Expression Of Peptidylarginine Deiminase 4 Research Articles

ATP induces PAD4 in renal proximal tubule cells via P2X7 receptor activation to exacerbate ischemic AKI.

We previously demonstrated that renal tubular peptidylarginine deiminase-4 (PAD4) is induced after ischemia-reperfusion (IR) injury and this induction of PAD4 exacerbates ischemic acute kidney injury (AKI) by promoting renal tubular inflammation and neutrophil infiltration. However, the mechanisms of renal tubular PAD4 induction after IR remain unknown. Here, we tested the hypothesis that ATP, a proinflammatory danger-associated molecular pattern (DAMP) ligand released from necrotic cells after IR injury, induces renal tubular PAD4 and exacerbates ischemic AKI via P2 purinergic receptor activation. ATP as well as ATPγS (a nonmetabolizable ATP analog) induced PAD4 mRNA, protein, and activity in human and mouse renal proximal tubule cells. Supporting the hypothesis that ATP induces renal tubular PAD4 via P2X7 receptor activation, A804598 (a selective P2X7 receptor antagonist) blocked the ATP-mediated induction of renal tubular PAD4 whereas BzATP (a selective P2X7 receptor agonist) mimicked the effects of ATP by inducing renal tubular PAD4 expression and activity. Moreover, ATP-mediated calcium influx in renal proximal tubule cells was blocked by A804598 and was mimicked by BzATP. P2X7 activation by BzATP also induced PAD4 expression and activity in mouse kidney in vivo. Finally, supporting a critical role for PAD4 in P2X7-mediated exacerbation of renal injury, BzATP exacerbated ischemic AKI in PAD4 wild-type mice but not in PAD4-deficient mice. Taken together, our studies show that ATP induces renal tubular PAD4 via P2X7 receptor activation to exacerbate renal tubular inflammation and injury after IR.

Expressions and clinicopathological significances of BMI-1 and PADI4 in esophageal squamous cell carcinoma

Objective To evaluate the expression levels of peptidylarginine deiminase 4 (PADI4) and B-cells pecific Moloney leukemia virus insert site-1 (BMI-1) in esophageal squamous cell carcinoma (ESCC) tissues and pericarcinous tissues. To explore the function and clinical significance in the development of ESCC and their association. Methods The expression levels of PADI4 and BMI-1 were measured by immunohistochemistry, Western blotting and quantitative real time PCR in ESCC tissues and pericarcinous tissues from 86 patients. The relationships between the expressions of PADI4 and BMI-1 and the clinicopathologic characte-ristics were analyzed. Results The immunohistochemistry showed that the expressions of PADI4 and BMI-1 in ESCC tissues (68.6% and 73.3%) were significantly higher than those in pericarcinous tissues (37.2% and 30.2%, χ2=17.011, P=0.000; χ2=31.876, P=0.000). Western blotting indicated that the levels of PADI4 and BMI-1 were higher than those in pericarcinous tissues (0.919±0.098 vs. 0.718±0.103, t=2.462, P=0.021; 0.975±0.074 vs. 0.717±0.071, t=2.640, P=0.014). The expressions of BMI-1 and PADI4 mRNA in ESCC tissues were higher than those in pericarcinous tissues, but the differences were not statistically significant (0.091±0.005 vs. 0.038±0.002, t=1.701, P=0.101; 0.114±0.075 vs. 0.048±0.003, t=1.499, P=0.146) by the quantitative real time PCR. The expression of PADI4 was correlated with lymph node metastasis (χ2=5.771, P=0.016), depth of invasion (χ2=6.672, P=0.010) and clinical stage (χ2=5.771, P=0.016). The BMI-1 gene expression had a correlation with lymph node metastasis (χ2=7.176, P=0.007), the differentiation degree (χ2=13.787, P=0.001) and clinical stage (χ2=7.176, P=0.007). In addition, there was a positive correlation between PADI4 and BMI-1 expression in ESCC by immunohistochemistry and quantitative real time PCR (r=0.214, P=0.047; r=0.534, P=0.005). Conclusion The expression levels of PADI4 and BMI-1 are significantly higher in ESCC compared to pericarcinous tissues. PADI4 and BMI-1 are positively correlated and may contribute to the diagnosis and prognosis of the ESCC. Key words: Esophageal neoplasms; Peptidylarginine deiminase 4; B-cells pecific Moloney leukemia virus insert site-1

Peptidyl arginine deiminase-4-deficient mice are protected against kidney and liver injury after renal ischemia and reperfusion.

