Abstract
Treatment of cells with phorbol ester, phorbol-12-myristate-13-acetate (PMA), triggers differentiation or apoptosis, depending on the cell type. In this study, we used an erythroblastic cell line, TF-1, to investigate the molecular mechanism that determines the cell fate in response to PMA exposure. Upon PMA treatment in the presence of serum or lysophosphatidic acid (LPA), TF-1 cells exhibited contraction followed by apoptosis. By contrast, under serum-free conditions, cells became adherent and survived after PMA treatment. Here, we show that the pathway of Rho kinase (ROCK)/myosin light chain (MLC) phosphorylation/myosin-mediated contraction was activated in PMA-induced apoptotic cells in serum-containing medium, but not in the adherent and survived cells. Pretreatment of cells with a specific ROCK inhibitor, Y27632, not only abrogated MLC phosphorylation and membrane contraction, but also prevented PMA-induced activation of caspase-3 and subsequent cell death, indicating that ROCK-dependent myosin-mediated contraction elicits an upstream signal required for caspase-3 activation in PMA-induced apoptosis. Interestingly, we further found that caspases-8 and -10 are the initiator caspases in PMA-induced apoptosis and a ROCK-dependent enhancement of specific complex formation between the Fas-associated death domain (FADD) and pro-caspase-10 in pro-apoptotic cells. In summary, these results revealed that, following PMA treatment, the upregulation of the RhoA/ROCK pathway contributes to a cellular context that switches-on myosin-mediated contraction, which provides a mechanism for triggering apoptotic induction mediated by caspase-8 and -10.
Highlights
Coordination and balance between cell survival and apoptosis is crucial for the normal development and homeostasis of multicellular organisms throughout adult life
On the basis of these data, we propose that activation of the RhoA/ROCK/myosin light chain (MLC) phosphorylation pathway in cooperation with PMA signaling in cells provides a cellular context that generates an initial membrane contraction, which in turn leads to activation of caspase-8 and -10 through a mechanism involving membrane receptor-mediated signaling
PMA-induced membrane contraction and MLC phosphorylation precede caspase-3 activation in TF-1 cells When exposed to PMA in serum-containing medium for 12 hours, TF-1 cells became shrunk and non-viable
Summary
Coordination and balance between cell survival and apoptosis is crucial for the normal development and homeostasis of multicellular organisms throughout adult life. Some cells commit to differentiation and others undergo renewal or apoptosis to maintain homeostasis. When exposed to external stimulation, such as phorbol-12-myristate13-acetate (PMA), which is an activator of protein kinase C (PKC), hematopoietic cells are induced to differentiate or undergo apoptosis depending on the cell type (Collins, 1987; Day et al, 1994; Garzotto et al, 1998; Gunji et al, 1992; Kizaki et al, 1989; Lotem et al, 1991). We have previously shown that lysophosphatidic acid (LPA) or serum promotes PMA-induced apoptosis in TF-1 cells by the RhoA-dependent pathway, whereas following PMA stimulation in serum-free medium, TF-1 cells survive and attach to the culture flask (Lai et al, 2001). We used this system to investigate further the detailed molecular mechanism by which the RhoA signal pathway is utilized to trigger the apoptotic process during PMA treatment
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