IL-22 Downregulates Peptidylarginine Deiminase-1 in Human Keratinocytes: Adding Another Piece to the IL-22 Puzzle in Epidermal Barrier Formation.

Increased presence of IL-22+ cells in the skin is a characteristic finding in skin barrier defects, such as psoriasis and atopic dermatitis. However, mechanistic insight into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here, we show reduced levels of peptidylarginine deiminase 1, an enzyme that converts peptidylarginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of peptidylarginine deiminase 1 in epidermal keratinocytes. Subsequently, total peptidylarginine deiminase activity and extent of protein deimination in keratinocytes treated with IL-22 were reduced together with a significant decrease in deimination of keratin 1 and FLG, both important for epidermal differentiation. Vitamin D and acitretin partly restored the peptidylarginine deiminase 1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.

Lipoproteins from Staphylococcus aureus Drive Neutrophil Extracellular Trap Formation in a TLR2/1- and PAD-Dependent Manner.

Neutrophils, polymorphonuclear leukocytes (PMN), play a critical role in the innate immune response to Staphylococcus aureus, a pathogen that continues to be associated with significant morbidity and mortality. Neutrophil extracellular trap (NET) formation is involved in ensnaring and killing of S. aureus, but this host-pathogen interaction also leads to host tissue damage. Importantly, NET components including neutrophil proteases are under consideration as therapeutic targets in a variety of disease processes. Although S. aureus lipoproteins are recognized to activate cells via TLRs, specific mechanisms of interaction with neutrophils are poorly delineated. We hypothesized that a lipoprotein-containing cell membrane preparation from methicillin-resistant S. aureus (MRSA-CMP) would elicit PMN activation, including NET formation. We investigated MRSA-CMP-elicited NET formation, regulated elastase release, and IL-8 production in human neutrophils. We studied PMN from healthy donors with or without a common single-nucleotide polymorphism in TLR1, previously demonstrated to impact TLR2/1 signaling, and used cell membrane preparation from both wild-type methicillin-resistant S. aureus and a mutant lacking palmitoylated lipoproteins (lgt). MRSA-CMP elicited NET formation, elastase release, and IL-8 production in a lipoprotein-dependent manner. TLR2/1 signaling was involved in NET formation and IL-8 production, but not elastase release, suggesting that MRSA-CMP-elicited elastase release is not mediated by triacylated lipoproteins. MRSA-CMP also primed neutrophils for enhanced NET formation in response to a subsequent stimulus. MRSA-CMP-elicited NET formation did not require Nox2-derived reactive oxygen species and was partially dependent on the activity of peptidyl arginine deiminase (PAD). In conclusion, lipoproteins from S. aureus mediate NET formation via TLR2/1 with clear implications for patients with sepsis.

Citrullination of pyruvate kinase M2 by PADI1 and PADI3 regulates glycolysis and cancer cell proliferation.

Chromodomain helicase DNA binding protein 4 (CHD4) is an ATPase subunit of the Nucleosome Remodelling and Deacetylation (NuRD) complex that regulates gene expression. CHD4 is essential for growth of multiple patient derived melanoma xenografts and for breast cancer. Here we show that CHD4 regulates expression of PADI1 (Protein Arginine Deiminase 1) and PADI3 in multiple cancer cell types modulating citrullination of arginine residues of the allosterically-regulated glycolytic enzyme pyruvate kinase M2 (PKM2). Citrullination of PKM2 R106 reprogrammes cross-talk between PKM2 ligands lowering its sensitivity to the inhibitors Tryptophan, Alanine and Phenylalanine and promoting activation by Serine. Citrullination thus bypasses normal physiological regulation by low Serine levels to promote excessive glycolysis and reduced cell proliferation. We further show that PADI1 and PADI3 expression is up-regulated by hypoxia where PKM2 citrullination contributes to increased glycolysis. We provide insight as to how conversion of arginines to citrulline impacts key interactions within PKM2 that act in concert to reprogramme its activity as an additional mechanism regulating this important enzyme.

