Iguratimod inhibits protein citrullination and inflammation by downregulating NBCe2 in patients with rheumatoid arthritis.

BACKGROUND: Bicarbonate has recently been identified as a crucial factor affecting peptidylarginine deiminase (PAD) activity; however, the mechanism underlying its role in rheumatoid arthritis (RA) remains unclear. Iguratimod (IGU), a small-molecule disease-modifying anti-rheumatic drug, requires further investigation. This study aimed to explore the mechanism by which bicarbonate affects citrullination and inflammation in RA and identify new targets for IGU. METHODS: We enrolled 20 patients with RA in the study. Sodium bicarbonate cotransporter 2 (NBCe2) was detected in the peripheral blood neutrophils and peripheral blood mononuclear cells (PBMCs) of these patients. The effects of varying concentrations of IGU, methotrexate (MTX), dexamethasone (DXM), and S0859 (an NBCe2 inhibitor) on NBCe2, PAD2, PAD4, and citrullinated histone H3 (cit-H3) levels in, migration ability of, and cytokine production from neutrophils and PBMCs were examined. RESULTS: Our findings showed that in patients with RA, citrullinated protein production by peripheral blood neutrophils instead of PBMCs, which showed higher NBCe2 expression levels, increased with an increase in the bicarbonate concentration. In addition, tumor necrosis factor-alpha (TNF-α) promoted NBCe2 expression in neutrophils from patients with RA. Furthermore, we revealed that the inhibitory effects of IGU on neutrophil NBCe2 and cit-H3 levels, degrees of inhibition of neutrophil and PBMC migration, and suppression of interleukin 6, TNF-α, and metalloproteinase-9 secretion from neutrophil-like differentiated HL-60 cells did not substantially differ from those of MTX, DXM, and S0859 at specific doses. CONCLUSIONS: Bicarbonate promotes protein citrullination and inflammation in RA via NBCe2, and IGU can downregulate NBCe2.

PAD4 controls tumor immunity via restraining the MHC class II machinery in macrophages.

Tumor-associated macrophages (TAMs) shape tumor immunity and therapeutic efficacy. However, it is poorly understood whether and how post-translational modifications (PTMs) intrinsically affect the phenotype and function of TAMs. Here, we reveal that peptidylarginine deiminase 4 (PAD4) exhibits the highest expression among common PTM enzymes in TAMs and negatively correlates with the clinical response to immune checkpoint blockade. Genetic and pharmacological inhibition of PAD4 in macrophages prevents tumor progression in tumor-bearing mouse models, accompanied by an increase in macrophage major histocompatibility complex (MHC) class II expression and T cell effector function. Mechanistically, PAD4 citrullinates STAT1 at arginine 121, thereby promoting the interaction between STAT1 and protein inhibitor of activated STAT1 (PIAS1), and the loss of PAD4 abolishes this interaction, ablating the inhibitory role of PIAS1 in the expression of MHC class II machinery in macrophages and enhancing T cell activation. Thus, the PAD4-STAT1-PIAS1 axis is an immune restriction mechanism in macrophages and may serve as a cancer immunotherapy target.

hRpn13 shapes the proteome and transcriptome through epigenetic factors HDAC8, PADI4, and transcription factor NF-κB p50.

The anti-cancer target hRpn13 is a proteasome substrate receptor. However, hRpn13-targeting molecules do not impair its interaction with proteasomes or ubiquitin, suggesting other critical cellular activities. We find that hRpn13 depletion causes correlated proteomic and transcriptomic changes, with pronounced effects in myeloma cells for cytoskeletal and immune response proteins and bone-marrow-specific arginine deiminase PADI4. Moreover, a PROTAC against hRpn13 co-depletes PADI4, histone deacetylase HDAC8, and DNA methyltransferase MGMT. PADI4 binds and citrullinates hRpn13 and proteasomes, and proteasomes from PADI4-inhibited myeloma cells exhibit reduced peptidase activity. When off proteasomes, hRpn13 can bind HDAC8, and this interaction inhibits HDAC8 activity. Further linking hRpn13 to transcription, its loss reduces nuclear factor κB (NF-κB) transcription factor p50, which proteasomes generate by cleaving its precursor protein. NF-κB inhibition depletes hRpn13 interactors PADI4 and HDAC8. Altogether, we find that hRpn13 acts dually in protein degradation and expression and that proteasome constituency and, in turn, regulation varies by cell type.

