Expression of protease-activated receptor (PAR)-2, but not other PARs, is regulated by inflammatory cytokines in rat astrocytes.

PubMed

Sokolova, Elena; Aleshin, Stepan; Reiser, Georg

2012-02-01

Protease-activated receptors (PARs) are widely expressed in the central nervous system (CNS) and are believed to play an important role in normal brain functioning as well as in development of various inflammatory and neurodegenerative disorders. Pathological conditions cause altered expression of PARs in brain cells and therefore altered responsiveness to PAR activation. The exact mechanisms of regulation of PAR expression are not well studied. Here, we evaluated in rat astrocytes the influence of LPS, pro-inflammatory cytokines TNFα and IL-1β and continuous PAR activation by PAR agonists on the expression levels of PARs. These stimuli are important in inflammatory and neurological disorders, where their levels are increased. We report that LPS as well as cytokines TNFα and IL-1β affected only the PAR-2 level, but their effects were opposite. LPS and TNFα increased the functional expression of PAR-2, whereas IL-1β down-regulated the functional response of PAR-2. Agonists of PAR-1 specifically increased mRNA level of PAR-2, but not protein level. Transcript levels of other PARs were not changed after PAR-1 activation. Stimulation of the cells with PAR-2 or PAR-4 agonists did not alter PAR levels. We found that up-regulation of PAR-2 is dependent on PKC activity, mostly via its Ca²⁺-sensitive isoforms. Two transcription factors, NFκB and AP-1, are involved in up-regulation of PAR-2. These findings provide new information about the regulation of expression of PAR subtypes in brain cells. This is of importance for targeting PARs, especially PAR-2, for the treatment of CNS disorders. Copyright © 2011 Elsevier Ltd. All rights reserved.

Identification of a Novel Regulatory Mechanism of Nutrient Transport Controlled by TORC1-Npr1-Amu1/Par32

PubMed Central

Boeckstaens, Mélanie; Merhi, Ahmad; Llinares, Elisa; Van Vooren, Pascale; Springael, Jean-Yves; Wintjens, René; Marini, Anna Maria

2015-01-01

Fine-tuning the plasma-membrane permeability to essential nutrients is fundamental to cell growth optimization. Nutritional signals including nitrogen availability are integrated by the TORC1 complex which notably regulates arrestin-mediated endocytosis of amino-acid transporters. Ammonium is a ubiquitous compound playing key physiological roles in many, if not all, organisms. In yeast, it is a preferred nitrogen source transported by three Mep proteins which are orthologues of the mammalian Rhesus factors. By combining genetic, kinetic, biochemical and cell microscopy analyses, the current study reveals a novel mechanism enabling TORC1 to regulate the inherent activity of ammonium transport proteins, independently of arrestin-mediated endocytosis, identifying the still functional orphan Amu1/Par32 as a selective regulator intermediate. We show that, under poor nitrogen supply, the TORC1 effector kinase' Npr1' promotes phosphorylation of Amu1/Par32 which appears mainly cytosolic while ammonium transport proteins are active. Upon preferred nitrogen supplementation, like glutamine or ammonium addition, TORC1 upregulation enables Npr1 inhibition and Amu1/Par32 dephosphorylation. In these conditions, as in Npr1-lacking cells, hypophosphorylated Amu1/Par32 accumulates at the cell surface and mediates the inhibition of specific ammonium transport proteins. We show that the integrity of a conserved repeated motif of Amu1/Par32 is required for the interaction with these transport proteins. This study underscores the diversity of strategies enabling TORC1-Npr1 to selectively monitor cell permeability to nutrients by discriminating between transporters to be degraded or transiently inactivated and kept stable at the plasma membrane. This study further identifies the function of Amu1/Par32 in acute control of ammonium transport in response to variations in nitrogen availability. PMID:26172854

Microtubules induce self-organization of polarized PAR domains in C. elegans zygotes

PubMed Central

Motegi, Fumio; Zonies, Seth; Hao, Yingsong; Cuenca, Adrian A.; Griffin, Erik; Seydoux, Geraldine

2011-01-01

A hallmark of polarized cells is the segregation of the PAR polarity regulators into asymmetric domains at the cell cortex1, 2. Antagonistic interactions involving two conserved kinases, atypical protein kinase C (aPKC) and PAR-1, have been implicated in polarity maintenance1, 2, but the mechanisms that initiate the formation of asymmetric PAR domains are not understood. Here, we describe one pathway used by the sperm-donated centrosome to polarize the PAR proteins in Caenorhabditis elegans zygotes. Before polarization, cortical aPKC excludes PAR-1 kinase and its binding partner PAR-2 by phosphorylation. During symmetry breaking, microtubules nucleated by the centrosome locally protect PAR-2 from phosphorylation by aPKC, allowing PAR-2 and PAR-1 to access the cortex nearest the centrosome. Cortical PAR-1 phosphorylates PAR-3, causing the PAR-3/aPKC complex to leave the cortex. Our findings illustrate how microtubules, independent of actin dynamics, stimulate the self-organization of PAR proteins by providing local protection against a global barrier imposed by aPKC. PMID:21983565

PARS: a web server for the prediction of Protein Allosteric and Regulatory Sites.

PubMed

Panjkovich, Alejandro; Daura, Xavier

2014-05-01

The regulation of protein activity is a key aspect of life at the molecular level. Unveiling its details is thus crucial to understanding signalling and metabolic pathways. The most common and powerful mechanism of protein-function regulation is allostery, which has been increasingly calling the attention of medicinal chemists due to its potential for the discovery of novel therapeutics. In this context, PARS is a simple and fast method that queries protein dynamics and structural conservation to identify pockets on a protein structure that may exert a regulatory effect on the binding of a small-molecule ligand.

Par-4 dependent modulation of cellular β-catenin by medicinal plant natural product derivative 3-azido Withaferin A.

PubMed

Amin, Hina; Nayak, Debasis; Ur Rasool, Reyaz; Chakraborty, Souneek; Kumar, Anmol; Yousuf, Khalid; Sharma, Parduman Raj; Ahmed, Zabeer; Sharma, Neelam; Magotra, Asmita; Mukherjee, Debaraj; Kumar, Lekha Dinesh; Goswami, Anindya

2016-05-01

Here, we provide evidences that natural product derivative 3-azido Withaferin A (3-AWA) abrogated EMT and invasion by modulating β-catenin localization and its transcriptional activity in the prostate as well as in breast cancer cells. This study, for the first time, reveals 3-AWA treatment consistently sequestered nuclear β-catenin and augmented its cytoplasmic pool as evidenced by reducing β-catenin transcriptional activity in these cells. Moreover, 3-AWA treatment triggered robust induction of pro-apoptotic intracellular Par-4, attenuated Akt activity and rescued Phospho-GSK3β (by Akt) to promote β-catenin destabilization. Further, our in vitro studies demonstrate that 3-AWA treatment amplified E-cadherin expression along with sharp downregulation of c-Myc and cyclin D1 proteins. Strikingly, endogenous Par-4 knock down by siRNA underscored 3-AWA mediated inhibition of nuclear β-catenin was Par-4 dependent and suppression of Par-4 activity, either by Bcl-2 or by Ras transfection, restored the nuclear β-catenin level suggesting Par-4 mediated β-catenin regulation was not promiscuous. In vivo results further demonstrated that 3-AWA was effective inhibitor of tumor growth and immunohistochemical studies indicated that increased expression of total β-catenin and decreased expression of phospho-β-catenin and Par-4 in breast cancer tissues as compared to normal breast tissue suggesting Par-4 and β-catenin proteins are mutually regulated and inversely co-related in normal as well as cancer condition. Thus, strategic regulation of intracellular Par-4 by 3-AWA in diverse cancers could be an effective tool to control cancer cell metastasis. Conclusively, this report puts forward a novel approach of controlling deregulated β-catenin signaling by 3-AWA induced Par-4 protein. © 2015 Wiley Periodicals, Inc.

Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins*

PubMed Central

Smith, Thomas H.; Coronel, Luisa J.; Li, Julia G.; Dores, Michael R.; Nieman, Marvin T.; Trejo, JoAnn

2016-01-01

Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Adaptor Protein Complex-2 (AP-2) and Epsin-1 Mediate Protease-activated Receptor-1 Internalization via Phosphorylation- and Ubiquitination-dependent Sorting Signals*

PubMed Central

Chen, Buxin; Dores, Michael R.; Grimsey, Neil; Canto, Isabel; Barker, Breann L.; Trejo, JoAnn

2011-01-01

Signaling by protease-activated receptor-1 (PAR1), a G protein-coupled receptor (GPCR) for thrombin, is regulated by desensitization and internalization. PAR1 desensitization is mediated by β-arrestins, like most classic GPCRs. In contrast, internalization of PAR1 occurs through a clathrin- and dynamin-dependent pathway independent of β-arrestins. PAR1 displays two modes of internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), where the μ2-adaptin subunit binds directly to a tyrosine-based motif localized within the receptor C-tail domain. However, AP-2 depletion only partially inhibits agonist-induced internalization of PAR1, suggesting a function for other clathrin adaptors in this process. Here, we now report that AP-2 and epsin-1 are both critical mediators of agonist-stimulated PAR1 internalization. We show that ubiquitination of PAR1 and the ubiquitin-interacting motifs of epsin-1 are required for epsin-1-dependent internalization of activated PAR1. In addition, activation of PAR1 promotes epsin-1 de-ubiquitination, which may increase its endocytic adaptor activity to facilitate receptor internalization. AP-2 also regulates activated PAR1 internalization via recognition of distal C-tail phosphorylation sites rather than the canonical tyrosine-based motif. Thus, AP-2 and epsin-1 are both required to promote efficient internalization of activated PAR1 and recognize discrete receptor sorting signals. This study defines a new pathway for internalization of mammalian GPCRs. PMID:21965661

Novel role for proteinase-activated receptor 2 (PAR2) in membrane trafficking of proteinase-activated receptor 4 (PAR4).

PubMed

Cunningham, Margaret R; McIntosh, Kathryn A; Pediani, John D; Robben, Joris; Cooke, Alexandra E; Nilsson, Mary; Gould, Gwyn W; Mundell, Stuart; Milligan, Graeme; Plevin, Robin

2012-05-11

Proteinase-activated receptors 4 (PAR(4)) is a class A G protein-coupled receptor (GPCR) recognized through the ability of serine proteases such as thrombin and trypsin to mediate receptor activation. Due to the irreversible nature of activation, a fresh supply of receptor is required to be mobilized to the cell surface for responsiveness to agonist to be sustained. Unlike other PAR subtypes, the mechanisms regulating receptor trafficking of PAR(4) remain unknown. Here, we report novel features of the intracellular trafficking of PAR(4) to the plasma membrane. PAR(4) was poorly expressed at the plasma membrane and largely retained in the endoplasmic reticulum (ER) in a complex with the COPI protein subunit β-COP1. Analysis of the PAR(4) protein sequence identified an arginine-based (RXR) ER retention sequence located within intracellular loop-2 (R(183)AR → A(183)AA), mutation of which allowed efficient membrane delivery of PAR(4). Interestingly, co-expression with PAR(2) facilitated plasma membrane delivery of PAR(4), an effect produced through disruption of β-COP1 binding and facilitation of interaction with the chaperone protein 14-3-3ζ. Intermolecular FRET studies confirmed heterodimerization between PAR(2) and PAR(4). PAR(2) also enhanced glycosylation of PAR(4) and activation of PAR(4) signaling. Our results identify a novel regulatory role for PAR(2) in the anterograde traffic of PAR(4). PAR(2) was shown to both facilitate and abrogate protein interactions with PAR(4), impacting upon receptor localization and cell signal transduction. This work is likely to impact markedly upon the understanding of the receptor pharmacology of PAR(4) in normal physiology and disease.

An interactive network of elastase, secretases, and PAR-2 protein regulates CXCR1 receptor surface expression on neutrophils.

PubMed

Bakele, Martina; Lotz-Havla, Amelie S; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C; Gersting, Soeren W; Hartl, Dominik

2014-07-25

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis.

An Interactive Network of Elastase, Secretases, and PAR-2 Protein Regulates CXCR1 Receptor Surface Expression on Neutrophils*

PubMed Central

Bakele, Martina; Lotz-Havla, Amelie S.; Jakowetz, Anja; Carevic, Melanie; Marcos, Veronica; Muntau, Ania C.; Gersting, Soeren W.; Hartl, Dominik

2014-01-01

CXCL8 (IL-8) recruits and activates neutrophils through the G protein-coupled chemokine receptor CXCR1. We showed previously that elastase cleaves CXCR1 and thereby impairs antibacterial host defense. However, the molecular intracellular machinery involved in this process remained undefined. Here we demonstrate by using flow cytometry, confocal microscopy, subcellular fractionation, co-immunoprecipitation, and bioluminescence resonance energy transfer that combined α- and γ-secretase activities are functionally involved in elastase-mediated regulation of CXCR1 surface expression on human neutrophils, whereas matrix metalloproteases are dispensable. We further demonstrate that PAR-2 is stored in mobilizable compartments in neutrophils. Bioluminescence resonance energy transfer and co-immunoprecipitation studies showed that secretases, PAR-2, and CXCR1 colocalize and physically interact in a novel protease/secretase-chemokine receptor network. PAR-2 blocking experiments provided evidence that elastase increased intracellular presenilin-1 expression through PAR-2 signaling. When viewed in combination, these studies establish a novel functional network of elastase, secretases, and PAR-2 that regulate CXCR1 expression on neutrophils. Interfering with this network could lead to novel therapeutic approaches in neutrophilic diseases, such as cystic fibrosis or rheumatoid arthritis. PMID:24914212

mTORC2 activation is regulated by the urokinase receptor (uPAR) in bladder cancer.

PubMed

Hau, Andrew M; Leivo, Mariah Z; Gilder, Andrew S; Hu, Jing-Jing; Gonias, Steven L; Hansel, Donna E

2017-01-01

Mammalian target of rapamycin complex 2 (mTORC2) has been identified as a major regulator of bladder cancer cell migration and invasion. Upstream pathways that mediate mTORC2 activation remain poorly defined. Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored membrane protein and known activator of cell-signaling. We identified increased uPAR expression in 94% of invasive human bladder cancers and in 54-71% of non-invasive bladder cancers, depending on grade. Normal urothelium was uPAR-immunonegative. Analysis of publicly available datasets identified uPAR gene amplification or mRNA upregulation in a subset of bladder cancer patients with reduced overall survival. Using biochemical approaches, we showed that uPAR activates mTORC2 in bladder cancer cells. Highly invasive bladder cancer cell lines, including T24, J82 and UM-UC-3 cells, showed increased uPAR mRNA expression and protein levels compared with the less aggressive cell lines, UROtsa and RT4. uPAR gene-silencing significantly reduced phosphorylation of Serine-473 in Akt, an mTORC2 target. uPAR gene-silencing also reduced bladder cancer cell migration and Matrigel invasion. S473 phosphorylation was observed by immunohistochemistry in human bladder cancers only when the tumors expressed high levels of uPAR. S473 phosphorylation was not controlled by uPAR in bladder cancer cell lines that are PTEN-negative; however, this result probably did not reflect altered mTORC2 regulation. Instead, PTEN deficiency de-repressed alternative kinases that phosphorylate S473. Our results suggest that uPAR and mTORC2 are components of a single cell-signaling pathway. Targeting uPAR or mTORC2 may be beneficial in patients with bladder cancer. Copyright © 2016. Published by Elsevier Inc.

Cortical PAR polarity proteins promote robust cytokinesis during asymmetric cell division

PubMed Central

Jordan, Shawn N.; Davies, Tim; Zhuravlev, Yelena; Dumont, Julien; Shirasu-Hiza, Mimi

2016-01-01

Cytokinesis, the physical division of one cell into two, is thought to be fundamentally similar in most animal cell divisions and driven by the constriction of a contractile ring positioned and controlled solely by the mitotic spindle. During asymmetric cell divisions, the core polarity machinery (partitioning defective [PAR] proteins) controls the unequal inheritance of key cell fate determinants. Here, we show that in asymmetrically dividing Caenorhabditis elegans embryos, the cortical PAR proteins (including the small guanosine triphosphatase CDC-42) have an active role in regulating recruitment of a critical component of the contractile ring, filamentous actin (F-actin). We found that the cortical PAR proteins are required for the retention of anillin and septin in the anterior pole, which are cytokinesis proteins that our genetic data suggest act as inhibitors of F-actin at the contractile ring. Collectively, our results suggest that the cortical PAR proteins coordinate the establishment of cell polarity with the physical process of cytokinesis during asymmetric cell division to ensure the fidelity of daughter cell formation. PMID:26728855

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration

PubMed Central

Wang, Shujie; Watanabe, Takashi; Matsuzawa, Kenji; Katsumi, Akira; Kakeno, Mai; Matsui, Toshinori; Ye, Feng; Sato, Kazuhide; Murase, Kiyoko; Sugiyama, Ikuko; Kimura, Kazushi; Mizoguchi, Akira; Ginsberg, Mark H.; Collard, John G.

2012-01-01

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration. PMID:23071154

Vps26B-retromer negatively regulates plasma membrane resensitization of PAR-2.

PubMed

Bugarcic, Andrea; Vetter, Irina; Chalmers, Silke; Kinna, Genevieve; Collins, Brett M; Teasdale, Rohan D

2015-11-01

Retromer is a trimeric complex composed of Vps26, Vps29, and Vps35 and has been shown to be involved in trafficking and sorting of transmembrane proteins within the endosome. The Vps26 paralog, Vps26B, defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. Although endosomally associated, Vps26B-retromer does not bind the established retromer transmembrane cargo protein, cation-independent mannose 6-phosphate receptor (CI-M6PR), indicating it has a distinct role to retromer containing the Vps26A paralog. In the present study we use the previously established Vps26B-expressing HEK293 cell model to address the role of Vps26B-retromer in trafficking of the protease activated G-protein coupled receptor PAR-2 to the plasma membrane. In these cells there is no apparent defect in the initial activation of the receptor, as evidenced by release of intracellular calcium, ERK1/2 signaling and endocytosis of activated receptor PAR-2 into degradative organelles. However, we observe a significant delay in plasma membrane repopulation of the protease activated G protein-coupled receptor PAR-2 following stimulation, resulting in a defect in PAR-2 activation after resensitization. Here we propose that PAR-2 plasma membrane repopulation is regulated by Vps26B-retromer, describing a potential novel role for this complex. © 2015 International Federation for Cell Biology.

Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

PubMed

Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

2015-07-01

Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation

PubMed Central

McLeod, Brett N.; Allison-Gamble, Gina E.; Barge, Madhuri T.; Tonthat, Nam K.; Schumacher, Maria A.; Hayes, Finbarr

2017-01-01

Abstract Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells. PMID:28034957

Conjugative DNA Transfer Is Enhanced by Plasmid R1 Partitioning Proteins

PubMed Central

Gruber, Christian J.; Lang, Silvia; Rajendra, Vinod K. H.; Nuk, Monika; Raffl, Sandra; Schildbach, Joel F.; Zechner, Ellen L.

2016-01-01

Bacterial conjugation is a form of type IV secretion used to transport protein and DNA directly to recipient bacteria. The process is cell contact-dependent, yet the mechanisms enabling extracellular events to trigger plasmid transfer to begin inside the cell remain obscure. In this study of plasmid R1 we investigated the role of plasmid proteins in the initiation of gene transfer. We find that TraI, the central regulator of conjugative DNA processing, interacts physically, and functionally with the plasmid partitioning proteins ParM and ParR. These interactions stimulate TraI catalyzed relaxation of plasmid DNA in vivo and in vitro and increase ParM ATPase activity. ParM also binds the coupling protein TraD and VirB4-like channel ATPase TraC. Together, these protein-protein interactions probably act to co-localize the transfer components intracellularly and promote assembly of the conjugation machinery. Importantly these data also indicate that the continued association of ParM and ParR at the conjugative pore is necessary for plasmid transfer to start efficiently. Moreover, the conjugative pilus and underlying secretion machinery assembled in the absence of Par proteins mediate poor biofilm formation and are completely dysfunctional for pilus specific R17 bacteriophage uptake. Thus, functional integration of Par components at the interface of relaxosome, coupling protein, and channel ATPases appears important for an optimal conformation and effective activation of the transfer machinery. We conclude that low copy plasmid R1 has evolved an active segregation system that optimizes both its vertical and lateral modes of dissemination. PMID:27486582

The C. elegans homolog of Drosophila Lethal giant larvae functions redundantly with PAR-2 to maintain polarity in the early embryo.

PubMed

Beatty, Alexander; Morton, Diane; Kemphues, Kenneth

2010-12-01

Polarity is essential for generating cell diversity. The one-cell C. elegans embryo serves as a model for studying the establishment and maintenance of polarity. In the early embryo, a myosin II-dependent contraction of the cortical meshwork asymmetrically distributes the highly conserved PDZ proteins PAR-3 and PAR-6, as well as an atypical protein kinase C (PKC-3), to the anterior. The RING-finger protein PAR-2 becomes enriched on the posterior cortex and prevents these three proteins from returning to the posterior. In addition to the PAR proteins, other proteins are required for polarity in many metazoans. One example is the conserved Drosophila tumor-suppressor protein Lethal giant larvae (Lgl). In Drosophila and mammals, Lgl contributes to the maintenance of cell polarity and plays a role in asymmetric cell division. We have found that the C. elegans homolog of Lgl, LGL-1, has a role in polarity but is not essential. It localizes asymmetrically to the posterior of the early embryo in a PKC-3-dependent manner, and functions redundantly with PAR-2 to maintain polarity. Furthermore, overexpression of LGL-1 is sufficient to rescue loss of PAR-2 function. LGL-1 negatively regulates the accumulation of myosin (NMY-2) on the posterior cortex, representing a possible mechanism by which LGL-1 might contribute to polarity maintenance.

A three-dimensional ParF meshwork assembles through the nucleoid to mediate plasmid segregation.

PubMed

McLeod, Brett N; Allison-Gamble, Gina E; Barge, Madhuri T; Tonthat, Nam K; Schumacher, Maria A; Hayes, Finbarr; Barillà, Daniela

2017-04-07

Genome segregation is a fundamental step in the life cycle of every cell. Most bacteria rely on dedicated DNA partition proteins to actively segregate chromosomes and low copy-number plasmids. Here, by employing super resolution microscopy, we establish that the ParF DNA partition protein of the ParA family assembles into a three-dimensional meshwork that uses the nucleoid as a scaffold and periodically shuttles between its poles. Whereas ParF specifies the territory for plasmid trafficking, the ParG partner protein dictates the tempo of ParF assembly cycles and plasmid segregation events by stimulating ParF adenosine triphosphate hydrolysis. Mutants in which this ParG temporal regulation is ablated show partition deficient phenotypes as a result of either altered ParF structure or dynamics and indicate that ParF nucleoid localization and dynamic relocation, although necessary, are not sufficient per se to ensure plasmid segregation. We propose a Venus flytrap model that merges the concepts of ParA polymerization and gradient formation and speculate that a transient, dynamic network of intersecting polymers that branches into the nucleoid interior is a widespread mechanism to distribute sizeable cargos within prokaryotic cells. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Coupling bimolecular PARylation biosensors with genetic screens to identify PARylation targets.

PubMed

Krastev, Dragomir B; Pettitt, Stephen J; Campbell, James; Song, Feifei; Tanos, Barbara E; Stoynov, Stoyno S; Ashworth, Alan; Lord, Christopher J

2018-05-22

Poly (ADP-ribose)ylation is a dynamic protein modification that regulates multiple cellular processes. Here, we describe a system for identifying and characterizing PARylation events that exploits the ability of a PBZ (PAR-binding zinc finger) protein domain to bind PAR with high-affinity. By linking PBZ domains to bimolecular fluorescent complementation biosensors, we developed fluorescent PAR biosensors that allow the detection of temporal and spatial PARylation events in live cells. Exploiting transposon-mediated recombination, we integrate the PAR biosensor en masse into thousands of protein coding genes in living cells. Using these PAR-biosensor "tagged" cells in a genetic screen we carry out a large-scale identification of PARylation targets. This identifies CTIF (CBP80/CBP20-dependent translation initiation factor) as a novel PARylation target of the tankyrase enzymes in the centrosomal region of cells, which plays a role in the distribution of the centrosomal satellites.

Novel role of prostate apoptosis response-4 tumor suppressor in B-cell chronic lymphocytic leukemia.

PubMed

McKenna, Mary K; Noothi, Sunil K; Alhakeem, Sara S; Oben, Karine Z; Greene, Joseph T; Mani, Rajeswaran; Perry, Kathryn L; Collard, James P; Rivas, Jacqueline R; Hildebrandt, Gerhard; Fleischman, Roger; Durbin, Eric B; Byrd, John C; Wang, Chi; Muthusamy, Natarajan; Rangnekar, Vivek M; Bondada, Subbarao

2018-04-25

Prostate apoptosis response-4 (Par-4), a pro-apoptotic tumor suppressor protein, is down regulated in many cancers including renal cell carcinoma, glioblastoma, endometrial and breast cancer. Par-4 induces apoptosis selectively in various types of cancer cells but not normal cells. We found that chronic lymphocytic leukemia (CLL) cells from human patients and from the Eµ-Tcl1 mice constitutively express Par-4 in greater amounts than normal B-1 or B-2 cells. Interestingly, knockdown of Par-4 in human CLL derived Mec-1 cells results in a robust increase in p21/WAF1 expression and decreased growth due to delayed G1 to S cell cycle transition. Lack of Par-4 also increased the expression of p21 and delayed CLL growth in Eμ-Tcl1 mice. Par-4 expression in CLL cells required constitutively active B-cell receptor (BCR) signaling, as inhibition of BCR signaling with FDA approved drugs caused a decrease in Par-4 mRNA and protein, and an increase in apoptosis. In particular, activities of Lyn, a Src family kinase, spleen tyrosine kinase and Bruton's tyrosine kinase are required for Par-4 expression in CLL cells, suggesting a novel regulation of Par-4 through BCR signaling. Together, these results suggest that Par-4 may play a novel pro-growth rather than pro-apoptotic role in CLL and could be targeted to enhance the therapeutic effects of BCR signaling inhibitors. Copyright © 2018 American Society of Hematology.

Mechanism of interleukin-13 production by granulocyte-macrophage colony-stimulating factor-dependent macrophages via protease-activated receptor-2.

PubMed

Yamaguchi, Rui; Yamamoto, Takatoshi; Sakamoto, Arisa; Ishimaru, Yasuji; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-06-01

Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway. Copyright © 2015 Elsevier Inc. All rights reserved.

Partitioning-defective Protein 6 (Par-6) Activates Atypical Protein Kinase C (aPKC) by Pseudosubstrate Displacement*

PubMed Central

Graybill, Chiharu; Wee, Brett; Atwood, Scott X.; Prehoda, Kenneth E.

2012-01-01

Atypical protein kinase C (aPKC) controls cell polarity by modulating substrate cortical localization. Aberrant aPKC activity disrupts polarity, yet the mechanisms that control aPKC remain poorly understood. We used a reconstituted system with purified components and a cultured cell cortical displacement assay to investigate aPKC regulation. We find that aPKC is autoinhibited by two domains within its NH2-terminal regulatory half, a pseudosubstrate motif that occupies the kinase active site, and a C1 domain that assists in this process. The Par complex member Par-6, previously thought to inhibit aPKC, is a potent activator of aPKC in our assays. Par-6 and aPKC interact via PB1 domain heterodimerization, and this interaction activates aPKC by displacing the pseudosubstrate, although full activity requires the Par-6 CRIB-PDZ domains. We propose that, along with its previously described roles in controlling aPKC localization, Par-6 allosterically activates aPKC to allow for high spatial and temporal control of substrate phosphorylation and polarization. PMID:22544755

The Sound of Silence: RNAi in Poly (ADP-Ribose) Research

PubMed Central

Blenn, Christian; Wyrsch, Philippe; Althaus, Felix R.

2012-01-01

Poly(ADP-ribosyl)-ation is a nonprotein posttranslational modification of proteins and plays an integral part in cell physiology and pathology. The metabolism of poly(ADP-ribose) (PAR) is regulated by its synthesis by poly(ADP-ribose) polymerases (PARPs) and on the catabolic side by poly(ADP-ribose) glycohydrolase (PARG). PARPs convert NAD+ molecules into PAR chains that interact covalently or noncovalently with target proteins and thereby modify their structure and functions. PAR synthesis is activated when PARP1 and PARP2 bind to DNA breaks and these two enzymes account for almost all PAR formation after genotoxic stress. PARG cleaves PAR molecules into free PAR and finally ADP-ribose (ADPR) moieties, both acting as messengers in cellular stress signaling. In this review, we discuss the potential of RNAi to manipulate the levels of PARPs and PARG, and consequently those of PAR and ADPR, and compare the results with those obtained after genetic or chemical disruption. PMID:24705085

Par-4-mediated recruitment of Amida to the actin cytoskeleton leads to the induction of apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Boosen, Meike; Vetterkind, Susanne; Koplin, Ansgar

Par-4 (prostate apoptosis response-4) sensitizes cells to apoptotic stimuli, but the exact mechanisms are still poorly understood. Using Par-4 as bait in a yeast two-hybrid screen, we identified Amida as a novel interaction partner, a ubiquitously expressed protein which has been suggested to be involved in apoptotic processes. Complex formation of Par-4 and Amida occurs in vitro and in vivo and is mediated via the C-termini of both proteins, involving the leucine zipper of Par-4. Amida resides mainly in the nucleus but displays nucleo-cytoplasmic shuttling in heterokaryons. Upon coexpression with Par-4 in REF52.2 cells, Amida translocates to the cytoplasm andmore » is recruited to actin filaments by Par-4, resulting in enhanced induction of apoptosis. The synergistic effect of Amida/Par-4 complexes on the induction of apoptosis is abrogated when either Amida/Par-4 complex formation or association of these complexes with the actin cytoskeleton is impaired, indicating that the Par-4-mediated relocation of Amida to the actin cytoskeleton is crucial for the pro-apoptotic function of Par-4/Amida complexes in REF52.2 cells. The latter results in enhanced phosphorylation of the regulatory light chain of myosin II (MLC) as has previously been shown for Par-4-mediated recruitment of DAP-like kinase (Dlk), suggesting that the recruitment of nuclear proteins involved in the regulation of apoptotic processes to the actin filament system by Par-4 represents a potent mechanism how Par-4 can trigger apoptosis.« less

Sir-two-homolog 2 (Sirt2) modulates peripheral myelination through polarity protein Par-3/atypical protein kinase C (aPKC) signaling.

PubMed

Beirowski, Bogdan; Gustin, Jason; Armour, Sean M; Yamamoto, Hiroyasu; Viader, Andreu; North, Brian J; Michán, Shaday; Baloh, Robert H; Golden, Judy P; Schmidt, Robert E; Sinclair, David A; Auwerx, Johan; Milbrandt, Jeffrey

2011-10-25

The formation of myelin by Schwann cells (SCs) occurs via a series of orchestrated molecular events. We previously used global expression profiling to examine peripheral nerve myelination and identified the NAD(+)-dependent deacetylase Sir-two-homolog 2 (Sirt2) as a protein likely to be involved in myelination. Here, we show that Sirt2 expression in SCs is correlated with that of structural myelin components during both developmental myelination and remyelination after nerve injury. Transgenic mice lacking or overexpressing Sirt2 specifically in SCs show delays in myelin formation. In SCs, we found that Sirt2 deacetylates Par-3, a master regulator of cell polarity. The deacetylation of Par-3 by Sirt2 decreases the activity of the polarity complex signaling component aPKC, thereby regulating myelin formation. These results demonstrate that Sirt2 controls an essential polarity pathway in SCs during myelin assembly and provide insights into the association between intracellular metabolism and SC plasticity.

Effect and mechanism of PAR-2 on the proliferation of esophageal cancer cells.

PubMed

Quanjun, D; Qingyu, Z; Qiliang, Z; Liqun, X; Jinmei, C; Ziquan, L; Shike, H

2016-11-01

Esophageal Cancer (EC) is a common malignant tumor occurred in the digestive tract. In this study, we investigated the mechanism of Protease Activated Receptor 2 (PAR-2) on the proliferation of esophageal cancer cell. Transfected esophageal cancer (EC) cell (PAR-2shRNA EC109) was established with low stable PAR-2 expression. EC109 cell was treated with PAR-2 agonist, PAR-2 anti-agonist and MAPK inhibitor respectively; Untreated EC109 cell (blank control) and PAR-2shRNA EC109 cell were used for analysis also. The mRNA expressions of PAR-2, ERK1, Cyclin D1, and c-fos in each group were detected by reverse transcript and polymerase chain reaction. Western blot was used to detect the protein expressions in each group. The cell growth curves were drawn to compare the cell growth. Compared with the blank control, the mRNA and protein expressions of PAR-2, Cyclin D1, and c-fos in PAR-2 agonist group increased significantly (p < 0.05), while decreased significantly in PAR-2shRNA EC109 cell and MAPK inhibitor group (p < 0.05). The mRNA expression of ERK1 and protein expression of p-ERK1 increased in PAR-2 agonist group, decreased in PAR-2shRNA EC109 cell and MAPK inhibitor group when compared with blank control (p < 0.05). The growth of cells was upward in PAR-2 agonist group at cell growth phase when compared with blank control, while decreased in PAR-2 shRNA EC109 cell and MAPK inhibitor group with statistical difference (p < 0.05). PAR-2 regulate cell proliferation through the MAPK pathway in esophageal carcinoma cell, and Cyclin D1, c-fos are involved in this process.

Par3L enhances colorectal cancer cell survival by inhibiting Lkb1/AMPK signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Taiyuan; Liu, Dongning; Lei, Xiong

Partitioning defective 3-like protein (Par3L) is a recently identified cell polarity protein that plays an important role in mammary stem cell maintenance. Previously, we showed that high expression of Par3L is associated with poor survival in malignant colorectal cancer (CRC), but the underlying mechanism remained unknown. To this end, we established a Par3L knockout colorectal cancer cell line using the CRISPR/Cas system. Interestingly, reduced proliferation, enhanced cell death and caspase-3 activation were observed in Par3L knockout (KO) cells as compared with wildtype (WT) cells. Consistent with previous studies, we showed that Par3L interacts with a tumor suppressor protein liver kinasemore » B1 (Lkb1). Moreover, Par3L depletion resulted in abnormal activation of Lkb1/AMPK signaling cascade. Knockdown of Lkb1 in these cells could significantly reduce AMPK activity and partially rescue cell death caused by Par3L knockdown. Furthermore, we showed that Par3L KO cells were more sensitive to chemotherapies and irradiation. Together, these results suggest that Par3L is essential for colorectal cancer cell survival by inhibiting Lkb1/AMPK signaling pathway, and is a putative therapeutic target for CRC. - Highlights: • Par3L knockout using the CRISPR/Cas system induces apoptosis in colorectal cancer cells. • Par3L interacts with Lkb1 and regulates the activity of AMPK signaling cascade. • Par3L knockout cells are more sensitive to treatment of different chemotherapy drugs and irradiation.« less

Regulation of long-term repopulating hematopoietic stem cells by EPCR/PAR1 signaling

PubMed Central

Gur-Cohen, Shiri; Kollet, Orit; Graf, Claudine; Esmon, Charles T.; Ruf, Wolfram; Lapidot, Tsvee

2016-01-01

The common developmental origin of endothelial and hematopoietic cells is manifested by coexpression of several cell surface receptors. Adult murine bone marrow (BM) long-term repopulating hematopoietic stem cells (LT-HSCs), endowed with the highest repopulation and self-renewal potential, express endothelial protein C receptor (EPCR), which is used as a marker to isolate them. EPCR/PAR1 signaling in endothelial cells has anticoagulant and anti-inflammatory roles, while thrombin/PAR1 signaling induces coagulation and inflammation. Recent studies define two new PAR1-mediated signaling cascades that regulate EPCR+ LT-HSC BM retention and egress. EPCR/PAR1 signaling facilitates LT-HSC BM repopulation, retention, survival, and chemotherapy resistance by restricting nitric oxide (NO) production, maintaining NOlow LT-HSC BM retention with increased VLA4 expression, affinity, and adhesion. Conversely, acute stress and clinical mobilization upregulate thrombin generation and activate different PAR1 signaling which overcomes BM EPCR+ LT-HSC retention, inducing their recruitment to the bloodstream. Thrombin/PAR1 signaling induces NO generation, TACE-mediated EPCR shedding, and upregulation of CXCR4 and PAR1, leading to CXCL12-mediated stem and progenitor cell mobilization. This review discusses new roles for factors traditionally viewed as coagulation related, which independently act in the BM to regulate PAR1 signaling in bone- and blood-forming progenitor cells, navigating their fate by controlling NO production. PMID:26928241

Increased Expression of Cathelicidin by Direct Activation of Protease-Activated Receptor 2: Possible Implications on the Pathogenesis of Rosacea

PubMed Central

Kim, Ji Young; Kim, Yoon Jee; Lim, Beom Jin; Sohn, Hyo Jung; Shin, Dongyun

2014-01-01

Purpose Recent findings of increased cathelicidin protein and its proteolytic fragments in rosacea suggest a pathogenic role for cathelicidin in this disease. The relationship between cathelicidin and protease-activated receptor 2 (PAR-2) is therefore of interest, as PAR-2, expressed principally in keratinocytes, regulates pro-inflammatory cytokine expression in the skin. The purpose of this study was to determine the relationship between expression of PAR-2 and cathelicidin in rosacea and to test the effect of direct PAR-2 activation on cathelicidin expression in keratinocytes. Materials and Methods Samples from 40 patients with clinicopathologic diagnosis of rosacea and facial skin tissue samples from 20 patients with no specific findings or milium without inflammation were retrieved. Intensities of immunohistochemical staining for PAR-2 and cathelicidin were compared between normal and rosacea-affected skin tissues. Additionally, correlations between PAR-2 and cathelicidin staining intensities within rosacea patients were analyzed. In cultured keratinocytes, changes in PAR-2, cathelicidin, and vascular endothelial growth factor (VEGF) mRNA and protein were analyzed after treatment with PAR-2 activating peptide (AP). Results Cathelicidin expression was significantly higher in rosacea skin tissues than in normal tissues (p<0.001), while PAR-2 expression was not significantly higher in rosacea tissues than in normal skin tissues. A positive correlation between PAR-2 and cathelicidin within rosacea samples was observed (R=0.330, p=0.037). After treatment of PAR-2 AP, both mRNA and protein levels for PAR-2, cathelicidin, and VEGF significantly increased in cultured keratinocytes, compared with PAR-2 control peptide treatment. Conclusion PAR-2 may participate in the pathogenesis of rosacea through activation of cathelicidin LL-37, a mediator of innate immune responses in the skin. PMID:25323904

Coagulation factor Xa drives tumor cells into apoptosis through BH3-only protein Bim up-regulation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Borensztajn, Keren S.; Bijlsma, Maarten F.; Groot, Angelique P.

2007-07-15

Coagulation Factor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulation factor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells withmore » up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.« less

Differential roles of kallikrein-related peptidase 6 in malignant transformation and ΔNp63β-mediated epithelial-mesenchymal transition of oral squamous cell carcinoma.

PubMed

Kaneko, Naoki; Kawano, Shintaro; Yasuda, Kaori; Hashiguchi, Yuma; Sakamoto, Taiki; Matsubara, Ryota; Goto, Yuichi; Jinno, Teppei; Maruse, Yasuyuki; Morioka, Masahiko; Hattori, Taichi; Tanaka, Shoichi; Tanaka, Hideaki; Kiyoshima, Tamotsu; Nakamura, Seiji

2017-12-01

We previously reported that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63β in oral squamous cell carcinoma (OSCC). In this study, DNA microarray analyses were performed using ΔNp63β-overexpressing OSCC cells to identify genes associated with ΔNp63β-mediated EMT. Thereby, we focused on kallikrein-related peptidase (KLK) 6, most up-regulated following ΔNp63β-overexpression, that activates protease-activated receptors (PARs). In RT-PCR analyses, ΔNp63 was positively associated with KLK6 and PAR2 and negatively with PAR1 in OSCC cells. By ΔNp63 knockdown, KLK6 and PAR2 expression was decreased and PAR1 was increased. Furthermore, KLK6 knockdown led to enhancing migration and invasion, and inhibiting proliferation, suggesting EMT-phenotypes. Although, in the KLK6 or PAR2 knockdown cells, phosphorylation of ERK was reduced, it was restored in the KLK6 knockdown OSCC cells treated with recombinant KLK6 proteins. Immunohistochemistry showed ΔNp63, KLK6, and PAR2 were more strongly expressed in the epithelial dysplasia and central region of OSCC than normal oral epithelium, whereas PAR1 expression was undetectable. Interestingly, at the invasive front of OSCC, ΔNp63, KLK6, and PAR2 were reduced, but PAR1 was elevated. In addition, the OSCC patients with decreasing KLK6 expression at the invasive front had more unfavourable prognosis. These results suggested differential roles of KLK6 in malignant transformation and EMT; high ΔNp63β expression up-regulates KLK6-PAR2 and down-regulates PAR1, inducing malignant transformation in oral epithelium with stimulating proliferation through ERK signal activation. Moreover, KLK6-PAR2 expression is down-regulated and PAR1 is up-regulated when ΔNp63β expression is decreased, leading to EMT with enhancing migration and invasion through ERK signal reduction at the invasive front. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cyclic strain increases protease-activated receptor-1 expression in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Frye, S. R.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Cyclic strain regulates many vascular smooth muscle cell (VSMC) functions through changing gene expression. This study investigated the effects of cyclic strain on protease-activated receptor-1 (PAR-1) expression in VSMCs and the possible signaling pathways involved, on the basis of the hypothesis that cyclic strain would enhance PAR-1 expression, reflecting increased thrombin activity. Uniaxial cyclic strain (1 Hz, 20%) of cells cultured on elastic membranes induced a 2-fold increase in both PAR-1 mRNA and protein levels. Functional activity of PAR-1, as assessed by cell proliferation in response to thrombin, was also increased by cyclic strain. In addition, treatment of cells with antioxidants or an NADPH oxidase inhibitor blocked strain-induced PAR-1 expression. Preincubation of cells with protein kinase inhibitors (staurosporine or Ro 31-8220) enhanced strain-increased PAR-1 expression, whereas inhibitors of NO synthase, tyrosine kinase, and mitogen-activated protein kinases had no effect. Cyclic strain in the presence of basic fibroblast growth factor induced PAR-1 mRNA levels beyond the effect of cyclic strain alone, whereas no additive effect was observed between cyclic strain and platelet-derived growth factor-AB. Our findings that cyclic strain upregulates PAR-1 mRNA expression but that shear stress downregulates this gene in VSMCs provide an opportunity to elucidate signaling differences by which VSMCs respond to different mechanical forces.

Par3 and aPKC regulate BACE1 endosome-to-TGN trafficking through PACS1.

PubMed

Sun, Miao; Zhang, Huaye

2017-12-01

The cleavage of amyloid precursor protein (APP) by β-site APP cleaving enzyme 1 (BACE1) is the rate-limiting step in beta amyloid generation during Alzheimer's disease (AD) pathogenesis. In AD brains, BACE1 is abnormally accumulated in endocytic compartments, where the acidic pH is optimal for its activity. However, mechanisms regulating the endosome-to-trans-Golgi network (TGN) retrieval of BACE1 remain unclear. Here, we show that partitioning defective 3 (Par3) facilitates BACE1 retrograde trafficking from endosomes to the TGN. Par3 functions through aPKC-mediated phosphorylation of BACE1 on Ser498, which in turn promotes the interaction between BACE1 and phosphofurin acidic cluster sorting protein 1 and facilitates the retrograde trafficking of BACE1 to the TGN. In human AD brains, there is a significant decrease in Ser498 phosphorylation of BACE1 suggesting that defective phosphorylation-dependent retrograde transport of BACE1 is important in AD pathogenesis. Together, our studies provide mechanistic insight into a novel role for Par3 and aPKC in regulating the retrograde endosome-to-TGN trafficking of BACE1 and shed light on the mechanisms of AD pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma*

PubMed Central

Johnson, Jeff J.; Miller, Daniel L.; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R.; Stack, M. Sharon

2016-01-01

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. PMID:26839311

Protease-activated Receptor-2 (PAR-2)-mediated Nf-κB Activation Suppresses Inflammation-associated Tumor Suppressor MicroRNAs in Oral Squamous Cell Carcinoma.

PubMed

Johnson, Jeff J; Miller, Daniel L; Jiang, Rong; Liu, Yueying; Shi, Zonggao; Tarwater, Laura; Williams, Russell; Balsara, Rashna; Sauter, Edward R; Stack, M Sharon

2016-03-25

Oral cancer is the sixth most common cause of death from cancer with an estimated 400,000 deaths worldwide and a low (50%) 5-year survival rate. The most common form of oral cancer is oral squamous cell carcinoma (OSCC). OSCC is highly inflammatory and invasive, and the degree of inflammation correlates with tumor aggressiveness. The G protein-coupled receptor protease-activated receptor-2 (PAR-2) plays a key role in inflammation. PAR-2 is activated via proteolytic cleavage by trypsin-like serine proteases, including kallikrein-5 (KLK5), or by treatment with activating peptides. PAR-2 activation induces G protein-α-mediated signaling, mobilizing intracellular calcium and Nf-κB signaling, leading to the increased expression of pro-inflammatory mRNAs. Little is known, however, about PAR-2 regulation of inflammation-related microRNAs. Here, we assess PAR-2 expression and function in OSCC cell lines and tissues. Stimulation of PAR-2 activates Nf-κB signaling, resulting in RelA nuclear translocation and enhanced expression of pro-inflammatory mRNAs. Concomitantly, suppression of the anti-inflammatory tumor suppressor microRNAs let-7d, miR-23b, and miR-200c was observed following PAR-2 stimulation. Analysis of orthotopic oral tumors generated by cells with reduced KLK5 expression showed smaller, less aggressive lesions with reduced inflammatory infiltrate relative to tumors generated by KLK5-expressing control cells. Together, these data support a model wherein KLK5-mediated PAR-2 activation regulates the expression of inflammation-associated mRNAs and microRNAs, thereby modulating progression of oral tumors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The 8th and 9th tandem spectrin-like repeats of utrophin cooperatively form a functional unit to interact with polarity-regulating kinase PAR-1b.

PubMed

Yamashita, Kazunari; Suzuki, Atsushi; Satoh, Yoshinori; Ide, Mariko; Amano, Yoshiko; Masuda-Hirata, Maki; Hayashi, Yukiko K; Hamada, Keisuke; Ogata, Kazuhiro; Ohno, Shigeo

2010-01-01

Utrophin is a widely expressed paralogue of dystrophin, the protein responsible for Duchenne muscular dystrophy. Utrophin is a large spectrin-like protein whose C-terminal domain mediates anchorage to a laminin receptor, dystroglycan (DG). The rod domain, composed of 22 spectrin-like repeats, connects the N-terminal actin-binding domain and the C-terminal DG binding domain, and thus mediates molecular linkage between intracellular F-actin and extracellular basement membrane. Previously, we demonstrated that a cell polarity-regulating kinase, PAR-1b, interacts with the utrophin-DG complex, and positively regulates the interaction between utrophin and DG. In this study, we demonstrate that the 8th and 9th spectrin-like repeats (R8 and R9) of utrophin cooperatively form a PAR-1b-interacting domain, and that Ser1258 within R9 is specifically phosphorylated by PAR-1b. Substitution of Ser1258 to alanine reduces the interaction between utrophin and DG, suggesting that the Ser1258 phosphorylation contributes to the stabilization of the utrophin-DG complex. Interestingly, PAR-1b also binds and phosphorylates R8-9 of dystrophin, and colocalizes with dystrophin at the skeletal muscle membrane. These results reveal a novel function of the rod domain of utrophin beyond that of a passive structural linker connecting the N- and C-terminal domain. Copyright 2009 Elsevier Inc. All rights reserved.

PAR-1/MARK: a kinase essential for maintaining the dynamic state of microtubules.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2012-01-01

The serine/threonine kinase, PAR-1, is an essential component of the evolutionary-conserved polarity-regulating system, PAR-aPKC system, which plays indispensable roles in establishing asymmetric protein distributions and cell polarity in various biological contexts (Suzuki, A. and Ohno, S. (2006). J. Cell Sci., 119: 979-987; Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). PAR-1 is also known as MARK, which phosphorylates classical microtubule-associated proteins (MAPs) and detaches MAPs from microtubules (Matenia, D. and Mandelkow, E.M. (2009). Trends Biochem. Sci., 34: 332-342). This MARK activity of PAR-1 suggests its role in microtubule (MT) dynamics, but surprisingly, only few studies have been carried out to address this issue. Here, we summarize our recent study on live imaging analysis of MT dynamics in PAR-1b-depleted cells, which clearly demonstrated the positive role of PAR-1b in maintaining MT dynamics (Hayashi, K., Suzuki, A., Hirai, S., Kurihara, Y., Hoogenraad, C.C., and Ohno, S. (2011). J. Neurosci., 31: 12094-12103). Importantly, our results further revealed the novel physiological function of PAR-1b in maintaining dendritic spine morphology in mature neurons.

Proteinase activated-receptors-associated signaling in the control of gastric cancer

PubMed Central

Sedda, Silvia; Marafini, Irene; Caruso, Roberta; Pallone, Francesco; Monteleone, Giovanni

2014-01-01

Gastric cancer (GC) is the fourth most common cancer in the world and the second cause of cancer-related death. Gastric carcinogenesis is a multifactorial process, in which environmental and genetic factors interact to activate multiple intracellular signals thus leading to uncontrolled growth and survival of GC cells. One such a pathway is regulated by proteinase activated-receptors (PARs), seven transmembrane-spanning domain G protein-coupled receptors, which comprise four receptors (i.e., PAR-1, PAR-2, PAR-3, and PAR-4) activated by various proteases. Both PAR-1 and PAR-2 are over-expressed on GC cells and their activation triggers and/or amplifies intracellular pathways, which sustain gastric carcinogenesis. There is also evidence that expression of either PAR-1 or PAR-2 correlates with depth of wall invasion and metastatic dissemination and inversely with the overall survival of patients. Consistently, data emerging from experimental models of GC suggest that both these receptors can be important targets for therapeutic interventions in GC patients. In contrast, PAR-4 levels are down-regulated in GC and correlate inversely with the aggressiveness of GC, thus suggesting a negative role of this receptor in the control of GC. In this article we review the available data on the expression and role of PARs in GC and discuss whether manipulation of PAR-driven signals may be useful for interfering with GC cell behavior. PMID:25232234

Protease Activated Receptor-2 Mediates Activated Protein C–Induced Cutaneous Wound Healing via Inhibition of p38

PubMed Central

Julovi, Sohel M.; Xue, Meilang; Dervish, Suat; Sambrook, Philip N.; March, Lyn; Jackson, Christopher John

2011-01-01

Activated protein C (APC) is a natural anticoagulant that exerts anti-inflammatory and cytoprotective properties mediated through the protease activated receptor (PAR)-1. APC can also proteolytically cleave PAR-2, although subsequent function is unknown. On the basis of recent evidence that APC promotes wound healing, the aim of this study was to determine whether APC acts through PARs to heal murine excisional wounds or to regulate human cultured keratinocyte function and to determine the signaling mechanisms. Topical administration of APC accelerated wound healing in wild-type mice and, unexpectedly, in PAR-1 knockout mice. PAR-2 knockout mice healed significantly slower than wild-type mice, and healing was not altered by adding APC, indicating that APC acts through PAR-2 to heal wounds. In cultured human primary keratinocytes, APC enhanced PAR-2, stimulated proliferation, activated phosphatidylinositol 3-kinase/Src/Akt, and inhibited phosphorylated (P)-p38. Inhibiting PAR-1 or PAR-2, by small-interfering RNA or blocking antibody, reversed APC-induced keratinocyte proliferation and Akt activation. Blocking PAR-2, but not PAR-1, reversed the inhibition of P-p38 by APC. Furthermore, inhibition of P-p38 accelerated wound healing in wild-type mice. In summary, although APC acts through both PAR-1 and PAR-2 to activate Akt and to increase keratinocyte proliferation, APC-induced murine wound healing depends on PAR-2 activity and inhibition of P-p38. PMID:21907694

Dimerization controls the lipid raft partitioning of uPAR/CD87 and regulates its biological functions

PubMed Central

Cunningham, Orla; Andolfo, Annapaola; Santovito, Maria Lisa; Iuzzolino, Lucia; Blasi, Francesco; Sidenius, Nicolai

2003-01-01

The urokinase-type plasminogen activator receptor (uPAR/CD87) is a glycosylphosphatidylinositol-anchored membrane protein with multiple functions in extracellular proteolysis, cell adhesion, cell migration and proliferation. We now report that cell surface uPAR dimerizes and that dimeric uPAR partitions preferentially to detergent-resistant lipid rafts. Dimerization of uPAR did not require raft partitioning as the lowering of membrane cholesterol failed to reduce dimerization and as a transmembrane uPAR chimera, which does not partition to lipid rafts, also dimerized efficiently. While uPA bound to uPAR independently of its membrane localization and dimerization status, uPA-induced uPAR cleavage was strongly accelerated in lipid rafts. In contrast to uPA, the binding of Vn occurred preferentially to raft- associated dimeric uPAR and was completely blocked by cholesterol depletion. PMID:14609946

PAR-2 regulates dental pulp inflammation associated with caries.

PubMed

Lundy, F T; About, I; Curtis, T M; McGahon, M K; Linden, G J; Irwin, C R; El Karim, I A

2010-07-01

Protease-activated receptors (PARs) are G-protein-coupled receptors that are activated enzymatically by proteolysis of an N-terminal domain. The cleavage and activation of PARs by serine proteases represent a novel mechanism by which such enzymes could influence the host inflammatory response. The aim of this study was to determine whether PAR-2 expression and activation were increased in dental caries. Using immunohistochemistry, we showed PAR-2 to be localized to pulp cells subjacent to caries lesions, but minimally expressed by healthy pulp tissue. Trypsin and the PAR-2 agonist (PAR2-AP) activated PAR-2 in an in vitro functional assay. Endogenous molecules present in pulp cell lysates from carious teeth specifically activated PAR-2, but those from healthy teeth failed to do so. The activation of PAR-2 in vitro was shown to increase the expression of the pro-inflammatory mediator cyclo-oxygenase-2 (COX-2), providing a mechanism whereby PAR-2 could modulate pulpal inflammation.

The human airway trypsin-like protease modulates the urokinase receptor (uPAR, CD87) structure and functions.

PubMed

Beaufort, Nathalie; Leduc, Dominique; Eguchi, Hiroshi; Mengele, Karin; Hellmann, Daniela; Masegi, Tsukio; Kamimura, Takashi; Yasuoka, Susumu; Fend, Falko; Chignard, Michel; Pidard, Dominique

2007-05-01

The human airway trypsin-like protease (HAT) is a respiratory epithelium-associated, type II transmembrane serine protease, which is also detected as an extracellular enzyme in lung fluids during airway inflammatory disorders. We have evaluated its capacity to affect the urokinase-type plasminogen activator receptor (uPAR), a membrane glycolipid-anchored, three-domain (D1D2D3) glycoprotein that plays a crucial role in innate immunity and inflammation by supporting cell migration and matrix degradation, with structure and biological properties that can be regulated via limited endoproteolysis. With the use of immunoblotting, flow immunocytometry, and ELISA analyses applied to a recombinant uPAR protein and to uPAR-expressing monocytic and human bronchial epithelial cells, it was shown that exposure of uPAR to soluble HAT in the range of 10-500 nM resulted in the proteolytic processing of the full-length (D1D2D3) into the truncated (D2D3) species, with cleavage occurring in the D1 to D2 linker sequence after arginine residues at position 83 and 89. Using immunohistochemistry, we found that both HAT and uPAR were expressed in the human bronchial epithelium. Moreover, transient cotransfection in epithelial cells showed that membrane coexpression of the two partners produced a constitutive and extensive shedding of the D1 domain, occurring for membrane-associated HAT concentrations in the nanomolar range. Because the truncated receptor was found to be unable to bind two of the major uPAR ligands, the adhesive matrix protein vitronectin and the serine protease urokinase, it thus appears that proteolytic regulation of uPAR by HAT is likely to modulate cell adherence and motility, as well as tissue remodeling during the inflammatory response in the airways.

Multiple roles of the coagulation protease cascade during virus infection.

PubMed

Antoniak, Silvio; Mackman, Nigel

2014-04-24

The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

Partitioning-Defective 1a/b Depletion Impairs Glomerular and Proximal Tubule Development.

PubMed

Akchurin, Oleh; Du, Zhongfang; Ramkellawan, Nadira; Dalal, Vidhi; Han, Seung Hyeok; Pullman, James; Müsch, Anne; Susztak, Katalin; Reidy, Kimberly J

2016-12-01

The kidney is a highly polarized epithelial organ that develops from undifferentiated mesenchyme, although the mechanisms that regulate the development of renal epithelial polarity are incompletely understood. Partitioning-defective 1 (Par1) proteins have been implicated in cell polarity and epithelial morphogenesis; however, the role of these proteins in the developing kidney has not been established. Therefore, we studied the contribution of Par1a/b to renal epithelial development. We examined the renal phenotype of newborn compound mutant mice carrying only one allele of Par1a or Par1b. Loss of three out of four Par1a/b alleles resulted in severe renal hypoplasia, associated with impaired ureteric bud branching. Compared with kidneys of newborn control littermates, kidneys of newborn mutant mice exhibited dilated proximal tubules and immature glomeruli, and the renal proximal tubular epithelia lacked proper localization of adhesion complexes. Furthermore, Par1a/b mutants expressed low levels of renal Notch ligand Jag1, activated Notch2, and Notch effecter Hes1. Together, these data demonstrate that Par1a/b has a key role in glomerular and proximal tubule development, likely via modulation of Notch signaling. Copyright © 2016 by the American Society of Nephrology.

Foxo-dependent Par-4 Upregulation Prevents Long-term Survival of Residual Cells Following PI3K-Akt Inhibition.

PubMed

Damrauer, Jeffrey S; Phelps, Stephanie N; Amuchastegui, Katie; Lupo, Ryan; Mabe, Nathaniel W; Walens, Andrea; Kroger, Benjamin R; Alvarez, James V

2018-04-01

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K-Akt-mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K-Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K-Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599-609. ©2018 AACR . ©2018 American Association for Cancer Research.

Differential regulation of protease activated receptor-1 and tissue plasminogen activator expression by shear stress in vascular smooth muscle cells

NASA Technical Reports Server (NTRS)

Papadaki, M.; Ruef, J.; Nguyen, K. T.; Li, F.; Patterson, C.; Eskin, S. G.; McIntire, L. V.; Runge, M. S.

1998-01-01

Recent studies have demonstrated that vascular smooth muscle cells are responsive to changes in their local hemodynamic environment. The effects of shear stress on the expression of human protease activated receptor-1 (PAR-1) and tissue plasminogen activator (tPA) mRNA and protein were investigated in human aortic smooth muscle cells (HASMCs). Under conditions of low shear stress (5 dyn/cm2), PAR-1 mRNA expression was increased transiently at 2 hours compared with stationary control values, whereas at high shear stress (25 dyn/cm2), mRNA expression was decreased (to 29% of stationary control; P<0.05) at all examined time points (2 to 24 hours). mRNA half-life studies showed that this response was not due to increased mRNA instability. tPA mRNA expression was decreased (to 10% of stationary control; P<0.05) by low shear stress after 12 hours of exposure and was increased (to 250% of stationary control; P<0.05) after 24 hours at high shear stress. The same trends in PAR-1 mRNA levels were observed in rat smooth muscle cells, indicating that the effects of shear stress on human PAR-1 were not species-specific. Flow cytometry and ELISA techniques using rat smooth muscle cells and HASMCs, respectively, provided evidence that shear stress exerted similar effects on cell surface-associated PAR-1 and tPA protein released into the conditioned media. The decrease in PAR-1 mRNA and protein had functional consequences for HASMCs, such as inhibition of [Ca2+] mobilization in response to thrombin stimulation. These data indicate that human PAR-1 and tPA gene expression are regulated differentially by shear stress, in a pattern consistent with their putative roles in several arterial vascular pathologies.

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines

PubMed Central

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-01-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics. PMID:22545177

A novel function of the cell polarity-regulating kinase PAR-1/MARK in dendritic spines.

PubMed

Hayashi, Kenji; Suzuki, Atsushi; Ohno, Shigeo

2011-11-01

Dendritic spines are postsynaptic structures that receive excitatory synaptic signals from presynaptic terminals in neurons. Because the morphology of spines has been considered to be a crucial factor for the efficiency of synaptic transmission, understanding the mechanisms regulating their morphology is important for neuroscience. Actin filaments and their regulatory proteins are known to actively maintain spine morphology; recent studies have also shown an essential role of microtubules (MTs). Live imaging of the plus-ends of MTs in mature neurons revealed that MTs stochastically enter spines and mediate accumulation of p140Cap, which regulates reorganization of actin filaments. However, the molecular mechanism by which MT dynamics is controlled has remained largely unknown. A cell polarity-regulating serine/threonine kinase, partitioning-defective 1 (PAR-1), phosphorylates classical MAPs and inhibits their binding to MTs. Because the interaction of MAPs with MTs can decrease MT dynamic instability, PAR-1 is supposed to activate MT dynamics through its MAP/MT affinity-regulating kinase (MARK) activity, although there is not yet any direct evidence for this. Here, we review recent findings on the localization of PAR-1b in the dendrites of mouse hippocampal neurons, and its novel function in the maintenance of mature spine morphology by regulating MT dynamics.

Divergent β-Arrestin-dependent Signaling Events Are Dependent upon Sequences within G-protein-coupled Receptor C Termini*

PubMed Central

Pal, Kasturi; Mathur, Maneesh; Kumar, Puneet; DeFea, Kathryn

2013-01-01

β-Arrestins are multifunctional adaptor proteins that, upon recruitment to an activated G-protein-coupled receptor, can promote desensitization of G-protein signaling and receptor internalization while simultaneously eliciting an independent signal. The result of β-arrestin signaling depends upon the activating receptor. For example, activation of two Gαq-coupled receptors, protease-activated receptor-2 (PAR2) and neurokinin-1 receptor (NK1R), results in drastically different signaling events. PAR2 promotes β-arrestin-dependent membrane-sequestered extracellular signal-regulated kinase (ERK1/2) activation, cofilin activation, and cell migration, whereas NK1R promotes nuclear ERK1/2 activation and proliferation. Using bioluminescence resonance energy transfer to monitor receptor/β-arrestin interactions in real time, we observe that PAR2 has a higher apparent affinity for both β-arrestins than does NK1R, recruits them at a faster rate, and exhibits more rapid desensitization of the G-protein signal. Furthermore, recruitment of β-arrestins to PAR2 does not require prior Gαq signaling events, whereas inhibition of Gαq signaling intermediates inhibits recruitment of β-arrestins to NK1R. Using chimeric receptors in which the C terminus of PAR2 is fused to the N terminus of NK1R and vice versa and a critical Ser/Thr mutant of PAR2, we demonstrate that interactions between β-arrestins and specific phosphoresidues in the C termini of each receptor are crucial for determining the rate and magnitude of β-arrestin recruitment as well as the ultimate signaling outcome. PMID:23235155

Proteinase-Activated Receptor 1 (PAR1) Regulates Leukemic Stem Cell Functions

PubMed Central

Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E.

2014-01-01

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1−/− hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1−/− leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance. PMID:24740120

Proteinase-Activated Receptor 1 (PAR1) regulates leukemic stem cell functions.

