Cell Surface Protein Disulfide Isomerase Research Articles

Protein Disulfide Isomerase and Extracellular Adherence Protein Cooperatively Potentiate Staphylococcal Invasion into Endothelial Cells.

Invasion of host cells is an important feature of Staphylococcus aureus. The main internalization pathway involves binding of the bacteria to host cells, e.g., endothelial cells, via a fibronectin (Fn) bridge between S. aureus Fn binding proteins and α5β1-integrin, followed by phagocytosis. The secreted extracellular adherence protein (Eap) has been shown to promote this cellular uptake pathway of not only S. aureus, but also of bacteria otherwise poorly taken up by host cells, such as Staphylococcus carnosus. The exact mechanisms are still unknown. Previously, we demonstrated that Eap induces platelet activation by stimulation of the protein disulfide isomerase (PDI), a catalyst of thiol-disulfide exchange reactions. Here, we show that Eap promotes PDI activity on the surface of endothelial cells, and that this contributes critically to Eap-driven staphylococcal invasion. PDI-stimulated β1-integrin activation followed by increased Fn binding to host cells likely accounts for the Eap-enhanced uptake of S. aureus into non-professional phagocytes. Additionally, Eap supports the binding of S. carnosus to Fn-α5β1 integrin, thereby allowing its uptake into endothelial cells. To our knowledge, this is the first demonstration that PDI is crucial for the uptake of bacteria into host cells. We describe a hitherto unknown function of Eap-the promotion of an enzymatic activity with subsequent enhancement of bacterial uptake-and thus broaden mechanistic insights into its importance as a driver of bacterial pathogenicity. IMPORTANCE Staphylococcus aureus can invade and persist in non-professional phagocytes, thereby escaping host defense mechanisms and antibiotic treatment. The intracellular lifestyle of S. aureus contributes to the development of infection, e.g., in infective endocarditis or chronic osteomyelitis. The extracellular adherence protein secreted by S. aureus promotes its own internalization as well as that of bacteria that are otherwise poorly taken up by host cells, such as Staphylococcus carnosus. In our study, we demonstrate that staphylococcal uptake by endothelial cells requires catalytic disulfide exchange activity by the cell-surface protein disulfide isomerase, and that this critical enzymatic function is enhanced by Eap. The therapeutic application of PDI inhibitors has previously been investigated in the context of thrombosis and hypercoagulability. Our results add another intriguing possibility: therapeutically targeting PDI, i.e., as a candidate approach to modulate the initiation and/or course of S. aureus infectious diseases.

Pathophysiological roles of cell surface and extracellular protein disulfide isomerase and their molecular mechanisms.

Protein disulfide isomerase (PDI) is the prototypic member of the thiol isomerase family that catalyses disulfide bond rearrangement. Initially identified in the endoplasmic reticulum as folding catalysts, PDI and other members in its family have also been widely reported to reside on the cell surface and in the extracellular matrix. Although how PDI is exported and retained on the cell surface remains a subject of debate, this unique pool of PDI is developing into an important mechanism underlying the redox regulation of protein sulfhydryls that are critical for the cellular activities under various disease conditions. This review aims to provide an overview of the pathophysiological roles of surface and extracellular PDI and their underlying molecular mechanisms. Understanding the involvement of extracellular PDI in these diseases will advance our knowledge in the molecular aetiology to facilitate the development of novel pharmacological strategies by specifically targeting PDI in extracellular compartments.

Protein Disulfide Isomerases Regulate IgE-Mediated Mast Cell Responses and Their Inhibition Confers Protective Effects During Food Allergy.