Chitosan nanomedicine containing RGD peptide and PAD4 inhibitor based on phenyl boronate coupling inhibition of primary tumor growth and lung metastasis.

Stimulus-responsive nanodrugs have been extensively studied and their structural changes in the cells are important for controlled intracellular drug release. Histone citrullination of peptidylarginine deiminase 4 (PAD4) regulates the expression of tumor suppressor genes. In our previous study, compounds such as YW3-56 (356) were developed as potent PAD4 inhibitors with excellent anti-tumor activity in vitro and in vivo. To enhance the antitumor activity and improve the bioavailability, we further optimized the structure by modifying the phenylboronic acid moiety to the PAD4 inhibitor (4B). Taking advantage of the oxidative stress responsiveness of the phenylboronic acid moiety, in this study, we covalently attached 4B to RGD sequence peptide modified chitosan (K-CRGDV) to construct this new oxidative stress responsive nanodrug (K-CRGDV-4B). The modification of RGD sequence peptide conferred the nanodrug the ability to actively target tumors. The release mechanism was verified by UV-Vis spectroscopy, NMR. The anti-tumor and anti-metastatic properties of K-CRGDV-4B were demonstrated by in vitro cytotoxicity assay and in vivo mouse Lewis lung cancer metastasis model. In addition, K-CRGDV-4B modulates the ratio of immune cells in LLC tumor-bearing mice. Immunosuppressive proteins such as PD1 were inhibited, while IFN-γ and IFN-ß, which are stimulators of tumor immune responses, were upregulated. Overall, K-CRGDV-4B is a stimulus-responsive nanodrug that responds to the tumor microenvironment by inhibiting PAD4 activity, blocking the formation of neutrophil extracellular traps (NETs), and improving the tumor immune microenvironment.

Investigating the cell membrane localization of PADI4 in breast cancer cells and inhibition of anti-PADI4 monoclonal antibody.

BACKGROUND: Peptidyl arginine deiminase 4 (PADI4) is a post-translational modification enzymecan that converts arginine in protein into citrulline in the presence of calcium ions, which is called citrullination. PADI4 has been reported to be expressed in the cytoplasm and nucleus in a variety of malignant tumors. Based on the GeneCards database and our previous research, it is speculated that PADI4 may also be expressed on the cell membrane. This study aimed to confirm the membrane expression of PADI4 and the effect of anti-PADI4 antibodies on cell membrane PADI4. This may be another mechanism of action of anti-PADI4 monoclonal antibodies in the treatment of breast cancer. METHODS: The subcellular localizations of PADI4 in MDA-MB-231 and MCF-7 breast cancer cells were determined by immunofluorescence, immunoelectron microscopy, and Western blot analysis. The tumor cells were treated with PADI4 antibody, and cell proliferation, migration, colony formation, apoptosis, glycolysis, and epithelial-mesenchymal transition (EMT) were measured as well as the expression of some essential tumor genes. RESULTS: PADI4 was not only localized in the nucleus and cytoplasm of breast cancer cells but was also detected on the cell membrane. Following PADI4 antibody treatment, cell proliferation, migration, colony formation, EMT, and ATP production through glycolysis were decreased, and the mRNA expression of MYC proto-oncogene (MYC), FAT atypical cadherin 1 (FAT1), nuclear factor kappa B subunit 1 (NFκB), and tumor necrosis factor (TNF-α) in breast cancer cells was downregulated, while the mRNA expression of tumor protein p63 (TP63) was upregulated. CONCLUSIONS: PADI4 is expressed on the cell membrane in breast cancer cells. Anti-PADI4 antibodies can affect the biological functions of cell membrane PADI4, including proliferation, migration, apoptosis, and glycolysis, thereby inhibiting tumor progression.