PubMed

Bäumer, Nicole; Krause, Annika; Köhler, Gabriele; Lettermann, Stephanie; Evers, Georg; Hascher, Antje; Bäumer, Sebastian; Berdel, Wolfgang E; Müller-Tidow, Carsten; Tickenbrock, Lara

2014-01-01

External signals that are mediated by specific receptors determine stem cell fate. The thrombin receptor PAR1 plays an important role in haemostasis, thrombosis and vascular biology, but also in tumor biology and angiogenesis. Its expression and function in hematopoietic stem cells is largely unknown. Here, we analyzed expression and function of PAR1 in primary hematopoietic cells and their leukemic counterparts. AML patients' blast cells expressed much lower levels of PAR1 mRNA and protein than CD34+ progenitor cells. Constitutive Par1-deficiency in adult mice did not affect engraftment or stem cell potential of hematopoietic cells. To model an AML with Par1-deficiency, we retrovirally introduced the oncogene MLL-AF9 in wild type and Par1-/- hematopoietic progenitor cells. Par1-deficiency did not alter initial leukemia development. However, the loss of Par1 enhanced leukemic stem cell function in vitro and in vivo. Re-expression of PAR1 in Par1-/- leukemic stem cells delayed leukemogenesis in vivo. These data indicate that Par1 contributes to leukemic stem cell maintenance.

Molecular networks implicated in speech-related disorders: FOXP2 regulates the SRPX2/uPAR complex.

PubMed

Roll, Patrice; Vernes, Sonja C; Bruneau, Nadine; Cillario, Jennifer; Ponsole-Lenfant, Magali; Massacrier, Annick; Rudolf, Gabrielle; Khalife, Manal; Hirsch, Edouard; Fisher, Simon E; Szepetowski, Pierre

2010-12-15

It is a challenge to identify the molecular networks contributing to the neural basis of human speech. Mutations in transcription factor FOXP2 cause difficulties mastering fluent speech (developmental verbal dyspraxia, DVD), whereas mutations of sushi-repeat protein SRPX2 lead to epilepsy of the rolandic (sylvian) speech areas, with DVD or with bilateral perisylvian polymicrogyria. Pathophysiological mechanisms driven by SRPX2 involve modified interaction with the plasminogen activator receptor (uPAR). Independent chromatin-immunoprecipitation microarray screening has identified the uPAR gene promoter as a potential target site bound by FOXP2. Here, we directly tested for the existence of a transcriptional regulatory network between human FOXP2 and the SRPX2/uPAR complex. In silico searches followed by gel retardation assays identified specific efficient FOXP2-binding sites in each of the promoter regions of SRPX2 and uPAR. In FOXP2-transfected cells, significant decreases were observed in the amounts of both SRPX2 (43.6%) and uPAR (38.6%) native transcripts. Luciferase reporter assays demonstrated that FOXP2 expression yielded a marked inhibition of SRPX2 (80.2%) and uPAR (77.5%) promoter activity. A mutant FOXP2 that causes DVD (p.R553H) failed to bind to SRPX2 and uPAR target sites and showed impaired down-regulation of SRPX2 and uPAR promoter activity. In a patient with polymicrogyria of the left rolandic operculum, a novel FOXP2 mutation (p.M406T) was found in the leucine-zipper (dimerization) domain. p.M406T partially impaired the FOXP2 regulation of SRPX2 promoter activity, whereas that of the uPAR promoter remained unchanged. Together with recently described FOXP2-CNTNAP2 and SRPX2/uPAR links, the FOXP2-SRPX2/uPAR network provides exciting insights into molecular pathways underlying speech-related disorders.

Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

PubMed Central

Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

2014-01-01

Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as providing the basis for selection of the appropriate genetic model organisms to study the physiology of the specific human PARP proteins. PMID:24865146

Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.

Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importancemore » of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.« less

Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes.

PubMed

Zuriaga, Elena; Romero, Carlos; Blanca, Jose Miguel; Badenes, Maria Luisa

2018-01-27

Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes. Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1. Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.

Urokinase-Type Plasminogen Activator Receptor Is Internalized by Different Mechanisms in Polarized and Nonpolarized Madin–Darby Canine Kidney Epithelial Cells

PubMed Central

Vilhardt, Frederik; Nielsen, Morten; Sandvig, Kirsten; van Deurs, Bo

1999-01-01

Accumulated data indicate that endocytosis of the glycosylphosphatidyl-inositol-anchored protein urokinase plasminogen activator receptor (uPAR) depends on binding of the ligand uPA:plasminogen activator inhibitor-1 (PAI-1) and subsequent interaction with internalization receptors of the low-density lipoprotein receptor family, which are internalized through clathrin-coated pits. This interaction is inhibited by receptor-associated protein (RAP). We show that uPAR with bound uPA:PAI-1 is capable of entering cells in a clathrin-independent process. First, HeLaK44A cells expressing mutant dynamin efficiently internalized uPA:PAI-1 under conditions in which transferrin endocytosis was blocked. Second, in polarized Madin–Darby canine kidney (MDCK) cells, which expressed human uPAR apically, the low basal rate of uPAR ligand endocytosis, which could not be inhibited by RAP, was increased by forskolin or phorbol ester (phorbol 12-myristate 13-acetate), which selectively up-regulate clathrin-independent endocytosis from the apical domain of epithelial cells. Third, in subconfluent nonpolarized MDCK cells, endocytosis of uPA:PAI-1 was only decreased marginally by RAP. At the ultrastructural level uPAR was largely excluded from clathrin-coated pits in these cells and localized in invaginated caveolae only in the presence of cross-linking antibodies. Interestingly, a larger fraction of uPAR in nonpolarized relative to polarized MDCK cells was insoluble in Triton X-100 at 0°C, and by surface labeling with biotin we also show that internalized uPAR was mainly detergent insoluble, suggesting a correlation between association with detergent-resistant membrane microdomains and higher degree of clathrin-independent endocytosis. Furthermore, by cryoimmunogold labeling we show that 5–10% of internalized uPAR in nonpolarized, but not polarized, MDCK cells is targeted to lysosomes by a mechanism that is regulated by ligand occupancy. PMID:9880335

Temporal regulation of epithelium formation mediated by FoxA, MKLP1, MgcRacGAP, and PAR-6

PubMed Central

Von Stetina, Stephen E.; Liang, Jennifer; Marnellos, Georgios; Mango, Susan E.

2017-01-01

To establish the animal body plan, embryos link the external epidermis to the internal digestive tract. In Caenorhabditis elegans, this linkage is achieved by the arcade cells, which form an epithelial bridge between the foregut and epidermis, but little is known about how development of these three epithelia is coordinated temporally. The arcade cell epithelium is generated after the epidermis and digestive tract epithelia have matured, ensuring that both organs can withstand the mechanical stress of embryo elongation; mistiming of epithelium formation leads to defects in morphogenesis. Using a combination of genetic, bioinformatic, and imaging approaches, we find that temporal regulation of the arcade cell epithelium is mediated by the pioneer transcription factor and master regulator PHA-4/FoxA, followed by the cytoskeletal regulator and kinesin ZEN-4/MKLP1 and the polarity protein PAR-6. We show that PHA-4 directly activates mRNA expression of a broad cohort of epithelial genes, including junctional factor dlg-1. Accumulation of DLG-1 protein is delayed by ZEN-4, acting in concert with its binding partner CYK-4/MgcRacGAP. Our structure–function analysis suggests that nuclear and kinesin functions are dispensable, whereas binding to CYK-4 is essential, for ZEN-4 function in polarity. Finally, PAR-6 is necessary to localize polarity proteins such as DLG-1 within adherens junctions and at the apical surface, thereby generating arcade cell polarity. Our results reveal that the timing of a landmark event during embryonic morphogenesis is mediated by the concerted action of four proteins that delay the formation of an epithelial bridge until the appropriate time. In addition, we find that mammalian FoxA associates with many epithelial genes, suggesting that direct regulation of epithelial identity may be a conserved feature of FoxA factors and a contributor to FoxA function in development and cancer. PMID:28539408

LIGR, a protease-activated receptor-2-derived peptide, enhances skin pigmentation without inducing inflammatory processes.

PubMed

Lin, Connie B; Chen, Nannan; Scarpa, Richard; Guan, Fei; Babiarz-Magee, Laura; Liebel, Frank; Li, Wen-Hwa; Kizoulis, Menas; Shapiro, Stanley; Seiberg, Miri

2008-04-01

The protease-activated receptor-2 (PAR-2) is a seven transmembrane G-protein-coupled receptor that could be activated by serine protease cleavage or by synthetic peptide agonists. We showed earlier that activation of PAR-2 with Ser-Leu-Ile-Gly-Arg-Leu-NH(2) (SLIGRL), a known PAR-2 activating peptide, induces keratinocyte phagocytosis and increases skin pigmentation, indicating that PAR-2 regulates pigmentation by controlling phagocytosis of melanosomes. Here, we show that Leu-Ile-Gly-Arg-NH(2) (LIGR) can also induce skin pigmentation. Both SLIGRL and LIGR increased melanin deposition in vitro and in vivo, and visibly darkened human skins grafted onto severe combined immuno-deficient (SCID) mice. Both SLIGRL and LIGR stimulated Rho-GTP activation resulting in keratinocyte phagocytosis. Interestingly, LIGR activates only a subset of the PAR-2 signaling pathways, and unlike SLIGRL, it does not induce inflammatory processes. LIGR did not affect many PAR-2 signaling pathways, including [Ca(2+)] mobilization, cAMP induction, the induction of cyclooxgenase-2 (COX-2) expression and the secretion of prostaglandin E2, interleukin-6 and -8. PAR-2 siRNA inhibited LIGR-induced phagocytosis, indicating that LIGR signals via PAR-2. Our data suggest that LIGR is a more specific regulator of PAR-2-induced pigmentation relative to SLIGRL. Therefore, enhancing skin pigmentation by topical applications of LIGR may result in a desired tanned-like skin color, without enhancing inflammatory processes, and without the need of UV exposure.

PAR1 activation affects the neurotrophic properties of Schwann cells.

PubMed

Pompili, Elena; Fabrizi, Cinzia; Somma, Francesca; Correani, Virginia; Maras, Bruno; Schininà, Maria Eugenia; Ciraci, Viviana; Artico, Marco; Fornai, Francesco; Fumagalli, Lorenzo

2017-03-01

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR. Copyright © 2017 Elsevier Inc. All rights reserved.

[Effect of ERK/AP-1 signaling pathway on proliferation of hepatoma cells induced by PAR-2 agonists].

PubMed

Zheng, Yan-min; Xie, Li-qun; Li, Xuan; Zhao, Jun-yan; Chen, Xiao-yi; Chen, Li; Zhou, Jing; Li, Fei

2009-12-01

To investigate the expression of protease activated receptor-2 (PAR-2) in human HepG2 hepatoma cells and elucidate the effects of trypsin and PAR-2 agonist peptide SLIGKV-NH(2) upon the proliferation of hepatoma cells and its intracellular signaling mechanism. PAR-2 protein and mRNA expression were detected by immunofluorescence and RT-PCR. The cells were treated with SLIGKV-NH(2), trypsin, reverse PAR-2 agonist peptide VKGILS-NH(2) or PD98059. The changes of cell cycle distribution were evaluated by flow cytometry. The proliferative potential of HepG2 cells was estimated by MTT. The changes of PAR-2, c-fos and PCNA mRNA expression were detected by RT-PCR. The changes of c-fos and PCNA protein expression were detected by Western blotting. PAR-2 protein and mRNA were expressed in HepG2 cells. PAR-2 mRNA expression (PAR-2/beta-actin) were 0.70 +/- 0.04 and 0.99 +/- 0.05 respectively in cells treated with trypsin and SLIGKV-NH(2). They were both significantly higher than that in the control group (0.35 +/- 0.05, F = 135.534, P < 0.01). Percent G(0)/G(1) phase of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly lower than those in the control group [(56.11 +/- 0.85)%, (57.85 +/- 0.46)% vs (79.12 +/- 0.67)%, both P < 0.01] Percent S phase, G(2)/M phase and proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH(2) were significantly elevated (P < 0.01). The proliferation-enhancing effects and the up-regulation of mRNA and protein of c-fos and PCNA induced by trypsin or SLIGKV-NH(2) were significantly blocked by pretreatment with PD98059 (P < 0.01). There was no statistical significance in proliferation of HepG2 cells between the reverse PAR-2 agonist peptide VKGILS-NH(2) and control group (P > 0.05). PAR-2 is expressed in HepG2 hepatoma cells. PAR-2 activation induced by trypsin or SLIGKV-NH(2) promotes the proliferation of HepG2 cells partially via the ERK/AP-1 pathway.

Interplay between Ubiquitin, SUMO, and Poly(ADP-Ribose) in the Cellular Response to Genotoxic Stress

PubMed Central

Pellegrino, Stefania; Altmeyer, Matthias

2016-01-01

Cells employ a complex network of molecular pathways to cope with endogenous and exogenous genotoxic stress. This multilayered response ensures that genomic lesions are efficiently detected and faithfully repaired in order to safeguard genome integrity. The molecular choreography at sites of DNA damage relies heavily on post-translational modifications (PTMs). Protein modifications with ubiquitin and the small ubiquitin-like modifier SUMO have recently emerged as important regulatory means to coordinate DNA damage signaling and repair. Both ubiquitylation and SUMOylation can lead to extensive chain-like protein modifications, a feature that is shared with yet another DNA damage-induced PTM, the modification of proteins with poly(ADP-ribose) (PAR). Chains of ubiquitin, SUMO, and PAR all contribute to the multi-protein assemblies found at sites of DNA damage and regulate their spatio-temporal dynamics. Here, we review recent advancements in our understanding of how ubiquitin, SUMO, and PAR coordinate the DNA damage response and highlight emerging examples of an intricate interplay between these chain-like modifications during the cellular response to genotoxic stress. PMID:27148359

Cathepsin B and uPAR regulate self-renewal of glioma-initiating cells through GLI-regulated Sox2 and Bmi1 expression

PubMed Central

Rao, Jasti S.

2013-01-01

Cancer-initiating cells comprise a heterogeneous population of undifferentiated cells with the capacity for self-renewal and high proliferative potential. We investigated the role of uPAR and cathepsin B in the maintenance of stem cell nature in glioma-initiating cells (GICs). Simultaneous knockdown of uPAR and cathepsin B significantly reduced the expression of CD133, Nestin, Sox2 and Bmi1 at the protein level and GLI1 and GLI2 at the messenger RNA level. Also, knockdown of uPAR and cathepsin B resulted in a reduction in the number of GICs as well as sphere size. These changes are mediated by Sox2 and Bmi1, downstream of hedgehog signaling. Addition of cyclopamine reduced the expression of Sox2 and Bmi1 along with GLI1 and GLI2 expression, induced differentiation and reduced subsphere formation of GICs thereby indicating that hedgehog signaling acts upstream of Sox2 and Bmi1. Further confirmation was obtained from increased luciferase expression under the control of a GLI-bound Sox2 and Bmi1 luciferase promoter. Simultaneous knockdown of uPAR and cathepsin B also reduced the expression of Nestin Sox2 and Bmi1 in vivo. Thus, our study highlights the importance of uPAR and cathepsin B in the regulation of malignant stem cell self-renewal through hedgehog components, Bmi1 and Sox2. PMID:23222817

Proteinase-activated receptor (PAR)-2 activation impacts bone resorptive properties of human osteoarthritic subchondral bone osteoblasts.

PubMed

Amiable, Nathalie; Tat, Steeve Kwan; Lajeunesse, Daniel; Duval, Nicolas; Pelletier, Jean-Pierre; Martel-Pelletier, Johanne; Boileau, Christelle

2009-06-01

In osteoarthritis (OA), the subchondral bone undergoes a remodelling process involving several factors synthesized by osteoblasts. In this study, we investigated the expression, production, modulation, and role of PAR-2 in human OA subchondral bone osteoblasts. PAR-2 expression and production were determined by real-time PCR and flow cytometry, respectively. PAR-2 modulation was investigated in OA subchondral bone osteoblasts treated with IL-1 beta (100 pg/ml), TNF-alpha (5 ng/ml), TGF-beta1 (10 ng/ml), PGE(2) (500 nM), IL-6 (10 ng/ml) and IL-17 (10 ng/ml). Membranous RANKL protein was assessed by flow cytometry, and OPG, MMP-1, MMP-9, MMP-13, IL-6 and intracellular signalling pathways by specific ELISAs. Bone resorptive activity was measured by using a co-culture model of human PBMC and OA subchondral bone osteoblasts. PAR-2 expression and production (p<0.05) were markedly increased when human OA subchondral bone osteoblasts were compared to normal. On OA osteoblasts, PAR-2 production was significantly increased by IL-1 beta, TNF-alpha and PGE(2). Activation of PAR-2 with a specific agonist, SLIGKV-NH(2), induced a significant up-regulation of MMP-1, MMP-9, IL-6, and membranous RANKL, but had no effect on MMP-13 or OPG production. Interestingly, bone resorptive activity was also significantly enhanced following PAR-2 activation. The PAR-2 effect was mediated by activation of the MAP kinases Erk1/2 and JNK. This study is the first to demonstrate that PAR-2 activation plays a role in OA subchondral bone resorption via an up-regulation of major bone remodelling factors. These results shed new light on the potential of PAR-2 as a therapeutic target in OA.

Post-Transcriptional Regulation of Urokinase-type Plasminogen Activator Receptor Expression in Lipopolysaccharide-induced Acute Lung Injury

PubMed Central

Bhandary, Yashodhar P.; Velusamy, Thirunavukkarasu; Shetty, Praveenkumar; Shetty, Rashmi S.; Idell, Steven; Cines, Douglas B.; Jain, Deepika; Bdeir, Khalil; Abraham, Edward; Tsuruta, Yuko; Shetty, Sreerama

2009-01-01

Rationale: Urokinase-type plasminogen activator (uPA) receptor (uPAR) is required for the recruitment of neutrophils in response to infection. uPA induces its own expression in lung epithelial cells, which involves its interaction with cell surface uPAR. Regulation of uPAR expression is therefore crucial for uPA-mediated signaling in infectious acute lung injury (ALI). Objectives: To determine the role of uPA in uPAR expression during ALI caused by sepsis. Methods: We used Western blot, Northern blot, Northwestern assay, and immunohistochemistry. Phosphate-buffered saline– and lipopolysaccharide (LPS)-treated wild-type and uPA−/− mice were used. Measurements and Main Results: Biological activities of uPA, including proteolysis, cell adhesion, migration, proliferation, and differentiation, are dependent on its association with uPAR. Bacterial endotoxin (LPS) is a major cause of pulmonary dysfunction and infection-associated mortality. The present study shows that LPS induces uPAR expression both in vitro and in vivo, and that the mechanism involves post-transcriptional stabilization of uPAR mRNA by reciprocal interaction of phosphoglycerate kinase (PGK) and heterogeneous nuclear ribonucleoprotein C (hnRNPC) with uPAR mRNA coding region and 3′ untranslated region determinants, respectively. The process involves tyrosine phosphorylation of PGK and hnRNPC. uPA−/− mice failed to induce uPAR expression after LPS treatment. In these mice, LPS treatment failed to alter the binding of PGK and hnRNPC protein with uPAR mRNA due to lack of tyrosine phosphorylation. Conclusions: Our study shows that induction of LPS-mediated uPAR expression is mediated through tyrosine phosphorylation of PGK and hnRNPC. This involves expression of uPA as an obligate intermediary. PMID:19029002

Loss of aPKCλ in Differentiated Neurons Disrupts the Polarity Complex but Does Not Induce Obvious Neuronal Loss or Disorientation in Mouse Brains

PubMed Central

Yamanaka, Tomoyuki; Tosaki, Asako; Kurosawa, Masaru; Akimoto, Kazunori; Hirose, Tomonori; Ohno, Shigeo; Hattori, Nobutaka; Nukina, Nobuyuki

2013-01-01

Cell polarity plays a critical role in neuronal differentiation during development of the central nervous system (CNS). Recent studies have established the significance of atypical protein kinase C (aPKC) and its interacting partners, which include PAR-3, PAR-6 and Lgl, in regulating cell polarization during neuronal differentiation. However, their roles in neuronal maintenance after CNS development remain unclear. Here we performed conditional deletion of aPKCλ, a major aPKC isoform in the brain, in differentiated neurons of mice by camk2a-cre or synapsinI-cre mediated gene targeting. We found significant reduction of aPKCλ and total aPKCs in the adult mouse brains. The aPKCλ deletion also reduced PAR-6β, possibly by its destabilization, whereas expression of other related proteins such as PAR-3 and Lgl-1 was unaffected. Biochemical analyses suggested that a significant fraction of aPKCλ formed a protein complex with PAR-6β and Lgl-1 in the brain lysates, which was disrupted by the aPKCλ deletion. Notably, the aPKCλ deletion mice did not show apparent cell loss/degeneration in the brain. In addition, neuronal orientation/distribution seemed to be unaffected. Thus, despite the polarity complex disruption, neuronal deletion of aPKCλ does not induce obvious cell loss or disorientation in mouse brains after cell differentiation. PMID:24391875

PAR1 signaling regulates the retention and recruitment of EPCR-expressing bone marrow hematopoietic stem cells

PubMed Central

Gur-Cohen, Shiri; Itkin, Tomer; Chakrabarty, Sagarika; Graf, Claudine; Kollet, Orit; Ludin, Aya; Golan, Karin; Kalinkovich, Alexander; Ledergor, Guy; Wong, Eitan; Niemeyer, Elisabeth; Porat, Ziv; Erez, Ayelet; Sagi, Irit; Esmon, Charles T; Ruf, Wolfram; Lapidot, Tsvee

2016-01-01

Retention of long-term repopulating hematopoietic stem cells (LT-HSCs) in the bone marrow is essential for hematopoiesis and for protection from myelotoxic injury. We report that signaling cascades that are traditionally viewed as coagulation-related also control retention of EPCR+ LT-HSCs in the bone marrow and their recruitment to the blood via two different protease activated receptor 1 (PAR1)-mediated pathways. Thrombin-PAR1 signaling induces nitric oxide (NO) production, leading to TACE-mediated EPCR shedding, enhanced CXCL12-CXCR4-induced motility, and rapid stem and progenitor cell mobilization. Conversely, bone marrow blood vessels provide a microenvironment enriched with protein C that retain EPCR+ LT-HSCs by limiting NO generation, reducing Cdc42 activity and enhancing VLA4 affinity and adhesion. Inhibition of NO production by activated protein C (aPC)-EPCR-PAR1 signaling reduces progenitor cell egress, increases NOlow bone marrow EPCR+ LT-HSCs retention and protects mice from chemotherapy-induced hematological failure and death. Our study reveals new roles for PAR1 and EPCR that control NO production to balance maintenance and recruitment of bone marrow EPCR+ LT-HSCs with clinical relevance. PMID:26457757

Prohibitin is involved in the activated internalization and degradation of protease-activated receptor 1.

PubMed

Wang, Yan-Jie; Guo, Xiao-Long; Li, Sheng-An; Zhao, Yu-Qi; Liu, Zi-Chao; Lee, Wen-Hui; Xiang, Yang; Zhang, Yun

2014-07-01

The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics. Copyright © 2014 Elsevier B.V. All rights reserved.

Potentiation of the P2X3 ATP receptor by PAR-2 in rat dorsal root ganglia neurons, through protein kinase-dependent mechanisms, contributes to inflammatory pain.

PubMed

Wang, Shenglan; Dai, Yi; Kobayashi, Kimiko; Zhu, Wanjun; Kogure, Yoko; Yamanaka, Hiroki; Wan, You; Zhang, Wensheng; Noguchi, Koichi

2012-08-01

Proinflammatory agents trypsin and mast cell tryptase cleave and activate protease-activated receptor-2 (PAR-2), which is expressed on sensory nerves and causes neurogenic inflammation. P2X3 is a subtype of the ionotropic receptors for adenosine 5'-triphosphate (ATP), and is mainly localized on nociceptors. Here, we show that a functional interaction of the PAR-2 and P2X3 in primary sensory neurons could contribute to inflammatory pain. PAR-2 activation increased the P2X3 currents evoked by α, β, methylene ATP in dorsal root ganglia (DRG) neurons. Application of inhibitors of either protein kinase C (PKC) or protein kinase A (PKA) suppressed this potentiation. Consistent with this, a PKC or PKA activator mimicked the PAR-2-mediated potentiation of P2X3 currents. In the in vitro phosphorylation experiments, application of a PAR-2 agonist failed to establish phosphorylation of the P2X3 either on the serine or the threonine site. In contrast, application of a PAR-2 agonist induced trafficking of the P2X3 from the cytoplasm to the plasma membrane. These findings indicate that PAR-2 agonists may potentiate the P2X3, and the mechanism of this potentiation is likely to be a result of translocation, but not phosphorylation. The functional interaction between P2X3 and PAR-2 was also confirmed by detection of the α, β, methylene-ATP-evoked extracellular signal-regulated kinases (ERK) activation, a marker of neuronal signal transduction in DRG neurons, and pain behavior. These results demonstrate a functional interaction of the protease signal with the ATP signal, and a novel mechanism through which protease released in response to tissue inflammation might trigger the sensation to pain through P2X3 activation. © 2012 The Authors. European Journal of Neuroscience © 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.

Astrocytes Secrete Exosomes Enriched with Proapoptotic Ceramide and Prostate Apoptosis Response 4 (PAR-4)

PubMed Central

Wang, Guanghu; Dinkins, Michael; He, Qian; Zhu, Gu; Poirier, Christophe; Campbell, Andrew; Mayer-Proschel, Margot; Bieberich, Erhard

2012-01-01

Amyloid protein is well known to induce neuronal cell death, whereas only little is known about its effect on astrocytes. We found that amyloid peptides activated caspase 3 and induced apoptosis in primary cultured astrocytes, which was prevented by caspase 3 inhibition. Apoptosis was also prevented by shRNA-mediated down-regulation of PAR-4, a protein sensitizing cells to the sphingolipid ceramide. Consistent with a potentially proapoptotic effect of PAR-4 and ceramide, astrocytes surrounding amyloid plaques in brain sections of the 5xFAD mouse (and Alzheimer disease patient brain) showed caspase 3 activation and were apoptotic when co-expressing PAR-4 and ceramide. Apoptosis was not observed in astrocytes with deficient neutral sphingomyelinase 2 (nSMase2), indicating that ceramide generated by nSMase2 is critical for amyloid-induced apoptosis. Antibodies against PAR-4 and ceramide prevented amyloid-induced apoptosis in vitro and in vivo, suggesting that apoptosis was mediated by exogenous PAR-4 and ceramide, potentially associated with secreted lipid vesicles. This was confirmed by the analysis of lipid vesicles from conditioned medium showing that amyloid peptide induced the secretion of PAR-4 and C18 ceramide-enriched exosomes. Exosomes were not secreted by nSMase2-deficient astrocytes, indicating that ceramide generated by nSMase2 is critical for exosome secretion. Consistent with the ceramide composition in amyloid-induced exosomes, exogenously added C18 ceramide restored PAR-4-containing exosome secretion in nSMase2-deficient astrocytes. Moreover, isolated PAR-4/ceramide-enriched exosomes were taken up by astrocytes and induced apoptosis in the absence of amyloid peptide. Taken together, we report a novel mechanism of apoptosis induction by PAR-4/ceramide-enriched exosomes, which may critically contribute to Alzheimer disease. PMID:22532571

Regulation of feto-maternal barrier by matriptase- and PAR-2-mediated signaling is required for placental morphogenesis and mouse embryonic survival.

PubMed

Szabo, Roman; Peters, Diane E; Kosa, Peter; Camerer, Eric; Bugge, Thomas H

2014-07-01

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival.

Regulation of Feto-Maternal Barrier by Matriptase- and PAR-2-Mediated Signaling Is Required for Placental Morphogenesis and Mouse Embryonic Survival

PubMed Central

Szabo, Roman; Peters, Diane E.; Kosa, Peter; Camerer, Eric; Bugge, Thomas H.

2014-01-01

The development of eutherian mammalian embryos is critically dependent on the selective bi-directional transport of molecules across the placenta. Here, we uncover two independent and partially redundant protease signaling pathways that include the membrane-anchored serine proteases, matriptase and prostasin, and the G protein-coupled receptor PAR-2 that mediate the establishment of a functional feto-maternal barrier. Mice with a combined matriptase and PAR-2 deficiency do not survive to term and the survival of matriptase-deficient mice heterozygous for PAR-2 is severely diminished. Embryos with the combined loss of PAR-2 and matriptase or PAR-2 and the matriptase partner protease, prostasin, uniformly die on or before embryonic day 14.5. Despite the extensive co-localization of matriptase, prostasin, and PAR-2 in embryonic epithelia, the overall macroscopic and histological analysis of the double-deficient embryos did not reveal any obvious developmental abnormalities. In agreement with this, the conditional deletion of matriptase from the embryo proper did not affect the prenatal development or survival of PAR-2-deficient mice, indicating that the critical redundant functions of matriptase/prostasin and PAR-2 are limited to extraembryonic tissues. Indeed, placentas of the double-deficient animals showed decreased vascularization, and the ability of placental epithelium to establish a functional feto-maternal barrier was severely diminished. Interestingly, molecular analysis suggested that the barrier defect was associated with a selective deficiency in the expression of the tight junction protein, claudin-1. Our results reveal unexpected complementary roles of matriptase-prostasin- and PAR-2-dependent proteolytic signaling in the establishment of placental epithelial barrier function and overall embryonic survival. PMID:25078604

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khoi, Pham Ngoc; Park, Jung Sun; Kim, Nam Ho

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promotermore » activity. The intracellular hydrogen peroxide (H{sub 2}O{sub 2}) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H{sub 2}O{sub 2} increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells. -- Highlights: ► Endothelial cells treated with nicotine displayed enhanced invasiveness. ► Nicotine induces uPAR expression and, in turn, stimulates invasiveness. ► MAPK/AP-1 and ROS/NF-κB signals are involved in nicotine-induced uPAR.« less

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF.

PubMed

Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

2005-04-06

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins.

Bacterial DNA segregation dynamics mediated by the polymerizing protein ParF

PubMed Central

Barillà, Daniela; Rosenberg, Mark F; Nobbmann, Ulf; Hayes, Finbarr

2005-01-01

Prokaryotic DNA segregation most commonly involves members of the Walker-type ParA superfamily. Here we show that the ParF partition protein specified by the TP228 plasmid is a ParA ATPase that assembles into extensive filaments in vitro. Polymerization is potentiated by ATP binding and does not require nucleotide hydrolysis. Analysis of mutations in conserved residues of the Walker A motif established a functional coupling between filament dynamics and DNA partitioning. The partner partition protein ParG plays two separable roles in the ParF polymerization process. ParF is unrelated to prokaryotic polymerizing proteins of the actin or tubulin families, but is a homologue of the MinD cell division protein, which also assembles into filaments. The ultrastructures of the ParF and MinD polymers are remarkably similar. This points to an evolutionary parallel between DNA segregation and cytokinesis in prokaryotic cells, and reveals a potential molecular mechanism for plasmid and chromosome segregation mediated by the ubiquitous ParA-type proteins. PMID:15775965

Looping and clustering model for the organization of protein-DNA complexes on the bacterial genome

NASA Astrophysics Data System (ADS)

Walter, Jean-Charles; Walliser, Nils-Ole; David, Gabriel; Dorignac, Jérôme; Geniet, Frédéric; Palmeri, John; Parmeggiani, Andrea; Wingreen, Ned S.; Broedersz, Chase P.

2018-03-01

The bacterial genome is organized by a variety of associated proteins inside a structure called the nucleoid. These proteins can form complexes on DNA that play a central role in various biological processes, including chromosome segregation. A prominent example is the large ParB-DNA complex, which forms an essential component of the segregation machinery in many bacteria. ChIP-Seq experiments show that ParB proteins localize around centromere-like parS sites on the DNA to which ParB binds specifically, and spreads from there over large sections of the chromosome. Recent theoretical and experimental studies suggest that DNA-bound ParB proteins can interact with each other to condense into a coherent 3D complex on the DNA. However, the structural organization of this protein-DNA complex remains unclear, and a predictive quantitative theory for the distribution of ParB proteins on DNA is lacking. Here, we propose the looping and clustering model, which employs a statistical physics approach to describe protein-DNA complexes. The looping and clustering model accounts for the extrusion of DNA loops from a cluster of interacting DNA-bound proteins that is organized around a single high-affinity binding site. Conceptually, the structure of the protein-DNA complex is determined by a competition between attractive protein interactions and loop closure entropy of this protein-DNA cluster on the one hand, and the positional entropy for placing loops within the cluster on the other. Indeed, we show that the protein interaction strength determines the ‘tightness’ of the loopy protein-DNA complex. Thus, our model provides a theoretical framework for quantitatively computing the binding profiles of ParB-like proteins around a cognate (parS) binding site.

Increased production of BDNF in colonic epithelial cells induced by fecal supernatants from diarrheic IBS patients.

PubMed

Wang, Peng; Chen, Fei-Xue; Du, Chao; Li, Chang-Qing; Yu, Yan-Bo; Zuo, Xiu-Li; Li, Yan-Qing

2015-05-22

Colonic brain-derived neurotrophic factor (BDNF) plays an essential role in pathogenesis of abdominal pain in diarrhea-predominant irritable bowel syndrome (IBS-D), but regulation on its expression remains unclear. We investigated the role of fecal supernatants (FSN) from IBS-D patients on regulating BDNF expression in colonic epithelial cells of human and mice. Using human Caco-2 cells, we found that IBS-D FSN significantly increased BDNF mRNA and protein levels compared to control FSN, which were remarkably suppressed by the serine protease inhibitor. To further explore the potential mechanisms, we investigated the impact of protease-activated receptor-2 (PAR-2) on BDNF expression. We found a significant increase in PAR-2 expression in Caco-2 after IBS-D FSN stimulation. Knockdown of PAR-2 significantly inhibited IBS-D FSN-induced upregulation of BDNF. Moreover, we found that phosphorylation of p38 MAPK, not NF-κB p65, contributed to PAR-2-mediated BDNF overexpression. To confirm these results, we intracolonically infused IBS-D or control FSN in mice and found that IBS-D FSN significantly elevated colonic BDNF and visceral hypersensitivity in mice, which were both suppressed by the inhibitor of serine protease or antagonist of PAR-2. Together, our data indicate that activation of PAR-2 signaling by IBS-D FSN promotes expression of colonic BDNF, thereby contributing to IBS-like visceral hypersensitivity.

Intercellular communications within the rat anterior pituitary. XVI: postnatal changes of distribution of S-100 protein positive cells, connexin 43 and LH-RH positive sites in the pars tuberalis of the rat pituitary gland. An immunohistochemical and electron microscopic study.

PubMed

Wada, Ikuo; Sakuma, Eisuke; Shirasawa, Nobuyuki; Wakabayashi, Kenjiro; Otsuka, Takanobu; Hattori, Kazuki; Yashiro, Takashi; Herbert, Damon C; Soji, Tsuyoshi

2014-02-01

The architecture of luteinizing hormone-releasing hormone (LH-RH) nerve ends and the S-100 protein containing folliculo-stellate cells forming gap junctions in the pars tuberalis is basically important in understanding the regulation of the hormone producing mechanism of anterior pituitary glands. In this study, intact male rats 5-60 days old were prepared for immunohistochemistry and electron microscopy. From immunostained sections, the S-100 containing cells in pars tuberalis were first detected on day 30 and increased in number to day 60; this was parallel to the immunohistochemical staining of gap junction protein, connexin 43. LH-RH positive sites were clearly observed on just behind the optic chiasm and on the root of pituitary stalk on day 30. On day 60, the width of layer increased, while follicles and gap junctions were frequently observed between agranular cells in 10 or more layers of pars tuberalis. In the present study, we investigated the sexual maturation of the anterior pituitary glands through the postnatal development of S-100 positive cells, connexin 43 and LH-RH nerves. It is suggested that the folliculo-stellate cell system including the LH-RH neurons in the pars tuberalis participates in the control of LH secretion along with the portal vein system. Copyright © 2013 Elsevier Ltd. All rights reserved.

Loss of Par-1a/MARK3/C-TAK1 kinase leads to reduced adiposity, resistance to hepatic steatosis, and defective gluconeogenesis.

PubMed

Lennerz, Jochen K; Hurov, Jonathan B; White, Lynn S; Lewandowski, Katherine T; Prior, Julie L; Planer, G James; Gereau, Robert W; Piwnica-Worms, David; Schmidt, Robert E; Piwnica-Worms, Helen

2010-11-01

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a(-/-) but not in control or Par-1b(-/-) mice. The intercrossing of Par-1a(-/-) with Par-1b(-/-) mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a(-/-) mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b(-/-) mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice.

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.

PubMed

Svensson, Katrin J; Kucharzewska, Paulina; Christianson, Helena C; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-08-09

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein-coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2-mediated activation of hypoxic ECs. We show that PAR-2-dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2-dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa-dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2-mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors.

RNA sequencing to determine the contribution of kinase receptor transactivation to G protein coupled receptor signalling in vascular smooth muscle cells.

PubMed

Kamato, Danielle; Bhaskarala, Venkata Vijayanand; Mantri, Nitin; Oh, Tae Gyu; Ling, Dora; Janke, Reearna; Zheng, Wenhua; Little, Peter J; Osman, Narin

2017-01-01

G protein coupled receptor (GPCR) signalling covers three major mechanisms. GPCR agonist engagement allows for the G proteins to bind to the receptor leading to a classical downstream signalling cascade. The second mechanism is via the utilization of the β-arrestin signalling molecule and thirdly via transactivation dependent signalling. GPCRs can transactivate protein tyrosine kinase receptors (PTKR) to activate respective downstream signalling intermediates. In the past decade GPCR transactivation dependent signalling was expanded to show transactivation of serine/threonine kinase receptors (S/TKR). Kinase receptor transactivation enormously broadens the GPCR signalling paradigm. This work utilizes next generation RNA-sequencing to study the contribution of transactivation dependent signalling to total protease activated receptor (PAR)-1 signalling. Transactivation, assessed as gene expression, accounted for 50 percent of the total genes regulated by thrombin acting through PAR-1 in human coronary artery smooth muscle cells. GPCR transactivation of PTKRs is approximately equally important as the transactivation of the S/TKR with 209 and 177 genes regulated respectively, via either signalling pathway. This work shows that genome wide studies can provide powerful insights into GPCR mediated signalling pathways.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells.

PubMed

Nomura, Kazuaki; Obata, Kazufumi; Keira, Takashi; Miyata, Ryo; Hirakawa, Satoshi; Takano, Ken-ichi; Kohno, Takayuki; Sawada, Norimasa; Himi, Tetsuo; Kojima, Takashi

2014-02-18

Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis.

Pseudomonas aeruginosa elastase causes transient disruption of tight junctions and downregulation of PAR-2 in human nasal epithelial cells

PubMed Central

2014-01-01

Background Pseudomonas aeruginosa causes chronic respiratory disease, and the elastase enzyme that it produces increases the permeability of airway epithelial cells owing to the disruption of tight junctions. P. aeruginosa is also implicated in prolonged chronic rhinosinusitis. However, the effects of P. aeruginosa elastase (PE) against the barrier formed by human nasal epithelial cells (HNECs) remain unknown. Methods To investigate the mechanisms involved in the disruption of tight junctions by PE in HNECs, primary cultures of HNECs transfected with human telomerase reverse transcriptase (hTERT-HNECs) were used. The hTERT-HNECs were pretreated with inhibitors of various signal transduction pathways, PKC, MAPK, p38MAPK, PI3K, JNK, NF-κB, EGF receptor, proteasome, COX1 and COX2 before treatment with PE. Some cells were pretreated with siRNA and agonist of protease activated receptor-2 (PAR-2) before treatment with PE. Expression and structures of tight junctions were determined by Western blotting, real-time PCR, immunostaining and freeze-fracture. Transepithelial electrical resistance (TER) was examined as the epithelial barrier function. Results PE treatment transiently disrupted the epithelial barrier and downregulated the transmembrane proteins claudin-1 and -4, occludin, and tricellulin, but not the scaffold PDZ-expression proteins ZO-1 and -2 and adherens junction proteins E-cadherin and β-catenin. The transient downregulation of tight junction proteins was controlled via distinct signal transduction pathways such as the PKC, MAPK, PI3K, p38 MAPK, JNK, COX-1 and -2, and NF-κB pathways. Furthermore, treatment with PE transiently decreased PAR-2 expression, which also regulated the expression of the tight junction proteins. Treatment with a PAR-2 agonist prevented the downregulation of the tight junction proteins after PE treatment in HNECs. Conclusions PE transiently disrupts tight junctions in HNECs and downregulates PAR-2. The transient disruption of tight junctions by PE might occur repeatedly during chronic rhinosinusitis. PMID:24548792

Expression Pattern of the Pro-apoptotic Gene PAR-4 During the Morphogenesis of MCF-10A Human Mammary Epithelial Cells.

PubMed

de Bessa Garcia, Simone A; Pereira, Michelly C; Nagai, Maria A

2010-12-21

The histological organization of the mammary gland involves a spatial interaction of epithelial and myoepithelial cells with the specialized basement membrane (BM), composed of extra-cellular matrix (ECM) proteins, which is disrupted during the tumorigenic process. The interactions between mammary epithelial cells and ECM components play a major role in mammary gland branching morphogenesis. Critical signals for mammary epithelial cell proliferation, differentiation, and survival are provided by the ECM proteins. Three-dimensional (3D) cell culture was developed to establish a system that simulates several features of the breast epithelium in vivo; 3D cell culture of the spontaneously immortalized cell line, MCF10A, is a well-established model system to study breast epithelial cell biology and morphogenesis. Mammary epithelial cells grown in 3D form spheroids, acquire apicobasal polarization, and form lumens that resemble acini structures, processes that involve cell death. Using this system, we evaluated the expression of the pro-apoptotic gene PAWR (PKC apoptosis WT1 regulator; also named PAR-4, prostate apoptosis response-4) by immunofluorescence and quantitative real time PCR (qPCR). A time-dependent increase in PAR-4 mRNA expression was found during the process of MCF10A acinar morphogenesis. Confocal microscopy analysis also showed that PAR-4 protein was highly expressed in the MCF10A cells inside the acini structure. During the morphogenesis of MCF10A cells in 3D cell culture, the cells within the lumen showed caspase-3 activation, indicating apoptotic activity. PAR-4 was only partially co-expressed with activated caspase-3 on these cells. Our results provide evidence, for the first time, that PAR-4 is differentially expressed during the process of MCF10A acinar morphogenesis.

DLG5 connects cell polarity and Hippo signaling protein networks by linking PAR-1 with MST1/2

PubMed Central

Kwan, Julian; Sczaniecka, Anna; Heidary Arash, Emad; Nguyen, Liem; Chen, Chia-Chun; Ratkovic, Srdjana; Klezovitch, Olga; Attisano, Liliana; McNeill, Helen; Emili, Andrew; Vasioukhin, Valeri

2016-01-01

Disruption of apical–basal polarity is implicated in developmental disorders and cancer; however, the mechanisms connecting cell polarity proteins with intracellular signaling pathways are largely unknown. We determined previously that membrane-associated guanylate kinase (MAGUK) protein discs large homolog 5 (DLG5) functions in cell polarity and regulates cellular proliferation and differentiation via undefined mechanisms. We report here that DLG5 functions as an evolutionarily conserved scaffold and negative regulator of Hippo signaling, which controls organ size through the modulation of cell proliferation and differentiation. Affinity purification/mass spectrometry revealed a critical role of DLG5 in the formation of protein assemblies containing core Hippo kinases mammalian ste20 homologs 1/2 (MST1/2) and Par-1 polarity proteins microtubule affinity-regulating kinases 1/2/3 (MARK1/2/3). Consistent with this finding, Hippo signaling is markedly hyperactive in mammalian Dlg5−/− tissues and cells in vivo and ex vivo and in Drosophila upon dlg5 knockdown. Conditional deletion of Mst1/2 fully rescued the phenotypes of brain-specific Dlg5 knockout mice. Dlg5 also interacts genetically with Hippo effectors Yap1/Taz. Mechanistically, we show that DLG5 inhibits the association between MST1/2 and large tumor suppressor homologs 1/2 (LATS1/2), uses its scaffolding function to link MST1/2 with MARK3, and inhibits MST1/2 kinase activity. These data reveal a direct connection between cell polarity proteins and Hippo, which is essential for proper development of multicellular organisms. PMID:28087714

Bioenergetic metabolites regulate base excision repair dependent cell death in response to DNA damage

PubMed Central

Tang, Jiang-bo; Goellner, Eva M.; Wang, Xiao-hong; Trivedi, Ram N.; Croix, Claudette M. St; Jelezcova, Elena; Svilar, David; Brown, Ashley R.; Sobol, Robert W.

2009-01-01

Base excision repair (BER) protein expression is important for resistance to DNA damage-induced cytotoxicity. Conversely, BER imbalance (Polß deficiency or repair inhibition) enhances cytotoxicity of radiation and chemotherapeutic DNA-damaging agents. Whereas inhibition of critical steps in the BER pathway result in the accumulation of cytotoxic DNA double-strand breaks, we report that DNA damage-induced cytotoxicity due to deficiency in the BER protein Polß triggers cell death dependent on PARP activation yet independent of poly(ADP-ribose) (PAR)-mediated AIF nuclear translocation or PARG, suggesting that cytotoxicity is not from PAR or PAR-catabolite signaling. Cell death is rescued by the NAD+ metabolite NMN and is synergistic with inhibition of NAD+ biosynthesis, demonstrating that DNA damage-induced cytotoxicity mediated via BER inhibition is primarily dependent on cellular metabolite bioavailability. We offer a mechanistic justification for the elevated alkylation-induced cytotoxicity of Polß deficient cells, suggesting a linkage between DNA repair, cell survival and cellular bioenergetics. PMID:20068071

Loss of Par-1a/MARK3/C-TAK1 Kinase Leads to Reduced Adiposity, Resistance to Hepatic Steatosis, and Defective Gluconeogenesis ▿

PubMed Central

Lennerz, Jochen K.; Hurov, Jonathan B.; White, Lynn S.; Lewandowski, Katherine T.; Prior, Julie L.; Planer, G. James; Gereau, Robert W.; Piwnica-Worms, David; Schmidt, Robert E.; Piwnica-Worms, Helen

2010-01-01

Par-1 is an evolutionarily conserved protein kinase required for polarity in worms, flies, frogs, and mammals. The mammalian Par-1 family consists of four members. Knockout studies of mice implicate Par-1b/MARK2/EMK in regulating fertility, immune homeostasis, learning, and memory as well as adiposity, insulin hypersensitivity, and glucose metabolism. Here, we report phenotypes of mice null for a second family member (Par-1a/MARK3/C-TAK1) that exhibit increased energy expenditure, reduced adiposity with unaltered glucose handling, and normal insulin sensitivity. Knockout mice were protected against high-fat diet-induced obesity and displayed attenuated weight gain, complete resistance to hepatic steatosis, and improved glucose handling with decreased insulin secretion. Overnight starvation led to complete hepatic glycogen depletion, associated hypoketotic hypoglycemia, increased hepatocellular autophagy, and increased glycogen synthase levels in Par-1a−/− but not in control or Par-1b−/− mice. The intercrossing of Par-1a−/− with Par-1b−/− mice revealed that at least one of the four alleles is necessary for embryonic survival. The severity of phenotypes followed a rank order, whereby the loss of one Par-1b allele in Par-1a−/− mice conveyed milder phenotypes than the loss of one Par-1a allele in Par-1b−/− mice. Thus, although Par-1a and Par-1b can compensate for one another during embryogenesis, their individual disruption gives rise to distinct metabolic phenotypes in adult mice. PMID:20733003

Trypsin Reduces Pancreatic Ductal Bicarbonate Secretion by Inhibiting CFTR Cl- channel and Luminal Anion Exchangers

PubMed Central

Pallagi, Petra; Venglovecz, Viktória; Rakonczay, Zoltán; Borka, Katalin; Korompay, Anna; Ózsvári, Béla; Judák, Linda; Sahin-Tóth, Miklós; Geisz, Andrea; Schnúr, Andrea; Maléth, József; Takács, Tamás; Gray, Mike A.; Argent, Barry E.; Mayerle, Julia; Lerch, Markus M.; Wittmann, Tibor; Hegyi, Péter

2012-01-01

Background & Aims The effects of trypsin on pancreatic ductal epithelial cells (PDEC) vary among species and depend on localization of proteinase-activated receptor-2 (PAR-2). Bicarbonate secretion is similar in human and guinea pig PDEC; we compared its localization in these cell types and isolated guinea pig ducts to study the effects of trypsin and a PAR-2 agonist on this process. Methods PAR-2 localization was analyzed by immunohistochemistry in guinea pig and human pancreatic tissue samples (from 15 patients with chronic pancreatitis and 15 without pancreatic disease). Functions of guinea pig PDEC were studied by microperfusion of isolated ducts, measurements of intracellular pH (pHi) and Ca2+ concentration [Ca2+]i, and patch clamp analysis. The effect of pH on trypsinogen autoactivation was assessed using recombinant human cationic trypsinogen. Results PAR-2 localized to the apical membrane of human and guinea pig PDEC. Trypsin increased [Ca2+]i and pHi, and inhibited secretion of bicarbonate by the luminal anion exchanger and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. Autoactivation of human cationic trypsinogen accelerated when the pH was reduced from 8.5 to 6.0. PAR-2 expression was strongly down-regulated, at transcriptional and protein levels, in the ducts of patients with chronic pancreatitis, consistent with increased activity of intraductal trypsin. Importantly, in PAR-2 knockout mice, the effects of trypsin were PAR-2 dependent. Conclusions Trypsin reduces pancreatic ductal bicarbonate secretion via PAR-2–dependent inhibition of the apical anion exchanger and the CFTR Cl- channel. This could contribute to the development of chronic pancreatitis, decreasing luminal pH and promoting premature activation of trypsinogen in the pancreatic ducts. PMID:21893120

Dishevelled-induced phosphorylation regulates membrane localization of Par1b

DOE Office of Scientific and Technical Information (OSTI.GOV)

Terabayashi, Takeshi; Funato, Yosuke; Miki, Hiroaki, E-mail: hmiki@protein.osaka-u.ac.jp

2008-10-31

Par1b is an evolutionarily conserved kinase that plays crucial roles in cell polarity. Controlling intracellular localization of Par1b is important for its biological activity. We previously reported that Wnt stimulation or expression of Dvl promotes accumulation of Par1b in the membrane (T. Terabayashi, T.J. Itoh, H. Yamaguchi, Y. Yoshimura, Y. Funato, S. Ohno, H. Miki, Polarity-Regulating Kinase Partitioning-Defective 1/Microtubule Affinity-Regulating Kinase 2 Negatively Regulates Development of Dendrites on Hippocampal Neurons, J. Neurosci. 27 (2007) 13098-13107). However, its molecular mechanism remains unclear. Here we show the importance of Par1b phosphorylation in the regulation of membrane localization. We find that Thr-324 ismore » phosphorylated in a Dvl-dependent manner. Interestingly, the conversion of Thr-324 to Glu results in a significant accumulation of Par1b in the membrane, without any effects on the kinase activity. Moreover, the phospho-mimicking Par1b mutant does not antagonistically function against Dvl in microtubule stabilization and neurite extension, although wildtype Par1b does. These results suggest that membrane accumulation of Par1b induced by Dvl is regulated by its phosphorylation status, which is important for Par1b to regulate the microtubule dynamics.« less

Par-4/THAP1 complex and Notch3 competitively regulated pre-mRNA splicing of CCAR1 and affected inversely the survival of T-cell acute lymphoblastic leukemia cells

PubMed Central

Lu, C; Li, J-Y; Ge, Z; Zhang, L; Zhou, G-P

2013-01-01

Although the intensification of therapy for children with T-cell acute lymphoblastic leukemia (T-ALL) has substantially improved clinical outcomes, T-ALL remains an important challenge in pediatric oncology. Here, we report that the cooperative synergy between prostate apoptosis response factor-4 (Par-4) and THAP1 induces cell cycle and apoptosis regulator 1 (CCAR1) gene expression and cellular apoptosis in human T-ALL cell line Jurkat cells, CEM cells and primary cultured neoplastic T lymphocytes from children with T-ALL. Par-4 and THAP1 collaborated to activate the promoter of CCAR1 gene. Mechanistic investigations revealed that Par-4 and THAP1 formed a protein complex by the interaction of their carboxyl termini, and THAP1 bound to CCAR1 promoter though its zinc-dependent DNA-binding domain at amino terminus. Par-4/THAP1 complex and Notch3 competitively bound to CCAR1 promoter and competitively modulated alternative pre-mRNA splicing of CCAR1, which resulted in two different transcripts and played an opposite role in T-ALL cell survival. Despite Notch3 induced a shift splicing from the full-length isoform toward a shorter form of CCAR1 mRNA by splicing factor SRp40 and SRp55, Par-4/THAP1 complex strongly antagonized this inductive effect. Our finding revealed a mechanistic rationale for Par-4/THAP1-induced apoptosis in T-ALL cells that would be of benefit to develop a new therapy strategy for T-ALL. PMID:23975424

Effects of tissue factor, PAR-2 and MMP-9 expression on human breast cancer cell line MCF-7 invasion.

PubMed

Lin, Zeng-Mao; Zhao, Jian-Xin; Duan, Xue-Ning; Zhang, Lan-Bo; Ye, Jing-Ming; Xu, Ling; Liu, Yin-Hua

2014-01-01

This study aimed to explore the expression of tissue factor (TF), protease activated receptor-2 (PAR-2), and matrix metalloproteinase-9 (MMP-9) in the MCF-7 breast cancer cell line and influence on invasiveness. Stable MCF-7 cells transfected with TF cDNA and with TF ShRNA were established. TF, PAR-2, and MMP-9 protein expression was analyzed using indirect immunofluorescence and invasiveness was evaluated using a cell invasion test. Effects of an exogenous PAR-2 agonist were also examined. TF protein expression significantly differed between the TF cDNA and TF ShRNA groups. MMP-9 protein expression was significantly correlated with TF protein expression, but PAR-2 protein expression was unaffected. The PAR- 2 agonist significantly enhanced MMP-9 expression and slightly increased TF and PAR-2 expression in the TF ShRNA group, but did not significantly affect protein expression in MCF-7 cells transfected with TF cDNA. TF and MMP-9 expression was positively correlated with the invasiveness of tumor cells. TF, PAR-2, and MMP-9 affect invasiveness of MCF-7 cells. TF may increase MMP-9 expression by activating PAR-2.

PAR-CLIP data indicate that Nrd1-Nab3-dependent transcription termination regulates expression of hundreds of protein coding genes in yeast

PubMed Central

2014-01-01

Background Nrd1 and Nab3 are essential sequence-specific yeast RNA binding proteins that function as a heterodimer in the processing and degradation of diverse classes of RNAs. These proteins also regulate several mRNA coding genes; however, it remains unclear exactly what percentage of the mRNA component of the transcriptome these proteins control. To address this question, we used the pyCRAC software package developed in our laboratory to analyze CRAC and PAR-CLIP data for Nrd1-Nab3-RNA interactions. Results We generated high-resolution maps of Nrd1-Nab3-RNA interactions, from which we have uncovered hundreds of new Nrd1-Nab3 mRNA targets, representing between 20 and 30% of protein-coding transcripts. Although Nrd1 and Nab3 showed a preference for binding near 5′ ends of relatively short transcripts, they bound transcripts throughout coding sequences and 3′ UTRs. Moreover, our data for Nrd1-Nab3 binding to 3′ UTRs was consistent with a role for these proteins in the termination of transcription. Our data also support a tight integration of Nrd1-Nab3 with the nutrient response pathway. Finally, we provide experimental evidence for some of our predictions, using northern blot and RT-PCR assays. Conclusions Collectively, our data support the notion that Nrd1 and Nab3 function is tightly integrated with the nutrient response and indicate a role for these proteins in the regulation of many mRNA coding genes. Further, we provide evidence to support the hypothesis that Nrd1-Nab3 represents a failsafe termination mechanism in instances of readthrough transcription. PMID:24393166

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Planar Cell Polarity Breaks the Symmetry of PAR Protein Distribution prior to Mitosis in Drosophila Sensory Organ Precursor Cells.

PubMed

Besson, Charlotte; Bernard, Fred; Corson, Francis; Rouault, Hervé; Reynaud, Elodie; Keder, Alyona; Mazouni, Khalil; Schweisguth, François

2015-04-20

During development, cell-fate diversity can result from the unequal segregation of fate determinants at mitosis. Polarization of the mother cell is essential for asymmetric cell division (ACD). It often involves the formation of a cortical domain containing the PAR complex proteins Par3, Par6, and atypical protein kinase C (aPKC). In the fly notum, sensory organ precursor cells (SOPs) divide asymmetrically within the plane of the epithelium and along the body axis to generate two distinct cells. Fate asymmetry depends on the asymmetric localization of the PAR complex. In the absence of planar cell polarity (PCP), SOPs divide with a random planar orientation but still asymmetrically, showing that PCP is dispensable for PAR asymmetry at mitosis. To study when and how the PAR complex localizes asymmetrically, we have used a quantitative imaging approach to measure the planar polarization of the proteins Bazooka (Baz, fly Par3), Par6, and aPKC in living pupae. By using imaging of functional GFP-tagged proteins with image processing and computational modeling, we find that Baz, Par6, and aPKC become planar polarized prior to mitosis in a manner independent of the AuroraA kinase and that PCP is required for the planar polarization of Baz, Par6, and aPKC during interphase. This indicates that a "mitosis rescue" mechanism establishes asymmetry at mitosis in PCP mutants. This study therefore identifies PCP as the initial symmetry-breaking signal for the planar polarization of PAR proteins in asymmetrically dividing SOPs. Copyright © 2015 Elsevier Ltd. All rights reserved.

Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

PubMed Central

Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

2015-01-01

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

Symmetry breaking and polarization of the C. elegans zygote by the polarity protein PAR-2.

PubMed

Zonies, Seth; Motegi, Fumio; Hao, Yingsong; Seydoux, Geraldine

2010-05-01

Polarization of the C. elegans zygote is initiated by ECT-2-dependent cortical flows, which mobilize the anterior PAR proteins (PAR-3, PAR-6 and PKC-3) away from the future posterior end of the embryo marked by the sperm centrosome. Here, we demonstrate the existence of a second, parallel and redundant pathway that can polarize the zygote in the absence of ECT-2-dependent cortical flows. This second pathway depends on the polarity protein PAR-2. We show that PAR-2 localizes to the cortex nearest the sperm centrosome even in the absence of cortical flows. Once on the cortex, PAR-2 antagonizes PAR-3-dependent recruitment of myosin, creating myosin flows that transport the anterior PAR complex away from PAR-2 in a positive-feedback loop. We propose that polarity in the C. elegans zygote is initiated by redundant ECT-2- and PAR-2-dependent mechanisms that lower PAR-3 levels locally, triggering a positive-feedback loop that polarizes the entire cortex.

Beta-arrestin inhibits CAMKKbeta-dependent AMPK activation downstream of protease-activated-receptor-2.

PubMed

Wang, Ping; Jiang, Yong; Wang, Yinsheng; Shyy, John Y; DeFea, Kathryn A

2010-09-21

Proteinase-activated-receptor-2 (PAR2) is a seven transmembrane receptor that can activate two separate signaling arms: one through Gαq and Ca2+ mobilization, and a second through recruitment of β-arrestin scaffolds. In some cases downstream targets of the Gαq/Ca2+ signaling arm are directly inhibited by β-arrestins, while in other cases the two pathways are synergistic; thus β-arrestins act as molecular switches capable of modifying the signal generated by the receptor. Here we demonstrate that PAR2 can activate adenosine monophosphate-activated protein kinase (AMPK), a key regulator of cellular energy balance, through Ca2+-dependent Kinase Kinase β (CAMKKβ), while inhibiting AMPK through interaction with β-arrestins. The ultimate outcome of PAR2 activation depended on the cell type studied; in cultured fibroblasts with low endogenous β-arrestins, PAR2 activated AMPK; however, in primary fat and liver, PAR2 only activated AMPK in β-arrestin-2-/- mice. β-arrestin-2 could be co-immunoprecipitated with AMPK and CAMKKβ under baseline conditions from both cultured fibroblasts and primary fat, and its association with both proteins was increased by PAR2 activation. Addition of recombinant β-arrestin-2 to in vitro kinase assays directly inhibited phosphorylation of AMPK by CAMKKβ on Thr172. Studies have shown that decreased AMPK activity is associated with obesity and Type II Diabetes, while AMPK activity is increased with metabolically favorable conditions and cholesterol lowering drugs. These results suggest a role for β-arrestin in the inhibition of AMPK signaling, raising the possibility that β-arrestin-dependent PAR2 signaling may act as a molecular switch turning a positive signal to AMPK into an inhibitory one.

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

NASA Astrophysics Data System (ADS)

Hu, Longhua; Vecchiarelli, Anthony G.; Mizuuchi, Kiyoshi; Neuman, Keir C.; Liu, Jian

The segregation of DNA prior to cell division is essential for faithful genetic inheritance. In many bacteria, segregation of the low-copy-number plasmids involves an active partition system composed of ParA ATPase and its stimulator protein ParB. Recent experiments suggest that ParA/ParB system motility is driven by a diffusion-ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. We develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB bound cargo. Paradoxically, the resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work sheds light on a new emergent phenomenon in which non-motor proteins work collectively via mechanochemical coupling to propel cargos -- an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

Plasmid partition system of the P1par family from the pWR100 virulence plasmid of Shigella flexneri.

PubMed

Sergueev, Kirill; Dabrazhynetskaya, Alena; Austin, Stuart

2005-05-01

P1par family members promote the active segregation of a variety of plasmids and plasmid prophages in gram-negative bacteria. Each has genes for ParA and ParB proteins, followed by a parS partition site. The large virulence plasmid pWR100 of Shigella flexneri contains a new P1par family member: pWR100par. Although typical parA and parB genes are present, the putative pWR100parS site is atypical in sequence and organization. However, pWR100parS promoted accurate plasmid partition in Escherichia coli when the pWR100 Par proteins were supplied. Unique BoxB hexamer motifs within parS define species specificities among previously described family members. Although substantially different from P1parS from the P1 plasmid prophage of E. coli, pWR100parS has the same BoxB sequence. As predicted, the species specificity of the two types proved identical. They also shared partition-mediated incompatibility, consistent with the proposed mechanistic link between incompatibility and species specificity. Among several informative sequence differences between pWR100parS and P1parS is the presence of a 21-bp insert at the center of the pWR100parS site. Deletion of this insert left much of the parS activity intact. Tolerance of central inserts with integral numbers of helical DNA turns reflects the critical topology of these sites, which are bent by binding the host IHF protein.

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif

PubMed Central

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-01-01

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon–helix–helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF ≈30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal ΔParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization. PMID:17261809

The tail of the ParG DNA segregation protein remodels ParF polymers and enhances ATP hydrolysis via an arginine finger-like motif.

PubMed

Barillà, Daniela; Carmelo, Emma; Hayes, Finbarr

2007-02-06

The ParF protein of plasmid TP228 belongs to the ubiquitous superfamily of ParA ATPases that drive DNA segregation in bacteria. ATP-bound ParF polymerizes into multistranded filaments. The partner protein ParG is dimeric, consisting of C-termini that interweave into a ribbon-helix-helix domain contacting the centromeric DNA and unstructured N-termini. ParG stimulates ATP hydrolysis by ParF approximately 30-fold. Here, we establish that the mobile tails of ParG are crucial for this enhancement and that arginine R19 within the tail is absolutely required for activation of ParF nucleotide hydrolysis. R19 is part of an arginine finger-like loop in ParG that is predicted to intercalate into the ParF nucleotide-binding pocket thereby promoting ATP hydrolysis. Significantly, mutations of R19 abrogated DNA segregation in vivo, proving that intracellular stimulation of ATP hydrolysis by ParG is a key regulatory process for partitioning. Furthermore, ParG bundles ParF-ATP filaments as well as promoting nucleotide-independent polymerization. The N-terminal flexible tail is required for both activities, because N-terminal DeltaParG polypeptides are defective in both functions. Strikingly, the critical arginine finger-like residue R19 is dispensable for ParG-mediated remodeling of ParF polymers, revealing that the ParG N-terminal tail possesses two separable activities in the interplay with ParF: a catalytic function during ATP hydrolysis and a mechanical role in modulation of polymerization. We speculate that activation of nucleotide hydrolysis via an arginine finger loop may be a conserved, regulatory mechanism of ParA family members and their partner proteins, including ParA-ParB and Soj-Spo0J that mediate DNA segregation and MinD-MinE that determine septum localization.