Long Noncoding RNA TINCR-Mediated Regulation of Acetyl-CoA Metabolism Promotes Nasopharyngeal Carcinoma Progression and Chemoresistance.

Frontier evidence suggests that dysregulation of long noncoding RNAs (lncRNA) is ubiquitous in all human tumors, indicating that lncRNAs might have essential roles in tumorigenesis. Therefore, an in-depth study of the roles of lncRNA in nasopharyngeal carcinoma (NPC) carcinogenesis might be helpful to provide novel therapeutic targets. Here we report that lncRNA TINCR was significantly upregulated in NPC and was associated positively with poor survival. Silencing TINCR inhibited NPC progression and cisplatin resistance. Mechanistically, TINCR bound ACLY and protected it from ubiquitin degradation to maintain total cellular acetyl-CoA levels. Accumulation of cellular acetyl-CoA promoted de novo lipid biosynthesis and histone H3K27 acetylation, which ultimately regulated the peptidyl arginine deiminase 1 (PADI1)-MAPK-MMP2/9 pathway. In addition, insulin-like growth factor 2 mRNA-binding protein 3 interacted with TINCR and slowed its decay, which partially accounted for TINCR upregulation in NPC. These findings demonstrate that TINCR acts as a crucial driver of NPC progression and chemoresistance and highlights the newly identified TINCR-ACLY-PADI1-MAPK-MMP2/9 axis as a potential therapeutic target in NPC. SIGNIFICANCE: TINCR-mediated regulation of a PADI1-MAPK-MMP2/9 signaling pathway plays a critical role in NPC progression and chemoresistance, marking TINCR as a viable therapeutic target in this disease.

Halogen Bonding Increases the Potency and Isozyme Selectivity of Protein Arginine Deiminase 1 Inhibitors.

Protein arginine deiminases (PADs) hydrolyze the side chain of arginine to form citrulline. Aberrant PAD activity is associated with rheumatoid arthritis, multiple sclerosis, lupus, and certain cancers. These pathologies established the PADs as therapeutic targets and multiple PAD inhibitors are known. Herein, we describe the first highly potent PAD1-selective inhibitors (1 and 19). Detailed structure-activity relationships indicate that their potency and selectivity is due to the formation of a halogen bond with PAD1. Importantly, these inhibitors inhibit histone H3 citrullination in HEK293TPAD1 cells and mouse zygotes with excellent potency. Based on this scaffold, we also developed a PAD1-selective activity-based probe that shows remarkable cellular efficacy and proteome selectivity. Based on their potency and selectivity we expect that 1 and 19 will be widely used chemical tools to understand PAD1 biology.

Peptidylarginine Deiminase Inhibitor Cl-Amidine Attenuates Cornification and Interferes with the Regulation of Autophagy in Reconstructed Human Epidermis.

Deimination, a post-translational modification catalyzed by a family of enzymes called peptidylarginine deiminases (PADs), is the conversion of arginine into citrulline residues in a protein. Deimination has been associated with numerous physiological and pathological processes. Our aim was to study its implication in the homeostasis of human epidermis, where three PADs are expressed, namely PAD1, 2, and 3. Three-dimensional reconstructed human epidermis (RHEs) were treated for 2 days with increased concentrations (0-800 µM) of Cl-amidine, a specific PAD inhibitor. Cl-amidine treatments inhibited deimination in a dose-dependent manner and were not cytotoxic for keratinocytes. At 800 µM , Cl-amidine was shown to reduce deimination by half, alter keratinocyte differentiation, decrease the number of corneocyte layers, significantly increase the number of transitional cells, induce clustering of mitochondria and of heterogeneous vesicles in the cytoplasm of granular keratinocytes, and upregulate the expression of autophagy proteins, including LC3-II, sestrin-2, and p62/SQSTM1. LC3 and PADs were further shown to partially co-localize in the upper epidermis. These results demonstrated that Cl-amidine treatments slow down cornification and alter autophagy in the granular layer. They suggest that PAD1 and/or PAD3 play a role in the constitutive epidermal autophagy process that appears as an important step in cornification.