Highly-tumor-targeted PAD4 inhibitors with PBA modification inhibit tumors in vivo by specifically inhibiting the PAD4-H3cit-NETs pathway in neutrophils.

As a new target for tumor therapy, PAD4 protein, shows excellent antitumor activity, and phenylboronic acid (PBA) could combine with sialic acid on the tumor surface to achieve dual targeting in situ and for metastatic tumors. The purpose of this study was therefore to modify PAD4 protein inhibitors with different phenylboronic acid groups in order to obtain highly-targeted PAD4 inhibitors. The activity and mechanism of these PBA-PAD4 inhibitors were studied in vitro by MTT assay, laser confocal analysis, and flow cytometry. The effects of the compounds on primary tumor and lung metastasis in mice were evaluated in vivo using a S180 sarcoma model and a 4T1 breast cancer model. In addition, cytometry mass (CyTOF) was used to analyze the immune microenvironment, and the results show that the PAD4 inhibitor 5i modified by m-PBA at the carboxyl terminal of ornithine skeleton had the best antitumor activity. In vitro evaluation of this activity revealed that 5i could not directly kill tumor cells but had a significant inhibitory effect on tumor cell metastasis. Further mechanism studies showed that 5i could be taken up by 4T1 cells in a time-dependent manner and distributed around the cell membrane but could not be taken up by normal cells. In addition, although 5i was distributed in the cytoplasm of tumor cells while in the nucleus of neutrophils, it could both decrease the histone 3 citrullination (H3cit) in the nucleus. In vivo 4T1 tumor-bearing mouse models, 5i inhibited breast cancer growth and metastasis in a concentration-dependent manner, and NET formation in tumor tissues was significantly reduced. In conclusion, PBA-PAD4 inhibitors show high targeting of tumor cells and good safety in vivo. By specifically inhibiting PAD4 protein in the neutrophil nucleus, PBA-PAD4 inhibitors also show excellent antitumor activity toward growth and metastasis in vivo, which provides a new idea for the design of highly-targeted PAD4 inhibitors.

Chloroquine reduces neutrophil extracellular trap (NET) formation through inhibition of peptidyl arginine deiminase 4 (PAD4).

Neutrophil extracellular traps (NETs) occur when chromatin is decondensed and extruded from the cell, generating a web-like structure. NETs have been implicated in the pathogenesis of several sterile disease states and thus are a potential therapeutic target. Various pathways have been shown to induce NETs, including autophagy, with several key enzymes being activated like peptidyl arginine deiminase 4 (PAD4), an enzyme responsible for citrullination of histones, allowing for DNA unwinding and subsequent release from the cell. Pre-clinical studies have already demonstrated that chloroquine (CQ) and hydroxychloroquine (HCQ) are able to reduce NETs and slow disease progression. The exact mechanism as to how these drugs reduce NETs has yet to be elucidated. CQ and HCQ decrease NET formation from various NET activators, independent of their autophagy inhibitory function. CQ and HCQ were found to inhibit PAD4 exclusively, in a dose-dependent manner, confirmed with reduced CitH3+ NETs after CQ or HCQ treatment. Circulating CitH3 levels were reduced in pancreatic cancer patients after HCQ treatment. In silico screening of PAD4 protein structure identified a likely binding site interaction at Arg639 for CQ and Trp347, Ser468, and Glu580 for HCQ. SPR analysis confirmed the binding of HCQ and CQ with PAD4 with KD values of 54.1 µM (CQ) and 88.1 µM (HCQ). This data provide evidence of direct PAD4 inhibition as a mechanism for CQ/HCQ inhibition of NETs. We propose that these drugs likely reduce NET formation through multiple mechanisms; the previously established TLR9 and autophagy inhibitory mechanism and the novel PAD4 inhibitory mechanism.

The armadillo-repeat domain of Plakophilin 1 binds to human enzyme PADI4.

