Search

Peptidylarginine deiminase IV (PADI4) posttranslationally converts peptidylarginine to citrulline. It plays an essential role in immune cell differentiation and apoptosis. A haplotype of single-nucleotide polymorphisms (SNPs) in PADI4 is functionally relevant as a rheumatoid arthritis (RA) gene. It could increase enzyme activity leading to raised levels of citrullinated protein and stimulating autoantibody. Previously, our study showed that inducible PADI4 causes haematopoietic cell death. Herein, we further investigate whether RA risk PADI4 haplotype (SNP PADI4; S55G, A82V and A112G) and the increase of its enzymatic activity induce apoptosis. In the tetracycline (Tet)-On Jurkat T cells, ionomycin (Ion) only treatment didn't induce apoptosis however it promoted inducible PADI4-decreased cell viability and -enhanced apoptosis. Through in vitro and in vivo PADI enzyme activity assay, we demonstrated that PADI4 enzyme activity of SNP PADI4 was higher than RA non-risk PADI4 haplotype (WT PADI4). The effect of SNP PADI4-induced apoptosis was superior to WT PADI4. In addition, both Ion and SNP PADI4 synergistically provoked apoptosis were compared with both Ion and WT PADI4. Concurrently, in the conditionally inducible SNP PADI4 cells of Ion treatment-induced apoptosis, not only the expression of Bcl-xL was down-regulated and Bax up-regulated, but also cytochrome c was released from mitochondria to cytoplasm in significant amounts. Western blotting data showed the increase in apoptosomal caspase activation during programmed cell death in the inducible SNP PADI4 cells subsequent to Ion treatment. These data demonstrated that both SNP PADI4 increasing their enzyme activity could enhance apoptosis through the mitochondrial pathway and further provide a conceivable explanation in the pathogenesis of RA following the upregulation of PADI4 activity in its SNPs.

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via posttranslational modification. One member of the family, PADI4, plays an important role in immune cell differentiation and cell death. To elucidate the participation of PADI4 in haematopoietic cell death, we examine whether inducible overexpression of PADI4 enhances the apoptotic cell death. PADI4 reduced the viability in a dose- and time-dependent manner of human leukemia HL-60 cells and human acute T leukemia Jurkat cells. The apoptosis-inducing activities were determined by nuclear condensation, DNA fragmentation, sub-G1 appearance, loss of mitochondrial membrane potential (delta psi(m)), release of mitochondrial cytochrome c into cytoplasm and proteolytic activation of caspase 9 and 3. Following PADI4 overexpression, cells arrest in G1 phase significantly before their entrance into apoptotic cell death. PADI4 increases tumor suppressor p53 and its downstream p21 to control cell cycle. In the detections of protein expression and kinase activity, all protein levels of cyclin-dependent kinases (CDKs) and cyclins are not reduced except cyclin D, however, CDK2 (G1 entry S phase) and CDK1 (G2 entry M phase) enzyme activities are inhibited by conditionally inducible PADI4. p53 also expands its other downstream Bax to induce cytochrome c release from mitochondria. According to these data, we suggest that PADI4 induces apoptosis mainly through cell cycle arrest and mitochondria-mediated pathway. Furthermore, p53 features in PADI4-induced apoptosis by increasing intracellular p21 to control cell cycle and by Bax accumulation to decline Bcl-2 function, destroy delta psi(m), release cytochrome c to cytoplasm and activate the caspase cascade.

Ornithine decarboxylase (ODC) plays an essential role in various biological functions, including cell proliferation, differentiation and cell death. However, how it prevents the cell apoptotic mechanism is still unclear. Previous studies have demonstrated that decreasing the activity of ODC by difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, causes the accumulation of intracellular reactive oxygen species (ROS) and cell arrest, thus inducing cell death. These findings might indicate how ODC exerts anti-oxidative and anti-apoptotic effects. In our study, tumor necrosis factor alpha (TNF-alpha) induced apoptosis in HL-60 and Jurkat T cells. The kinetic studies revealed that the TNF-alpha -induced apoptotic process included intracellular ROS generation (as early as 1 h after treatment), the activation of caspase 8 (3 h), the cleavage of Bid (3 h) and the disruption of mitochondrial membrane potential (Delta psi(m)) (6 h). Furthermore, ROS scavengers, such as glutathione (GSH) and catalase, maintained Delta psi(m) and prevented apoptosis upon treatment. Putrescine and overexpression of ODC had similar effects as ROS scavengers in decreasing intracellular ROS and preventing the disruption of Delta psi(m) and apoptosis. Inhibition of ODC by DFMO in HL-60 cells only could increase ROS generation, but did not disrupt Delta psi(m) or induce apoptosis. However, DFMO enhanced the accumulation of ROS, disruption of Delta psi(m) and apoptosis when cells were treated with TNF-alpha . ODC overexpression avoided the decline of Bcl-2, prevented cytochrome c release from mitochondria and inhibited the activation of caspase 8, 9 and 3. Overexpression of Bcl-2 maintained Delta psi(m) and prevented apoptosis, but could not reduce ROS until four hours after TNF-alpha treatment. According to these data, we suggest that TNF-alpha induces apoptosis mainly by a ROS-dependent, mitochondria-mediated pathway. Furthermore, ODC prevents TNF-alpha -induced apoptosis by decreasing intracellular ROS to avoid Bcl-2 decline, maintain Delta psi(m), prevent cytochrome c release and deactivate the caspase cascade pathway.