The thiol isomerase, protein disulfide isomerase (PDI), plays important intracellular roles during protein folding, maintaining cellular function and viability. Recent studies suggest novel roles for extracellular cell surface PDI in enhancing cellular activation and promoting their function. Moreover, a number of food-derived substances have been shown to regulate cellular PDI activity and alter disease progression. We hypothesized that PDI may have similar roles during mast cell-mediated allergic responses and examined its effects on IgE-induced mast cell activity during cell culture and food allergy. Mast cells were activated via IgE and antigen and the effects of PDI inhibition on mast cell activation were assessed. The effects of PDI blockade in vivo were examined by treating mice with the irreversible PDI inhibitor, PACMA-31, in an ovalbumin-induced model of food allergy. The role of dietary PDI modulators was investigated using various dietary compounds including curcumin and quercetin-3-rutinoside (rutin). PDI expression was observed on resting mast cell surfaces, intracellularly, and in the intestines of allergic mice. Furthermore, enhanced secretion of extracellular PDI was observed on mast cell membranes during IgE and antigen activation. Insulin turbidimetric assays demonstrated that curcumin is a potent PDI inhibitor and pre-treatment of mast cells with curcumin or established PDI inhibitors such as bacitracin, rutin or PACMA-31, resulted in the suppression of IgE-mediated activation and the secretion of various cytokines. This was accompanied by decreased mast cell proliferation, FcεRI expression, and mast cell degranulation. Similarly, treatment of allergic BALB/c mice with PACMA-31 attenuated the development of food allergy resulting in decreased allergic diarrhea, mast cell activation, and fewer intestinal mast cells. The production of TH2-specific cytokines was also suppressed. Our observations suggest that PDI catalytic activity is essential in the regulation of mast cell activation, and that its blockade may benefit patients with allergic inflammation.

Binding of Host Cell Surface Protein Disulfide Isomerase by Anaplasma phagocytophilum Asp14 Enables Pathogen Infection.

Diverse intracellular pathogens rely on eukaryotic cell surface disulfide reductases to invade host cells. Pharmacologic inhibition of these enzymes is cytotoxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for disrupting this common infection strategy. Anaplasma phagocytophilum invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium's adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identified protein disulfide isomerase (PDI) as an Asp14 binding partner. Coimmunoprecipitation confirmed the interaction and validated it to be Asp14 C terminus dependent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disulfide bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to productively infect myeloid-specific-PDI conditional-knockout mice, marking the first demonstration of in vivo microbial dependency on PDI for infection. Mutational analyses identified the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulfide bonds that it reduces, which enables cellular and in vivo infection.IMPORTANCEAnaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging potentially fatal disease and the second-most common tick-borne illness in the United States. Treatment options are limited, and no vaccine exists. Due to the bacterium's obligatory intracellular lifestyle, A. phagocytophilum survival and pathogenesis are predicated on its ability to enter host cells. Understanding its invasion mechanism will yield new targets for preventing bacterial entry and, hence, disease. We report a novel entry pathway in which the A. phagocytophilum outer membrane protein Asp14 binds host cell surface protein disulfide isomerase via specific C-terminal residues to promote reduction of bacterial surface disulfide bonds, which is critical for cellular invasion and productive infection in vivo Targeting the Asp14 C terminus could be used to prevent/treat granulocytic anaplasmosis. Our findings have broad implications, as a thematically similar approach could be applied to block infection by other intracellular microbes that exploit cell surface reductases.

Thiol/disulfide exchange occurs in rotavirus structural proteins during contact with intestinal villus cell surface.

Protein disulfide isomerase (PDI) is an enzyme that catalyzes disulfide bond reduction or formation and rearrangements of disulfide bridges, and also functions as a chaperone. During entry of some of the viruses PDI participates in thiol-disulfide exchange. Previous reports show that rotavirus entry is interfered by impermeant thiol/disulfide exchange inhibitors and antibodies against PDI. Our objective was to assess the interaction between PDI and triple-layered particles (TLPs) from rotavirus strains ECwt and RRV and from a human rotavirus isolate (HI) during the early steps of virus entry in a system of isolated small intestinal villi. Purified soluble PDI was incubated with either isolated intestinal villi or cell membrane-enriched fractions in the presence or absence of thiol/disulfide inhibitors such as bacitracin, DTNB or N- ethylmaleimide followed by the assessment of the PDI interactions with TLPs and rotavirus structural proteins in terms of their redox state changes. Soluble and membrane-bound PDI was found to interact with TLPs from all the rotaviruses assayed and also with the isolated structural proteins represented by the recombinant rVP5* (a tryptic cleavage product of VP4), rVP6 and the native VP7. PDI interaction with TLPs and rotavirus structural proteins was decreased by the presence of thiol/disulfide exchange inhibitors. Interactions of cell membrane-enriched fractions with TLPs produced rearrangements in the disulfide bridges of rotavirus structural proteins. We conclude that PDI interacts with rotavirus virions through redox reactions that could facilitate the rotavirus entry into the host cell. Keywords: cell surface PDI; thiol-disulfide exchange; rotavirus TLPs; virus entry; bacitracin; DTNB.