Plakophilin 1 (PKP1), a member of the armadillo repeat family of proteins, is a key structural component of cell-cell adhesion scaffolds, although it can also be found in other cell locations, including the cytoplasm and the nucleus. PADI4 (peptidyl-arginine deiminase 4) is one of the human isoforms of a family of enzymes engaged in the conversion of arginine to citrulline, and is present in monocytes, macrophages, granulocytes, and in several types of cancer cells. It is the only family member observed both within the nucleus and the cytoplasm under ordinary conditions. We studied the binding of the armadillo domain of PKP1 (ARM-PKP1) with PADI4, by using several biophysical methods, namely fluorescence, far-ultraviolet (far-UV) circular dichroism (CD), isothermal titration calorimetry (ITC), and molecular simulations; furthermore, binding was also tested by Western-blot (WB) analyses. Our results show that there was binding between the two proteins, with a dissociation constant in the low micromolar range (∼ 1 µM). Molecular modelling provided additional information on the possible structure of the binding complex, and especially on the binding hot-spot predicted for PADI4. This is the first time that the interaction between these two proteins has been described and studied. Our findings could be of importance to understand the development of tumors, where PKP1 and PADI4 are involved. Moreover, our findings pave the way to describe the formation of neutrophil extracellular traps (NETs), whose construction is modulated by PADI4, and which mediate the proteolysis of cell-cell junctions where PKP1 intervenes.

Human Enzyme PADI4 Binds to the Nuclear Carrier Importin α3.

PADI4 is a peptidyl-arginine deiminase (PADI) involved in the conversion of arginine to citrulline. PADI4 is present in macrophages, monocytes, granulocytes, and several cancer cells. It is the only PADI family member observed within both the nucleus and the cytoplasm. PADI4 has a predicted nuclear localization sequence (NLS) comprising residues Pro56 to Ser83, to allow for nuclear translocation. Recent predictors also suggest that the region Arg495 to Ile526 is a possible NLS. To understand how PADI4 is involved in cancer, we studied the ability of intact PADI4 to bind importin α3 (Impα3), a nuclear transport factor that plays tumor-promoting roles in several cancers, and its truncated species (ΔImpα3) without the importin-binding domain (IBB), by using fluorescence, circular dichroism (CD), and isothermal titration calorimetry (ITC). Furthermore, the binding of two peptides, encompassing the first and the second NLS regions, was also studied using the same methods and molecular docking simulations. PADI4 interacted with both importin species, with affinity constants of ~1-5 µM. The isolated peptides also interacted with both importins. The molecular simulations predict that the anchoring of both peptides takes place in the major binding site of Impα3 for the NLS of cargo proteins. These findings suggest that both NLS regions were essentially responsible for the binding of PADI4 to the two importin species. Our data are discussed within the framework of a cell mechanism of nuclear transport that is crucial in cancer.

Changes in Tumor Necrosis Factor α (TNFα) and Peptidyl Arginine Deiminase 4 (PAD-4) Levels in Serum of General Treated Psoriatic Patients.

Psoriasis is an autoimmune disease in which the disturbed dependencies between lymphocytes, dendritic cells, keratinocytes and neutrophils play the most important role. One of them is the overproduction of neutrophil extracellular traps (NETs). The release of NETs can be induced by pathogens, as well as antibodies and immune complexes, cytokines and chemokines, including TNFα. The first step of the NET creation is the activation of peptidyl arginine deiminase 4 (PAD-4). PAD-4 seems to be responsible for citrullination of histones and chromatin decondensation, but the data on PAD-4 in NETs is inconclusive. Thus, the current study aimed to determine PAD-4 and TNFα levels in the serum of psoriatic patients by ELISA and observe the response of these factors to systemic (anti-17a, anti-TNFα and methotrexate) therapies. Increased levels of both PAD-4 and its main stimulus factor TNFα in pre-treatment patients have been reported along with the concentrations of proteins correlated with disease severity (PASI, BSA). Before treatment, the irregularities in the case of anti-nuclear antibodies level (ANA) were also observed. All of the applied therapies led to a decrease in PAD-4 and TNFα levels after 12 weeks. The most significant changes, both in protein concentrations as well as in scale scores, were noted with anti-TNFα therapy (adalimumab and infliximab). This phenomenon may be associated with the inhibition of TNFα production at different stages of psoriasis development, including NET creation. The obtained data suggest the participation of PAD-4 in the activation of neutrophils to produce NETs in psoriasis, which may create opportunities for modern therapies with PAD inhibitors. However, further exploration of gene and protein expression in psoriatic skin is needed.

