Cell volume regulation is a vital system for cellular activities. When perturbed by hypoosmotic or hyperosmotic stress, cells immediately induce the cell volume recovery system, regulatory volume decrease (RVD) or regulatory volume increase (RVI), respectively. In contrast to the knowledge about effector molecules, the molecular mechanisms linking osmosensing to RVD/RVI induction remain unknown. Additionally, few reciprocal responders in the bidirectional osmotic stress response have been identified. We previously reported that ASK3 bidirectionally switches its kinase activity under osmotic stress. Herein we demonstrate that ASK3 controls both RVD and RVI under osmotic stress. Using a high-content genome-wide small interfering RNA (siRNA) screen, we identify PP6 as a direct ASK3 inactivator. Furthermore, PP6 rapidly interacts with ASK3 in an osmolality-dependent manner, and it inactivates ASK3 to induce RVI and, thereby, cell survival under hyperosmotic stress. These findings suggest that the PP6-ASK3 interaction is a core module in the bidirectional osmotic stress response.

Human promyelocytic HL-60 cells can be differentiated into macrophage-like cells by treatment with 12-O-tetra decanoylphorbol-13-acetate (TPA). Certain 5' upstream regions of the zinc finger protein (ZNF)-encoding genes contain duplicated GGAA motifs, which are frequently found in the TPA-responding gene promoter regions. To examine transcriptional responses to TPA, 5'flanking regions of human zinc finger CCCH-type containing, antiviral, ZNF252, ZNF343, ZNF555, ZNF782 and zinc finger nfx-1-type containing 1 (ZNFX1) genes were isolated by polymerase chain reaction (PCR) and ligated into a multiple-cloning site of the pGL4.10[luc2] vector. Transient transfection and a luciferase assay revealed that the ZNFX1 promoter most prominently responded to the TPA treatment. Deletion and point mutation experiments indicated that the duplicated GGAA motif in the 100-bp region positively responded to TPA. In addition, reverse transcription-quantitative PCR and western blotting showed that the mRNA and protein of ZNFX1 accumulate during the differentiation of HL-60 cells. These results indicated that expression of the TPA-inducible ZNFX1 gene, which belongs to the group of interferon-responsive genes, is regulated by the cis-action of the duplicated GGAA motif.