We previously demonstrated that renal peptidyl arginine deiminase-4 (PAD4) is induced after renal ischemia and reperfusion (I/R) injury and exacerbates acute kidney injury (AKI) by increasing the renal tubular inflammatory response. Here, we tested whether genetic ablation of PAD4 attenuates renal injury and inflammation after I/R in mice. After renal I/R, PAD4 wild-type mice develop severe AKI with large increases in plasma creatinine, neutrophil infiltration, as well as significant renal tubular necrosis, apoptosis, and proinflammatory cytokine generation. In contrast, PAD4-deficient mice are protected against ischemic AKI with reduced real tubular neutrophil infiltration, renal tubular necrosis, and apoptosis. In addition, hepatic injury and inflammation observed in PAD4 wild-type mice after renal I/R are significantly attenuated in PAD4-deficient mice. We also show that increased renal tubular PAD4 expression after renal I/R is associated with translocation of PAD4 from the nucleus to the cytosol. Consistent with PAD4 cytosolic translocation, we show increased renal tubular cytosolic peptidyl-citrullination after ischemic AKI. Mechanistically, recombinant PAD4 treatment increased nuclear translocation of NF-κB in cultured human as well as murine proximal tubule cells that is inhibited by a PAD4 inhibitor (2-chloroamidine). Taken together, our studies further support the hypothesis that renal tubular PAD4 plays a critical role in renal I/R injury by increasing the renal tubular inflammatory response and neutrophil infiltration after renal I/R perhaps by interacting with the proinflammatory transcription factor NF-κB in the cytosol and promoting its nuclear translocation.

Abstract 24: NETosis is Associated with Abdominal Aortic Aneurysm Rupture

Objectives: Abdominal aortic aneurysm (AAA) rupture (RAAA) is associated with high mortality. Intraluminal thrombus and associated inflammation have been implicated in RAAA. We sought to characterize neutrophil extracellular trap (NET) formation, or NETosis, associated with a rat RAAA model that we previously developed. Methods: To stimulate AAA formation, infrarenal abdominal aortas of male SD rats were infused with porcine pancreatic elastase (PPE): active (AAA, n=21) and heat-inactivated (control, n=14). Aortas were harvested 3, 6, and 14d post PPE exposure. To stimulate RAAA, a separate group of animals was administered β-aminopropionitrile (BAPN, n=12) daily. Those animals that did not rupture (NRAAA-14d, n=6) were harvested 14d post PPE exposure. Paraffin embedded sections were analyzed using immunofluorescence. To evaluate evidence of primarily neutrophil activity and NET generation, myeloperoxidase (MPO) and peptidylarginine deiminase 4 (PAD4) were quantified utilizing Cy3 and Alexa-488 secondary antibodies, respectively. To determine the presence of NETs, citrullinated histone H3 (H3Cit, Cy3) was visualized. DAPI was utilized to detect nuclei. Aortic sections were imaged with fluorescent microscopy, and quantification of percent area stained (PAS) was performed by threshold analysis. Results: Greater MPO PAS was observed for AAA-6d compared with control-6d (p=0.03). Greater PAD4 PAS was observed for AAA-3d (p=0.02) and -6d (p=0.05) compared with control-3d and -6d, respectively. Greater H3Cit PAS was observed for AAA-6d (p<0.0001) and -14d (p=0.001) compared with control-6d and -14d, respectively. In the setting of rupture stimulation, greater H3Cit PAS was observed for RAAA compared with AAA-14d (p=0.001) and NRAAA-14d (p=0.0002). Conclusions: Increased PAD4 expression by AAAs compared with controls at 3 and 6d post PPE exposure, results in increased H3Cit expression at 6 and 14d post PPE exposure. Comparable H3Cit expression by AAA-6d and RAAA demonstrates the acute nature of this rupture model; however, greater H3Cit expression by RAAAs suggests a role for NETosis in RAAA. Further evaluation of NETosis as it pertains to human RAAA is necessary. Therapeutic inhibition of NETosis may slow AAA progression and prevent RAAA.

