Abstract
Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. However, the physiological mechanisms underlying cellular quiescence remain largely unknown. In the present study, we used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We found that Vmem is hyperpolarized and that the intracellular chloride concentration is high in diapause embryos, whereas Vmem is depolarized and intracellular chloride concentration is reduced in postdiapause embryos and during further embryonic development. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Ar-CFTR knockdown and GlyH-101-mediated chemical inhibition of Ar-CFTR produced diapause embryos having a high Vmem and intracellular chloride concentration, whereas control Artemia embryos released free-swimming nauplius larvae. Transcriptome analysis of embryos at different developmental stages revealed that proliferation, differentiation, and metabolism are suppressed in diapause embryos and restored in postdiapause embryos. Combined with RNA sequencing (RNA-Seq) of GlyH-101-treated MCF-7 breast cancer cells, these analyses revealed that CFTR inhibition down-regulates the Wnt and Aurora Kinase A (AURKA) signaling pathways and up-regulates the p53 signaling pathway. Our findings provide insight into CFTR-mediated regulation of cellular quiescence and Vmem in the Artemia model.
Highlights
Cellular quiescence, a reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance
The present study demonstrates that Vmem was hyperpolarized and the intracellular chloride concentration was high in diapause embryos, whereas Vmem was depolarized and the intracellular chloride concentration was reduced in postdiapause embryos and during further embryonic development
Western blotting analysis showed that expression of cell proliferation markers, phosphorylation histone H3 at Ser10 and phosphorylation of retinoblastoma at Thr356, was greatly reduced in diapause and postdiapause embryos (Fig. 1B)
Summary
A reversible state in which growth, proliferation, and other cellular activities are arrested, is important for self-renewal, differentiation, development, regeneration, and stress resistance. We used embryos of the crustacean Artemia in the diapause stage, in which these embryos remain quiescent for prolonged periods, as a model to explore the relationship between cell-membrane potential (Vmem) and quiescence. We identified and characterized the chloride ion channel protein cystic fibrosis transmembrane conductance regulator (CFTR) of Artemia (Ar-CFTR) and found that its expression is silenced in quiescent cells of Artemia diapause embryos but remains constant in all other embryonic stages. Hyperpolarization of the resting Vmem is accompanied by decreased proliferation of astrocytes isolated from the developing spinal cord and hippocampus of rats [13, 14] These observations led us to speculate that Vmem plays a critical role in cellular quiescence.
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