Anti-phospholipid antibodies are elevated and functionally active in chronic rhinosinusitis with nasal polyps.

BACKGROUND: Polyps from patients with chronic rhinosinusitis with nasal polyps (CRSwNP) contain increased levels of autoreactive antibodies, B cells and fibrin deposition. Anti-phospholipid antibodies (APA) are autoantibodies known to cause thrombosis but have not been implicated in chronic rhinosinusitis (CRS). OBJECTIVE: To compare APA levels (anti-cardiolipin, anti-phosphatidylethanolamine (anti-PE), and anti-ß2 -glycoprotein (anti-B2GP)) in nasal polyp (NP) tissue with tissue from control and CRS without nasal polyp (CRSsNP) patients, we tested whether NP antibodies affect coagulation, and correlate APAs with anti-dsDNA IgG and markers of coagulation. METHODS: Patient specimens were assayed for APA IgG, anti-dsDNA IgG and thrombin-anti-thrombin (TaT) complex by ELISA. Antibodies from a subset of specimens were tested for modified activated partial thromboplastin time (aPTT) measured on an optical-mechanical coagulometer. RESULTS: Anti-cardiolipin IgG in NP was 5-fold higher than control tissue (p < .0001). NP antibodies prolonged aPTT compared to control tissue antibodies at 400 µg/mL (36.7 s vs. 33.8 s, p = .024) and 600 µg/mL (40.9 s vs. 34.7 s, p = .0037). Anti-PE IgG antibodies were increased in NP (p = .027), but anti-B2GP IgG was not significantly higher (p = .084). All APAs correlated with anti-dsDNA IgG levels, which were also elevated (R = .77, .71 and .54, respectively, for anti-cardiolipin, anti-PE, and anti-B2GP; all p < .001), but only anti-cardiolipin (R = .50, p = .0185) and anti-PE (R = 0.45, p = .037) correlated with TaT complex levels. CONCLUSIONS: APA IgG antibodies are increased in NP and correlate with autoreactive tissue antibodies. NP antibodies have in vitro anti-coagulant activity similar to those observed in anti-phospholipid syndrome, suggesting that they may have pro-coagulant effects in polyp tissue.

Early endosome as a pathogenic target for antiphosphatidylethanolamine antibodies.

Phosphatidylethanolamine (PE) is a major phospholipid species with important roles in membrane trafficking and reorganization. Accumulating clinical data indicate that the presence of circulating antibodies against PE is positively correlated with the symptoms of antiphospholipid syndromes (APS), including thrombosis and repeated pregnancy loss. However, PE is generally sequestered inside a normal resting cell, and the mechanism by which circulating anti-PE antibodies access cellular PE remains unknown. The studies presented here were conducted with synthetic PE-binding agents, plasma samples from patients with anti-PE autoimmunity, and purified anti-PE antibodies. The results suggest that the cellular vulnerability to anti-PE antibodies may be mediated by the binding of PE molecules in the membrane of the early endosome. Endosomal PE binding led to functional changes in endothelial cells, including declines in proliferation and increases in the production of reactive oxygen species, as well as the expression of inflammatory molecules. Collectively, our findings provide insight into the etiology of anti-PE autoimmunity and, because endosomes are of central importance in almost all types of cells, could have important implications for a wide range of biological processes.

The K-Ras effector p38γ MAPK confers intrinsic resistance to tyrosine kinase inhibitors by stimulating EGFR transcription and EGFR dephosphorylation.