Photochemical Control of Protein Arginine Deiminase (PAD) Activity.

Protein arginine deiminases (PADs) play an important role in the pathogenesis of various diseases, including rheumatoid arthritis, multiple sclerosis, lupus, ulcerative colitis, and breast cancer. Therefore, the development of PAD inhibitors has drawn significant research interest in recent years. Herein, we describe the development of the first photoswitchable PAD inhibitors. These compounds possess an azobenzene photoswitch to optically control PAD activity. Screening of a series of inhibitors structurally similar to BB-Cl-amidine afforded compounds 1 and 2 as the most promising candidates for the light-controlled inhibition of PAD2; the cis isomer of 1 is 10-fold more potent than its trans isomer, whereas the trans isomer of 2 is 45-fold more potent than the corresponding cis isomer. The altered inhibitory potency upon photoisomerization has been confirmed in a competitive activity-based protein profiling (ABPP) assay. Further investigations indicate that the trans isomer of 2 is an irreversible inhibitor, whereas the cis isomer acts as a competitive inhibitor. In cells, the trans isomer of compound 1 is completely inactive, whereas the cis isomer inhibits histone H3-citrullination in a dose-dependent manner. Taken together, 1 serves as the foundation for developing photopharmaceuticals that can be activated at the desired tissue, using light, to treat diseases where PAD activity is dysregulated.

PAD1 promotes epithelial-mesenchymal transition and metastasis in triple-negative breast cancer cells by regulating MEK1-ERK1/2-MMP2 signaling.

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.

Peptidylarginine deiminase 1-catalyzed histone citrullination is essential for early embryo development.

Peptidylarginine deiminase (PADI) enzymes are increasingly being associated with the regulation of chromatin structure and gene activity via histone citrullination. As one of the PADI family members, PADI1 has been mainly reported to be expressed in the epidermis and uterus, where the protein in keratinocytes is thought to promote differentiation by citrullinating filament proteins. However, the roles of PADI1 in preimplantation development have not been addressed. Using a PADI1-specific inhibitor and Padi1-morpholino knockdown, we found that citrullination of histone tails at H4R3 and H3R2/8/17 were markedly reduced in the 2- and 4-cell embryos. Consistent with this observation, early embryo development was also arrested at the 4-cell stage upon depletion of PADI1 or inhibition of PADI1 enzyme activity. Additionally, by employing 5-ethynyl uridine (EU) incorporation analysis, ablation of PADI1 function led to a dramatic decrease in overall transcriptional activity, correlating well with the reduced levels of phosphorylation of RNA Pol II at Ser2 observed at 2- or 4-cell stage of embryos under Padi1 knockdown or inhibiting PADI1. Thus, our data reveal a novel function of PADI1 during early embryo development transitions by catalyzing histone tail citrullination, which facilitates early embryo genome transactivation.

Monomeric Form of Peptidylarginine Deiminase Type I Revealed by X-ray Crystallography and Small-Angle X-ray Scattering.

Peptidylarginine deiminase (PAD; EC 3.5.3.15) is a post-translational modification enzyme that catalyzes the conversion of arginine in protein molecules to a citrulline residue in a Ca(2+)-dependent manner. In this study, we determined the structure of an active form of human PAD1 crystallized in the presence of Ca(2+) at 3.2-Å resolution. Although human PAD2 and PAD4 isozymes were previously reported to form a head-to-tail homodimer, it is still unknown whether this quaternary structure is common to other PAD isozymes. The asymmetric unit of the crystal contained two PAD1 molecules; however, the head-to-tail dimeric form was not found. Small-angle X-ray scattering analyses revealed PAD1 to be a monomer in solution, while PAD3 was dimerized with a structure similar to PAD2 and PAD4. PAD1 was apparently different from the crystal structures of PAD2 and PAD4, with an elongated N-terminal loop that appears to prevent the formation of the homodimer. Of interest, the N-terminal loop occupied the substrate binding site of the adjacent PAD1 molecules in the crystal. Deimination of S100A3 peptides in vitro implied that PAD isozymes recognize the quaternary structure of S100A3. The substrate-accessible monomeric structure brought about by the extension of its N terminus may partly account for the highest tolerant substrate recognition of PAD1. This is the first ever report on the molecular structure of PAD1 demonstrating the unique monomeric form of the PAD isozyme.

