Protein Disulfide Isomerase Expression Research Articles

Endothelium but Not Platelet-Derived Protein Disulfide Isomerase Is Required for Fibrin Generation during Thrombus Formation in Vivo.

We have previously reported that protein disulfide isomerase is required in wild-type mice for platelet thrombus formation and fibrin generation in an in vivo laser injury model of thrombosis (Cho et al. J. Clin. Invest. , 2008; 118:1123–31). Fibrin deposition after laser injury to the vessel wall in Par4−/− mice, lacking the G protein-coupled platelet thrombin receptor, is independent of platelets or requires minimal platelet activation or accumulation (Vandendries et al. Proc. Natl. Acad. Sci. , 2007; 104:288–92). However, protein disulfide isomerase inhibitors have a dramatic effect on fibrin accumulation in Par4− mice, suggesting that these inhibitors may function by a platelet independent mechanism. Here, we compare the contributions of endothelium and platelet-derived protein disulfide isomerase to fibrin generation in the mouse laser injury model of thrombosis. In vitro studies using cultured human umbilical vein endothelial cells and human aortic endothelial cells show that protein disulfide isomerase can be secreted rapidly into the culture medium from these cells upon thrombin stimulation. Using intravital microscopy, we observe that protein disulfide isomerase is not detectable on the vessel wall prior to laser injury but can be detected on the injured cremaster arteriolar wall and in the developing thrombus very rapidly after laser induced injury in the live mouse. The median integrated fluorescence intensity for protein disulfide isomerase in wild-type mice was compared to wild-type mice injected with 10ug/g mouse of Integrilin, an inhibitor of platelet activation and platelet thrombus formation, and thus, an inhibitor of the contribution of platelet derived protein disulfide isomerase to thrombus formation. Protein disulfide isomerase expression was similar in both treated and untreated animals upto 30 seconds post-laser injury. After 30 seconds, the expression of protein disulfide isomerase in integrilin treated mice was significantly decreased compared to that in untreated mice, indicating that the initial protein disulfide isomerase was derived from the endothelium and later additional protein disulfide isomerase was derived from the platelets following their accumulation in the developing thrombus. Fibrin deposition, a measure of thrombin generation was comparable in wild-type mice that had been treated with Integrilin or treated with a control buffer, suggesting that endothelial-derived protein disulfide isomerase was sufficient for fibrin generation. The rate and amount of fibrin generation was indistinguishable in both groups. Furthermore, inhibition of the protein disulfide isomerase with the function blocking monoclonal antibody RL-90 (3ug/g mouse) eliminated any fibrin deposition in wild-type mice that had been treated with Integrilin. Taken together, these data indicate that endothelium-derived protein disulfide isomerase is necessary to support fibrin deposition in vivo in our laser injury model of thrombus formation. The initial protein disulfide isomerase expressed at the site of injury is derived from endothelial cells but platelets activated at the site of thrombus formation contribute, amplify and sustain protein disulfide isomerase expression.

A critical role for extracellular protein disulfide isomerase during thrombus formation in mice

Thiol isomerases, including protein disulfide isomerase (PDI), catalyze disulfide oxidation, reduction, and isomerization, thereby playing an important role in protein synthesis. To determine whether extracellular PDI mediates thrombus formation in an animal model, PDI expression, platelet accumulation, and fibrin generation were monitored in the blood vessels of mice by intravital fluorescence microscopy following laser-induced arteriolar injury. A time-dependent increase in PDI was observed in murine thrombi following injury. Infusion of the PDI inhibitor bacitracin or a blocking monoclonal antibody against PDI inhibited platelet thrombus formation and fibrin generation. Fibrin deposition is normal in mice lacking the G protein-coupled platelet receptor Par4, although there is no stable accumulation of platelets. Infusion of monoclonal antibodies against PDI into the circulation of Par4(-/-) mice prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required in vivo in mice for both fibrin generation and platelet thrombus formation.