Mutations in K-Ras and epidermal growth factor receptor (EGFR) are mutually exclusive, but it is not known how K-Ras activation inactivates EGFR, leading to resistance of cancer cells to anti-EGFR therapy. Here, we report that the K-Ras effector p38γ MAPK confers intrinsic resistance to small molecular tyrosine kinase inhibitors (TKIs) by concurrently stimulating EGFR gene transcription and protein dephosphorylation. We found that p38γ increases EGFR transcription by c-Jun-mediated promoter binding and stimulates EGFR dephosphorylation via activation of protein-tyrosine phosphatase H1 (PTPH1). Silencing the p38γ/c-Jun/PTPH1 signaling network increased sensitivities to TKIs in K-Ras mutant cells in which EGFR knockdown inhibited growth. Similar results were obtained with the p38γ-specific pharmacological inhibitor pirfenidone. These results indicate that in K-Ras mutant cancers, EGFR activity is regulated by the p38γ/c-Jun/PTPH1 signaling network, whose disruption may be a novel strategy to restore the sensitivity to TKIs.

A One-Step Staining Probe for Phosphatidylethanolamine.

Phosphatidylethanolamine (PE) is an abundant phospholipid in cellular membranes, but relatively little is known about the kinetics of PE in biological membrane systems. Characterizing PE on a cellular level has been challenging owing to a lack of proper molecular tools. The lantibiotic duramycin and its structural analogue, cinnamycin, are currently the only known polypeptides that have an established stereospecific structure for binding membrane PE with high affinity and high specificity. These lantibiotics are recognized for their potential as molecular probes for studying PE kinetics in various membranes. However, owing to their antibiotic nature, duramycin and cinnamycin exhibit appreciable levels of cytotoxicity at low micromolar concentrations in cultured mammalian cells by inducing membrane distortion and possible PE redistribution. These issues can potentially complicate study design and data interpretation. Here, we report the construction of a molecular probe consisting of duramycin attached to the C terminus of green fluorescent protein (GFP) by a PEG linker at a stoichiometry of 1. The construct retained specific binding toward PE and essentially no cytotoxicity compared to native duramycin. The biological utilities of this probe were demonstrated in a number of cellular staining studies involving PE dynamics. The availability of a one-step, nontoxic molecular probe for PE will enable characterization of the biology of this important phospholipid.

In Vivo Mapping of Myocardial Injury Outside the Infarct Zone: Tissue at an Intermediate Pathological State.

BACKGROUND: The goal was to determine the feasibility of mapping the injured-but-not-infarcted myocardium using 99mTc-duramycin in the postischemic heart, with spatial information for its characterization as a pathophysiologically intermediate tissue, which is neither normal nor infarcted. METHODS AND RESULTS: Coronary occlusion was conducted in Sprague Dawley rats with preconditioning and 30-minute ligation. In vivo single-photon emission computed tomography was acquired after 3 hours (n=6) using 99mTc-duramycin, a phosphatidylethanolamine-specific radiopharmaceutical. The 99mTc-duramycin+ areas were compared with infarct and area-at-risk (n=8). Cardiomyocytes and endothelial cells were isolated for gene expression profiling. Cardiac function was measured with echocardiography (n=6) at 4 weeks. In vivo imaging with 99mTc-duramycin identified the infarct (3.9±2.4% of the left ventricle and an extensive area 23.7±2.2% of the left ventricle) with diffuse signal outside the infarct, which is pathologically between normal and infarcted (apoptosis 1.8±1.6, 8.9±4.2, 13.6±3.8%; VCAM-1 [vascular cell adhesion molecule 1] 3.2±0.8, 9.8±4.1, 15.9±4.2/mm2; tyrosine hydroxylase 14.9±2.8, 8.6±4.4, 5.6±2.2/mm2), with heterogeneous changes including scattered micronecrosis, wavy myofibrils, hydropic change, and glycogen accumulation. The 99mTc-duramycin+ tissue is quantitatively smaller than the area-at-risk (26.7% versus 34.4% of the left ventricle, P=0.008). Compared with infarct, gene expression in the 99mTc-duramycin+-noninfarct tissue indicated a greater prosurvival ratio (BCL2/BAX [B-cell lymphoma 2/BCL2-associated X] 7.8 versus 5.7 [cardiomyocytes], 3.7 versus 3.2 [endothelial]), and an upregulation of ion channels in electrophysiology. There was decreased contractility at 4 weeks (regional fractional shortening -8.6%, P<0.05; circumferential strain -52.9%, P<0.05). CONCLUSIONS: The injured-but-not-infarcted tissue, being an intermediate zone between normal and infarct, is mapped in vivo using phosphatidylethanolamine-based imaging. The intermediate zone contributes significantly to cardiac dysfunction.

p38γ Mitogen-activated protein kinase signals through phosphorylating its phosphatase PTPH1 in regulating ras protein oncogenesis and stress response.