The Prominent Role of Hematopoietic Peptidyl Arginine Deiminase 4 in Arthritis: Collagen- and Granulocyte Colony-Stimulating Factor-Induced Arthritis Model in C57BL/6 Mice.

OBJECTIVE: Genome-wide association studies have connected PADI4, encoding peptidylarginine deiminase 4 (PAD4), with rheumatoid arthritis (RA). PAD4 promotes neutrophil extracellular trap (NET) formation. This study was undertaken to investigate the origin of PAD4 and the importance of NET formation in a C57BL/6 mouse model of arthritis. METHODS: To permit the effective use of C57BL/6 mice in the collagen-induced arthritis (CIA) model, we introduced the administration of granulocyte colony-stimulating factor (G-CSF) for 4 consecutive days in conjunction with the booster immunization on day 21. Mice with global Padi4 deficiency (Padi4-/- ) and mice with hematopoietic lineage-specific Padi4 deficiency (Padi4Vav1Cre/+ ) were evaluated in the model. RESULTS: G-CSF significantly increased the incidence and severity of CIA. G-CSF-treated mice showed elevated citrullinated histone H3 (Cit-H3) levels in plasma, while vehicle-treated mice did not. Immunofluorescence microscopy revealed deposition of Cit-H3 in synovial tissue in G-CSF-treated mice. Padi4-/- mice developed less severe arthritis and had lower levels of serum interleukin-6 and plasma Cit-H3, lower levels of Cit-H4 in synovial tissue, and less bone erosion on micro-computed tomography than Padi4+/+ mice in the G-CSF-modified CIA model. Similarly, Padi4Vav1Cre/+ mice developed less severe arthritis, compared with Padi4fl/fl mice, and presented the same phenotype as Padi4-/- mice. CONCLUSION: We succeeded in developing an arthritis model suitable for use in C57BL/6 mice that is fully compliant with high animal welfare standards. We observed a >90% incidence of arthritis in male mice and detectable NET markers. This model, with some features consistent with human RA, demonstrates that hematopoietic PAD4 is an important contributor to arthritis development and may prove useful in future RA research.

Biochemical and biophysical characterization of PADI4 supports its involvement in cancer.

PADI4 (protein-arginine deiminase, also known as protein l-arginine iminohydrolase) is one of the human isoforms of a family of Ca2+-dependent proteins catalyzing the conversion of arginine to citrulline. Although the consequences of this process, known as citrullination, are not fully understood, all PADIs have been suggested to play essential roles in development and cell differentiation. They have been found in a wide range of cells and tissues and, among them, PADI4 is present in macrophages, monocytes, granulocytes and cancer cells. In this work, we focused on the biophysical features of PADI4 and, more importantly, how its expression was altered in cancer cells. Firstly, we described the different expression patterns of PADI4 in various cancer cell lines and its colocalization with the tumor-related protein p53. Secondly, we carried out a biophysical characterization of PADI4, by using a combination of biophysical techniques and in silico molecular dynamics simulations. Our biochemical results suggest the presence of several forms of PADI4 with different subcellular localizations, depending on the cancer cell line. Furthermore, PADI4 could have a major role in tumorigenesis by regulating p53 expression in certain cancer cell lines. On the other hand, the native structure of PADI4 was strongly pH-dependent both in the absence or presence of Ca2+, and showed two pH-titrations at basic and acidic pH values. Thus, there was a narrow pH range (from 6.5 to 8.0) where the protein was dimeric and had a native structure, supporting its role in histones citrullination. Thermal denaturations were always two-state, but guanidinium-induced ones showed that PADI4 unfolded through at least one intermediate. Our simulation results suggest that the thermal melting of PADI4 structure was rather homogenous throughout its sequence. The overall results are discussed in terms of the functional role of PADI4 in the development of cancer.