ParA and ParB coordinate chromosome segregation with cell elongation and division during Streptomyces sporulation

PubMed Central

Donczew, Magdalena; Mackiewicz, Paweł; Wróbel, Agnieszka; Flärdh, Klas; Zakrzewska-Czerwińska, Jolanta

2016-01-01

In unicellular bacteria, the ParA and ParB proteins segregate chromosomes and coordinate this process with cell division and chromosome replication. During sporulation of mycelial Streptomyces, ParA and ParB uniformly distribute multiple chromosomes along the filamentous sporogenic hyphal compartment, which then differentiates into a chain of unigenomic spores. However, chromosome segregation must be coordinated with cell elongation and multiple divisions. Here, we addressed the question of whether ParA and ParB are involved in the synchronization of cell-cycle processes during sporulation in Streptomyces. To answer this question, we used time-lapse microscopy, which allows the monitoring of growth and division of single sporogenic hyphae. We showed that sporogenic hyphae stop extending at the time of ParA accumulation and Z-ring formation. We demonstrated that both ParA and ParB affect the rate of hyphal extension. Additionally, we showed that ParA promotes the formation of massive nucleoprotein complexes by ParB. We also showed that FtsZ ring assembly is affected by the ParB protein and/or unsegregated DNA. Our results indicate the existence of a checkpoint between the extension and septation of sporogenic hyphae that involves the ParA and ParB proteins. PMID:27248800

ARTD1/PARP1 negatively regulates glycolysis by inhibiting hexokinase 1 independent of NAD+ depletion

PubMed Central

Fouquerel, Elise; Goellner, Eva M.; Yu, Zhongxun; Gagné, Jean-Philippe; de Moura, Michelle Barbi; Feinstein, Tim; Wheeler, David; Redpath, Philip; Li, Jianfeng; Romero, Guillermo; Migaud, Marie; Van Houten, Bennett; Poirier, Guy G.; Sobol, Robert W.

2014-01-01

Summary ARTD1 (PARP1) is a key enzyme involved in DNA repair by synthesizing poly(ADP-ribose) (PAR) in response to strand breaks and plays an important role in cell death following excessive DNA damage. ARTD1-induced cell death is associated with NAD+ depletion and ATP loss, however the molecular mechanism of ARTD1-mediated energy collapse remains elusive. Using real-time metabolic measurements, we directly compared the effects of ARTD1 activation and direct NAD+ depletion. We found that ARTD1-mediated PAR synthesis, but not direct NAD+ depletion, resulted in a block to glycolysis and ATP loss. We then established a proteomics based PAR-interactome after DNA damage and identified hexokinase 1 (HK1) as a PAR binding protein. HK1 activity is suppressed following nuclear ARTD1 activation and binding by PAR. These findings help explain how prolonged activation of ARTD1 triggers energy collapse and cell death, revealing new insight on the importance of nucleus to mitochondria communication via ARTD1 activation. PMID:25220464

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system.

PubMed

Hu, Longhua; Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Neuman, Keir C; Liu, Jian

2015-12-22

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA-nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos-an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria.

Par-aPKC-dependent and -independent mechanisms cooperatively control cell polarity, Hippo signaling, and cell positioning in 16-cell stage mouse embryos.

PubMed

Hirate, Yoshikazu; Hirahara, Shino; Inoue, Ken-Ichi; Kiyonari, Hiroshi; Niwa, Hiroshi; Sasaki, Hiroshi

2015-10-01

In preimplantation mouse embryos, the Hippo signaling pathway plays a central role in regulating the fates of the trophectoderm (TE) and the inner cell mass (ICM). In early blastocysts with more than 32 cells, the Par-aPKC system controls polarization of the outer cells along the apicobasal axis, and cell polarity suppresses Hippo signaling. Inactivation of Hippo signaling promotes nuclear accumulation of a coactivator protein, Yap, leading to induction of TE-specific genes. However, whether similar mechanisms operate at earlier stages is not known. Here, we show that slightly different mechanisms operate in 16-cell stage embryos. Similar to 32-cell stage embryos, disruption of the Par-aPKC system activated Hippo signaling and suppressed nuclear Yap and Cdx2 expression in the outer cells. However, unlike 32-cell stage embryos, 16-cell stage embryos with a disrupted Par-aPKC system maintained apical localization of phosphorylated Ezrin/Radixin/Moesin (p-ERM), and the effects on Yap and Cdx2 were weak. Furthermore, normal 16-cell stage embryos often contained apolar cells in the outer position. In these cells, the Hippo pathway was strongly activated and Yap was excluded from the nuclei, thus resembling inner cells. Dissociated blastomeres of 8-cell stage embryos form polar-apolar couplets, which exhibit different levels of nuclear Yap, and the polar cell engulfed the apolar cell. These results suggest that cell polarization at the 16-cell stage is regulated by both Par-aPKC-dependent and -independent mechanisms. Asymmetric cell division is involved in cell polarity control, and cell polarity regulates cell positioning and most likely controls Hippo signaling. © The Authors Development, Growth & Differentiation published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Society of Developmental Biologists.

Proteolytic activation of the protease-activated receptor (PAR)-2 by the glycosylphosphatidylinositol-anchored serine protease testisin.

PubMed

Driesbaugh, Kathryn H; Buzza, Marguerite S; Martin, Erik W; Conway, Gregory D; Kao, Joseph P Y; Antalis, Toni M

2015-02-06

Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca(2+) mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Effects of TGF-β1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

PubMed

Chang, Mei-Chi; Chang, Hsiao-Hua; Lin, Po-Shuan; Huang, Yu-An; Chan, Chiu-Po; Tsai, Yi-Ling; Lee, Shen-Yang; Jeng, Po-Yuan; Kuo, Han-Yueh; Yeung, Sin-Yuet; Jeng, Jiiang-Huei

2018-04-01

Transforming growth factor-β1 (TGF-β1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-β1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-β1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-β1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-β1 slightly inhibited cell growth that was reversed by SB431542. TGF-β1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-β1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-β1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-β1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-β1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-β1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-β1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

A family of ParA-like ATPases promotes cell pole maturation by facilitating polar localization of chemotaxis proteins

PubMed Central

Ringgaard, Simon; Schirner, Kathrin; Davis, Brigid M.; Waldor, Matthew K.

2011-01-01

Stochastic processes are thought to mediate localization of membrane-associated chemotaxis signaling clusters in peritrichous bacteria. Here, we identified a new family of ParA-like ATPases (designated ParC [for partitioning chemotaxis]) encoded within chemotaxis operons of many polar-flagellated γ-proteobacteria that actively promote polar localization of chemotaxis proteins. In Vibrio cholerae, a single ParC focus is found at the flagellated old pole in newborn cells, and later bipolar ParC foci develop as the cell matures. The cell cycle-dependent redistribution of ParC occurs by its release from the old pole and subsequent relocalization at the new pole, consistent with a “diffusion and capture” model for ParC dynamics. Chemotaxis proteins encoded in the same cluster as ParC have a similar unipolar-to-bipolar transition; however, they reach the new pole after the arrival of ParC. Cells lacking ParC exhibit aberrantly localized foci of chemotaxis proteins, reduced chemotaxis, and altered motility, which likely accounts for their enhanced colonization of the proximal small intestine in an animal model of cholera. Collectively, our findings indicate that ParC promotes the efficiency of chemotactic signaling processes. In particular, ParC-facilitated development of a functional chemotaxis apparatus at the new pole readies this site for its development into a functional old pole after cell division. PMID:21764856

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

PubMed

van der Merwe, Jacques Q; Moreau, France; MacNaughton, Wallace K

2009-06-01

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

Proteinase-Activated Receptor-2 Sensitivity of Amplified TRPA1 Activity in Skeletal Muscle Afferent Nerves and Exercise Pressor Reflex in Rats with Femoral Artery Occlusion

PubMed Central

Xing, Jihong; Li, Jianhua

2017-01-01

Background/Aims Limb ischemia occurs in peripheral artery disease (PAD). Sympathetic nerve activity (SNA) that regulates blood flow directed to the ischemic limb is exaggerated during exercise in this disease, and transient receptor potential channel A1 (TRPA1) in thin-fiber muscle afferents contributes to the amplified sympathetic response. The purpose of the present study was to determine the role of proteinase-activated receptor-2 (PAR2) in regulating abnormal TRPA1 function and the TRPA1-mediated sympathetic component of the exercise pressor reflex. Methods A rat model of femoral artery ligation was employed to study PAD. Dorsal root ganglion (DRG) tissues were obtained to examine the protein levels of PAR2 using western blot analysis. Current responses induced by activation of TRPA1 in skeletal muscle DRG neurons were characterized using whole-cell patch clamp methods. The blood pressure response to static exercise (i.e., muscle contraction) and stimulation of TRPA1 was also examined after a blockade of PAR2. Results The expression of PAR2 was amplified in DRG neurons of the occluded limb, and PAR2 activation with SL-NH2 (a PAR2 agonist) increased the amplitude of TRPA1 currents to a greater degree in DRG neurons of the occluded limb. Moreover, FSLLRY-NH2 (a PAR antagonist) injected into the arterial blood supply of the hindlimb muscles significantly attenuated the pressor response to muscle contraction and TRPA1 stimulation in rats with occluded limbs. Conclusions The PAR2 signal in muscle sensory nerves contributes to the amplified exercise pressor reflex via TRPA1 mechanisms in rats with femoral artery ligation. These findings provide a pathophysiological basis for autonomic responses during exercise activity in PAD, which may potentially aid in the development of therapeutic approaches for improvement of blood flow in this disease. PMID:29131007

Directed and persistent movement arises from mechanochemistry of the ParA/ParB system

PubMed Central

Hu, Longhua; Vecchiarelli, Anthony G.; Mizuuchi, Kiyoshi; Neuman, Keir C.; Liu, Jian

2015-01-01

The segregation of DNA before cell division is essential for faithful genetic inheritance. In many bacteria, segregation of low-copy number plasmids involves an active partition system composed of a nonspecific DNA-binding ATPase, ParA, and its stimulator protein ParB. The ParA/ParB system drives directed and persistent movement of DNA cargo both in vivo and in vitro. Filament-based models akin to actin/microtubule-driven motility were proposed for plasmid segregation mediated by ParA. Recent experiments challenge this view and suggest that ParA/ParB system motility is driven by a diffusion ratchet mechanism in which ParB-coated plasmid both creates and follows a ParA gradient on the nucleoid surface. However, the detailed mechanism of ParA/ParB-mediated directed and persistent movement remains unknown. Here, we develop a theoretical model describing ParA/ParB-mediated motility. We show that the ParA/ParB system can work as a Brownian ratchet, which effectively couples the ATPase-dependent cycling of ParA–nucleoid affinity to the motion of the ParB-bound cargo. Paradoxically, this resulting processive motion relies on quenching diffusive plasmid motion through a large number of transient ParA/ParB-mediated tethers to the nucleoid surface. Our work thus sheds light on an emergent phenomenon in which nonmotor proteins work collectively via mechanochemical coupling to propel cargos—an ingenious solution shaped by evolution to cope with the lack of processive motor proteins in bacteria. PMID:26647183

Activated protein C promotes breast cancer cell migration through interactions with EPCR and PAR-1

PubMed Central

Beaulieu, Lea M.; Church, Frank C.

2014-01-01

Activated protein C (APC) is a serine protease that regulates thrombin (IIa) production through inactivation of blood coagulation factors Va and VIIIa. APC also has non-hemostatic functions related to inflammation, proliferation, and apoptosis through various mechanisms. Using two breast cancer cell lines, MDA-MB-231 and MDA-MB-435, we investigated the role of APC in cell chemotaxis and invasion. Treatment of cells with increasing APC concentrations (1–50 μg/ml) increased invasion and chemotaxis in a concentration-dependent manner. Only the active form of APC increased invasion and chemotaxis of the MDA-MB-231 cells when compared to 3 inactive APC derivatives. Using a modified “checkerboard” analysis, APC was shown to only affect migration when plated with the cells; therefore, APC is not a chemoattractant. Blocking antibodies to endothelial protein C receptor (EPCR) and protease-activated receptor-1 (PAR-1) attenuated the effects of APC on chemotaxis in the MDA-MB-231 cells. Finally, treatment of the MDA-MB-231 cells with the proliferation inhibitor, Na butyrate, showed that APC did not increase migration by increasing cell number. Therefore, APC increases invasion and chemotaxis of cells by binding to the cell surface and activating specific signaling pathways through EPCR and PAR-1. PMID:17254565

Mammalian aPKC/Par polarity complex mediated regulation of epithelial division orientation and cell fate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vorhagen, Susanne; Niessen, Carien M., E-mail: carien.niessen@uni-koeln.de

2014-11-01

Oriented cell division is a key regulator of tissue architecture and crucial for morphogenesis and homeostasis. Balanced regulation of proliferation and differentiation is an essential property of tissues not only to drive morphogenesis but also to maintain and restore homeostasis. In many tissues orientation of cell division is coupled to the regulation of differentiation producing daughters with similar (symmetric cell division, SCD) or differential fate (asymmetric cell division, ACD). This allows the organism to generate cell lineage diversity from a small pool of stem and progenitor cells. Division orientation and/or the ratio of ACD/SCD need to be tightly controlled. Lossmore » of orientation or an altered ratio can promote overgrowth, alter tissue architecture and induce aberrant differentiation, and have been linked to morphogenetic diseases, cancer and aging. A key requirement for oriented division is the presence of a polarity axis, which can be established through cell intrinsic and/or extrinsic signals. Polarity proteins translate such internal and external cues to drive polarization. In this review we will focus on the role of the polarity complex aPKC/Par3/Par6 in the regulation of division orientation and cell fate in different mammalian epithelia. We will compare the conserved function of this complex in mitotic spindle orientation and distribution of cell fate determinants and highlight common and differential mechanisms in which this complex is used by tissues to adapt division orientation and cell fate to the specific properties of the epithelium.« less

The multi-PDZ domain protein-1 (MUPP-1) expression regulates cellular levels of the PALS-1/PATJ polarity complex.

PubMed

Assémat, Emeline; Crost, Emmanuelle; Ponserre, Marion; Wijnholds, Jan; Le Bivic, Andre; Massey-Harroche, Dominique

2013-10-15

MUPP-1 (multi-PDZ domain protein-1) and PATJ (PALS-1-associated tight junction protein) proteins are closely related scaffold proteins and bind to many common interactors including PALS-1 (protein associated with Lin seven) a member of the Crumbs complex. Our goal is to understand how MUPP-1 and PATJ and their interaction with PALS-1 are regulated in the same cells. We have shown that in MCF10A cells there are at least two different and co-existing complexes, PALS-1/MUPP-1 and PALS-1/PATJ. Surprisingly, MUPP-1 levels inversely correlated with PATJ protein levels by acting on the stabilization of the PATJ/PALS-1 complex. Upon MUPP-1 depletion, the increased amounts of PATJ are in part localized at the migrating front of MCF10A cells and are able to recruit more PAR3 (partition defective 3). All together these data indicate that a precise balance between MUPP-1 and PATJ is achieved in epithelial cells by regulating their association with PALS-1. © 2013 Elsevier Inc. All rights reserved.

Loss of Axonal Mitochondria Promotes Tau-Mediated Neurodegeneration and Alzheimer's Disease–Related Tau Phosphorylation Via PAR-1

PubMed Central

Iijima-Ando, Kanae; Sekiya, Michiko; Suzuki, Emiko; Lu, Bingwei; Iijima, Koichi M.

2012-01-01

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi–mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD–related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD. PMID:22952452

Splicing alterations in human renal allografts: detection of a new splice variant of protein kinase Par1/Emk1 whose expression is associated with an increase of inflammation in protocol biopsies of transplanted patients.

PubMed

Hueso, Miguel; Beltran, Violeta; Moreso, Francesc; Ciriero, Eva; Fulladosa, Xavier; Grinyó, Josep Maria; Serón, Daniel; Navarro, Estanis

2004-05-24

Protein kinase Emk1/Par1 (GenBank accession no. X97630) has been identified as a regulator of the immune system homeostasis. Since immunological factors are critical for the development of chronic allograft nephropathy (CAN), we reasoned that expression of Par1/Emk1 could be altered in kidney allografts undergoing CAN. In this paper, we have analysed the association among renal allograft lesions and expression of Par1/Emk1, studied by RT-PCR on total RNA from 51 protocol biopsies of transplanted kidneys, five normal kidneys, and five dysfunctional allografts. The most significant result obtained has been the detection of alterations in the normal pattern of alternative splicing of the Par1/Emk1 transcript, alterations that included loss of expression of constitutively expressed isoforms, and the inclusion of a cryptic exon to generate a new Emk1 isoform (Emk1C). Expression of Emk1C was associated with an increase in the extension of the interstitial infiltrate (0.88+/-0.33 in Emk1C([+]) vs. 0.41+/-0.50 in Emk1C([-]); P<0.011), and with a trend to display higher interstitial scarring (0.66+/-0.70 vs. 0.29+/-0.52; P=0.09) in protocol biopsies when evaluated according to the Banff schema. Moreover, a higher mean arterial pressure (MAP) was also observed (110+/-11 vs. 99+/-11 mm Hg; P=0.012). From these results we propose that Par1/Emk1 could have a role in the development of CAN in kidney allografts.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

PubMed

Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

2014-05-29

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Cytoprotective signaling by activated protein C requires protease-activated receptor-3 in podocytes

PubMed Central

Madhusudhan, Thati; Wang, Hongjie; Straub, Beate K.; Gröne, Elisabeth; Zhou, Qianxing; Shahzad, Khurrum; Müller-Krebs, Sandra; Schwenger, Vedat; Gerlitz, Bruce; Grinnell, Brian W.; Griffin, John H.; Reiser, Jochen; Gröne, Hermann-Josef; Esmon, Charles T.; Nawroth, Peter P.

2012-01-01

The cytoprotective effects of activated protein C (aPC) are well established. In contrast, the receptors and signaling mechanism through which aPC conveys cytoprotection in various cell types remain incompletely defined. Thus, within the renal glomeruli, aPC preserves endothelial cells via a protease-activated receptor-1 (PAR-1) and endothelial protein C receptor-dependent mechanism. Conversely, the signaling mechanism through which aPC protects podocytes remains unknown. While exploring the latter, we identified a novel aPC/PAR-dependent cytoprotective signaling mechanism. In podocytes, aPC inhibits apoptosis through proteolytic activation of PAR-3 independent of endothelial protein C receptor. PAR-3 is not signaling competent itself as it requires aPCinduced heterodimerization with PAR-2 (human podocytes) or PAR-1 (mouse podocytes). This cytoprotective signaling mechanism depends on caveolin-1 dephosphorylation. In vivo aPC protects against lipopolysaccharide-induced podocyte injury and proteinuria. Genetic deletion of PAR-3 impairs the nephroprotective effect of aPC, demonstrating the crucial role of PAR-3 for aPC-dependent podocyte protection. This novel, aPC-mediated interaction of PARs demonstrates the plasticity and cell-specificity of cytoprotective aPC signaling. The evidence of specific, dynamic signaling complexes underlying aPC-mediated cytoprotection may allow the design of cell type specific targeted therapies. PMID:22117049

Cleavage of the urokinase receptor (uPAR) on oral cancer cells: regulation by transforming growth factor - β1 (TGF-β1) and potential effects on migration and invasion.

PubMed

Magnussen, Synnove Norvoll; Hadler-Olsen, Elin; Costea, Daniela Elena; Berg, Eli; Jacobsen, Cristiane Cavalcanti; Mortensen, Bente; Salo, Tuula; Martinez-Zubiaurre, Inigo; Winberg, Jan-Olof; Uhlin-Hansen, Lars; Svineng, Gunbjorg

2017-05-19

Urokinase plasminogen activator (uPA) receptor (uPAR) is up-regulated at the invasive tumour front of human oral squamous cell carcinoma (OSCC), indicating a role for uPAR in tumour progression. We previously observed elevated expression of uPAR at the tumour-stroma interface in a mouse model for OSCC, which was associated with increased proteolytic activity. The tumour microenvironment regulated uPAR expression, as well as its glycosylation and cleavage. Both full-length- and cleaved uPAR (uPAR (II-III)) are involved in highly regulated processes such as cell signalling, proliferation, migration, stem cell mobilization and invasion. The aim of the current study was to analyse tumour associated factors and their effect on uPAR cleavage, and the potential implications for cell proliferation, migration and invasion. Mouse uPAR was stably overexpressed in the mouse OSCC cell line AT84. The ratio of full-length versus cleaved uPAR as analysed by Western blotting and its regulation was assessed by addition of different protease inhibitors and transforming growth factor - β1 (TGF-β1). The role of uPAR cleavage in cell proliferation and migration was analysed using real-time cell analysis and invasion was assessed using the myoma invasion model. We found that when uPAR was overexpressed a proportion of the receptor was cleaved, thus the cells presented both full-length uPAR and uPAR (II-III). Cleavage was mainly performed by serine proteases and urokinase plasminogen activator (uPA) in particular. When the OSCC cells were stimulated with TGF-β1, the production of the uPA inhibitor PAI-1 was increased, resulting in a reduction of uPAR cleavage. By inhibiting cleavage of uPAR, cell migration was reduced, and by inhibiting uPA activity, invasion was reduced. We could also show that medium containing soluble uPAR (suPAR), and cleaved soluble uPAR (suPAR (II-III)), induced migration in OSCC cells with low endogenous levels of uPAR. These results show that soluble factors in the tumour microenvironment, such as TGF-β1, PAI-1 and uPA, can influence the ratio of full length and uPAR (II-III) and thereby potentially effect cell migration and invasion. Resolving how uPAR cleavage is controlled is therefore vital for understanding how OSCC progresses and potentially provides new targets for therapy.

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

PubMed

Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Small Molecules Engage Hot Spots through Cooperative Binding To Inhibit a Tight Protein-Protein Interaction.

PubMed

Liu, Degang; Xu, David; Liu, Min; Knabe, William Eric; Yuan, Cai; Zhou, Donghui; Huang, Mingdong; Meroueh, Samy O

2017-03-28

Protein-protein interactions drive every aspect of cell signaling, yet only a few small-molecule inhibitors of these interactions exist. Despite our ability to identify critical residues known as hot spots, little is known about how to effectively engage them to disrupt protein-protein interactions. Here, we take advantage of the ease of preparation and stability of pyrrolinone 1, a small-molecule inhibitor of the tight interaction between the urokinase receptor (uPAR) and its binding partner, the urokinase-type plasminogen activator uPA, to synthesize more than 40 derivatives and explore their effect on the protein-protein interaction. We report the crystal structure of uPAR bound to previously discovered pyrazole 3 and to pyrrolinone 12. While both 3 and 12 bind to uPAR and compete with a fluorescently labeled peptide probe, only 12 and its derivatives inhibit the full uPAR·uPA interaction. Compounds 3 and 12 mimic and engage different hot-spot residues on uPA and uPAR, respectively. Interestingly, 12 is involved in a π-cation interaction with Arg-53, which is not considered a hot spot. Explicit-solvent molecular dynamics simulations reveal that 3 and 12 exhibit dramatically different correlations of motion with residues on uPAR. Free energy calculations for the wild-type and mutant uPAR bound to uPA or 12 show that Arg-53 interacts with uPA or with 12 in a highly cooperative manner, thereby altering the contributions of hot spots to uPAR binding. The direct engagement of peripheral residues not considered hot spots through π-cation or salt-bridge interactions could provide new opportunities for enhanced small-molecule engagement of hot spots to disrupt challenging protein-protein interactions.

Immunoregulatory Protein Profiles of Necrotizing Enterocolitis versus Spontaneous Intestinal Perforation in Preterm Infants

PubMed Central

Leung, Fiona Wan Lun; Lam, Hugh Simon; Tam, Yuk Him; To, Ka Fai; Cheung, Hon Ming; Leung, Kam Tong; Poon, Terence Chuen Wai; Lee, Kim Hung; Li, Karen; Fok, Tai Fai

2012-01-01

Necrotizing enterocolitis (NEC) and spontaneous intestinal perforation (SIP) are the most common acute surgical emergencies associated with high morbidity and mortality in preterm infants. We aimed to compare the profiles of immunoregulatory proteins and identify novel mediators in plasma of NEC and SIP infants. We also investigated the expression of target genes in resected intestinal tissues and an enterocyte cell line. Using Cytokine Antibody Array assay, we reported the first comparative profiles of immunoregulatory proteins in plasma of NEC and SIP infants, and showed that dysregulated proteins belonged to functionally diversified categories, including pro- and anti-inflammation, angiogenesis, cell growth, wound healing, anti-apoptosis, cell adhesion and extracellular matrix reorganization. Validation by ELISA confirmed significantly higher concentrations of interleukin (IL)-6, angiopoietin (Ang)-2, soluble type II interleukin-1 receptor (sIL-1RII), and soluble urokinase-type plasminogen activator receptor (suPAR) in NEC infants compared with gestational age-matched control, and a lower level of an epidermal growth factor receptor, secreted form of receptor tyrosine-protein kinase ErbB3 (sErbB3), compared with SIP infants. mRNA expressions of IL1-RII and uPAR were up-regulated in resected bowel tissues from NEC infants, indicating that immunoregulation also occurred at the cellular level. In FHs-74 Int cells, Ang-2, IL1-RII and uPAR mRNA expressions were significantly induced by the combined treatment with lipopolysaccharide (LPS) and platelet activating factor (PAF). Our study provided plasmatic signatures of immunoregulatory proteins in NEC and SIP infants, and demonstrated involvement of multiple functional pathways. The magnitude of changes in these proteins was significantly more extensive in NEC infants, reflecting the different nature of injury and/or severity of inflammation. We speculate that dysregulation of IL-6, Ang-2, IL-1RII and uPAR occurred at both systemic and cellular levels, and probably mediated via LPS and endogeneous PAF signals. Such exaggerated immunologic responses may account for the high morbidity and mortality in NEC compared with SIP patients. PMID:22606320

Accessory factors promote AlfA-dependent plasmid segregation by regulating filament nucleation, disassembly, and bundling

PubMed Central

Polka, Jessica K.; Kollman, Justin M.; Mullins, R. Dyche

2014-01-01

In bacteria, some plasmids are partitioned to daughter cells by assembly of actin-like proteins (ALPs). The best understood ALP, ParM, has a core set of biochemical properties that contributes to its function, including dynamic instability, spontaneous nucleation, and bidirectional elongation. AlfA, an ALP that pushes plasmids apart in Bacillus, relies on a different set of underlying properties to segregate DNA. AlfA elongates unidirectionally and is not dynamically unstable; its assembly and disassembly are regulated by a cofactor, AlfB. Free AlfB breaks up AlfA bundles and promotes filament turnover. However, when AlfB is bound to the centromeric DNA sequence, parN, it forms a segrosome complex that nucleates and stabilizes AlfA filaments. When reconstituted in vitro, this system creates polarized, motile comet tails that associate by antiparallel filament bundling to form bipolar, DNA-segregating spindles. PMID:24481252

Genomic structure of the human D-site binding protein (DBP) gene

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shutler, G.; Glassco, T.; Kang, Xiaolin

1996-06-15

The human gene for the D-Site Binding Protein (DBP) has been sequenced and characterized. This gene is a member of the b/ZIP family of transcription factors and is one of three genes forming the PAR sub-family. DBP has been implicated in the diurnal regulation of a variety of liver-specific genes. Examination of the genomic structure of DBP reveals that the gene is divided into four exons and is contained within a relatively compact region of approximately 6 kb. These exons appear to correspond to functional divisions the DBP protein. Exon 1 contains a long 5{prime} UTR, and conservation between themore » rat and the human genes of the presence of small open reading frames within this region suggests that is may play a role in translational control. Exon 2 contains a limited region of similarity to the other PAR domain genes, which may be part of a potential activation domain. Exon 3 contains the PAR domain and differs by only 1 of 71 amino acids between rat and human. Exon 4, containing both the basic and the leucine zipper domains, is likewise highly conserved. The overall degree of homology between the rat and the human cDNA sequences is 82% for the nucleic acid sequence and 92% for the protein sequence. comparison of the rat and human proximal promoters reveals extensive sequence conservation, with two previously characterized DNA binding sites being conserved at the functional and sequence levels. 31 refs., 4 figs.« less

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells

PubMed Central

Alfano, Daniela; Gorrasi, Anna; Li Santi, Anna; Ricci, Patrizia; Montuori, Nunzia; Selleri, Carmine; Ragno, Pia

2015-01-01

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo. PMID:26082201

MtPAR MYB transcription factor acts as an on switch for proanthocyanidin biosynthesis in Medicago truncatula

PubMed Central

Verdier, Jerome; Zhao, Jian; Torres-Jerez, Ivone; Ge, Shujun; Liu, Chenggang; He, Xianzhi; Mysore, Kirankumar S.; Dixon, Richard A.; Udvardi, Michael K.

2012-01-01

MtPAR (Medicago truncatula proanthocyanidin regulator) is an MYB family transcription factor that functions as a key regulator of proanthocyanidin (PA) biosynthesis in the model legume Medicago truncatula. MtPAR expression is confined to the seed coat, the site of PA accumulation. Loss-of-function par mutants contained substantially less PA in the seed coat than the wild type, whereas levels of anthocyanin and other specialized metabolites were normal in the mutants. In contrast, massive accumulation of PAs occurred when MtPAR was expressed ectopically in transformed hairy roots of Medicago. Transcriptome analysis of par mutants and MtPAR-expressing hairy roots, coupled with yeast one-hybrid analysis, revealed that MtPAR positively regulates genes encoding enzymes of the flavonoid–PA pathway via a probable activation of WD40-1. Expression of MtPAR in the forage legume alfalfa (Medicago sativa) resulted in detectable levels of PA in shoots, highlighting the potential of this gene for biotechnological strategies to increase PAs in forage legumes for reduction of pasture bloat in ruminant animals. PMID:22307644

Characterization of protein dynamics in asymmetric cell division by scanning fluorescence correlation spectroscopy.

PubMed

Petrásek, Zdenek; Hoege, Carsten; Mashaghi, Alireza; Ohrt, Thomas; Hyman, Anthony A; Schwille, Petra

2008-12-01

The development and differentiation of complex organisms from the single fertilized egg is regulated by a variety of processes that all rely on the distribution and interaction of proteins. Despite the tight regulation of these processes with respect to temporal and spatial protein localization, exact quantification of the underlying parameters, such as concentrations and distribution coefficients, has so far been problematic. Recent experiments suggest that fluorescence correlation spectroscopy on a single molecule level in living cells has great promise in revealing these parameters with high precision. The optically challenging situation in multicellular systems such as embryos can be ameliorated by two-photon excitation, where scattering background and cumulative photobleaching is limited. A more severe problem is posed by the large range of molecular mobilities observed at the same time, as standard FCS relies strongly on the presence of mobility-induced fluctuations. In this study, we overcame the limitations of standard FCS. We analyzed in vivo polarity protein PAR-2 from eggs of Caenorhabditis elegans by beam-scanning FCS in the cytosol and on the cortex of C. elegans before asymmetric cell division. The surprising result is that the distribution of PAR-2 is largely uncoupled from the movement of cytoskeletal components of the cortex. These results call for a more systematic future investigation of the different cortical elements, and show that the FCS technique can contribute to answering these questions, by providing a complementary approach that can reveal insights not obtainable by other techniques.

Huntingtin Is Required for Epithelial Polarity through RAB11A-Mediated Apical Trafficking of PAR3-aPKC

PubMed Central

Elias, Salah; McGuire, John Russel; Yu, Hua; Humbert, Sandrine

2015-01-01

The establishment of apical-basolateral polarity is important for both normal development and disease, for example, during tumorigenesis and metastasis. During this process, polarity complexes are targeted to the apical surface by a RAB11A-dependent mechanism. Huntingtin (HTT), the protein that is mutated in Huntington disease, acts as a scaffold for molecular motors and promotes microtubule-based dynamics. Here, we investigated the role of HTT in apical polarity during the morphogenesis of the mouse mammary epithelium. We found that the depletion of HTT from luminal cells in vivo alters mouse ductal morphogenesis and lumen formation. HTT is required for the apical localization of PAR3-aPKC during epithelial morphogenesis in virgin, pregnant, and lactating mice. We show that HTT forms a complex with PAR3, aPKC, and RAB11A and ensures the microtubule-dependent apical vesicular translocation of PAR3-aPKC through RAB11A. We thus propose that HTT regulates polarized vesicular transport, lumen formation and mammary epithelial morphogenesis. PMID:25942483

The Cell Cycle Regulator CCDC6 Is a Key Target of RNA-Binding Protein EWS

PubMed Central

Duggimpudi, Sujitha; Larsson, Erik; Nabhani, Schafiq; Borkhardt, Arndt; Hoell, Jessica I

2015-01-01

Genetic translocation of EWSR1 to ETS transcription factor coding region is considered as primary cause for Ewing sarcoma. Previous studies focused on the biology of chimeric transcription factors formed due to this translocation. However, the physiological consequences of heterozygous EWSR1 loss in these tumors have largely remained elusive. Previously, we have identified various mRNAs bound to EWS using PAR-CLIP. In this study, we demonstrate CCDC6, a known cell cycle regulator protein, as a novel target regulated by EWS. siRNA mediated down regulation of EWS caused an elevated apoptosis in cells in a CCDC6-dependant manner. This effect was rescued upon re-expression of CCDC6. This study provides evidence for a novel functional link through which wild-type EWS operates in a target-dependant manner in Ewing sarcoma. PMID:25751255

PAR-2 agonists induce contraction of murine small intestine through neurokinin receptors.

PubMed

Zhao, Aiping; Shea-Donohue, Terez

2003-10-01

Protease-activated receptor-2 (PAR-2) is a G protein-coupled receptor and is expressed throughout the gut. It is well known that PAR-2 participates in the regulation of gastrointestinal motility; however, the results are inconsistent. The present study investigated the effect and mechanism of PAR-2 activation on murine small intestinal smooth muscle function in vitro. Both trypsin and PAR-2-activating peptide SLIGRL induced a small relaxation followed by a concentration-dependent contraction. The sensitivity to trypsin was greater than that to SLIGRL (EC50 = 0.03 vs. 40 microM), but maximal responses were similar (12.3 +/- 1.6 vs. 13.7 +/- 1.3 N/cm2). Trypsin-evoked contraction (1 microM) exhibited a rapid desensitization, whereas the desensitization of response to SLIGRL was less even at high concentration (50 microM). Atropine had no effect on PAR-2 agonist-induced contractions. In contrast, TTX and capsaicin significantly attenuated those contractions, implicating a neurogenic mechanism that may involve capsaicin-sensitive sensory nerves. Furthermore, contractions induced by trypsin and SLIGRL were reduced by neurokinin receptor NK1 antagonist SR-140333 or NK2 antagonist SR-48968 alone or were further reduced by combined application of SR-140333 and SR-48968, indicating the involvement of neurokinin receptors. In addition, desensitizing neurokinin receptors with substance P and/or neurokinin A decreased the PAR-2 agonist-evoked contraction. We concluded that PAR-2 agonists induced a contraction of murine intestinal smooth muscle that was mediated by nerves. The excitatory effect is also dependent on sensory neural pathways and requires both NK1 and NK2 receptors.

A Community Standard Format for the Representation of Protein Affinity Reagents*

PubMed Central

Gloriam, David E.; Orchard, Sandra; Bertinetti, Daniela; Björling, Erik; Bongcam-Rudloff, Erik; Borrebaeck, Carl A. K.; Bourbeillon, Julie; Bradbury, Andrew R. M.; de Daruvar, Antoine; Dübel, Stefan; Frank, Ronald; Gibson, Toby J.; Gold, Larry; Haslam, Niall; Herberg, Friedrich W.; Hiltke, Tara; Hoheisel, Jörg D.; Kerrien, Samuel; Koegl, Manfred; Konthur, Zoltán; Korn, Bernhard; Landegren, Ulf; Montecchi-Palazzi, Luisa; Palcy, Sandrine; Rodriguez, Henry; Schweinsberg, Sonja; Sievert, Volker; Stoevesandt, Oda; Taussig, Michael J.; Ueffing, Marius; Uhlén, Mathias; van der Maarel, Silvère; Wingren, Christer; Woollard, Peter; Sherman, David J.; Hermjakob, Henning

2010-01-01

Protein affinity reagents (PARs), most commonly antibodies, are essential reagents for protein characterization in basic research, biotechnology, and diagnostics as well as the fastest growing class of therapeutics. Large numbers of PARs are available commercially; however, their quality is often uncertain. In addition, currently available PARs cover only a fraction of the human proteome, and their cost is prohibitive for proteome scale applications. This situation has triggered several initiatives involving large scale generation and validation of antibodies, for example the Swedish Human Protein Atlas and the German Antibody Factory. Antibodies targeting specific subproteomes are being pursued by members of Human Proteome Organisation (plasma and liver proteome projects) and the United States National Cancer Institute (cancer-associated antigens). ProteomeBinders, a European consortium, aims to set up a resource of consistently quality-controlled protein-binding reagents for the whole human proteome. An ultimate PAR database resource would allow consumers to visit one on-line warehouse and find all available affinity reagents from different providers together with documentation that facilitates easy comparison of their cost and quality. However, in contrast to, for example, nucleotide databases among which data are synchronized between the major data providers, current PAR producers, quality control centers, and commercial companies all use incompatible formats, hindering data exchange. Here we propose Proteomics Standards Initiative (PSI)-PAR as a global community standard format for the representation and exchange of protein affinity reagent data. The PSI-PAR format is maintained by the Human Proteome Organisation PSI and was developed within the context of ProteomeBinders by building on a mature proteomics standard format, PSI-molecular interaction, which is a widely accepted and established community standard for molecular interaction data. Further information and documentation are available on the PSI-PAR web site. PMID:19674966

Tissue Factor-Factor VIIa Complex Triggers Protease Activated Receptor 2-Dependent Growth Factor Release and Migration in Ovarian Cancer

PubMed Central

Chanakira, Alice; Westmark, Pamela R.; Ong, Irene M.; Sheehan, John P.

2017-01-01

Objective Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC. Methods TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expression were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists. Results Relative mRNA expression consistently demonstrated PAR-2>PAR-1≫PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4–10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present. Conclusions Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation. PMID:28148395

PH motifs in PAR1&2 endow breast cancer growth.

PubMed

Kancharla, A; Maoz, M; Jaber, M; Agranovich, D; Peretz, T; Grisaru-Granovsky, S; Uziely, B; Bar-Shavit, R

2015-11-24

Although emerging roles of protease-activated receptor1&2 (PAR1&2) in cancer are recognized, their underlying signalling events are poorly understood. Here we show signal-binding motifs in PAR1&2 that are critical for breast cancer growth. This occurs via the association of the pleckstrin homology (PH) domain with Akt/PKB as a key signalling event of PARs. Other PH-domain signal-proteins such as Etk/Bmx and Vav3 also associate with PAR1 and PAR2 through their PH domains. PAR1 and PAR2 bind with priority to Etk/Bmx. A point mutation in PAR2, H349A, but not in R352A, abrogates PH-protein association and is sufficient to markedly reduce PAR2-instigated breast tumour growth in vivo and placental extravillous trophoblast (EVT) invasion in vitro. Similarly, the PAR1 mutant hPar1-7A, which is unable to bind the PH domain, reduces mammary tumours and EVT invasion, endowing these motifs with physiological significance and underscoring the importance of these previously unknown PAR1 and PAR2 PH-domain-binding motifs in both pathological and physiological invasion processes.

The tight junction protein ZO-2 and Janus kinase 1 mediate intercellular communications in vascular smooth muscle cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tkachuk, Natalia; Tkachuk, Sergey; Patecki, Margret

2011-07-08

Highlights: {yields} The tight junction protein ZO-2 associates with Jak1 in vascular smooth muscle cells via ZO-2 N-terminal fragment. {yields} Jak1 mediates ZO-2 tyrosine phosphorylation and ZO-2 localization to the sites of homotypic intercellular contacts. {yields} The urokinase receptor uPAR regulates ZO-2/Jak1 functional association. {yields} The ZO-2/Jak1/uPAR signaling complex is required for vascular smooth muscle cells functional network formation. -- Abstract: Recent evidence points to a multifunctional role of ZO-2, the tight junction protein of the MAGUK (membrane-associated guanylate kinase-like) family. Though ZO-2 has been found in cell types lacking tight junction structures, such as vascular smooth muscle cells (VSMC),more » little is known about ZO-2 function in these cells. We provide evidence that ZO-2 mediates specific homotypic cell-to-cell contacts between VSMC. Using mass spectrometry we found that ZO-2 is associated with the non-receptor tyrosine kinase Jak1. By generating specific ZO-2 constructs we further found that the N-terminal fragment of ZO-2 molecule is responsible for this interaction. Adenovirus-based expression of Jak1 inactive mutant demonstrated that Jak1 mediates ZO-2 tyrosine phosphorylation. By means of RNA silencing, expression of Jak1 mutant form and fluorescently labeled ZO-2 fusion protein we further specified that active Jak1, but not Jak1 inactive mutant, mediates ZO-2 localization to the sites of intercellular contacts. We identified the urokinase receptor uPAR as a pre-requisite for these cellular events. Functional requirement of the revealed signaling complex for VSMC network formation was confirmed in experiments using Matrigel and in contraction assay. Our findings imply involvement of the ZO-2 tight junction independent signaling complex containing Jak1 and uPAR in VSMC intercellular communications. This mechanism may contribute to vascular remodeling in occlusive cardiovascular diseases and in arteriogenesis.« less

Expression of Par3 polarity protein correlates with poor prognosis in ovarian cancer.

PubMed

Nakamura, Hiroe; Nagasaka, Kazunori; Kawana, Kei; Taguchi, Ayumi; Uehara, Yuriko; Yoshida, Mitsuyo; Sato, Masakazu; Nishida, Haruka; Fujimoto, Asaha; Inoue, Tomoko; Adachi, Katsuyuki; Nagamatsu, Takeshi; Arimoto, Takahide; Oda, Katsutoshi; Osuga, Yutaka; Fujii, Tomoyuki

2016-11-17

Previous studies have shown that the cell polarity protein partitioning defective 3 (Par3) plays an essential role in the formation of tight junctions and definition of apical-basal polarity. Aberrant function of this protein has been reported to be involved in epithelial-mesenchymal transition (EMT) and cancer invasion. The aim of this study was to examine the functional mechanism of Par3 in ovarian cancer. First, we investigated the association between Par3 expression level and survival of 50 ovarian cancer patients. Next, we conducted an in vitro analysis of ovarian cancer cell lines, focusing on the cell line JHOC5, to investigate Par3 function. To investigate the function of Par3 in invasion, the IL-6/STAT3 pathway was analyzed upon Par3 knockdown with siRNA. The effect of siRNA treatment was assessed by qPCR, ELISA, and western blotting. Invasiveness and cell proliferation following treatment with siRNA against Par3 were investigated using Matrigel chamber, wound healing, and cell proliferation assays. Expression array data for ovarian cancer patient samples revealed low Par3 expression was significantly associated with good prognosis. Univariate analysis of clinicopathological factors revealed significant association between high Par3 levels and peritoneal dissemination at the time of diagnosis. Knockdown of Par3 in JHOC5 cells suppressed cell invasiveness, migration, and cell proliferation with deregulation of IL-6/STAT3 activity. Taken together, these results suggest that Par3 expression is likely involved in ovarian cancer progression, especially in peritoneal metastasis. The underlying mechanism may be that Par3 modulates IL-6 /STAT3 signaling. Here, we propose that the expression of Par3 in ovarian cancer may control disease outcome.

The intracellular carboxyl tail of the PAR-2 receptor controls intracellular signaling and cell death.

PubMed

Zhu, Zhihui; Stricker, Rolf; Li, Rong yu; Zündorf, Gregor; Reiser, Georg

2015-03-01

The protease-activated receptors are a group of unique G protein-coupled receptors, including PAR-1, PAR-2, PAR-3 and PAR-4. PAR-2 is activated by multiple trypsin-like serine proteases, including trypsin, tryptase and coagulation proteases. The clusters of phosphorylation sites in the PAR-2 carboxyl tail are suggested to be important for the binding of adaptor proteins to initiate intracellular signaling to Ca(2+) and mitogen-activated protein kinases. To explore the functional role of PAR-2 carboxyl tail in controlling intracellular Ca(2+), ERK and AKT signaling, a series of truncated mutants containing different clusters of serines/threonines were generated and expressed in HEK293 cells. Firstly, we observed that lack of the complete C-terminus of PAR-2 in a mutated receptor gave a relatively low level of localization on the cell plasma membrane. Secondly, the shortened carboxyl tail containing 13 amino acids was sufficient for receptor internalization. Thirdly, the cells expressing truncation mutants showed deficits in their capacity to couple to intracellular Ca(2+) and ERK and AKT signaling upon trypsin challenge. In addition, HEK293 cells carrying different PAR-2 truncation mutants displayed decreased levels of cell survival after long-lasting trypsin stimulation. In summary, the PAR-2 carboxyl tail was found to control the receptor localization, internalization, intracellular Ca(2+) responses and signaling to ERK and AKT. The latter can be considered to be important for cell death control.

Evidence that compatibility of closely related replicons in Clostridium perfringens depends on linkage to parMRC-like partitioning systems of different subfamilies.

PubMed

Watts, Thomas D; Johanesen, Priscilla A; Lyras, Dena; Rood, Julian I; Adams, Vicki

2017-05-01

Clostridium perfringens produces an extensive repertoire of toxins and extracellular enzymes, many of which are intimately involved in the progression of disease and are encoded by genes on conjugative plasmids. In addition, many C. perfringens strains can carry up to five of these conjugative toxin or antimicrobial resistance plasmids, each of which has a similar 35kb backbone. This conserved backbone includes the tcp conjugation locus and the central control region (CCR), which encodes genes involved in plasmid regulation, replication and partitioning, including a parMRC partitioning locus. Most conjugative plasmids in C. perfringens have a conserved replication protein, raising questions as to how multiple, closely related plasmids are maintained within a single strain. Bioinformatics analysis has highlighted the presence of at least 10 different parMRC partitioning system families (parMRC A-J ) in these plasmids, with differences in amino acid sequence identity between each ParM family ranging from 15% to 54%. No two plasmids that encode genes belonging to the same partitioning family have been observed in a single strain, suggesting that these families represent the basis for plasmid incompatibility. In an attempt to validate the proposed parMRC incompatibility groups, genetically marked C. perfringens plasmids encoding identical parMRC C or parMRC D homologues or different combinations of parMRC A , parMRC C and parMRC D family homologues were introduced into a single strain via conjugation. The stability of each plasmid was determined using an incompatibility assay in which the plasmid profile of each strain was monitored over the course of two days in the absence of direct selection. The results showed that plasmids with identical parMRC C or parMRC D homologues were incompatible and could not coexist in the absence of external selection. By contrast, plasmids that encoded different parMRC homologues were compatible and could coexist in the same cell in the absence of selection, with the exception of strains housing parMRC C and parMRC D combinations, which showed a minor incompatibility phenotype. In conclusion, we have provided the first direct evidence of plasmid incompatibility in Clostridium spp. and have shown experimentally that the compatibility of conjugative C. perfringens plasmids correlates with the presence of parMRC-like partitioning systems of different phylogenetic subfamilies. Copyright © 2017 Elsevier Inc. All rights reserved.

Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis

NASA Astrophysics Data System (ADS)

Davis, Shaun M.; Thomas, Amanda L.; Nomie, Krystle J.; Huang, Longwen; Dierick, Herman A.

2014-02-01

Aggressive behaviour is widespread throughout the animal kingdom. However, its mechanisms are poorly understood, and the degree of molecular conservation between distantly related species is unknown. Here we show that knockdown of tailless (tll) increases aggression in Drosophila, similar to the effect of its mouse orthologue Nr2e1. Tll localizes to the adult pars intercerebralis (PI), which shows similarity to the mammalian hypothalamus. Knockdown of tll in the PI is sufficient to increase aggression and is rescued by co-expressing human NR2E1. Knockdown of Atrophin, a Tll co-repressor, also increases aggression, and both proteins physically interact in the PI. tll knockdown-induced aggression is fully suppressed by blocking neuropeptide processing or release from the PI. In addition, genetically activating PI neurons increases aggression, mimicking the aggression-inducing effect of hypothalamic stimulation. Together, our results suggest that a transcriptional control module regulates neuropeptide signalling from the neurosecretory cells of the brain to control aggressive behaviour.

Orderly Replication and Segregation of the Four Replicons of Burkholderia cenocepacia J2315

PubMed Central

Kamgoué, Alain; Murray, Heath; Pasta, Franck

2016-01-01

Bacterial genomes typically consist of a single chromosome and, optionally, one or more plasmids. But whole-genome sequencing reveals about ten per-cent of them to be multipartite, with additional replicons which by size and indispensability are considered secondary chromosomes. This raises the questions of how their replication and partition is managed without compromising genome stability and of how such genomes arose. Vibrio cholerae, with a 1 Mb replicon in addition to its 3 Mb chromosome, is the only species for which maintenance of a multipartite genome has been investigated. In this study we have explored the more complex genome of Burkholderia cenocepacia (strain J2315). It comprises an extra replicon (c2) of 3.21 Mb, comparable in size to the3.87Mb main chromosome (c1), another extra replicon(c3) of 0.87 Mb and a plasmid of 0.09 Mb. The replication origin of c1 is typically chromosomal and those of c2 and c3 are plasmid-like; all are replicated bidirectionally. Fluorescence microscopy of tagged origins indicates that all initiate replication at mid-cell and segregate towards the cell quarter positions sequentially, c1-c2-p1/c3. c2 segregation is as well-phased with the cell cycle as c1, implying that this plasmid-like origin has become subject to regulation not typical of plasmids; in contrast, c3 segregates more randomly through the cycle. Disruption of individual Par systems by deletion of parAB or by addition of parS sites showed each Par system to govern the positioning of its own replicon only. Inactivation of c1, c2 and c3 Par systems not only reduced growth rate, generated anucleate cells and compromised viability but influenced processes beyond replicon partition, notably regulation of replication, chromosome condensation and cell size determination. In particular, the absence of the c1 ParA protein altered replication of all three chromosomes, suggesting that the partition system of the main chromosome is a major participant in the choreography of the cell cycle. PMID:27428258

Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

PubMed Central

2013-01-01

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

Urokinase links plasminogen activation and cell adhesion by cleavage of the RGD motif in vitronectin.

PubMed

De Lorenzi, Valentina; Sarra Ferraris, Gian Maria; Madsen, Jeppe B; Lupia, Michela; Andreasen, Peter A; Sidenius, Nicolai

2016-07-01

Components of the plasminogen activation system including urokinase (uPA), its inhibitor (PAI-1) and its cell surface receptor (uPAR) have been implicated in a wide variety of biological processes related to tissue homoeostasis. Firstly, the binding of uPA to uPAR favours extracellular proteolysis by enhancing cell surface plasminogen activation. Secondly, it promotes cell adhesion and signalling through binding of the provisional matrix protein vitronectin. We now report that uPA and plasmin induces a potent negative feedback on cell adhesion through specific cleavage of the RGD motif in vitronectin. Cleavage of vitronectin by uPA displays a remarkable receptor dependence and requires concomitant binding of both uPA and vitronectin to uPAR Moreover, we show that PAI-1 counteracts the negative feedback and behaves as a proteolysis-triggered stabilizer of uPAR-mediated cell adhesion to vitronectin. These findings identify a novel and highly specific function for the plasminogen activation system in the regulation of cell adhesion to vitronectin. The cleavage of vitronectin by uPA and plasmin results in the release of N-terminal vitronectin fragments that can be detected in vivo, underscoring the potential physiological relevance of the process. © 2016 The Authors.

Ranking prediction model using the competition record of Ladies Professional Golf Association players.

PubMed

Chae, Jin Seok; Park, Jin; So, Wi-Young

2017-07-28

The purpose of this study was to suggest a ranking prediction model using the competition record of the Ladies Professional Golf Association (LPGA) players. The top 100 players on the tour money list from the 2013-2016 US Open were analyzed in this model. Stepwise regression analysis was conducted to examine the effect of performance and independent variables (i.e., driving accuracy, green in regulation, putts per round, driving distance, percentage of sand saves, par-3 average, par-4 average, par-5 average, birdies average, and eagle average) on dependent variables (i.e., scoring average, official money, top-10 finishes, winning percentage, and 60-strokes average). The following prediction model was suggested:Y (Scoring average) = 55.871 - 0.947 (Birdies average) + 4.576 (Par-4 average) - 0.028 (Green in regulation) - 0.012 (Percentage of sand saves) + 2.088 (Par-3 average) - 0.026 (Driving accuracy) - 0.017 (Driving distance) + 0.085 (Putts per round)Y (Official money) = 6628736.723 + 528557.907 (Birdies average) - 1831800.821 (Par-4 average) + 11681.739 (Green in regulation) + 6476.344 (Percentage of sand saves) - 688115.074 (Par-3 average) + 7375.971 (Driving accuracy)Y (Top-10 finish%) = 204.462 + 12.562 (Birdies average) - 47.745 (Par-4 average) + 1.633 (Green in regulation) - 5.151 (Putts per round) + 0.132 (Percentage of sand saves)Y (Winning percentage) = 49.949 + 3.191 (Birdies average) - 15.023 (Par-4 average) + 0.043 (Percentage of sand saves)Y (60-strokes average) = 217.649 + 13.978 (Birdies average) - 44.855 (Par-4 average) - 22.433 (Par-3 average) + 0.16 (Green in regulation)Scoring of the above five prediction models and the prediction of golf ranking in the 2016 Women's Golf Olympic competition in Rio revealed a significant correlation between the predicted and real ranking (r = 0.689, p < 0.001) and between the predicted and the real average score (r = 0.653, p < 0.001). Our ranking prediction model using LPGA data may help coaches and players to identify which players are likely to participate in Olympic and World competitions, based on their performance.

MARK2/EMK1/Par-1Balpha phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells.

PubMed

Ducharme, Nicole A; Hales, Chadwick M; Lapierre, Lynne A; Ham, Amy-Joan L; Oztan, Asli; Apodaca, Gerard; Goldenring, James R

2006-08-01

Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1Balpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.

Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

PubMed Central

Taylor, James A.; Pastrana, Cesar L.; Butterer, Annika; Pernstich, Christian; Gwynn, Emma J.; Sobott, Frank; Moreno-Herrero, Fernando; Dillingham, Mark S.

2015-01-01

The segregation of many bacterial chromosomes is dependent on the interactions of ParB proteins with centromere-like DNA sequences called parS that are located close to the origin of replication. In this work, we have investigated the binding of Bacillus subtilis ParB to DNA in vitro using a variety of biochemical and biophysical techniques. We observe tight and specific binding of a ParB homodimer to the parS sequence. Binding of ParB to non-specific DNA is more complex and displays apparent positive co-operativity that is associated with the formation of larger, poorly defined, nucleoprotein complexes. Experiments with magnetic tweezers demonstrate that non-specific binding leads to DNA condensation that is reversible by protein unbinding or force. The condensed DNA structure is not well ordered and we infer that it is formed by many looping interactions between neighbouring DNA segments. Consistent with this view, ParB is also able to stabilize writhe in single supercoiled DNA molecules and to bridge segments from two different DNA molecules in trans. The experiments provide no evidence for the promotion of non-specific DNA binding and/or condensation events by the presence of parS sequences. The implications of these observations for chromosome segregation are discussed. PMID:25572315

A Conserved Mode of Protein Recognition and Binding in a ParD−ParE Toxin−Antitoxin Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dalton, Kevin M.; Crosson, Sean

2010-05-06

Toxin-antitoxin (TA) systems form a ubiquitous class of prokaryotic proteins with functional roles in plasmid inheritance, environmental stress response, and cell development. ParDE family TA systems are broadly conserved on plasmids and bacterial chromosomes and have been well characterized as genetic elements that promote stable plasmid inheritance. We present a crystal structure of a chromosomally encoded ParD-ParE complex from Caulobacter crescentus at 2.6 {angstrom} resolution. This TA system forms an {alpha}{sub 2}{beta}{sub 2} heterotetramer in the crystal and in solution. The toxin-antitoxin binding interface reveals extensive polar and hydrophobic contacts of ParD antitoxin helices with a conserved recognition and bindingmore » groove on the ParE toxin. A cross-species comparison of this complex structure with related toxin structures identified an antitoxin recognition and binding subdomain that is conserved between distantly related members of the RelE/ParE toxin superfamily despite a low level of overall primary sequence identity. We further demonstrate that ParD antitoxin is dimeric, stably folded, and largely helical when not bound to ParE toxin. Thus, the paradigmatic model in which antitoxin undergoes a disorder-to-order transition upon toxin binding does not apply to this chromosomal ParD-ParE TA system.« less

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling.

PubMed

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L; Hille, Bertil; Koh, Duk-Su

2016-03-01

Activated Gq protein-coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein-tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein-coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2. © 2016 Jung et al.

The chitinase-like protein YKL-40 increases mucin5AC production in human bronchial epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Chunyi; Li, Qi; Zhou, Xiangdong, E-mail: zxd999@263.net

2013-11-01

Mucus overproduction is an important feature in patients with chronic inflammatory airway diseases. However, the regulatory mechanisms that mediate excessive mucin production remain elusive. Recently, the level of YKL-40, a chitinase-like protein, has been found to be significantly increased in chronic inflammatory airway diseases and has been shown to be associated with the severity of these diseases. In this study, we sought to explore the effect of YKL-40 on mucin5AC (MUC5AC) production in chronic inflammatory airway diseases and the potential signaling pathways involved in this process. We found that elevated YKL-40 levels increased the mRNA and protein expression of MUC5ACmore » in a dose- and time-dependent manner, in association with the phosphorylation of extracellular signal-regulated kinase (ERK) and nuclear factor κB (NF-κB), reflecting their activation. These responses were significantly suppressed by the knockdown of protease-activating receptor 2 (PAR2) with specific small interfering RNA or the inhibitors of ERK and NF-κB. YKL-40-induced MUC5AC overproduction was also effectively attenuated by the inhibitor of focal adhesion kinase (FAK). Taken together, these results imply that YKL-40 can stimulate excessive MUC5AC production through PAR2- and FAK-mediated mechanisms. - Highlights: • MUC5AC is the major secreted mucin in chronic inflammatory airway diseases. • YKL-40 is a prototype of the chitinase-like protein in mammals. • YKL-40 is an active player in chronic inflammatory airway diseases. • YKL-40 can increase MUC5AC production via PAR2-mediated pathway. • FAK is another candidate to mediate YKL-40-induced MUC5AC overexpression.« less

Analysis of ParB-centromere interactions by multiplex SPR imaging reveals specific patterns for binding ParB in six centromeres of Burkholderiales chromosomes and plasmids

PubMed Central

Pillet, Flavien; Passot, Fanny Marie

2017-01-01

Bacterial centromeres–also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA—the motor protein, segregates the sister replicons. Closely related families of partition systems, called Bsr, were identified on the chromosomes and large plasmids of the multi-chromosomal bacterium Burkholderia cenocepacia and other species from the order Burkholeriales. The centromeres of the Bsr partition families are 16 bp palindromes, displaying similar base compositions, notably a central CG dinucleotide. Despite centromeres bind the cognate ParB with a narrow specificity, weak ParB-parS non cognate interactions were nevertheless detected between few Bsr partition systems of replicons not belonging to the same genome. These observations suggested that Bsr partition systems could have a common ancestry but that evolution mostly erased the possibilities of cross-reactions between them, in particular to prevent replicon incompatibility. To detect novel similarities between Bsr partition systems, we have analyzed the binding of six Bsr parS sequences and a wide collection of modified derivatives, to their cognate ParB. The study was carried out by Surface Plasmon Resonance imaging (SPRi) mulitplex analysis enabling a systematic survey of each nucleotide position within the centromere. We found that in each parS some positions could be changed while maintaining binding to ParB. Each centromere displays its own pattern of changes, but some positions are shared more or less widely. In addition from these changes we could speculate evolutionary links between these centromeres. PMID:28562673

Analysis of ParB-centromere interactions by multiplex SPR imaging reveals specific patterns for binding ParB in six centromeres of Burkholderiales chromosomes and plasmids.

PubMed

Pillet, Flavien; Passot, Fanny Marie; Pasta, Franck; Anton Leberre, Véronique; Bouet, Jean-Yves

2017-01-01

Bacterial centromeres-also called parS, are cis-acting DNA sequences which, together with the proteins ParA and ParB, are involved in the segregation of chromosomes and plasmids. The specific binding of ParB to parS nucleates the assembly of a large ParB/DNA complex from which ParA-the motor protein, segregates the sister replicons. Closely related families of partition systems, called Bsr, were identified on the chromosomes and large plasmids of the multi-chromosomal bacterium Burkholderia cenocepacia and other species from the order Burkholeriales. The centromeres of the Bsr partition families are 16 bp palindromes, displaying similar base compositions, notably a central CG dinucleotide. Despite centromeres bind the cognate ParB with a narrow specificity, weak ParB-parS non cognate interactions were nevertheless detected between few Bsr partition systems of replicons not belonging to the same genome. These observations suggested that Bsr partition systems could have a common ancestry but that evolution mostly erased the possibilities of cross-reactions between them, in particular to prevent replicon incompatibility. To detect novel similarities between Bsr partition systems, we have analyzed the binding of six Bsr parS sequences and a wide collection of modified derivatives, to their cognate ParB. The study was carried out by Surface Plasmon Resonance imaging (SPRi) mulitplex analysis enabling a systematic survey of each nucleotide position within the centromere. We found that in each parS some positions could be changed while maintaining binding to ParB. Each centromere displays its own pattern of changes, but some positions are shared more or less widely. In addition from these changes we could speculate evolutionary links between these centromeres.

15-PGDH/15-KETE plays a role in hypoxia-induced pulmonary vascular remodeling through ERK1/2-dependent PAR-2 pathway.

PubMed

Wei, Liuping; Yu, Xiufeng; Shi, Hengyuan; Zhang, Bo; Lian, Mingming; Li, Jing; Shen, Tingting; Xing, Yan; Zhu, Daling

2014-07-01

We have established that 15-hydroxyeicosatetraenoic acid is an important factor in regulation of pulmonary vascular remodeling (PVR) associated with hypoxia-induced pulmonary hypertension (PH), which is further metabolized by 15-hydroxyprostaglandin dehydrogenase (15-PGDH) to form 15-ketoeicosatetraenoic acid (15-KETE). However, the role of 15-PGDH and 15-KETE on PH has not been identified. The purpose of this study was to investigate whether 15-PGDH/15-KETE pathway regulates hypoxia-induced PVR in PH and to characterize the underlying mechanisms. To accomplish this, Immunohistochemistry, Ultra Performance Liquid Chromatography, Western blot, bromodeoxyuridine incorporation and cell cycle analysis were preformed. Our results showed that the levels of 15-PGDH expression and endogenous 15-KETE were drastically elevated in the lungs of humans with PH and hypoxic PH rats. Hypoxia stimulated pulmonary arterial smooth muscle cell (PASMC) proliferation, which seemed to be due to the increased 15-PGDH/15-KETE. 15-PGDH/15-KETE pathway was also capable of stimulating the cell cycle progression and promoting the cell cycle-related protein expression. Furthermore, 15-KETE-promoted cell cycle progression and proliferation in PASMCs depended on protease-activated receptor 2 (PAR-2). ERK1/2 signaling was likely required for 15-PGDH/15-KETE-induced PAR-2 expression under hypoxia. Our study indicates that 15-PGDH/15-KETE stimulates the cell cycle progression and proliferation of PASMCs involving ERK1/2-mediated PAR-2 expression, and contributes to hypoxia-induced PVR. Copyright © 2014 Elsevier Inc. All rights reserved.