Implication of heat shock proteins in rotavirus entry into Reh cells.

The mechanisms of rotavirus entry into the target cell are described as a multi-step event in which the virions are bound to sialic acid (SA), followed by interaction with heat shock cognate protein 70 (Hsc70), some integrins and protein disulfide isomerase (PDI). However, the cell surface receptor molecules facilitating the entry of tumor cell-adapted rotavirus are not completely characterized. Using infection blocking assays with antibodies to some heat shock proteins (HSPs) and also some inhibitors of these cellular proteins, we were able to identify the cell surface Hsp90, Hsp70, Hsc70, Hsp60, Hsp40, PDI and integrin β3 as receptors of tumor cell-adapted rotavirus in Reh cells. Furthermore, the results also indicated that these rotavirus receptors are associated with lipid microdomains (rafts). Our findings provide evidence that rotavirus tropism for these human acute lymphocytic leukemia cells is explained by the relatively high expression of some HSPs in rafts. The results shown here encourage further research aim at evaluating the potential use of rotaviruses as an oncolytic agent for the treatment of some cancers. Keywords: heat shock proteins; rotavirus; cell receptor; cancer; oncolytic virus.

Galectin-9 binds to O-glycans on protein disulfide isomerase.

Changes in the T cell surface redox environment regulate critical cell functions, such as cell migration, viral entry and cytokine production. Cell surface protein disulfide isomerase (PDI) contributes to the regulation of T cell surface redox status. Cell surface PDI can be released into the extracellular milieu or can be internalized by T cells. We have found that galectin-9, a soluble lectin expressed by T cells, endothelial cells and dendritic cells, binds to and retains PDI on the cell surface. While endogenous galectin-9 is not required for basal cell surface PDI expression, exogenous galectin-9 mediated retention of cell surface PDI shifted the disulfide/thiol equilibrium on the T cell surface. O-glycans on PDI are required for galectin-9 binding, and PDI recognition appears to be specific for galectin-9, as galectin-1 and galectin-3 do not bind PDI. Galectin-9 is widely expressed by immune and endothelial cells in inflamed tissues, suggesting that T cells would be exposed to abundant galectin-9, in cis and in trans, in infectious or autoimmune conditions.

HPW-RX40 prevents human platelet activation by attenuating cell surface protein disulfide isomerases

Protein disulfide isomerase (PDI) present at platelet surfaces has been considered to play an important role in the conformational change and activation of the integrin glycoprotein IIb/IIIa (GPIIb/IIIa) and thus enhances platelet aggregation. Growing evidences indicated that platelet surface PDI may serve as a potential target for developing of a new class of antithrombotic agents. In the present study, we investigated the effects of HPW-RX40, a chemical derivative of β-nitrostyrene, on platelet activation and PDI activity. HPW-RX40 inhibited platelet aggregation, GPIIb/IIIa activation, and P-selectin expression in human platelets. Moreover, HPW-RX40 reduced thrombus formation in human whole blood under flow conditions, and protects mice from FeCl3-induced carotid artery occlusion. HPW-RX40 inhibited the activity of recombinant PDI family proteins (PDI, ERp57, and ERp5) as well as suppressed cell surface PDI activity of platelets in a reversible manner. Exogenous addition of PDI attenuated the inhibitory effect of HPW-RX40 on GPIIb/IIIa activation. Structure-based molecular docking simulations indicated that HPW-RX40 binds to the active site of PDI by forming hydrogen bonds. In addition, HPW-RX40 neither affected the cell viability nor induced endoplasmic reticulum stress in human cancer A549 and MDA-MB-231 cells. Taken together, our results suggest that HPW-RX40 is a reversible and non-cytotoxic PDI inhibitor with antiplatelet effects, and it may have a potential for development of novel antithrombotic agents.

