Protein Disulfide Isomerase A1 Research Articles

Protein disulfide isomerase-mediated transcriptional upregulation of Nox1 contributes to vascular dysfunction in hypertension.

Nox1 signaling is a causal key element in arterial hypertension. Recently, we identified protein disulfide isomerase A1 (PDI) as a novel regulatory protein that regulates Nox1 signaling in VSMCs. Spontaneously hypertensive rats (SHR) have increased levels of PDI in mesenteric resistance arteries compared with Wistar controls; however, its consequences remain unclear. Herein, we investigated the role of PDI in mediating Nox1 transcriptional upregulation and its effects on vascular dysfunction in hypertension. We demonstrate that PDI contributes to the development of hypertension via enhanced transcriptional upregulation of Nox1 in vascular smooth muscle cells (VSMCs). We show for the first time that PDI sulfenylation by hydrogen peroxide contributes to EGFR activation in hypertension via increased shedding of epidermal growth factor-like ligands. PDI also increases intracellular calcium levels, and contractile responses induced by ANG II. PDI silencing or pharmacological inhibition in VSMCs significantly decreases EGFR activation and Nox1 transcription. Overexpression of PDI in VSMCs enhances ANG II-induced EGFR activation and ATF1 translocation to the nucleus. Mechanistically, PDI increases ATF1-induced Nox1 transcription and enhances the contractile responses to ANG II. Herein we show that ATF1 binding to Nox1 transcription putative regulatory regions is augmented by PDI. Altogether, we provide evidence that HB-EGF in SHR resistance vessels promotes the nuclear translocation of ATF1, under the control of PDI, and thereby induces Nox1 gene expression and increases vascular reactivity. Thus, PDI acts as a thiol redox-dependent enhancer of vascular dysfunction in hypertension and could represent a novel therapeutic target for the treatment of this disease.

Protein disulfide isomerase A1 regulates fenestration dynamics in primary mouse liver sinusoidal endothelial cells (LSECs)

Protein disulfide isomerases (PDIs) are involved in many intracellular and extracellular processes, including cell adhesion and cytoskeletal reorganisation, but their contribution to the regulation of fenestrations in liver sinusoidal endothelial cells (LSECs) remains unknown. Given that fenestrations are supported on a cytoskeleton scaffold, this study aimed to investigate whether endothelial PDIs regulate fenestration dynamics in primary mouse LSECs.PDIA3 and PDIA1 were found to be the most abundant among PDI isoforms in LSECs. Taking advantage of atomic force microscopy, the effects of PDIA1 or PDIA3 inhibition on the fenestrations in LSECs were investigated using a classic PDIA1 inhibitor (bepristat) and novel aromatic N-sulfonamides of aziridine-2-carboxylic acid derivatives as PDIA1 (C-3389) or PDIA3 (C-3399) inhibitors. The effect of PDIA1 inhibition on liver perfusion was studied in vivo using dynamic contrast-enhanced magnetic resonance imaging. Additionally, PDIA1 inhibitors were examined in vitro in LSECs for effects on adhesion, cytoskeleton organisation, bioenergetics, and viability.Inhibition of PDIA1 with bepristat or C-3389 significantly reduced the number of fenestrations in LSECs, while inhibition of PDIA3 with C-3399 had no effect. Moreover, the blocking of free thiols by the cell-penetrating N-ethylmaleimide, but not by the non-cell-penetrating 4-chloromercuribenzenesulfonate, resulted in LSEC defenestration. Inhibition of PDIA1 did not affect LSEC adhesion, viability, and bioenergetics, nor did it induce a clear-cut rearrangement of the cytoskeleton. However, PDIA1-dependent defenestration was reversed by cytochalasin B, a known fenestration stimulator, pointing to the preserved ability of LSECs to form new pores. Importantly, systemic inhibition of PDIA1 in vivo affected intra-parenchymal uptake of contrast agent in mice consistent with LSEC defenestration.These results revealed the role of intracellular PDIA1 in the regulation of fenestration dynamics in LSECs, and in maintaining hepatic sinusoid homeostasis.