Zinc is the modulator of the calcium-dependent activation of post-translationally acting thiol-enzymes in autoimmune diseases.

UNLABELLED: Post-translational modifications of proteins can generate antigenic conformations that may cause autoimmune diseases in persons with specific HLA-haplotypes. Monocytes and macrophages, attracted to an inflamed site, can release post-translationally acting enzymes, such as transglutaminases and peptidylarginine deiminases. In vivo, the activation of these enzymes is crucial for the further course of event. Our hypothesis is that zinc modulates the activation of these calcium-dependent thiol-enzymes. Persons with celiac disease carry antibodies against deamidated dietary gluten and against transglutaminase type 2. Similarly, antibodies against citrulline-containing peptides and against peptidylarginine deiminase are detected in patients with rheumatoid arthritis. Thus, in two major autoimmune diseases, antibodies are detected against post-translationally modified proteins and against the thiol-enzymes responsible for catalyzing the modifications. In vitro, physiological concentrations of zinc reversibly inhibit the calcium-dependent activation of transglutaminases. Zinc attenuates the calcium-induced increase in affinity between transglutaminase 2 and serum from patients with celiac disease. Peptidylarginine deiminases are also inhibited by zinc. Moreover, zinc is rapidly redistributed in animals when an infection is induced. This pathway starting with an unspecific inflammation and ending up with an immune reaction against a specific tissue constitutes a theme with variations in other autoimmune diseases, such as dermatitis herpetiformis, multiple sclerosis, and type 1 diabetes. Inhibitors against transglutaminases and peptidylarginine deiminases have a great pharmacological potential. Interestingly, a large portion of the population may have been exposed to such an inhibitor. The primary metabolite of ethanol, acetaldehyde, can probably function as an irreversible inhibitor of these enzymes by forming a hemithioacetal with the thiol group of the active site. Not surprisingly, epidemiological studies have shown that alcohol is beneficial in rheumatoid arthritis. We predict that a similar situation will be observed in multiple sclerosis. The affinity of chelators such as EDTA and EGTA for Zn(2+) is three orders of magnitude greater than that for Ca(2+). This frequently overlooked complication imposes problems in biomedical research since a restoration of the zinc level can never be achieved in a blood sample which has been anti-coagulated by calcium chelators. The new synthetic direct thrombin inhibitors may offer a better way of preventing coagulation in vitro. CONCLUSIONS: Post-translational modifications are of potential interest in autoimmune diseases. The in vivo activation of calcium-dependent thiol-enzymes catalyzing these alterations, such as the transglutaminases and the peptidylarginine deiminases, is crucial for this pathway. According to our hypothesis, zinc is the modulator of this key function.

Mechanistic studies of protein arginine deiminase 2: evidence for a substrate-assisted mechanism.

Citrullination, which is catalyzed by protein arginine deiminases (PADs 1-4 and 6), is a post-translational modification (PTM) that effectively neutralizes the positive charge of a guanidinium group by its replacement with a neutral urea. Given the sequence similarity of PAD2 across mammalian species and the genomic organization of the PAD2 gene, PAD2 is predicted to be the ancestral homologue of the PADs. Although PAD2 has long been known to play a role in myelination, it has only recently been linked to other cellular processes, including gene transcription and macrophage extracellular trap formation. For example, PAD2 deiminates histone H3 at R26, and this PTM leads to the increased transcription of more than 200 genes under the control of the estrogen receptor. Given that our understanding of PAD2 biology remains incomplete, we initiated mechanistic studies on this enzyme to aid the development of PAD2-specific inhibitors. Herein, we report that the substrate specificity and calcium dependence of PAD2 are similar to those of PADs 1, 3, and 4. However, unlike those isozymes, PAD2 appears to use a substrate-assisted mechanism of catalysis in which the positively charged substrate guanidinium depresses the pKa of the nucleophilic cysteine. By contrast, PADs 1, 3, and 4 use a reverse-protonation mechanism. These mechanistic differences will aid the development of isozyme-specific inhibitors.