Evaluation of galectin-3, peptidylarginine deiminase-4, and tumor necrosis factor-α levels in gingival crevicular fluid for periodontal health, gingivitis, and Stage III Grade C periodontitis: A pilot study.

BACKGROUND: Comparing the gingival crevicular fluid (GCF) levels of galectin-3, peptidylarginine deiminase 4 (PAD4) and tumor necrosis factor-alpha (TNF-α) in individuals with stage III grade C periodontitis and gingivitis and with healthy periodontium was the purpose of this clinical research. METHODS: Sixty systemically healthy and non-smoker individuals consisting of stage III grade C periodontitis (group S3P/n = 20), gingivitis (group G/n = 20), and periodontally healthy (group HP/n = 20) were recruited for this research. Clinical parameters such as probing depth, clinical attachment level, gingival index, plaque index, and bleeding on probing were recorded in periodontal charts. Enzyme-linked immunosorbent assay method was used in evaluating the GCF levels of galectin-3, PAD4, and TNF-α for study groups. RESULTS: The GCF galectin-3 total amount was highest in group S3P compared with group G and group HP (P <0.05). Its total amount was also higher in group G compared with group HP (P <0.05). The GCF PAD4 total amount was higher in group S3P compared with group HP (P <0.05) but was similar with group G (P >0.05). Its total amounts were also similar in group G and group HP (P >0.05). The GCF TNF-α total amounts were similar in group S3P and group G (P >0.05) but significantly greater than the group HP (P ˂0.05). The GCF galectin-3, PAD4, and TNF-α concentrations were lower in the group S3P and group G compared with the group HP (P <0.05). There were significant positive correlations between GCF galectin-3 total amount and all clinical parameters (P ˂0.01) and also between GCF galectin-3 and TNF-α total amounts (P ˂0.01). There was no correlation between PAD4 and clinical parameters, or between PAD4 and TNF-α (P >0.05). CONCLUSIONS: Galectin-3 and PAD4 may be involved in the periodontal disease pathogenesis considering the elevated levels of these molecules in periodontal disease. These biomarkers may be used in the diagnosis of periodontal diseases.

Peptidylarginine deiminases 4 as a promising target in drug discovery.

Peptidylarginine deaminase 4 (PAD4) is a crucial post-translational modifying enzyme catalyzing the conversion of arginine into citrulline residues, and mediating the formation of neutrophil extracellular traps (NETs). PAD4 plays a vital role in the occurrence and development of cardiovascular diseases, autoimmune diseases, and various tumors. Therefore, PAD4 is considered as a promising drug target for disease diagnosis and treatment. More and more efforts are devoted to developing highly efficient and selective PAD4 inhibitors via high-throughput screening, structure-based drug design and structure-activity relationship study. This article outlined the physiological and pathological functions of PAD4, and corresponding representative small molecule inhibitors reported in recent years.

The regulation of macrophage polarization by hypoxia-PADI4 coordination in Rheumatoid arthritis.

BACKGROUND: Hypoxia, a common feature of rheumatoid arthritis (RA), induces the over-expression of peptidyl arginine deiminase 4 (PADI4) in fibroblast-like synoviocytes (FLSs) and macrophages. However, the roles of PADI4 and its inducer hypoxia in the regulation of macrophage polarization remain unclear. This study aimed to investigate the role of hypoxia-PADI4 for macrophage polarization in RA patients. METHODS: Synovial tissue (ST) and synovial fluid (SF) were collected from 3 OA patients and 6 RA patients. The distribution of M1 and M2 in ST and cytokines in SF were examined by immunohistochemical analysis and Bio-Plex immunoassays. THP-1 macrophages and BMDM polarization were determined under normoxic (21% oxygen) or hypoxic (3% oxygen) conditions. The effects of PADI4 on macrophages were determined by transfection of adenovirus vector-coated PADI4 (AdPADI4) and the use of PADI4 inhibitor. Finally, the roles of PADI4 in joint synovial lesions on macrophage polarization were investigated in collagen-induced arthritis (CIA) rats. RESULTS: We found increased macrophage polarization of M1 and M2 in the RA ST, compared with OA ST. The ratio of M1/M2 for RA and OA was 1.633 ± 0.1443 and 2.544 ± 0.4429, respectively. The concentration of M1- and M2-type cytokines was higher in RA than that in OA patients. Hypoxia contributed to the increase of the gene and protein expression of M1 and M2 markers. M1- but not M2-type gene expression showed a positive relationship with PADI4 expressionwhile the level of expression of M2-type genes showed no significant difference. The degree of joint swelling and destruction was effectively alleviated, and the number of macrophages especially M1 decreased in CIA rats after down-regulating PADI4 expression. CONCLUSION: Hypoxia is responsible for the co-polarization of M1 and M2. Hypoxia-associated PADI4 is responsible for M1 macrophage activation, implying that the inflammatory environment can be eased by decreasing PADI4 expression and improving the hypoxic environment.