A Conformational-Dependent Interdomain Redox Relay at the Core of Protein Disulfide Isomerase Activity.

Aims: Protein disulfide isomerases (PDIs) are a family of chaperones resident in the endoplasmic reticulum (ER). In addition to holdase function, some members catalyze disulfide bond formation and isomerization, a crucial step for native folding and prevention of aggregation of misfolded proteins. PDIs are characterized by an arrangement of thioredoxin-like domains, with the canonical protein disulfide isomerase A1 (PDIA1) organized as four thioredoxin-like domains forming a horseshoe with two active sites, a and a', at the extremities. We aimed to clarify important aspects underlying the catalytic cycle of PDIA1 in the context of the full pathways of oxidative protein folding operating in the ER. Results: Using two fluorescent redox sensors, redox green fluorescent protein 2 (roGFP2) and HyPer (circularly permutated yellow fluorescent protein containing the regulatory domain of the H2O2-sensing protein OxyR), either unfolded or native, as client substrates, we identified the N-terminal a active site of PDIA1 as the main oxidant of thiols. From there, electrons can flow to the C-terminal a' active site, with the redox-dependent conformational flexibility of PDIA1 allowing the formation of an interdomain disulfide bond. The a' active site then acts as a crossing point to redirect electrons to ER downstream oxidases or back to client proteins to reduce scrambled disulfide bonds. Innovation and Conclusions: The two active sites of PDIA1 work cooperatively as an interdomain redox relay mechanism that explains PDIA1 oxidative activity to form native disulfides and PDIA1 reductase activity to resolve scrambled disulfides. This mechanism suggests a new rationale for shutting down oxidative protein folding under ER redox imbalance. Whether it applies to physiological substrates in cells remains to be shown.

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204.

Redox processes can modulate vascular pathophysiology. The endoplasmic reticulum redox chaperone protein disulfide isomerase A1 (PDIA1) is overexpressed during vascular proliferative diseases, regulating thrombus formation, endoplasmic reticulum stress adaptation, and structural remodeling. However, both protective and deleterious vascular effects have been reported for PDIA1, depending on the cell type and underlying vascular condition. Further understanding of this question is hampered by the poorly studied mechanisms underlying PDIA1 expression regulation. Here, we showed that PDIA1 mRNA and protein levels were upregulated (average 5-fold) in the intima and media/adventitia following partial carotid ligation (PCL). Our search identified that miR-204-5p and miR-211-5p (miR-204/211), two broadly conserved miRNAs, share PDIA1 as a potential target. MiR-204/211 was downregulated in vascular layers following PCL. In isolated endothelial cells, gain-of-function experiments of miR-204 with miR mimic decreased PDIA1 mRNA while having negligible effects on markers of endothelial activation/stress response. Similar effects were observed in vascular smooth muscle cells (VSMCs). Furthermore, PDIA1 downregulation by miR-204 decreased levels of the VSMC contractile differentiation markers. In addition, PDIA1 overexpression prevented VSMC dedifferentiation by miR-204. Collectively, we report a new mechanism for PDIA1 regulation through miR-204 and identify its relevance in a model of vascular disease playing a role in VSMC differentiation. This mechanism may be regulated in distinct stages of atherosclerosis and provide a potential therapeutic target.

Activity-Based Protein Profiling Identifies Protein Disulfide-Isomerases as Target Proteins of the Volatile Salinilactones.