Protease-Activated Receptor 2 Activation Inhibits N-Type Ca2+ Currents in Rat Peripheral Sympathetic Neurons

PubMed Central

Kim, Young-Hwan; Ahn, Duck-Sun; Kim, Myeong Ok; Joeng, Ji-Hyun; Chung, Seungsoo

2014-01-01

The protease-activated receptor (PAR)-2 is highly expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although several mechanisms have been suggested to explain PAR-2-induced hypotension, the precise mechanism remains to be elucidated. To investigate this possibility, we investigated the effects of PAR-2 activation on N-type Ca2+ currents (ICa-N) in isolated neurons of the celiac ganglion (CG), which is involved in the sympathetic regulation of mesenteric artery vascular tone. PAR-2 agonists irreversibly diminished voltage-gated Ca2+ currents (ICa), measured using the patch-clamp method, in rat CG neurons, whereas thrombin had little effect on ICa. This PAR-2-induced inhibition was almost completely prevented by ω-CgTx, a potent N-type Ca2+ channel blocker, suggesting the involvement of N-type Ca2+ channels in PAR-2-induced inhibition. In addition, PAR-2 agonists inhibited ICa–N in a voltage-independent manner in rat CG neurons. Moreover, PAR-2 agonists reduced action potential (AP) firing frequency as measured using the current-clamp method in rat CG neurons. This inhibition of AP firing induced by PAR-2 agonists was almost completely prevented by ω-CgTx, indicating that PAR-2 activation may regulate the membrane excitability of peripheral sympathetic neurons through modulation of N-type Ca2+ channels. In conclusion, the present findings demonstrate that the activation of PAR-2 suppresses peripheral sympathetic outflow by modulating N-type Ca2+ channel activity, which appears to be involved in PAR-2-induced hypotension, in peripheral sympathetic nerve terminals. PMID:25410909

RNA interactome capture in yeast.

PubMed

Beckmann, Benedikt M

2017-04-15

RNA-binding proteins (RBPs) are key players in post-transcriptional regulation of gene expression in eukaryotic cells. To be able to unbiasedly identify RBPs in Saccharomyces cerevisiae, we developed a yeast RNA interactome capture protocol which employs RNA labeling, covalent UV crosslinking of RNA and proteins at 365nm wavelength (photoactivatable-ribonucleoside-enhanced crosslinking, PAR-CL) and finally purification of the protein-bound mRNA. The method can be easily implemented in common workflows and takes about 3days to complete. Next to a comprehensive explanation of the method, we focus on our findings about the choice of crosslinking in yeast and discuss the rationale of individual steps in the protocol. Copyright © 2016. Published by Elsevier Inc.

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development.

PubMed

Yu, Cao Guo; Harris, Tony J C

2012-09-01

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D-enzymes that produce phosphatidic acid-reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.

Trefoil factor 3 (TFF3) from human breast milk activates PAR-2 receptors, of the intestinal epithelial cells HT-29, regulating cytokines and defensins.

PubMed

Barrera, G J; Tortolero, G Sanchez

2016-01-01

Trefoil factors are effector molecules in gastrointestinal tract physiology. Each one improves healing of the gastrointestinal tract. Trefoil factors may be grouped into three classes: the gastric peptides (TFF1), spasmolytic peptide (TFF2) and intestinal trefoil factor (TFF3). Significant amounts of TFF3 are present in human breast milk. Previously, we have reported that trefoil factor 3 isolated from human breast milk produces down regulation of cytokines and promotes human beta defensins expression in intestinal epithelial cells. This study aimed to determine the molecular mechanism involved. Here we showed that the presence of TFF3 strongly correlated with protease activated receptors 2 (PAR-2) activation in human intestinal cells. Intracellular calcium ((Ca2+)i)mobilization was induced by the treatment with: 1) TFF3, 2) synthetic PAR-2 agonist peptide. The co-treatment with a synthetic PAR-2 antagonist peptide and TFF3 eliminates the latter's effect. Additionally, we demonstrated the existence of interactions among TFF3 and PAR-2 receptors through far Western blot and co-precipitation. Finally, down regulation of PAR-2 by siRNA resulted in a decrease of TFF3 induced intracellular (Ca2+)i mobilization, cytokine regulation and defensins expression. These findings suggest that TFF3 activates intestinal cells through PAR-2 (Fig. 4, Ref. 19).

Elongation factor-2 kinase regulates TG2/β1 integrin/Src/uPAR pathway and epithelial–mesenchymal transition mediating pancreatic cancer cells invasion

PubMed Central

Ashour, Ahmed A; Gurbuz, Nilgun; Alpay, Sultan Neslihan; Abdel-Aziz, Abdel-Aziz H; Mansour, Ahmed M; Huo, Longfei; Ozpolat, Bulent

2014-01-01

Pancreatic ductal adenocarcinoma is one of the lethal cancers with extensive local tumour invasion, metastasis, early systemic dissemination and poorest prognosis. Thus, understanding the mechanisms regulating invasion/metastasis and epithelial–mesenchymal transition (EMT), is the key for developing effective therapeutic strategies for pancreatic cancer (PaCa). Eukaryotic elongation factor-2 kinase (eEF-2K) is an atypical kinase that we found to be highly up-regulated in PaCa cells. However, its role in PaCa invasion/progression remains unknown. Here, we investigated the role of eEF-2K in cellular invasion, and we found that down-regulation of eEF-2K, by siRNA or rottlerin, displays impairment of PaCa cells invasion/migration, with significant decreases in the expression of tissue transglutaminase (TG2), the multifunctional enzyme implicated in regulation of cell attachment, motility and survival. These events were associated with reductions in β1 integrin/uPAR/MMP-2 expressions as well as decrease in Src activity. Furthermore, inhibition of eEF-2K/TG2 axis suppresses the EMT, as demonstrated by the modulation of the zinc finger transcription factors, ZEB1/Snail, and the tight junction proteins, claudins. Importantly, while eEF-2K silencing recapitulates the rottlerin-induced inhibition of invasion and correlated events, eEF-2K overexpression, by lentivirus-based expression system, suppresses such rottlerin effects and potentiates PaCa cells invasion/migration capability. Collectively, our results show, for the first time, that eEF-2K is involved in regulation of the invasive phenotype of PaCa cells through promoting a new signalling pathway, which is mediated by TG2/β1 integrin/Src/uPAR/MMP-2, and the induction of EMT biomarkers which enhance cancer cell motility and metastatic potential. Thus, eEF-2K could represent a novel potential therapeutic target in pancreatic cancer. PMID:25215932

Dbl3 drives Cdc42 signaling at the apical margin to regulate junction position and apical differentiation

PubMed Central

Zihni, Ceniz; Munro, Peter M.G.; Elbediwy, Ahmed; Keep, Nicholas H.; Terry, Stephen J.; Harris, John

2014-01-01

Epithelial cells develop morphologically characteristic apical domains that are bordered by tight junctions, the apical–lateral border. Cdc42 and its effector complex Par6–atypical protein kinase c (aPKC) regulate multiple steps during epithelial differentiation, but the mechanisms that mediate process-specific activation of Cdc42 to drive apical morphogenesis and activate the transition from junction formation to apical differentiation are poorly understood. Using a small interfering RNA screen, we identify Dbl3 as a guanine nucleotide exchange factor that is recruited by ezrin to the apical membrane, that is enriched at a marginal zone apical to tight junctions, and that drives spatially restricted Cdc42 activation, promoting apical differentiation. Dbl3 depletion did not affect junction formation but did affect epithelial morphogenesis and brush border formation. Conversely, expression of active Dbl3 drove process-specific activation of the Par6–aPKC pathway, stimulating the transition from junction formation to apical differentiation and domain expansion, as well as the positioning of tight junctions. Thus, Dbl3 drives Cdc42 signaling at the apical margin to regulate morphogenesis, apical–lateral border positioning, and apical differentiation. PMID:24379416

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia.

PubMed

Gu, Y; Groome, L J; Alexander, J S; Wang, Y

2012-10-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial-specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. Copyright © 2012 Elsevier Ltd. All rights reserved.

PAR-2 triggers placenta-derived protease-induced altered VE-cadherin reorganization at endothelial junctions in preeclampsia

PubMed Central

Gu, Yang; Groome, Lynn J.; Alexander, J. Steven; Wang, Yuping

2014-01-01

PAR-2 is a G-protein coupled protease receptor whose activation in endothelial cells (ECs) is associated with increased solute permeability. VE-cadherin is an endothelial specific junction protein, which exhibits a disorganized distribution at cell junction during inflammation and is a useful indicator of endothelial barrier dysfunction. In the present study, we tested the hypothesis that PAR-2 activation mediates placenta-derived chymotrypsin-like protease (CLP)-induced endothelial junction disturbance and permeability in preeclampsia (PE). PAR-2 and VE-cadherin were examined by immunofluorescent staining. Specific CLP-induced PAR-2 activation and altered VE-cadherin distribution was assessed following depletion of protease chymotrypsin in the placental conditioned medium and after PAR-2 siRNA. VE-cadherin assembly was determined by treating cells with protease chymotrypsin and/or the specific PAR-2 agonist SLIGKV-NH2. Our results showed: 1) placental conditioned medium not only disturbed VE-cadherin distribution at cell junctions but also activated PAR-2 in ECs; 2) PAR-2 siRNA blocked the placental conditioned medium induced PAR-2 upregulation and disorganization of VE-cadherin at cell junctions; 3) PAR-2 agonist induced PAR-2 activation and VE-cadherin reorganization were dose-dependent; and 4) PAR-2 agonist could stimulate ERK1/2 activation. These results strongly suggest that proteases produced by the placenta elicit endothelial barrier dysfunction via a PAR-2 signaling regulatory mechanism in PE. PMID:22840244

Proton assisted recoupling and protein structure determination

NASA Astrophysics Data System (ADS)

de Paëpe, Gaël; Lewandowski, Józef R.; Loquet, Antoine; Böckmann, Anja; Griffin, Robert G.

2008-12-01

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between 13C or 15N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between 1H-13C (or 1H-15N) couplings to mediate zero quantum 13C-13C (or 15N-15N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C1+/-C2-/+HZ for 13C-13C recoupling experiments (or N1+/-N2-/+HZ for 15N-15N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with 13C-13C spectra of uniformly 13C, 15N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2×10.4 kDa). The spectra, which were acquired at high MAS frequencies (ωr2π>20 kHz) and magnetic fields (750-900 MHz 1H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A˚) 13C-13C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

Protease and Protease-Activated Receptor-2 Signaling in the Pathogenesis of Atopic Dermatitis

PubMed Central

Lee, Sang Eun; Jeong, Se Kyoo

2010-01-01

Proteases in the skin are essential to epidermal permeability barrier homeostasis. In addition to their direct proteolytic effects, certain proteases signal to cells by activating protease-activated receptors (PARs), the G-protein-coupled receptors. The expression of functional PAR-2 on human skin and its role in inflammation, pruritus, and skin barrier homeostasis have been demonstrated. Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by genetic barrier defects and allergic inflammation, which is sustained by gene-environmental interactions. Recent studies have revealed aberrant expression and activation of serine proteases and PAR-2 in the lesional skin of AD patients. The imbalance between proteases and protease inhibitors associated with genetic defects in the protease/protease inhibitor encoding genes, increase in skin surface pH, and exposure to proteolytically active allergens contribute to this aberrant protease/PAR-2 signaling in AD. The increased protease activity in AD leads to abnormal desquamation, degradation of lipid-processing enzymes and antimicrobial peptides, and activation of primary cytokines, thereby leading to permeability barrier dysfunction, inflammation, and defects in the antimicrobial barrier. Moreover, up-regulated proteases stimulate PAR-2 in lesional skin of AD and lead to the production of cytokines and chemokines involved in inflammation and immune responses, itching sensation, and sustained epidermal barrier perturbation with easier allergen penetration. In addition, PAR-2 is an important sensor for exogenous danger molecules, such as exogenous proteases from various allergens, and plays an important role in AD pathogenesis. Together, these findings suggest that protease activity or PAR-2 may be a future target for therapeutic intervention for the treatment of AD. PMID:20879045

Diallylsulfide attenuates excessive collagen production and apoptosis in a rat model of bleomycin induced pulmonary fibrosis through the involvement of protease activated receptor-2

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kalayarasan, Srinivasan, E-mail: kalaivasanbio@gmail.com; Sriram, Narayanan; Soumyakrishnan, Syamala

Pulmonary fibrosis (PF) can be a devastating lung disease. It is primarily caused by inflammation leading to severe damage of the alveolar epithelial cells. The pathophysiology of PF is not yet been clearly defined, but studying lung parenchymal injury by involving reactive oxygen species (ROS) through the activation of protease activated receptor-2 (PAR-2) may provide promising results. PAR-2 is a G-protein coupled receptor is known to play an important role in the development of PF. In this study, we investigated the inhibitory role of diallylsulfide (DAS) against ROS mediated activation of PAR-2 and collagen production accompanied by epithelial cell apoptosis.more » Bleomycin induced ROS levels may prompt to induce the expression of PAR-2 as well as extracellular matrix proteins (ECM), such as MMP 2 and 9, collagen specific proteins HSP-47, α-SMA, and cytokines IL-6, and IL-8RA. Importantly DAS treatment effectively decreased the expression of all these proteins. The inhibitory effect of DAS on profibrotic molecules is mediated by blocking the ROS level. To identify apoptotic signaling as a mediator of PF induction, we performed apoptotic protein expression, DNA fragmentation analysis and ultrastructural details of the lung tissue were performed. DAS treatment restored all these changes to near normalcy. In conclusion, treatment of PF bearing rats with DAS results in amelioration of the ROS production, PAR-2 activation, ECM production, collagen synthesis and alveolar epithelial cell apoptosis during bleomycin induction. We attained the first evidence that treatment of DAS decreases the ROS levels and may provide a potential therapeutic effect attenuating bleomycin induced PF. - Highlights: • DAS inhibits PAR-2 activity; bleomycin stimulates PAR-2 activity. • Increase in PAR-2 activity is correlated with pulmonary fibrosis • DAS reduces pro-inflammatory activity linked to facilitating pulmonary fibrosis. • DAS inhibits apoptosis of alveolar epithelial cells.« less

Genetic Interaction of Centrosomin and Bazooka in Apical Domain Regulation in Drosophila Photoreceptor

PubMed Central

Chen, Geng; Rogers, Alicia K.; League, Garrett P.; Nam, Sang-Chul

2011-01-01

Background Cell polarity genes including Crumbs (Crb) and Par complexes are essential for controlling photoreceptor morphogenesis. Among the Crb and Par complexes, Bazooka (Baz, Par-3 homolog) acts as a nodal component for other cell polarity proteins. Therefore, finding other genes interacting with Baz will help us to understand the cell polarity genes' role in photoreceptor morphogenesis. Methodology/Principal Findings Here, we have found a genetic interaction between baz and centrosomin (cnn). Cnn is a core protein for centrosome which is a major microtubule-organizing center. We analyzed the effect of the cnn mutation on developing eyes to determine its role in photoreceptor morphogenesis. We found that Cnn is dispensable for retinal differentiation in eye imaginal discs during the larval stage. However, photoreceptors deficient in Cnn display dramatic morphogenesis defects including the mislocalization of Crumbs (Crb) and Bazooka (Baz) during mid-stage pupal eye development, suggesting that Cnn is specifically required for photoreceptor morphogenesis during pupal eye development. This role of Cnn in apical domain modulation was further supported by Cnn's gain-of-function phenotype. Cnn overexpression in photoreceptors caused the expansion of the apical Crb membrane domain, Baz and adherens junctions (AJs). Conclusions/Significance These results strongly suggest that the interaction of Baz and Cnn is essential for apical domain and AJ modulation during photoreceptor morphogenesis, but not for the initial photoreceptor differentiation in the Drosophila photoreceptor. PMID:21253601

Matrix metalloproteases and PAR1 activation

PubMed Central

Austin, Karyn M.; Covic, Lidija

2013-01-01

Cardiovascular diseases, including atherothrombosis, are the leading cause of morbidity and mortality in the United States, Europe, and the developed world. Matrix metalloproteases (MMPs) have recently emerged as important mediators of platelet and endothelial function, and atherothrombotic disease. Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor that is classically activated through cleavage of the N-terminal exodomain by the serine protease thrombin. Most recently, 2 MMPs have been discovered to have agonist activity for PAR1. Unexpectedly, MMP-1 and MMP-13 cleave the N-terminal exodomain of PAR1 at noncanonical sites, which result in distinct tethered ligands that activate G-protein signaling pathways. PAR1 exhibits metalloprotease-specific signaling patterns, known as biased agonism, that produce distinct functional outputs by the cell. Here we contrast the mechanisms of canonical (thrombin) and noncanonical (MMP) PAR1 activation, the contribution of MMP-PAR1 signaling to diseases of the vasculature, and the therapeutic potential of inhibiting MMP-PAR1 signaling with MMP inhibitors, including atherothrombotic disease, in-stent restenosis, heart failure, and sepsis. PMID:23086754

Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

PubMed

Martinez-Moreno, Julio M; Herencia, Carmen; Montes de Oca, Addy; Muñoz-Castañeda, Juan R; Rodríguez-Ortiz, M Encarnación; Díaz-Tocados, Juan M; Peralbo-Santaella, Esther; Camargo, Antonio; Canalejo, Antonio; Rodriguez, Mariano; Velasco-Gimena, Francisco; Almaden, Yolanda

2016-03-01

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs. © FASEB.

Macelignan inhibits melanosome transfer mediated by protease-activated receptor-2 in keratinocytes.

PubMed

Choi, Eun-Jung; Kang, Young-Gyu; Kim, Jaekyung; Hwang, Jae-Kwan

2011-01-01

Skin pigmentation is the result of melanosome transfer from melanocytes to keratinocytes. Protease-activated receptor-2 (PAR-2) is a key mediator of melanosome transfer, which occurs as the melanocyte extends its dendrite toward surrounding keratinocytes that take up melanosomes by phagocytosis. We investigated the effects of macelignan isolated from Myristica fragrans HOUTT. (nutmeg) on melanosome transfer and the regulation of PAR-2 in human keratinocytes (HaCaT). HaCaT cells stimulated by the PAR-2-activating peptide Ser-Leu-Ile-Gly-Arg-Leu-NH₂ (SLIGRL) were treated with macelignan; PAR-2 expression was then determined by reverse transcription-polymerase chain reaction (RT-PCR), Western blot, and immunocytochemistry. We evaluated the effects of macelignan on calcium mobilization and keratinocyte phagocytosis. In addition, B16F10 melanoma cells and keratinocytes were co-cultured to assess the effects of macelignan on prostaglandin E₂ (PGE₂) secretion and subsequent dendrite formation. Macelignan decreased HaCaT PAR-2 mRNA and protein levels in a dose-dependent manner. Furthermore, macelignan markedly reduced intracellular calcium mobilization and significantly downregulated keratinocyte phagocytosis, as shown by decreased ingestion of Escherichia coli bioparticles and fluorescent microspheres. In co-culture experiments, macelignan reduced keratinocyte PGE₂ secretion, thereby preventing dendrite formation in B16F10 melanoma cells compared with SLIGRL-treated controls. Macelignan inhibits melanosome transfer by downregulating PAR-2, thereby reducing keratinocyte phagocytosis and PGE₂ secretion, which in turn inhibits dendrite formation in B16F10 melanoma cells. Taken together, our findings suggest that macelignan could be used as a natural depigmenting agent to ameliorate hyperpigmentation.

Effect of steroid hormones on Bufo arenarum oviduct. Ultrastructural study.

PubMed

Medina, Marcela Fátima; Crespo, Claudia Alejandra; Ramos, Inés; Cisint, Susana Beatriz; Fernández, Silvia Nélida

2007-06-01

The endocrine regulation of the mucosa of the oviductal pars convoluta was analyzed by ultrastructural studies demonstrating that ovariectomy, together with a decrease in ovarian steroids circulating levels, caused a marked regression in this portion of Bufo arenarum oviduct. Twenty-five days after ovariectomy, a decrease in the depth of the epithelial and glandular layers was observed due to the notable loss of secretory cells, whose number was clearly smaller than in nonovariectomized females. The remaining secretory cells showed involution signs, with few secretory granules in their cytoplasm, little endoplasmic reticulum near poorly developed Golgi complexes and a large amount of lipid droplets. Cells in an advanced autolysis state were found in the lumen. These characteristics evidence a nonfunctional state of the pars convoluta. Treatment with 5alpha-dihydrotestosterone (DHT) completely reversed the ovariectomy effect, inducing pars convoluta growths and restoring the characteristics of epithelial and glandular secretory cells in the whole pars convoluta, with micrographs similar to the control. These same effects were observed after treatment with estradiol-17beta (E2), progesterone (P) o E(2)+P in the glandular layer of the whole pars convoluta, but only in the epithelial layer of the most anterior region of this duct. In the secretory cells of other segments these treatments induced the formation of granules of high electron density and homogeneous aspect. Each steroid had a particular effect on the pars convoluta. Although E2 and DHT induced the development of the organoids involved in the proteins biosynthesis, P and DHT acted as secretagogues. (c) 2007 Wiley-Liss, Inc.

PAR2 (Protease-Activated Receptor 2) Deficiency Attenuates Atherosclerosis in Mice.

PubMed

Jones, Shannon M; Mann, Adrien; Conrad, Kelsey; Saum, Keith; Hall, David E; McKinney, Lisa M; Robbins, Nathan; Thompson, Joel; Peairs, Abigail D; Camerer, Eric; Rayner, Katey J; Tranter, Michael; Mackman, Nigel; Owens, A Phillip

2018-06-01

PAR2 (protease-activated receptor 2)-dependent signaling results in augmented inflammation and has been implicated in the pathogenesis of several autoimmune conditions. The objective of this study was to determine the effect of PAR2 deficiency on the development of atherosclerosis. PAR2 mRNA and protein expression is increased in human carotid artery and mouse aortic arch atheroma versus control carotid and aortic arch arteries, respectively. To determine the effect of PAR2 deficiency on atherosclerosis, male and female low-density lipoprotein receptor-deficient ( Ldlr -/- ) mice (8-12 weeks old) that were Par2 +/+ or Par2 -/- were fed a fat- and cholesterol-enriched diet for 12 or 24 weeks. PAR2 deficiency attenuated atherosclerosis in the aortic sinus and aortic root after 12 and 24 weeks. PAR2 deficiency did not alter total plasma cholesterol concentrations or lipoprotein distributions. Bone marrow transplantation showed that PAR2 on nonhematopoietic cells contributed to atherosclerosis. PAR2 deficiency significantly attenuated levels of the chemokines Ccl2 and Cxcl1 in the circulation and macrophage content in atherosclerotic lesions. Mechanistic studies using isolated primary vascular smooth muscle cells showed that PAR2 deficiency is associated with reduced Ccl2 and Cxcl1 mRNA expression and protein release into the supernatant resulting in less monocyte migration. Our results indicate that PAR2 deficiency is associated with attenuation of atherosclerosis and may reduce lesion progression by blunting Ccl2 - and Cxcl1 -induced monocyte infiltration. © 2018 American Heart Association, Inc.

Urokinase-type plasminogen activator receptor (uPAR) ligation induces a raft-localized integrin signaling switch that mediates the hypermotile phenotype of fibrotic fibroblasts.

PubMed

Grove, Lisa M; Southern, Brian D; Jin, Tong H; White, Kimberly E; Paruchuri, Sailaja; Harel, Efrat; Wei, Ying; Rahaman, Shaik O; Gladson, Candece L; Ding, Qiang; Craik, Charles S; Chapman, Harold A; Olman, Mitchell A

2014-05-02

The urokinase-type plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-linked membrane protein with no cytosolic domain that localizes to lipid raft microdomains. Our laboratory and others have documented that lung fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) exhibit a hypermotile phenotype. This study was undertaken to elucidate the molecular mechanism whereby uPAR ligation with its cognate ligand, urokinase, induces a motile phenotype in human lung fibroblasts. We found that uPAR ligation with the urokinase receptor binding domain (amino-terminal fragment) leads to enhanced migration of fibroblasts on fibronectin in a protease-independent, lipid raft-dependent manner. Ligation of uPAR with the amino-terminal fragment recruited α5β1 integrin and the acylated form of the Src family kinase, Fyn, to lipid rafts. The biological consequences of this translocation were an increase in fibroblast motility and a switch of the integrin-initiated signal pathway for migration away from the lipid raft-independent focal adhesion kinase pathway and toward a lipid raft-dependent caveolin-Fyn-Shc pathway. Furthermore, an integrin homologous peptide as well as an antibody that competes with β1 for uPAR binding have the ability to block this effect. In addition, its relative insensitivity to cholesterol depletion suggests that the interactions of α5β1 integrin and uPAR drive the translocation of α5β1 integrin-acylated Fyn signaling complexes into lipid rafts upon uPAR ligation through protein-protein interactions. This signal switch is a novel pathway leading to the hypermotile phenotype of IPF patient-derived fibroblasts, seen with uPAR ligation. This uPAR dependent, fibrotic matrix-selective, and profibrotic fibroblast phenotype may be amenable to targeted therapeutics designed to ameliorate IPF.

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

PubMed

Xie, Meng; Roy, Richard

2015-01-01

Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress.

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease

PubMed Central

Spinale, Joann M.; Mariani, Laura H.; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X.; Wong, Hetty N.; Troost, Jonathan P.; Gadegbeku, Crystal A.; Gipson, Debbie S.; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B.

2014-01-01

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 hours. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multi-center observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared to other diagnoses. Thus, these results do not support a pathological role for suPAR in FSGS. PMID:25354239

A reassessment of soluble urokinase-type plasminogen activator receptor in glomerular disease.

PubMed

Spinale, Joann M; Mariani, Laura H; Kapoor, Shiv; Zhang, Jidong; Weyant, Robert; Song, Peter X; Wong, Hetty N; Troost, Jonathan P; Gadegbeku, Crystal A; Gipson, Debbie S; Kretzler, Matthias; Nihalani, Deepak; Holzman, Lawrence B

2015-03-01

It has been suggested that soluble urokinase receptor (suPAR) is a causative circulating factor for and a biomarker of focal and segmental glomerulosclerosis (FSGS). Here we undertook validation of these assumptions in both mouse and human models. Injection of recombinant suPAR in wild-type mice did not induce proteinuria within 24 h. Moreover, a disease phenotype was not seen in an inducible transgenic mouse model that maintained elevated suPAR concentrations for 6 weeks. Plasma and urine suPAR concentrations were evaluated as clinical biomarkers in 241 patients with glomerular disease from the prospective, longitudinal multicenter observational NEPTUNE cohort. The serum suPAR concentration at baseline inversely correlated with estimated glomerular filtration rate (eGFR) and the urine suPAR/creatinine ratio positively correlated with the urine protein/creatinine ratio. After adjusting for eGFR and urine protein, neither the serum nor urine suPAR level was an independent predictor of FSGS histopathology. A multivariable mixed-effects model of longitudinal data evaluated the association between the change in serum suPAR concentration from baseline with eGFR. After adjusting for baseline suPAR concentration, age, gender, proteinuria, and time, the change in suPAR from baseline was associated with eGFR, but this association was not different for patients with FSGS as compared with other diagnoses. Thus these results do not support a pathological role for suPAR in FSGS.

Biological Effects of Activating Distinct ErbB Receptor Dimers in Polarized Growth Arrested Epithelia

DTIC Science & Technology

2006-09-01

deregulating the function of Par protein complex, we made the unexpected observation that overexpression of Par6 induced growth- factor independent...predisposition factors for human cancer [8] and the human papillomavirus protein E6, targets scribble for degradation[9]. It has also been shown that Par6...vivo and thus is an excellent model to study the important factors in the initiation of the oncogenic process. However, activation of ErbB1 does not

Matrix metalloproteinase-10 is upregulated by thrombin in endothelial cells and increased in patients with enhanced thrombin generation.

PubMed

Orbe, Josune; Rodríguez, José A; Calvayrac, Olivier; Rodríguez-Calvo, Ricardo; Rodríguez, Cristina; Roncal, Carmen; Martínez de Lizarrondo, Sara; Barrenetxe, Jaione; Reverter, Juan C; Martínez-González, José; Páramo, José A

2009-12-01

Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

uPA/uPAR system activation drives a glycolytic phenotype in melanoma cells.

PubMed

Laurenzana, Anna; Chillà, Anastasia; Luciani, Cristina; Peppicelli, Silvia; Biagioni, Alessio; Bianchini, Francesca; Tenedini, Elena; Torre, Eugenio; Mocali, Alessandra; Calorini, Lido; Margheri, Francesca; Fibbi, Gabriella; Del Rosso, Mario

2017-09-15

In this manuscript, we show the involvement of the uPA/uPAR system in the regulation of aerobic glycolysis of melanoma cells. uPAR over-expression in human melanoma cells controls an invasive and glycolytic phenotype in normoxic conditions. uPAR down-regulation by siRNA or its uncoupling from integrins, and hence from integrin-linked tyrosine kinase receptors (IL-TKRs), by an antagonist peptide induced a striking inhibition of the PI3K/AKT/mTOR/HIF1α pathway, resulting into impairment of glucose uptake, decrease of several glycolytic enzymes and of PKM2, a checkpoint that controls metabolism of cancer cells. Further, binding of uPA to uPAR regulates expression of molecules that govern cell invasion, including extracellular matrix metallo-proteinases inducer (EMPPRIN) and enolase, a glycolytyc enzyme that also serves as a plasminogen receptor, thus providing a common denominator between tumor metabolism and phenotypic invasive features. Such effects depend on the α5β1-integrin-mediated uPAR connection with EGFR in melanoma cells with engagement of the PI3K-mTOR-HIFα pathway. HIF-1α trans-activates genes whose products mediate tumor invasion and glycolysis, thus providing the common denominator between melanoma metabolism and its invasive features. These findings unveil a unrecognized interaction between the invasion-related uPAR and IL-TKRs in the control of glycolysis and disclose a new pharmacological target (i.e., uPAR/IL-TKRs axis) for the therapy of melanoma. © 2017 UICC.

[Experimental study on repair of the defect of the pars interarticularis in rat with bone morphogenetic protein and fibrin glue].

PubMed

Nakamura, T

1992-07-01

The possibility of repairing the defect of the pars interarticularis (pars defect) with Bone Morphogenetic Protein (BMP) and fibrin glue was studied. The pars defect established in the 5th lumbar vertebra of Wistar rat was treated with surgical implantation of a composite consisting of BMP, fibrin glue and autologous cancellous bone. At 3, 6, 9 and 12 weeks after implantation, the osteoinductive activity in the pars defect was observed histologically and compared with that of other composite implants such as BMP with fibrin glue, autologous cancellous bone alone and autologous cancellous bone with fibrin glue. Although perfect bone fusion was not obtained with any of the composites employed, a significant increase in bone formation was seen in a composite of BMP, fibrin glue and autologous cancellous bone (p less than 0.01) as compared with that seen in the others. Consequently, implantation of BMP and fibrin glue combined with some biomaterials which support osteo-induction of BMP and stabilize the pars defect might be successfully applied to repair the pars defect.

SURFING BIOLOGICAL SURFACES: EXPLOITING THE NUCLEOID FOR PARTITION AND TRANSPORT IN BACTERIA

PubMed Central

Vecchiarelli, Anthony G.; Mizuuchi, Kiyoshi; Funnell, Barbara E.

2012-01-01

The ParA family of ATPases are responsible for transporting bacterial chromosomes, plasmids, and large protein machineries. ParAs pattern the nucleoid in vivo, but how patterning functions or is exploited in transport is of considerable debate. Here we discuss the process of self-organization into patterns on the bacterial nucleoid and explore how it relates to the molecular mechanism of ParA action. We review ParA-mediated DNA partition as a general mechanism of how ATP-driven protein gradients on biological surfaces can result in spatial organization on a mesoscale. We also discuss how the nucleoid acts as a formidable diffusion barrier for large bodies in the cell, and make the case that the ParA family evolved to overcome the barrier by exploiting the nucleoid as a matrix for movement. PMID:22934804

Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development

PubMed Central

Yu, Cao Guo; Harris, Tony J. C.

2012-01-01

Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D—enzymes that produce phosphatidic acid—reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity. PMID:22833561

A Heme-responsive Regulator Controls Synthesis of Staphyloferrin B in Staphylococcus aureus*♦

PubMed Central

Laakso, Holly A.; Marolda, Cristina L.; Pinter, Tyler B.; Stillman, Martin J.; Heinrichs, David E.

2016-01-01

Staphylococcus aureus possesses a multitude of mechanisms by which it can obtain iron during growth under iron starvation conditions. It expresses an effective heme acquisition system (the iron-regulated surface determinant system), it produces two carboxylate-type siderophores staphyloferrin A and staphyloferrin B (SB), and it expresses transporters for many other siderophores that it does not synthesize. The ferric uptake regulator protein regulates expression of genes encoding all of these systems. Mechanisms of fine-tuning expression of iron-regulated genes, beyond simple iron regulation via ferric uptake regulator, have not been uncovered in this organism. Here, we identify the ninth gene of the sbn operon, sbnI, as encoding a ParB/Spo0J-like protein that is required for expression of genes in the sbn operon from sbnD onward. Expression of sbnD–I is drastically decreased in an sbnI mutant, and the mutant does not synthesize detectable SB during early phases of growth. Thus, SB-mediated iron acquisition is impaired in an sbnI mutant strain. We show that the protein forms dimers and tetramers in solution and binds to DNA within the sbnC coding region. Moreover, we show that SbnI binds heme and that heme-bound SbnI does not bind DNA. Finally, we show that providing exogenous heme to S. aureus growing in an iron-free medium results in delayed synthesis of SB. This is the first study in S. aureus that identifies a DNA-binding regulatory protein that senses heme to control gene expression for siderophore synthesis. PMID:26534960

An Apical MRCK-driven Morphogenetic Pathway Controls Epithelial Polarity

PubMed Central

Zihni, Ceniz; Vlassaks, Evi; Terry, Stephen; Carlton, Jeremy; Leung, Thomas King Chor; Olson, Michael; Pichaud, Franck; Balda, Maria Susana; Matter, Karl

2017-01-01

Polarized epithelia develop distinct cell surface domains, with the apical membrane acquiring characteristic morphological features such as microvilli. Cell polarization is driven by polarity determinants including the evolutionarily conserved partitioning defective (PAR) proteins that are separated into distinct cortical domains. PAR protein segregation is thought to be a consequence of asymmetric actomyosin contractions. The mechanism of activation of apically polarized actomyosin contractility is unknown. Here we show that the Cdc42 effector MRCK activates Myosin-II at the apical pole to segregate aPKC-Par6 from junctional Par3, defining the apical domain. Apically polarized MRCK-activated actomyosin contractility is reinforced by cooperation with aPKC-Par6 downregulating antagonistic RhoA-driven junctional actomyosin contractility, and drives polarization of cytosolic brush border determinants and apical morphogenesis. MRCK-activated polarized actomyosin contractility is required for apical differentiation and morphogenesis in vertebrate epithelia and Drosophila photoreceptors. Our results identify an apical origin of actomyosin-driven morphogenesis that couples cytoskeletal reorganization to PAR polarity signalling. PMID:28825699

Evolution and tinkering: what do a protein kinase, a transcriptional regulator and chromosome segregation/cell division proteins have in common?

PubMed

Derouiche, Abderahmane; Shi, Lei; Kalantari, Aida; Mijakovic, Ivan

2016-02-01

In this study, we focus on functional interactions among multi-domain proteins which share a common evolutionary origin. The examples we develop are four Bacillus subtilis proteins, which all possess an ATP-binding Walker motif: the bacterial tyrosine kinase (BY-kinase) PtkA, the chromosome segregation protein Soj (ParA), the cell division protein MinD and a transcription regulator SalA. These proteins have arisen via duplication of the ancestral ATP-binding domain, which has undergone fusions with other functional domains in the process of divergent evolution. We point out that these four proteins, despite having very different physiological roles, engage in an unusually high number of binary functional interactions. Namely, MinD attracts Soj and PtkA to the cell pole, and in addition, activates the kinase function of PtkA. SalA also activates the kinase function of PtkA, and it gets phosphorylated by PtkA as well. The consequence of this phosphorylation is the activation of SalA as a transcriptional repressor. We hypothesize that these functional interactions remain preserved during divergent evolution and represent a constraint on the process of evolutionary "tinkering", brought about by fusions of different functional domains.

Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA

PubMed Central

Walther, Rhian F.; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J.; Pichaud, Franck

2016-01-01

Summary The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. PMID:27052178

A novel mutation in the P2Y12 receptor and a function-reducing polymorphism in protease-activated receptor 1 in a patient with chronic bleeding.

PubMed

Patel, Y M; Lordkipanidzé, M; Lowe, G C; Nisar, S P; Garner, K; Stockley, J; Daly, M E; Mitchell, M; Watson, S P; Austin, S K; Mundell, S J

2014-05-01

The study of patients with bleeding problems is a powerful approach in determining the function and regulation of important proteins in human platelets. We have identified a patient with a chronic bleeding disorder expressing a homozygous P2RY(12) mutation, predicting an arginine to cysteine (R122C) substitution in the G-protein-coupled P2Y(12) receptor. This mutation is found within the DRY motif, which is a highly conserved region in G-protein-coupled receptors (GPCRs) that is speculated to play a critical role in regulating receptor conformational states. To determine the functional consequences of the R122C substitution for P2Y(12) function. We performed a detailed phenotypic analysis of an index case and affected family members. An analysis of the variant R122C P2Y(12) stably expressed in cells was also performed. ADP-stimulated platelet aggregation was reduced as a result of a significant impairment of P2Y(12) activity in the patient and family members. Cell surface R122C P2Y(12) expression was reduced both in cell lines and in platelets; in cell lines, this was as a consequence of agonist-independent internalization followed by subsequent receptor trafficking to lysosomes. Strikingly, members of this family also showed reduced thrombin-induced platelet activation, owing to an intronic polymorphism in the F2R gene, which encodes protease-activated receptor 1 (PAR-1), that has been shown to be associated with reduced PAR-1 receptor activity. Our study is the first to demonstrate a patient with deficits in two stimulatory GPCR pathways that regulate platelet activity, further indicating that bleeding disorders constitute a complex trait. © 2014 International Society on Thrombosis and Haemostasis.

Surfing biological surfaces: exploiting the nucleoid for partition and transport in bacteria.

PubMed

Vecchiarelli, Anthony G; Mizuuchi, Kiyoshi; Funnell, Barbara E

2012-11-01

The ParA family of ATPases is responsible for transporting bacterial chromosomes, plasmids and large protein machineries. ParAs pattern the nucleoid in vivo, but how patterning functions or is exploited in transport is of considerable debate. Here we discuss the process of self-organization into patterns on the bacterial nucleoid and explore how it relates to the molecular mechanism of ParA action. We review ParA-mediated DNA partition as a general mechanism of how ATP-driven protein gradients on biological surfaces can result in spatial organization on a mesoscale. We also discuss how the nucleoid acts as a formidable diffusion barrier for large bodies in the cell, and make the case that the ParA family evolved to overcome the barrier by exploiting the nucleoid as a matrix for movement. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.

Numb regulates cell–cell adhesion and polarity in response to tyrosine kinase signalling

PubMed Central

Wang, Zezhou; Sandiford, Shelley; Wu, Chenggang; Li, Shawn Shun-Cheng

2009-01-01

Epithelial-mesenchymal transition (EMT), which can be caused by aberrant tyrosine kinase signalling, marks epithelial tumour progression and metastasis, yet the underlying molecular mechanism is not fully understood. Here, we report that Numb interacts with E-cadherin (E-cad) through its phosphotyrosine-binding domain (PTB) and thereby regulates the localization of E-cad to the lateral domain of epithelial cell–cell junction. Moreover, Numb engages the polarity complex Par3–aPKC–Par6 by binding to Par3 in polarized Madin-Darby canine kidney cells. Intriguingly, after Src activation or hepatocyte growth factor (HGF) treatment, Numb decouples from E-cad and Par3 and associates preferably with aPKC–Par6. Binding of Numb to aPKC is necessary for sequestering the latter in the cytosol during HGF-induced EMT. Knockdown of Numb by small hairpin RNA caused a basolateral-to-apicolateral translocation of E-cad and β-catenin accompanied by elevated actin polymerization, accumulation of Par3 and aPKC in the nucleus, an enhanced sensitivity to HGF-induced cell scattering, a decrease in cell–cell adhesion, and an increase in cell migration. Our work identifies Numb as an important regulator of epithelial polarity and cell–cell adhesion and a sensor of HGF signalling or Src activity during EMT. PMID:19609305

Thrombin Receptors and Protease-Activated Receptor-2 in Human Placentation

PubMed Central

O’Brien, Peter J.; Koi, Hideki; Parry, Samuel; Brass, Lawrence F.; Strauss, Jerome F.; Wang, Li-Peng; Tomaszewski, John E.; Christenson, Lane K.

2003-01-01

Proteolysis of the thrombin receptor, protease activated receptor-1 (PAR1), may enhance normal and pathological cellular invasion, and indirect evidence suggests that activation of PAR1 expressed by invasive extravillous trophoblasts (EVTs) influences human placentation. Here we describe PAR1, PAR2, and PAR3 protein distribution in the developing human placenta and implicate PAR1 and PAR2 activation in functions central to EVT invasion. PAR1, PAR2, and PAR3 are expressed in cultured 8- to 13-week-old EVTs, and in situ in 18- to 20-week-old placental syncytiotrophoblasts and invasive trophoblasts. Thrombin, but not the PAR2 agonist peptide SLIGKV, inhibited proliferation in cultured EVTs, although both agonists stimulated phosphoinositide hydrolysis and EVT invasion through Matrigel barriers. Thrombin-induced phosphoinositide hydrolysis was completely inhibited and the thrombin effect on proliferation was prevented when PAR1 cleavage was first blocked with specific monoclonal antibodies, indicating that PAR1 is the predominant thrombin receptor on EVTs. Together these results support a role for PAR1, and potentially PAR2 and PAR3 in the invasive phase of human placentation. PMID:14507634

Unaltered Angiogenesis-Regulating Activities of Platelets in Mild Type 2 Diabetes Mellitus despite a Marked Platelet Hyperreactivity.

PubMed

Miao, Xinyan; Zhang, Wei; Huang, Zhangsen; Li, Nailin

2016-01-01

Type 2 diabetes mellitus (T2DM) is associated with platelet dysfunction and impaired angiogenesis. Aim of the study is to investigate if platelet dysfunction might hamper platelet angiogenic activities in T2DM patients. Sixteen T2DM patients and gender/age-matched non-diabetic controls were studied. Flow cytometry and endothelial colony forming cell (ECFC) tube formation on matrigel were used to assess platelet reactivity and angiogenic activity, respectively. Thrombin receptor PAR1-activating peptide (PAR1-AP) induced higher platelet P-selectin expression, and evoked more rapid and intense platelet annexin V binding in T2DM patients, seen as a more rapid increase of annexin V+ platelets (24.3±6.4% vs 12.6±3.8% in control at 2 min) and a higher elevation (30.9±5.1% vs 24.3±3.0% at 8 min). However, PAR1-AP and PAR4-AP induced similar releases of angiogenic regulators from platelets, and both stimuli evoked platelet release of platelet angiogenic regulators to similar extents in T2DM and control subjects. Thus, PAR1-stimulated platelet releasate (PAR1-PR) and PAR4-PR similarly enhanced capillary-like network/tube formation of ECFCs, and the enhancements did not differ between T2DM and control subjects. Direct supplementation of platelets to ECFCs at the ratio of 1:200 enhanced ECFC tube formation even more markedly, leading to approximately 100% increases of the total branch points of ECFC tube formation, for which the enhancements were also similar between patients and controls. In conclusion, platelets from T2DM subjects are hyperreactive. Platelet activation induced by high doses of PAR1-AP, however, results in similar releases of angiogenic regulators in mild T2DM and control subjects. Platelets from T2DM and control subjects also demonstrate similar enhancements on ECFC angiogenic activities.

Human papillomavirus (HPV)-18 E6 oncoprotein interferes with the epithelial cell polarity Par3 protein.

PubMed

Facciuto, Florencia; Bugnon Valdano, Marina; Marziali, Federico; Massimi, Paola; Banks, Lawrence; Cavatorta, Ana Laura; Gardiol, Daniela

2014-05-01

High-risk human papillomavirus (HPV) infection is the principal risk factor for the development of cervical cancer. The HPV E6 oncoprotein has the ability to target and interfere with several PSD-95/DLG/ZO-1 (PDZ) domain-containing proteins that are involved in the control of cell polarity. This function can be significant for E6 oncogenic activity because a deficiency in cell polarisation is a marker of tumour progression. The establishment and control of polarity in epithelial cells depend on the correct asymmetrical distribution of proteins and lipids at the cell borders and on specialised cell junctions. In this report, we have investigated the effects of HPV E6 protein on the polarity machinery, with a focus on the PDZ partitioning defective 3 (Par3) protein, which is a key component of tight junctions (TJ) and the polarity network. We demonstrate that E6 is able to bind and induce the mislocalisation of Par3 protein in a PDZ-dependent manner without significant reduction in Par3 protein levels. In addition, the high-risk HPV-18 E6 protein promotes a delay in TJ formation when analysed by calcium switch assays. Taken together, the data presented in this study contribute to our understanding of the molecular mechanism by which HPVs induce the loss of cell polarity, with potential implications for the development and progression of HPV-associated tumours. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Protease-activated receptor 2 in dorsal root ganglion contributes to peripheral sensitization of bone cancer pain.

PubMed

Liu, S; Liu, Y-P; Yue, D-M; Liu, G-J

2014-03-01

Treating bone cancer pain continues to be a major clinical challenge, and the underlying mechanisms of bone cancer pain remain elusive. Protease-activated receptor 2 (PAR2) has been reported to be involved in neurogenic inflammation, nociceptive pain and hyperalgesia. Here, we investigated the role of PAR2 in bone cancer pain development. Expression of PAR2, mechanical allodynia, thermal hyperalgesia and neurochemical alterations induced by bone cancer pain were analysed in male, adult C3H/HeJ mice with tumour cell implantation (TCI). To investigate the contribution of PAR2 to bone cancer pain, PAR2 antagonist peptide and PAR2 knockout mice were used. TCI produced bone cancer-related pain behaviours. Production and persistence of these pain behaviours were well correlated with TCI-induced up-regulation of PAR2 in sciatic nerve and dorsal root ganglia (DRG). PAR2 knockout and spinal administration of PAR2 antagonist peptide prevented and/or reversed bone cancer-related pain behaviours and associated neurochemical changes in DRG and dorsal horn (DH). TCI also induced proteases release in tumour-bearing tibia, sciatic nerve and DRG. Plantar injection of supernatant from sarcoma cells induced PAR2 up-regulation and intracellular calcium [Ca(2+) ]i increase in DRG, and calcitonin gene-related peptide accumulation in DH, as well as significant thermal and mechanical hyperalgesia, which were all in PAR2-dependent manners. These findings suggest that PAR2 may be a key mediator for peripheral sensitization of bone cancer pain. Inhibiting PAR2 activation, especially during the early phase, may be a new therapy for preventing/suppressing development of bone cancer pain. © 2013 European Pain Federation - EFIC®

Physiological factors that regulate skin pigmentation

PubMed Central

Yamaguchi, Yuji; Hearing, Vincent J.

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1 and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2 and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including α-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. PMID:19449448

Activated protein C (APC) can increase bone anabolism via a protease-activated receptor (PAR)1/2 dependent mechanism.

PubMed

Shen, Kaitlin; Murphy, Ciara M; Chan, Ben; Kolind, Mille; Cheng, Tegan L; Mikulec, Kathy; Peacock, Lauren; Xue, Meilang; Park, Sang-Youel; Little, David G; Jackson, Chris J; Schindeler, Aaron

2014-12-01

Activated Protein C (APC) is an anticoagulant with strong cytoprotective properties that has been shown to promote wound healing. In this study APC was investigated for its potential orthopedic application using a Bone Morphogenetic Protein 2 (rhBMP-2) induced ectopic bone formation model. Local co-administration of 10 µg rhBMP-2 with 10 µg or 25 µg APC increased bone volume at 3 weeks by 32% (N.S.) and 74% (p<0.01) compared to rhBMP-2 alone. This was associated with a significant increase in CD31+ and TRAP+ cells in tissue sections of ectopic bone, consistent with enhanced vascularity and bone turnover. The actions of APC are largely mediated by its receptors endothelial protein C receptor (EPCR) and protease-activated receptors (PARs). Cultured pre-osteoblasts and bone nodule tissue sections were shown to express PAR1/2 and EPCR. When pre-osteoblasts were treated with APC, cell viability and phosphorylation of ERK1/2, Akt, and p38 were increased. Inhibition with PAR1 and sometimes PAR2 antagonists, but not with EPCR blocking antibodies, ameliorated the effects of APC on cell viability and kinase phosphorylation. These data indicate that APC can affect osteoblast viability and signaling, and may have in vivo applications with rhBMP-2 for bone repair. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity.

PubMed

Daley, William P; Gervais, Elise M; Centanni, Samuel W; Gulfo, Kathryn M; Nelson, Deirdre A; Larsen, Melinda

2012-01-01

The basement membrane is crucial for epithelial tissue organization and function. However, the mechanisms by which basement membrane is restricted to the basal periphery of epithelial tissues and the basement membrane-mediated signals that regulate coordinated tissue organization are not well defined. Here, we report that Rho kinase (ROCK) controls coordinated tissue organization by restricting basement membrane to the epithelial basal periphery in developing mouse submandibular salivary glands, and that ROCK inhibition results in accumulation of ectopic basement membrane throughout the epithelial compartment. ROCK-regulated restriction of PAR-1b (MARK2) localization in the outer basal epithelial cell layer is required for basement membrane positioning at the tissue periphery. PAR-1b is specifically required for basement membrane deposition, as inhibition of PAR-1b kinase activity prevents basement membrane deposition and disrupts overall tissue organization, and suppression of PAR-1b together with ROCK inhibition prevents interior accumulations of basement membrane. Conversely, ectopic overexpression of wild-type PAR-1b results in ectopic interior basement membrane deposition. Significantly, culture of salivary epithelial cells on exogenous basement membrane rescues epithelial organization in the presence of ROCK1 or PAR-1b inhibition, and this basement membrane-mediated rescue requires functional integrin β1 to maintain epithelial cell-cell adhesions. Taken together, these studies indicate that ROCK1/PAR-1b-dependent regulation of basement membrane placement is required for the coordination of tissue polarity and the elaboration of tissue structure in the developing submandibular salivary gland.

Self-Regulation of a Chiropractic Association through Participatory Action Research

ERIC Educational Resources Information Center

Sheppard, Lorraine A.; Jorgensen, Anna Maria S.; Crowe, Michael J.

2012-01-01

Participatory action research (PAR) can be used in the health professions to redefine their roles. This study investigated a small health professional group, the members of The Chiropractic Association Singapore (TCAS), by using a PAR method; researchers and participants gained insights into the self-regulation of a health profession. A…

Pathway-Specific Aggregate Biomarker Risk Score Is Associated With Burden of Coronary Artery Disease and Predicts Near-Term Risk of Myocardial Infarction and Death

PubMed Central

Ghasemzadeh, Nima; Hayek, Salim S.; Ko, Yi-An; Eapen, Danny J.; Patel, Riyaz S.; Manocha, Pankaj; Kassem, Hatem Al; Khayata, Mohamed; Veledar, Emir; Kremastinos, Dimitrios; Thorball, Christian W.; Pielak, Tomasz; Sikora, Sergey; Zafari, A. Maziar; Lerakis, Stamatios; Sperling, Laurence; Vaccarino, Viola; Epstein, Stephen E.; Quyyumi, Arshed A.

2018-01-01

Background Inflammation, coagulation, and cell stress contribute to atherosclerosis and its adverse events. A biomarker risk score (BRS) based on the circulating levels of biomarkers C-reactive protein, fibrin degradation products, and heat shock protein-70 representing these 3 pathways was a strong predictor of future outcomes. We investigated whether soluble urokinase plasminogen activator receptor (suPAR), a marker of immune activation, is predictive of outcomes independent of the aforementioned markers and whether its addition to a 3-BRS improves risk reclassification. Methods and Results C-reactive protein, fibrin degradation product, heat shock protein-70, and suPAR were measured in 3278 patients undergoing coronary angiography. The BRS was calculated by counting the number of biomarkers above a cutoff determined using the Youden’s index. Survival analyses were performed using models adjusted for traditional risk factors. A high suPAR level ≥3.5 ng/mL was associated with all-cause death and myocardial infarction (hazard ratio, 1.83; 95% confidence interval, 1.43–2.35) after adjustment for risk factors, C-reactive protein, fibrin degradation product, and heat shock protein-70. Addition of suPAR to the 3-BRS significantly improved the C statistic, integrated discrimination improvement, and net reclassification index for the primary outcome. A BRS of 1, 2, 3, or 4 was associated with a 1.81-, 2.59-, 6.17-, and 8.80-fold increase, respectively, in the risk of death and myocardial infarction. The 4-BRS was also associated with severity of coronary artery disease and composite end points. Conclusions SuPAR is independently predictive of adverse outcomes, and its addition to a 3-BRS comprising C-reactive protein, fibrin degradation product, and heat shock protein-70 improved risk reclassification. The clinical utility of using a 4-BRS for risk prediction and management of patients with coronary artery disease warrants further study. PMID:28280039

Pathway-Specific Aggregate Biomarker Risk Score Is Associated With Burden of Coronary Artery Disease and Predicts Near-Term Risk of Myocardial Infarction and Death.

PubMed

Ghasemzedah, Nima; Hayek, Salim S; Ko, Yi-An; Eapen, Danny J; Patel, Riyaz S; Manocha, Pankaj; Al Kassem, Hatem; Khayata, Mohamed; Veledar, Emir; Kremastinos, Dimitrios; Thorball, Christian W; Pielak, Tomasz; Sikora, Sergey; Zafari, A Maziar; Lerakis, Stamatios; Sperling, Laurence; Vaccarino, Viola; Epstein, Stephen E; Quyyumi, Arshed A

2017-03-01

Inflammation, coagulation, and cell stress contribute to atherosclerosis and its adverse events. A biomarker risk score (BRS) based on the circulating levels of biomarkers C-reactive protein, fibrin degradation products, and heat shock protein-70 representing these 3 pathways was a strong predictor of future outcomes. We investigated whether soluble urokinase plasminogen activator receptor (suPAR), a marker of immune activation, is predictive of outcomes independent of the aforementioned markers and whether its addition to a 3-BRS improves risk reclassification. C-reactive protein, fibrin degradation product, heat shock protein-70, and suPAR were measured in 3278 patients undergoing coronary angiography. The BRS was calculated by counting the number of biomarkers above a cutoff determined using the Youden's index. Survival analyses were performed using models adjusted for traditional risk factors. A high suPAR level ≥3.5 ng/mL was associated with all-cause death and myocardial infarction (hazard ratio, 1.83; 95% confidence interval, 1.43-2.35) after adjustment for risk factors, C-reactive protein, fibrin degradation product, and heat shock protein-70. Addition of suPAR to the 3-BRS significantly improved the C statistic, integrated discrimination improvement, and net reclassification index for the primary outcome. A BRS of 1, 2, 3, or 4 was associated with a 1.81-, 2.59-, 6.17-, and 8.80-fold increase, respectively, in the risk of death and myocardial infarction. The 4-BRS was also associated with severity of coronary artery disease and composite end points. SuPAR is independently predictive of adverse outcomes, and its addition to a 3-BRS comprising C-reactive protein, fibrin degradation product, and heat shock protein-70 improved risk reclassification. The clinical utility of using a 4-BRS for risk prediction and management of patients with coronary artery disease warrants further study. © 2017 American Heart Association, Inc.

Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model

PubMed Central

Romeo Cardeillac, Carlos J.; Cal Castillo, Karina B.; Vilchez Larrea, Salomé C.; Sotelo Sosa, José R.; Folle Ungo, Gustavo A.; Fernández Villamil, Silvia H.

2017-01-01

Background Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. Methods Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. Results We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. Discussion The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR stabilizing the AJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer’s, Parkinson’s and Hungtington’s diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently, the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated. PMID:28503382

CTCF regulates the human p53 gene through direct interaction with its natural antisense transcript, Wrap53

PubMed Central

Saldaña-Meyer, Ricardo; González-Buendía, Edgar; Guerrero, Georgina; Narendra, Varun; Bonasio, Roberto; Recillas-Targa, Félix; Reinberg, Danny

2014-01-01

The multifunctional CCCTC-binding factor (CTCF) protein exhibits a broad range of functions, including that of insulator and higher-order chromatin organizer. We found that CTCF comprises a previously unrecognized region that is necessary and sufficient to bind RNA (RNA-binding region [RBR]) and is distinct from its DNA-binding domain. Depletion of cellular CTCF led to a decrease in not only levels of p53 mRNA, as expected, but also those of Wrap53 RNA, an antisense transcript originated from the p53 locus. PAR-CLIP-seq (photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation [PAR-CLIP] combined with deep sequencing) analyses indicate that CTCF binds a multitude of transcripts genome-wide as well as to Wrap53 RNA. Apart from its established role at the p53 promoter, CTCF regulates p53 expression through its physical interaction with Wrap53 RNA. Cells harboring a CTCF mutant in its RBR exhibit a defective p53 response to DNA damage. Moreover, the RBR facilitates CTCF multimerization in an RNA-dependent manner, which may bear directly on its role in establishing higher-order chromatin structures in vivo. PMID:24696455

Blockade of protease-activated receptor-4 (PAR4) provides robust antithrombotic activity with low bleeding.

PubMed

Wong, Pancras C; Seiffert, Dietmar; Bird, J Eileen; Watson, Carol A; Bostwick, Jeffrey S; Giancarli, Mary; Allegretto, Nick; Hua, Ji; Harden, David; Guay, Jocelyne; Callejo, Mario; Miller, Michael M; Lawrence, R Michael; Banville, Jacques; Guy, Julia; Maxwell, Brad D; Priestley, E Scott; Marinier, Anne; Wexler, Ruth R; Bouvier, Michel; Gordon, David A; Schumacher, William A; Yang, Jing

2017-01-04

Antiplatelet agents are proven efficacious treatments for cardiovascular and cerebrovascular diseases. However, the existing drugs are compromised by unwanted and sometimes life-threatening bleeding that limits drug usage or dosage. There is a substantial unmet medical need for an antiplatelet drug with strong efficacy and low bleeding risk. Thrombin is a potent platelet agonist that directly induces platelet activation via the G protein (heterotrimeric guanine nucleotide-binding protein)-coupled protease-activated receptors PAR1 and PAR4. A PAR1 antagonist is approved for clinical use, but its use is limited by a substantial bleeding risk. Conversely, the potential of PAR4 as an antiplatelet target has not been well characterized. Using anti-PAR4 antibodies, we demonstrated a low bleeding risk and an effective antithrombotic profile with PAR4 inhibition in guinea pigs. Subsequently, high-throughput screening and an extensive medicinal chemistry effort resulted in the discovery of BMS-986120, an orally active, selective, and reversible PAR4 antagonist. In a cynomolgus monkey arterial thrombosis model, BMS-986120 demonstrated potent and highly efficacious antithrombotic activity. BMS-986120 also exhibited a low bleeding liability and a markedly wider therapeutic window compared to the standard antiplatelet agent clopidogrel tested in the same nonhuman primate model. These preclinical findings define the biological role of PAR4 in mediating platelet aggregation. In addition, they indicate that targeting PAR4 is an attractive antiplatelet strategy with the potential to treat patients at a high risk of atherothrombosis with superior safety compared with the current standard of care. Copyright © 2017, American Association for the Advancement of Science.

Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy.

PubMed

Rwibasira Rudinga, Gamariel; Khan, Ghulam Jilany; Kong, Yi

2018-02-14

Cardiovascular diseases (CVDs) are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs), including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR). Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

Contributions of protein kinases and β-arrestin to termination of protease-activated receptor 2 signaling

PubMed Central

Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi; Asbury, Charles L.; Hille, Bertil

2016-01-01

Activated Gq protein–coupled receptors (GqPCRs) can be desensitized by phosphorylation and β-arrestin binding. The kinetics and individual contributions of these two mechanisms to receptor desensitization have not been fully distinguished. Here, we describe the shut off of protease-activated receptor 2 (PAR2). PAR2 activates Gq and phospholipase C (PLC) to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol and inositol trisphosphate (IP3). We used fluorescent protein–tagged optical probes to monitor several consequences of PAR2 signaling, including PIP2 depletion and β-arrestin translocation in real time. During continuous activation of PAR2, PIP2 was depleted transiently and then restored within a few minutes, indicating fast receptor activation followed by desensitization. Knockdown of β-arrestin 1 and 2 using siRNA diminished the desensitization, slowing PIP2 restoration significantly and even adding a delayed secondary phase of further PIP2 depletion. These effects of β-arrestin knockdown on PIP2 recovery were prevented when serine/threonine phosphatases that dephosphorylate GPCRs were inhibited. Thus, PAR2 may continuously regain its activity via dephosphorylation when there is insufficient β-arrestin to trap phosphorylated receptors. Similarly, blockers of protein kinase C (PKC) and G protein–coupled receptor kinase potentiated the PIP2 depletion. In contrast, an activator of PKC inhibited receptor activation, presumably by augmenting phosphorylation of PAR2. Our interpretations were strengthened by modeling. Simulations supported the conclusions that phosphorylation of PAR2 by protein kinases initiates receptor desensitization and that recruited β-arrestin traps the phosphorylated state of the receptor, protecting it from phosphatases. Speculative thinking suggested a sequestration of phosphatidylinositol 4-phosphate 5 kinase (PIP5K) to the plasma membrane by β-arrestin to explain why knockdown of β-arrestin led to secondary depletion of PIP2. Indeed, artificial recruitment of PIP5K removed the secondary loss of PIP2 completely. Altogether, our experimental and theoretical approaches demonstrate roles and dynamics of the protein kinases, β-arrestin, and PIP5K in the desensitization of PAR2. PMID:26927499

Postsynaptic density 95 (PSD-95) serine 561 phosphorylation regulates a conformational switch and bidirectional dendritic spine structural plasticity.

PubMed

Wu, Qian; Sun, Miao; Bernard, Laura P; Zhang, Huaye

2017-09-29

Postsynaptic density 95 (PSD-95) is a major synaptic scaffolding protein that plays a key role in bidirectional synaptic plasticity, which is a process important for learning and memory. It is known that PSD-95 shows increased dynamics upon induction of plasticity. However, the underlying structural and functional changes in PSD-95 that mediate its role in plasticity remain unclear. Here we show that phosphorylation of PSD-95 at Ser-561 in its guanylate kinase (GK) domain, which is mediated by the partitioning-defective 1 (Par1) kinases, regulates a conformational switch and is important for bidirectional plasticity. Using a fluorescence resonance energy transfer (FRET) biosensor, we show that a phosphomimetic mutation of Ser-561 promotes an intramolecular interaction between GK and the nearby Src homology 3 (SH3) domain, leading to a closed conformation, whereas a non-phosphorylatable S561A mutation or inhibition of Par1 kinase activity decreases SH3-GK interaction, causing PSD-95 to adopt an open conformation. In addition, S561A mutation facilitates the interaction between PSD-95 and its binding partners. Fluorescence recovery after photobleaching imaging reveals that the S561A mutant shows increased stability, whereas the phosphomimetic S561D mutation increases PSD-95 dynamics at the synapse. Moreover, molecular replacement of endogenous PSD-95 with the S561A mutant blocks dendritic spine structural plasticity during chemical long-term potentiation and long-term depression. Endogenous Ser-561 phosphorylation is induced by synaptic NMDA receptor activation, and the SH3-GK domains exhibit a Ser-561 phosphorylation-dependent switch to a closed conformation during synaptic plasticity. Our results provide novel mechanistic insight into the regulation of PSD-95 in dendritic spine structural plasticity through phosphorylation-mediated regulation of protein dynamics and conformation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Pak4 Is Required during Epithelial Polarity Remodeling through Regulating AJ Stability and Bazooka Retention at the ZA.