Novel antiplatelet role for a protein disulfide isomerase‐targeted peptide: evidence of covalent binding to the C‐terminal CGHC redox motif

Background Protein disulfide isomerase (PDI) plays a major role in platelet aggregation, and its inhibitors have emerged as novel antithrombotic drugs. In previous work, we designed a peptide based on a PDI redox motif (CGHC) that inhibited both PDI reductase activity and PDI-modulated superoxide generation by neutrophil Nox2. Thus, we hypothesized that this peptide would also inhibit platelet aggregation by association with surface PDI. Methods Three peptides were used: CxxC, containing the PDI redox motif; Scr, presenting a scrambled sequence of the same residues and AxxA, with cysteines replaced by alanine. These peptides were tested under platelet aggregation and flow cytometry protocols to identify their possible antiplatelet activity. We labeled membrane free thiol and electrospray ionization liquid chromatography tandem mass spectrometry to test for an interaction. Results CxxC decreased platelet aggregation in a dose-dependent manner, being more potent at lower agonist concentrations, whereas neither AxxA nor Scr peptides exerted any effect. CxxC decreased aIIbb3 activation, but had no effect on the other markers. CxxC also decreased cell surface PDI pulldown without interfering with the total thiol protein content. Finally, we detected the addition of one CxxC molecule to reduced PDI through binding to Cys400 through mass spectrometry. Interestingly, CxxC did not react with oxidized PDI. Discussion CxxC has consistently shown its antiplatelet effects, both in PRP and washed platelets, corroborated by decreased aIIbb3 activation. The probable mechanism of action is through a mixed dissulphide bond with Cys400 of PDI, which has been shown to be essential for PDI's actions. Conclusion In summary, our data support antiplatelet activity for CxxC through binding to Cys400 in the PDI a0 domain, which can be further exploited as a model for sitedriven antithrombotic agent development.

3,4-Methylenedioxy-β-nitrostyrene inhibits adhesion and migration of human triple-negative breast cancer cells by suppressing β1 integrin function and surface protein disulfide isomerase.

Triple negative breast cancer (TNBC) exhibits an aggressive clinical course by high metastatic potential. It is known that integrin-mediated cell adhesion and migration are important for cancer metastasis. In the present study, a synthetic compound, 3, 4-methyenedioxy-β-nitrostyrene (MNS), significantly inhibited adhesion of TNBC cell lines to different extracellular matrix (ECM) components. The antimetastatic capacity of MNS was also observed through reducing TNBC cells migration and invasion without affecting cell viability. Confocal microscopy revealed that MNS disrupted the formation of focal adhesion complex and actin stress fiber networks. Consistent with this finding, MNS inhibited phosphorylation of focal adhesion kinase (FAK) and paxillin as detected by Western blot analysis. In exploring the underlying mechanism, we found that MNS inhibited phosphorylation of FAK as a result of reducing β1 integrin activation and clustering. A cell-impermeable dithiol reagent, 2, 3-dimercaptopropane-1-sulfonic acid abrogated all of MNS's actions, indicating that MNS may react with thiol groups of cell surface proteins that are involved in regulation of β1 integrin function as well as cell adhesion and migration. Cell surface protein disulfide isomerase (PDI) has been reported to be essential for the affinity modulation of β integrins. We also demonstrated that MNS inhibited PDI activity both in a pure enzyme system and in intact cancer cells. Taken together, our results suggest that MNS inhibits invitro metastatic properties of TNBC cells through suppression of β1 integrin activation and focal adhesion signaling. Moreover, inhibition of surface PDI may contribute, at least in part, to the actions of MNS. These results suggest that MNS has a potential to be developed as an anticancer agent for treatment of TNBC.

Lung cancer chemotherapy agents increase procoagulant activity via protein disulfide isomerase-dependent tissue factor decryption.

Lung cancer patients undergoing chemotherapy have an elevated risk for thrombosis. However, the mechanisms by which chemotherapy agents increase the risk for thrombosis remains unclear. The aim of this study was to determine the mechanism(s) by which lung cancer chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel elicit increased tissue factor activity on endothelial cells, A549 cells, and monocytes. Tissue factor activity, tissue factor antigen, and phosphatidylserine exposure were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Cell surface protein disulfide isomerase (PDI) and cell surface free thiol levels were measured on HUVEC and A549 non-small cell lung carcinoma cells. Treatment of HUVECs, A549 cells, and monocytes with lung cancer chemotherapy significantly increased cell surface tissue factor activity. However, elevated tissue factor antigen levels were observed only on cisplatin-treated and gemcitabine-treated monocytes. Cell surface levels of phosphatidylserine were increased on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Chemotherapy also resulted in increased cell surface levels of PDI and reduced cell surface free thiol levels. Glutathione treatment and PDI inhibition, but not phosphatidylserine inhibition, attenuated tissue factor activity. Furthermore, increased tissue factor activity was reversed by reducing cysteines with dithiothreitol. These studies are the first to demonstrate that lung cancer chemotherapy agents increase procoagulant activity on endothelial cells and A549 cells by tissue factor decryption through a disulfide bond formation in a PDI-dependent mechanism.