The salinilactones, volatile marine natural products secreted from Salinispora arenicola, feature a unique [3.1.0]-lactone ring system and cytotoxic activities through a hitherto unknown mechanism. To find their molecular target, an activity-based protein profiling with a salinilactone-derived probe is applied that disclosed the protein disulfide-isomerases (PDIs) as the dominant mammalian targets of salinilactones, and thioredoxin (TRX1) as secondary target. The inhibition of protein disulfide-isomerase A1 (PDIA1) and TRX1 is confirmed by biochemical assays with recombinant proteins, showing that (1S,5R)-salinilactone B is more potent than its (1R,5S)-configured enantiomer. The salinilactones bound covalently to C53 and C397, the catalytically active cysteines of the isoform PDIA1 according to tandem mass spectrometry. Reactions with a model substrate demonstrated that the cyclopropyl group is opened by an attack of the thiol at C6. Fluorophore labeling experiments showed the cell permeability of a salinilactone-BODIPY (dipyrrometheneboron difluoride) conjugate and its co-localization with PDIs in the endoplasmic reticulum. The study is one of the first to pinpoint a molecular target for a volatile microbial natural product, and it demonstrates that salinilactones can achieve high selectivity despite their small size and intrinsic reactivity.

Vascular protein disulfide isomerase A1 mediates endothelial dysfunction induced by angiotensin II in mice.

Protein disulfide isomerases (PDIs) are involved in platelet aggregation and intravascular thrombosis, but their role in regulating endothelial function is unclear. Here, we characterized the involvement of vascular PDIA1 in angiotensin II (Ang II)-induced endothelial dysfunction in mice. Endothelial dysfunction was induced in C57BL/6JCmd male mice via Ang II subcutaneous infusion, and PDIA1 was inhibited with bepristat. Endothelial function was assessed invivo with magnetic resonance imaging and exvivo with a myography, while arterial stiffness was measured as pulse wave velocity. Nitric oxide (NO) bioavailability was measured in the aorta (spin-trapping electron paramagnetic resonance) and plasma (NO2 - and NO3 - levels). Oxidative stress, eNOS uncoupling (DHE-based aorta staining), and thrombin activity (thrombin-antithrombin complex; calibrated automated thrombography) were evaluated. The inhibition of PDIA1 by bepristat in Ang II-treated mice prevented the impairment of NO-dependent vasodilation in the aorta as evidenced by the response to acetylcholine invivo, increased systemic NO bioavailability and the aortic NO production, and decreased vascular stiffness. Bepristat's effect on NO-dependent function was recapitulated exvivo in Ang II-induced endothelial dysfunction in isolated aorta. Furthermore, bepristat diminished the Ang II-induced eNOS uncoupling and overproduction of ROS without affecting thrombin activity. In Ang II-treated mice, the inhibition of PDIA1 normalized the NO-ROS balance, prevented endothelial eNOS uncoupling, and, thereby, improved vascular function. These results indicate the importance of vascular PDIA1 in regulating endothelial function, but further studies are needed to elucidate the details of the mechanisms involved.

Conditional hepatocyte ablation of PDIA1 uncovers indispensable roles in both APOB and MTTP folding to support VLDL secretion

ObjectivesThe assembly and secretion of hepatic very low-density lipoprotein (VLDL) plays pivotal roles in hepatic and plasma lipid homeostasis. Protein disulfide isomerase A1 (PDIA1/P4HB) is a molecular chaperone whose functions are essential for protein folding in the endoplasmic reticulum. Here we investigated the physiological requirement in vivo for PDIA1 in maintaining VLDL assembly and secretion. MethodsPdia1/P4hb was conditionally deleted in adult mouse hepatocytes and the phenotypes characterized. Mechanistic analyses in primary hepatocytes determined how PDIA1 ablation alters MTTP synthesis and degradation as well as altering synthesis and secretion of Apolipoprotein B (APOB), along with complementary expression of intact PDIA1 vs a catalytically inactivated PDIA1 mutant. ResultsHepatocyte-specific deletion of Pdia1/P4hb inhibited hepatic MTTP expression and dramatically reduced VLDL production, leading to severe hepatic steatosis and hypolipidemia. Pdia1-deletion did not affect mRNA expression or protein stability of MTTP but rather prevented Mttp mRNA translation. We demonstrate an essential role for PDIA1 in MTTP synthesis and function and show that PDIA1 interacts with APOB in an MTTP-independent manner via its molecular chaperone function to support APOB folding and secretion. ConclusionsPDIA1 plays indispensable roles in APOB folding, MTTP synthesis and activity to support VLDL assembly. Thus, like APOB and MTTP, PDIA1 is an obligatory component of hepatic VLDL production.