Crystallization and preliminary X-ray crystallographic analysis of human peptidylarginine deiminase type I.

Peptidylarginine deiminase (PAD) catalyzes the post-translational conversion of peptidylarginine to peptidylcitrulline in the presence of calcium ions. Among the five known human PAD isozymes (PAD1-4 and PAD6), PAD1 exhibits the broadest substrate specificity. Crystals of PAD1 obtained using polyethylene glycol 3350 as a precipitant diffracted to 3.70 Å resolution using synchrotron radiation. Two PAD1 molecules were contained in the asymmetric unit and the crystals belonged to space group P6(1), with unit-cell parameters a = b = 90.3, c = 372.3 Å. The solvent content was 58.2%.

Myocardial citrullination in rheumatoid arthritis: a correlative histopathologic study.

INTRODUCTION: The aim of this study was to explore the presence and localization of myocardial citrullination in samples from rheumatoid arthritis (RA) patients compared to rheumatic and non-rheumatic disease control groups. METHODS: Archived myocardial samples obtained during autopsy from 1995 to 2009 were assembled into four groups: RA; scleroderma; fatal myocarditis; and non-rheumatic disease controls. Samples were examined by immunohistochemistry (IHC) for the presence and localization of citrullination and peptidyl arginine deiminase enzymes (PADs) by a single cardiovascular pathologist blinded to disease group and clinical characteristics. RESULTS: Myocardial samples from seventeen RA patients were compared with those from fourteen controls, five fatal myocarditis patients, and ten scleroderma patients. Strong citrullination staining was detected exclusively in the myocardial interstitium in each of the groups. However, average and peak anti-citrulline staining was 59% and 44% higher, respectively, for the RA group compared to the combined non-RA groups (P < 0.05 for both comparisons). Myocardial fibrosis did not differ between the groups. In contrast to citrullination, PADs 1 to 3 and 6 were detected in cardiomyocytes (primarily PADs 1 and 3), resident inflammatory cells (primarily PADs 2 and 4), and, to a smaller extent, in endothelial cells and vascular smooth muscle cells. PAD staining did not co-localize with anti-citrulline staining in the interstitium and did not vary by disease state. CONCLUSIONS: Staining for citrullination was higher in the myocardial interstitium of RA compared to other disease states, a finding that could link autoimmunity to the known increase in myocardial dysfunction and heart failure in RA.

An intronic enhancer driven by NF-κB contributes to transcriptional regulation of peptidylarginine deiminase type I gene in human keratinocytes.

Peptidylarginine deiminases (PADs) catalyze the conversion of protein-bound arginine to citrulline residues. In human epidermis, where filaggrin is the main deiminated protein, three PADs are detected with specific patterns of expression depending on the keratinocyte (KC) differentiation state. Previous characterizations of the PAD-encoding gene promoters have shown that proximal regulation alone is not sufficient to explain this specificity of expression. In this work, we describe an evolutionarily highly conserved nucleotide segment located in the first intron of the PAD1 gene (PADI1). Luciferase reporter assays showed that it enhances the activity of the PADI1 promoter, in a calcium- and orientation-independent manner. Mutation of a putative NF-κB cis-element markedly reduced its enhancer activity, which also confirmed its potential regulatory function. Chromatin immunoprecipitation assays evidenced the binding of both p65 and p50 NF-κB subunits to the cis-element, and RNA interference inhibition assays confirmed that NF-κB contributes to the PADI1 transcriptional control. Furthermore, the intronic enhancer and promoter of PADI1 potentially interact through chromatin looping, as indicated by chromosome conformation capture assays. Our findings provide evidence that an NF-κB-mediated signaling pathway is involved in PADI1 regulation in human epidermal KCs.

Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3.

