Abstract
Neutrophils are deprived of proliferative capacity and have a tightly controlled lifespan to avoid their persistence at the site of injury. We have recently described that the proliferating cell nuclear antigen (PCNA), a nuclear factor involved in DNA replication and repair of proliferating cells, is a key regulator of neutrophil survival. In neutrophils, PCNA was localized exclusively in the cytoplasm due to its nuclear-to-cytoplasmic relocalization during granulocytic differentiation. We showed here that leptomycin B, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin, inhibited PCNA relocalization during granulocytic differentiation of HL-60 and NB4 promyelocytic cell lines and of human CD34(+) primary cells. Using enhanced green fluorescent protein fusion constructs, we have demonstrated that PCNA relocalization involved a nuclear export signal (NES) located from Ile-11 to Ile-23 in the PCNA sequence. However, this NES, located at the inner face of the PCNA trimer, was not functional in wild-type PCNA, but instead, was fully active and leptomycin B-sensitive in the monomeric PCNAY114A mutant. To test whether a defect in PCNA cytoplasmic relocalization would affect its antiapoptotic activity in mature neutrophils, a chimeric PCNA fused with the SV40 nuclear localization sequence (NLS) was generated to preclude its cytoplasmic localization. As expected, neutrophil-differentiated PLB985 cells expressing ectopic SV40NLS-PCNA had an increased nuclear PCNA as compared with cells expressing wild-type PCNA. Accordingly, the nuclear PCNA mutant did not show any antiapoptotic activity as compared with wild-type PCNA. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES.
Highlights
proliferating cell nuclear antigen (PCNA) is exclusively cytoplasmic in neutrophils and inhibits their apoptosis
We examined whether LMB, an inhibitor of the chromosome region maintenance 1 (CRM1) exportin that requires a leucine-rich sequence referred to as an nuclear export signal (NES) [20], could increase nuclear localization of PCNA during alltrans-retinoic acid (ATRA)-induced granulocytic differentiation in the promyelocytic NB4 cells
We previously reported that redistribution of PCNA from the nucleus to the cytoplasm occurred during granulocytic differentiation of human CD34ϩ primary cells [7]
Summary
PCNA is exclusively cytoplasmic in neutrophils and inhibits their apoptosis. Results: Nuclear export of PCNA is mediated by a CRM1-dependent nuclear export sequence (NES) and is pivotal for its antiapoptotic activity in mature neutrophils. Nuclear-to-cytoplasmic relocalization that occurred during myeloid differentiation is essential for PCNA antiapoptotic activity in mature neutrophils and is dependent on the newly identified monomerization-dependent PCNA NES. PCNA overexpression rendered neutrophil-differentiated PLB985 cells significantly more resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or gliotoxin-induced apoptosis, and PCNA siRNA sensitized them to apoptosis [7] These results identify cytoplasmic PCNA as a key regulator of neutrophil lifespan, in a manner independent of the cell cycle, exerting its antiapoptotic activity by associating to procaspases to prevent their activation [7]. We investigated whether modulation of PCNA nuclear-tocytoplasmic relocalization would directly affect granulocytic differentiation or its antiapoptotic activity in mature neutrophils
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