[Effect of PADI4 on the Expression of Inflammatory Cytokines During NB4 Cells Differentiation].

OBJECTIVE: To investigate the expression of peptidylarginine deiminase 4 (PADI4) during the process of differentiation into granulocyte of NB4 cells induced by all-trans-retinoic acid (ATRA) and whether PADI4 is involved in the inflammatory cytokines expression. METHODS: Granulocyte differentiation model of NB4 cells induced by ATRA was established. The cell morphology changes were observed by Wright-Giemsa staining. The expression of cell differentiation marker CD11b was analyzed by flow cytometry. The mRNA and protein expression of PADI4 was detected by RT-PCR and Western blot, respectively. The expression of tumor necrosis factor (TNF) α and interleukin (IL) 1ß was analyzed by ELISA, and also examined with the knockdown of PADI4 expression by siRNA. RESULTS: After NB4 cells induced by ATRA, the cytoplasm increased and the ratio of nuclear to cytoplasmic was reduced. Nuclear dented, and rod-shaped nucleus, lobulated phenomenon increased (P<0.05). Flow cytometry analysis results showed that the cell surface molecule CD11b expression increased (P<0.01). RT-PCR and Western blot showed the expression of PADI4 increased at both transcriptional and translational levels during the process of the differentiation. ELISA showed TNF-α and IL-1ß secretion increased in differentiated macrophages, while they could be inhibited by PADI4-specific siRNA. CONCLUSION: During the differentiation into granulocyte of NB4 cells induced by ATRA, PADI4 expression increased. Furthermore, PADI4 appeared to play a critical role in inflammatory cytokines secretion.

DNAse-dependent, NET-independent pathway of thrombus formation in vivo.

The contribution of NETs (neutrophil extracellular traps) to thrombus formation has been intensively documented in both arterial and venous thrombosis in mice. We previously demonstrated that adenosine triphosphate (ATP)-activated neutrophils play a key role in initiating the tissue factor-dependent activation of the coagulation cascade, leading to thrombus formation following laser-induced injury. Here, we investigated the contribution of NETs to thrombus formation in a laser-induced injury model. In vivo, treatment of mice with DNase-I significantly inhibited the accumulation of polymorphonuclear neutrophils at the site of injury, neutrophil elastase secretion, and platelet thrombus formation within seconds following injury. Surprisingly, electron microscopy of the thrombus revealed that neutrophils present at the site of laser-induced injury did not form NETs. In vitro, ATP, the main neutrophil agonist present at the site of laser-induced injury, induced the overexpression of PAD4 and CitH3 but not NETosis. However, compared to no treatment, the addition of DNase-I was sufficient to cleave ATP and adenosine diphosphate (ADP) in adenosine. Human and mouse platelet aggregation by ADP and neutrophil activation by ATP were also significantly reduced in the presence of DNase-I. We conclude that following laser-induced injury, neutrophils but not NETs are involved in thrombus formation. Treatment with DNase-I induces the hydrolysis of ATP and ADP, leading to the generation of adenosine and the inhibition of thrombus formation in vivo.

Antithrombin Together with NETs Inhibitor Protected Against Postoperative Adhesion Formation in Mice.

