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Apoptosis inhibitory activity of cytoplasmic p21(Cip1/WAF1) in monocytic differentiation
1Department of Virology, The National Children's Medical Research Center, 3-35-31, Taishido, Setagaya-ku, Tokyo, 154, Japan.
Abstract:
p21(Cip1/WAF1) inhibits cell-cycle progression by binding to G1 cyclin/CDK complexes and proliferating cell nuclear antigen (PCNA) through its N- and C-terminal domains, respectively. The cell-cycle inhibitory activity of p21(Cip1/WAF1) is correlated with its nuclear localization. Here, we report a novel cytoplasmic localization of p21(Cip1/WAF1) in peripheral blood monocytes (PBMs) and in U937 cells undergoing monocytic differentiation by in vitro treatment with vitamin D3 or ectopic expression of p21(Cip1/WAF1), and analyze the biological consequences of this cytoplasmic expression. U937 cells which exhibit nuclear p21(Cip1/WAF1) demonstrated G1 cell-cycle arrest and subsequently differentiated into monocytes. The latter event was associated with a cytoplasmic expression of nuclear p21(Cip1/WAF1), concomitantly with a resistance to various apoptogenic stimuli. Biochemical analysis showed that cytoplasmic p21(Cip1/WAF1) forms a complex with the apoptosis signal-regulating kinase 1 (ASK1) and inhibits stress-activated MAP kinase cascade. Expression of a deletion mutant of p21(Cip1/WAF1) lacking the nuclear localization signal (DeltaNLS-p21) did not induce cell cycle arrest nor monocytic differentiation, but led to an apoptosis-resistant phenotype, mediated by binding to and inhibition of the stress-activated ASK1 activity. Thus, cytoplasmic p21(Cip1/WAF1) itself acted as an inhibitor of apoptosis. Our findings highlight the different functional roles of p21(Cip1/WAF1), which are determined by its intracellular distribution and are dependent on the stage of differentiation.
Insights
The protein p21 (also known as Cip1/WAF1) can inhibit cell division when in the nucleus or prevent cell death when in the cytoplasm. Its location determines its function in cell differentiation and apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Cancer Research
Background:
- p21(Cip1/WAF1) is a key regulator of the cell cycle, typically inhibiting progression by binding to cyclin/CDK complexes.
- Nuclear localization of p21(Cip1/WAF1) is traditionally associated with its cell-cycle inhibitory function.
- The role of p21(Cip1/WAF1) in cytoplasmic compartments and its impact on cellular processes remain less understood.
Purpose of the Study:
- To investigate the novel cytoplasmic localization of p21(Cip1/WAF1) in monocytes and U937 cells.
- To analyze the biological consequences of cytoplasmic p21(Cip1/WAF1) expression during monocytic differentiation.
- To elucidate the mechanism by which cytoplasmic p21(Cip1/WAF1) influences apoptosis and cell survival.
Main Methods:
- Induction of monocytic differentiation in U937 cells using vitamin D3 or ectopic p21(Cip1/WAF1) expression.
- Analysis of p21(Cip1/WAF1) localization (nuclear vs. cytoplasmic) using biochemical and cellular assays.
- Investigation of cell-cycle progression, differentiation, and apoptosis resistance.
- Biochemical analysis of protein complex formation, specifically involving p21(Cip1/WAF1) and ASK1.
- Utilizing a deletion mutant (DeltaNLS-p21) to assess the role of nuclear localization signal.
Main Results:
- A novel cytoplasmic localization of p21(Cip1/WAF1) was observed in peripheral blood monocytes (PBMs) and differentiating U937 cells.
- Nuclear p21(Cip1/WAF1) induced G1 cell-cycle arrest and monocytic differentiation.
- Cytoplasmic p21(Cip1/WAF1) was associated with resistance to apoptosis and formed a complex with ASK1, inhibiting the stress-activated MAP kinase cascade.
- A mutant lacking nuclear localization (DeltaNLS-p21) promoted apoptosis resistance by inhibiting ASK1, without inducing cell cycle arrest or differentiation.
Conclusions:
- Intracellular distribution of p21(Cip1/WAF1) dictates its distinct functional roles.
- Cytoplasmic p21(Cip1/WAF1) acts as a direct inhibitor of apoptosis by modulating the ASK1/MAPK pathway.
- The stage of cellular differentiation influences the localization and functional outcomes of p21(Cip1/WAF1).