DNAJB12 and DNJB14 are non-redundant Hsp40 redox chaperones involved in endoplasmic reticulum protein reflux

BackgroundThe endoplasmic reticulum (ER) transmembrane chaperones DNAJB12(B12) and DNAJB14(B14) are cofactors that cooperate with cytosolic Heat Shock-70 protein (HSC70) facilitating folding/degradation of nascent membrane proteins and supporting the ER-membrane penetration of viral particles. Here, we assessed structural/functional features of B12/B14 with respect to their regulation by ER stress and their involvement in ER stress-mediated protein reflux. MethodsWe investigated the effect of Unfolded Protein Response(UPR)-eliciting drugs on the expression/regulation of B12-B14 and their roles in ER-to-cytosol translocation of Protein Disulfide Isomerase-A1(PDI). ResultsWe show that B12 and B14 are similar but do not seem redundant. They share predicted structural features and show high homology of their cytosolic J-domains, while their ER-lumen DUF1977 domains are quite dissimilar. Interactome analysis suggested that B12/B14 associate with different biological processes. UPR activation did not significantly impact on B12 gene expression, while B14 transcripts were up-regulated. Meanwhile, B12 and B14 (33.4 kDa isoform) protein levels were degraded by the proteasome upon acute reductive challenge. Also, B12 degradation was impaired upon sulfenic-acid trapping by dimedone. We originally report that knockdown of B12/B14 and their cytosolic partner SGTA in ER-stressed cells significantly impaired the amount of the ER redox-chaperone PDI in a cytosolic-enriched fraction. Additionally, B12 but not B14 overexpression increased PDI relocalization in non-stressed cells. Conclusions and general significanceOur findings reveal that B12/B14 regulation involves thiol redox processes that may impact on their stability and possibly on physiological effects. Furthermore, we provide novel evidence that these proteins are involved in UPR-induced ER protein reflux.

Interaction between Protein Disulfide Isomerase-A1 and Enolase-1 contributes to plasmin activation in vascular smooth muscle cells

Interaction between Protein Disulfide Isomerase-A1 and Enolase-1 contributes to plasmin activation in vascular smooth muscle cells

Endoplasmic reticulum - plasma membrane contact sites as a potential hub for Protein Disulfide Isomerase-A1 (PDIA1) / NADPH oxidase cross-talk.

Endoplasmic reticulum - plasma membrane contact sites as a potential hub for Protein Disulfide Isomerase-A1 (PDIA1) / NADPH oxidase cross-talk.

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204 and controls vascular smooth muscle cell differentiation

Disturbed flow regulates protein disulfide isomerase A1 expression via microRNA-204 and controls vascular smooth muscle cell differentiation

Structural and functional characterization of the alternative splicing isoform P4HB-021 of the human protein disulfide isomerase-A1 (P4HB) gene.

Structural and functional characterization of the alternative splicing isoform P4HB-021 of the human protein disulfide isomerase-A1 (P4HB) gene.