BACKGROUND/AIMS: Postoperative adhesions may induce adverse outcomes in patients. Adhesion formation is initiated by fibrin accumulation at the surgical site which is followed by local neutrophilia and the establishment of neutrophil extracellular traps (NET). Previous reports have suggested that the preventive efficacy of reagents designed to reduce postoperative adhesion is inversely correlated with neutrophilia and NET production. Antithrombin (AT) is a natural inhibitor of thrombin, a key factor in coagulation. Here, we evaluate whether treatment with AT and/or NET inhibitors prevent or reduce postoperative adhesion formation in mice. METHODS: Mice were treated with AT and/or NET inhibitors before and/or after cecum cauterization and their adhesion scores were evaluated on day 7 post-operation. Immunochemistry/ immunofluorescence analyses were also performed and we used GSK484, an inhibitor of peptidyl arginine deiminase 4 (PAD4), as the NET inhibitor. RESULTS: AT or GSK484 partially rescued postoperative adhesion formation in mice. AT prevented thrombin-induced plasminogen activator inhibitor 1 and interleukin-6 expression in mesothelial cells in vitro. However, AT could not prevent neutrophilia or NETs formation around the injured serosa. Finally, we investigated a combination of AT and a PAD4 inhibitor and found that this could inhibit almost all adhesion formation in these animals. Since AT-inactivating proteases are liberated following NET release, they might dampen the biological action of the AT treatment. This suggests that NET inhibitors might allow AT to exert its full action in the surgically injured serosa. CONCLUSION: Combined treatment with AT and GSK484 may effectively attenuate postoperative adhesion production in mice.

Factors Associated with Promoted Proliferation of Osteosarcoma by Peptidylarginine Deiminase 4.

Osteosarcoma is the most common type of bone malignancy, and the pathogenesis has not been entirely elucidated yet. An important deimination modification enzyme PADI4 (peptidylarginine deiminase 4) has attracted much attention in recent years for its important function in several kinds of human tumors. However, the role of PADI4 on osteosarcoma tumorigenesis remains largely unrevealed. Here, we first assessed the effect of PADI4 on osteosarcoma proliferation by the CCK8 method and colony formation assay. Ectopically expressing PADI4 positively regulates the colony formation capacity of both U2OS and Saos-2 cells. Furthermore, we explored the related mechanism and showed that PADI4 could stimulate Wnt/ß-catenin and MEK/ERK signaling in both U2OS and Saos-2 cells. Then, we detected expression of PADI4 in human tissues of osteosarcoma and revealed that differential expression of PADI4 was associated with tumorigenesis of osteosarcoma. Last, we performed the in vivo experiment in nude mice and results also showed PADI4 could affect the tumor growth. In conclusion, this work revealed that PADI4 could upregulate the proliferation of osteosarcoma, mainly via the Wnt/ß-catenin and MEK/ERK signaling pathway. This study gives us new insight into the regulation mechanism of osteosarcoma proliferation and highlights PADI4 as a promising target for osteosarcoma diagnosis and treatment.

Discovery of new inhibitor for the protein arginine deiminase type 4 (PAD4) by rational design of α-enolase-derived peptides.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease affecting about 0.24 % of the world population. Protein arginine deiminase type 4 (PAD4) is believed to be responsible for the occurrence of RA by catalyzing citrullination of proteins. The citrullinated proteins act as autoantigens by stimulating an immune response. Citrullinated α-enolase has been identified as one of the autoantigens for RA. Hence, α-enolase serves as a suitable template for design of potential peptide inhibitors against PAD4. The binding affinity of α-enolase-derived peptides and PAD4 was virtually determined using PatchDock and HADDOCK docking programs. Synthesis of the designed peptides was performed using a solid phase peptide synthesis method. The inhibitory potential of each peptide was determined experimentally by PAD4 inhibition assay and IC50 measurement. PAD4 assay data show that the N-P2 peptide is the most favourable substrate among all peptides. Further modification of N-P2 by changing the Arg residue to canavanine [P2 (Cav)] rendered it an inhibitor against PAD4 by reducing the PAD4 activity to 35 % with IC50 1.39 mM. We conclude that P2 (Cav) is a potential inhibitor against PAD4 and can serve as a starting point for the development of even more potent inhibitors.