Cell Surface Protein Disulfide Isomerase Regulates Natriuretic Peptide Generation of Cyclic Guanosine Monophosphate

RationaleThe family of natriuretic peptides (NPs), including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), exert important and diverse actions for cardiovascular and renal homeostasis. The autocrine and paracrine functions of the NPs are primarily mediated through the cellular membrane bound guanylyl cyclase-linked receptors GC-A (NPR-A) and GC-B (NPR-B). As the ligands and receptors each contain disulfide bonds, a regulatory role for the cell surface protein disulfide isomerase (PDI) was investigated.ObjectiveWe utilized complementary in vitro and in vivo models to determine the potential role of PDI in regulating the ability of the NPs to generate its second messenger, cyclic guanosine monophosphate.Methods and ResultsInhibition of PDI attenuated the ability of ANP, BNP and CNP to generate cGMP in human mesangial cells (HMCs), human umbilical vein endothelial cells (HUVECs), and human aortic smooth muscle cells (HASMCs), each of which were shown to express PDI. In LLC-PK1 cells, where PDI expression was undetectable by immunoblotting, PDI inhibition had a minimal effect on cGMP generation. Addition of PDI to cultured LLC-PK1 cells increased intracellular cGMP generation mediated by ANP. Inhibition of PDI in vivo attenuated NP-mediated generation of cGMP by ANP. Surface Plasmon Resonance demonstrated modest and differential binding of the natriuretic peptides with immobilized PDI in a cell free system. However, PDI was shown to co-localize on the surface of cells with GC-A and GC-B by co-immunoprecpitation and immunohistochemistry.ConclusionThese data demonstrate for the first time that cell surface PDI expression and function regulate the capacity of natriuretic peptides to generate cGMP through interaction with their receptors.

Abstract 580: Lung Cancer Chemotherapy Agents Increase Procoagulant Activity via Protein Disulfide Isomerase-Dependent Tissue Factor Decryption

Background: Lung cancer patients undergoing chemotherapy have an elevated risk for thrombosis. However, the mechanisms by which chemotherapy agents increase the risk for thrombosis remains unclear. Objectives: To determine the mechanism(s) by which lung cancer chemotherapy agents cisplatin, carboplatin, gemcitabine, and paclitaxel elicit increased tissue factor (TF) activity on endothelial cells, A549 cells, and monocytes. Methods: TF activity, TF antigen, and phosphatidylserine exposure were measured on chemotherapy-treated human umbilical vein endothelial cells (HUVEC), A549 cells, and monocytes. Cell surface protein disulfide isomerase (PDI) and cell surface free-thiol levels were measured on HUVEC and A549 non-small cell lung carcinoma cells. Results: Treatment of HUVECs, A549 cells, and monocytes with lung cancer chemotherapy significantly increased cell surface TF activity. However, elevated TF antigen levels were observed only on cisplatin- and gemcitabine-treated monocytes. Cell surface levels of phosphatidylserine were increased on HUVEC and monocytes treated with cisplatin/gemcitabine combination therapy. Chemotherapy also resulted in increased cell surface levels of PDI and reduced cell surface free thiol levels. Glutathione treatment and PDI inhibition, but not phosphatidylserine inhibition attenuated TF activity. Furthermore, increased TF activity was reversed by reducing cysteines with dithiothreitol. Conclusions: These studies are the first to demonstrate that lung cancer chemotherapy agents increase procoagulant activity on endothelial cells, blood monocytes, and A549 cells by TF decryption through a disulfide bond formation in a PDI-dependent mechanism.