Downregulation of protein disulfide isomerase in sepsis and its role in tumor necrosis factor-alpha release

IntroductionProtein disulfide isomerase (PDI) is an important factor for the protein modification step in the post-translational event. PDI plays an essential role in cell survival under various stress conditions. It has been reported that PDI can serve as a negative regulator of nuclear factor-kappa-B (NF-κB) and that it can inhibit lipopolysaccharide (LPS)-induced proinflammatory cytokine production in macrophages. Thus, PDI may be an intracellular anti-inflammatory molecule. Although we have previously shown that Kupffer cell-derived proinflammatory cytokines cause liver injury in sepsis, the effect of sepsis on PDI expression as well as the effect of PDI inhibition on cytokine production have not been investigated. We therefore hypothesized that sepsis downregulates PDI expression and that the inhibition of PDI promotes proinflammatory cytokine production.MethodAdult male rats were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia (continuous infusion of 1 μg/kg body weight LPS by an osmotic pump) for 20 hours. Hepatic tissues were collected and PDI gene expression was determined. In additional experiments, cells from a macrophage-like cell line, RAW 264.7, were treated with 100 ng/mL LPS for 4 hours and protein expressions were measured. RAW 264.7 cells were also treated with bacitracin, a specific PDI inhibitor, for 24 hours, and tumor necrosis factor-alpha (TNF-α) gene and protein expression as well as its release in the cell supernatant were determined. To further confirm the beneficial effect of PDI in sepsis, RAW 264.7 cells were transfected with PDI short interfering RNA (siRNA) and PDI gene expression and TNF-α release were measured by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.ResultsPDI gene expression was significantly decreased by 28% and 69% at 20 hours after CLP or LPS infusion, respectively. LPS also decreased PDI protein expression by 33% in RAW 264.7 cells. Incubation of RAW 264.7 cells with bacitracin significantly increased TNF-α gene expression and TNF-α release as well as its cellular levels in a dose-dependent manner. Transfection of RAW 264.7 cells with PDI siRNA produced an average 36.8% inhibition of the PDI gene expression. This downregulation was correlated with a 3.19-fold increase in TNF-α release into the cell supernatant.ConclusionTaken together, these results suggest that downregulation of PDI by sepsis significantly increases proinflammatory cytokine production. Thus, prevention of PDI downregulation in sepsis may be a novel approach to attenuate hyperinflammation and to reduce tissue injury under such conditions.

Protein Disulfide Isomerase Is Required for Fibrin Generation and Platelet Thrombus Formation In Vivo.

Thiol isomerases catalyze disulfide oxidation, reduction and isomerization, playing an important role during protein synthesis. Recent studies suggest a role for protein disulfide isomerase (PDI), a prototype of the thiol isomerase family, in platelet function and regulation of tissue factor activity (Essex and Li. Curr Drug Targets. 2006; Chen and Hogg. J Thromb Haemost. 2006). To determine the role of intravascular PDI during thrombus formation, PDI expression, platelet accumulation, and fibrin generation were monitored following laser-induced arteriolar injury in the mouse cremaster muscle by intravital fluorescence microscopy. PDI antigen exhibited a time-dependent increase in the developing thrombus after vessel wall injury and remained associated with the thrombus. Infusion of bacitracin, a non-specific inhibitor of thiol isomerases, into the circulation inhibited platelet thrombus formation and fibrin generation in a dose-dependent manner. Infusion of a function-blocking monoclonal antibody to PDI (RL90) into the circulation of a wild type mouse also resulted in dose-dependent inhibition of platelet accumulation and fibrin generation. To determine whether PDI inhibits fibrin formation by blocking tissue factor activation, or by preventing platelet activation and the development of the membrane surface that is required for assembly of the tenase and the prothrombinase complex in vivo, we explored fibrin formation in mice lacking protease-activated receptor-4 (Par4). Although there is no stable accumulation of platelets and no platelet activation, fibrin formation is normal in the Par4 null mouse (Vandendries et al, Proc Natl Acad Sci USA. 2007), suggesting that fibrin generation in the laser-induced vessel injury model is independent of platelet activation. Infusion of the function-blocking anti-PDI antibody (RL90) into the circulation of a Par4 null mouse prior to vessel wall injury inhibited fibrin generation. These results indicate that PDI is required to generate tissue factor in a form that leads to thrombin generation and fibrin formation during thrombus development and is required for thrombus formation.