Evidence for a protective role of Protein Disulfide Isomerase-A1 against aortic dissection

Background and aimsRedox signaling is involved in the pathophysiology of aortic aneurysm/dissection. Protein Disulfide Isomerases and its prototype PDIA1 are thiol redox chaperones mainly from endoplasmic reticulum (ER), while PDIA1 cell surface pool redox-regulates thrombosis, cytoskeleton remodeling and integrin activation, which are mechanisms involved in aortic disease. Here we investigate the roles of PDIA1 in aortic dissection. MethodsInitially, we assessed the outcome of aortic aneurysm/dissection in transgenic PDIA1-overexpressing FVB mice using a model of 28-day exposure to lysyl oxidase inhibitor BAPN plus angiotensin-II infusion. In a second protocol, we assessed the effects of PDIA1 inhibitor isoquercetin (IQ) against aortic dissection in C57BL/6 mice exposed to BAPN for 28 days. ResultsTransgenic PDIA1 overexpression associated with ca. 50% (p = 0.022) decrease (vs.wild-type) in mortality due to abdominal aortic rupture and protected against elastic fiber breaks in thoracic aorta. Conversely, exposure of mice to IQ increased thoracic aorta dissection-related mortality rates, from ca. 18%–50% within 28-days (p = 0.019); elastic fiber disruption and collagen deposition were also enhanced. The structurally-related compound diosmetin, which does not inhibit PDI, had negligible effects. In parallel, stretch-tension curves indicated that IQ amplified a ductile-type of biomechanical failure vs. control or BAPN-exposed mice aortas. IQ-induced effects seemed unassociated with nonspecific antioxidant effects or ER stress. In both models, echocardiographic analysis of surviving mice suggested that aortic rupture was dissociated from progressive dilatation. ConclusionsOur data indicate a protective role of PDIA1 against aortic dissection/rupture and potentially uncovers a novel integrative mechanism coupling redox and biomechanical homeostasis in vascular remodeling.

Endoplasmic Reticulum Stress-Regulated Chaperones as a Serum Biomarker Panel for Parkinson’s Disease

Examination of post-mortem brain tissues has previously revealed a strong association between Parkinson’s disease (PD) pathophysiology and endoplasmic reticulum (ER) stress. Evidence in the literature regarding the circulation of ER stress-regulated factors released from neurons provides a rationale for investigating ER stress biomarkers in the blood to aid diagnosis of PD. The levels of ER stress-regulated proteins in serum collected from 29 PD patients and 24 non-PD controls were measured using enzyme-linked immunosorbent assays. A panel of four biomarkers, protein disulfide-isomerase A1, protein disulfide-isomerase A3, mesencephalic astrocyte-derived neurotrophic factor, and clusterin, together with age and gender had higher ability (area under the curve 0.64, sensitivity 66%, specificity 57%) and net benefit to discriminate PD patients from the non-PD group compared with other analyzed models. Addition of oligomeric and total α-synuclein to the model did not improve the diagnostic power of the biomarker panel. We provide evidence that ER stress-regulated proteins merit further investigation for their potential as diagnostic biomarkers of PD.Graphical

Abstract 12127: Protein Disulfide Isomerase A1 as Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Background: VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. However, role of PDIA1 in ROS-dependent VEGFR2 signaling and angiogenesis is entirely unknown. Results: Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or early endosome marker Rab5, but not late endosome marker Rab7, at the perinuclear region in human ECs in response to VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration (43.3%), proliferation (52.8%) and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Experiments using “redox-dead” mutant PDIA1 with replacement of the active four Cys residues with Ser revealed that VEGF-induced PDIA1 CysOH formation promoted angiogenic responses by increasing phosphorylation of VEGFR2 at Y1175 via oxidative inactivation of PTP1B. In vivo, Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic CD31 positive ECs of ischemic muscles, and that limb perfusion recovery and neovascularization (48%) in response to ischemic injury were significantly impaired in EC-specific Pdia1 conditional knockout mice. Conclusion: PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs via oxidative inactivation of PTP1B, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases

Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

Protein Disulfide Isomerase A1 functions as a Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Protein Disulfide Isomerase A1 functions as a Novel Redox Sensor in VEGFR2 Signaling and Angiogenesis

Sustained overexpression of Protein Disulfide Isomerase-A1 induces profound mitochondrial reorganization and a differentiated vascular smooth muscle phenotype

Sustained overexpression of Protein Disulfide Isomerase-A1 induces profound mitochondrial reorganization and a differentiated vascular smooth muscle phenotype

Protein disulfide isomerase A1‑associated pathways inthedevelopment of stratified breast cancer therapies.

