisms Regulate TIMP-1 Expression at Different Stages of Phorbol Ester-Mediated Differentiation of U937 Cells

Abstract

Upon exposure to 12-O-tetradecanoylphorbol 13-acetate (PMA), promonocyte-like U937 cells differentiate into macrophage-like cells and begin to express certain metalloproteinases and TIMP-1. We report here that distinct mechanisms regulate TIMP-1 production in PMA-treated U937 cells. TIMP-1 protein and steady-state mRNA levels increased about 10-fold in PMA-differentiated cells compared to undifferentiated cells. TIMP-1 transcription increased about 2.5-fold, but this stimulation was not detected until at least 48 h post-PMA. In contrast, the half-life for TIMP-1 mRNA increased about 3-fold and was detected at 8 h post-PMA. Using in vitro translation assays, we found that TIMP-1 mRNA from PMA-differentiated cells translated about 5-fold less efficiently than that from basal cells, suggesting structural differences in TIMP-1 mRNA in basal and differentiated U937 cells. Although primer extension and RNase protection analyses showed 5‘ heterogeneity of TIMP-1 transcripts, all forms were equally stimulated in response to PMA-mediated differentiation. The poly(A) tail length of TIMP-1 mRNA, however, was longer in PMA-treated cells. Our findings suggested that up-regulation of TIMP-1 expression in PMA-treated U937 cells is mediated early by enhanced TIMP-1 mRNA stability, possibly related to increased poly(A) tail length, and later by an increase in transcription rate.