Abstract 163: Cell-Surface Protein Disulfide Isomerase Shapes Vascular Architecture to Counteract Inward Remodeling

BACKGROUND: Vascular remodeling orchestrates a complex network of signaling pathways responsible for pathological changes in many vascular diseases such as atherosclerosis. We investigated the role of endoplasmic reticulum chaperone Protein Disulfide Isomerase(PDI) and cell-surface PDI (ecPDI) pool in vascular caliber and architecture during vascular repair after injury(AI). Methods and Results: After rabbit iliac artery balloon injury, there was marked increase in PDI mRNA and protein (25-fold vs. basal at day 14AI), with increase in both intracellular and ecPDI. Silencing PDI by siRNA (organ culture) induced ER stress augmentation and apoptosis). PDI knockdown also upregulated proliferation marker PCNA and decreased differentiation marker calponin-C. EcPDI neutralization in vivo with PDIAb-containing perivascular gel from days 12-14AI promoted ca.25% decrease in vascular caliber at arteriography and similar decreases in total vessel circumference at optical coherence tomography, without changing neointima, indicating increased constrictive remodeling. EcPDI neutralization promoted marked changes in collagen and cytoskeleton architecture, with inverted fiber orientation and induction of thicker collagen fibrils. Collagen type I and III were decreased after ecPDI inhibition in arteries 14 days AI. Furthermore, ecPDI inhibition prevents injury-increased hydrogen peroxide generation and decreases arterial nitrate (NO3-) levels. In human coronary atheromas, PDI expression inversely correlated with constrictive remodeling. There was decreased PDI expression in media and intima from plaques exhibiting inward remodeling and, conversely, enhanced PDI expression in media of plaques exhibiting outward remodeling. CONCLUSIONS: Thus, strongly-expressed PDI after injury reshapes matrix and cytoskeleton architecture to support an anticonstrictive remodeling effect. Such findings suggest an important role for PDI in lumen maintenance during vascular remodeling.

Endo-PDI is required for TNFα-induced angiogenesis

Protein disulfide isomerase (PDI) and its homologs are oxidoreductases facilitating protein folding in the ER. Endo-PDI (also termed ERp46) is highly expressed in endothelial cells. It belongs to the PDI family but its physiological function is largely unknown. We studied the role of Endo-PDI in endothelial angiogenic responses. Stimulation of human umbilical vein endothelial cells (with TNFα (10ng/ml) increased ERK1/2 phosphorylation. This effect was largely attenuated by Endo-PDI siRNA, whereas JNK and p38 MAP kinase phosphorylation was Endo-PDI independent. Similarly, TNFα-stimulated NF-κB signaling determined by IκBα degradation as well as TNFα-induced ICAM expression was unaffected by Endo-PDI siRNA. The action of Endo-PDI was not mediated by extracellular thiol exchange or cell surface PDI as demonstrated by nonpermeative inhibitors and PDI-neutralizing antibody. Moreover, exogenously added PDI failed to restore ERK1/2 activation after Endo-PDI knockdown. This suggests that Endo-PDI acts intracellularly potentially by maintaining the Ras/Raf/MEK/ERK pathway. Indeed, knockdown of Endo-PDI attenuated Ras activation measured by G-LISA and Raf phosphorylation. ERK activation influences gene expression by the transcriptional factor AP-1, which controls MMP-9 and cathepsin B, two proteases required for angiogenesis. TNFα-stimulated MMP-9 and cathepsin B induction was reduced by silencing of Endo-PDI. Accordingly, inhibition of cathepsin B or Endo-PDI siRNA blocked the TNFα-stimulated angiogenic response in the spheroid outgrowth assays. Moreover ex vivo tube formation and in vivo Matrigel angiogenesis in response to TNFα were attenuated by Endo-PDI siRNA. In conclusion, our study establishes Endo-PDI as a novel, important mediator of AP-1-driven gene expression and endothelial angiogenic function.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase

AbstractLymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.