Expression of peptidylarginine deiminase 4 and protein tyrosine phosphatase nonreceptor type 22 in the synovium of collagen-induced arthritis rats.

To study the expression level of peptidylarginine deiminase 4 (PADI4) and protein tyrosine phosphatase nonreceptor type 22 (PTPN22) in the synovium of rat model of collagen-induced arthritis, and to explore their possible therapeutic role in rheumatoid arthritis. Thirty-two female Wistar rats weighing 100±20 g were randomly assigned into 3-week collagen-induced arthritis (CIA) model group (n=8), 4-week CIA model group (n=8), 6-week CIA model group (n=8), and the control group (n=8). The body weight changes of each group were recorded. The expression levels of PADI4 and PTPN22 were detected and compared by the methods of immunohistochemical staining and Western blot. Arthritis of rat began to form 14 days after sensitization and the joint swelling reached peak at 28 days. The weights of the rats slowly grew both in CIA model groups and the control group. Immunohistochemical staining results showed that the positive expression of PADI4 and PTPN22 was mainly located in cartilage peripheral mononuclear cells, the cytoplasm of infiltrated cells, and bone marrow cavity. There were significant differences in the optical density of PADI4 and PTPN22 among CIA model groups and the control group (PADI4, 0.2898±0.012, 0.2982±0.022, 0.2974±0.031, 0.2530±0.013 in 3-week CIA model, 4-week CIA model, 6-week CIA model and control groups; PTPN22, 0.2723±0.004, 0.2781±0.010, 0.2767±0.008, 0.2422±0.019; all P <0.05). The expression bands of PADI4 were observed in Western blot 3 weeks after initial immunization, the thickest in the 4th week, and decreased in the 6th week. The expression bands of PTPN2 were observed at all the time points, with no obvious time-dependent trend. PADI4 and PTPN22 are obviously correlated with CIA in rat model. PADI4 is expressed at early stage of the disease, while the expression of PTPN22 sustains throughout the course.

PAD4 regulates proliferation of multipotent haematopoietic cells by controlling c-myc expression

Peptidylarginine deiminase 4 (PAD4) functions as a transcriptional coregulator by catalyzing the conversion of histone H3 arginine residues to citrulline residues. Although the high level of PAD4 expression in bone marrow cells suggests its involvement in haematopoiesis, its precise contribution remains unclear. Here we show that PAD4, which is highly expressed in lineage− Sca-1+ c-Kit+ (LSK) cells of mouse bone marrow compared with other progenitor cells, controls c-myc expression by catalyzing the citrullination of histone H3 on its promoter. Furthermore, PAD4 is associated with lymphoid enhancer-binding factor 1 and histone deacetylase 1 at the upstream region of the c-myc gene. Supporting these findings, LSK cells, especially multipotent progenitors, in PAD4-deficient mice show increased proliferation in a cell-autonomous fashion compared with those in wild-type mice. Together, our results strongly suggest that PAD4 regulates the proliferation of multipotent progenitors in the bone marrow by controlling c-myc expression.

Extracellular Chromatin Traps Interconnect Cell Biology, Microbiology, and Immunology

Extracellular Chromatin Traps Interconnect Cell Biology, Microbiology, and Immunology

Negative Regulation of NIP45 Function by Peptidylarginine Deminase 4 (87.28)

