p53, the most commonly mutated tumor suppressor gene in human cancers, is a master regulator of apoptosis in many types of cells. Recently, protein phosphatase-1 (PP1) has emerged as a key phosphatase of p53, which modulates the interaction of p53 with its regulatory protein mouse double minute 2 (MDM2) and transcriptional activity. In the present study, we demonstrate the potential role of PP1 nuclear targeting subunit (PNUTS) in regulating the phosphorylation and apoptotic activities of p53. Hypoxia significantly increased mRNA and protein expression of PNUTS in various cell lines concomitantly with increases in p53. Promoter analysis confirmed the presence of hypoxia response elements in the promoter region of the PNUTS gene, which respond to hypoxia and forced expression of hypoxia-inducible factor 1 alpha. Overexpression of PNUTS markedly increased cell death in response to hypoxia, with increased expression of Bax, an apoptosis-related gene induced by p53. Consistently, PNUTS increased the nuclear localization, phosphorylation, and transcriptional activity of p53 as well as the ubiquitin-dependent proteosomal degradation of MDM2. However, the W401A mutant form of PNUTS, which is incapable of binding to PP1, failed to induce these events. Taken together, our findings suggest that PNUTS may play an important role in controlling cell death in response to cellular stresses such as hypoxia through the post-translational modification of p53 and MDM2.

The enzymatic activity of phospholipase D (PLD) is known to be essential for cell survival and protection from apoptosis. However, the mechanisms regulating PLD activity during apoptosis remain unknown. Here we report that cleavage of PLD1 by caspases facilitates p53-mediated apoptosis. Cleavage of PLD1 into an N-terminal fragment (NF-PLD1) and a C-terminal fragment at the amino-acid sequence, DDVD 545 , led to a reduction in PLD1 activity. However, a caspase-resistant mutant form of PLD1 retained significant levels of enzymatic activity and apoptotic function as compared to wild-type PLD1. Exogenous NF-PLD1 expression induced apoptosis through a dominant-negative effect on the activity of endogenous PLD1. During apoptosis, a small fraction of PLD1 is cleaved by caspases in a p53-independent manner and NF-PLD1 amplifies apoptotic signaling through inhibition of the remaining PLD1 activity. As PLD1 suppresses the ATM-Chk2-p53 pathway, elimination of PLD1 activity through NF-PLD1 or si-RNA against PLD1 increases apoptosis in a p53-dependent manner. Taken together, our results reveal that cleavage of PLD1 by caspases promotes apoptosis via modulation of the p53-dependent cell death pathway.

Due to an imbalance in the MMP:TIMP ratio determined a tissue damage in arthritis, it is hypothesized that polymorphic variations of the TIMP genes are associated with regulation of the MMP:TIMP balance. To test this hypothesis, the presence of single nucleotide polymorphisms (SNPs) located in the human TIMP-2 and TIMP-4 genes was confirmed in the Korean RA and OA patients. We performed a case-control study comprising 109 unrelated Korean OA patients, 177 unrelated Korean RA patients and 175 healthy subjects. There were statistically significant differences in the genotype distribution and allele frequencies of the C/T polymorphism of TIMP-4 gene between OA and control groups (P = 0.0002 and P = 0.001, respectively). However, no significant association between TIMP-2 polymorphisms and OA was observed. Also, no difference was observed when allele or genotype frequencies of both TIMP-2 and TIMP-4 gene polymorphisms were compared between RA and controls. We demonstrated that the C/T polymorphism which is located on the 3'-untranslational regions of the TIMP-4 gene might be associated with susceptibility to OA patients.