Evaluation of the transcription level of the protein disulfide isomerase in different stages from Ancylostoma caninum with a real-time PCR assay

The protein disulfide isomerase (PDI) is a ubiquitous protein, which contributes in building disulfide bridges. In the work presented here, the expression of the PDI in different stages of the canine hookworm Ancylostoma caninum was investigated. Third-stage larvae (L3), adults, as well as serum-stimulated and hypobiotic L3 were used. For quantification of the PDI gene transcription, a real-time PCR was used establishing a hybridization probe (TaqMantrade mark probes) for detection of PDI copy numbers in different populations. 18S ribosomal ribonucleic acid (rRNA) was used as a housekeeping gene for normalization. The results show differences in the transcription level of the investigated A. caninum populations: The serum-stimulated larvae representing the switch to parasitism showed the highest PDI expression. The hypobiotic larvae representing a resting stage showed the lowest expression level. Male adults showed an elevated expression compared to female adult worms. The L3 expression level was just below the serum-stimulated population. This work confirms the upregulated gene expression of PDI during host penetration and invasion.

Identification of Protein Disulfide Isomerase as a Cardiomyocyte Survival Factor in Ischemic Cardiomyopathy

The aim of the study was to analyze the molecular mechanisms activated during postinfarction remodeling in human hearts. The molecular mechanisms of initial response to ischemic insult in the heart and the pathways involved in compensation and remodeling are still largely unknown. Up-regulation or down-regulation of gene expression in the human viable peri-infarct (vs. remote) myocardial region was investigated by complementary deoxyribonucleic acid array technology and confirmed at a single-gene/protein level with reverse transcriptase polymerase chain reaction and immunohistochemistry. An in vitro model of cardiomyocyte hypoxia in HL1 cells was used to validate anti-apoptotic effects of the candidate gene/protein and to assess the associated downstream cascade. Finally, a mouse model of myocardial infarction was used to test the in vivo effects of exogenous transfection with the candidate gene/protein. Protein disulfide isomerase (PDI), a member of the unfolded protein response, is 3-fold up-regulated in the viable peri-infarct myocardial region, and in a postmortem model, its expression is significantly inversely correlated with apoptotic rate and with presence of heart failure (HF) and biventricular dilatation. Induced PDI expression in HL1 cells conferred protection from hypoxia-induced apoptosis. Adenoviral-mediated PDI gene transfer to the mouse heart resulted in 2.5-fold smaller infarct size, significantly reduced cardiomyocyte apoptosis in the peri-infarct region, and smaller left ventricular end-diastolic diameter versus mice treated with a transgene-null adenoviral vector. These results suggest that PDI promotes survival after ischemic damage and that zinc-superoxide dismutase is one of the PDI molecular targets. Pharmacological modulation of this pathway might prove useful for future prevention and treatment of HF.

Effect of doxycycline‐regulated protein disulfide isomerase expression on the specific productivity of recombinant CHO cells: Thrombopoietin and antibody

Protein disulfide isomerase (PDI), one of the ER-resident molecular chaperones, forms and isomerizes disulfide bonds. This study attempts to investigate the effect of PDI expression level on specific productivity (q) of recombinant Chinese hamster ovary (rCHO) cells producing thrombopoietin (TPO) and antibody (Ab). To regulate the PDI expression level, the Tet-Off system was introduced in TPO and Ab producing CHO cells, and stable Tet-Off cells (TPO-Tet-Off and Ab-Tet-Off) were screened using the luciferase assay. The doxycycline-regulated PDI expression system in Tet-Off rCHO cells (Tet-TPO-PDI and Tet-Ab-PDI) was established by the cotransfection of pTRE-PDI and pTK-Hyg expression vector into TPO-Tet-Off and Ab-Tet-Off cells, respectively. Subsequent screening was done by Western blot analysis of PDI and an enzyme-linked immunosorbent assay of the secreted TPO and antibody. We cultured two Tet-TPO-PDI and two Tet-Ab-PDI clones, and all these clones showed an average of 2.5-fold increase in PDI expression when compared to the basal level. In both these cell lines the PDI expression was tightly controlled by various concentrations of doxycycline. The q of TPO (q(TPO)) was unaffected but that of antibody producing cells was increased by 15-27% due to the PDI expression level.