Phosphatase plays a crucial role in determining cellular fate by inactivating its substrate kinase, but it is not known whether a kinase can vice versa phosphorylate its phosphatase to execute this function. Protein-tyrosine phosphatase H1 (PTPH1) is a specific phosphatase of p38γ mitogen-activated protein kinase (MAPK) through PDZ binding, and here, we show that p38γ is also a PTPH1 kinase through which it executes its oncogenic activity and regulates stress response. PTPH1 was identified as a substrate of p38γ by unbiased proteomic analysis, and its resultant phosphorylation at Ser-459 occurs in vitro and in vivo through their complex formation. Genetic and pharmacological analyses showed further that Ser-459 phosphorylation is directly regulated by Ras signaling and is important for Ras, p38γ, and PTPH1 oncogenic activity. Moreover, experiments with physiological stimuli revealed a novel stress pathway from p38γ to PTPH1/Ser-459 phosphorylation in regulating cell growth and cell death by a mechanism dependent on cellular environments but independent of canonical MAPK activities. These results thus reveal a new mechanism by which a MAPK regulates Ras oncogenesis and stress response through directly phosphorylating its phosphatase.

Phosphorylation and stabilization of topoisomerase IIα protein by p38γ mitogen-activated protein kinase sensitize breast cancer cells to its poisons.

Cancer drugs suppress tumor cell growth by inhibiting specific cellular targets. However, most drugs also activate several cellular nonspecific stress pathways, and the implications of these off-target effects are mostly unknown. Here, we report that p38γ, but not p38α, MAPK is specifically activated by treatment of breast cancer cells with topoisomerase II (Topo II) drugs, whereas paclitaxel (Taxol) does not have this effect. The activated p38γ in turn phosphorylates and stabilizes Topo IIα protein, and this enhances the growth inhibition by Topo II drugs. Moreover, p38γ activity was shown to be necessary and sufficient for Topo IIα expression, the drug-p38γ-Topo IIα axis is only detected in intrinsically sensitive but not resistant cells, and p38γ is co-overexpressed with Topo IIα protein in primary breast cancers. These results reveal a new paradigm in which p38γ actively regulates the drug-Topo IIα signal transduction, and this may be exploited to increase the therapeutic activity of Topo II drugs.

p38gamma MAPK cooperates with c-Jun in trans-activating matrix metalloproteinase 9.

Mitogen-activated protein kinases (MAPKs) regulate gene expression through transcription factors. However, the precise mechanisms in this critical signal event are largely unknown. Here, we show that the transcription factor c-Jun is activated by p38gamma MAPK, and the activated c-Jun then recruits p38gamma as a cofactor into the matrix metalloproteinase 9 (MMP9) promoter to induce its trans-activation and cell invasion. This signaling event was initiated by hyperexpressed p38gamma that led to increased c-Jun synthesis, MMP9 transcription, and MMP9-dependent invasion through p38gamma interacting with c-Jun. p38gamma requires phosphorylation and its C terminus to bind c-Jun, whereas both c-Jun and p38gamma are required for the trans-activation of MMP9. The active p38gamma/c-Jun/MMP9 pathway also exists in human colon cancer, and there is a coupling of increased p38gamma and MMP9 expression in the primary tissues. These results reveal a new paradigm in which a MAPK acts both as an activator and a cofactor of a transcription factor to regulate gene expression leading to an invasive response.

The outcome of ELISA for antiphosphatidylethanolamine antibodies is dependent on the composition of phosphatidylethanolamine.