PubMed

Walther, Rhian F; Nunes de Almeida, Francisca; Vlassaks, Evi; Burden, Jemima J; Pichaud, Franck

2016-04-05

The ability of epithelial cells to assemble into sheets relies on their zonula adherens (ZA), a circumferential belt of adherens junction (AJ) material, which can be remodeled during development to shape organs. Here, we show that during ZA remodeling in a model neuroepithelial cell, the Cdc42 effector P21-activated kinase 4 (Pak4/Mbt) regulates AJ morphogenesis and stability through β-catenin (β-cat/Arm) phosphorylation. We find that β-catenin phosphorylation by Mbt, and associated AJ morphogenesis, is needed for the retention of the apical determinant Par3/Bazooka at the remodeling ZA. Importantly, this retention mechanism functions together with Par1-dependent lateral exclusion of Par3/Bazooka to regulate apical membrane differentiation. Our results reveal an important functional link between Pak4, AJ material morphogenesis, and polarity remodeling during organogenesis downstream of Par3. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Oncoprotein AEG-1 is an endoplasmic reticulum RNA-binding protein whose interactome is enriched in organelle resident protein-encoding mRNAs.

PubMed

Hsu, Jack C-C; Reid, David W; Hoffman, Alyson M; Sarkar, Devanand; Nicchitta, Christopher V

2018-05-01

Astrocyte elevated gene-1 (AEG-1), an oncogene whose overexpression promotes tumor cell proliferation, angiogenesis, invasion, and enhanced chemoresistance, is thought to function primarily as a scaffolding protein, regulating PI3K/Akt and Wnt/β-catenin signaling pathways. Here we report that AEG-1 is an endoplasmic reticulum (ER) resident integral membrane RNA-binding protein (RBP). Examination of the AEG-1 RNA interactome by HITS-CLIP and PAR-CLIP methodologies revealed a high enrichment for endomembrane organelle-encoding transcripts, most prominently those encoding ER resident proteins, and within this cohort, for integral membrane protein-encoding RNAs. Cluster mapping of the AEG-1/RNA interaction sites demonstrated a normalized rank order interaction of coding sequence >5' untranslated region, with 3' untranslated region interactions only weakly represented. Intriguingly, AEG-1/membrane protein mRNA interaction sites clustered downstream from encoded transmembrane domains, suggestive of a role in membrane protein biogenesis. Secretory and cytosolic protein-encoding mRNAs were also represented in the AEG-1 RNA interactome, with the latter category notably enriched in genes functioning in mRNA localization, translational regulation, and RNA quality control. Bioinformatic analyses of RNA-binding motifs and predicted secondary structure characteristics indicate that AEG-1 lacks established RNA-binding sites though shares the property of high intrinsic disorder commonly seen in RBPs. These data implicate AEG-1 in the localization and regulation of secretory and membrane protein-encoding mRNAs and provide a framework for understanding AEG-1 function in health and disease. © 2018 Hsu et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

On the role of PDZ domain-encoding genes in Drosophila border cell migration.

PubMed

Aranjuez, George; Kudlaty, Elizabeth; Longworth, Michelle S; McDonald, Jocelyn A

2012-11-01

Cells often move as collective groups during normal embryonic development and wound healing, although the mechanisms governing this type of migration are poorly understood. The Drosophila melanogaster border cells migrate as a cluster during late oogenesis and serve as a powerful in vivo genetic model for collective cell migration. To discover new genes that participate in border cell migration, 64 out of 66 genes that encode PDZ domain-containing proteins were systematically targeted by in vivo RNAi knockdown. The PDZ domain is one of the largest families of protein-protein interaction domains found in eukaryotes. Proteins that contain PDZ domains participate in a variety of biological processes, including signal transduction and establishment of epithelial apical-basal polarity. Targeting PDZ proteins effectively assesses a larger number of genes via the protein complexes and pathways through which these proteins function. par-6, a known regulator of border cell migration, was a positive hit and thus validated the approach. Knockdown of 14 PDZ domain genes disrupted migration with multiple RNAi lines. The candidate genes have diverse predicted cellular functions and are anticipated to provide new insights into the mechanisms that control border cell movement. As a test of this concept, two genes that disrupted migration were characterized in more detail: big bang and the Dlg5 homolog CG6509. We present evidence that Big bang regulates JAK/STAT signaling, whereas Dlg5/CG6509 maintains cluster cohesion. Moreover, these results demonstrate that targeting a selected class of genes by RNAi can uncover novel regulators of collective cell migration.

A new class of orthosteric uPAR·uPA small-molecule antagonists are allosteric inhibitors of the uPAR·vitronectin interaction.

PubMed

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O

2015-06-19

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR·uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 μM and inhibited the tight uPAR·uPAATF interaction with an IC50 of 18 μM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR·uPA interface, also inhibited the distal VTN·uPAR interaction. In cell culture, 7 blocked the uPAR·uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTNSMB·uPAR interaction through more favorable electrostatics and entropy. Disruption of the uPAR·VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB·uPAR interaction was less favorable in the VTNSMB·uPAR·7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology.

Actin dynamics regulate immediate PAR-2-dependent responses to acute epidermal permeability barrier abrogation.

PubMed

Roelandt, Truus; Heughebaert, Carol; Verween, Gunther; Giddelo, Christina; Verbeken, Gilbert; Pirnay, Jean-Paul; Devos, Daniel; Crumrine, Debra; Roseeuw, Diane; Elias, Peter M; Hachem, Jean-Pierre

2011-02-01

Lamellar body (LB) secretion and terminal differentiation of stratum granulosum (SG) cells are signaled by both protease activated receptor-2 (PAR-2) and caveolin-1 (cav-1). To address the early dynamics of LB secretion, we examined cytoskeletal remodeling of keratinocytes in 3 mouse models following acute barrier abrogation: hairless mice, PAR-2 knockout (-/-) and cav-1 -/-. Under basal conditions, globular (G)-actin accumulates in SG cells cytosol, while filamentous (F)-actin is restricted to peri-membrane domains. Barrier abrogation induces the apical movement of F-actin and the retreat of the SG-G-actin front, paralleled by upstream cytoskeletal kinases activation. This phenomenon was both enhanced by PAR-2 agonist, and inhibited by cytochalasin-D and in PAR-2 knockout mice. We found that plasma membrane conformational changes causing LB secretion are controlled by PAR-2-dependent cytoskeletal rearrangements. We next addressed the interaction dynamics between cytoskeleton and plasma membrane following PAR-2-induced actin stress fiber formation in both cav-1 -/- and wildtype cells. Actin stress fiber formation is increased in cav-1 -/- cells prior to and following PAR-2 agonist peptide-treatment, while absence of cav-1 inhibits E-cadherin-mediated cell-to-cell adhesion. PAR-2 drives cytoskeletal/plasma membrane dynamics that regulate early LB secretion following barrier abrogation, stress fiber formation and keratinocyte adhesion. Copyright © 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

HAT1 induces lung cancer cell apoptosis via up regulating Fas.

PubMed

Han, Na; Shi, Lei; Guo, Qiuyun; Sun, Wei; Yu, Yang; Yang, Li; Zhang, Xiaoxi; Zhang, Mengxian

2017-10-27

The dysfunction of apoptosis is one of the factors contributing to lung cancer (LC) growth. Histone acetyltransferase HAT1 can up regulate cell apoptosis. This study aims to investigate the mechanism by which HAT1 induces LC cell (LCC) apoptosis via up regulating the expression of Fas. In this study, the surgically removed human LC tissues were collected. LCCs were isolated from the LC tissues and analyzed for the expression of HAT1 and Fas by RT-qPCR and Western blotting. We observed that the expression of Fas was negatively correlated with PAR2 in LCCs. Activation of PAR2 suppressed the expression of Fas in normal lung epithelial cells. The expression of HAT1 was lower and positively correlated with Fas expression and negatively correlated with PAR2 expression in LCCs. Activation of PAR2 suppressed Fas expression in lung epithelial cells via inhibiting HAT1. Restoration of HAT1 expression restored Fas expression in LCCs and induced LCC apoptosis. In conclusion, less expression of HAT1 in LCCs was associated with the pathogenesis of LC. Up regulation of HAT1 expression in LCCs can induce LCCs apoptosis, which may be a potential novel therapy for the treatment of LC.

Matrix metalloproteinase-2: A key regulator in coagulation proteases mediated human breast cancer progression through autocrine signaling.

PubMed

Das, Kaushik; Prasad, Ramesh; Ansari, Shabbir Ahmed; Roy, Abhishek; Mukherjee, Ashis; Sen, Prosenjit

2018-06-02

Cell invasion is attributed to the synthesis and secretion of proteolytically active matrix-metalloproteinases (MMPs) by tumor cells to degrade extracellular matrix (ECM) and promote metastasis. The role of protease-activated receptor 2 (PAR2) in human breast cancer migration/invasion via MMP-2 up-regulation remains ill-defined; hence we investigated whether TF-FVIIa/trypsin-mediated PAR2 activation induces MMP-2 expression in human breast cancer. MMP-2 expression and the signaling mechanisms were analyzed by western blotting and RT-PCR. MMP-2 activity was measured by gelatin zymography. Cell invasion was analyzed by transwell invasion assay whereas; wound healing assay was performed to understand the cell migratory potential. Here, we highlight that TF-FVIIa/trypsin-mediated PAR2 activation leads to enhanced MMP-2 expression in human breast cancer cells contributing to tumor progression. Knock-down of PAR2 abrogated TF-FVIIa/trypsin-induced up-regulation of MMP-2. Again, genetic manipulation of AKT or inhibition of NF-ĸB suggested that PAR2-mediated enhanced MMP-2 expression is dependent on the PI3K-AKT-NF-ĸB pathway. We also reveal that TF, PAR2, and MMP-2 are over-expressed in invasive breast carcinoma tissues as compared to normal. Knock-down of MMP-2 significantly impeded TF-FVIIa/trypsin-induced cell invasion. Further, we report that MMP-2 activates p38 MAPK-MK2-HSP27 signaling axis that leads to actin polymerization and induces cell migration. Pharmacological inhibition of p38 MAPK or MK2 attenuates MMP-2-induced cell migration. The study delineates a novel signaling pathway by which PAR2-induced MMP-2 expression regulates human breast cancer cell migration/invasion. Understanding these mechanistic details will certainly help to identify crucial targets for therapeutic interventions in breast cancer metastasis. Copyright © 2018. Published by Elsevier Masson SAS.

PAR(2) expression in peripheral blood monocytes of patients with rheumatoid arthritis.

PubMed

Crilly, A; Burns, E; Nickdel, M B; Lockhart, J C; Perry, M E; Ferrell, P W; Baxter, D; Dale, J; Dunning, L; Wilson, H; Nijjar, J S; Gracie, J A; Ferrell, W R; McInnes, I B

2012-06-01

Proteinase-activated receptor 2 (PAR(2)) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functional significance of PAR(2) expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR(2) on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Patients with RA had elevated but variable surface expression of PAR(2) on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86-4.10%) vs 0.06% (0.03-0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR(2) expression in patients with RA compared with controls (3.05% (0.36-11.82%) vs 0.08% (0.02-0.28%), p<0.0001). For both subsets, PAR(2) expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR(2) expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR(2) expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR(2) expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR(2) agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). These findings are consistent with a pathogenic role for PAR(2) in RA.

Estradiol attenuates EGF-induced rapid uPAR mobilization and cell migration via the G-protein-coupled receptor 30 in ovarian cancer cells.

PubMed

Henic, Emir; Noskova, Vera; Høyer-Hansen, Gunilla; Hansson, Stefan; Casslén, Bertil

2009-02-01

Epidermal growth factor (EGF) stimulates proliferation and migration in ovarian cancer cells, and high tumor expression of the EGF system correlates with poor prognosis. Epidermal growth factor upregulates urokinase plasminogen activator receptor (uPAR) on the cell surface via 3 distinct mechanisms: rapid mobilization of uPAR from detergent-resistant domains, increased mRNA, and decreased degradation. G-protein-coupled receptor 30 (GPR30) is a newly identified membrane estrogen receptor (ER).The objective of this study was to explore the effects of 17beta-estradiol (E(2)) on uPAR expression and cell migration in ovarian cancer cells and further to identify the ER involved.We used 7 ovarian cancer cell lines, cell migration assay, cellular binding of (125)I-uPA, cellular degradation of (125)I-uPA/PAI-1 complex, enzyme-linked immunosorbent assay for uPAR, solid-phase enzyme immunoassay for ERalpha, and quantitative polymerase chain reaction. Estradiol attenuates the stimulatory effect of EGF on cell migration and uPAR expression. Specifically, E(2) reduces the very rapid increase of detergent extractable uPAR, which occurs within minutes of EGF stimulation and probably represents mobilization of uPAR from detergent-resistant domains such as lipid rafts. Estradiol influenced neither the amount of uPAR mRNA nor the rate of uPAR degradation or solubilization. The nuclear ER antagonists ICI 182780 and tamoxifen, which are GPR30 agonists, as well as the specifically constructed GPR30 agonist G1, mimicked the effect of E(2) on uPAR expression and cell migration. OVCAR-3 cells express mRNA for GPR30.Estradiol attenuates EGF-induced mobilization of ligated uPAR from detergent-resistant domains and subsequent migration in ovarian cancer cells. The response to various ER ligands indicates that this effect is mediated via the membrane ER GPR30.

PAR2 expression in peripheral blood monocytes of patients with rheumatoid arthritis

PubMed Central

Crilly, A; Burns, E; Nickdel, M B; Lockhart, J C; Perry, M E; Ferrell, P W; Baxter, D; Dale, J; Dunning, L; Wilson, H; Nijjar, J S; Gracie, J A; Ferrell, W R; McInnes, I B

2012-01-01

Objectives Proteinase-activated receptor 2 (PAR2) is a G protein-coupled receptor activated by serine proteinases with proinflammatory activity. A study was undertaken to investigate the presence and functional significance of PAR2 expression on rheumatoid arthritis (RA)-derived leucocyte subsets. Methods Venous blood was obtained from patients with RA and osteoarthritis (OA) as well as healthy control subjects. Surface expression of PAR2 on peripheral blood mononuclear cells (PBMCs) was analysed by flow cytometry and interleukin 6 (IL-6) generation by ELISA. Results Patients with RA had elevated but variable surface expression of PAR2 on CD14+ monocytes compared with control subjects (median (1st to 3rd quartiles) 1.76% (0.86–4.10%) vs 0.06% (0.03–0.81%), p<0.0001). CD3+ T cells showed a similar pattern with significantly higher PAR2 expression in patients with RA compared with controls (3.05% (0.36–11.82%) vs 0.08% (0.02–0.28%), p<0.0001). For both subsets, PAR2 expression was significantly higher (p<0.00001) in patients with high levels of disease activity: PAR2 expression for both CD14+ and CD3+ cells correlated to C reactive protein and erythrocyte sedimentation rate. Furthermore, in a cohort of patients with newly diagnosed RA, elevated PAR2 expression in both CD14+ and CD3+ cells was significantly reduced 3 months after methotrexate or sulfasalazine treatment and this reduction correlated significantly with the reduction in the 28-joint Disease Activity Scale score (p<0.05). PAR2 expression on cells from patients with OA was low, similar to levels seen in control subjects. Generation of IL-6 by monocytes in response to a selective PAR2 agonist was significantly greater in patients with RA than in patients with OA and control subjects (p<0.05). Conclusions These findings are consistent with a pathogenic role for PAR2 in RA. PMID:22294633

Cortical geometry may influence placement of interface between Par protein domains in early Caenorhabditis elegans embryos.

PubMed

Dawes, Adriana T; Iron, David

2013-09-21

During polarization, proteins and other polarity determinants segregate to the opposite ends of the cell (the poles) creating biochemically and dynamically distinct regions. Embryos of the nematode worm Caenorhabditis elegans (C. elegans) polarize shortly after fertilization, creating distinct regions of Par protein family members. These regions are maintained through to first cleavage when the embryo divides along the plane specified by the interface between regions, creating daughter cells with different protein content. In wild type single cell embryos the interface between these Par protein regions is reliably positioned at approximately 60% egg length, however, it is not known what mechanisms are responsible for specifying the position of the interface. In this investigation, we use two mathematical models to investigate the movement and positioning of the interface: a biologically based reaction-diffusion model of Par protein dynamics, and the analytically tractable perturbed Allen-Cahn equation. When we numerically simulate the models on a static 2D domain with constant thickness, both models exhibit a persistently moving interface that specifies the boundary between distinct regions. When we modify the simulation domain geometry, movement halts and the interface is stably positioned where the domain thickness increases. Using asymptotic analysis with the perturbed Allen-Cahn equation, we show that interface movement depends explicitly on domain geometry. Using a combination of analytic and numeric techniques, we demonstrate that domain geometry, a historically overlooked aspect of cellular simulations, may play a significant role in spatial protein patterning during polarization. Copyright © 2013 Elsevier Ltd. All rights reserved.

A systematic analysis of factors localized to damaged chromatin reveals PARP-dependent recruitment of transcription factors

PubMed Central

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C.; Westbrook, Thomas F.; Harper, J. Wade; Elledge, Stephen J.

2015-01-01

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify new DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the ALS candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a PARP-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors and >70% of randomly tested transcription factors localized to sites of DNA damage and approximately 90% were PARP-dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding domain-dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP-dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. PMID:26004182

The potential regulatory roles of NAD(+) and its metabolism in autophagy.

PubMed

Zhang, Dong-Xia; Zhang, Jia-Ping; Hu, Jiong-Yu; Huang, Yue-Sheng

2016-04-01

(Macro)autophagy mediates the bulk degradation of defective organelles, long-lived proteins and protein aggregates in lysosomes and plays a critical role in cellular and tissue homeostasis. Defective autophagy processes have been found to contribute to a variety of metabolic diseases. However, the regulatory mechanisms of autophagy are not fully understood. Increasing data indicate that nicotinamide adenine nucleotide (NAD(+)) homeostasis correlates intimately with autophagy. NAD(+) is a ubiquitous coenzyme that functions primarily as an electron carrier of oxidoreductase in multiple redox reactions. Both NAD(+) homeostasis and its metabolism are thought to play critical roles in regulating autophagy. In this review, we discuss how the regulation of NAD(+) and its metabolism can influence autophagy. We focus on the regulation of NAD(+)/NADH homeostasis and the effects of NAD(+) consumption by poly(ADP-ribose) (PAR) polymerase-1 (PARP-1), NAD(+)-dependent deacetylation by sirtuins and NAD(+) metabolites on autophagy processes and the underlying mechanisms. Future studies should provide more direct evidence for the regulation of autophagy processes by NAD(+). A better understanding of the critical roles of NAD(+) and its metabolites on autophagy will shed light on the complexity of autophagy regulation, which is essential for the discovery of new therapeutic tools for autophagy-related diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Physiological factors that regulate skin pigmentation.

PubMed

Yamaguchi, Yuji; Hearing, Vincent J

2009-01-01

More than 150 genes have been identified that affect skin color either directly or indirectly, and we review current understanding of physiological factors that regulate skin pigmentation. We focus on melanosome biogenesis, transport and transfer, melanogenic regulators in melanocytes, and factors derived from keratinocytes, fibroblasts, endothelial cells, hormones, inflammatory cells, and nerves. Enzymatic components of melanosomes include tyrosinase, tyrosinase-related protein 1, and dopachrome tautomerase, which depend on the functions of OA1, P, MATP, ATP7A, and BLOC-1 to synthesize eumelanins and pheomelanins. The main structural component of melanosomes is Pmel17/gp100/Silv, whose sorting involves adaptor protein 1A (AP1A), AP1B, AP2, and spectrin, as well as a chaperone-like component, MART-1. During their maturation, melanosomes move from the perinuclear area toward the plasma membrane. Microtubules, dynein, kinesin, actin filaments, Rab27a, melanophilin, myosin Va, and Slp2-a are involved in melanosome transport. Foxn1 and p53 up-regulate skin pigmentation via bFGF and POMC derivatives including alpha-MSH and ACTH, respectively. Other critical factors that affect skin pigmentation include MC1R, CREB, ASP, MITF, PAX3, SOX9/10, LEF-1/TCF, PAR-2, DKK1, SCF, HGF, GM-CSF, endothelin-1, prostaglandins, leukotrienes, thromboxanes, neurotrophins, and neuropeptides. UV radiation up-regulates most factors that increase melanogenesis. Further studies will elucidate the currently unknown functions of many other pigment genes/proteins. (c) 2009 International Union of Biochemistry and Molecular Biology, Inc.

Expression of protease-activated receptor-2 (PAR-2) in patients with nasopharyngeal carcinoma: correlation with clinicopathological features and prognosis.

PubMed

Li, Zhi; Bian, Li-Juan; Li, Yang; Liang, Ying-Jie; Liang, Hui-Zhen

2009-01-01

We aimed at determining whether the expression of protease-activated receptor 2 (PAR-2) is involved in the progression of nasopharyngeal carcinoma (NPC) and correlated with latent membrane protein 1 (LMP-1), matrix metalloproteinases-9 (MMP9), and angiogenesis of tumor. PAR-2, LMP-1, and MMP9 expressions were detected in 57 biopsies of primary NPC by immunohistochemistry. The presence of Epstein-Barr virus (EBV) was determined using EBER in situ hybridization, and intratumoral microvessels were highlighted by staining endothelial cells for anti-CD34. The correlations with immunostainings and clinicopathological factors, as well as the follow-up data of patients, were analyzed statistically. Strong expression of PAR-2 in 61.4% (35/57) of the biopsies was correlated with extensive lymph node metastasis and advanced stage of NPC. The patients with PAR-2/LMP-1 or PAR-2/MMP9 dual high-expression tumors had a significant worse prognosis than those with single protein high expression and dual low or negative expression tumors (P=0.013 and 0.004, respectively). Angiogenesis in the tumor is related to overall survival of NPC patients (P=0.001), and exhibits strong PAR-2 expression or LMP-1 expression in tumors associated with increased intratumoral microvessel density (P=0.026 and 0.006, respectively). PAR-2 is a possible mediator cooperating with LMP-1 and MMP9 to influence the progression of NPC by inducing angiogenesis and promoting lymph node metastasis.

Rap1, Canoe and Mbt cooperate with Bazooka to promote zonula adherens assembly in the fly photoreceptor

PubMed Central

Burki, Mubarik

2018-01-01

ABSTRACT In Drosophila epithelial cells, apical exclusion of Bazooka (the Drosophila Par3 protein) defines the position of the zonula adherens (ZA), which demarcates the apical and lateral membrane and allows cells to assemble into sheets. Here, we show that the small GTPase Rap1, its effector Canoe (Cno) and the Cdc42 effector kinase Mushroom bodies tiny (Mbt), converge in regulating epithelial morphogenesis by coupling stabilization of the adherens junction (AJ) protein E-Cadherin and Bazooka retention at the ZA. Furthermore, our results show that the localization of Rap1, Cno and Mbt at the ZA is interdependent, indicating that their functions during ZA morphogenesis are interlinked. In this context, we find the Rap1-GEF Dizzy is enriched at the ZA and our results suggest that it promotes Rap1 activity during ZA morphogenesis. Altogether, we propose the Dizzy, Rap1 and Cno pathway and Mbt converge in regulating the interface between Bazooka and AJ material to promote ZA morphogenesis. PMID:29507112

Getting to the core of cadherin complex function in Caenorhabditis elegans.

PubMed

Hardin, Jeff

2015-01-01

The classic cadherin-catenin complex (CCC) mediates cell-cell adhesion in metazoans. Although substantial insights have been gained by studying the CCC in vertebrate tissue culture, analyzing requirements for and regulation of the CCC in vertebrates remains challenging. Caenorhabditis elegans is a powerful system for connecting the molecular details of CCC function with functional requirements in a living organism. Recent data, using an "angstroms to embryos" approach, have elucidated functions for key residues, conserved across all metazoans, that mediate cadherin/β-catenin binding. Other recent work reveals a novel, potentially ancestral, role for the C. elegans p120ctn homologue in regulating polarization of blastomeres in the early embryo via Cdc42 and the partitioning-defective (PAR)/atypical protein kinase C (aPKC) complex. Finally, recent work suggests that the CCC is trafficked to the cell surface via the clathrin adaptor protein complex 1 (AP-1) in surprising ways. These studies continue to underscore the value of C. elegans as a model system for identifying conserved molecular mechanisms involving the CCC.

Sam68 Is Required for DNA Damage Responses via Regulating Poly(ADP-ribosyl)ation

PubMed Central

Hodgson, Andrea; Wier, Eric M.; Wen, Matthew G.; Kamenyeva, Olena; Xia, Xue; Koo, Lily Y.

2016-01-01

The rapid and robust synthesis of polymers of adenosine diphosphate (ADP)-ribose (PAR) chains, primarily catalyzed by poly(ADP-ribose) polymerase 1 (PARP1), is crucial for cellular responses to DNA damage. However, the precise mechanisms through which PARP1 is activated and PAR is robustly synthesized are not fully understood. Here, we identified Src-associated substrate during mitosis of 68 kDa (Sam68) as a novel signaling molecule in DNA damage responses (DDRs). In the absence of Sam68, DNA damage-triggered PAR production and PAR-dependent DNA repair signaling were dramatically diminished. With serial cellular and biochemical assays, we demonstrated that Sam68 is recruited to and significantly overlaps with PARP1 at DNA lesions and that the interaction between Sam68 and PARP1 is crucial for DNA damage-initiated and PARP1-conferred PAR production. Utilizing cell lines and knockout mice, we illustrated that Sam68-deleted cells and animals are hypersensitive to genotoxicity caused by DNA-damaging agents. Together, our findings suggest that Sam68 plays a crucial role in DDR via regulating DNA damage-initiated PAR production. PMID:27635653

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar.

PubMed

Aisiku, Omozuanvbo; Peters, Christian G; De Ceunynck, Karen; Ghosh, Chandra C; Dilks, James R; Fustolo-Gunnink, Susanna F; Huang, Mingdong; Dockendorff, Chris; Parikh, Samir M; Flaumenhaft, Robert

2015-03-19

Protease-activated receptor-1 (PAR1) couples the coagulation cascade to platelet activation during myocardial infarction and to endothelial inflammation during sepsis. This receptor demonstrates marked signaling bias. Its activation by thrombin stimulates prothrombotic and proinflammatory signaling, whereas its activation by activated protein C (APC) stimulates cytoprotective and antiinflammatory signaling. A challenge in developing PAR1-targeted therapies is to inhibit detrimental signaling while sparing beneficial pathways. We now characterize a novel class of structurally unrelated small-molecule PAR1 antagonists, termed parmodulins, and compare the activity of these compounds to previously characterized compounds that act at the PAR1 ligand-binding site. We find that parmodulins target the cytoplasmic face of PAR1 without modifying the ligand-binding site, blocking signaling through Gαq but not Gα13 in vitro and thrombus formation in vivo. In endothelium, parmodulins inhibit prothrombotic and proinflammatory signaling without blocking APC-mediated pathways or inducing endothelial injury. In contrast, orthosteric PAR1 antagonists such as vorapaxar inhibit all signaling downstream of PAR1. Furthermore, exposure of endothelial cells to nanomolar concentrations of vorapaxar induces endothelial cell barrier dysfunction and apoptosis. These studies demonstrate how functionally selective antagonism can be achieved by targeting the cytoplasmic face of a G-protein-coupled receptor to selectively block pathologic signaling while preserving cytoprotective pathways. © 2015 by The American Society of Hematology.

Cell painting with an engineered EPCR to augment the protein C system.

PubMed

Bouwens, Eveline A M; Stavenuiter, Fabian; Mosnier, Laurent O

2015-11-25

The protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC's effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR's bioavailability via "cell painting." The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400 % of wild-type cells after 2 hours and remained > 200 % for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalised PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signalling. Therefore, EPCR-GPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR- depleted and -deficient cells.

SHOX Haploinsufficiency as a Cause of Syndromic and Nonsyndromic Short Stature

PubMed Central

Fukami, Maki; Seki, Atsuhito; Ogata, Tsutomu

2016-01-01

SHOX in the short arm pseudoautosomal region (PAR1) of sex chromosomes is one of the major growth genes in humans. SHOX haploinsufficiency results in idiopathic short stature and Léri-Weill dyschondrosteosis and is associated with the short stature of patients with Turner syndrome. The SHOX protein likely controls chondrocyte apoptosis by regulating multiple target genes including BNP,Fgfr3, Agc1, and Ctgf. SHOX haploinsufficiency frequently results from deletions and duplications in PAR1 involving SHOX exons and/or the cis-acting enhancers, while exonic point mutations account for a small percentage of cases. The clinical severity of SHOX haploinsufficiency reflects hormonal conditions rather than mutation types. Growth hormone treatment seems to be beneficial for cases with SHOX haploinsufficiency, although the long-term outcomes of this therapy require confirmation. Future challenges in SHOX research include elucidating its precise function in the developing limbs, identifying additional cis-acting enhancers, and determining optimal therapeutic strategies for patients. PMID:27194967

Predicting Mortality in African Americans With Type 2 Diabetes Mellitus: Soluble Urokinase Plasminogen Activator Receptor, Coronary Artery Calcium, and High-Sensitivity C-Reactive Protein.

PubMed

Hayek, Salim S; Divers, Jasmin; Raad, Mohamad; Xu, Jianzhao; Bowden, Donald W; Tracy, Melissa; Reiser, Jochen; Freedman, Barry I

2018-05-01

Type 2 diabetes mellitus is a major risk factor for cardiovascular disease; however, outcomes in individual patients vary. Soluble urokinase plasminogen activator receptor (suPAR) is a bone marrow-derived signaling molecule associated with adverse cardiovascular and renal outcomes in many populations. We characterized the determinants of suPAR in African Americans with type 2 diabetes mellitus and assessed whether levels were useful for predicting mortality beyond clinical characteristics, coronary artery calcium (CAC), and high-sensitivity C-reactive protein (hs-CRP). We measured plasma suPAR levels in 500 African Americans with type 2 diabetes mellitus enrolled in the African American-Diabetes Heart Study. We used Kaplan-Meier curves and Cox proportional hazards models adjusting for clinical characteristics, CAC, and hs-CRP to examine the association between suPAR and all-cause mortality. Last, we report the change in C-statistics comparing the additive values of suPAR, hs-CRP, and CAC to clinical models for prediction of mortality. The suPAR levels were independently associated with female sex, smoking, insulin use, decreased kidney function, albuminuria, and CAC. After a median 6.8-year follow-up, a total of 68 deaths (13.6%) were recorded. In a model incorporating suPAR, CAC, and hs-CRP, only suPAR was significantly associated with mortality (hazard ratio 2.66, 95% confidence interval 1.63-4.34). Addition of suPAR to a baseline clinical model significantly improved the C-statistic for all-cause death (Δ0.05, 95% confidence interval 0.01-0.10), whereas addition of CAC or hs-CRP did not. In African Americans with type 2 diabetes mellitus, suPAR was strongly associated with mortality and improved risk discrimination metrics beyond traditional risk factors, CAC and hs-CRP. Studies addressing the clinical usefulness of measuring suPAR concentrations are warranted. © 2018 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley.

A New Class of Orthosteric uPAR•uPA Small-Molecule Antagonists Are Allosteric Inhibitors of the uPAR•Vitronectin Interaction

PubMed Central

Liu, Degang; Zhou, Donghui; Wang, Bo; Knabe, William Eric; Meroueh, Samy O.

2015-01-01

The urokinase receptor (uPAR) is a GPI-anchored cell surface receptor that is at the center of an intricate network of protein-protein interactions. Its immediate binding partners are the serine proteinase urokinase (uPA), and vitronectin (VTN), a component of the extracellular matrix. uPA and VTN bind at distinct sites on uPAR to promote extracellular matrix degradation and integrin signaling, respectively. Here, we report the discovery of a new class of pyrrolone small-molecule inhibitors of the tight ∼1 nM uPAR•uPA protein-protein interaction. These compounds were designed to bind to the uPA pocket on uPAR. The highest affinity compound, namely 7, displaced a fluorescently-labeled α-helical peptide (AE147-FAM) with an inhibition constant Ki of 0.7 µM and inhibited the tight uPAR•uPAATF interaction with an IC50 of 18 µM. Biophysical studies with surface plasmon resonance showed that VTN binding is highly dependent on uPA. This cooperative binding was confirmed as 7, which binds at the uPAR•uPA interface, also inhibited the distal VTN•uPAR interaction. In cell culture, 7 blocked the uPAR•uPA interaction in uPAR-expressing human embryonic kidney (HEK-293) cells, and impaired cell adhesion to VTN, a process that is mediated by integrins. As a result, 7 inhibited integrin signaling in MDA-MB-231 cancer cells as evidenced by a decrease in focal adhesion kinase (FAK) phosphorylation and Rac1 GTPase activation. Consistent with these results, 7 blocked breast MDA-MB-231 cancer cell invasion with IC50 values similar to those observed in ELISA and surface plasmon resonance competition studies. Explicit-solvent molecular dynamics simulations show that the cooperativity between uPA and VTN is attributed to stabilization of uPAR motion by uPA. In addition, free energy calculations revealed that uPA stabilizes the VTN•uPARSMB interaction through more favorable electrostatics and entropy. Disruption of the uPAR•VTNSMB interaction by 7 is consistent with the cooperative binding to uPAR by uPA and VTN. Interestingly, the VTNSMB•uPAR interaction was less favorable in the VTNSMB•uPAR•7 complex suggesting potential cooperativity between 7 and VTN. Compound 7 provides an excellent starting point for the development of more potent derivatives to explore uPAR biology. PMID:25671694

Formation of a PKCζ/β-catenin complex in endothelial cells promotes angiopoietin-1–induced collective directional migration and angiogenic sprouting

PubMed Central

Oubaha, Malika; Lin, Michelle I.; Margaron, Yoran; Filion, Dominic; Price, Emily N.; Zon, Leonard I.; Côté, Jean-François

2012-01-01

Angiogenic sprouting requires that cell-cell contacts be maintained during migration of endothelial cells. Angiopoietin-1 (Ang-1) and vascular endothelial growth factor act oppositely on endothelial cell junctions. We found that Ang-1 promotes collective and directional migration and, in contrast to VEGF, induces the formation of a complex formed of atypical protein kinase C (PKC)-ζ and β-catenin at cell-cell junctions and at the leading edge of migrating endothelial cells. This complex brings Par3, Par6, and adherens junction proteins at the front of migrating cells to locally activate Rac1 in response to Ang-1. The colocalization of PKCζ and β-catenin at leading edge along with PKCζ-dependent stabilization of cell-cell contacts promotes directed and collective endothelial cell migration. Consistent with these results, down-regulation of PKCζ in endothelial cells alters Ang-1–induced sprouting in vitro and knockdown in developing zebrafish results in intersegmental vessel defects caused by a perturbed directionality of tip cells and by loss of cell contacts between tip and stalk cells. These results reveal that PKCζ and β-catenin function in a complex at adherens junctions and at the leading edge of migrating endothelial cells to modulate collective and directional migration during angiogenesis. PMID:22936663

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA.

PubMed

Liro, Małgorzata J; Rose, Lesilee S

2016-11-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. Copyright © 2016 by the Genetics Society of America.

Mitotic Spindle Positioning in the EMS Cell of Caenorhabditis elegans Requires LET-99 and LIN-5/NuMA

PubMed Central

Liro, Małgorzata J.; Rose, Lesilee S.

2016-01-01

Asymmetric divisions produce daughter cells with different fates, and thus are critical for animal development. During asymmetric divisions, the mitotic spindle must be positioned on a polarized axis to ensure the differential segregation of cell fate determinants into the daughter cells. In many cell types, a cortically localized complex consisting of Gα, GPR-1/2, and LIN-5 (Gαi/Pins/Mud, Gαi/LGN/NuMA) mediates the recruitment of dynactin/dynein, which exerts pulling forces on astral microtubules to physically position the spindle. The conserved PAR polarity proteins are known to regulate both cytoplasmic asymmetry and spindle positioning in many cases. However, spindle positioning also occurs in response to cell signaling cues that appear to be PAR-independent. In the four-cell Caenorhabditis elegans embryo, Wnt and Mes-1/Src-1 signaling pathways act partially redundantly to align the spindle on the anterior/posterior axis of the endomesodermal (EMS) precursor cell. It is unclear how those extrinsic signals individually contribute to spindle positioning and whether either pathway acts via conserved spindle positioning regulators. Here, we genetically test the involvement of Gα, LIN-5, and their negative regulator LET-99, in transducing EMS spindle positioning polarity cues. We also examined whether the C. elegans ortholog of another spindle positioning regulator, DLG-1, is required. We show that LET-99 acts in the Mes-1/Src-1 pathway for spindle positioning. LIN-5 is also required for EMS spindle positioning, possibly through a Gα- and DLG-1-independent mechanism. PMID:27672093

Photoperiod regulates multiple gene expression in the suprachiasmatic nuclei and pars tuberalis of the Siberian hamster (Phodopus sungorus).

PubMed

Johnston, Jonathan D; Ebling, Francis J P; Hazlerigg, David G

2005-06-01

Photoperiod regulates the seasonal physiology of many mammals living in temperate latitudes. Photoperiodic information is decoded by the master circadian clock in the suprachiasmatic nuclei (SCN) of the hypothalamus and then transduced via pineal melatonin secretion. This neurochemical signal is interpreted by tissues expressing melatonin receptors (e.g. the pituitary pars tuberalis, PT) to drive physiological changes. In this study we analysed the photoperiodic regulation of the circadian clockwork in the SCN and PT of the Siberian hamster. Female hamsters were exposed to either long or short photoperiod for 8 weeks and sampled at 2-h intervals across the 24-h cycle. In the SCN, rhythmic expression of the clock genes Per1, Per2, Cry1, Rev-erbalpha, and the clock-controlled genes arginine vasopressin (AVP) and d-element binding protein (DBP) was modulated by photoperiod. All of these E-box-containing genes tracked dawn, with earlier peak mRNA expression in long, compared to short, photoperiod. This response occurred irrespective of the presence of additional regulatory cis-elements, suggesting photoperiodic regulation of SCN gene expression through a common E-box-related mechanism. In long photoperiod, expression of Cry1 and Per1 in the PT tracked the onset and offset of melatonin secretion, respectively. However, whereas Cry1 tracked melatonin onset in short period, Per1 expression was not detectably rhythmic. We therefore propose that, in the SCN, photoperiodic regulation of clock gene expression primarily occurs via E-boxes, whereas melatonin-driven signal transduction drives the phasing of a subset of clock genes in the PT, independently of the E-box.

Different strains of Propionibacterium acnes modulate differently the cutaneous innate immunity.

PubMed

Jasson, Fiona; Nagy, Istvan; Knol, Anne Chantal; Zuliani, Thomas; Khammari, Amir; Dréno, Brigitte

2013-09-01

Acne is a chronic inflammatory illness of the pilosebaceous follicle where innate immunity plays a central role. In acne, the density of Propionibacterium acnes is increased in the pilosebaceous unit. We hypothesized that the severity of acne is not only dependent on the proliferation of P. acnes but also dependent on the pro-inflammatory potential of P. acnes strains and consequently constitutes potential triggering factor for acne scarring. We investigated pro-inflammatory potential of five different strains of P. acnes and P. avidum in skin explants and the preventive effect of zinc gluconate. The expression of immune markers was studied by immunohistochemistry, RT-qPCR and ELISA. P. acnes strains modulate differently the expression of immune markers both at gene and at protein levels. P. acnes type III had the highest pro-inflammatory potential by up-regulating the expression of PAR-2, TNF-alpha, MMP-13 and TIMP-2, whereas P. avidum had the weakest by up-regulating only MMP-13 and TIMP-2. Preincubation of zinc gluconate, which is a modulator of innate immunity, down-regulates the expression of most immune markers induced by P. acnes, PAR-2, TIMP-2, up-regulates MMP-1, TIMP-1. Our results demonstrate that different P. acnes strains have different inflammatory potential targeting markers of cutaneous innate immunity, and that inflammatory potential can be down-regulated by zinc gluconate. As such, the inflammatory potential of P. acnes strains on acne skin may influence the severity of inflammatory acne lesions and scars. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Protease-activated receptor 1 and 2 contribute to angiotensin II-induced activation of adventitial fibroblasts from rat aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

He, Rui-Qing; Tang, Xiao-Feng; Zhang, Bao-Li

Adventitial fibroblasts (AFs) can be activated by angiotensin II (Ang II) and exert pro-fibrotic and pro-inflammatory effects in vascular remodeling. Protease-activated receptor (PAR) 1 and 2 play a significant role in fibrogenic and inflammatory diseases. The present study hypothesized that PAR1 and PAR2 are involved in Ang II-induced AF activation and contribute to adventitial remodeling. We found that direct activation of PAR1 and PAR2 with PAR1-AP and PAR2-AP led to AF activation, including proliferation and differentiation of AFs, extracellular matrix synthesis, as well as production of pro-fibrotic cytokine TGF-β and pro-inflammatory cytokines IL-6 and MCP-1. Furthermore, PAR1 and PAR2 mediatedmore » Ang II-induced AF activation, since both PAR1 and PAR2 antagonists inhibited Ang II-induced proliferation, migration, differentiation, extracellular matrix synthesis and production of pro-fibrotic and pro-inflammatory cytokines in AFs. Finally, mechanistic study showed that Ang II, via Ang II type I receptor (AT1R), upregulated both PAR1 and PAR2 expression, and transactivated PAR1 and PAR2, as denoted by internalization of both proteins. In conclusion, our results suggest that PAR1 and PAR2 play a critical role in Ang II-induced AF activation, and this may contribute to adventitia-related pathological changes. - Highlights: • Direct activation of PAR1 and PAR2 led to adventitial fibroblast (AF) activation. • PAR1 and PAR2 antagonists attenuated Ang II-induced AF activation. • Ang II induced the upregulation and transactivation of PAR1/PAR2 in AFs.« less

Urokinase and its Receptors in Chronic Kidney Disease

PubMed Central

Zhang, Guoqiang; Eddy, Allison A.

2011-01-01

Since the recognition that plasminogen activator inhibitor-1 (PAI-1) is a powerful profibrotic molecule, there has been considerable interest in deciphering the extent to which this effect is mediated by its ability to inhibit serine proteases with downstream effects on fibrogenesis. This review will summarize current knowledge about the serine protease urokinase-type plasminogen activator and its high affinity receptor uPAR/CD87 as it pertains to chronic kidney disease (CKD) progression. An emerging theme is that the effects of PAI-1 and uPAR appear to be organ- and site-specific. Normal kidney tubules produce a large quantity of uPA that is secreted into the urinary space. Activity levels increase during CKD presumably due to new sources of production by macrophages and fibroblasts. By activating hepatocyte growth factor and degrading fibrinogen uPA may have anti-fibrotic effects. However CKD severity after experimental ureteral obstruction is not altered by endogenous uPA deficiency. Beneficial effects of exogenous uPA have been reported in experimental models of fibrosis in the lung and liver but CKD awaits exploration. Absent in normal kidneys uPAR is expressed by both renal parenchymal cells and inflammatory cells in a variety of pathological states. Such expression appears beneficial based on studies performed in uPAR-deficient mice. The uPAR promotes bacterial clearance in infectious diseases. In CKD uPAR expression is associated with high uPA activity but its most important effect appears to be due to scavenging activities and effects on cell recruitment and migration. Although uPAR itself is a non-signaling receptor, it interacts with a variety of co-receptors to modify cellular behavior. Best known are interactions with the low-density lipoprotein receptor-related protein (LRP-1) that lead to PAI-1 endocytosis and degradation, and interactions with several integrins to regulate matrix-dependent cell migration. Contacts with the receptor for the complement C5a component and the interleukin −6 receptor gp130 are examples of other recently recognized interactions. In addition to uPA, vitronectin and high molecular weight kininogen are alternate uPAR ligands that could be implicated in CKD progression. uPAR may also be shed from cell membranes. This soluble form (suPAR) has been detected in plasma and urine and is known to be a chemoattractant for leukocytes that express the formyl-peptide-receptor-like receptor 1/lipoxin A4 receptor. In addition to uPAR several other receptors, including some of the uPAR co-receptors, may also bind directly to uPA and activate cell signaling pathways. The roles of these newer uPAR ligands and uPA receptors are just beginning to be investigated. Since many of them are expressed in the kidney, their potential participation in CKD pathogenesis will be of interest. PMID:18508599

Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2–mediated heparin-binding EGF signaling in endothelial cells

PubMed Central

Svensson, Katrin J.; Kucharzewska, Paulina; Christianson, Helena C.; Sköld, Stefan; Löfstedt, Tobias; Johansson, Maria C.; Mörgelin, Matthias; Bengzon, Johan; Ruf, Wolfram; Belting, Mattias

2011-01-01

Highly malignant tumors, such as glioblastomas, are characterized by hypoxia, endothelial cell (EC) hyperplasia, and hypercoagulation. However, how these phenomena of the tumor microenvironment may be linked at the molecular level during tumor development remains ill-defined. Here, we provide evidence that hypoxia up-regulates protease-activated receptor 2 (PAR-2), i.e., a G-protein–coupled receptor of coagulation-dependent signaling, in ECs. Hypoxic induction of PAR-2 was found to elicit an angiogenic EC phenotype and to specifically up-regulate heparin-binding EGF-like growth factor (HB-EGF). Inhibition of HB-EGF by antibody neutralization or heparin treatment efficiently counteracted PAR-2–mediated activation of hypoxic ECs. We show that PAR-2–dependent HB-EGF induction was associated with increased phosphorylation of ERK1/2, and inhibition of ERK1/2 phosphorylation attenuated PAR-2–dependent HB-EGF induction as well as EC activation. Tissue factor (TF), i.e., the major initiator of coagulation-dependent PAR signaling, was substantially induced by hypoxia in several types of cancer cells, including glioblastoma; however, TF was undetectable in ECs even at prolonged hypoxia, which precludes cell-autonomous PAR-2 activation through TF. Interestingly, hypoxic cancer cells were shown to release substantial amounts of TF that was mainly associated with secreted microvesicles with exosome-like characteristics. Vesicles derived from glioblastoma cells were found to trigger TF/VIIa–dependent activation of hypoxic ECs in a paracrine manner. We provide evidence of a hypoxia-induced signaling axis that links coagulation activation in cancer cells to PAR-2–mediated activation of ECs. The identified pathway may constitute an interesting target for the development of additional strategies to treat aggressive brain tumors. PMID:21788507

The DNA binding parvulin Par17 is targeted to the mitochondrial matrix by a recently evolved prepeptide uniquely present in Hominidae

PubMed Central

Kessler, Daniel; Papatheodorou, Panagiotis; Stratmann, Tina; Dian, Elke Andrea; Hartmann-Fatu, Cristina; Rassow, Joachim; Bayer, Peter; Mueller, Jonathan Wolf

2007-01-01

Background The parvulin-type peptidyl prolyl cis/trans isomerase Par14 is highly conserved in all metazoans. The recently identified parvulin Par17 contains an additional N-terminal domain whose occurrence and function was the focus of the present study. Results Based on the observation that the human genome encodes Par17, but bovine and rodent genomes do not, Par17 exon sequences from 10 different primate species were cloned and sequenced. Par17 is encoded in the genomes of Hominidae species including humans, but is absent from other mammalian species. In contrast to Par14, endogenous Par17 was found in mitochondrial and membrane fractions of human cell lysates. Fluorescence of EGFP fusions of Par17, but not Par14, co-localized with mitochondrial staining. Par14 and Par17 associated with isolated human, rat and yeast mitochondria at low salt concentrations, but only the Par17 mitochondrial association was resistant to higher salt concentrations. Par17 was imported into mitochondria in a time and membrane potential-dependent manner, where it reached the mitochondrial matrix. Moreover, Par17 was shown to bind to double-stranded DNA under physiological salt conditions. Conclusion Taken together, the DNA binding parvulin Par17 is targeted to the mitochondrial matrix by the most recently evolved mitochondrial prepeptide known to date, thus adding a novel protein constituent to the mitochondrial proteome of Hominidae. PMID:17875217

Cell painting with an engineered EPCR to augment the protein C system

PubMed Central

Bouwens, Eveline A. M.; Stavenuiter, Fabian; Mosnier, Laurent O.

2016-01-01

The protein C (PC) system conveys beneficial anticoagulant and cytoprotective effects in numerous in vivo disease models. The endothelial protein C receptor (EPCR) plays a central role in these pathways as cofactor for PC activation and by enhancing activated protein C (APC)-mediated protease-activated receptor (PAR) activation. During inflammatory disease, expression of EPCR on cell membranes is often diminished thereby limiting PC activation and APC’s effects on cells. Here a caveolae-targeting glycosylphosphatidylinositol (GPI)-anchored EPCR (EPCR-GPI) was engineered to restore EPCR’s bioavailability via “cell painting.” The painting efficiency of EPCR-GPI on EPCR-depleted endothelial cells was time- and dose-dependent. The EPCR-GPI bioavailability after painting was long lasting since EPCR surface levels reached 400% of wild-type cells after 2 hours and remained >200% for 24 hours. EPCR-GPI painting conveyed APC binding to EPCR-depleted endothelial cells where EPCR was lost due to shedding or shRNA. EPCR painting normalized PC activation on EPCR-depleted cells indicating that EPCR-GPI is functional active on painted cells. Caveolin-1 lipid rafts were enriched in EPCR after painting due to the GPI-anchor targeting caveolae. Accordingly, EPCR painting supported PAR1 and PAR3 cleavage by APC and augmented PAR1-dependent Akt phosphorylation by APC. Thus, EPCR-GPI painting achieved physiological relevant surface levels on endothelial cells, restored APC binding to EPCR-depleted cells, supported PC activation, and enhanced APC-mediated PAR cleavage and cytoprotective signaling. Therefore, EPCR-GPI provides a novel tool to restore the bioavailability and functionality of EPCR on EPCR-depleted and deficient cells. PMID:26272345

Soluble Urokinase-Type Plasminogen Activator Receptor in Black Americans with CKD.

PubMed

Luo, Shengyuan; Coresh, Josef; Tin, Adrienne; Rebholz, Casey M; Chen, Teresa K; Hayek, Salim S; Tracy, Melissa; Lipkowitz, Michael S; Appel, Lawrence J; Levey, Andrew S; Inker, Lesley A; Reiser, Jochen; Grams, Morgan Erika

2018-06-14

Black Americans with and without APOL1 kidney disease risk variants face high risk of ESKD. Soluble urokinase-type plasminogen activator receptor (suPAR), a circulating signaling protein and marker of immune activation, constitutes a promising biomarker of CKD-associated risks. We aimed to quantify the associations between serum suPAR concentration and adverse outcomes in Black Americans with and without APOL1 kidney disease risk variants, over and above iodine-125 iothalamate measured GFR and proteinuria. Using data from the African-American Study of Kidney Disease and Hypertension, a multicenter clinical trial followed by a cohort phase with a median total follow-up of 9.7 years (interquartile range, 6.5-10.9 years), we examined the associations of suPAR with CKD progression (defined as doubling of serum creatinine or ESKD), ESKD, worsening proteinuria (defined as pre-ESKD doubling of 24-hour urine protein-to-creatinine ratio to ≥220 mg/g), and all-cause death. At baseline, the median suPAR was 4462 pg/ml, mean measured GFR was 46 ml/min per 1.73 m 2 , and median 24-hour urine protein-to-creatinine ratio was 80 mg/g. After controlling for baseline demographics, randomization arm, GFR, proteinuria, APOL1 risk status, and clinical risk factors, there was a 1.26-times higher risk for CKD progression per SD higher baseline log-transformed suPAR (hazard ratio [HR], 1.26; 95% confidence interval [95% CI], 1.11 to 1.43; P <0.001). Higher suPAR was also independently associated with risk of ESKD (HR, 1.36; 95% CI, 1.17 to 1.58; P <0.001) and death (HR, 1.25; 95% CI, 1.08 to 1.45; P =0.003). suPAR was only associated with worsening proteinuria in patients with two APOLI risk alleles (HR, 1.46; 95% CI, 1.08 to 1.99; P =0.02). Higher suPAR was associated with various adverse outcomes in Black Americans with CKD, with and without APOL1 kidney disease risk variants, independently of proteinuria and GFR. Copyright © 2018 by the American Society of Nephrology.

TRYPTASE/PAR-2 INTERACTIONS INDUCE SELECTIVE MAPK SIGNALING AND COLLAGEN SYNTHESIS BY CARDIAC FIBROBLASTS

PubMed Central

McLarty, Jennifer L.; Meléndez, Giselle C.; Brower, Gregory L.; Janicki, Joseph S.; Levick, Scott P.

2012-01-01

The mast cell product, tryptase, has recently been implicated in fibrosis in the hypertensive heart. Tryptase has been shown to mediate non-cardiac fibroblast function via activation of protease activated receptor-2 and subsequent activation of the mitogen-activated protein kinase pathway, including extracellular signal-regulated kinase1/2. Therefore, we hypothesized that this pathway may be a mechanism leading to fibrosis in the hypertensive heart. Isolated adult cardiac fibroblasts were treated with tryptase, which induced activation of extracellular signal-regulated kinase1/2 via protease activated receptor-2. Blockade of protease activated receptor-2 with FSLLRY (10 μM) and inhibition of the extracellular signal-regulated kinase pathway with PD98059 (10 μM) prevented collagen synthesis in isolated cardiac fibroblasts stimulated with tryptase. p38 mitogen activated protein kinase and stress-activated protein/c-Jun N-terminal kinase were not activated by tryptase. Cardiac fibroblasts isolated from spontaneously hypertensive rats showed this same pattern of activation and treatment of spontaneously hypertensive rats with FSLLRY prevented fibrosis in these animals indicating the in vivo applicability of the cultured fibroblast findings. Also, tryptase induced a myofibroblastic phenotype indicated by elevations in α smooth muscle actin and ED-A fibronectin. Thus, the results from this study demonstrate the importance of tryptase for inducing a cardiac myofibroblastic phenotype, ultimately leading to the development of cardiac fibrosis through the activation of the extracellular signal-regulated kinase pathway. Specifically, tryptase causes cardiac fibroblasts to increase collagen synthesis via a mechanism involving activation of protease activated receptor-2 and subsequent induction of extracellular signal-regulated kinase signaling. PMID:21730297

Activation of blood coagulation in cancer: implications for tumour progression

PubMed Central

Lima, Luize G.; Monteiro, Robson Q.

2013-01-01

Several studies have suggested a role for blood coagulation proteins in tumour progression. Herein, we discuss (1) the activation of the blood clotting cascade in the tumour microenvironment and its impact on primary tumour growth; (2) the intravascular activation of blood coagulation and its impact on tumour metastasis and cancer-associated thrombosis; and (3) antitumour therapies that target blood-coagulation-associated proteins. Expression levels of the clotting initiator protein TF (tissue factor) have been correlated with tumour cell aggressiveness. Simultaneous TF expression and PS (phosphatidylserine) exposure by tumour cells promote the extravascular activation of blood coagulation. The generation of blood coagulation enzymes in the tumour microenvironment may trigger the activation of PARs (protease-activated receptors). In particular, PAR1 and PAR2 have been associated with many aspects of tumour biology. The procoagulant activity of circulating tumour cells favours metastasis, whereas the release of TF-bearing MVs (microvesicles) into the circulation has been correlated with cancer-associated thrombosis. Given the role of coagulation proteins in tumour progression, it has been proposed that they could be targets for the development of new antitumour therapies. PMID:23889169

Loss of partitioning-defective-3/isotype-specific interacting protein (par-3/ASIP) in the elongating spermatid of RA175 (IGSF4A/SynCAM)-deficient mice.

PubMed

Fujita, Eriko; Tanabe, Yuko; Hirose, Tomonori; Aurrand-Lions, Michel; Kasahara, Tadashi; Imhof, Beat A; Ohno, Shigeo; Momoi, Takashi

2007-12-01

IGSF4a/RA175/SynCAM (RA175) and junctional adhesion molecules (Jams) are members of the immunoglobulin superfamily with a PDZ-binding domain at their C termini. Deficiency of Ra175 (Ra175(-/-)) as well as Jam-C deficiency (Jam-C(-/-)) causes the defect of the spermatid differentiation, oligo-astheno-teratozoospermia. Ra175(-/-) elongating spermatids fail to mature further, whereas Jam-C(-/-) round spermatids lose cell polarity, and most of Jam-C(-/-) elongated spermatids are completely lost. RA175 and Jam-C seem to have similar but distinct functional roles during spermatid differentiation. Here we show that the cell polarity protein Par-3 with PDZ domains, a binding partner of Jams, is one of the associated proteins of the cytoplasmic region of RA175 in testis. Par-3 and Jam-C are partly co-localized with RA175 in the elongating and elongated spermatids; their distributions overlapped with that of RA175 on the tips of the dorsal region of the head of the elongating spermatid (steps 9 to 12) in the wild type. In the Ra175(-/-) elongating spermatid, Par-3 was absent, and Jam-C was absent or abnormally localized. The RA175 formed a ternary complex with Jam-C via interaction with Par-3. The lack of the ternary complex in the Ra175(-/-) elongating spermatid may cause the defect of the specialized adhesion structures, resulting in the oligo-astheno-teratozoospermia.

Proteinase-activated receptor 2 and disease biomarkers in cerebrospinal fluid in cases with autopsy-confirmed prion diseases and other neurodegenerative diseases.

PubMed

Rohan, Zdenek; Smetakova, Magdalena; Kukal, Jaromir; Rusina, Robert; Matej, Radoslav

2015-03-31

Proteinase-activated receptor 2 (PAR-2) has been shown to promote both neurotoxic and neuroprotective effects. Similarly, other routinely used nonspecific markers of neuronal damage can be found in cerebrospinal fluid (CSF) and can be used as biomarkers for different neurodegenerative disorders. Using enzyme-linked immunosorbent assays and western blotting we assessed PAR-2, total-tau, phospho-tau, beta-amyloid levels, and protein 14-3-3 in the CSF of former patients who had undergone a neuropathological autopsy after death and who had been definitively diagnosed with a prion or other neurodegenerative disease. We did not find any significant correlation between levels of PAR-2 and other biomarkers, nor did we find any differences in PAR-2 levels between prion diseases and other neurodegenerative conditions. However, we confirmed that very high total-tau levels were significantly associated with definitive prion diagnoses and exhibited greater sensitivity and specificity than protein 14-3-3, which is routinely used as a marker. Our study showed that PAR-2, in CSF, was not specifically altered in prion diseases compared to other neurodegenerative conditions. Our results also confirmed that very high total-tau protein CSF levels were significantly associated with a definitive Creutzfeldt-Jakob disease (CJD) diagnosis and should be routinely tested as a diagnostic marker. Observed individual variability in CSF biomarkers provide invaluable feedback from neuropathological examinations even in "clinically certain" cases.

Family-wide analysis of poly(ADP-ribose) polymerase activity

PubMed Central

Uchima, Lilen; Rood, Jenny; Zaja, Roko; Hay, Ronald T.; Ahel, Ivan; Chang, Paul

2014-01-01

The poly(ADP-ribose) polymerase (PARP) protein family generates ADP-ribose (ADPr) modifications onto target proteins using NAD+ as substrate. Based on the composition of three NAD+ coordinating amino acids, the H-Y-E motif, each PARP is predicted to generate either poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR). However, the reaction product of each PARP has not been clearly defined, and is an important priority since PAR and MAR function via distinct mechanisms. Here we show that the majority of PARPs generate MAR, not PAR, and demonstrate that the H-Y-E motif is not the sole indicator of PARP activity. We identify automodification sites on seven PARPs, and demonstrate that MAR and PAR generating PARPs modify similar amino acids, suggesting that the sequence and structural constraints limiting PARPs to MAR synthesis do not limit their ability to modify canonical amino acid targets. In addition, we identify cysteine as a novel amino acid target for ADP-ribosylation on PARPs. PMID:25043379

Increased protein oxidation in human substantia nigra pars compacta in comparison with basal ganglia and prefrontal cortex measured with an improved dinitrophenylhydrazine assay.

PubMed

Floor, E; Wetzel, M G

1998-01-01

The dopaminergic phenotype of neurons in human substantia nigra deteriorates during normal aging, and loss of these neurons is prominent in Parkinson's disease. These degenerative processes are hypothesized to involve oxidative stress. To compare oxidative stress in the nigra and related regions, we measured carbonyl modifications of soluble proteins in postmortem samples of substantia nigra, basal ganglia, and prefrontal cortex from neurologically normal subjects, using an improved 2,4-dinitrophenylhydrazine assay. The protein carbonyl content was found to be about twofold higher in substantia nigra pars compacta than in the other regions. To further analyze this oxidative damage, the distribution of carbonyl groups on soluble proteins was determined by western immunoblot analysis. This method revealed that carbonyl content of the major proteins in each region was linearly dependent on molecular weight. This distribution raises the possibility that protein carbonyl content is controlled by a size-dependent mechanism in vivo. Our results suggest that oxidative stress is elevated in human substantia nigra pars compacta in comparison with other regions and that oxidative damage is higher within the dopaminergic neurons. Elevated oxidative damage may contribute to the degeneration of nigral dopaminergic neurons in aging and in Parkinson's disease.

Protease-activated receptor 2 activation of myeloid dendritic cells regulates allergic airway inflammation

PubMed Central

2011-01-01

Background A common characteristic of allergens is that they contain proteases that can activate protease-activated receptor (PAR-2); however the mechanism by which PAR-2 regulates allergic airway inflammation is unclear. Methods Mice (wild type and PAR-2-deficient) were sensitized using German cockroach (GC) feces (frass), the isolated protease from GC frass, or through adoptive transfer of GC frass-treated bone marrow-derived dendritic cells (BMDC) and measurements of airway inflammation (cellular infiltration, cytokine expression, and mucin production), serum IgE levels and airway hyperresponsiveness (AHR) were assessed. BMDC were cultured, treated with GC frass and assessed for cytokine production. PAR-2 expression on pulmonary mDCs was determined by flow cytometry. Results Exposure to GC frass induced AHR and airway inflammation in wild type mice; however PAR-2-deficient mice had significantly attenuated responses. To directly investigate the role of the protease, we isolated the protease from GC frass and administered the endotoxin-free protease into the airways of mice in the presence of OVA. GC frass proteases were sufficient to promote the development of AHR, serum IgE, and Th2 cytokine production. PAR-2 expression on mDC was upregulated following GC frass exposure, but the presence of a functional PAR-2 did not alter antigen uptake. To determine if PAR-2 activation led to differential cytokine production, we cultured BMDC in the presence of GM-CSF and treated these cells ex vivo with GC frass. PAR-2-deficient BMDC released significantly less IL-6, IL-23 and TNFα compared to BMDC from wild type mice, suggesting PAR-2 activation was important in Th2/Th17 skewing cytokine production. To determine the role for PAR-2 on mDCs on the initiation of allergic airway inflammation, BMDCs from wild type and PAR-2-deficient mice were treated in the presence or absence of GC frass and then adoptively transferred into the airway of wild type mice. Importantly, GC frass-stimulated wild type BMDCs were sufficient to induce AHR and allergic airway inflammation, while GC frass-stimulated PAR-2-deficient BMDC had attenuated responses. Conclusions Together these data suggest an important role for allergen activation of PAR-2 on mDCs in mediating Th2/Th17 cytokine production and allergic airway responses. PMID:21936897

Comparative in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes clinical isolates with alterations in GyrA and ParC proteins.

PubMed

Brisse, S; Milatovic, D; Fluit, A C; Verhoef, J; Martin, N; Scheuring, S; Köhrer, K; Schmitz, F J

1999-08-01

The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes.