Protein Disulfide Isomerase Expression Is Related to the Invasive Properties of Malignant Glioma

By serial transplantation of human glioblastoma biopsies into the brain of immunodeficient nude rats, two different tumor phenotypes were obtained. Initially, the transplanted xenografts displayed a highly invasive phenotype that showed no signs of angiogenesis. By serial transplantation in animals, the tumors changed to a less invasive, predominantly angiogenic phenotype. To identify novel proteins related to the invasive phenotype, the xenografts were analyzed using a global proteomics approach. One of the identified proteins was protein disulfide isomerase (PDI) A6 precursor. PDI is a chaperone protein that mediates integrin-dependent cell adhesion. It is both present in the cytosol and at the cell surface. We show that PDI is strongly expressed on invasive glioma cells, in both xenografts and at the invasive front of human glioblastomas. Using an in vitro migration assay, we also show that PDI is expressed on migrating glioma cells. To determine the functional significance of PDI in cell migration, we tested the effect of a PDI inhibitor, bacitracin, and a PDI monoclonal antibody on glioma cell migration and invasion in vitro. Both tumor spheroids derived from human glioblastoma xenografts in nude rat brain and cell line spheroids were used. The PDI antibody, as well as bacitracin, inhibited tumor cell migration and invasion. The anti-invasive effect of bacitracin was reversible after withdrawal of the inhibitor, indicating a specific, nontoxic effect. In conclusion, using a global proteomics approach, PDI was identified to play an important role in glioma cell invasion, and its action was effectively inhibited by bacitracin.

Regulation of NAD(P)H Oxidase by Associated Protein Disulfide Isomerase in Vascular Smooth Muscle Cells

NAD(P)H oxidase, the main source of reactive oxygen species in vascular cells, is known to be regulated by redox processes and thiols. However, the nature of thiol-dependent regulation has not been established. Protein disulfide isomerase (PDI) is a dithiol/disulfide oxidoreductase chaperone of the thioredoxin superfamily involved in protein processing and translocation. We postulated that PDI regulates NAD(P)H oxidase activity of rabbit aortic smooth muscle cells (VSMCs). Western blotting confirmed robust PDI expression and shift to membrane fraction after incubation with angiotensin II (AII, 100 nm, 6 h). In VSMC membrane fraction, PDI antagonism with bacitracin, scrambled RNase, or neutralizing antibody led to 26-83% inhibition (p < 0.05) of oxidase activity. AII incubation led to significant increase in oxidase activity, accompanied by a 6-fold increase in PDI refolding isomerase activity. AII-induced NAD(P)H oxidase activation was inhibited by 57-71% with antisense oligonucleotide against PDI (PDIasODN). Dihydroethidium fluorescence showed decreased superoxide generation due to PDIasODN. Confocal microscopy showed co-localization between PDI and the oxidase subunits p22(phox), Nox1, and Nox4. Co-immunoprecipitation assays supported spatial association between PDI and oxidase subunits p22(phox), Nox1, and Nox4 in VSMCs. Moreover, in HEK293 cells transfected with green fluorescent protein constructs for Nox1, Nox2, and Nox4, each of these subunits co-immunoprecipitated with PDI. Akt phosphorylation, a known downstream pathway of AII-driven oxidase activation, was significantly reduced by PDIasODN. These results suggest that PDI closely associates with NAD(P)H oxidase and acts as a novel redox-sensitive regulatory protein of such enzyme complex, potentially affecting subunit traffic/assembling.