Inhibitory Autoantibodies to Protein Disulfide Isomerase From Systemic Lupus Erythematosus Patients Are Prothrombotic

AbstractAbstract 392Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by loss of self-tolerance, aberrant autoantibody production and multiple organ damage. Treating the disease symptoms (flares) has remarkably improved the survival of SLE patients, but thrombosis remains a major negative prognostic factor. Currently available tests do not predict thrombotic events; hence there is a need for identification of novel biomarkers of thrombosis associated with SLE and characterization of the relevant pathophysiologic mechanisms. We have shown that inhibition of cell-surface protein disulfide isomerase (PDI) can regulate the thrombotic properties of the endothelium in vitro, through exposure of phosphatidylserine (PS). We therefore hypothesized that inhibitory autoantibodies to PDI may promote or enhance the prothrombotic phenotype in SLE.We developed a quantitative ELISA to measure the titers of anti-PDI autoantibodies present in the sera and performed a cross-sectional analysis of sera from 217 SLE patients and 99 healthy controls enrolled in the Oklahoma Lupus Cohort. Low levels of PDI autoreactivity were detected in 54 out of 99 healthy controls tested, with a median of 4.185 AU and an interquartile range (IQR) of 2.25–4.18. Anti-PDI autoantibodies were detected in 107 out of 217 SLE sera tested, with significantly higher PDI autoreactivity (median 5.982, IQR 2.43–11.23, p = 0.042 Mann-Whitney test). We defined elevated anti-PDI titers as those to be higher than the 95th percentile of serum anti-PDI levels in the control group. Thirty-five out of the 217 SLE patients (16%) exhibited anti-PDI titers above the 95th percentile cut-off, an incidence significantly higher (p=0.0057, Fisher’s exact test) than the control group (5/99, 5% by cut-off definition). Interestingly, the incidence of patients with elevated anti-PDI autoreactivity was doubled in SLE patients with secondary antiphospholipid syndrome (APS), 20 out of 89 (22%), as compared to SLE patients without APS (15 out of 128, 11%, p = 0.04 Fisher’s exact test). Accordingly, we found a significant correlation between the titers of anti-PDI autoantibodies and the anti-cardiolipin IgG levels (Spearman r= 0.212, P = 0.003), indicating a link with the presence of antiphospholipid antibodies. Since the APS diagnosis involves both the detection of antiphospholipid antibodies and the presence of clinical thrombotic events, this might be an indication that anti-PDI autoantibodies are associated with thrombosis in SLE.Clinical history revealed that SLE patients with elevated PDI autoreactivity had higher SLE Disease Activity Index (SLEDAI) scores than those with normal anti-PDI levels (P = 0.0256, Mann-Whitney test), indicating a correlation between anti-PDI levels and disease activity. We observed significant inverse correlations between anti-PDI titers and complement levels as measured by the total hemolytic complement assay (Spearman r = −0.134, P = 0.049), C3 levels (Spearman r = −0.169, P = 0.013) and C4 levels (Spearman r = −0.215, P < 0.001). While the biological significance of this correlation is under investigation, activation of the classical complement pathway on the surface of PS-exposing cells has been documented in the literature.We have shown that PDI inhibition on endothelial surface promotes PS exposure and a thrombotic phenotype. We tested the inhibitory properties of anti-PDI autoantibodies and found that 50% of the SLE sera with elevated anti-PDI titers inhibited PDI reductase activity in vitro, thus indicating that these autoantibodies can modulate PDI function. Furthermore, affinity-purified anti-PDI autoantibodies from SLE sera were able to induce a prothrombotic phenotype on endothelial cells in vitro as evidenced by tissue factor decryption and enhanced prothrombinase activity, a direct indicator that anti-PDI autoantibodies induce anionic phospholipid exposure in vascular cells.In conclusion, we found an increased incidence of elevated anti-PDI titers in SLE, especially in patients with antiphospholipid syndrome, that correlate with disease activity. These autoantibodies inhibit PDI enzymatic activity and are prothrombotic in vitro. To our knowledge this is the first report of autoantibodies directly inducing the exposure of anionic phospholipids on vascular cells with mechanistic implications for the pathophysiology of SLE and APS. Disclosures:No relevant conflicts of interest to declare.