Comparative In Vitro Activities of Ciprofloxacin, Clinafloxacin, Gatifloxacin, Levofloxacin, Moxifloxacin, and Trovafloxacin against Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes Clinical Isolates with Alterations in GyrA and ParC Proteins

PubMed Central

Brisse, Sylvain; Milatovic, Dana; Fluit, Ad C.; Verhoef, Jan; Martin, Nele; Scheuring, Sybille; Köhrer, Karl; Schmitz, Franz-Josef

1999-01-01

The in vitro activities of ciprofloxacin, clinafloxacin, gatifloxacin, levofloxacin, moxifloxacin, and trovafloxacin were tested against 72 ciprofloxacin-resistant and 28 ciprofloxacin-susceptible isolates of Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, and Enterobacter aerogenes. Irrespective of the alterations in GyrA and ParC proteins, clinafloxacin exhibited greater activity than all other fluoroquinolones tested against K. pneumoniae and E. aerogenes. PMID:10428935

Rap1 and Canoe/afadin are essential for establishment of apical–basal polarity in the Drosophila embryo

PubMed Central

Choi, Wangsun; Harris, Nathan J.; Sumigray, Kaelyn D.; Peifer, Mark

2013-01-01

The establishment and maintenance of apical–basal cell polarity is critical for assembling epithelia and maintaining organ architecture. Drosophila embryos provide a superb model. In the current view, apically positioned Bazooka/Par3 is the initial polarity cue as cells form during cellularization. Bazooka then helps to position both adherens junctions and atypical protein kinase C (aPKC). Although a polarized cytoskeleton is critical for Bazooka positioning, proteins mediating this remained unknown. We found that the small GTPase Rap1 and the actin-junctional linker Canoe/afadin are essential for polarity establishment, as both adherens junctions and Bazooka are mispositioned in their absence. Rap1 and Canoe do not simply organize the cytoskeleton, as actin and microtubules become properly polarized in their absence. Canoe can recruit Bazooka when ectopically expressed, but they do not obligatorily colocalize. Rap1 and Canoe play continuing roles in Bazooka localization during gastrulation, but other polarity cues partially restore apical Bazooka in the absence of Rap1 or Canoe. We next tested the current linear model for polarity establishment. Both Bazooka and aPKC regulate Canoe localization despite being “downstream” of Canoe. Further, Rap1, Bazooka, and aPKC, but not Canoe, regulate columnar cell shape. These data reshape our view, suggesting that polarity establishment is regulated by a protein network rather than a linear pathway. PMID:23363604

G protein-coupled receptors: bridging the gap from the extracellular signals to the Hippo pathway.

PubMed

Zhou, Xin; Wang, Zhen; Huang, Wei; Lei, Qun-Ying

2015-01-01

The Hippo pathway is crucial in organ size control, whereas its dysregulation contributes to organ degeneration or tumorigenesis. The kinase cascade of MST1/2 and LATS1/2 and the coupling transcription co-activators YAP/TAZ represent the core components of the Hippo pathway. Extensive studies have identified a number of upstream regulators of the Hippo pathway, including contact inhibition, mechanic stress, extracellular matrix stiffness, cytoskeletal rearrangement, and some molecules of cell polarity and cell junction. However, how the diffuse extracellular signals regulate the Hippo pathway puzzles the researchers for a long time. Unexpectedly, recent elegant studies demonstrated that stimulation of some G protein-coupled receptors (GPCRs), such as lysophosphatidic acid receptor, sphingosine-1-phosphate receptor, and the protease activated receptor PAR1, causes potent YAP/TAZ dephosphorylation and activation by promoting actin cytoskeleton assemble. In this review, we briefly describe the components of the Hippo pathway and focus on the recent progress with respect to the regulation of the Hippo pathway by GPCRs and G proteins in cancer cells. In addition, we also discuss the potential therapeutic roles targeting the Hippo pathway in human cancers. © The Author 2014. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

A Systematic Analysis of Factors Localized to Damaged Chromatin Reveals PARP-Dependent Recruitment of Transcription Factors.

PubMed

Izhar, Lior; Adamson, Britt; Ciccia, Alberto; Lewis, Jedd; Pontano-Vaites, Laura; Leng, Yumei; Liang, Anthony C; Westbrook, Thomas F; Harper, J Wade; Elledge, Stephen J

2015-06-09

Localization to sites of DNA damage is a hallmark of DNA damage response (DDR) proteins. To identify DDR factors, we screened epitope-tagged proteins for localization to sites of chromatin damaged by UV laser microirradiation and found >120 proteins that localize to damaged chromatin. These include the BAF tumor suppressor complex and the amyotrophic lateral sclerosis (ALS) candidate protein TAF15. TAF15 contains multiple domains that bind damaged chromatin in a poly-(ADP-ribose) polymerase (PARP)-dependent manner, suggesting a possible role as glue that tethers multiple PAR chains together. Many positives were transcription factors; > 70% of randomly tested transcription factors localized to sites of DNA damage, and of these, ∼90% were PARP dependent for localization. Mutational analyses showed that localization to damaged chromatin is DNA-binding-domain dependent. By examining Hoechst staining patterns at damage sites, we see evidence of chromatin decompaction that is PARP dependent. We propose that PARP-regulated chromatin remodeling at sites of damage allows transient accessibility of DNA-binding proteins. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Regulation of DNA repair mechanism in human glioma xenograft cells both in vitro and in vivo in nude mice.

PubMed

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H; Rao, Jasti S

2011-01-01

Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point.

Regulation of DNA Repair Mechanism in Human Glioma Xenograft Cells both In Vitro and In Vivo in Nude Mice

PubMed Central

Ponnala, Shivani; Veeravalli, Krishna Kumar; Chetty, Chandramu; Dinh, Dzung H.; Rao, Jasti S.

2011-01-01

Background Glioblastoma Multiforme (GBM) is the most lethal form of brain tumor. Efficient DNA repair and anti-apoptotic mechanisms are making glioma treatment difficult. Proteases such as MMP9, cathepsin B and urokinase plasminogen activator receptor (uPAR) are over expressed in gliomas and contribute to enhanced cancer cell proliferation. Non-homologous end joining (NHEJ) repair mechanism plays a major role in double strand break (DSB) repair in mammalian cells. Methodology/Principal Findings Here we show that silencing MMP9 in combination with uPAR/cathepsin B effects NHEJ repair machinery. Expression of DNA PKcs and Ku70/80 at both mRNA and protein levels in MMP9-uPAR (pMU) and MMP9-cathepsin B (pMC) shRNA-treated glioma xenograft cells were reduced. FACS analysis showed an increase in apoptotic peak and proliferation assays revealed a significant reduction in the cell population in pMU- and pMC-treated cells compared to untreated cells. We hypothesized that reduced NHEJ repair led to DSBs accumulation in pMU- and pMC-treated cells, thereby initiating cell death. This hypothesis was confirmed by reduced Ku70/Ku80 protein binding to DSB, increased comet tail length and elevated γH2AX expression in treated cells compared to control. Immunoprecipitation analysis showed that EGFR-mediated lowered DNA PK activity in treated cells compared to controls. Treatment with pMU and pMC shRNA reduced the expression of DNA PKcs and ATM, and elevated γH2AX levels in xenograft implanted nude mice. Glioma cells exposed to hypoxia and irradiation showed DSB accumulation and apoptosis after pMU and pMC treatments compared to respective controls. Conclusion/Significance Our results suggest that pMU and pMC shRNA reduce glioma proliferation by DSB accumulation and increase apoptosis under normoxia, hypoxia and in combination with irradiation. Considering the radio- and chemo-resistant cancers favored by hypoxia, our study provides important therapeutic potential of MMP9, uPAR and cathepsin B shRNA in the treatment of glioma from clinical stand point. PMID:22022560

Par3 integrates Tiam1 and phosphatidylinositol 3-kinase signaling to change apical membrane identity

PubMed Central

Ruch, Travis R.; Bryant, David M.; Mostov, Keith E.; Engel, Joanne N.

2017-01-01

Pathogens can alter epithelial polarity by recruiting polarity proteins to the apical membrane, but how a change in protein localization is linked to polarity disruption is not clear. In this study, we used chemically induced dimerization to rapidly relocalize proteins from the cytosol to the apical surface. We demonstrate that forced apical localization of Par3, which is normally restricted to tight junctions, is sufficient to alter apical membrane identity through its interactions with phosphatidylinositol 3-kinase (PI3K) and the Rac1 guanine nucleotide exchange factor Tiam1. We further show that PI3K activity is required upstream of Rac1, and that simultaneously targeting PI3K and Tiam1 to the apical membrane has a synergistic effect on membrane remodeling. Thus, Par3 coordinates the action of PI3K and Tiam1 to define membrane identity, revealing a signaling mechanism that can be exploited by human mucosal pathogens. PMID:27881661

PAR-2 receptor-induced effects on human eccrine sweat gland cells.

PubMed

L Bovell, Douglas; Kofler, Barbara; Lang, Roland

2009-01-01

Serine proteases can induce cell signaling by stimulating G-protein-coupled receptors, called proteinase-activated receptors (PAR's) on a variety of epithelial cells. While PAR-2, one such receptor, activates cell signaling in a secretory cell line derived from human sweat glands, there was no information on their presence and effects on intact sweat glands. PAR-2 presence and activation of eccrine sweat glands isolated from human skin samples was investigated using Western blot analysis, immunohistochemistry, electron microscopy (EM) and Ca(2+) imaging. Anti-human PAR-2 antibody demonstrated the presence of these receptors in eccrine sweat glands. EM showed that PAR-2 activation resulted in degranulation of secretory cells. Ca(2+) imaging using PAR-2 activators demonstrated a two phase increase in [Ca(2+)](i) which was dependent on extracellular Ca(2+) for the second phase, and that the response could be blocked by prior incubation with xestospongin, the IP(3) receptor blocker. The results demonstrated that PAR-2 receptors are present in human sweat gland secretory cells and that these receptors are functionally active and can induce changes associated with secretory events in eccrine glands.

Protease Activated Receptor-2 Contributes to Heart Failure

PubMed Central

Antoniak, Silvio; Sparkenbaugh, Erica M.; Tencati, Michael; Rojas, Mauricio; Mackman, Nigel; Pawlinski, Rafal

2013-01-01

Heart failure is a major clinical problem worldwide. Previous studies have demonstrated an important role for G protein-coupled receptors, including protease-activated receptors (PARs), in the pathology of heart hypertrophy and failure. Activation of PAR-2 on cardiomyocytes has been shown to induce hypertrophic growth in vitro. PAR-2 also contributes to myocardial infarction and heart remodeling after ischemia/reperfusion injury. In this study, we found that PAR-2 induced hypertrophic growth of cultured rat neonatal cardiomyocytes in a MEK1/2 and p38 dependent manner. In addition, PAR-2 activation on mouse cardiomyocytes increased expression of the pro-fibrotic chemokine MCP-1. Furthermore, cardiomyocyte-specific overexpression of PAR-2 in mice induced heart hypertrophy, cardiac fibrosis, inflammation and heart failure. Finally, in a mouse model of myocardial infarction induced by permanent ligation of the left anterior descending coronary artery, PAR-2 deficiency attenuated heart remodeling and improved heart function independently of its contribution to the size of the initial infarct. Taken together, our data indicate that PAR-2 signaling contributes to the pathogenesis of hypertrophy and heart failure. PMID:24312345

Self-organization principles of intracellular pattern formation.

PubMed

Halatek, J; Brauns, F; Frey, E

2018-05-26

Dynamic patterning of specific proteins is essential for the spatio-temporal regulation of many important intracellular processes in prokaryotes, eukaryotes and multicellular organisms. The emergence of patterns generated by interactions of diffusing proteins is a paradigmatic example for self-organization. In this article, we review quantitative models for intracellular Min protein patterns in Escherichia coli , Cdc42 polarization in Saccharomyces cerevisiae and the bipolar PAR protein patterns found in Caenorhabditis elegans By analysing the molecular processes driving these systems we derive a theoretical perspective on general principles underlying self-organized pattern formation. We argue that intracellular pattern formation is not captured by concepts such as 'activators', 'inhibitors' or 'substrate depletion'. Instead, intracellular pattern formation is based on the redistribution of proteins by cytosolic diffusion, and the cycling of proteins between distinct conformational states. Therefore, mass-conserving reaction-diffusion equations provide the most appropriate framework to study intracellular pattern formation. We conclude that directed transport, e.g. cytosolic diffusion along an actively maintained cytosolic gradient, is the key process underlying pattern formation. Thus the basic principle of self-organization is the establishment and maintenance of directed transport by intracellular protein dynamics.This article is part of the theme issue 'Self-organization in cell biology'. © 2018 The Authors.

Quail egg homogenate alleviates food allergy induced eosinophilic esophagitis like disease through modulating PAR-2 transduction pathway in peanut sensitized mice.

PubMed

Lianto, Priscilia; Han, Shiwen; Li, Xinrui; Ogutu, Fredrick Onyango; Zhang, Yani; Fan, Zhuoyan; Che, Huilian

2018-01-18

The present pharmacotherapy for eosinophilic esophagitis (EoE) fundamentally depend on inhaled corticosteroids. Despite the fact that oral intake of topical steroids can be successful in restricting EoE-related inflammation, there are concerns with respect to the long term utilization of steroids, especially in kids. In the current research, we assess the effect of quail egg, which is reportedly a known serine protease inhibitor, on symptomatology and immune responses in a peanut-sensitized mouse model of food allergy induced EoE. Daily oral treatment with quail egg attenuated mice symptomatology and immune response. Treatment with quail egg inhibited antigen-prompted increments in mouse tryptase and eosinophil cationic protein (ECP) in serum and eosinophil in inflamed tissues like oesophagus, lung, and digestive system. Quail egg treatment resulted in decreased antibody specific IgE and IgG1 and a variety of inflammatory genes that were abnormally expressed in EoE. Other effects included increased IL-10, decreased PAR-2 activation and NF-kB p65 in inflamed tissues. Our results suggest that quail egg treatment may have therapeutic potential in attenuating the symptoms of food allergy induced EoE like disease through regulating PAR-2 downstream pathway by blocking the activation of the transcription factor NF-kB p65 activity.

PAR-1 and PAR-2 Expression Is Enhanced in Inflamed Odontoblast Cells.

PubMed

Alvarez, M M P; Moura, G E; Machado, M F M; Viana, G M; de Souza Costa, C A; Tjäderhane, L; Nader, H B; Tersariol, I L S; Nascimento, F D

2017-12-01

Protease-activated receptors (PARs) are G protein-coupled receptors, which are activated by proteolytical cleavage of the amino-terminus and act as sensors for extracellular proteases. We hypothesized that PAR-1 and PAR-2 can be modulated by inflammatory stimulus in human dental pulp cells. PAR-1 and PAR-2 gene expression in human pulp tissue and MDPC-23 cells were analyzed by quantitative polymerase chain reaction. Monoclonal PAR-1 and PAR-2 antibodies were used to investigate the cellular expression of these receptors using Western blot, flow cytometry, and confocal microscopy in MDPC-23 cells. Immunofluorescence assays of human intact and carious teeth were performed to assess the presence of PAR-1 and PAR-2 in the dentin-pulp complex. The results show for the first time that human odontoblasts and MDPC-23 cells constitutively express PAR-1 and PAR-2. PAR-2 activation increased significantly the messenger RNA expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MMP-14 in MDPC-23 cells ( P < 0.05), while the expression of these enzymes decreased significantly in the PAR-1 agonist group ( P < 0.05). The high-performance liquid chromatography and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry analysis showed the presence of MMP-13 activity cleaving PAR-1 at specific, noncanonical site TLDPRS 42 ↓F 43 LL in human dental pulp tissues. Also, we detected a presence of a trypsin-like activity cleaving PAR-2 at canonical site SKGR 20 ↓S 21 LIGRL in pulp tissues. Confocal microscopy analysis of human dentin-pulp complex showed intense positive staining of PAR-1 and PAR-2 in the odontoblast processes in dentinal tubules of carious teeth compared to intact ones. The present results support the hypothesis of activation of the upregulated PAR-1 and PAR-2 by endogenous proteases abundant during the inflammatory response in dentin-pulp complex.

Protease-activated receptor (PAR)2, but not PAR1, is involved in collateral formation and anti-inflammatory monocyte polarization in a mouse hind limb ischemia model.

PubMed

van den Hengel, Lisa G; Hellingman, Alwine A; Nossent, Anne Yael; van Oeveren-Rietdijk, Annemarie M; de Vries, Margreet R; Spek, C Arnold; van Zonneveld, Anton Jan; Reitsma, Pieter H; Hamming, Jaap F; de Boer, Hetty C; Versteeg, Henri H; Quax, Paul H A

2013-01-01

In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model. PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered. PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.

Expression of protease-activated-receptor 2 (PAR-2) in human esophageal mucosa.

PubMed

Inci, Kamuran; Edebo, Anders; Olbe, Lars; Casselbrant, Anna

2009-01-01

The role of duodenal reflux in gastroesophageal reflux disease (GERD) containing bile salts and pancreatic enzymes (with special attention to trypsin) is still under discussion. Proteinase-activated receptors (PARs) are a novel family and PAR-2 is a unique member of this family because it is activated by trypsin. The aim of the present study was to examine the presence and the position of the PAR-2 receptor in human esophageal mucosa in different subgroups of GERD. Distal biopsies taken from healthy controls, patients with erosive reflux disease (ERD), patients with specialized intestinal metaplasia (SIM) and adenocarcinoma were analyzed for the PAR-2 receptor with reverse-transcription polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. Gene transcripts for the PAR-2 receptor were found in all groups, with increased levels in SIM patients compared to controls. However, this visual pattern was not seen for the protein expression of the PAR-2 receptor showing no apparent quantitative differences between the groups. Immunohistochemistry revealed distinct staining for the PAR-2 receptor in the luminal part of the esophageal epithelium. The localization of the PAR-2 receptor indicates that the receptor can be cleaved and activated by trypsin in duodenogastric esophageal refluxate. The data thus suggest that the trypsin-PAR-2 pathway may be involved in the pathogenesis of GERD.

Loss of Partitioning-Defective-3/Isotype-Specific Interacting Protein (Par-3/ASIP) in the Elongating Spermatid of RA175 (IGSF4A/SynCAM)-Deficient Mice

PubMed Central

Fujita, Eriko; Tanabe, Yuko; Hirose, Tomonori; Aurrand-Lions, Michel; Kasahara, Tadashi; Imhof, Beat A.; Ohno, Shigeo; Momoi, Takashi

2007-01-01

IGSF4a/RA175/SynCAM (RA175) and junctional adhesion molecules (Jams) are members of the immunoglobulin superfamily with a PDZ-binding domain at their C termini. Deficiency of Ra175 (Ra175−/−) as well as Jam-C deficiency (Jam-C−/−) causes the defect of the spermatid differentiation, oligo-astheno-teratozoospermia. Ra175−/− elongating spermatids fail to mature further, whereas Jam-C−/− round spermatids lose cell polarity, and most of Jam-C−/− elongated spermatids are completely lost. RA175 and Jam-C seem to have similar but distinct functional roles during spermatid differentiation. Here we show that the cell polarity protein Par-3 with PDZ domains, a binding partner of Jams, is one of the associated proteins of the cytoplasmic region of RA175 in testis. Par-3 and Jam-C are partly co-localized with RA175 in the elongating and elongated spermatids; their distributions overlapped with that of RA175 on the tips of the dorsal region of the head of the elongating spermatid (steps 9 to 12) in the wild type. In the Ra175−/− elongating spermatid, Par-3 was absent, and Jam-C was absent or abnormally localized. The RA175 formed a ternary complex with Jam-C via interaction with Par-3. The lack of the ternary complex in the Ra175−/− elongating spermatid may cause the defect of the specialized adhesion structures, resulting in the oligo-astheno-teratozoospermia. PMID:18055550

The protease-activated receptor-2 upregulates keratinocyte phagocytosis.

PubMed

Sharlow, E R; Paine, C S; Babiarz, L; Eisinger, M; Shapiro, S; Seiberg, M

2000-09-01

The protease-activated receptor-2 (PAR-2) belongs to the family of seven transmembrane domain receptors, which are activated by the specific enzymatic cleavage of their extracellular amino termini. Synthetic peptides corresponding to the tethered ligand domain (SLIGRL in mouse, SLIGKV in human) can activate PAR-2 without the need for receptor cleavage. PAR-2 activation is involved in cell growth, differentiation and inflammatory processes, and was shown to affect melanin and melanosome ingestion by human keratinocytes. Data presented here suggest that PAR-2 activation may regulate human keratinocyte phagocytosis. PAR-2 activation by trypsin, SLIGRL or SLIGKV increased the ability of keratinocytes to ingest fluorescently labeled microspheres or E. coli K-12 bioparticles. This PAR-2 mediated increase in keratinocyte phagocytic capability correlated with an increase in actin polymerization and *-actinin reorganization, cell surface morphological changes and increased soluble protease activity. Moreover, addition of serine protease inhibitors downmodulated both the constitutive and the PAR-2 mediated increases in phagocytosis, suggesting that serine proteases mediate this functional activity in keratinocytes. PAR-2 involvement in keratinocyte phagocytosis is a novel function for this receptor.

Discovery of potent peptide-mimetic antagonists for the human thrombin receptor, protease-activated receptor-1 (PAR-1).

PubMed

Maryanoff, Bruce E; Zhang, Han-Cheng; Andrade-Gordon, Patricia; Derian, Claudia K

2003-03-01

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G-protein-coupled receptors, which are enzymatically cleaved to expose a new extracellular N-terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g. platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. By using a de novo design approach, we have discovered a series of potent heterocycle-based peptide-miimetic antagonists of PAR-1, exemplified by advanced leads RWJ-56110 (22) and RWJ-58259 (32). These compounds are potent, selective PAR-1 antagonists, devoid of PAR-1 agonist and thrombin inhibitory activity: they bind to PAR-1, interfere with calcium mobilization and cellular functions associated with PAR-1, and do not affect PAR-2, PAR-3, or PAR-4. RWJ-56110 was determined to be a direct inhibitor of PAR-1 activation and internalization, without affecting PAR-1 N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, but not in human platelets; whereas, at high concentrations of TRAP-6, RWJ-56110 blocked activation responses in both cell types. This result is consistent with the presence of another thrombin receptor on human platelets, namely PAR-4. RWJ-56110 and RWJ-58259 clearly interrupt the binding of a tethered ligand to its receptor. RWJ-58259 demonstrated antirestenotic activity in a rat balloon angioplasty model and antithrombotic activity in a cynomolgus monkey arterial injury model. Such PAR-1 antagonists should not only serve as useful tools to delineate the physiological and pathophysiological roles of PAR-1, but also may have therapeutic potential for treating thrombosis and restenosis in humans.

Kallikrein-related peptidase 4 (KLK4) initiates intracellular signaling via protease-activated receptors (PARs). KLK4 and PAR-2 are co-expressed during prostate cancer progression.

PubMed

Ramsay, Andrew J; Dong, Ying; Hunt, Melanie L; Linn, MayLa; Samaratunga, Hemamali; Clements, Judith A; Hooper, John D

2008-05-02

Kallikrein-related peptidase 4 (KLK4) is one of the 15 members of the human KLK family and a trypsin-like, prostate cancer-associated serine protease. Signaling initiated by trypsin-like serine proteases are transduced across the plasma membrane primarily by members of the protease-activated receptor (PAR) family of G protein-coupled receptors. Here we show, using Ca(2+) flux assays, that KLK4 signals via both PAR-1 and PAR-2 but not via PAR-4. Dose-response analysis over the enzyme concentration range 0.1-1000 nM indicated that KLK4-induced Ca(2+) mobilization via PAR-1 is more potent than via PAR-2, whereas KLK4 displayed greater efficacy via the latter PAR. We confirmed the specificity of KLK4 signaling via PAR-2 using in vitro protease cleavage assays and anti-phospho-ERK1/2/total ERK1/2 Western blot analysis of PAR-2-overexpressing and small interfering RNA-mediated receptor knockdown cell lines. Consistently, confocal microscopy analyses indicated that KLK4 initiates loss of PAR-2 from the cell surface and receptor internalization. Immunohistochemical analysis indicated the co-expression of agonist and PAR-2 in primary prostate cancer and bone metastases, suggesting that KLK4 signaling via this receptor will have pathological relevance. These data provide insight into KLK4-mediated cell signaling and suggest that signals induced by this enzyme via PARs may be important in prostate cancer.

Urokinase-receptor (u-PAR): an essential player in multiple games of cancer: a review on its role in tumor progression, invasion, metastasis, proliferation/dormancy, clinical outcome and minimal residual disease.

PubMed

Laufs, Stephanie; Schumacher, Jens; Allgayer, Heike

2006-08-01

The relevance of the u-PA system in mediating tumor-associated proteolysis, invasion and metastasis, amongst other phenomena associated with tumor progression, has been clearly demonstrated in diverse cancer entities. This review will update on the biological and clinical relevance of the urokinase-receptor (u-PAR). Specifically, the article focuses on the potential importance of u-PAR for the development of minimal residual disease in solid cancer, and in this context reviews the biological relevance of the u-PAR for tumor cell dormancy. Furthermore, transcriptional mechanisms regulating u-PAR in vitro and in vivo, and their potential clinical and therapeutic relevance in gastrointestinal cancers, are elucidated.

Transient early neurotrophin release and delayed inflammatory cytokine release by microglia in response to PAR-2 stimulation.

PubMed

Chen, Chen-Wen; Chen, Qian-Bo; Ouyang, Qing; Sun, Ji-Hu; Liu, Fang-Ting; Song, Dian-Wen; Yuan, Hong-Bin

2012-06-25

Activated microglia exerts both beneficial and deleterious effects on neurons, but the signaling mechanism controlling these distinct responses remain unclear. We demonstrated that treatment of microglial cultures with the PAR-2 agonist, 2-Furoyl-LIGRLO-NH2, evoked early transient release of BDNF, while sustained PAR-2 stimulation evoked the delayed release of inflammatory cytokines (IL-1 β and TNF-α) and nitric oxide. Culture medium harvested during the early phase (at 1 h) of microglial activation induced by 2-Furoyl-LIGRLO-NH2 (microglial conditioned medium, MCM) had no deleterious effects on cultured neurons, while MCM harvested during the late phase (at 72 h) promoted DNA fragmentation and apoptosis as indicated by TUNEL and annexin/PI staining. Blockade of PAR-1 during the early phase of PAR-2 stimulation enhanced BDNF release (by 11%, small but significant) while a PAR-1 agonist added during the late phase (24 h after 2-Furoyl-LIGRLO-NH2 addition) suppressed the release of cytokines and NO. The neuroprotective and neurotoxic effects of activated microglial exhibit distinct temporal profiles that are regulated by PAR-1 and PAR-2 stimulation. It may be possible to facilitate neuronal recovery and repair by appropriately timed stimulation and inhibition of microglial PAR-1 and PAR-2 receptors.

Protease-Activated Receptor-2 Is Associated with Terminal Differentiation of Epidermis and Eccrine Sweat Glands

PubMed Central

Shin, Yong-Sup; Kim, Hyung Won; Kim, Chang Deok; Kim, Hyun-Woo; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Ko, Young-Kwon

2015-01-01

Background Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin. Objective To evaluate the basic physiological role of PAR-2 in skin. Methods We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes. Results Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker. Conclusion Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands. PMID:26273149

Protease-Activated Receptor-2 Is Associated with Terminal Differentiation of Epidermis and Eccrine Sweat Glands.

PubMed

Shin, Yong-Sup; Kim, Hyung Won; Kim, Chang Deok; Kim, Hyun-Woo; Park, Jin Woon; Jung, Sunggyun; Lee, Jeung-Hoon; Ko, Young-Kwon; Lee, Young Ho

2015-08-01

Protease-activated receptor 2 (PAR-2) participates in various biological activities, including the regulation of epidermal barrier homeostasis, inflammation, pain perception, and melanosome transfer in the skin. To evaluate the basic physiological role of PAR-2 in skin. We investigated PAR-2 expression in human epidermis, skin tumors, and cultured epidermal cells using western blot and immunohistochemical analysis. Additionally, we examined the effect of the PAR-2 agonist, SLIGRL-NH2, on cultured keratinocytes. Strong PAR-2 immunoreactivity was observed in the granular layer of normal human skin and the acrosyringium of the eccrine sweat glands. In contrast, weak PAR-2 immunoreactivity was seen in the granular layer of callused skin and in the duct and gland cells of the eccrine sweat glands. Interestingly, PAR-2 immunoreactivity was very weak or absent in the tumor cells of squamous cell carcinoma (SCC) and syringoma. PAR-2 was detected in primary keratinocytes and SV-40T-transformed human epidermal keratinocytes (SV-HEKs), an immortalized keratinocyte cell line, but not in SCC12 cells. SV-HEKs that were fully differentiated following calcium treatment displayed higher PAR-2 expression than undifferentiated SV-HEKs. Treatment of cultured SV-HEKs with PAR-2 agonist increased loricrin and filaggrin expression, a terminal differentiation marker. Our data suggest that PAR-2 is associated with terminal differentiation of epidermis and eccrine sweat glands.

Impacts of diurnal variation of ultraviolet-B and photosynthetically active radiation on phycobiliproteins of the hot-spring cyanobacterium Nostoc sp. strain HKAR-2.

PubMed

Kannaujiya, Vinod K; Sinha, Rajeshwar P

2017-01-01

The effects of diurnal variation of photosynthetically active radiation (PAR; 400-700 nm) and ultraviolet-B (UV-B; 280-315 nm) radiation on phycobiliproteins (PBPs) and photosynthetic pigments (PP) have been studied in the hot-spring cyanobacterium Nostoc sp. strain HKAR-2. The variations in PBPs and PP were monitored by alternating light and dark under PAR, UV-B, and PAR + UV-B radiations over a period of 25 h. There was a decline in the amount of Chl a and PBPs during light periods of UV-B and PAR + UV-B and an increase during dark periods showing a circadian rhythm by destruction and resynthesis of pigment-protein complex. However, a marked induction in carotenoids was recorded during light periods of the same radiations. Moreover, the ratio of Chl a/PE and Chl a/PC was increased in dark periods showing the resynthesis of bleached Chl a. The wavelength shift in emission fluorescence of PBPs toward shorter wavelengths further indicated the bleaching and destruction of PBPs during light periods. Oxidative damage upon exposure to PAR, UV-B, and PAR + UV-B was alleviated by induction of antioxidative enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX). The studied cyanobacterium exhibits a significant increase in the activities of SOD, CAT, and APX upon exposure to UV-B and PAR + UV-B radiations. The results indicate that pigment-protein composition of Nostoc sp. stain HKAR-2 was significantly altered during diurnal variation of light/radiation, which might play an important role in optimization for their productivity in a particular cyanobacterium.

Analysis of mutations in DNA gyrase and topoisomerase IV of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones.

PubMed

Piccinelli, Giorgio; Gargiulo, Franco; Biscaro, Valeria; Caccuri, Francesca; Caruso, Arnaldo; De Francesco, Maria Antonia

2017-01-01

This study aims to determine the prevalence of fluoroquinolone resistance of Ureaplasma biovars and serovars isolated from urogenital clinical samples and determine the underlying molecular mechanism for quinolone resistance for all resistant isolates. Of 105 samples confirmed as positive for U. urealyticum/U. parvum, 85 were resistant to quinolones by the Mycoplasma-IST2 kit. However, only 43 out of 85 quinolone resistant isolates had amino acid substitutions in GyrA, GyrB, ParC and ParE proteins underlining that this assay have mis-identified as fluoroquinolone resistant 42 isolates. The known ParC E87K and ParC S83L mutations were found in 1 and 10 isolates, respectively. An original mutation of ureaplasmal ParC (E87Q, 1 isolate) was found. Furthermore, we found a ParE R448K mutation in one isolate, already described. Among the additional alterations detected, the most prevalent mutation found was L176F in GyrA protein in 18 isolates with single infection and in 3 isolates with mixed ureaplasma infections. Mutations in GyrB (E502Q, 4 isolates), ParE (Q412K, Q412P, Q412T, 3 independent isolates), whose role is unknown, were also found. Other sporadic mutations in the four genes were identified. This investigation is the result of monitoring the data for molecular fluoroquinone resistance in Ureaplasma spp. in Italy. Resulting that this acquired resistance is high and that continued local epidemiological studies are essential to monitor and document their antimicrobial resistance trends. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulation of skin pigmentation and thickness by dickkopf 1 (DKK1)

PubMed Central

Yamaguchi, Yuji; Morita, Akimichi; Maeda, Akira; Hearing, Vincent J.

2009-01-01

Dickkopf 1 (DKK1), an inhibitor of Wnt signaling, not only functions as a head inducer during development, but also regulates joint remodeling and bone formation, which suggests roles for DKK1 in the pathogenesis of rheumatoid arthritis and multiple myeloma. We recently demonstrated that levels of DKK1 in palmoplantar dermal fibroblasts are physiologically higher than those observed in non-palmoplantar dermal fibroblasts. Thus, the DKK1-rich mesenchyme in palmoplantar dermis affects the overlying epithelium and induces a palmoplantar phenotype in the epidermis. More specifically, DKK1 suppresses melanocyte function and growth via the regulation of microphthalmia-associated transcription factor (MITF) and β-catenin. Furthermore, DKK1 induces the expression of keratin 9 and α-Kelch-like ECT2 interacting protein (αKLEIP) but down-regulates the expression of β-catenin, glycogen synthase kinase 3β, protein kinase C and proteinase-activated receptor-2 (PAR-2) in keratinocytes. Treatment of reconstructed skin with DKK1 reproduces the hypopigmentation and thickening of skin via Wnt/β-catenin signaling. These studies elucidate why human palmoplantar skin is thicker and paler than non-palmoplantar skin via the secretion of DKK1 by fibroblasts that affect the overlying epidermis. Thus, DKK1 may be useful for reducing skin pigmentation and for thickening photo-aged skin and palmoplantar wounds caused by diabetes mellitus and rheumatic skin diseases. PMID:19675559

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages

PubMed Central

Schumacher, Maria A.; Tonthat, Nam K; Lee, Jeehyun; Rodriguez-Castañeda, Fernando A.; Chinnam, Naga babu; Kalliomaa-Sanford, Anne K.; Ng, Irene W.; Barge, Madhuri T.; Shaw, Porsha L.R.; Barillà, Daniela

2016-01-01

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system employs three proteins; ParA, an atypical ParB adaptor and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The ParB C-domain harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, suggesting the possible conservation of DNA segregation principles across the three domains of life. PMID:26339031

A peptide inhibitor of the urokinase/urokinase receptor system inhibits alteration of the blood-retinal barrier in diabetes.

PubMed

Navaratna, Deepti; Menicucci, Gina; Maestas, Joann; Srinivasan, Ramprasad; McGuire, Paul; Das, Arup

2008-09-01

One of the major complications of diabetes is the alteration of the blood-retinal barrier, leading to retinal edema and consequent vision loss. The aim of this study was to evaluate the role of the urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system in the regulation of retinal vascular permeability. Biochemical, molecular, and histological techniques were used to examine the role of uPA and uPAR in the regulation of retinal vascular permeability in diabetic rats and cultured retinal endothelial cells. The increased retinal vascular permeability in diabetic rats was associated with a decrease in vascular endothelial (VE) -cadherin expression in retinal vessels. Treatment with the uPA/uPAR-inhibiting peptide (A6) was shown to reduce diabetes-induced permeability and the loss of VE-cadherin. The increased permeability of cultured cells in response to advanced glycation end products (AGEs) was significantly inhibited with A6. Treatment of endothelial cells with specific matrix metalloproteinases or AGEs resulted in loss of VE-cadherin from the cell surface, which could be inhibited by A6. uPA/uPAR physically interacts with AGEs/receptor for advanced glycation end products on the cell surface and regulates its activity. uPA and its receptor uPAR play important roles in the alteration of the blood-retinal barrier through proteolytic degradation of VE-cadherin. The ability of A6 to block retinal vascular permeability in diabetes suggests a potential therapeutic approach for the treatment of diabetic macular edema.

Proteinase-activated receptor 2 (PAR(2)) in cholangiocarcinoma (CCA) cells: effects on signaling and cellular level.

PubMed

Kaufmann, Roland; Hascher, Alexander; Mussbach, Franziska; Henklein, Petra; Katenkamp, Kathrin; Westermann, Martin; Settmacher, Utz

2012-12-01

In this study, we demonstrate functional expression of the proteinase-activated receptor 2 (PAR(2)), a member of a G-protein receptor subfamily in primary cholangiocarcinoma (PCCA) cell cultures. Treatment of PCCA cells with the serine proteinase trypsin and the PAR(2)-selective activating peptide, furoyl-LIGRLO-NH(2), increased migration across a collagen membrane barrier. This effect was inhibited by a PAR(2)-selective pepducin antagonist peptide (P2pal-18S) and it was also blocked with the Met receptor tyrosine kinase (Met) inhibitors SU 11274 and PHA 665752, the MAPKinase inhibitors PD 98059 and SL 327, and the Stat3 inhibitor Stattic. The involvement of Met, p42/p44 MAPKinases and Stat3 in PAR(2)-mediated PCCA cell signaling was further supported by the findings that trypsin and the PAR(2)-selective agonist peptide, 2-furoyl-LIGRLO-NH(2), stimulated activating phosphorylation of these signaling molecules in cholangiocarcinoma cells. With our results, we provide a novel signal transduction module in cholangiocarcinoma cell migration involving PAR(2)-driven activation of Met, p42/p44 MAPKinases and Stat3.

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1

PubMed Central

Valdés, Pamela; Mercado, Gabriela; Vidal, Rene L.; Molina, Claudia; Parsons, Geoffrey; Court, Felipe A.; Martinez, Alexis; Galleguillos, Danny; Armentano, Donna; Schneider, Bernard L.; Hetz, Claudio

2014-01-01

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD. PMID:24753614

Protease-Activated Receptor-2 Deficiency Attenuates Atherosclerotic Lesion Progression and Instability in Apolipoprotein E-Deficient Mice

PubMed Central

Zuo, Pengfei; Zuo, Zhi; Zheng, Yueyue; Wang, Xin; Zhou, Qianxing; Chen, Long; Ma, Genshan

2017-01-01

Inflammatory mechanisms are involved in the process of atherosclerotic plaque formation and rupture. Accumulating evidence suggests that protease-activated receptor (PAR)-2 contributes to the pathophysiology of chronic inflammation on the vasculature. To directly examine the role of PAR-2 in atherosclerosis, we generated apolipoprotein E/PAR-2 double-deficient mice. Mice were fed with high-fat diet for 12 weeks starting at ages of 6 weeks. PAR-2 deficiency attenuated atherosclerotic lesion progression with reduced total lesion area, reduced percentage of stenosis and reduced total necrotic core area. PAR-2 deficiency increased fibrous cap thickness and collagen content of plaque. Moreover, PAR-2 deficiency decreased smooth muscle cell content, macrophage accumulation, matrix metallopeptidase-9 expression and neovascularization in plaque. Relative quantitative PCR assay using thoracic aorta revealed that PAR-2 deficiency reduced mRNA expression of inflammatory molecules, such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1, tumor necrosis factor (TNF)-α and monocyte chemoattractant protein (MCP)-1. In vitro experiment, we found that PAR-2 deficiency reduced mRNA expression of interferon-γ, interleukin-6, TNF-α and MCP-1 in macrophage under unstimulated and lipopolysaccharide-stimulated conditions. These results suggest that PAR-2 deficiency attenuates the progression and instability of atherosclerotic plaque. PMID:28959204

Protease-Activated Receptor 4 Variant p.Tyr157Cys Reduces Platelet Functional Responses and Alters Receptor Trafficking.

PubMed

Norman, Jane E; Cunningham, Margaret R; Jones, Matthew L; Walker, Mary E; Westbury, Sarah K; Sessions, Richard B; Mundell, Stuart J; Mumford, Andrew D

2016-05-01

Protease-activated receptor 4 (PAR4) is a key regulator of platelet reactivity and is encoded by F2RL3, which has abundant rare missense variants. We aimed to provide proof of principle that rare F2LR3 variants potentially affect platelet reactivity and responsiveness to PAR1 antagonist drugs and to explore underlying molecular mechanisms. We identified 6 rare F2RL3 missense variants in 236 cardiac patients, of which the variant causing a tyrosine 157 to cysteine substitution (Y157C) was predicted computationally to have the greatest effect on PAR4 structure. Y157C platelets from 3 cases showed reduced responses to PAR4-activating peptide and to α-thrombin compared with controls, but no reduction in responses to PAR1-activating peptide. Pretreatment with the PAR1 antagonist vorapaxar caused lower residual α-thrombin responses in Y157C platelets than in controls, indicating greater platelet inhibition. HEK293 cells transfected with a PAR4 Y157C expression construct had reduced PAR4 functional responses, unchanged total PAR4 expression but reduced surface expression. PAR4 Y157C was partially retained in the endoplasmic reticulum and displayed an expression pattern consistent with defective N-glycosylation. Mutagenesis of Y322, which is the putative hydrogen bond partner of Y157, also reduced PAR4 surface expression in HEK293 cells. Reduced PAR4 responses associated with Y157C result from aberrant anterograde surface receptor trafficking, in part, because of disrupted intramolecular hydrogen bonding. Characterization of PAR4 Y157C establishes that rare F2RL3 variants have the potential to markedly alter platelet PAR4 reactivity particularly after exposure to therapeutic PAR1 antagonists. © 2016 American Heart Association, Inc.

Protease Activated Receptor-2 Expression and Function in Asthmatic Bronchial Smooth Muscle

PubMed Central

Gilbert, Guillaume; Carvalho, Gabrielle; Trian, Thomas; Ozier, Annaig; Gillibert-Duplantier, Jennifer; Ousova, Olga; Maurat, Elise; Thumerel, Matthieu; Quignard, Jean-François; Girodet, Pierre-Olivier; Marthan, Roger; Berger, Patrick

2014-01-01

Asthmatic bronchial smooth muscle (BSM) is characterized by structural remodeling associated with mast cell infiltration displaying features of chronic degranulation. Mast cell-derived tryptase can activate protease activated receptor type-2 (PAR-2) of BSM cells. The aims of the present study were (i) to evaluate the expression of PAR-2 in both asthmatic and non asthmatic BSM cells and, (ii) to analyze the effect of prolonged stimulation of PAR-2 in asthmatic BSM cells on cell signaling and proliferation. BSM cells were obtained from both 33 control subjects and 22 asthmatic patients. PAR-2 expression was assessed by flow cytometry, western blot and quantitative RT-PCR. Calcium response, transduction pathways and proliferation were evaluated before and following PAR-2 stimulation by SLIGKV-NH2 or trypsin for 1 to 3 days. Asthmatic BSM cells expressed higher basal levels of functional PAR-2 compared to controls in terms of mRNA, protein expression and calcium response. When PAR-2 expression was increased by means of lentivirus in control BSM cells to a level similar to that of asthmatic cells, PAR-2-induced calcium response was then similar in both types of cell. However, repeated PAR-2 stimulations increased the proliferation of asthmatic BSM cells but not that of control BSM cells even following lentiviral over-expression of PAR-2. Such an increased proliferation was related to an increased phosphorylation of ERK in asthmatic BSM cells. In conclusion, we have demonstrated that asthmatic BSM cells express increased baseline levels of functional PAR-2. This higher basal level of PAR-2 accounts for the increased calcium response to PAR-2 stimulation, whereas the increased proliferation to repeated PAR-2 stimulation is related to increased ERK phosphorylation. PMID:24551046

In vitro and in vivo inhibition of proangiogenic retinal phenotype by an antisense oligonucleotide downregulating uPAR expression.

PubMed

Lulli, Matteo; Cammalleri, Maurizio; Granucci, Irene; Witort, Ewa; Bono, Silvia; Di Gesualdo, Federico; Lupia, Antonella; Loffredo, Rosa; Casini, Giovanni; Dal Monte, Massimo; Capaccioli, Sergio

2017-08-26

Neoangiogenesis is the main pathogenic event involved in a variety of retinal diseases. It has been recently demonstrated that inhibiting the urokinase-type plasminogen activator receptor (uPAR) results in reduced angiogenesis in a mouse model of oxygen-induced retinopathy (OIR), establishing uPAR as a therapeutic target in proliferative retinopathies. Here, we evaluated in cultured human retinal endothelial cells (HRECs) and in OIR mice the potential of a specific antisense oligodeoxyribonucleotide (ASO) in blocking the synthesis of uPAR and in providing antiangiogenic effects. uPAR expression in HRECs was inhibited by lipofection with the phosphorotioated 5'-CGGCGGGTGACCCATGTG-3' ASO-uPAR, complementary to the initial translation site of uPAR mRNA. Inhibition of uPAR expression via ASO-uPAR was evaluated in HRECs by analyzing VEGF-induced tube formation and migration. In addition, the well-established and reproducible murine OIR model was used to induce retinal neovascularization in vivo. OIR mice were injected intraperitoneally with ASO-uPAR and retinopathy was evaluated considering the extent of the avascular area in the central retina and neovascular tuft formation. The ASO-uPAR specifically decreased uPAR mRNA and protein levels in HRECs and mitigated VEGF-induced tube formation and cell migration. Noteworthy, in OIR mice ASO-uPAR administration reduced both the avascular area and the formation of neovascular tufts. In conclusion, although the extrapolation of these experimental findings to the clinic is not straightforward, ASO-uPAR may be considered a potential therapeutic tool for treatment of proliferative retinal diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

The role of protease activation of inflammation in allergic respiratory diseases.

PubMed

Reed, Charles E; Kita, Hirohito

2004-11-01

Extracellular endogenous proteases, as well as exogenous proteases from mites and molds, react with cell-surface receptors in the airways to generate leukocyte infiltration and to amplify the response to allergens. Stimulation leads to increased intracellular Ca ++ and gene transcription. The most thoroughly investigated receptors, protease-activated receptors (PARs), are 7-transmembrane proteins coupled to G proteins. PARs are widely distributed on the cells of the airways, where they contribute to the inflammation characteristic of allergic diseases. PAR stimulation of epithelial cells opens tight junctions, causes desquamation, and produces cytokines, chemokines, and growth factors. They degranulate eosinophils and mast cells. Proteases contract bronchial smooth muscle and cause it to proliferate. PARs also promote maturation, proliferation, and collagen production of fibroblast precursors and mature fibroblasts. PAR-2, apparently the most important of the 4 PARs that have been characterized, is increased on the epithelium of patients with asthma. Trypsin, a product of injured epithelial cells, and mast cell tryptase are potent activators of PAR-2. Mast cell chymase activates PAR-1. Proteases from mites and molds appear to act through similar receptors. They amplify IgE production to allergens, degranulate eosinophils, and can generate inflammation, even in the absence of IgE. Proteases produced by Aspergillus species to support its growth are presumably responsible for the exuberant IgE, IgG, and granulomatous response of allergic bronchopulmonary aspergillosis. Similar proteases from molds germinating on the respiratory mucosa have been recently been implicated in the pathogenesis of chronic hyperplastic rhinitis and polyps and, by extension, of intrinsic asthma. Finally, proteases from mites and fungi growing in damp, water-damaged buildings might be the basis for the increased prevalence in these buildings of rhinitis, asthma, and other respiratory diseases. Future research promises to promote our understanding of the pathogenesis of allergic respiratory diseases and point the way to new therapies.

31 CFR 360.10 - Amounts which may be purchased.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 31 Money and Finance:Treasury 2 2011-07-01 2011-07-01 false Amounts which may be purchased. 360.10 Section 360.10 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL... follows: (a) General annual limitation, $5,000 (par value). (b) Special limitation, $4,000 (par value...

31 CFR 360.10 - Amounts which may be purchased.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 31 Money and Finance:Treasury 2 2012-07-01 2012-07-01 false Amounts which may be purchased. 360.10 Section 360.10 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL... follows: (a) General annual limitation, $5,000 (par value). (b) Special limitation, $4,000 (par value...

Poly(ADP-ribosyl)ation is recognized by ECT2 during mitosis.

PubMed

Li, Mo; Bian, Chunjing; Yu, Xiaochun

2014-01-01

Poly(ADP-ribosyl)ation is an unique posttranslational modification and required for spindle assembly and function during mitosis. However, the molecular mechanism of poly(ADP-ribose) (PAR) in mitosis remains elusive. Here, we show the evidence that PAR is recognized by ECT2, a key guanine nucleotide exchange factor in mitosis. The BRCT domain of ECT2 directly binds to PAR both in vitro and in vivo. We further found that α-tubulin is PARylated during mitosis. PARylation of α-tubulin is recognized by ECT2 and recruits ECT2 to mitotic spindle for completing mitosis. Taken together, our study reveals a novel mechanism by which PAR regulates mitosis.

Cross-reactivity among non-specific lipid-transfer proteins from food and pollen allergenic sources.

PubMed

Morales, María; López-Matas, M Ángeles; Moya, Raquel; Carnés, Jerónimo

2014-12-15

Non-specific lipid-transfer proteins (nsLTPs) are a family of pan-allergens present in foods and pollen. However, sequence homology among them is limited. The objective of this study was to evaluate the IgE-mediated cross-reactivity between nsLTPs from different sources and evaluate the allergenic properties of LTPs from peach (Pru p 3) and pellitory (Par j 1/Par j 2), major fruit and pollen allergens. Both proteins were purified and characterised. Cross-reactivity studies among nsLTPs from different foods and pollens were performed by immunoblot inhibition using sera specific to peach or pellitory pollen. Cross-reactivity with Pru p 3 was observed in hazelnut, onion, corn, peanut and apple while in pollens, none of the extracts was inhibited with Par j 1/2. In conclusion, Pru p 3 did not inhibit LTPs from most fruits. Therefore, although Pru p 3 covers the largest number of epitopes, diagnosis with only this allergen may not detect all LTP sensitivities. Copyright © 2014 Elsevier Ltd. All rights reserved.

Effect of Par Frying on Composition and Texture of Breaded and Battered Catfish

PubMed Central

Woods, Kristin; Lea, Jeanne M.; Brashear, Suzanne S.; Boue, Stephen M.; Daigle, Kim W.; Bett-Garber, Karen L.

2018-01-01

Catfish is often consumed as a breaded and battered fried product; however, there is increasing interest in breaded and battered baked products as a healthier alternative. Par frying can improve the texture properties of breaded and battered baked products, but there are concerns about the increase in lipid uptake from par frying. The objective of this study was to examine the effect of different batters (rice, corn, and wheat) and the effect of par frying on the composition and texture properties of baked catfish. Catfish fillets were cut strips and then coated with batters, which had similar viscosities. Half of the strips were par fried in 177 °C vegetable oil for 1 min and the other half were not par fried. Samples were baked at 177 °C for 25 min. Analysis included % batter adhesion, cooking loss, protein, lipid, ash, and moisture, plus hardness and fracture quality measured using a texture analyzer. A trained sensory panel evaluated both breading and flesh texture attributes. Results found the lipid content of par fried treatments were significantly higher for both corn and wheat batters than for non-par fried treatments. Sensory analysis indicated that the texture of the coatings in the par fried treatments were significantly greater for hardness attributes. Fillet flakiness was significantly greater in the par fried treatments and corn-based batters had moister fillet strips compared to the wheat flour batters. Texture analyzer hardness values were higher for the par fried treatments. PMID:29570660

Combination with CK19 Might Increase the Prognostic Power of Hep Par 1 in Hepatocellular Carcinoma after Curative Resection.

PubMed

Jin, Ye; Liang, Zhi-Yong; Zhou, Wei-Xun; Zhou, Li

2017-07-31

Hepatocyte Paraffin 1 (Hep Par 1) and cytokeratin 19 (CK19) were shown to be associated with post-surgical prognosis of hepatocellular carcinoma (HCC). However, further validation might be needed. Besides, their combined evaluation has not been reported. The present study was designed to address the issues. Expressions of Hep Par 1 and CK19 were detected using tissue microarray-based immunohistochemical staining in 79 patients with HCC underwent curative hepatectomy. Their associations with cliniopathologic variables, overall and recurrence-free survival were analyzed. Hep Par 1 was highly expressed in 61 patients (77.2%), whereas CK19 was positive in 8 patients (10.1%). Moreover, expressions of these two proteins were all associated with tumor-node-metastasis (TNM) stage and vascular invasion. It was found that high Hep Par 1 expression was univariately associated with good overall and recurrence-free survival, while CK19 was marginally prognostic. Also in univariate analyses, combination of the two markers more effectively predicted for long-term prognosis in HCC than Hep Par 1 did. However, neither Hep Par 1 nor Hep Par 1/CK19 was multivariately significant. Finally, Hep Par 1/CK19 combined with TNM stage might obtain more satisfactory outcome prediction, especially for overall survival. Combination of CK19 with Hep Par 1 might have higher prognostic power, which might be further improved by adding TNM stage, than Hep Par 1 alone, in resected HCC. Of course, subsequent confirmation is necessary.

Mechanisms of CDC-42 activation during contact-induced cell polarization.

PubMed

Chan, Emily; Nance, Jeremy

2013-04-01

Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure-function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts.

Mechanisms of CDC-42 activation during contact-induced cell polarization

PubMed Central

Chan, Emily; Nance, Jeremy

2013-01-01

Summary Polarization of early embryos provides a foundation to execute essential patterning and morphogenetic events. In Caenorhabditis elegans, cell contacts polarize early embryos along their radial axis by excluding the cortical polarity protein PAR-6 from sites of cell contact, thereby restricting PAR-6 to contact-free cell surfaces. Radial polarization requires the cortically enriched Rho GTPase CDC-42, which in its active form recruits PAR-6 through direct binding. The Rho GTPase activating protein (RhoGAP) PAC-1, which localizes specifically to cell contacts, triggers radial polarization by inactivating CDC-42 at these sites. The mechanisms responsible for activating CDC-42 at contact-free surfaces are unknown. Here, in an overexpression screen of Rho guanine nucleotide exchange factors (RhoGEFs), which can activate Rho GTPases, we identify CGEF-1 and ECT-2 as RhoGEFs that act through CDC-42 to recruit PAR-6 to the cortex. We show that ECT-2 and CGEF-1 localize to the cell surface and that removing their activity causes a reduction in levels of cortical PAR-6. Through a structure–function analysis, we show that the tandem DH-PH domains of CGEF-1 and ECT-2 are sufficient for GEF activity, but that regions outside of these domains target each protein to the cell surface. Finally, we provide evidence suggesting that the N-terminal region of ECT-2 may direct its in vivo preference for CDC-42 over another known target, the Rho GTPase RHO-1. We propose that radial polarization results from a competition between RhoGEFs, which activate CDC-42 throughout the cortex, and the RhoGAP PAC-1, which inactivates CDC-42 at cell contacts. PMID:23424200

Cryo-EM reconstruction of AlfA from Bacillus subtilis reveals the structure of a simplified actin-like filament at 3.4-Å resolution.

PubMed

Szewczak-Harris, Andrzej; Löwe, Jan

2018-03-27

Low copy-number plasmid pLS32 of Bacillus subtilis subsp. natto contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence parN Similar to the ParMRC partitioning system from Escherichia coli plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and parN Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.

The expression and activation of protease-activated receptor-2 correlate with skin color.

PubMed

Babiarz-Magee, Laura; Chen, Nannan; Seiberg, Miri; Lin, Connie B

2004-06-01

Skin color results from the production and distribution of melanin in the epidermis. The protease-activated receptor-2 (PAR-2), expressed on keratinocytes but not on melanocytes, is involved in melanosome uptake via phagocytosis, and modulation of PAR-2 activation affects skin color. The pattern of melanosome distribution within the epidermis is skin color-dependent. In vitro, this distribution pattern is regulated by the ethnic origin of the keratinocytes, not the melanocytes. Therefore, we hypothesized that PAR-2 may play a role in the modulation of pigmentation in a skin type-dependent manner. We examined the expression of PAR-2 and its activator, trypsin, in human skins with different pigmentary levels. Here we show that PAR-2 and trypsin are expressed in higher levels, and are differentially localized in highly pigmented, relative to lightly pigmented skins. Moreover, highly pigmented skins exhibit an increase in PAR-2-specific protease cleavage ability. Microsphere phagocytosis was more efficient in keratinocytes from highly pigmented skins, and PAR-2 induced phagocytosis resulted in more efficient microsphere ingestion and more compacted microsphere organization in dark skin-derived keratinocytes. These results demonstrate that PAR-2 expression and activity correlate with skin color, suggesting the involvement of PAR-2 in ethnic skin color phenotypes.

Serine protease allergen favours Th2 responses via PAR-2 and STAT3 activation in murine model.

PubMed

Agrawal, K; Arora, N

2018-03-01

Protease activity of Per a 10 favours Th2 responses by differential regulation of IL-12p70 and IL-23 cytokine subunits. This study aimed to elucidate the underlying mechanism of differential regulation of IL-12p70 and IL-23. PAR-2 activation was blocked in murine model by administering SAM11 before each sensitization. CD11c + p-STAT3 + cells were measured in lungs by flow cytometry. BMDCs were pretreated with SAM11 or isotype control or stattic and stimulated with Per a 10. p-STAT3 levels were measured using Western blot. Transcript levels of IL-12p35, IL-12/23p40 and IL-23p19 were measured using RT-PCR. Cytokine levels were analysed using ELISA. Protease activity of Per a 10 increased p-STAT3 levels in mouse lungs, which was reduced upon PAR-2 blockage. Percentage of p-STAT3 + CD11c + cells was higher in Per a 10-administered mice and was reduced upon PAR-2 blockage. IL-12p35 and IL-12p70 levels were higher, and IL-23p19 and IL-23 levels were lower in both SAM11-treated mice and BMDCs indicating a role of PAR-2-mediated signalling. IL-4, TSLP, IL-17A, EPO activity, total cell count and specific IgE and IgG1 levels were lower in SAM11-administered mice. Inhibiting STAT3 activation via stattic also leads to lower levels of IL-23p19 and IL-23 and higher levels of IL-12p35. Per a 10 leads to PAR-2 activation on BMDCs resulting in downstream activation of STAT3 to regulate the balance between IL-12/IL-23 subunits causing a cytokine milieu rich in IL-23 to favour Th2 polarization. © 2017 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

The role of protease-activated receptors PAR-1 and PAR-2 in the repair of 16HBE 14o(-) epithelial cell monolayers in vitro.

PubMed

Ewen, D; Clarke, S L; Smith, J R; Berger, C; Salmon, G; Trevethick, M; Shute, J K

2010-03-01

We recently reported that repair following mechanical wounding of epithelial cell layers in vitro is dependent on fibrin formation and the activity of locally expressed coagulation cascade proteins. Serine proteases of the coagulation cascade are an important group of protease-activated receptor (PAR) activators and PAR-1 to 4 are expressed by the normal bronchial epithelium. We tested the hypothesis that activation of PAR-1 and PAR-2 by coagulation cascade proteases stimulates epithelial repair via effects on fibrin formation. Using mechanically wounded 16HBE 14o(-) epithelial cell layers in culture, we investigated the effect of PAR-1 and PAR-2 agonist peptides, control partially scrambled peptides and PAR-neutralizing antibodies on the rate of repair and fibrin formation. Coagulation factors in culture supernatants were measured by immunoblot. RT-PCR was used to investigate PAR-1, PAR-2 and PGE2 receptor (EP-1 to EP-4) expression in this model and qRT-PCR to quantify responses to wounding. Additionally, we investigated the effect of exogenously added factor Xa (FXa) and neutrophil elastase and the influence of PGE2 and indomethacin on the repair response. PAR-1 and PAR-2 peptide agonists stimulated the rate of repair and enhanced the formation of a fibrin provisional matrix to support the repair process. Conversely, PAR-neutralizing antibodies inhibited repair. Under serum-free culture conditions, 16HBE 14o(-) cells expressed EP-2 and EP-3, but not EP-1 or EP-4, receptors. Wounding induced an increased expression of EP-3 but did not alter EP-2, PAR-1 or PAR-2 expression. In the absence of PAR agonists, there was no evidence for a role for PGE2 in fibrin formation or the repair process. Indomethacin attenuated fibrin formation in wounded cultures only in the presence of the PAR-2 peptide. FXa stimulated epithelial repair while neutrophil elastase reduced the levels of coagulation factors and inhibited repair. Locally expressed serine proteases of the coagulation cascade activate PAR-1 and PAR-2 to enhance fibrin formation and bronchial epithelial repair.

Centromere Binding and Evolution of Chromosomal Partition Systems in the Burkholderiales

PubMed Central

Passot, Fanny M.; Calderon, Virginie; Fichant, Gwennaele; Lane, David

2012-01-01

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species. PMID:22522899

Centromere binding and evolution of chromosomal partition systems in the Burkholderiales.

PubMed

Passot, Fanny M; Calderon, Virginie; Fichant, Gwennaele; Lane, David; Pasta, Franck

2012-07-01

How split genomes arise and evolve in bacteria is poorly understood. Since each replicon of such genomes encodes a specific partition (Par) system, the evolution of Par systems could shed light on their evolution. The cystic fibrosis pathogen Burkholderia cenocepacia has three chromosomes (c1, c2, and c3) and one plasmid (pBC), whose compatibility depends on strictly specific interactions of the centromere sequences (parS) with their cognate binding proteins (ParB). However, the Par systems of B. cenocepacia c2, c3, and pBC share many features, suggesting that they arose within an extended family. Database searching revealed seven subfamilies of Par systems like those of B. cenocepacia. All are from plasmids and secondary chromosomes of the Burkholderiales, which reinforces the proposal of an extended family. The subfamily of the Par system of B. cenocepacia c3 includes plasmid variants with parS sequences divergent from that of c3. Using electrophoretic mobility shift assay (EMSA), we found that ParB-c3 binds specifically to centromeres of these variants, despite high DNA sequence divergence. We suggest that the Par system of B. cenocepacia c3 has preserved the features of an ancestral system. In contrast, these features have diverged variably in the plasmid descendants. One such descendant is found both in Ralstonia pickettii 12D, on a free plasmid, and in Ralstonia pickettii 12J, on a plasmid integrated into the main chromosome. These observations suggest that we are witnessing a plasmid-chromosome interaction from which a third chromosome will emerge in a two-chromosome species.

Transient early neurotrophin release and delayed inflammatory cytokine release by microglia in response to PAR-2 stimulation

PubMed Central

2012-01-01

Activated microglia exerts both beneficial and deleterious effects on neurons, but the signaling mechanism controlling these distinct responses remain unclear. We demonstrated that treatment of microglial cultures with the PAR-2 agonist, 2-Furoyl-LIGRLO-NH2, evoked early transient release of BDNF, while sustained PAR-2 stimulation evoked the delayed release of inflammatory cytokines (IL-1β and TNF-α) and nitric oxide. Culture medium harvested during the early phase (at 1 h) of microglial activation induced by 2-Furoyl-LIGRLO-NH2 (microglial conditioned medium, MCM) had no deleterious effects on cultured neurons, while MCM harvested during the late phase (at 72 h) promoted DNA fragmentation and apoptosis as indicated by TUNEL and annexin/PI staining. Blockade of PAR-1 during the early phase of PAR-2 stimulation enhanced BDNF release (by 11%, small but significant) while a PAR-1 agonist added during the late phase (24 h after 2-Furoyl-LIGRLO-NH2 addition) suppressed the release of cytokines and NO. The neuroprotective and neurotoxic effects of activated microglial exhibit distinct temporal profiles that are regulated by PAR-1 and PAR-2 stimulation. It may be possible to facilitate neuronal recovery and repair by appropriately timed stimulation and inhibition of microglial PAR-1 and PAR-2 receptors. PMID:22731117

Structures of archaeal DNA segregation machinery reveal bacterial and eukaryotic linkages.