Comparative proteomic analysis identifies protein disulfide isomerase and peroxiredoxin 1 as new players involved in embryonic interdigital cell death

In this study, we used comparative proteomics to identify proteins that were involved in the regulation of interdigital cell death. The protein profiles of embryonic day (E) 12.5 and 13.5 mouse hindlimb interdigital tissues were compared to identify proteins that were differentially expressed. The interdigital cells are irreversibly committed to programmed cell death (PCD) at E13.5, whereas they are developmentally plastic at E12.5. We established that protein disulfide isomerase (PDI) expression was up-regulated at E13.5, while peroxiredoxin 1 (Prdx1) expression was down-regulated at this time point. Semiquantitative reverse transcriptase-polymerase chain reaction and Western blot analyses confirmed the data obtained from the two-dimensional electrophoresis gels. Furthermore, we were able to up-regulate PDI expression by manipulating the E12.5 interdigital tissues to die during culture, although this up-regulation was not possible when cell survival was promoted. In addition, we could inhibit interdigital cell death and expression of proapoptotic genes (Bmp-4 and Bambi) by treating interdigital tissues with PDI antibodies and bacitracin (a PDI enzyme inhibitor). These findings suggested that PDI was involved in the activation and maintenance of interdigital cell death. Conversely, we determined that Prdx1 expression was maintained when interdigital cultures were manipulated to survive but down-regulated when the cultures were permitted to die. The result suggested that Prdx1 was involved in maintaining interdigital cell survival. However, we were unable to induce interdigital cell death by means of RNA interference-mediated silencing of Prdx1 expression, indicating that Prdx1 down-regulation is not sufficient for PCD to occur. Proteomic analysis of the Prdx1 knock-down cells revealed that the level of NF-kappaB inhibitor epsilon (IkappaBepsilon) was dramatically reduced. Furthermore, we found an increase in NFkappaB activation and reactive oxygen species (ROS) levels in the cytoplasm as a result of Prdx1 knockdown. We also found that silencing Prdx1 made the interdigital cells more susceptible to ROS-induced cell death. Taken together, our study identifies two new players in interdigital cell death and highlights that PCD is regulated by a delicate balance of proapoptotic and survival-promoting activities.

Protein disulfide isomerase suppresses the transcriptional activity of NF-κB

We report here that the transcriptional activity of NF-κB is negatively regulated by protein disulfide isomerase (PDI). Over-expression of PDI in RAW 264.7 cells strongly suppressed the LPS-induced production of inflammatory cytokines as well as NF-κB-dependent luciferase activity. This negative regulation of NF-κB was reversed by bacitracin, a PDI inhibitor. Interestingly, NF-κB/DNA complex formation and phosphorylation of NF-κB subunits was intact in PDI-expressing cells following stimulation with LPS. In addition, PDI and another redox regulator, thioredoxin (TRX), had opposite effects on NF-κB-dependent gene expression: activation of the NF-κB pathway by TRX was suppressed by expression of PDI in a dose-dependent manner. Finally, PDI expression was induced by the anti-inflammatory cytokine IL-10, and IL-10-mediated inhibition of LPS-induced IL-6 expression was reduced by bacitracin. These findings clearly demonstrate that PDI is a negative regulator of NF-κB, and may act downstream of IL-10 in this signaling pathway.

A defined level of protein disulfide isomerase expression is required for optimal secretion of thaumatin by Aspegillus awamori.

Thaumatin, a 22-kDa protein containing eight disulfide bonds, is secreted by the filamentous fungus Aspergillus awamori at levels which are dependent upon the extent of overexpression of protein disulfide isomerase (PDIA). Additional copies of the PDIA-encoding gene pdiA were introduced into a strain of A. awamori that expresses a cassette encoding thaumatin. Transformants with different levels of pdiA mRNA and measured PDIA levels were chosen for examination of the impact that PDIA levels had on thaumatin secretion. The secretion of two native proteins, alpha-amylase and acid phosphatase, was also examined in relation to varying levels of PDIA. Over a range of PDIA levels of 1-8, relative to the native level in strains with just one copy of the pdiA gene, the fraction of alpha-amylase and acid phosphatase in the total secreted protein was unaffected. In contrast, a peak level of thaumatin, about 5-fold higher than in the strain with one copy of pdiA, was found in strains with a relative PDIA level of between two and four. Improved thaumatin production was confirmed in 5-1 fermenters using a strain of A. awamori with six pdiA gene copies, containing 3.2-fold higher levels of PDIA than wild-type strains.

Cell-surface protein disulfide isomerase catalyzes transnitrosation and regulates intracellular transfer of nitric oxide.