Protein citrullination has been shown to regulate numerous physiological pathways (e.g., the innate immune response and gene transcription) and is, when dysregulated, known to be associated with numerous human diseases, including cancer, rheumatoid arthritis, and multiple sclerosis. This modification, also termed deimination, is catalyzed by a group of enzymes called the protein arginine deiminases (PADs). In mammals, there are five PAD family members (i.e., PADs 1, 2, 3, 4, and 6) that exhibit tissue-specific expression patterns and vary in their subcellular localization. The kinetic characterization of PAD4 was recently reported, and these efforts guided the development of the two most potent PAD4 inhibitors (i.e., F- and Cl-amidine) known to date. In addition to being potent PAD4 inhibitors, we show here that Cl-amidine also exhibits a strong inhibitory effect against PADs 1 and 3, thus indicating its utility as a pan PAD inhibitor. Given the increasing number of diseases in which dysregulated PAD activity has been implicated, the development of PAD-selective inhibitors is of paramount importance. To aid that goal, we characterized the catalytic mechanism and substrate specificity of PADs 1 and 3. Herein, we report the results of these studies, which suggest that, like PAD4, PADs 1 and 3 employ a reverse protonation mechanism. Additionally, the substrate specificity studies provided critical information that aided the identification of PAD3-selective inhibitors. These compounds, denoted F4- and Cl4-amidine, are the most potent PAD3 inhibitors ever described.

Long-range enhancer differentially regulated by c-Jun and JunD controls peptidylarginine deiminase-3 gene in keratinocytes.

Long-range cis elements are critical regulators of transcription, particularly for clustered paralogous genes. Such are the five PADI genes in 1p35-36 encoding peptidylarginine deiminases, which catalyze deimination, a Ca2+-dependent post-translational modification. Deimination has been implicated in the pathophysiology of severe human diseases such as multiple sclerosis and rheumatoid arthritis. The PADI genes present different expression patterns. PADI1-3 are expressed in the epidermis, with increased expression levels in the most differentiated keratinocytes. Previous studies on PADI proximal promoters failed to explain such specificity of expression. We identified a conserved intergenic sequence in the PADI locus (IG1), which may play a role in PADI transcriptional regulation. In this work, we identified two DNase I.hypersensitive sites located in IG1, PAD intergenic enhancer segment 1 (PIE-S1) and PIE-S2, which act in synergy as a bipartite enhancer of the PADI3 and probably PADI1 promoters in normal human epidermal keratinocytes differentiated by a high-calcium-containing medium (1.5 mM). PIE-S1 and PIE-S2 present all the hallmarks of transcriptional enhancers: orientation-independence, copy-number dependence and cell-type specificity. PIE-S1 and PIE-S2 comprise conserved putative binding sites for MIBP1/RFX1 and activator protein 1, respectively. Deletion mutant screening revealed that these sites are crucial for the enhancer activity. Furthermore, chromatin immunoprecipitation assays evidenced differential binding of JunD or c-Jun on the activator protein 1 site depending on the cell differentiation state. Our results reveal the molecular bases of the expression specificity of PADI1 and PADI3 during keratinocyte differentiation through a long-range enhancer and support a model of PADI gene regulation depending on c-Jun-JunD competition.

Gene expression profiling identifies genes predictive of oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is associated with substantial mortality and morbidity. To identify potential biomarkers for the early detection of invasive OSCC, we compared the gene expressions of incident primary OSCC, oral dysplasia, and clinically normal oral tissue from surgical patients without head and neck cancer or preneoplastic oral lesions (controls), using Affymetrix U133 2.0 Plus arrays. We identified 131 differentially expressed probe sets using a training set of 119 OSCC patients and 35 controls. Forward and stepwise logistic regression analyses identified 10 successive combinations of genes which expression differentiated OSCC from controls. The best model included LAMC2, encoding laminin-gamma2 chain, and COL4A1, encoding collagen, type IV alpha1 chain. Subsequent modeling without these two markers showed that COL1A1, encoding collagen, type I alpha1 chain, and PADI1, encoding peptidyl arginine deiminase, type 1, could also distinguish OSCC from controls. We validated these two models using an internal independent testing set of 48 invasive OSCC and 10 controls and an external testing set of 42 head and neck squamous cell carcinoma cases and 14 controls (GEO GSE6791), with sensitivity and specificity above 95%. These two models were also able to distinguish dysplasia (n = 17) from control (n = 35) tissue. Differential expression of these four genes was confirmed by quantitative reverse transcription-PCR. If confirmed in larger studies, the proposed models may hold promise for monitoring local recurrence at surgical margins and the development of second primary oral cancer in patients with OSCC.