OBJECTIVE: The presence of circulating autoantibodies against phosphatidylethanolamine (PE) has been shown to be positively associated with symptoms of antiphospholipid syndromes (APS). However, the current ELISA-based tests for antiphosphatidylethanolamine (aPE) antibodies remain inconsistent and controversial. The term PE refers to a collection of phospholipids that have phosphorylethanolamine head group as a common structural feature, but can vary in fatty acids with diverse physicochemical properties. The present study was to investigate, using synthetic positionally symmetrical PE species as a model system, the impact of PE structural variations on aPE ELISA. METHODS: Single and combinations of synthetic PE species, including 16:0 (fatty acid length:degree of unsaturation), 18:0, 18:1, 20:4 and 22:6, were screened with ELISA using serum samples from aPE patients and compared with chicken egg PE. There were a total of 37 aPE patient serum samples, including 11 cofactor-independent IgM, 14 ABP-independent IgG and 12 ABP-dependent aPE serum samples (3 IgM, 8 IgG and 1 IgA). The ELISA conditions were investigated for different isotypes and cofactor dependence. Based on the initial screening, adjustments in phospholipid compositions were made for achieving optimal OD readings. Finally, we isolated total IgG from aPE sera to validate different antigenic preferences. RESULTS: The antigenic preference was different among immunoglobulin isotypes and between cofactor-dependent versus cofactor-independent aPE antibodies. More specifically, 18:1 PE was a preferred antigen for cofactor-dependent aPE, whereas 20:4 PE was the preferred antigen for cofactor-independent IgG aPE. In contrast, cofactor-independent IgM aPE appeared to have a general preference for a more complex PE combination with longer fatty acids and a higher degree of unsaturation. CONCLUSION: The present data indicated that the outcome of aPE ELISA was dependent on the composition and physicochemical properties of PE antigens. The discovery that aPE antibodies may have different antigenic preferences could shed light on the nature of their binding interactions.

An efficient method of constructing homologous recom binant baculovirus with PCR-amplified fragments.

This paper describes a rapid method of constructing homologous recombinant baculovirus inE. coli with PCR-amplified fragments. By using this method, the traditional steps of constructing transfer vector are omitted. The method is based on phage λ red system which can promote the recombination between the homologous fragments with the length above 36 bp. Taking HaSNPV as an example, this paper describes the rapid recombination process by using chloramphenicol resistance gene (Cm ( R )) to replaceorf135 in HaSNPV genome. A pair of primers with length of 60 bp was synthesized, in which 40 bp was homologous to the each end sequence oforf135, and the rest 20 bp was homologous to the each end sequence ofCm ( R ). By using these primers, a linear fragment containing the completeCm ( R ) gene between 40 bp of homologous arms oforf135 was generated by PCR with the plasmid pKD3 which containsCm ( R ) as the template. By transforming the linear fragment into theE. coli containing the bacterial artificial chromosome of HaSNPV and with the help of a plasmid expressing λ recombinase, the recombinants on which the homologue replacement had taken place were selected by chloramphenicol resistance. This method greatly shortens the process of constructing recombinant baculovirus since the process was performed inE. coli and does not need to construct transfer vectors. It can be further used for gene replacement and gene deletion of other large viral genomes.

Membrane phospholipid redistribution in cancer micro-particles and implications in the recruitment of cationic protein factors.