The oxidoreductase protein disulfide isomerase A1 (PDIA1) functions as a cofactor for many transcription factors including estrogen receptor α (ERα), nuclear factor (NF)-κB, nuclear factor erythroid 2-like 2 (NRF2) and regulates the protein stability of the tumor suppressor p53. Taking this into account we hypothesized that PDIA1, by differentially modulating the gene expression of a diverse subset of genes in the ERα-positive vs. the ERα-negative breast cancer cells, might modify dissimilar pathways in the two types of breast cancer. This hypothesis was investigated using RNA-seq data from PDIA1-silenced MCF-7 (ERα-positive) and MDA-MB-231 (ERα-negative) breast cancer cells treated with either interferon γ (IFN-γ) or etoposide (ETO), and the obtained data were further analyzed using a variety of bioinformatic tools alongside clinical relevance assessment via Kaplan-Meier patient survival curves. The results highlighted the dual role of PDIA1 in suppressing carcinogenesis in the ERα(+) breast cancer patients by negatively regulating the response to reactive oxygen species (ROS) and promoting carcinogenesis by inducing cell cycle progression. In the ERα(−) breast cancer patients, PDIA1 prevented tumor development by modulating NF-κB and p53 activity and cell migration and induced breast cancer progression through control of cytokine signaling and the immune response. The findings reported in this study shed light on the differential pathways regulating carcinogenesis in ERα(+) and ERα(−) breast cancer patients and could help identify therapeutic targets selectively effective in ERα(+) vs. ERα(−) patients.

CXCL1-Triggered PAD4 Cytoplasmic Translocation Enhances Neutrophil Adhesion through Citrullination of PDIA1.

Aims: Vascular inflammation is critical for the development and progression of atherosclerosis. Previously, we reported that neutrophils adhere to the vascular endothelium in low-density lipoprotein receptor null mice fed a high-fat diet through hypercitrullination of histone H3 by peptidylarginine deiminase 4 (PAD4) in neutrophils. However, the involvement of PAD4 and citrullination of proteins other than histone H3 in neutrophil adhesion is not well known. In this study, we investigated the function of PAD4 and identified citrullinated proteins during vascular inflammation. Methods: We pefformed flow assay under physiological flow conditions using differentiated HL-60 (dHL-60) cells stimulated with CXCL1 and human umbilical vein endothelial cells (HUVECs). Furthermore, phalloidin stain for dHL-60 stimulated with CXCL1 to observe F-actin polymerization and immunohistochemistry for the activated β2-integrin was conducted. To identify a target of citrullination in the cytoplasm of dHL-60 cells, liquid chromatography-mass spectrometry (LC-MS/MS) for dHL-60 stimulated with CXCL1 was performed. Results: Inhibition or knockdown of PAD4 significantly decreased adhesion of under physiological flow conditions. Thr-Asp-F-amidine trifluoroacetate salt (TDFA), a PAD4 inhibitor, inhibited cytoplasmic translocation of PAD4 by CXCL1. TDFA or knockdown of PAD4 significantly decreased expression of β2-integrin and F-actin polymerization activated by CXCL1. Moreover, LC-MS/MS identified protein disulfide isomerase A1 (PDIA1) as a target of citrullination in the cytoplasm of dHL-60 cells. Knockdown of PDIA1 significantly decreased adhesion of dHL-60 cells to HUVECs, expression of β2-integrin, and F-actin polymerization. Conclusions: Cytoplasmic translocation of PAD4 by CXCL1 induces neutrophil adhesion to vascular endothelial cells and citrullination of PDIA1.