PubMed

Schumacher, Maria A; Tonthat, Nam K; Lee, Jeehyun; Rodriguez-Castañeda, Fernando A; Chinnam, Naga Babu; Kalliomaa-Sanford, Anne K; Ng, Irene W; Barge, Madhuri T; Shaw, Porsha L R; Barillà, Daniela

2015-09-04

Although recent studies have provided a wealth of information about archaeal biology, nothing is known about the molecular basis of DNA segregation in these organisms. Here, we unveil the machinery and assembly mechanism of the archaeal Sulfolobus pNOB8 partition system. This system uses three proteins: ParA; an atypical ParB adaptor; and a centromere-binding component, AspA. AspA utilizes a spreading mechanism to create a DNA superhelix onto which ParB assembles. This supercomplex links to the ParA motor, which contains a bacteria-like Walker motif. The C domain of ParB harbors structural similarity to CenpA, which dictates eukaryotic segregation. Thus, this archaeal system combines bacteria-like and eukarya-like components, which suggests the possible conservation of DNA segregation principles across the three domains of life. Copyright © 2015, American Association for the Advancement of Science.

Down-regulated PAR-2 is associated in part with interrupted melanosome transfer in pigmented basal cell epithelioma.

PubMed

Sakuraba, Kazuko; Hayashi, Nobukazu; Kawashima, Makoto; Imokawa, Genji

2004-08-01

In pigmented basal cell epithelioma (BCE), there seems to be an abnormal transfer of melanized melanosomes from proliferating melanocytes to basaloid tumor cells. In this study, the interruption of that melanosome transfer was studied with special respect to the altered function of a phagocytic receptor, protease-activated receptor (PAR)-2 in the basaloid tumor cells. We used electron microscopy to clarify the disrupted transfer at the ultrastructural level and then performed immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) to examine the regulation of a phagocytic receptor, PAR-2, expressed on basaloid tumor cells. Electron microscopic analysis revealed that basaloid tumor cells of pigmented BCE have a significantly lower population of melanosomes ( approximately 16.4%) than do normal keratinocytes located in the perilesional normal epidermis ( approximately 91.0%). In contrast, in pigmented seborrheic keratosis (SK), a similarly pigmented epidermal tumor, the distribution of melanin granules does not differ between the lesional ( approximately 93.9%) and the perilesional normal epidermis ( approximately 92.2 %), indicating that interrupted melanosome transfer occurs in BCE but not in all pigmented epithelial tumors. RT-PCR analysis demonstrated that the expression of PAR-2 mRNA transcripts in basaloid cells is significantly decreased in pigmented BCE compared with the perilesional normal epidermis. In contrast, in pigmented SK, where melanosome transfer to basaloid tumor cells is not interrupted, the expression of PAR-2 mRNA transcripts is comparable between the basaloid tumor cells and the perilesional normal epidermis. Immunohistochemistry demonstrated that basaloid cells in pigmented BCE have less immunostaining for PAR-2 than do keratinocytes in the perilesional normal epidermis whereas in pigmented SK, there is no difference in immunostaining for PAR-2 between the basaloid tumor and the perilesional normal epidermis. These findings suggest that the decreased expression of PAR-2 in the basaloid cells is associated in part with the observed interruption of melanosome transfer in pigmented BCE.

Proteinase-activated receptor-4 evoked colorectal analgesia in mice: an endogenously activated feed-back loop in visceral inflammatory pain.

PubMed

Annaházi, A; Dabek, M; Gecse, K; Salvador-Cartier, C; Polizzi, A; Rosztóczy, A; Róka, R; Theodorou, V; Wittmann, T; Bueno, L; Eutamene, H

2012-01-01

Activation of proteinase-activated receptor-4 (PAR-4) from the colonic lumen has an antinociceptive effect to colorectal distension (CRD) in mice in basal conditions. We aimed to determine the functional localization of the responsible receptors and to test their role in two different hyperalgesia models. Mice received PAR-4 activating peptide (PAR-4-AP, AYPGKF-NH(2)) or vehicle intraperitoneally (IP), and abdominal EMG response to CRD was measured. The next group received PAR-4-AP intracolonically (IC) with or without 2,4,6-triaminopyrimidine, a chemical tight junction blocker, before CRD. The SCID mice were used to test the role of lymphocytes in the antihyperalgesic effect. The effects of PAR-4-AP and PAR-4-antagonist (P4pal-10) were evaluated in water avoidance stress (WAS) model and low grade 2,4,6-trinitrobenzene sulfonic acid (TNBS) colitis. Spinal Fos protein expression was visualized by immunohistochemistry. The antinociceptive effect of PAR-4-AP disappeared when was administrered IP, or with the blockade of colonic epithelial tight junctions, suggesting that PAR-4-AP needs to reach directly the nerve terminals in the colon. The CRD-induced spinal Fos overexpression was reduced by 43% by PAR-4-AP. The PAR-4-AP was antihyperalgesic in both hyperalgesia models and in mice with impaired lymphocytes. The PAR-4-antagonist significantly increased the TNBS, but not the WAS-induced colonic hyperalgesia. The antinociceptive effect of PAR-4-AP depends on its penetration to the colonic mucosa. The PAR-4 activation is endogenously involved as a feedback loop to attenuate inflammatory colonic hyperalgesia to CRD. © 2011 Blackwell Publishing Ltd.

The protease-activated receptor 2 regulates pigmentation via keratinocyte-melanocyte interactions.

PubMed

Seiberg, M; Paine, C; Sharlow, E; Andrade-Gordon, P; Costanzo, M; Eisinger, M; Shapiro, S S

2000-01-10

Close association exists between melanocytes, the pigment melanin-producing cells in the body, and their neighboring keratinocytes. Keratinocytes are the pigment recipients and skin pigmentation is the result of this interaction. While the chemical basis of melanin production (melanogenesis) is well documented, the molecular mechanism of melanosome transfer needs to be elucidated. We are now providing first evidence that the protease-activated receptor 2 (PAR-2) expressed on keratinocytes, but not on melanocytes, is involved in melanosome transfer and therefore may regulate pigmentation. Activation of PAR-2 with trypsin or with the peptide agonist SLIGRL induced pigmentation in both two- and three-dimensional cocultures of keratinocytes and melanocytes, but not in cocultures that were spatially separated, indicating the need for intimate cell-cell contact. Topical application of SLIGRL on human skin transplanted on SCID mice resulted in a visible skin darkening. Histological examination revealed increased deposits of melanin in the keratinocytes. Inhibition of PAR-2 activation by RWJ-50353, a serine protease inhibitor, resulted in depigmentation and changes in expression of melanogenic-specific genes. Keratinocyte-melanocyte contact was essential for this depigmenting effect. Topical application of this inhibitor induced lightening of the dark skin Yucatan swine, which was confirmed by histochemical analysis. The results presented here suggest a novel mechanism for the regulation of pigmentation, mediated by the activation or inhibition of the keratinocyte receptor PAR-2. Copyright 2000 Academic Press.

ADAP interactions with talin and kindlin promote platelet integrin αIIbβ3 activation and stable fibrinogen binding

PubMed Central

Kang, Jian; Kahner, Bryan; Ye, Feng; Ginsberg, Mark H.; Shattil, Sanford J.

2014-01-01

ADAP is a hematopoietic-restricted adapter protein that promotes integrin activation and is a carrier for other adapter proteins, Src kinase–associated phosphoprotein 1 (SKAP1) and SKAP2. In T lymphocytes, SKAP1 is the ADAP-associated molecule that activates integrins through direct linkages with Rap1 effectors (regulator of cell adhesion and polarization enriched in lymphoid tissues; Rap1-interacting adapter molecule). ADAP also promotes integrin αIIbβ3 activation in platelets, which lack SKAP1, suggesting an ADAP integrin–regulatory pathway different from those in lymphocytes. Here we characterized a novel association between ADAP and 2 essential integrin-β cytoplasmic tail-binding proteins involved in αIIbβ3 activation, talin and kindlin-3. Glutathione S-transferase pull-downs identified distinct regions in ADAP necessary for association with kindlin or talin. ADAP was physically proximal to talin and kindlin-3 in human platelets, as assessed biochemically, and by immunofluorescence microscopy and proximity ligation. Relative to wild-type mouse platelets, ADAP-deficient platelets exhibited reduced co-localization of talin with αIIbβ3, and reduced irreversible fibrinogen binding in response to a protease activated receptor 4 (PAR4) thrombin receptor agonist. When ADAP was heterologously expressed in Chinese hamster ovary cells co-expressing αIIbβ3, talin, PAR1, and kindlin-3, it associated with an αIIbβ3/talin complex and enabled kindlin-3 to promote agonist-dependent ligand binding to αIIbβ3. Thus, ADAP uniquely promotes activation of and irreversible fibrinogen binding to platelet αIIbβ3 through interactions with talin and kindlin-3. PMID:24523237

Aspergillus fumigatus Increased PAR-2 Expression and Elevated Proinflammatory Cytokines Expression Through the Pathway of PAR-2/ERK1/2 in Cornea.

PubMed

Niu, Yawen; Zhao, Guiqiu; Li, Cui; Lin, Jing; Jiang, Nan; Che, Chengye; Zhang, Jie; Xu, Qiang

2018-01-01

To determine the role of protease-activated receptor-2 (PAR-2) in cornea infected by Aspergillus fumigatus. PAR-2 was tested in normal and infected corneas of C57BL/6 mice. Mice corneas were infected with A. fumigatus with or without pretreatment of PAR-2 antagonist (FSLLRY-NH2). Polymorphonuclear neutrophilic leukocytes (PMNs) were stimulated with 75% ethanol-killed A. fumigatus with or without pretreatment of FSLLRY-NH2. Disease severity was documented by clinical score and photographs with a slit lamp. PCR, Western blot, and ELISA tested expression of PAR-2, IL-1β, TNF-α, IFN-γ, MIP-2, and p-ERK1/2. PMN infiltration was assessed by myeloperoxidase assay and immunofluorescent staining. PAR-2 expression was significantly elevated by A. fumigatus, whereas the upregulation was significantly inhibited by FSLLRY-NH2 in mice corneas. FSLLRY-NH2 decreased disease response, PMN infiltration, and proinflammatory cytokine expression compared with infected control. In PMNs, PAR-2 expression was also significantly increased by A. fumigatus, which was significantly inhibited by FSLLRY-NH2. FSLLRY-NH2 significantly inhibited proinflammatory cytokine protein expression, as compared with that in infected control cells, which may be modified by p-ERK1/2. These data provide evidence that A. fumigatus increased PAR-2 expression and elevated disease, PMN infiltration, and proinflammatory cytokine expression through PAR-2, which may be modified by p-ERK1/2.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fahrer, Joerg, E-mail: joerg.fahrer@uni-ulm.de; Wagner, Silvia; Buerkle, Alexander

Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin didmore » not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.« less

A high-protein diet during hospitalization is associated with an accelerated decrease in soluble urokinase plasminogen activator receptor levels in acutely ill elderly medical patients with SIRS.

PubMed

Tavenier, Juliette; Haupt, Thomas H; Andersen, Aino L; Buhl, Sussi F; Langkilde, Anne; Andersen, Jens R; Jensen, Jens-Erik B; Pedersen, Mette M; Petersen, Janne; Andersen, Ove

2017-05-01

Acute illness and hospitalization in elderly individuals are often accompanied by the systemic inflammatory response syndrome (SIRS) and malnutrition, both associated with wasting and mortality. Nutritional support and resistance training were shown to increase muscle anabolism and reduce inflammation in healthy elderly. We hypothesized that nutritional support and resistance training would accelerate the resolution of inflammation in hospitalized elderly patients with SIRS. Acutely admitted patients aged >65 years with SIRS were randomized to an intervention consisting of a high-protein diet (1.7 g/kg per day) during hospitalization, and daily protein supplement (18.8 g) and 3 weekly resistance training sessions for 12 weeks after discharge (Intervention, n=14), or to standard-care (Control, n=15). Plasma levels of the inflammatory biomarkers soluble urokinase plasminogen activator receptor (suPAR), interleukin-6, C-reactive protein (CRP), and albumin were measured at admission, discharge, and 4 and 13 weeks after discharge. The Intervention group had an earlier decrease in suPAR levels than the Control group: -15.4% vs. +14.5%, P=.007 during hospitalization, and -2.4% vs. -28.6%, P=.007 between discharge and 4 weeks. There were no significant effects of the intervention on the other biomarkers. All biomarkers improved significantly between admission and 13 weeks, although with different kinetics (suPAR: -22%, interleukin-6: -86%, CRP: -89%, albumin: +11%). Nutritional support during hospitalization was associated with an accelerated decrease in suPAR levels, whereas the combined nutrition and resistance training intervention after discharge did not appear to affect the inflammatory state. Our results indicate that improved nutritional care during hospitalization may accelerate recovery in acutely ill elderly medical patients. Copyright © 2017 Elsevier Inc. All rights reserved.

Expression of the Ly6/uPAR-domain proteins C4.4A and Haldisin in non-invasive and invasive skin lesions.

PubMed

Kriegbaum, Mette C; Clausen, Ole P F; Lærum, Ole D; Ploug, Michael

2015-02-01

C4.4A and Haldisin belong to the Ly6/uPAR/α-neurotoxin protein domain family. They exhibit highly regulated expression profiles in normal epidermis, where they are confined to early (C4.4A) and late (Haldisin) squamous differentiation. We have now explored if dysregulated expressions occur in non-invasive and invasive skin lesions. In non-invasive lesions, their expression signatures were largely maintained as defined by that of normal epidermis. The scenario was, however, markedly different in the progression towards invasive squamous cell carcinomas. In its non-invasive stage (carcinoma in situ), a pronounced attenuation of C4.4A expression was observed, but upon transition to malignant invasive squamous cell carcinomas, the invasive fronts regained high expression of C4.4A. A similar progression was observed for the early stages of benign infiltrating keratoacanthomas. Interestingly, this transition was accompanied by a shift in the predominant association of C4.4A expression with CK1/10 in the normal epidermis to CK5/14 in the invasive lesions. In contrast, Haldisin expression maintained its confinement to the most-differentiated cells and was hardly expressed in the invasive lesions. Because this altered expression of C4.4A was seen in the invasive front of benign (keratoacanthomas) and malignant (squamous cell carcinomas) neoplasms, we propose that this transition of expression is primarily related to the invasive process. © The Author(s) 2014.

PAR-2, IL-4R, TGF-β and TNF-α in bronchoalveolar lavage distinguishes extrinsic allergic alveolitis from sarcoidosis.

PubMed

Matěj, Radoslav; Smětáková, Magdalena; Vašáková, Martina; Nováková, Jana; Sterclová, Martina; Kukal, Jaromír; Olejár, Tomáš

2014-08-01

Sarcoidosis (SARC) and extrinsic allergic alveolitis (EAA) share certain markers, making a differential diagnosis difficult even with histopathological investigation. In lung tissue, proteinase-activated receptor-2 (PAR-2) is primarily investigated with regard to epithelial and inflammatory perspectives. Varying levels of certain chemokines can be a useful tool for distinguishing EAA and SARC. Thus, in the present study, differences in the levels of transforming growth factor (TGF)-β1, tumor necrosis factor (TNF)-α, interleukin-4 receptor (IL-4R) and PAR-2 in bronchoalveolar lavage fluid (BALF) were compared, using an ELISA method, between 14 patients with EAA and six patients with SARC. Statistically significant higher levels of IL-4R, PAR-2 and the PAR-2/TGF-β1 and PAR-2/TNF-α ratios were observed in EAA patients as compared with SARC patients. Furthermore, the ratios of TNF-α/total protein, TGF-β1/PAR-2 and TNF-α/PAR-2 were significantly lower in EAA patients than in SARC patients. The results indicated a higher detection of PAR-2 in EAA samples in association with TNF-α and TGF-β levels. As EAA and PAR-2 in parallel belong to the Th2-mediated pathway, the results significantly indicated an association between this receptor and etiology. In addition, the results indicated that SARC is predominantly a granulomatous inflammatory disease, thus, higher levels of TNF-α are observed. Therefore, the detection of PAR-2 and investigated chemokines in BALF may serve as a useful tool in the differential diagnosis between EAA and SARC.

Enhanced proliferation of human hepatoma cells by PAR-2 agonists via the ERK/AP-1 pathway.

PubMed

Xie, Liqun; Zheng, Yanmin; Li, Xuan; Zhao, Junyan; Chen, Xiaoyi; Chen, Li; Zhou, Jing; Hai, Ou; Li, Fei

2012-11-01

To investigate the expression and role of PAR-2 in the proliferation of the human hepatoma cell line HepG2, PAR-2 protein and mRNA expression were evaluated by immuno-histochemistry, immunofluorescence and RT-PCR analysis. The signaling pathways downstream of PAR-2 activation that lead to hepatoma cell proliferation were analyzed. The results showed that PAR-2 is expressed in human hepatoma cells and PAR-2 mRNA expression was found to be upregulated in cells treated with trypsin or SLIGKV-NH2 (P<0.001). The proliferation rate of HepG2 cells treated with trypsin or SLIGKV-NH2 was significantly increased (P<0.001). The percentage of S phase, G2/M phase and the proliferation index (PI) of HepG2 cells treated with trypsin or SLIGKV-NH2 were significantly elevated (P<0.001). The proliferative responses of HepG2 to trypsin and SLIGKV-NH2 were associated with the upregulation of c-fos and PCNA, which were significantly blocked by PD98059 pretreatment. In conclusion, our results indicate that PAR-2 enhances proliferation of human hepatoma cells possibly via the ERK/AP-1 pathway.

Poly(ADP-ribose) polymerases covalently modify strand break termini in DNA fragments in vitro

PubMed Central

Talhaoui, Ibtissam; Lebedeva, Natalia A.; Zarkovic, Gabriella; Saint-Pierre, Christine; Kutuzov, Mikhail M.; Sukhanova, Maria V.; Matkarimov, Bakhyt T.; Gasparutto, Didier; Saparbaev, Murat K.; Lavrik, Olga I.; Ishchenko, Alexander A.

2016-01-01

Poly(ADP-ribose) polymerases (PARPs/ARTDs) use nicotinamide adenine dinucleotide (NAD+) to catalyse the synthesis of a long branched poly(ADP-ribose) polymer (PAR) attached to the acceptor amino acid residues of nuclear proteins. PARPs act on single- and double-stranded DNA breaks by recruiting DNA repair factors. Here, in in vitro biochemical experiments, we found that the mammalian PARP1 and PARP2 proteins can directly ADP-ribosylate the termini of DNA oligonucleotides. PARP1 preferentially catalysed covalent attachment of ADP-ribose units to the ends of recessed DNA duplexes containing 3′-cordycepin, 5′- and 3′-phosphate and also to 5′-phosphate of a single-stranded oligonucleotide. PARP2 preferentially ADP-ribosylated the nicked/gapped DNA duplexes containing 5′-phosphate at the double-stranded termini. PAR glycohydrolase (PARG) restored native DNA structure by hydrolysing PAR-DNA adducts generated by PARP1 and PARP2. Biochemical and mass spectrometry analyses of the adducts suggested that PARPs utilise DNA termini as an alternative to 2′-hydroxyl of ADP-ribose and protein acceptor residues to catalyse PAR chain initiation either via the 2′,1″-O-glycosidic ribose-ribose bond or via phosphodiester bond formation between C1′ of ADP-ribose and the phosphate of a terminal deoxyribonucleotide. This new type of post-replicative modification of DNA provides novel insights into the molecular mechanisms underlying biological phenomena of ADP-ribosylation mediated by PARPs. PMID:27471034

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma

PubMed Central

Shrestha Palikhe, Nami; Nahirney, Drew; Laratta, Cheryl; Gandhi, Vivek Dipak; Vethanayagam, Dilini; Bhutani, Mohit; Mayers, Irvin

2015-01-01

Background Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied. Methods We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry. Results Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations. Conclusions PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma. PMID:26658828

Increased Protease-Activated Receptor-2 (PAR-2) Expression on CD14++CD16+ Peripheral Blood Monocytes of Patients with Severe Asthma.

PubMed

Shrestha Palikhe, Nami; Nahirney, Drew; Laratta, Cheryl; Gandhi, Vivek Dipak; Vethanayagam, Dilini; Bhutani, Mohit; Mayers, Irvin; Cameron, Lisa; Vliagoftis, Harissios

2015-01-01

Protease-Activated Receptor-2 (PAR-2), a G protein coupled receptor activated by serine proteases, is widely expressed in humans and is involved in inflammation. PAR-2 activation in the airways plays an important role in the development of allergic airway inflammation. PAR-2 expression is known to be upregulated in the epithelium of asthmatic subjects, but its expression on immune and inflammatory cells in patients with asthma has not been studied. We recruited 12 severe and 24 mild/moderate asthmatics from the University of Alberta Hospital Asthma Clinics and collected baseline demographic information, medication use and parameters of asthma severity. PAR-2 expression on blood inflammatory cells was analyzed by flow cytometry. Subjects with severe asthma had higher PAR-2 expression on CD14++CD16+ monocytes (intermediate monocytes) and also higher percentage of CD14++CD16+PAR-2+ monocytes (intermediate monocytes expressing PAR-2) in blood compared to subjects with mild/moderate asthma. Receiver operating characteristics (ROC) curve analysis showed that the percent of CD14++CD16+PAR-2+ in peripheral blood was able to discriminate between patients with severe and those with mild/moderate asthma with high sensitivity and specificity. In addition, among the whole populations, subjects with a history of asthma exacerbations over the last year had higher percent of CD14++CD16+ PAR-2+ cells in peripheral blood compared to subjects without exacerbations. PAR-2 expression is increased on CD14++CD16+ monocytes in the peripheral blood of subjects with severe asthma and may be a biomarker of asthma severity. Our data suggest that PAR-2 -mediated activation of CD14++CD16+ monocytes may play a role in the pathogenesis of severe asthma.

Pharmacophore-based virtual screening, biological evaluation and binding mode analysis of a novel protease-activated receptor 2 antagonist

NASA Astrophysics Data System (ADS)

Cho, Nam-Chul; Seo, Seoung-Hwan; Kim, Dohee; Shin, Ji-Sun; Ju, Jeongmin; Seong, Jihye; Seo, Seon Hee; Lee, Iiyoun; Lee, Kyung-Tae; Kim, Yun Kyung; No, Kyoung Tai; Pae, Ae Nim

2016-08-01

Protease-activated receptor 2 (PAR2) is a G protein-coupled receptor, mediating inflammation and pain signaling in neurons, thus it is considered to be a potential therapeutic target for inflammatory diseases. In this study, we performed a ligand-based virtual screening of 1.6 million compounds by employing a common-feature pharmacophore model and two-dimensional similarity search to identify a new PAR2 antagonist. The common-feature pharmacophore model was established based on the biological screening results of our in-house library. The initial virtual screening yielded a total number of 47 hits, and additional biological activity tests including PAR2 antagonism and anti-inflammatory effects resulted in a promising candidate, compound 43, which demonstrated an IC50 value of 8.22 µM against PAR2. In next step, a PAR2 homology model was constructed using the crystal structure of the PAR1 as a template to explore the binding mode of the identified ligands. A molecular docking method was optimized by comparing the binding modes of a known PAR2 agonist GB110 and antagonist GB83, and applied to predict the binding mode of our hit compound 43. In-depth docking analyses revealed that the hydrophobic interaction with Phe2435.39 is crucial for PAR2 ligands to exert antagonistic activity. MD simulation results supported the predicted docking poses that PAR2 antagonist blocked a conformational rearrangement of Na+ allosteric site in contrast to PAR2 agonist that showed Na+ relocation upon GPCR activation. In conclusion, we identified new a PAR2 antagonist together with its binding mode, which provides useful insights for the design and development of PAR2 ligands.

IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity In vivo and operator binding in vitro.

PubMed

Jagura-Burdzy, G; Kostelidou, K; Pole, J; Khare, D; Jones, A; Williams, D R; Thomas, C M

1999-05-01

The korAB operon of broad-host-range plasmid RK2 encodes five genes, two of which, incC and korB, belong to the parA and parB families, respectively, of genome partitioning functions. Both korB and a third gene, korA, are responsible for coordinate regulation of operons encoding replication, transfer, and stable inheritance functions. Overexpression of incC alone caused rapid displacement of RK2. Using two different reporter systems, we show that incC modulates the action of KorB. Using promoter fusions to the reporter gene xylE, we show that incC potentiates the repression of transcription by korB. This modulation of korB activity was only observed with incC1, which encodes the full-length IncC (364 amino acids [aa]), whereas no effect was observed with incC2, which encodes a polypeptide of 259 aa that lacks the N-terminal 105 aa. Using bacterial extracts with IncC1 and IncC2 or IncC1 purified through the use of a His6 tail and Ni-agarose chromatography, we showed that IncC1 potentiates the binding of KorB to DNA at representative KorB operators. The ability of IncC to stabilize KorB-DNA complexes suggests that these two proteins work together in the global regulation of many operons on the IncP-1 genomes, as well in plasmid partitioning.

Suppression of Peripheral Sympathetic Activity Underlies Protease-Activated Receptor 2-Mediated Hypotension

PubMed Central

Kim, Young-Hwan; Ahn, Duck-Sun; Joeng, Ji-Hyun

2014-01-01

Protease-activated receptor (PAR)-2 is expressed in endothelial cells and vascular smooth muscle cells. It plays a crucial role in regulating blood pressure via the modulation of peripheral vascular tone. Although some reports have suggested involvement of a neurogenic mechanism in PAR-2-induced hypotension, the accurate mechanism remains to be elucidated. To examine this possibility, we investigated the effect of PAR-2 activation on smooth muscle contraction evoked by electrical field stimulation (EFS) in the superior mesenteric artery. In the present study, PAR-2 agonists suppressed neurogenic contractions evoked by EFS in endothelium-denuded superior mesenteric arterial strips but did not affect contraction elicited by the external application of noradrenaline (NA). However, thrombin, a potent PAR-1 agonist, had no effect on EFS-evoked contraction. Additionally, ω-conotoxin GVIA (CgTx), a selective N-type Ca2+ channel (ICa-N) blocker, significantly inhibited EFS-evoked contraction, and this blockade almost completely occluded the suppression of EFS-evoked contraction by PAR-2 agonists. Finally, PAR-2 agonists suppressed the EFS-evoked overflow of NA in endothelium-denuded rat superior mesenteric arterial strips and this suppression was nearly completely occluded by ω-CgTx. These results suggest that activation of PAR-2 may suppress peripheral sympathetic outflow by modulating activity of ICa-N which are located in peripheral sympathetic nerve terminals, which results in PAR-2-induced hypotension. PMID:25598663

31 CFR 359.55 - How are redemption values calculated for book-entry Series I savings bonds?

Code of Federal Regulations, 2010 CFR

2010-07-01

... prorated to the book-entry par investment amount for the corresponding issue and redemption dates... to $25.04; calculated value of $25.045 rounds to $25.05. [Book-entry par investment ÷ 100] × [CRV... for book-entry Series I savings bonds? 359.55 Section 359.55 Money and Finance: Treasury Regulations...

Etude de l'apprentissage par la lecture d'etudiants en contexte d'apprentissage par problemes (APP) (A Study of Student Text-Learning Strategy).

ERIC Educational Resources Information Center

Cartier, Sylvie

2002-01-01

Examined strategies reported by students during reading to develop knowledge in their area of professional training. Discovered the reading, encoding, self-regulation, and resource management strategies used most often by students, which were not sufficient to attain effective learning. Less frequently used strategies added nuances to the…

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer.

PubMed

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-10-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer.

Effects of silenced PAR-2 on cell proliferation, invasion and metastasis of esophageal cancer

PubMed Central

Chen, Jinmei; Xie, Liqun; Zheng, Yanmin; Liu, Caihong

2017-01-01

The present study aimed to investigate the effect of protease-activated receptor 2 (PAR-2) on cell proliferation, invasion and metastasis in the esophageal EC109 cell line. Two short hairpin RNA (shRNA) expression plasmids were constructed based on the PAR-2 mRNA sequence in humans, and they were transfected into the EC109 esophageal cancer cell line, and the stable interference cell line (shRNA-PAR-2 EC109) was obtained by puromycin selection. Following transfection of PAR-2 shRNA-1, PAR-2 expression was significantly downregulated in mRNA level and protein level in EC109 cells (P<0.05). The proliferation of EC109 cells transfected with PAR-2 shRNA was significantly lower than the negative control group (P<0.05). At 24, 48 and 72 h, the ratio of proliferation inhibition was 15.92, 24.89 and 32.28%, respectively. Compared with the control group, S-phase arrest was observed in cells transfected with shRNA-PAR-2. The ratio of cells in the S phase was 32.79±4.06, 26.54±1.37 and 33.45±2.46% at 24, 48 and 72 h, respectively. For invasion, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.05). For metastasis assay, the number of invasive cells was significantly lower in shRNA-PAR2-2 cells compared with the control group (P<0.01). In the present study, the PAR-2 shRNA plasmid was constructed successfully, which can significantly downregulate PAR-2 expression in EC109 cells. Subsequent to silencing of PAR-2, the proliferation of EC109 cells was inhibited and the capabilities of invasion and migration were reduced. It is indicated that PAR-2 may be a potential target in esophageal cancer. PMID:28943918

Mechanism of the asymmetric activation of the MinD ATPase by MinE

PubMed Central

Park, Kyung-Tae; Wu, Wei; Lovell, Scott; Lutkenhaus, Joe

2012-01-01

Summary MinD is a component of the Min system involved in the spatial regulation of cell division. It is an ATPase in the MinD/ParA/Mrp deviant Walker A motif family which is within the P loop GTPase superfamily. Its ATPase activity is stimulated by MinE, however, the mechanism of this activation is unclear. MinD forms a symmetric dimer with two binding sites for MinE, however, a recent model suggested that MinE occupying one site was sufficient for ATP hydrolysis. By generating heterodimers with one binding site for MinE we show that one binding site is sufficient for stimulation of the MinD ATPase. Furthermore, comparison of structures of MinD and related proteins led us to examine the role of N45 in the switch I region. An asparagine at this position is conserved in four of the deviant Walker A motif subfamilies (MinD, chromosomal ParAs, Get3 and FleN) and we find that N45 in MinD is essential for MinE stimulated ATPase activity and suggest that it is a key residue affected by MinE binding. PMID:22651575

New PARP targets for cancer therapy

PubMed Central

Vyas, Sejal; Chang, Paul

2015-01-01

Poly(ADP-ribose) polymerases (PARPs) modify target proteins post-translationally with poly(ADP-ribose) (PAR) or mono(ADP-ribose) (MAR) using NAD+ as substrate. The best-studied PARPs generate PAR modifications and include PARP1 and the tankyrase PARP5a, both of which are targets for cancer therapy with inhibitors in either clinical trials or preclinical development. There are 15 additional PARPs, the majority of which modify proteins with MAR, and their biology is less well understood. Recent data identify potentially cancer relevant functions for these PARPs, indicating that we need to understand more about these PARPs in order to target them effectively. PMID:24898058

Persistent kallikrein 5 activation induces atopic dermatitis-like skin architecture independent of PAR2 activity.

PubMed

Zhu, Yanan; Underwood, Joanne; Macmillan, Derek; Shariff, Leila; O'Shaughnessy, Ryan; Harper, John I; Pickard, Chris; Friedmann, Peter S; Healy, Eugene; Di, Wei-Li

2017-11-01

Upregulation of kallikreins (KLKs) including KLK5 has been reported in atopic dermatitis (AD). KLK5 has biological functions that include degrading desmosomal proteins and inducing proinflammatory cytokine secretion through protease-activated receptor 2 (PAR2). However, due to the complex interactions between various cells in AD inflamed skin, it is difficult to dissect the precise and multiple roles of upregulated KLK5 in AD skin. We investigated the effect of upregulated KLK5 on the expression of epidermal-related proteins and cytokines in keratinocytes and on skin architecture. Lesional and nonlesional AD skin biopsies were collected for analysis of morphology and protein expression. The relationship between KLK5 and barrier-related molecules was investigated using an ex vivo dermatitis skin model with transient KLK5 expression and a cell model with persistent KLK5 expression. The influence of upregulated KLK5 on epidermal morphology was investigated using an in vivo skin graft model. Upregulation of KLK5 and abnormal expression of desmoglein 1 (DSG1) and filaggrin, but not PAR2 were identified in AD skin. PAR2 was increased in response to transient upregulation of KLK5, whereas persistently upregulated KLK5 did not show this effect. Persistently upregulated KLK5 degraded DSG1 and stimulated secretion of IL-8, IL-10, and thymic stromal lymphopoietin independent of PAR2 activity. With control of higher KLK5 activity by the inhibitor sunflower trypsin inhibitor G, restoration of DSG1 expression and a reduction in AD-related cytokine IL-8, thymic stromal lymphopoietin, and IL-10 secretion were observed. Furthermore, persistently elevated KLK5 could induce AD-like skin architecture in an in vivo skin graft model. Persistently upregulated KLK5 resulted in AD-like skin architecture and secretion of AD-related cytokines from keratinocytes in a PAR2 independent manner. Inhibition of KLK5-mediated effects may offer potential as a therapeutic approach in AD. Copyright © 2017 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

1,25-Dihydroxyvitamin D(3) Inhibits Podocyte uPAR Expression and Reduces Proteinuria

PubMed Central

Liu, Shuangxin; Xie, Shaoting; Yang, Yun; Ma, Juan; Deng, Yujun; Wang, Wenjian; Xu, Lixia; Li, Ruizhao; Zhang, Li; Yu, Chunping; Shi, Wei

2013-01-01

Background Accumulating studies have demonstrated that 1,25-Dihydroxyvitamin D(3) (1,25(OH)2D3) reduces proteinuria and protects podocytes from injury. Recently, urokinase receptor (uPAR) and its soluble form have been shown to cause podocyte injury and focal segmental glomerulosclerosis (FSGS). Here, our findings showed that 1,25(OH)2D3 did inhibit podocyte uPAR expression and attenuate proteinuria and podocyte injury. Methodology/Principal Findings In this study, the antiproteinuric effect of 1,25(OH)2D3 was examined in the lipopolysaccharide mice model of transient proteinuria (LPS mice) and in the 5/6 nephrectomy rat FSGS model(NTX rats). uPAR protein expression were tested by flow cytometry, immune cytochemistry and western blot analysis, and uPAR mRNA expression by real-time quantitative PCR in cultured podocytes and kidney glomeruli isolated from mice and rats. Podocyte motility was observed by transwell migration assay and wound healing assay. Podocyte foot processes effacement was identified by transmission electron microscopy. We found that 1,25(OH)2D3 inhibited podocyte uPAR mRNA and protein synthesis in LPS-treated podocytes, LPS mice and NTX rats, along with 1,25(OH)2D3 reducing proteinuria in NTX rats and LPS mice.1,25(OH)2D3 reduced glomerulosclerosis in NTX rats and alleviated podocyte foot processes effacement in LPS mice. Transwell migration assay and wound healing assay showed that LPS-induced podocyte motility, irrespective of random or directed motility, were substantially reduced by 1,25(OH)2D3. Conclusions/Significance Our results demonstrated that 1,25(OH)2D3 inhibited podocyte uPAR expression in vitro and in vivo, which may be an unanticipated off target effect of 1,25(OH)2D3 and explain its antiproteinuric effect in the 5/6 nephrectomy rat FSGS model and the LPS mouse model of transient proteinuria. PMID:23741418

PARP-1 may be involved in hydroquinone-induced apoptosis by poly ADP-ribosylation of ZO-2

PubMed Central

Liu, Jiaxian; Yuan, Qian; Ling, Xiaoxuan; Tan, Qiang; Liang, Hairong; Chen, Jialong; Lin, Lianzai; Xiao, Yongmei; Chen, Wen; Liu, Linhua; Tang, Huanwen

2017-01-01

Hydroquinone (HQ), a major reactive metabolite of benzene, contributes to benzene-induced leukemia. The molecular mechanisms that underlie this activity remain to be elucidated. Poly ADP-ribosylation (PARylation) is a type of reversible posttranslational modification that is performed by enzymes in the PAR polymerase (PARP) family and mediates different biological processes, including apoptosis. Zona occludens 2 (ZO-2) is a tight junction scaffold protein, which is involved in cell proliferation and apoptosis. The present study investigated the activity and mechanisms regulated by PARP-1 during HQ-induced apoptosis using TK6 lymphoblastoid cells and PARP-1-silenced TK6 cells. The results revealed that exposure to 10 µM HQ for 72 h induced apoptosis in TK6 cells and that apoptosis was attenuated in PARP-1-silenced TK6 cells. In cells treated with HQ, inhibition of PARP-1 increased the expression of B cell leukemia/lymphoma 2 (Bcl-2), increased ATP production and reduced reactive oxygen species (ROS) production relative to the levels observed in cells treated with HQ alone. Co-localization of ZO-2 and PAR (or PARP-1 protein) was determined using immunofluorescence confocal microscopy. The findings of the present study revealed that ZO-2 was PARylated via an interaction with PARP-1, which was consistent with an analysis of protein expression that was performed using western blot analysis, which determined that ZO-2 protein expression was upregulated in HQ-treated control cells and downregulated in HQ-treated PARP-1-silenced TK6 cells. These findings indicated that prolonged exposure to a low dose of HQ induced TK6 cells to undergo apoptosis, whereas inhibiting PARP-1 attenuates cellular apoptosis by activating Bcl-2 and energy-saving processes and reducing ROS. The present study determined that PARP-1 was involved in HQ-induced apoptosis by PARylation of ZO-2. PMID:28983606

Intrastrain heterogeneity of the mgpB gene in Mycoplasma genitalium is extensive in vitro and in vivo and suggests that variation is generated via recombination with repetitive chromosomal sequences.

PubMed

Iverson-Cabral, Stefanie L; Astete, Sabina G; Cohen, Craig R; Rocha, Eduardo P C; Totten, Patricia A

2006-07-01

Mycoplasma genitalium is associated with reproductive tract disease in women and may persist in the lower genital tract for months, potentially increasing the risk of upper tract infection and transmission to uninfected partners. Despite its exceptionally small genome (580 kb), approximately 4% is composed of repeated elements known as MgPar sequences (MgPa repeats) based on their homology to the mgpB gene that encodes the immunodominant MgPa adhesin protein. The presence of these MgPar sequences, as well as mgpB variability between M. genitalium strains, suggests that mgpB and MgPar sequences recombine to produce variant MgPa proteins. To examine the extent and generation of diversity within single strains of the organism, we examined mgpB variation within M. genitalium strain G-37 and observed sequence heterogeneity that could be explained by recombination between the mgpB expression site and putative donor MgPar sequences. Similarly, we analyzed mgpB sequences from cervical specimens from a persistently infected woman (21 months) and identified 17 different mgpB variants within a single infecting M. genitalium strain, confirming that mgpB heterogeneity occurs over the course of a natural infection. These observations support the hypothesis that recombination occurs between the mgpB gene and MgPar sequences and that the resulting antigenically distinct MgPa variants may contribute to immune evasion and persistence of infection.

The Role of PAR2 in TGF-β1-Induced ERK Activation and Cell Motility

PubMed Central

Ungefroren, Hendrik; Witte, David; Fiedler, Christian; Gädeken, Thomas; Kaufmann, Roland; Lehnert, Hendrik

2017-01-01

Background: Recently, the expression of proteinase-activated receptor 2 (PAR2) has been shown to be essential for activin receptor-like kinase 5 (ALK5)/SMAD-mediated signaling and cell migration by transforming growth factor (TGF)-β1. However, it is not known whether activation of non-SMAD TGF-β signaling (e.g., RAS–RAF–MEK–extracellular signal-regulated kinase (ERK) signaling) is required for cell migration and whether it is also dependent on PAR2. Methods: RNA interference was used to deplete cells of PAR2, followed by xCELLigence technology to measure cell migration, phospho-immunoblotting to assess ERK1/2 activation, and co-immunoprecipitation to detect a PAR2–ALK5 physical interaction. Results: Inhibition of ERK signaling with the MEK inhibitor U0126 blunted the ability of TGF-β1 to induce migration in pancreatic cancer Panc1 cells. ERK activation in response to PAR2 agonistic peptide (PAR2–AP) was strong and rapid, while it was moderate and delayed in response to TGF-β1. Basal and TGF-β1-dependent ERK, but not SMAD activation, was blocked by U0126 in Panc1 and other cell types indicating that ERK activation is downstream or independent of SMAD signaling. Moreover, cellular depletion of PAR2 in HaCaT cells strongly inhibited TGF-β1-induced ERK activation, while the biased PAR2 agonist GB88 at 10 and 100 µM potentiated TGF-β1-dependent ERK activation and cell migration. Finally, we provide evidence for a physical interaction between PAR2 and ALK5. Our data show that both PAR2–AP- and TGF-β1-induced cell migration depend on ERK activation, that PAR2 expression is crucial for TGF-β1-induced ERK activation, and that the functional cooperation of PAR2 and TGF-β1 involves a physical interaction between PAR2 and ALK5. PMID:29261154

Protease-Activated Receptor 4 Induces Bladder Pain through High Mobility Group Box-1

PubMed Central

Kouzoukas, Dimitrios E.; Ma, Fei; Meyer-Siegler, Katherine L.; Westlund, Karin N.; Hunt, David E.; Vera, Pedro L.

2016-01-01

Pain is the significant presenting symptom in Interstitial Cystitis/Painful Bladder Syndrome (IC/PBS). Activation of urothelial protease activated receptor 4 (PAR4) causes pain through release of urothelial macrophage migration inhibitory factor (MIF). High Mobility Group Box-1 (HMGB1), a chromatin-binding protein, mediates bladder pain (but not inflammation) in an experimental model (cyclophosphamide) of cystitis. To determine if PAR4-induced bladder hypersensitivity depends on HMGB1 downstream, we tested whether: 1) bladder PAR4 stimulation affected urothelial HMGB1 release; 2) blocking MIF inhibited urothelial HMGB1 release; and 3) blocking HMGB1 prevented PAR4-induced bladder hypersensitivity. HMGB1 release was examined in immortalized human urothelial cultures (UROtsa) exposed to PAR4-activating peptide (PAR4-AP; 100 μM; 2 hours) or scrambled control peptide. Female C57BL/6 mice, pretreated with a HMGB1 inhibitor (glycyrrhizin: 50 mg/kg; ip) or vehicle, received intravesical PAR4-AP or a control peptide (100 μM; 1 hour) to determine 1) HMGB1 levels at 1 hour in the intravesical fluid (released HMGB1) and urothelium, and 2) abdominal hypersensitivity to von Frey filament stimulation 24 hours later. We also tested mice pretreated with a MIF blocker (ISO-1: 20 mg/kg; ip) to determine whether MIF mediated PAR4-induced urothelial HMGB1 release. PAR4-AP triggered HMGB1 release from human (in vitro) and mice (in vivo) urothelial cells. Intravesical PAR4 activation elicited abdominal hypersensitivity in mice that was prevented by blocking HMGB1. MIF inhibition prevented PAR4-mediated HMGB1 release from mouse urothelium. Urothelial MIF and HGMB1 represent novel targets for therapeutic intervention in bladder pain conditions. PMID:27010488

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro.

PubMed

Zhao, Li-Ping; Xu, Tian-Min; Kan, Mu-Jie; Xiao, Ye-Chen; Cui, Man-Hua

2016-05-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings.

A novel uPAg-KPI fusion protein inhibits the growth and invasion of human ovarian cancer cells in vitro

PubMed Central

ZHAO, LI-PING; XU, TIAN-MIN; KAN, MU-JIE; XIAO, YE-CHEN; CUI, MAN-HUA

2016-01-01

Urokinase-type plasminogen activator (uPA) acts by breaking down the basement membrane and is involved in cell proliferation, migration and invasion. These actions are mediated by binding to the uPA receptor (uPAR) via its growth factor domain (GFD). The present study evaluated the effects of uPAg-KPI, a fusion protein of uPA-GFD and a kunitz protease inhibitor (KPI) domain that is present in the amyloid β-protein precursor. Using SKOV-3 cells, an ovarian cancer cell line, we examined cell viability, migration, invasion and also protein expression. Furthermore, we examined wound healing, and migration and invasion using a Transwell assay. Our data showed that uPAg-KPI treatment reduced the viability of ovarian cancer SKOV-3 cells in both a concentration and time-dependent manner by arresting tumor cells at G1/G0 phase of the cell cycle. The IC50 of uPAg-KPI was 0.5 µg/µl after 48 h treatment. At this concentration, uPAg-KPI also inhibited tumor cell colony formation, wound closure, as well as cell migration and invasion capacity. At the protein level, western blot analysis demonstrated that uPAg-KPI exerted no significant effect on the expression of total extracellular signal-regulated kinase (ERK)1/ERK2 and AKT, whereas it suppressed levels of phosphorylated ERK1/ERK2 and AKT. Thus, we suggest that this novel uPAg-KPI fusion protein reduced cell viability, colony formation, wound healing and the invasive ability of human ovarian cancer SKOV-3 cells in vitro by regulating ERK and AKT signaling. Further studies using other cell lines will confirm these findings. PMID:27035617

Myosin II Motors and F-Actin Dynamics Drive the Coordinated Movement of the Centrosome and Soma during CNS Glial-Guided Neuronal Migration

DOE Office of Scientific and Technical Information (OSTI.GOV)

Solecki, Dr. David; Trivedi, Dr. Niraj; Govek, Eve-Ellen

2009-01-01

Lamination of cortical regions of the vertebrate brain depends on glial-guided neuronal migration. The conserved polarity protein Par6{alpha} localizes to the centrosome and coordinates forward movement of the centrosome and soma in migrating neurons. The cytoskeletal components that produce this unique form of cell polarity and their relationship to polarity signaling cascades are unknown. We show that F-actin and Myosin II motors are enriched in the neuronal leading process and that Myosin II activity is necessary for leading process actin dynamics. Inhibition of Myosin II decreased the speed of centrosome and somal movement, whereas Myosin II activation increased coordinated movement.more » Ectopic expression or silencing of Par6{alpha} inhibited Myosin II motors by decreasing Myosin light-chain phosphorylation. These findings suggest leading-process Myosin II may function to 'pull' the centrosome and soma forward during glial-guided migration by a mechanism involving the conserved polarity protein Par6{alpha}.« less

Downregulation of protein tyrosine phosphatase PTPL1 alters cell cycle and upregulates invasion-related genes in prostate cancer cells.

PubMed

Castilla, Carolina; Flores, M Luz; Conde, José M; Medina, Rafael; Torrubia, Francisco J; Japón, Miguel A; Sáez, Carmen

2012-04-01

PTPL1, a non-receptor type protein tyrosine phosphatase, has been involved in the regulation of apoptosis and invasiveness of various tumour cell types, but its role in prostate cancer remained to be investigated. We report here that downregulation of PTPL1 by small interfering RNA in PC3 cells decreases cell proliferation and concomitantly reduces the expression of cell cycle-related proteins such as cyclins E and B1, PCNA, PTTG1 and phospho-histone H3. PTPL1 downregulation also increases the invasion ability of PC3 cells through Matrigel coated membranes. cDNA array of PTPL1-silenced PC3 cells versus control cells showed an upregulation of invasion-related genes such as uPA, uPAR, tPA, PAI-1, integrin α6 and osteopontin. This increased expression was also confirmed in PTPL1-silenced DU145 prostate cancer cells by quantitative real time PCR and western blot. These findings suggest that PTPL1 is an important mediator of central cellular processes such as proliferation and invasion.

Activation of PAR-2 elicits NO-dependent and CGRP-independent dilation of the dural artery.

PubMed

Bhatt, Deepak K; Ploug, Kenneth B; Ramachandran, Roshni; Olesen, Jes; Gupta, Saurabh

2010-06-01

The goal of this study was to determine the vascular effects of protease-activated receptor-2 (PAR-2) activation in the rat cranial vasculature. The role of PAR-2 in pain and inflammatory conditions has been established but the information available on its effects and receptor distribution in the trigeminal vascular axis is limited. We studied the dilatory function and expression of PAR-2 in the neuro-vascular circuit, critical in migraine pathogenesis. We also investigated the interaction of PAR-2 with calcitonin gene-related peptide (CGRP) and dural mast cells. We used an improved model of intravital microscopy on the closed cranial window in rats to study the vascular effects of PAR-2 activating peptides (PAR-2 APs; SLIGRL-NH(2), 2-Furoyl-LIGRLO-NH(2)) in the dural vasculature. Measurement of immunoreactive CGRP in skull halves and in trigeminal nucleus caudalis was done by using an enzyme-linked immunosorbent assay. We also analyzed the presence of PAR-2 in different migraine relevant tissues by quantitative real-time PCR and Western blot analysis. PAR-2 APs and trypsin induced a dose-dependent increase in dural artery diameter. The topical application of a nonspecific nitric oxide synthase (NOS) inhibitor, L-N(G)-Nitroarginine methyl ester, attenuated SLIGRL-NH(2) responses. Olcegepant, a CGRP receptor antagonist, did not a have significant effect on the SLIGRL-NH(2) responses, though exogenous CGRP responses were completely blocked. There was no significant release of CGRP from skull halves incubated with SLIGRL-NH(2) as compared with those incubated with the corresponding negative peptide. Chronic mast cell degranulation did not change the vascular effects of PAR-2 APs. mRNA and protein expression of PAR-2 were found throughout trigeminovasuclar axis. PAR-2 activation leads to vasodilation of dural arteries and these responses are partially mediated by nitric oxide. As PAR-2 is present throughout trigeminovasuclar axis, it may have a role in migraine pathogenesis, independent of CGRP and mast cell mediated mechanism.

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

PubMed Central

Ando, Seijitsu; Otani, Hitomi; Yagi, Yasuhiro; Kawai, Kenzo; Araki, Hiromasa; Fukuhara, Shirou; Inagaki, Chiyoko

2007-01-01

Background Proteinase-activated receptors (PARs; PAR1–4) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR4, a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR4 stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. Methods EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). Results Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR4 agonist peptide (AYPGKF-NH2, 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR4 stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR4-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR4-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR4-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR4 stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. Conclusion PAR4 stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation. PMID:17433115

Structure and function of Helicobacter pylori CagA, the first-identified bacterial protein involved in human cancer

PubMed Central

HATAKEYAMA, Masanori

2017-01-01

Chronic infection with Helicobacter pylori cagA-positive strains is the strongest risk factor of gastric cancer. The cagA gene-encoded CagA protein is delivered into gastric epithelial cells via bacterial type IV secretion, where it undergoes tyrosine phosphorylation at the Glu-Pro-Ile-Tyr-Ala (EPIYA) motifs. Delivered CagA then acts as a non-physiological scaffold/hub protein by interacting with multiple host signaling molecules, most notably the pro-oncogenic phosphatase SHP2 and the polarity-regulating kinase PAR1/MARK, in both tyrosine phosphorylation-dependent and -independent manners. CagA-mediated manipulation of intracellular signaling promotes neoplastic transformation of gastric epithelial cells. Transgenic expression of CagA in experimental animals has confirmed the oncogenic potential of the bacterial protein. Structural polymorphism of CagA influences its scaffold function, which may underlie the geographic difference in the incidence of gastric cancer. Since CagA is no longer required for the maintenance of established gastric cancer cells, studying the role of CagA during neoplastic transformation will provide an excellent opportunity to understand molecular processes underlying “Hit-and-Run” carcinogenesis. PMID:28413197

76 FR 34019 - Requirements for Taxpayers Filing Form 5472

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-10

... CFR U.S.C. 7805 * * * Par. 2. Section 1.6038A-1 is amended by revising paragraph (n)(2) to read as... elsewhere in this same issue of the Federal Register]. * * * * * Par. 3. Section 1.6038A-2 is amended by... regulations remove the requirement contained in Sec. 1.6038A-2(d) and Sec. 1.6038A-2(e) that a duplicate Form...

31 CFR 328.1 - Scope of regulations.

Code of Federal Regulations, 2011 CFR

2011-07-01

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31 CFR 328.1 - Scope of regulations.

Code of Federal Regulations, 2012 CFR

2012-07-01

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31 CFR 328.1 - Scope of regulations.

Code of Federal Regulations, 2010 CFR

2010-07-01

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31 CFR 328.1 - Scope of regulations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance: Treasury 2 2014-07-01 2014-07-01 false Scope of regulations. 328.1 Section 328.1 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL SERVICE... to their maturity or call date for redemption at par and application of the entire proceeds in...

31 CFR 328.1 - Scope of regulations.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 31 Money and Finance:Treasury 2 2013-07-01 2013-07-01 false Scope of regulations. 328.1 Section 328.1 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FISCAL SERVICE... to their maturity or call date for redemption at par and application of the entire proceeds in...

Long Dynamics Simulations of Proteins Using Atomistic Force Fields and a Continuum Representation of Solvent Effects: Calculation of Structural and Dynamic Properties

PubMed Central

Li, Xianfeng; Hassan, Sergio A.; Mehler, Ernest L.

2006-01-01

Long dynamics simulations were carried out on the B1 immunoglobulin-binding domain of streptococcal protein G (ProtG) and bovine pancreatic trypsin inhibitor (BPTI) using atomistic descriptions of the proteins and a continuum representation of solvent effects. To mimic frictional and random collision effects, Langevin dynamics (LD) were used. The main goal of the calculations was to explore the stability of tens-of-nanosecond trajectories as generated by this molecular mechanics approximation and to analyze in detail structural and dynamical properties. Conformational fluctuations, order parameters, cross correlation matrices, residue solvent accessibilities, pKa values of titratable groups, and hydrogen-bonding (HB) patterns were calculated from all of the trajectories and compared with available experimental data. The simulations comprised over 40 ns per trajectory for ProtG and over 30 ns per trajectory for BPTI. For comparison, explicit water molecular dynamics simulations (EW/MD) of 3 ns and 4 ns, respectively, were also carried out. Two continuum simulations were performed on each protein using the CHARMM program, one with the all-atom PAR22 representation of the protein force field (here referred to as PAR22/LD simulations) and the other with the modifications introduced by the recently developed CMAP potential (CMAP/LD simulations). The explicit solvent simulations were performed with PAR22 only. Solvent effects are described by a continuum model based on screened Coulomb potentials (SCP) reported earlier, i.e., the SCP-based implicit solvent model (SCP–ISM). For ProtG, both the PAR22/LD and the CMAP/LD 40-ns trajectories were stable, yielding Cα root mean square deviations (RMSD) of about 1.0 and 0.8 Å respectively along the entire simulation time, compared to 0.8 Å for the EW/MD simulation. For BPTI, only the CMAP/LD trajectory was stable for the entire 30-ns simulation, with a Cα RMSD of ≈ 1.4 Å, while the PAR22/LD trajectory became unstable early in the simulation, reaching a Cα RMSD of about 2.7 Å and remaining at this value until the end of the simulation; the Cα RMSD of the EW/MD simulation was about 1.5 Å. The source of the instabilities of the BPTI trajectories in the PAR22/LD simulations was explored by an analysis of the backbone torsion angles. To further validate the findings from this analysis of BPTI, a 35-ns SCP–ISM simulation of Ubiquitin (Ubq) was carried out. For this protein, the CMAP/LD simulation was stable for the entire simulation time (Cα RMSD of ≈1.0 Å), while the PAR22/LD trajectory showed a trend similar to that in BPTI, reaching a Cα RMSD of ≈1.5 Å at 7 ns. All the calculated properties were found to be in agreement with the corresponding experimental values, although local deviations were also observed. HB patterns were also well reproduced by all the continuum solvent simulations with the exception of solvent-exposed side chain–side chain (sc–sc) HB in ProtG, where several of the HB interactions observed in the crystal structure and in the EW/MD simulation were lost. The overall analysis reported in this work suggests that the combination of an atomistic representation of a protein with a CMAP/CHARMM force field and a continuum representation of solvent effects such as the SCP–ISM provides a good description of structural and dynamic properties obtained from long computer simulations. Although the SCP–ISM simulations (CMAP/LD) reported here were shown to be stable and the properties well reproduced, further refinement is needed to attain a level of accuracy suitable for more challenging biological applications, particularly the study of protein–protein interactions. PMID:15959866

Activation of PAR-1/NADPH oxidase/ROS signaling pathways is crucial for the thrombin-induced sFlt-1 production in extravillous trophoblasts: possible involvement in the pathogenesis of preeclampsia.

PubMed

Huang, Qi-Tao; Chen, Jian-Hong; Hang, Li-Lin; Liu, Shi-San; Zhong, Mei

2015-01-01

Preeclampsia was characterized by excessive thrombin generation in placentas and previous researches showed that thrombin could enhance soluble Fms-like tyrosine kinase 1 (sFlt-1) expression in first trimester trophoblasts. However, the detailed mechanism for the sFlt-1 over-production induced by thrombin was largely unknown. The purpose of this study was to explore the possible signaling pathway of thrombin-induced sFlt-1 production in extravillous trophoblasts (EVT). An EVT cell line (HRT-8/SVneo) was treated with various concentrations of thrombin. The mRNA expression and protein secretion of sFlt-1 in EVT were detected with real-time polymerase chain reaction and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production were determined by DCFH-DA. Exposure of EVT to thrombin induced increased intracellular ROS generation and overexpression of sFlt-1 at both mRNA and protein levels in a dose dependent manner. Short interfering RNA (siRNA) directed against PAR-1 or apocynin (an inhibitor of NADPH oxidase) could decrease the intracellular ROS generation and subsequently suppressed the production of sFlt-1 at mRNA and protein levels. Our results suggested that thrombin increased sFlt-1 production in EVT via the PAR-1 /NADPH oxidase /ROS signaling pathway. This also highlights the PAR-1 / NADPH oxidase / ROS pathway might be a potential therapeutic target for the prevention of preeclampsia in the future. © 2015 S. Karger AG, Basel.

Leukemia inhibitory factor promote trophoblast invasion via urokinase-type plasminogen activator receptor in preeclampsia.

PubMed

Zheng, Qin; Dai, Kuixing; Cui, Xinyuan; Yu, Ming; Yang, Xuesong; Yan, Bin; Liu, Shuai; Yan, Qiu

2016-05-01

Preeclampsia is a pregnancy-related syndrome which can cause perinatal mortality and morbidity. Inadequate invasion by trophoblast cells may lead to poor perfusion of the placenta, even result in preeclampsia. Understanding the molecular mechanisms underlying placentation facilitates the better intervention of preeclampsia. Urokinase-type plasminogen activator receptor (uPAR) is involved in the physiological and pathological processes. Leukemia inhibitory factor (LIF) is an important regulator in the establishment of pregnancy. However, the expression of uPAR in preeclamptic patients and its relationship with LIF remains unclear. In the current study, we found that the level of uPAR was relatively lower in the placentas from preeclamptic patients as compared with normal pregnant women. LIF promoted trophoblast cell outgrowth by upregulating uPAR in an explants culture, and LIF also enhanced migration and invasion potential through uPAR in trophoblast JAR and JEG-3 cell lines, and with increased gelatinolytic activities of matrix metalloproteinase 2 (MMP-2). The effect of LIF and uPAR on trophoblast migration and invasion was mediated by PI3K/AKT signaling pathway. Our data indicates the roles of LIF in promoting trophoblast migration and invasion through uPAR and suggest that abnormal expression of uPAR might be associated with the etiology of preeclampsia. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

uPAR and Cathepsin B Downregulation Induces Apoptosis by Targeting Calcineurin A to BAD via Bcl-2 in Glioma

PubMed Central

Malla, Rama Rao; Gopinath, Sreelatha; Gondi, Christopher S.; Alapati, Kiranmai; Dinh, Dzung H.; Tsung, Andrew J.; Rao, Jasti S.

2011-01-01

Cathepsin B and urokinase plasminogen activator receptor (uPAR) are postulated to play key roles in glioma invasion. Calcineurin is one of the key regulators of mitochondrial-dependent apoptosis, but its mechanism is poorly understood. Hence, we studied subcellular localization of calcineurin after transcriptional downregulation of uPAR and cathepsin B in glioma. In the present study, efficient downregulation of uPAR and cathepsin B increased the translocation of calcineurin A from the mitochondria to the cytosol, decreased pBAD (S136) expression and its interaction with 14-3-3ζ, and increased the interaction of BAD with Bcl-Xl. Co-depletion of uPAR and cathepsin B induced mitochondrial translocation of BAD and caspase 3 as well as PARP activation, cytochrome c and SMAC release. These effects were inhibited by FK506 (10 μM), a specific inhibitor of calcineurin. Calcineurin A was co-localized and also co-immunoprecipitated with Bcl-2. This interaction decreased with co-depletion of uPAR and cathepsin B and also with Bcl-2 inhibitor, HA 14-1 (20 μg/mL). Altered localization and interaction of calcineurin A with Bcl-2 was also observed in vivo when uPAR and cathepsin B were downregulated. In conclusion, downregulation of uPAR and cathepsin B induced apoptosis by targeting calcineurin A to BAD via Bcl-2 in glioma. PMID:21964739

Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation

PubMed Central

Ambegaokar, Surendra S.; Jackson, George R.

2011-01-01

A functional genetic screen using loss-of-function and gain-of-function alleles was performed to identify modifiers of tau-induced neurotoxicity using the 2N/4R (full-length) isoform of wild-type human tau expressed in the fly retina. We previously reported eye pigment mutations, which create dysfunctional lysosomes, as potent modifiers; here, we report 37 additional genes identified from ∼1900 genes screened, including the kinases shaggy/GSK-3beta, par-1/MARK, CamKI and Mekk1. Tau acts synergistically with Mekk1 and p38 to down-regulate extracellular regulated kinase activity, with a corresponding decrease in AT8 immunoreactivity (pS202/T205), suggesting that tau can participate in signaling pathways to regulate its own kinases. Modifiers showed poor correlation with tau phosphorylation (using the AT8, 12E8 and AT270 epitopes); moreover, tested suppressors of wild-type tau were equally effective in suppressing toxicity of a phosphorylation-resistant S11A tau construct, demonstrating that changes in tau phosphorylation state are not required to suppress or enhance its toxicity. Genes related to autophagy, the cell cycle, RNA-associated proteins and chromatin-binding proteins constitute a large percentage of identified modifiers. Other functional categories identified include mitochondrial proteins, lipid trafficking, Golgi proteins, kinesins and dynein and the Hsp70/Hsp90-organizing protein (Hop). Network analysis uncovered several other genes highly associated with the functional modifiers, including genes related to the PI3K, Notch, BMP/TGF-β and Hedgehog pathways, and nuclear trafficking. Activity of GSK-3β is strongly upregulated due to TDP-43 expression, and reduced GSK-3β dosage is also a common suppressor of Aβ42 and TDP-43 toxicity. These findings suggest therapeutic targets other than mitigation of tau phosphorylation. PMID:21949350

Role of the parCBA Operon of the Broad-Host-Range Plasmid RK2 in Stable Plasmid Maintenance

PubMed Central

Easter, Carla L.; Schwab, Helmut; Helinski, Donald R.