Abstract IL-4 production in Th2 lymphocytes is regulated by the nuclear factor of activated T-cells (NFAT) and NFAT-interacting protein 45kDa (NIP45). Recently, we showed that arginine methylation of NIP45 modulates its interaction with NFAT and augments IL-4 transcription. Here we report that NIP45 methylation is negatively regulated by peptidylarginine-deiminase 4 (PAD4) via deimination of arginine residues and generation of the atypical amino acid citrulline. NIP45 loses its ability to become methylated after deimination by PAD4. Ectopic expression of PAD4 impairs the association of NIP45 with NFAT and profoundly inhibits IL-4 promoter activity. Whereas, knockdown of PAD4 expression in Th2 cells by a PAD4-directed shRNA-expressing retrovirus leads to elevated IL-4 production. Interestingly, a PAD4 variant is association with rheumatoid arthritis (RA) in a Japanese population. Indeed, many autoantibodies in RA are directed against citrullinated proteins. It has been proposed that PAD4 is linked to RA because citrullination of peptides leads to a breakdown of self-tolerance to self-antigens. Our data suggest that PAD4 may also influence RA development through regulation of the cytokine milieu. Modification of NIP45 by PAD4 provides a novel negative regulatory mechanism whereby NFAT and NIP45 control IL-4 and potentially other cytokines production.

The inhibition of antithrombin by peptidylarginine deiminase 4 may contribute to pathogenesis of rheumatoid arthritis

The gene for peptidylarginine deiminase 4 (PADI4) has been found to be closely associated with rheumatoid arthritis (RA). Peptidylarginine deiminase (PADI) catalyses the post-translational modification of peptidylarginine to citrulline, a reaction known as citrullination. PADI extracted from rabbit muscle has been reported to citrullinate antithrombin, a principal plasma inhibitor of thrombin. Thrombin is known to induce angiogenesis, fibrin formation and inflammation, the primary events of the RA joint. Here, we investigate whether human PADI4 can inhibit antithrombin by catalysing antithrombin citrullination and how the enzyme is involved in RA pathogenesis. Antithrombin was incubated with recombinant PADI4 protein, and the inactivation of antithrombin was determined by reduction of its thrombin-inhibiting activity. Citrullination of antithrombin was detected by western blotting and enzyme-linked immunosorbent assay (ELISA). In addition, the citrullination level, activity and concentration of antithrombin in RA plasma were investigated by sandwich ELISA. Incubation of antithrombin with PADI4 resulted in loss of thrombin-inhibitory activity and in citrullination of antithrombin. RA plasma showed higher levels of citrullinated antithrombin than controls with non-arthritis disease and healthy individuals. The results indicate that PADI4 could inactivate antithrombin through citrullination. The abnormal expression or activation of PADI4 in RA synovium is suggested to be responsible for the high level of citrullinated antithrombin in RA plasma. Local inhibition of antithrombin activity in RA synovium might lead to the excessive angiogenesis, fibrin deposition and inflammation of the tissue.

Localization of peptidylarginine deiminase 4 (PADI4) and citrullinated protein in synovial tissue of rheumatoid arthritis.

Peptidylarginine deiminases (PADIs) convert peptidylarginine into citrulline via post-translational modification. Anti-citrullinated peptide antibodies are highly specific for rheumatoid arthritis (RA). Our genome-wide case-control study of single-nucleotide polymorphisms found that the PADI4 gene polymorphism is closely associated with RA. Here, we localized the expression of PADI4 and the citrullinated protein product in synovial RA tissue. We used immunohistochemistry, double immunofluorescent labelling and western blotting. We found that PADI4 is extensively expressed in T cells, B cells, macrophages, neutrophils, fibroblast-like cells and endothelial cells in the lining and sublining areas of the RA synovium. We also found extracellular and intracellular expression of PADI4 in fibrin deposits with loose tissue structures where apoptosis was widespread. Unlike PADI4, citrullinated protein generally appeared in fibrin deposits that were abundant in the RA synovium. The citrullinated fibrin aggregate was immunoreactive against immunoglobulin (Ig) A and IgM, but not IgG and IgE. Although a little PADI4 was expressed in osteoarthritic and normal synovial tissues, significant citrullination was undetectable. The results showed that PADI4 is mainly distributed in cells of various haematopoietic lineages and expressed at high levels in the inflamed RA synovium. The co-localization of PADI4, citrullinated protein and apoptotic cells in fibrin deposits suggests that PADI4 is responsible for fibrin citrullination and is involved in apoptosis. The immunoreactivity of citrullinated fibrin with IgA and IgM in the RA synovium supports the notion that citrullinated fibrin is a potential antigen of RA autoimmunity.