Role of Protein Disulfide Isomerase during vascular repair after injury

Endoplasmic reticulum(ER) redox chaperone protein disulfide isomerase(PDI) regulates vascular/phagocytic NADPH oxidase and supports cell migration. We investigated the role of PDI during vascular repair after injury(AI) induced by balloon in rabbit iliac artery. There was marked increase of PDI mRNA and protein(5–10‐fold) at 4, 7 and 14 days AI vs. intact control(CT). PDI immunostaining was greater in intima = neo‐endothelium &gt; media. Increased cell‐surface PDI was also evident. ER stress‐related KDEL chaperones also increased with similar time‐course AI. PDI siRNA(siPDI) transfection in cultured vessel rings collected 14 days AI enhanced KDEL expression vs. scrambled siRNA(siScr) (siScr 2.1±0.9 vs. siPDI 5.0±2.3‐fold vs. CT, p&lt;0.05), apoptosis (siScr 4.6±0.2 vs. siPDI 6.2±0.9 %TUNEL + nuclei, p&lt;0.05) and proliferation marker PCNA (siScr 1.2±0.3 vs. siPDI 4.0 ±0.2 AU, p&lt;0.05), and decreased differentiation marker calponin‐C (siScr 0.52±0.04 vs. siPDI 0.36 ±0.04 AU, p&lt;0.05). siPDI in CT rings did not alter such variables. PCR array analysis showed analogous pattern of mRNA changes. Also, siPDI 14 days AI upregulated Nox1 and downregulated Nox4 NADPH oxidase, while siPDI attenuated oxidant production (in situ hydroethidine) only in CT vessels. Thus, strongly‐overexpressed PDI 14 days AI protects against apoptosis and ER stress and sustains VSMC differentiation. Support: FAPESP, CNPq/INCT Redoxoma

Endothelial cell surface expression of protein disulfide isomerase activates β1 and β3 integrins and facilitates dengue virus infection

Infection with dengue virus (DENV) causes diseases ranging from mild dengue fever to severe hemorrhage or shock syndrome. DENV infection of endothelial cells may cause cell apoptosis or vascular leakage and result in clinical illness of hemorrhage. However, the endothelial cell molecules involved in DENV infection and the mechanisms governing virus-cell interactions are still uncertain. Since protein disulfide isomerase (PDI) reducing function at the cell surface was shown to be required for entry of certain viruses and bacteria, we explored the role of PDI expressed on endothelial cell surface in DENV infection. Using siRNA to knock down PDI, DENV infection was reduced which could be reversed by treating cells with a reducing agent Tris(2-carboxyethyl)phosphine hydrochloride (TCEP). DENV-induced PDI surface expression was mediated through the Lys-Asp-Glu-Leu (KDEL) receptor-Src family kinase signal pathway. Furthermore, cell surface PDI colocalized with β1 and β3 integrins after DENV infection, and the activation of integrins was blocked by PDI inhibition. Finally, blockade of PDI inhibited DENV entry into endothelial cells. Our findings suggest a novel mechanism whereby surface PDI which causes integrin activation is involved in DENV entry, and DENV infection further increases PDI surface expression at later time points. These findings may have implications for anti-DENV drug design.

Thrombin-induced shedding of tumour endothelial marker 5 and exposure of its RGD motif are regulated by cell-surface protein disulfide-isomerase

TEM5 (tumour endothelial marker 5; also known as GPR124) is an adhesion G-protein-coupled receptor containing a cryptic RGD motif in its extracellular domain. TEM5 is expressed in endothelial cells and pericytes during angiogenesis. In the present paper, we report that thrombin mediates shedding of an N-terminal TEM5 fragment of 60 kDa (termed N60) containing the RGD motif in an open conformation. Thrombin directly cleaved rsTEM5 (recombinant soluble TEM5) 5 and 34 residues downstream of the RGD motif, resulting in formation of N60 and its C-terminal counterpart (termed C50). Interestingly, N60 derived from thrombin cleavage of rsTEM5 was covalently linked to C50 by disulfide bonds, whereas N60 shed from thrombin-treated cells was not associated with its membrane-bound C-terminal counterpart. Inhibition of the reducing function of cell-surface PDI (protein disulfide-isomerase) abrogated thrombin-induced N60 shedding. Conversely, addition of reduced PDI enhanced N60 shedding. Furthermore, thrombin cleavage of rsTEM5 was increased by reduced PDI and resulted in dissociation of the N60-C50 heterodimer. We conclude that PDI regulates thrombin-induced shedding of N60 and exposure of the TEM5 RGD motif by catalysing the reduction of crucial disulfide bonds of TEM5 on the cell surface. Binding of N60 to RGD-dependent integrins may modulate cellular functions such as adhesion and migration during angiogenesis.