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In response to treatment with phorbol-12-myristate-13-acetate (PMA), the half-population of erythromyeloblast D2 cells, a cytokine-independent variant of TF-1 cells, displayed adhesion and differentiated into a monocyte/macrophage-like morphology, while the other half-population remained in suspension and underwent apoptosis. Expression of the cell cycle inhibitor p21Cip1/Waf1 was induced after PMA treatment in the adherent cells but not in the proapoptotic cells. We investigated the mechanism responsible for the impairment of p21Cip1/Waf1 induction in PMA-induced proapoptotic cells. We demonstrated that in PMA-induced adherent cells, upregulation of p21Cip1/Waf1 requires the activation and nuclear translocation of phosphorylated extracellular signal-regulated kinase (phospho-ERK). Although ERK was phosphorylated to comparable levels in PMA-induced proapoptotic and adherent cells, nuclear distribution of phospho-ERK was seen only in the adherent, not in the proapoptotic cells. We also found that only PMA-induced proapoptotic cells contained the phosphorylated form of myosin light chain, which is dependent on Rho-associated kinase (ROCK) activation, and that expression of a dominant-active form of ROCK suppressed activation of the p21Cip1/Waf1 promoter during PMA induction. Finally, we demonstrated that inhibition of ROCK restores nuclear distribution of phospho-ERK and activation of p21Cip1/Waf1 expression. Based on these findings, we propose that a ROCK-mediated signal is involved in interfering with the process of ERK-mediated p21Cip1/Waf1 induction in PMA-induced proapoptotic TF-1 and D2 cells

When exposed to PMA, the erythroblastic cell line TF-1 and its cytokine-independent variant D2 cells can be induced to undergo differentiation and apoptosis. In this study we investigated the mechanism responsible for the differential responses to PMA induction and show that serum present in the medium has a major role in promoting PMA-induced apoptosis in TF-1 and D2 cells. Interestingly, lysophosphatidic acid (LPA) could replace serum to co-operate with PMA in inducing apoptosis via the Rho-dependent pathway. The expression of a constitutively active form of RhoA also increased PMA-induced apoptosis. However, by inhibiting adhesion, most cells underwent PMA-induced apoptosis even in the absence of LPA or serum, indicating that adhesion is required for PMA-induced differentiation. Given that LPA could prevent adhesion for cells maintained in the serum-free medium, here we propose that RhoA has a switching role in determining whether TF-1 and D2 cells undergo differentiation or apoptosis in response to PMA, by modulating cell adhesion.

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