Cancer cell-derived micro-particles (MPs) play important regulatory roles on cellular and system levels. These activities are attributed in part to protein factors carried by MPs. However, recruitment strategies for sequestering certain protein factors in MPs are poorly understood. In the current study, using exogenous and endogenously expressed phospholipid-binding probes, we investigated the distribution of membrane phospholipids in MPs as a potential mechanism for electrostatically enriching cationic protein factors in MPs. We detected a significant level of externalised phosphatidylethanolamine (PE) at the outer surface of MPs. This was accompanied, in the inner leaflet of the MP membrane, by a greater density of negatively charged phospholipids, particularly phosphatidylserine (PS). The local enrichment of PS in the inner surface of MPs was correlated with an elevated presence of small GTPases in a polybasic region (PBR)-dependent fashion. By employing a series of RhoA derivatives, including constitutively active and RhoA derivatives lacking a PBR, we could demonstrate that the congregation of RhoA in MPs was dependent on the presence of the PBR. A chimer with the fusion of PBR sequence alone to GFP significantly enhanced GFP localisation in MPs, indicative of a positive contribution of electrostatic interactions in RhoA recruitment to MPs. Using in silico thermodynamic simulations, we characterised the electrostatic interactions between PBR and anionic lipid membrane surface. In summary, the redistribution of membrane phospholipids in MPs has an impact on the local ionic density, and is likely a contributing factor in the electrostatic recruitment of membrane-associated proteins to MPs in a PBR-dependent fashion.

Identification of a ternary protein-complex as a therapeutic target for K-Ras-dependent colon cancer.

A cancer phenotype is driven by several proteins and targeting a cluster of functionally interdependent molecules should be more effective for therapeutic intervention. This is specifically important for Ras-dependent cancer, as mutated (MT) Ras is non-druggable and targeting its interaction with effectors may be essential for therapeutic intervention. Here, we report that a protein-complex activated by the Ras effector p38γ MAPK is a novel therapeutic target for K-Ras-dependent colon cancer. Unbiased proteomic screening and immune-precipitation analyses identified p38γ interaction with heat shock protein 90 (Hsp90) and K-Ras in K-Ras MT, but not wild-type (WT), colon cancer cells, indicating a role of this complex in Ras-dependent growth. Further experiments showed that this complex requires p38γ and Hsp90 activity to maintain MT, but not WT, K-Ras protein expression. Additional studies demonstrated that this complex is activated by p38γ-induced Hsp90 phosphorylation at S595, which is important for MT K-Ras stability and for K-Ras dependent growth. Of most important, pharmacologically inhibition of Hsp90 or p38γ activity disrupts the complex, decreases K-Ras expression, and selectively inhibits the growth of K-Ras MT colon cancer in vitro and in vivo. These results demonstrated that the p38γ-activated ternary complex is a novel therapeutic target for K-Ras-dependent colon cancer.

PTPH1 dephosphorylates and cooperates with p38gamma MAPK to increase ras oncogenesis through PDZ-mediated interaction.

Protein phosphatases are believed to coordinate with kinases to execute biological functions, but examples of such integrated activities, however, are still missing. In this report, we have identified protein tyrosine phosphatase H1 (PTPH1) as a specific phosphatase for p38gamma mitogen-activated protein kinase (MAPK) and shown their cooperative oncogenic activity through direct binding. p38gamma, a Ras effector known to act independent of its phosphorylation, was first shown to require its unique PDZ-binding motif to increase Ras transformation. Yeast two-hybrid screening and in vitro and in vivo analyses further identified PTPH1 as a specific p38gamma phosphatase through PDZ-mediated binding. Additional experiments showed that PTPH1 itself plays a role in Ras-dependent malignant growth in vitro and/or in mice by a mechanism depending on its p38gamma-binding activity. Moreover, Ras increases both p38gamma and PTPH1 protein expression and there is a coupling of increased p38gamma and PTPH1 protein expression in primary colon cancer tissues. These results reveal a coordinative oncogenic activity of a MAPK with its specific phosphatase and suggest that PDZ-mediated p38gamma/PTPH1 complex may be a novel target for Ras-dependent malignancies.

Control of TANK-binding kinase 1-mediated signaling by the gamma(1)34.5 protein of herpes simplex virus 1.