Since thiols can undergo nitrosation and the cell membrane is rich in thiol-containing proteins, we considered the possibility that membrane surface thiols may regulate cellular entry of NO. Recently, protein disulfide isomerase (PDI), a protein that catalyzes thio-disulfide exchange reactions, has been found on the cell-surface membrane. We hypothesized that cell-surface PDI reacts with NO, catalyzes S-nitrosation reactions, and facilitates NO transfer from the extracellular to intracellular compartment. We observed that PDI catalyzes the S-nitrosothiol-dependent oxidation of the heme group of myoglobin (15-fold increase in the rate of oxidation compared with control), and that NO reduces the activity of PDI by 73.1 +/- 21.8% (P < 0.005). To assess the role of PDI in the cellular action of NO, we inhibited human erythroleukemia (HEL) cell-surface PDI expression using an antisense phosphorothioate oligodeoxynucleotide directed against PDI mRNA. This oligodeoxynucleotide decreased cell-surface PDI content by 74.1 +/- 9.3% and PDI folding activity by 46.6 +/- 3.5% compared with untreated or "scrambled" phosphorothioate oligodeoxynucleotide-treated cells (P < 0.0001). This decrease in cell-surface PDI was associated with a significant decrease in cyclic guanosine monophosphate (cGMP) generation after S-nitrosothiol exposure (65.4 +/- 26.7% reduction compared with control; P < 0.05), with no effect on cyclic adenosine monophosphate (cAMP) generation after prostaglandin E1 exposure. These data demonstrate that the cellular entry of NO involves a transnitrosation mechanism catalyzed by cell-surface PDI. These observations suggest a unique mechanism by which extracellular NO gains access to the intracellular environment.

Purification, Characterization, and Intracellular Localization of Glycosylated Protein Disulfide Isomerase from Wheat Grains

Wheat (Triticum aestivum) storage proteins fold and assemble into complexes that are linked by intra- and intermolecular disulfide bonds, but it is not yet clear whether these processes are spontaneous or require the assistance of endoplasmic reticulum (ER)-resident enzymes and molecular chaperones. Aiming to unravel these processes, we have purified and characterized the enzyme protein disulfide isomerase (PDI) from wheat endosperm, as well as studied its developmental expression and intracellular localization. This ER-resident enzyme was previously shown to be involved in the formation of disulfide bonds in secretory proteins. Wheat PDI appears as a 60-kD glycoprotein and is among the most abundant proteins within the ER of developing grains. PDI is notably upregulated in developing endosperm in comparison to embryos, leaves, and roots. In addition, the increase in PDI expression in grains appears at relatively early stages of development, preceding the onset of storage protein accumulation by several days. Subcellular localization analysis and immunogold labeling of electron micrographs showed that PDI is not only present in the lumen of the ER but is also co-localized with the storage proteins in the dense protein bodies. These observations are consistent with the hypothesis that PDI is involved in the assembly of wheat storage proteins within the ER.

The stress response in Chinese hamster ovary cells. Regulation of ERp72 and protein disulfide isomerase expression and secretion.

Expression of the glucose-regulated proteins (GRPs), GRP78 and GRP94, is induced by a variety of stress conditions including treatment of cells with tunicamycin or the calcium ionophore A23187. The stimulus for induction of these resident endoplasmic reticulum (ER) proteins appears to be accumulation of misfolded or underglycosylated protein within the ER. We have studied the induction of mRNAs encoding two other resident ER proteins, ERp72 and protein disulfide isomerase (PDI), during the stress response in Chinese hamster ovary cells. ERp72 shares amino acid sequence homology with PDI within the presumed catalytic active sites. ERp72 mRNA and, to a lesser degree, PDI mRNA were induced by treatment of Chinese hamster ovary cells with tunicamycin or A23187. These results identify ERp72 as a member of the GRP family. Stable high level overproduction of ERp72 or PDI from recombinant expression vectors did not alter the constitutive or induced expression of other GRPs. High level overexpression resulted in secretion of the overproduced protein specifically but not other resident ER proteins. This suggests that the ER retention mechanism is mediated by more specific interactions than just KDEL sequence recognition.