Crucial roles of MZF1 and Sp1 in the transcriptional regulation of the peptidylarginine deiminase type I gene (PADI1) in human keratinocytes.

Peptidylarginine deiminases (PADs) catalyze the conversion of protein-bound arginine residues into citrulline residues in a calcium-dependent manner. The PAD1 gene (PADI1) is expressed in a few tissues, including the epidermis, where the protein is detected with a higher level in the more differentiated keratinocytes. Using quantitative reverse transcription-PCR experiments, we show that PADI1 mRNAs are more abundant in keratinocytes cultured with 1.2 than 0.15 mM calcium. We cloned and characterized the promoter region using human keratinocytes transfected with variously deleted fragments of the 5'-upstream region of PADI1 coupled to the luciferase gene. We found that as few as 195 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Mutations of MZF1- or Sp1-binding sites markedly reduced PADI1 promoter activity. Chromatin immunoprecipitation assays revealed that MZF1 and Sp1/Sp3 bind to this region in vivo. Furthermore, MZF1 or Sp1 small interfering RNAs (siRNAs) effectively diminished PADI1 expression in keratinocytes cultured in both low- and high-calcium-containing medium. In addition, the expression of MZF1 and PAD1 increased in parallel when normal human epidermal keratinocytes underwent differentiation. These data indicate that MZF1 and Sp1/Sp3 binding to the promoter region drive the PADI1 expression.

Peptidyl arginine deiminase type 2 (PAD-2) and PAD-4 but not PAD-1, PAD-3, and PAD-6 are expressed in rheumatoid arthritis synovium in close association with tissue inflammation.

OBJECTIVE: Autoantibodies to citrullinated proteins (ACPAs) are specific for rheumatoid arthritis (RA) and probably are involved in its pathophysiology. Citrullyl residues, posttranslationally generated by peptidyl arginine deiminase (PAD), are indispensable components of ACPA-targeted epitopes. The aim of this study was to identify which PAD isotypes are expressed in the synovial tissue (ST) of patients with RA and are involved in the citrullination of fibrin, the major synovial target of ACPAs. METHODS: Expression of all PAD isotypes, including the recently described PAD type 6 (PAD-6), was explored by reverse transcription-polymerase chain reaction and immunoblotting, first in blood-derived mononuclear leukocytes from healthy donors, then in ST samples from 16 patients with RA and 11 control patients (4 with other arthritides and 7 with osteoarthritis [OA]). In ST samples from patients with RA, PADs were localized by immunohistochemistry. RESULTS: In lymphocytic and monocytic cells and, similarly, in ST samples from patients with RA, the PAD-2, PAD-4, and PAD-6 genes were found to be transcribed, but only PAD-2 and PAD-4 enzymes were detected. PAD-2 was also expressed in ST from control patients, including those with OA, while PAD-4 was preferentially expressed in ST from patients with other arthritides. In RA, the expression levels of PAD-2 and PAD-4 were correlated with the intensity of inflammation (cell infiltration, hypervascularization, and synovial lining hyperplasia), and both enzymes were demonstrable within or in the vicinity of citrullinated fibrin deposits. CONCLUSION: PAD-2 and PAD-4 are the only PAD isotypes expressed in the ST of patients with RA and those with other arthritides. Inflammatory cells are a major source, but PAD-4 also comes from hyperplastic synoviocytes. Both isotypes are probably involved in the citrullination of fibrin.