TANK-binding kinase 1 (TBK1) is a key component of Toll-like receptor-dependent and -independent signaling pathways. In response to microbial components, TBK1 activates interferon regulatory factor 3 (IRF3) and cytokine expression. Here we show that TBK1 is a novel target of the gamma(1)34.5 protein, a virulence factor whose expression is regulated in a temporal fashion. Remarkably, the gamma(1)34.5 protein is required to inhibit IRF3 phosphorylation, nuclear translocation, and the induction of antiviral genes in infected cells. When expressed in mammalian cells, the gamma(1)34.5 protein forms complexes with TBK1 and disrupts the interaction of TBK1 and IRF3, which prevents the induction of interferon and interferon-stimulated gene promoters. Down-regulation of TBK1 requires the amino-terminal domain. In addition, unlike wild type virus, a herpes simplex virus mutant lacking gamma(1)34.5 replicates efficiently in TBK1(-/-) cells but not in TBK1(+/+) cells. Addition of exogenous interferon restores the antiviral activity in both TBK1(-/-) and TBK(+/+) cells. Hence, control of TBK1-mediated cell signaling by the gamma(1)34.5 protein contributes to herpes simplex virus infection. These results reveal that TBK1 plays a pivotal role in limiting replication of a DNA virus.

p38alpha antagonizes p38gamma activity through c-Jun-dependent ubiquitin-proteasome pathways in regulating Ras transformation and stress response.

p38 MAPK family consists of four isoform proteins (alpha, beta, gamma, and delta) that are activated by the same stimuli, but the information about how these proteins act together to yield a biological response is missing. Here we show a feed-forward mechanism by which p38alpha may regulate Ras transformation and stress response through depleting its family member p38gamma protein via c-Jun-dependent ubiquitin-proteasome pathways. Analyses of MAPK kinase 6 (MKK6)-p38 fusion proteins showed that constitutively active p38alpha (MKK6-p38alpha) and p38gamma (MKK6-p38gamma) stimulates and inhibits c-Jun phosphorylation respectively, leading to a distinct AP-1 regulation. Depending on cell type and/or stimuli, p38alpha phosphorylation results in either Ras-transformation inhibition or a cell-death escalation that invariably couples with a decrease in p38gamma protein expression. p38gamma, on the other hand, increases Ras-dependent growth or inhibits stress induced cell-death independent of phosphorylation. In cells expressing both proteins, p38alpha phosphorylation decreases p38gamma protein expression, whereas its inhibition increases cellular p38gamma concentrations, indicating an active role of p38alpha phosphorylation in negatively regulating p38gamma protein expression. Mechanistic analyses show that p38alpha requires c-Jun activation to deplete p38gamma proteins by ubiquitin-proteasome pathways. These results suggest that p38alpha may, upon phosphorylation, act as a gatekeeper of the p38 MAPK family to yield a coordinative biological response through disrupting its antagonistic p38gamma family protein.

Deletion of a Helicoverpa armigera nucleopolyhedrovirus gene encoding a virion structural protein (ORF107) increases the budded virion titre and reduces in vivo infectivity.

The open reading frame Ha107 of Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus (HearNPV) encodes a putative protein of 51 kDa with homologues in a few group II NPVs and a granulovirus. Ha107 was transcribed as polyadenylated transcripts in infected HzAM1 insect cells. The transcripts were initiated at two distinct locations, one upstream of Ha106 (superoxide dismutase gene, sod) and the second upstream of Ha107. By Western blot analysis, two forms of the HA107 protein were detected in infected cells, a major polypeptide of 48 kDa and a minor one of 51 kDa. Western blot and immunoelectron microscopy analyses further showed that the HA107 protein was associated with the nucleocapsids of both budded virions (BVs) and occlusion-derived virions. A Ha107 knockout virus expressing enhanced green fluorescent protein and polyhedrin was constructed using bacmid technology. A one-step virus growth curve indicated that the BV titre of the knockout virus was significantly higher than that of the parental virus and a Ha107 repair virus. Bioassays indicated that the knockout virus was able to infect third-instar H. armigera larvae; however, its median lethal dose (LD50) was significantly higher than those of the parental virus and Ha107 repair virus. These data indicate that Ha107 encodes a non-essential structural protein of HearNPV virions and that deletion of this gene increases the BV titre and LD50 of the occluded virus.