1998-01-01

The par region of the stably maintained broad-host-range plasmid RK2 is organized as two divergent operons, parCBA and parDE, and a cis-acting site. parDE encodes a postsegregational killing system, and parCBA encodes a resolvase (ParA), a nuclease (ParB), and a protein of unknown function (ParC). The present study was undertaken to further delineate the role of the parCBA region in the stable maintenance of RK2 by first introducing precise deletions in the three genes and then assessing the abilities of the different constructs to stabilize RK2 in three strains of Escherichia coli and two strains of Pseudomonas aeruginosa. The intact parCBA operon was effective in stabilizing a conjugation-defective RK2 derivative in E. coli MC1061K and RR1 but was relatively ineffective in E. coli MV10Δlac. In the two strains in which the parCBA operon was effective, deletions in parB, parC, or both parB and parC caused an approximately twofold reduction in the stabilizing ability of the operon, while a deletion in the parA gene resulted in a much greater loss of parCBA activity. For P. aeruginosa PAO1161Rifr, the parCBA operon provided little if any plasmid stability, but for P. aeruginosa PAC452Rifr, the RK2 plasmid was stabilized to a substantial extent by parCBA. With this latter strain, parA and res alone were sufficient for stabilization. The cer resolvase system of plasmid ColE1 and the loxP/Cre system of plasmid P1 were tested in comparison with the parCBA operon. We found that, not unlike what was previously observed with MC1061K, cer failed to stabilize the RK2 plasmid with par deletions in strain MV10Δlac, but this multimer resolution system was effective in stabilizing the plasmid in strain RR1. The loxP/Cre system, on the other hand, was very effective in stabilizing the plasmid in all three E. coli strains. These observations indicate that the parA gene, along with its res site, exhibits a significant level of plasmid stabilization in the absence of the parC and parB genes but that in at least one E. coli strain, all three genes are required for maximum stabilization. It cannot be determined from these results whether or not the stabilization effects seen with parCBA or the cer and loxP/Cre systems are strictly due to a reduction in the level of RK2 dimers and an increase in the number of plasmid monomer units or if these systems play a role in a more complex process of plasmid stabilization that requires as an essential step the resolution of plasmid dimers. PMID:9811663

The role of protease-activated receptor-2 on pulmonary neutrophils in the innate immune response to cockroach allergen

PubMed Central

2012-01-01

Background Serine proteases in German cockroach (GC) have been shown to mediate allergic airway inflammation through the activation of protease activated receptor (PAR)-2. Neutrophils play an important role in regulating the innate immune response, and are recruited into the airways following GC frass exposure. As such, we investigated the role of PAR-2 in airway neutrophil recruitment, activation and cytokine production following allergen exposure. Methods Wild type and PAR-2-deficient mice were administered a single intratracheal instillation of PBS or GC frass and neutrophil recruitment, expression of PAR-2, CD80, CD86, and MHC class II were assessed by flow cytometry and levels of tumor necrosis factor (TNF)α was assessed by ELISA. Uptake of AlexaFluor 405-labeled GC frass by neutrophils was performed by flow cytometry. Results Neutrophil recruitment in the lung and airways following GC frass exposure was significantly decreased in PAR-2-deficient mice compared to wild type mice. GC frass exposure increased the level of PAR-2 on pulmonary neutrophils and increased numbers of PAR-2-positive neutrophils were found in the lungs; however PAR-2 did not play a role in meditating allergen uptake. Comparing wild type and PAR-2-deficient mice, we found that a single exposure to GC frass increased levels of CD80 and CD86 on pulmonary neutrophils, an effect which was independent of PAR-2 expression. Neutrophils isolated from the whole lungs of naïve PAR-2-deficient mice treated ex vivo with GC frass produced significantly less TNFα than in similarly treated wild type neutrophils. Lastly, neutrophils were isolated from the bronchoalveolar lavage fluid of wild type and PAR-2-deficient mice following a single intratracheal exposure to GC frass. Airway neutrophils from PAR-2-deficient mice released substantially decreased levels of TNFα, suggesting a role for PAR-2 in neutrophil-derived cytokine production. Conclusions Together these data suggest PAR-2 expression can be upregulated on lung neutrophils following allergen exposure and the consequence is altered release of TNFα which could drive the early innate immune response. PMID:22954301

Topoisomerase I (TopA) Is Recruited to ParB Complexes and Is Required for Proper Chromosome Organization during Streptomyces coelicolor Sporulation

PubMed Central

Szafran, Marcin; Skut, Patrycja; Ditkowski, Bartosz; Ginda, Katarzyna; Chandra, Govind; Zakrzewska-Czerwińska, Jolanta

2013-01-01

Streptomyces species are bacteria that resemble filamentous fungi in their hyphal mode of growth and sporulation. In Streptomyces coelicolor, the conversion of multigenomic aerial hyphae into chains of unigenomic spores requires synchronized septation accompanied by segregation of tens of chromosomes into prespore compartments. The chromosome segregation is dependent on ParB protein, which assembles into an array of nucleoprotein complexes in the aerial hyphae. Here, we report that nucleoprotein ParB complexes are bound in vitro and in vivo by topoisomerase I, TopA, which is the only topoisomerase I homolog found in S. coelicolor. TopA cannot be eliminated, and its depletion inhibits growth and blocks sporulation. Surprisingly, sporulation in the TopA-depleted strain could be partially restored by deletion of parB. Furthermore, the formation of regularly spaced ParB complexes, which is a prerequisite for proper chromosome segregation and septation during the development of aerial hyphae, has been found to depend on TopA. We hypothesize that TopA is recruited to ParB complexes during sporulation, and its activity is required to resolve segregating chromosomes. PMID:23913317

High density of tryptase-positive mast cells in human colorectal cancer: a poor prognostic factor related to protease-activated receptor 2 expression

PubMed Central

Malfettone, Andrea; Silvestris, Nicola; Saponaro, Concetta; Ranieri, Girolamo; Russo, Antonio; Caruso, Stefano; Popescu, Ondina; Simone, Giovanni; Paradiso, Angelo; Mangia, Anita

2013-01-01

Tryptase(+) mast cells (MCs), abundant in the invasive front of tumours, contribute to tissue remodelling. Indeed, protease-activated receptor-2 (PAR-2) activation by MC-tryptase is considered an oncogenic event in colorectal cancer (CRC). Recently, we have suggested NHERF1 as a potential new marker in CRC. In this study, we aimed to determine the distribution of tryptase(+) MCs and PAR-2 and to examine the relationship between PAR-2 and NHERF1, investigating their reputed usefulness as tumour markers. We studied a cohort of 115 CRC specimens including primary cancer (C) and adjacent normal mucosa (NM) by immunohistochemical double staining, analyzing the protein expression of MC-tryptase, PAR-2 and cytoplasmic NHERF1. MC density was higher in NM than in C. Tumours with high TNM stage and poor grade showed the highest MC density. A higher PAR-2 immunoreactivity characterized tumours most infiltrated by MCs compared with samples with low MC density. Furthermore, PAR-2 overexpression was associated with advanced TNM stage, poor grade and lymphovascular invasion (LVI). A positive correlation existed between tryptase(+) MC density and PAR-2 expression. Cytoplasmic NHERF1 was higher in C than in NM and overexpressing tumours resulted associated with nodal and distant metastases, poor grade and LVI. PAR-2 correlated with cytoplasmic NHERF1 and the PAR-2(+)/cytoplasmic NHERF1(+) expression immunophenotype identified tumours associated with unfavourable prognosis and aggressive clinical parameters. Our data indicate that the high density of tryptase(+) MCs at invasive margins of tumours was associated with advanced stages of CRC and was strongly correlated with PAR-2 expression. PMID:23991686

Caspase-3 mediated release of SAC domain containing fragment from Par-4 is necessary for the sphingosine-induced apoptosis in Jurkat cells

PubMed Central

2013-01-01

Background Prostate apoptosis response-4 (Par-4) is a tumor-suppressor protein that selectively activates and induces apoptosis in cancer cells, but not in normal cells. The cancer specific pro-apoptotic function of Par-4 is encoded in its centrally located SAC (Selective for Apoptosis induction in Cancer cells) domain (amino acids 137–195). The SAC domain itself is capable of nuclear entry, caspase activation, inhibition of NF-κB activity, and induction of apoptosis in cancer cells. However, the precise mechanism(s) of how the SAC domain is released from Par-4, in response to apoptotic stimulation, is not well explored. Results In this study, we demonstrate for the first time that sphingosine (SPH), a member of the sphingolipid family, induces caspase-dependant cleavage of Par-4, leading to the release of SAC domain containing fragment from it. Par-4 is cleaved at the EEPD131G site on incubation with caspase-3 in vitro, and by treating cells with several anti-cancer agents. The caspase-3 mediated cleavage of Par-4 is blocked by addition of the pan-caspase inhibitor z-VAD-fmk, caspase-3 specific inhibitor Ac-DEVD-CHO, and by introduction of alanine substitution for D131 residue. Moreover, suppression of SPH-induced Akt dephosphorylation also abrogated the caspase dependant cleavage of Par-4. Conclusion Evidence provided here shows that Par-4 is cleaved by caspase-3 during SPH-induced apoptosis. Cleavage of Par-4 leads to the generation of SAC domain containing fragment which may possibly be essential and sufficient to induce or augment apoptosis in cancer cells. PMID:23442976

The inhibitory effect of tongxieyaofang on rats with post infectious irritable bowel syndrome through regulating colonic par-2 receptor.

PubMed

Hu, Xuguang; Zhang, Xiaojun; Han, Bin; Bei, Weijian

2013-10-02

The aims of this study were to evaluate the effect and mechanism of a traditional Chinese medicine formula: Tongxieyaofang (TXYF) on Rats with Post Infectious Irritable Bowel Syndrome (PI-IBS). SD male rats in adult were used to model PI-IBS and treated with TXYF at three dosage for 14 consecutive days, and then visceral sensation and the frequency of stool in PI-IBS rats were investigated. In addition, the contents of SP, TNF- α and IL-6 in colonic mucosal were analyzed by ELISA. Moreover faecal serine protease activity and PAR-2 mRNA expression were measured by ultraviolet spectrophotometry and RT-PCR, respectively. Our study showed that TXYF attenuated visceral hyperalgesia and inhibited stool frequency in Campylobacter-stimulated Post Infectious Irritable Bowel Syndrome (PI-IBS) rats. Furthermore, TXYF decreased the colonic SP, TNF- α and IL-6 content in PI-IBS rats. In addition, the up-regulated colonic mucosa PAR-2 mRNA expression in PI-IBS rats was significantly suppressed by orally TXYF. TXYF attenuated PI-IBS symptom by attenuating behavioral hyperalgesia and anti-diarrhea, the underlying mechanism was mediated by inhibiting PAR-2 receptor expression, reducing the levels of SP, TNF- α and IL-6 in colonic mucosa and decreasing faecal serine protease activity.

Identification of Residues of the Kid Toxin Involved in Autoregulation of the parD System

PubMed Central

Lemonnier, Marc; Santos-Sierra, Sandra; Pardo-Abarrio, Consolación; Díaz-Orejas, Ramón

2004-01-01

The toxin-antitoxin system parD (kis kid) of plasmid R1 is coregulated by the coordinated action of its two gene products. Here we describe the isolation and the in vivo characterization of three single-amino-acid changes in the Kid toxin, G4E, C74Y, and E91K, that affect the coregulatory activity but preserve the toxicity of the protein. PMID:14679244

Surgical intestinal manipulation increases gene expression of TrkA, CGRP, and PAR-2 IN dorsal root ganglia in the rat.

PubMed

Berdún, S; Rychter, J; Vergara, P

2016-06-01

Surgical handling of the bowel evokes degranulation of peritoneal mast cells (PMC). Nonetheless, role of PMCs in postoperative ileus (POI) is somewhat controversial. We aimed to investigate if intestinal manipulation elicits changes in afferent mediators related to MC activation and alteration of gastrointestinal (GI) motility. Postoperative ileus was induced by intestinal manipulation in Sprague-Dawley rats. Additionally, compound 48/80 (C48/80) and ketotifen were used to modulate MC activity. Rat mast cell protease 6 (RMCP-6, ELISA) release was determined in peritoneal lavage 20 min after intestinal manipulation. At 24 h, GI transit was determined. Gene expression of calcitonin gene-related peptide (CGRP), protease-activated receptor-2 (PAR-2), nerve growth factor (NGF), and TrkA receptor was determined (PCR) in dorsal root ganglia (DRG). Ileal wall inflammation was assessed by myeloperoxidase (MPO) activity, interleukin-6 expression (IL-6). Intestinal manipulation and exposure to C48/80-induced degranulation of PMCs delayed GI transit and up-regulated IL-6 and MPO activity. Intestinal manipulation, but not C48/80, up-regulated CGRP, PAR-2, and NGF/TrkA in DRGs. Ketotifen only improved gastric emptying and fecal output. Up-regulation of CGRP and TrkA expression in DRG was not prevented by ketotifen. Postoperative ileus is accompanied by activation of CGRP, NGF-TrkA, and PAR-2 in DRGs. Our results suggest that these mediators could be a target in further POI studies in order to find new therapeutic targets for this medical condition. © 2016 John Wiley & Sons Ltd.

Cancer-associated fibroblasts promote cancer cell growth through a miR-7-RASSF2-PAR-4 axis in the tumor microenvironment

PubMed Central

Yan, Ming; Li, Rongrong; Chen, Gang; Zhang, Jianjun; Chen, Wantao

2017-01-01

Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, play an important role in cancer progression but little is known about how CAFs affect tumorigenesis and development. MicroRNAs (miRNAs) are small non-coding RNAs that can negatively regulate target mRNA expression at post-transcriptional levels. In head and neck cancer (HNC), our analysis of miRNA arrays showed that miR-7, miR-196 and miR-335 were significantly up-regulated in CAFs when compared with their paired normal fibroblasts (NFs). FAP, α-SMA and FSP, specific markers of CAFs, were significantly expressed in CAFs. Functionally, exogenous expression of miR-7 in NFs induced a functional conversion of NFs into CAFs. In contrast, inhibition of miR-7 expression in CAFs could induce a functional conversion of CAFs into NFs. Our study demonstrated that overexpression of miR-7 in NFs significantly increased the migration activity and growth rates of cancer cells in co-culture experiments. Mechanistically, we confirmed that the RASSF2-PAR-4 axis was mainly responsible for miR-7 functions in CAFs using bioinformatics methods. Overexpression of miR-7 in CAFs led to down-regulation of RASSF2, which dramatically decreased the secretion of PAR-4 from CAFs and then enhanced the proliferation and migration of the co-cultured cancer cells. Thus, these results reveal that the inactivation of the RASSF2-PAR-4 axis controlled by miR-7 may be a novel strategy for gene therapy in HNCs. PMID:27901488

In Vivo Fluorescence Correlation and Cross-Correlation Spectroscopy

NASA Astrophysics Data System (ADS)

Mütze, Jörg; Ohrt, Thomas; Petrášek, Zdeněk; Schwille, Petra

In this manuscript, we describe the application of Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and scanning FCS (sFCS) to two in vivo systems. In the first part, we describe the application of two-photon standard and scanning FCS in Caenorhabditis elegans embryos. The differentiation of a single fertilized egg into a complex organism in C. elegans is regulated by a number of protein-dependent processes. The oocyte divides asymmetrically into two daughter cells of different developmental fate. Two of the involved proteins, PAR-2 and NMY-2, are studied. The second investigated system is the mechanism of RNA interference in human cells. An EGFP based cell line that allows to study the dynamics and localization of the RNA-induced silencing complex (RISC) with FCS in vivo is created, which has so far been inaccessible with other experimental methods. Furthermore, Fluorescence Cross-Correlation Spectroscopy is employed to highlight the asymmetric incorporation of labeled siRNAs into RISC.

31 CFR 337.12 - Applicability of TREASURY DIRECT regulations.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 31 Money and Finance: Treasury 2 2014-07-01 2014-07-01 false Applicability of TREASURY DIRECT regulations. 337.12 Section 337.12 Money and Finance: Treasury Regulations Relating to Money and Finance... subject to call, may be called for redemption, in whole or in part, at par and accrued interest, on any...

Chromosomal localization and cDNA cloning of the human DBP and TEF genes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Khatib, Z.A.; Inaba, T.; Valentine, M.

1994-09-15

The authors have isolated cDNA and genomic clones and determined the human chromosome positions of two genes encoding transcription factors expressed in the liver and the pituitary gland: albumin D-site-binding protein (DBP) and thyrotroph embryonic factor (TEF). Both proteins have been identified as members of the PAR (proline and acidic amino acid-rich) subfamily of bZIP transcription factors in the rat, but human homologues have not been characterized. Using a fluorescence in situ hybridization technique, the DBP locus was assigned to chromosome 19q13, and TEF to chromosome 22q13. Each assignment was confirmed by means of human chromosome segregation in somatic cellmore » hybrids. Coding sequences of DBP and TEF, extending beyond the bZIP domain to the PAR region, were highly conserved in both human-human and interspecies comparisons. Conservation of the exon-intron boundaries of each bZIP domain-encoding exon suggested derivation from a common ancestral gene. DBP and TEF mRNAs were expressed in all tissues and cell lines examined, including brain, lung, liver, spleen, and kidney. Knowledge of the human chromosome locations of these PAR proteins will facilitate studies to assess their involvement in carcinogenesis and other fundamental biological processes. 37 refs., 5 figs., 1 tab.« less

Resveratrol inhibits proteinase-activated receptor-2-induced release of soluble vascular endothelial growth factor receptor-1 from human endothelial cells

PubMed Central

Al-Ani, Bahjat

2013-01-01

We recently reported that (i) activation of the proinflammatory receptor, proteinase-activated receptor-2 (PAR-2) caused the release of an important biomarker in preeclampsia, soluble vascular endothelial growth factor receptor-1 (sVEGFR-1, also known as sFlt-1) from human umbilical vein endothelial cells (HUVECs), and (ii) that the anti-oxidant and anti-inflammatory agent, resveratrol, is capable of inhibiting the proinflammatory cytokine-induced sVEGFR-1 release from human placenta. Based on these findings and because PAR-2 is upregulated by proinflammatory cytokines, we sought to determine whether resveratrol can inhibit PAR-2-induced sVEGFR-1 release. PAR-2 expressing cells, HUVECs and human embryonic kidney cells (HEK-293) transfected with a human VEGFR-1 promoter-luciferase reporter construct were incubated with PAR-2-activating peptide and/or resveratrol. Cell supernatants were assayed for sVEGFR-1 by enzyme-linked immunosorbent assay (ELISA), and VEGFR-1 promoter-luciferase assay was performed on the harvested cell lysates. Preincubation of HEK-293 cells with resveratrol significantly inhibited PAR-2-induced VEGFR-1 promoter activity without affecting cell viability as assessed by MTT assay. The addition of resveratrol also blocked PAR-2-mediated sVEGFR-1 release from HUVECs. The present study demonstrates that resveratrol suppressed both VEGFR-1 promoter activity and sVEGFR-1 protein release induced by PAR-2 activation, which further endorses our recent findings of a potential therapeutic role for resveratrol in preeclampsia. PMID:26933402

Coincident regulation of PKCδ in human platelets by phosphorylation of Tyr311 and Tyr565 and phospholipase C signalling

PubMed Central

Hall, Kellie J.; Jones, Matthew L.; Poole, Alastair W.

2007-01-01

PKC (protein kinase C)δ plays a complex role in platelets, having effects on both positive and negative signalling functions. It is phosphorylated on tyrosine residues in response to thrombin and collagen, and it has recently been shown that Tyr311 is phosphorylated in response to PAR (protease-activated receptor) 1 and PAR4 receptor activation. In the present study, we show that Tyr311 and Tyr565 are phosphorylated in response to thrombin, and have examined the interplay between phosphorylation and the classical lipid-mediated activation of PKCδ. Phosphorylation of both Tyr311 and Tyr565 is dependent on Src kinase and PLC (phospholipase C) activity in response to thrombin. Importantly, direct allosteric activation of PKCδ with PMA also induced phosphorylation of Tyr311 and Tyr565, and this was dependent on the activity of Src kinases, but not PLC. Membrane recruitment of PKCδ is essential for phosphorylation of this tyrosine residue, but tyrosine phosphorylation is not required for membrane recruitment of PKCδ. Both thrombin and PMA induce recruitment of PKCδ to the membrane, and for thrombin, this recruitment is a PLC-dependent process. In order to address the functional role of tyrosine residue phosphorylation of PKCδ, we demonstrate that phosphorylation can potentiate the activity of the kinase, although phosphorylation does not play a role in membrane recruitment of the kinase. PKCδ is therefore regulated in a coincident fashion, PLC-dependent signals recruiting it to the plasma membrane and by phosphorylation on tyrosine residues, potentiating its activity. PMID:17570831

Protease-Activated Receptor 2, Dipeptidyl Peptidase I, and Proteases Mediate Clostridium difficile Toxin A Enteritis

PubMed Central

COTTRELL, GRAEME S.; AMADESI, SILVIA; PIKIOS, STELLA; CAMERER, ERIC; WILLARDSEN, J. ADAM; MURPHY, BRETT R.; CAUGHEY, GEORGE H.; WOLTERS, PAUL J.; COUGHLIN, SHAUN R.; PETERSON, ANDERS; KNECHT, WOLFGANG; POTHOULAKIS, CHARALABOS; BUNNETT, NIGEL W.; GRADY, EILEEN F.

2008-01-01

Background & Aims We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. Results TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%−28% and tissue and fluid myeloperoxidase by 31%−71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms. PMID:17570216

Specific Distribution of the Autophagic Protein GABARAPL1/GEC1 in the Developing and Adult Mouse Brain and Identification of Neuronal Populations Expressing GABARAPL1/GEC1

PubMed Central

Le Grand, Jaclyn Nicole; Bon, Karine; Fraichard, Annick; Zhang, Jianhua; Jouvenot, Michèle; Risold, Pierre-Yves; Boyer-Guittaut, Michaël; Delage-Mourroux, Régis

2013-01-01

Macroautophagy is a highly conserved cellular degradation process, regulated by autophagy-related (atg) factors, in which a double membrane autophagosome engulfs cytoplasmic components to target them for degradation. In yeast, the Atg8 protein is indispensable for autophagosome formation. In mammals, this is complicated by the presence of six Atg8 homologues grouped into the GABARAP and MAP1LC3 subfamilies. Although these proteins share a high similarity, their transcript expression, regulation and protein interactions differ, suggesting they may display individual properties and specific functions. GABARAPL1/GEC1 is a member of the GABARAP subfamily and its mRNA is the most highly expressed Atg8 homologue in the central nervous system. Consequently, we performed an in depth study of GABARAPL1 distribution in the developing and adult murine brain. Our results show that GABARAPL1 brain expression is visible as early as embryonic day 11 and progressively increases to a maximum level in the adult. Immunohistochemical staining was detected in both fibers and immature neurons in embryos but was restrained to neurons in adult tissue. By E17, intense punctate-like structures were visible and these accumulated in cortical primary neurons treated with the autophagosome/lysosome fusion inhibitor Bafilomycin A1 (Baf A1), suggesting that they represent autophagosomes. Finally, GABARAPL1 expression was particularly intense in motoneurons in the embryo and in neurons involved in somatomotor and neuroendocrine functions in the adult, particularly in the substantia nigra pars compacta, a region affected in Parkinson's disease. Our study of cerebral GABARAPL1 protein expression provides insight into its role in the development and homeostasis of the mouse brain. PMID:23690988

Fully human antibodies against the Protease-Activated Receptor-2 (PAR-2) with anti-inflammatory activity.

PubMed

Giblin, Patricia; Boxhammer, Rainer; Desai, Sudha; Kroe-Barrett, Rachel; Hansen, Gale; Ksiazek, John; Panzenbeck, Maret; Ralph, Kerry; Schwartz, Racheline; Zimmitti, Clare; Pracht, Catrin; Miller, Sandra; Magram, Jeanne; Litzenburger, Tobias

2011-01-01

PAR-2 belongs to a family of G-protein coupled Protease-Activated Receptors (PAR) which are activated by specific proteolytic cleavage in the extracellular N-terminal region. PAR-2 is activated by proteases such as trypsin, tryptase, proteinase 3, factor VIIa, factor Xa and is thought to be a mediator of inflammation and tissue injury, where elevated levels of proteases are found. Utilizing the HuCAL GOLD® phage display library we generated fully human antibodies specifically blocking the protease cleavage site in the N-terminal domain. In vitro affinity optimization resulted in antibodies with up to 1000-fold improved affinities relative to the original parental antibodies with dissociation constants as low as 100 pM. Corresponding increases in potency were observed in a mechanistic protease cleavage assay. The antibodies effectively inhibited PAR-2 mediated intracellular calcium release and cytokine secretion in various cell types stimulated with trypsin. In addition, the antibodies demonstrated potent inhibition of trypsin induced relaxation of isolated rat aortic rings ex vivo. In a short term mouse model of inflammation, the trans vivo DTH model, anti-PAR-2 antibodies showed inhibition of the inflammatory swelling response. In summary, potent inhibitors of PAR-2 were generated which allow further assessment of the role of this receptor in inflammation and evaluation of their potential as therapeutic agents.

Optical heterogeneous bioassay for the detection of the inflammatory biomarker suPAR

NASA Astrophysics Data System (ADS)

Tombelli, S.; Trono, C.; Adinolfi, B.; Chiavaioli, F.; Giannetti, A.; Eugen-Olsen, J.; Bernini, R.; Grimaldi, I. A.; Persichetti, G.; Testa, G.; Baldini, F.

2015-03-01

Soluble urokinase plasminogen activator receptor (suPAR) is an inflammatory protein present in blood and a marker of disease presence, severity and prognosis. A heterogeneous sandwich assay is proposed for quantifying suPAR by employing a capture antibody from rat and a biotinylated detection antibody from mouse. Optical detection was achieved by a successive exposure of the biotinylated sandwich to streptavidin labelled with ATTO647N. The heterogeneous assay was implemented on a multichannel polymethylmetacrylate (PMMA) optical biochip, potentially capable of the simultaneous detection of more than one analyte. Capture antibody was immobilized on the PMMA surface of the microfluidic channel and the assay was performed with the following protocol: i) surface blocking with BSA, ii) incubation with suPAR or PBS, iii) incubation with biotinylated suPAR detection Ab and iv) incubation with streptavidin-ATTO647N. Promising preliminary results were obtained with this protocol. Moreover, an improved optical setup is proposed which avoids the mechanical scanning of the chip and consequently the in-series fluorescence excitation and read out, allowing the simultaneous measurement of the fluorescence on all the channels of the microfluidic chip.

Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl Polarity Complex Promotes Epithelial Proliferation via Overlapping Mechanisms

PubMed Central

Schimizzi, Gregory V.; Maher, Meghan T.; Loza, Andrew J.; Longmore, Gregory D.

2016-01-01

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin. PMID:27454609

Disruption of the Cdc42/Par6/aPKC or Dlg/Scrib/Lgl Polarity Complex Promotes Epithelial Proliferation via Overlapping Mechanisms.

PubMed

Schimizzi, Gregory V; Maher, Meghan T; Loza, Andrew J; Longmore, Gregory D

2016-01-01

The establishment and maintenance of apical-basal polarity is a defining characteristic and essential feature of functioning epithelia. Apical-basal polarity (ABP) proteins are also tumor suppressors that are targeted for disruption by oncogenic viruses and are commonly mutated in human carcinomas. Disruption of these ABP proteins is an early event in cancer development that results in increased proliferation and epithelial disorganization through means not fully characterized. Using the proliferating Drosophila melanogaster wing disc epithelium, we demonstrate that disruption of the junctional vs. basal polarity complexes results in increased epithelial proliferation via distinct downstream signaling pathways. Disruption of the basal polarity complex results in JNK-dependent proliferation, while disruption of the junctional complex primarily results in p38-dependent proliferation. Surprisingly, the Rho-Rok-Myosin contractility apparatus appears to play opposite roles in the regulation of the proliferative phenotype based on which polarity complex is disrupted. In contrast, non-autonomous Tumor Necrosis Factor (TNF) signaling appears to suppress the proliferation that results from apical-basal polarity disruption, regardless of which complex is disrupted. Finally we demonstrate that disruption of the junctional polarity complex activates JNK via the Rho-Rok-Myosin contractility apparatus independent of the cortical actin regulator, Moesin.

The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation.

PubMed

Mahale, Sagar; Kumar, Megha; Sharma, Amit; Babu, Aswini; Ranjan, Shashi; Sachidanandan, Chetana; Mylavarapu, Sivaram V S

2016-12-23

Cytoplasmic dynein 1 is a multi-protein intracellular motor essential for mediating several mitotic functions, including the establishment of proper spindle orientation. The functional relevance and mechanistic distinctions between two discrete dynein subpopulations distinguished only by Light Intermediate Chain (LIC) homologues, LIC1 and LIC2 is unknown during mitosis. Here, we identify LIC2-dynein as the major mediator of proper spindle orientation and uncover its underlying molecular mechanism. Cortically localized dynein, essential for maintaining correct spindle orientation, consists majorly of LIC2-dynein, which interacts with cortical 14-3-3 ε- ζ and Par3, conserved proteins required for orienting the spindle. LIC2-dynein is also responsible for the majority of dynein-mediated asymmetric poleward transport of NuMA, helping focus microtubule minus ends. In addition, LIC2-dynein dominates in equatorially aligning chromosomes at metaphase and in regulating mitotic spindle length. Key mitotic functions of LIC2 were remarkably conserved in and essential for early embryonic divisions and development in zebrafish. Thus LIC2-dynein exclusively engages with two major cortical pathways to govern spindle orientation. Overall, we identify a novel selectivity of molecular interactions between the two LICs in mitosis as the underlying basis for their uneven distribution of labour in ensuring proper spindle orientation.

77 FR 75016 - Guidance Regarding Deduction and Capitalization of Expenditures Related to Tangible Property

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-19

...: Authority: 26 U.S.C. 7805 * * * Section 1.168(i)-1 also issued under 26 U.S.C. 168(i)(4). * * * 0 Par. 2... for Sec. 1.168(i)-1T(m). 0 Par. 3. Section 1.168(i)-1 is amended by revising paragraphs (l)(2) and (l... redesignated paragraphs (m)(2) and (m)(3) for the general asset account regulations as in effect before TD 9564...

A balance between TFPI and thrombin-mediated platelet activation is required for murine embryonic development

PubMed Central

Ellery, Paul E. R.; Maroney, Susan A.; Cooley, Brian C.; Luyendyk, James P.; Zogg, Mark; Weiler, Hartmut

2015-01-01

Tissue factor pathway inhibitor (TFPI) is a critical anticoagulant protein present in endothelium and platelets. Mice lacking TFPI (Tfpi−/−) die in utero from disseminated intravascular coagulation. They are rescued by concomitant tissue factor (TF) deficiency, demonstrating that TFPI modulates TF function in vivo. Recent studies have found TFPI inhibits prothrombinase activity during the initiation of coagulation and limits platelet accumulation during thrombus formation, implicating TFPI in modulating platelet procoagulant activity. To examine whether altered platelet function would compensate for the lack of TFPI and rescue TFPI-null embryonic lethality, Tfpi+/− mice lacking the platelet thrombin receptor, protease activated receptor 4 (PAR4; Par4−/−), or its coreceptor, PAR3, were mated. PAR3 deficiency did not rescue Tfpi−/− embryos, but >40% of expected Tfpi−/−:Par4−/− offspring survived to adulthood. Adult Tfpi−/−:Par4−/− mice did not exhibit overt thrombosis. However, they had focal sterile inflammation with fibrin(ogen) deposition in the liver and elevated plasma thrombin-antithrombin complexes, indicating activation of coagulation at baseline. Tfpi−/−:Par4−/− mice have platelet and fibrin accumulation similar to Par4−/− mice following venous electrolytic injury but were more susceptible than Par4−/− mice to TF-induced pulmonary embolism. In addition, ∼30% of the Tfpi−/−:Par4−/− mice were born with short tails. Tfpi−/−:Par4−/− mice are the first adult mice described that lack TFPI with unaltered TF. They demonstrate that TFPI physiologically modulates thrombin-dependent platelet activation in a manner that is required for successful embryonic development and identify a role for TFPI in dampening intravascular procoagulant stimuli that lead to thrombin generation, even in the absence of thrombin-mediated platelet activation. PMID:25954015

Protease-activated receptor-4 and purinergic receptor P2Y12 dimerize, co-internalize, and activate Akt signaling via endosomal recruitment of β-arrestin.

PubMed

Smith, Thomas H; Li, Julia G; Dores, Michael R; Trejo, JoAnn

2017-08-18

Vascular inflammation and thrombosis require the concerted actions of several different agonists, many of which act on G protein-coupled receptors (GPCRs). GPCR dimerization is a well-established phenomenon that can alter protomer function. In platelets and other cell types, protease-activated receptor-4 (PAR4) has been shown to dimerize with the purinergic receptor P2Y12 to coordinate β-arrestin-mediated Akt signaling, an important mediator of integrin activation. However, the mechanism by which the PAR4-P2Y12 dimer controls β-arrestin-dependent Akt signaling is not known. We now report that PAR4 and P2Y12 heterodimer internalization is required for β-arrestin recruitment to endosomes and Akt signaling. Using bioluminescence resonance energy transfer, immunofluorescence microscopy, and co-immunoprecipitation in cells expressing receptors exogenously and endogenously, we demonstrate that PAR4 and P2Y12 specifically interact and form dimers expressed at the cell surface. We also found that activation of PAR4 but not of P2Y12 drives internalization of the PAR4-P2Y12 heterodimer. Remarkably, activated PAR4 internalization was required for recruitment of β-arrestin to endocytic vesicles, which was dependent on co-expression of P2Y12. Interestingly, stimulation of the PAR4-P2Y12 heterodimer promotes β-arrestin and Akt co-localization to intracellular vesicles. Moreover, activated PAR4-P2Y12 internalization is required for sustained Akt activation. Thus, internalization of the PAR4-P2Y12 heterodimer is necessary for β-arrestin recruitment to endosomes and Akt signaling and lays the foundation for examining whether blockade of PAR4 internalization reduces integrin and platelet activation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Quercetin Has Antimetastatic Effects on Gastric Cancer Cells via the Interruption of uPA/uPAR Function by Modulating NF-κb, PKC-δ, ERK1/2, and AMPKα.

PubMed

Li, Hai; Chen, Chen

2018-06-01

Gastric cancer (GC) is a malignancy with few effective treatment options after metastasis occurs. Quercetin (Qu) intake has been associated with reduced incidence and slow development of GC, probably due to its anti-proliferative and apoptotic effects, but it is unclear whether Qu can inhibit the metastatic activity. The urokinase plasminogen activator (uPA)/uPA receptor (uPAR) system plays an important role in cancer metastasis. In this study, we measured both uPA activity and uPAR expression in GC and pericarcinous tissues, and we investigated the correlation between uPAR expression and the migratory and invasive activities of various GC cell lines. GC BGC823 and AGS cells were subjected to treatment with 10 μM Qu for 72 hours and uPAR knockdown, alone or in combination, before evaluating cell metastasis. The results showed that uPA activity and uPAR expression were higher in GC tissues than in pericarcinous tissues. Migratory and invasive activities of GC cell lines positively correlated with uPAR expression. Qu treatment decreased BGC823 and AGS cell migration and invasion, accompanied by reduced uPA and uPAR protein expression. Both Qu treatment and uPAR knockdown decreased matrix metalloproteinase-2 and -9 activity and blocked Pak1-Limk1-cofilin signaling. Qu treatment was associated with inhibition of NF-κb, PKC-δ, and ERK1/2, and with AMPKα activation. Specific inhibitors of NF-κb, PKC, and ERK1/2, and an AMPKα activator suppressed uPA and uPAR expression in GC cells. Collectively, Qu showed an antimetastatic effect on GC cells via the interruption of uPA/uPAR function and modulation of NF-κb, PKC-δ, ERK1/2, and AMPKα. This suggests that Qu is a promising agent against GC metastasis.

OVER-EXPRESSION OF THE THROMBIN RECEPTOR (PAR-1) IN THE PLACENTA IN PREECLAMPSIA: A MECHANISM FOR THE INTERSECTION OF COAGULATION AND INFLAMMATION

PubMed Central

EREZ, OFFER; ROMERO, ROBERTO; KIM, SUNG-SU; KIM, JUNG-SUN; KIM, YEON MEE; WILDMAN, DEREK E; THAN, NANDOR GABOR; MAZAKI-TOVI, SHALI; GOTSCH, FRANCESCA; PINELES, BETH; KUSANOVIC, JUAN PEDRO; ESPINOZA, JIMMY; MITTAL, POOJA; MAZOR, MOSHE; HASSAN, SONIA S.; KIM, CHONG JAI

2008-01-01

Objective Preeclampsia (PE) is characterized by excessive thrombin generation that has been implicated in the multiple organ damage associated with the disease. The biological effects of thrombin on coagulation and inflammation are mediated by protease activated receptor-1 (PAR-1), a G-protein coupled receptor. The aim of this study was to determine whether preterm preeclampsia (PE) is associated with changes in placental expression of PAR-1. Study design This cross-sectional study included two groups matched for gestational age at delivery: 1) patients with preterm PE (<37 weeks of gestation; n=26) and 2) a control group of patients with preterm labor without intraamniotic infection (n=26). Placental tissue microarrays were immunostained for PAR-1. Immunoreactivity of PAR-1 in the villous trophoblasts was graded as negative, weak-positive, or strong-positive. Results 1) The proportion of cases with strong PAR-1 immunoreactivity was significantly higher in placentas of patients with preeclampsia than in placentas from the control group [37.5% (9/24) vs. 8.7% (2/23); p=0.036, respectively]. 2) PAR-1 immunoreactivity was found in the cellular compartments of the placental villous tree, mainly in villous trophoblasts and stromal endothelial cells. 3) PAR-1 was detected in 92.3% (24/26) of the placentas of women with preeclampsia and in 88.5% (23/26) of the placentas from the control group (p=1). Conclusion Placentas from pregnancies complicated by preterm PE had a significantly higher frequency of strong PAR-1 expression than placentas from women with spontaneous PTL. This observation is consistent with a role for PAR-1 as a mediator of the effect of thrombin on coagulation and inflammation in preeclampsia. We propose that the effects of thrombin in PE are due to increased thrombin generation and higher expression of PAR-1, the major receptor for this enzyme. PMID:18570113

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

PubMed

Kreutter, Guillaume; Kassem, Mohamad; El Habhab, Ali; Baltzinger, Philippe; Abbas, Malak; Boisrame-Helms, Julie; Amoura, Lamia; Peluso, Jean; Yver, Blandine; Fatiha, Zobairi; Ubeaud-Sequier, Geneviève; Kessler, Laurence; Toti, Florence

2017-11-01

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f β-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or β-cells were applied to H 2 O 2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in β-cells. MP from aPC-treated EC (eM aPC ) exhibited high EPCR and annexin A1 content, protected β-cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H 2 O 2 -treated rat islets with increased viability (62% versus 48% H 2 O 2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

PARP1 expression, activity and ex vivo sensitivity to the PARP inhibitor, talazoparib (BMN 673), in chronic lymphocytic leukaemia

PubMed Central

Herriott, Ashleigh; Tudhope, Susan J.; Junge, Gesa; Rodrigues, Natalie; Patterson, Miranda J.; Woodhouse, Laura; Lunec, John; Hunter, Jill E.; Mulligan, Evan A.; Cole, Michael; Allinson, Lisa M.; Wallis, Jonathan P.; Marshall, Scott; Wang, Evelyn; Curtin, Nicola J.; Willmore, Elaine

2015-01-01

In chronic lymphocytic leukemia (CLL), mutation and loss of p53 and ATM abrogate DNA damage signalling and predict poorer response and shorter survival. We hypothesised that poly (ADP-ribose) polymerase (PARP) activity, which is crucial for repair of DNA breaks induced by oxidative stress or chemotherapy, may be an additional predictive biomarker and a target for therapy with PARP inhibitors. We measured PARP activity in 109 patient-derived CLL samples, which varied widely (192 – 190052 pmol PAR/106 cells) compared to that seen in healthy volunteer lymphocytes (2451 – 7519 pmol PAR/106 cells). PARP activity was associated with PARP1 protein expression and endogenous PAR levels. PARP activity was not associated with p53 or ATM loss, Binet stage, IGHV mutational status or survival, but correlated with Bcl-2 and Rel A (an NF-kB subunit). Levels of 8-hydroxy-2′-deoxyguanosine in DNA (a marker of oxidative damage) were not associated with PAR levels or PARP activity. The potent PARP inhibitor, talazoparib (BMN 673), inhibited CD40L-stimulated proliferation of CLL cells at nM concentrations, independently of Binet stage or p53/ATM function. PARP activity is highly variable in CLL and correlates with stress-induced proteins. Proliferating CLL cells (including those with p53 or ATM loss) are highly sensitive to the PARP inhibitor talazoparib. PMID:26539646

Agonists of proteinase-activated receptor 1 induce plasma extravasation by a neurogenic mechanism.

PubMed

de Garavilla, L; Vergnolle, N; Young, S H; Ennes, H; Steinhoff, M; Ossovskaya, V S; D'Andrea, M R; Mayer, E A; Wallace, J L; Hollenberg, M D; Andrade-Gordon, P; Bunnett, N W

2001-08-01

Thrombin, generated in the circulation during injury, cleaves proteinase-activated receptor 1 (PAR1) to stimulate plasma extravasation and granulocyte infiltration. However, the mechanism of thrombin-induced inflammation in intact tissues is unknown. We hypothesized that thrombin cleaves PAR1 on sensory nerves to release substance P (SP), which interacts with the neurokinin 1 receptor (NK1R) on endothelial cells to cause plasma extravasation. PAR1 was detected in small diameter neurons known to contain SP in rat dorsal root ganglia by immunohistochemistry and in situ hybridization. Thrombin and the PAR1 agonist TFLLR-NH(2) (TF-NH(2)) increased [Ca(2+)](i) >50% of cultured neurons (EC(50)s 24 mu ml(-1) and 1.9 microM, respectively), assessed using Fura-2 AM. The PAR1 agonist completely desensitized responses to thrombin, indicating that thrombin stimulates neurons through PAR1. Injection of TF-NH(2) into the rat paw stimulated a marked and sustained oedema. An NK1R antagonist and ablation of sensory nerves with capsaicin inhibited oedema by 44% at 1 h and completely by 5 h. In wild-type but not PAR1(-/-) mice, TF-NH(2) stimulated Evans blue extravasation in the bladder, oesophagus, stomach, intestine and pancreas by 2 - 8 fold. Extravasation in the bladder, oesophagus and stomach was abolished by an NK1R antagonist. Thus, thrombin cleaves PAR1 on primary spinal afferent neurons to release SP, which activates the NK1R on endothelial cells to stimulate gap formation, extravasation of plasma proteins, and oedema. In intact tissues, neurogenic mechanisms are predominantly responsible for PAR1-induced oedema.

Ketorolac reduces spinal astrocytic activation and PAR1 expression associated with attenuation of pain after facet joint injury.

PubMed

Dong, Ling; Smith, Jenell R; Winkelstein, Beth A

2013-05-15

Chronic neck pain affects up to 70% of persons, with the facet joint being the most common source. Intra-articular injection of the non-steroidal anti-inflammatory drug ketorolac reduces post-operative joint-mediated pain; however, the mechanism of its attenuation of facet-mediated pain has not been evaluated. Protease-activated receptor-1 (PAR1) has differential roles in pain maintenance depending on the type and location of painful injury. This study investigated if the timing of intra-articular ketorolac injection after painful cervical facet injury affects behavioral hypersensitivity by modulating spinal astrocyte activation and/or PAR1 expression. Rats underwent a painful joint distraction and received an injection of ketorolac either immediately or 1 day later. Separate control groups included injured rats with a vehicle injection at day 1 and sham operated rats. Forepaw mechanical allodynia was measured for 7 days, and spinal cord tissue was immunolabeled for glial fibrillary acidic protein (GFAP) and PAR1 expression in the dorsal horn on day 7. Ketorolac administered on day 1 after injury significantly reduced allodynia (p=0.0006) to sham levels, whereas injection immediately after the injury had no effect compared with vehicle. Spinal astrocytic activation followed behavioral responses and was significantly decreased (p=0.009) only for ketorolac given at day 1. Spinal PAR1 (p=0.0025) and astrocytic PAR1 (p=0.012) were significantly increased after injury. Paralleling behavioral data, astrocytic PAR1 was returned to levels in sham only when ketorolac was administered on day 1. Yet, spinal PAR1 was significantly reduced (p<0.0001) by ketorolac independent of timing. Spinal astrocyte expression of PAR1 appears to be associated with the maintenance of facet-mediated pain.

Ketorolac Reduces Spinal Astrocytic Activation and PAR1 Expression Associated with Attenuation of Pain after Facet Joint Injury

PubMed Central

Dong, Ling; Smith, Jenell R.

2013-01-01

Abstract Chronic neck pain affects up to 70% of persons, with the facet joint being the most common source. Intra-articular injection of the non-steroidal anti-inflammatory drug ketorolac reduces post-operative joint-mediated pain; however, the mechanism of its attenuation of facet-mediated pain has not been evaluated. Protease-activated receptor-1 (PAR1) has differential roles in pain maintenance depending on the type and location of painful injury. This study investigated if the timing of intra-articular ketorolac injection after painful cervical facet injury affects behavioral hypersensitivity by modulating spinal astrocyte activation and/or PAR1 expression. Rats underwent a painful joint distraction and received an injection of ketorolac either immediately or 1 day later. Separate control groups included injured rats with a vehicle injection at day 1 and sham operated rats. Forepaw mechanical allodynia was measured for 7 days, and spinal cord tissue was immunolabeled for glial fibrillary acidic protein (GFAP) and PAR1 expression in the dorsal horn on day 7. Ketorolac administered on day 1 after injury significantly reduced allodynia (p=0.0006) to sham levels, whereas injection immediately after the injury had no effect compared with vehicle. Spinal astrocytic activation followed behavioral responses and was significantly decreased (p=0.009) only for ketorolac given at day 1. Spinal PAR1 (p=0.0025) and astrocytic PAR1 (p=0.012) were significantly increased after injury. Paralleling behavioral data, astrocytic PAR1 was returned to levels in sham only when ketorolac was administered on day 1. Yet, spinal PAR1 was significantly reduced (p<0.0001) by ketorolac independent of timing. Spinal astrocyte expression of PAR1 appears to be associated with the maintenance of facet-mediated pain. PMID:23126437

Inscuteable Regulates the Pins-Mud Spindle Orientation Pathway

PubMed Central

Mauser, Jonathon F.; Prehoda, Kenneth E.

2012-01-01

During asymmetric cell division, alignment of the mitotic spindle with the cell polarity axis ensures that the cleavage furrow separates fate determinants into distinct daughter cells. The protein Inscuteable (Insc) is thought to link cell polarity and spindle positioning in diverse systems by binding the polarity protein Bazooka (Baz; aka Par-3) and the spindle orienting protein Partner of Inscuteable (Pins; mPins or LGN in mammals). Here we investigate the mechanism of spindle orientation by the Insc-Pins complex. Previously, we defined two Pins spindle orientation pathways: a complex with Mushroom body defect (Mud; NuMA in mammals) is required for full activity, whereas binding to Discs large (Dlg) is sufficient for partial activity. In the current study, we have examined the role of Inscuteable in mediating downstream Pins-mediated spindle orientation pathways. We find that the Insc-Pins complex requires Gαi for partial activity and that the complex specifically recruits Dlg but not Mud. In vitro competition experiments revealed that Insc and Mud compete for binding to the Pins TPR motifs, while Dlg can form a ternary complex with Insc-Pins. Our results suggest that Insc does not passively couple polarity and spindle orientation but preferentially inhibits the Mud pathway, while allowing the Dlg pathway to remain active. Insc-regulated complex assembly may ensure that the spindle is attached to the cortex (via Dlg) before activation of spindle pulling forces by Dynein/Dynactin (via Mud). PMID:22253744

TGF-beta inhibits IL-1beta-activated PAR-2 expression through multiple pathways in human primary synovial cells.

PubMed

Tsai, Shin-Han; Sheu, Ming-Thau; Liang, Yu-Chih; Cheng, Hsiu-Tan; Fang, Sheng-Shiung; Chen, Chien-Ho

2009-10-23

To investigate the mechanism how Transforming growth factor-beta(TGF-beta) represses Interleukin-1beta (IL-1beta)-induced Proteinase-Activated Receptor-2 (PAR-2) expression in human primary synovial cells (hPSCs). Human chondrocytes and hPSCs isolated from cartilages and synovium of Osteoarthritis (OA) patients were cultured with 10% fetal bovine serum media or serum free media before treatment with IL-1beta, TGF-beta1, or Connective tissue growth factor (CTGF). The expression of PAR-2 was detected using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting. Collagen zymography was performed to assess the activity of Matrix metalloproteinases-13 (MMP-13). It was demonstrated that IL-1beta induces PAR-2 expression via p38 pathway in hPSCs. This induction can be repressed by TGF-beta and was observed to persist for at least 48 hrs, suggesting that TGF-beta inhibits PAR-2 expression through multiple pathways. First of all, TGF-beta was able to inhibit PAR-2 activity by inhibiting IL-1beta-induced p38 signal transduction and secondly the inhibition was also indirectly due to MMP-13 inactivation. Finally, TGF-beta was able to induce CTGF, and in turn CTGF represses PAR-2 expression by inhibiting IL-1beta-induced phospho-p38 level. TGF-beta could prevent OA from progression with the anabolic ability to induce CTGF production to maintain extracellular matrix (ECM) integrity and to down regulate PAR-2 expression, and the anti-catabolic ability to induce Tissue inhibitors of metalloproteinase-3 (TIMP-3) production to inhibit MMPs leading to avoid PAR-2 over-expression. Because IL-1beta-induced PAR-2 expressed in hPSCs might play a significantly important role in early phase of OA, PAR-2 repression by exogenous TGF-beta or other agents might be an ideal therapeutic target to prevent OA from progression.

Localization of amylin-like immunoreactivity in melanocyte-stimulating hormone-containing cells of the pars intermedia but not those of the pars distalis in the axolotl (Ambystoma mexicanum) pituitary.

PubMed

Suzuki, Hirohumi; Yamamoto, Toshiharu

2016-04-01

Immunohistochemical techniques were employed to investigate the distribution of amylin-like immunoreactivity in the axolotl (Ambystoma mexicanum) pituitary. Amylin-immunoreactive cells were observed in the pars intermedia, and these cells were found to be immunoreactive for α-melanocyte-stimulating hormone (αMSH) as well. In contrast, αMSH-immunoreactive cells in the pars distalis were immuno-negaitive for amylin. These light microscopic findings were confirmed by immunoelectron microscopy. Amylin-immunoreactive signals were located on the haloes of presumable secretory granules in association with αMSH-immunoreactive signals in the amylin-positive cells. However, in the pars distalis, the αMSH-positive cells did not contain amylin-immunoreactive secretory granules. Western blot analysis of axolotl pituitary extracts revealed the labeling of a protein band at approximately 10.5-kDa by the anti-rat amylin serum, which was not labeled by the anti-αMSH antibody. These findings indicate that amylin secreted from MSH-producing cells in the pars intermedia may modulate MSH secretion in an autocrine fashion and may participate in MSH functions such as fatty homeostasis together with MSH. Copyright © 2016 Elsevier GmbH. All rights reserved.

Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

PubMed Central

XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

2015-01-01

The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P<0.01), but not in the Val-Lys-Gly-Ile-Leu-Ser group (P>0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

Could soluble urokinase plasminogen receptor (suPAR) be used as a diagnostic biomarker for ventilator-associated pneumonia?

PubMed

Sunnetcioglu, Aysel; Sunnetcioglu, Mahmut; Adıyaman, Fırat; Binici, Irfan; Soyoral, Lokman

2017-11-01

Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker that is increasingly used for evaluation of systemic inflammation. This study was performed to investigate whether suPAR may possess a diagnostic value in patients with ventilator-associated pneumonia (VAP). This clinical study was performed in the anesthesia intensive care units (ICUs) of our university. In addition to descriptive data, WBC, serum levels of C-reactive protein (CRP) and suPAR prior to and after development of VAP were noted and compared in 31 patients (22 men, 9 women) diagnosed with VAP (Study Group) and 19 patients without VAP (Control Group) in ICU (14 men, 5 women). The suPAR (P = 0.023), CRP (P = 0.037), WBCs (P = 0.024) in patients with VAP were significantly higher than patients without VAP. There was no remarkable difference in terms of WBCs (P = 0.052) and suPAR levels (P = 0.616) between groups on the first day of connection to mechanical ventilator. The suPAR and CRP levels in patients with VAP were significantly higher than prior to development of VAP (P = 0.001 for both). Area under curve value after diagnosis of pneumonia was found 0.248 (P = 0.002). To conclude, our results suggest that suPAR can be a useful diagnostic biomarker in patients with VAP. However, clinical trials on larger series are warranted to explore the clinical significance more accurately. © 2016 John Wiley & Sons Ltd.

Cathepsin S Cleavage of Protease-Activated Receptor-2 on Endothelial Cells Promotes Microvascular Diabetes Complications

PubMed Central

Kumar VR, Santhosh; Darisipudi, Murthy N.; Steiger, Stefanie; Devarapu, Satish Kumar; Tato, Maia; Kukarni, Onkar P.; Mulay, Shrikant R.; Thomasova, Dana; Popper, Bastian; Demleitner, Jana; Zuchtriegel, Gabriele; Reichel, Christoph; Cohen, Clemens D.; Lindenmeyer, Maja T.; Liapis, Helen; Moll, Solange; Reid, Emma; Stitt, Alan W.; Schott, Brigitte; Gruner, Sabine; Haap, Wolfgang; Ebeling, Martin; Hartmann, Guido

2016-01-01

Endothelial dysfunction is a central pathomechanism in diabetes-associated complications. We hypothesized a pathogenic role in this dysfunction of cathepsin S (Cat-S), a cysteine protease that degrades elastic fibers and activates the protease-activated receptor-2 (PAR2) on endothelial cells. We found that injection of mice with recombinant Cat-S induced albuminuria and glomerular endothelial cell injury in a PAR2-dependent manner. In vivo microscopy confirmed a role for intrinsic Cat-S/PAR2 in ischemia–induced microvascular permeability. In vitro transcriptome analysis and experiments using siRNA or specific Cat-S and PAR2 antagonists revealed that Cat-S specifically impaired the integrity and barrier function of glomerular endothelial cells selectively through PAR2. In human and mouse type 2 diabetic nephropathy, only CD68+ intrarenal monocytes expressed Cat-S mRNA, whereas Cat-S protein was present along endothelial cells and inside proximal tubular epithelial cells also. In contrast, the cysteine protease inhibitor cystatin C was expressed only in tubules. Delayed treatment of type 2 diabetic db/db mice with Cat-S or PAR2 inhibitors attenuated albuminuria and glomerulosclerosis (indicators of diabetic nephropathy) and attenuated albumin leakage into the retina and other structural markers of diabetic retinopathy. These data identify Cat-S as a monocyte/macrophage–derived circulating PAR2 agonist and mediator of endothelial dysfunction–related microvascular diabetes complications. Thus, Cat-S or PAR2 inhibition might be a novel strategy to prevent microvascular disease in diabetes and other diseases. PMID:26567242

Possible role of rivaroxaban in attenuating pressure-overload-induced atrial fibrosis and fibrillation.

PubMed

Kondo, Hidekazu; Abe, Ichitaro; Fukui, Akira; Saito, Shotaro; Miyoshi, Miho; Aoki, Kohei; Shinohara, Tetsuji; Teshima, Yasushi; Yufu, Kunio; Takahashi, Naohiko

2018-03-01

Coagulation factor Xa (FXa) promotes thrombus formation and exacerbates inflammation via activation of protease-activated receptor (PAR)-2. We tested the hypothesis of whether administration of direct oral anticoagulant, rivaroxaban, would attenuate transverse aortic constriction (TAC)-induced atrial inflammatory fibrosis and vulnerability to atrial fibrillation (AF) in mice. Ten-week-old male CL57/B6 mice were divided into a sham-operation (CNT) group and TAC-surgery group. These two groups were then subdivided into vehicle (VEH) and rivaroxaban (RVX) treatment (30μg/g/day) groups. We assessed PAR-2 expression in response to TAC-related stimulation using rat cultured cells. TAC-induced left atrial thrombus formation was not observed in the TAC-RVX group. Cardiac PAR-2 upregulation was observed in both TAC groups. In the quantitative analysis of mRNA levels, cardiac PAR-2 upregulation was attenuated in the TAC-RVX group compared to TAC-VEH group. In histological evaluation, the TAC-VEH group showed cardiac inhomogeneous interstitial fibrosis and abundant infiltration of macrophages, which were attenuated by RVX administration. Electrophysiological examination revealed that AF duration in the TAC group was shortened by RVX administration. TAC-induced protein overexpression of monocyte chemoattractant protein-1, and mRNA overexpression of tumor necrosis factor-α, interleukin (IL)-1β and IL-6 in the left atrium was suppressed by RVX treatment. In cardiac fibroblasts, persistent intermittent stretch upregulated PAR-2, which was suppressed by RVX pre-incubation. These observations demonstrate that coagulation FXa inhibitor probably has a cardioprotective effect against pressure-overload-induced atrial remodeling. Copyright © 2017 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

Mature and progenitor endothelial cells perform angiogenesis also under protease inhibition: the amoeboid angiogenesis.

PubMed

Chillà, Anastasia; Margheri, Francesca; Biagioni, Alessio; Del Rosso, Mario; Fibbi, Gabriella; Laurenzana, Anna

2018-04-03

Controlling vascular growth is a challenging aim for the inhibition of tumor growth and metastasis. The amoeboid and mesenchymal types of invasiveness are two modes of migration interchangeable in cancer cells: the Rac-dependent mesenchymal migration requires the activity of proteases; the Rho-ROCK-dependent amoeboid motility is protease-independent and has never been described in endothelial cells. A cocktail of physiologic inhibitors (Ph-C) of serine-proteases, metallo-proteases and cysteine-proteases, mimicking the physiological environment that cells encounter during their migration within the angiogenesis sites was used to induce amoeboid style migration of Endothelial colony forming cells (ECFCs) and mature endothelial cells (ECs). To evaluate the mesenchymal-ameboid transition RhoA and Rac1 activation assays were performed along with immunofluorescence analysis of proteins involved in cytoskeleton organization. Cell invasion was studied in Boyden chambers and Matrigel plug assay for the in vivo angiogenesis. In the present study we showed in both ECFCs and ECs, a decrease of activated Rac1 and an increase of activated RhoA upon shifting of cells to the amoeboid conditions. In presence of Ph-C inhibitors both cell lines acquired a round morphology and Matrigel invasion was greatly enhanced with respect to that observed in the absence of protease inhibition. We also observed that the urokinase-plasminogen-activator (uPAR) receptor silencing and uPAR-integrin uncoupling with the M25 peptide abolished both mesenchymal and amoeboid angiogenesis of ECFCs and ECs in vitro and in vivo, indicating a role of the uPAR-integrin-actin axis in the regulation of amoeboid angiogenesis. Furthermore, under amoeboid conditions endothelial cells seem to be indifferent to VEGF stimulation, which induces an amoeboid signaling pattern also in mesenchymal conditions. Here we first provide a data set disclosing that endothelial cells can move and differentiate into vascular structures in vitro and in vivo also in the absence of proteases activity, performing a new type of neovascularization: the "amoeboid angiogenesis". uPAR is indispensable for ECs and ECFCs to perform an efficient amoeboid angiogenesis. Therefore, uPAR silencing or the block of its integrin-interaction, together with standard treatment against VEGF, could be a possible solution for angiogenesis inhibition.

The effects of splicing variant of PXR PAR-2 on CYP3A4 and MDR1 mRNA expressions.

PubMed

Liu, Yan; Ji, Wei; Yin, You; Fan, Lan; Zhang, Jian; Yun, Huang; Wang, Nianci; Li, Qing; Wei, Zhang; Ouyang, Dongshen; Zhou, Hong-Hao

2009-05-01

PAR-2(SV1), a splicing variant of PXR, has similar activity as PXR wild type. Currently, a 6bp-deletion variant ((-133)GAGAAG(-128)) in promoter region of PAR-2(SV1) was reported, which could diminish the hPAR-2 promote activity in HepG2 cells. The distribution and functions of 6bp-deletion in Chinese were investigated. The PXR genotype was analyzed from 56 liver samples and 177 blood samples. Then the mRNA expression of PAR-2(SV1), total PXR, CYP3A4 and MDR1 were quantitatively analyzed by real-time PCR. The allelic frequencies of 6bp-deletion were 22.4%, 38.4% and 23.7%, in blood of Chinese healthy (n=177), hepatic carcinoma samples (n=33) and calculus of bile duct ones (n=23) respectively. PAR-2(SV1) transcript represented approximately 15.3% of the total PXR transcripts in all liver samples. The 6bp-deletion cut down PAR-2(SV1) mRNA and total PXR mRNA transcriptional expression, and then led to down regulations of MDR1 and CYP3A4. PAR-2(SV1) plays an important role in total PXR mRNA expression. The 6bp-deletion affects the PAR-2(SV1) expression greatly, and then contributes to the adjustment of expression and function of total PXR. Thus it leads to the changed target gene expressions, which may partly explain interindividual variations in CYP3A4 and MDR1. And these phenomena suggest that individuals with 6bp-deletion are prone to carcinoma when exposed to toxicity.

Regulation of the Incorporation of Tissue Factor into Microparticles by Serine Phosphorylation of the Cytoplasmic Domain of Tissue Factor*

PubMed Central

Collier, Mary E. W.; Ettelaie, Camille

2011-01-01

The mechanisms that regulate the incorporation and release of tissue factors (TFs) into cell-derived microparticles are as yet unidentified. In this study, we have explored the regulation of TF release into microparticles by the phosphorylation of serine residues within the cytoplasmic domain of TF. Wild-type and mutant forms of TF, containing alanine and aspartate substitutions at Ser253 and Ser258, were overexpressed in coronary artery and dermal microvascular endothelial cells and microparticle release stimulated with PAR2 agonist peptide (PAR2-AP). The release of TF antigen and activity was then monitored. In addition, the phosphorylation state of the two serine residues within the released microparticles and the cells was monitored for 150 min. The release of wild-type TF as procoagulant microparticles peaked at 90 min and declined thereafter in both cell types. The TF within these microparticles was phosphorylated at Ser253 but not at Ser258. Aspartate substitution of Ser253 resulted in rapid release of TF antigen but not activity, whereas TF release was reduced and delayed by alanine substitution of Ser253 or aspartate substitution of Ser258. Alanine substitution of Ser258 prolonged the release of TF following PAR2-AP activation. The release of TF was concurrent with phosphorylation of Ser253 and was followed by dephosphorylation at 120 min and phosphorylation of Ser258. We propose a sequential mechanism in which the phosphorylation of Ser253 through PAR2 activation results in the incorporation of TF into microparticles, simultaneously inducing Ser258 phosphorylation. Phosphorylation of Ser258 in turn promotes the dephosphorylation of Ser253 and suppresses the release of TF. PMID:21310953

Copper and Copper Proteins in Parkinson's Disease

PubMed Central

Rivera-Mancia, Susana; Diaz-Ruiz, Araceli; Tristan-Lopez, Luis; Rios, Camilo

2014-01-01

Copper is a transition metal that has been linked to pathological and beneficial effects in neurodegenerative diseases. In Parkinson's disease, free copper is related to increased oxidative stress, alpha-synuclein oligomerization, and Lewy body formation. Decreased copper along with increased iron has been found in substantia nigra and caudate nucleus of Parkinson's disease patients. Copper influences iron content in the brain through ferroxidase ceruloplasmin activity; therefore decreased protein-bound copper in brain may enhance iron accumulation and the associated oxidative stress. The function of other copper-binding proteins such as Cu/Zn-SOD and metallothioneins is also beneficial to prevent neurodegeneration. Copper may regulate neurotransmission since it is released after neuronal stimulus and the metal is able to modulate the function of NMDA and GABA A receptors. Some of the proteins involved in copper transport are the transporters CTR1, ATP7A, and ATP7B and the chaperone ATOX1. There is limited information about the role of those biomolecules in the pathophysiology of Parkinson's disease; for instance, it is known that CTR1 is decreased in substantia nigra pars compacta in Parkinson's disease and that a mutation in ATP7B could be associated with Parkinson's disease. Regarding copper-related therapies, copper supplementation can represent a plausible alternative, while copper chelation may even aggravate the pathology. PMID:24672633

Effect of Thrombin on Human Amnion Mesenchymal Cells, Mouse Fetal Membranes, and Preterm Birth*

PubMed Central

Mogami, Haruta; Keller, Patrick W.; Shi, Haolin; Word, R. Ann

2014-01-01

Here, we investigated the effects of thrombin on matrix metalloproteinases (MMPs) and prostaglandin (PG) synthesis in fetal membranes. Thrombin activity was increased in human amnion from preterm deliveries. Treatment of mesenchymal, but not epithelial, cells with thrombin resulted in increased MMP-1 and MMP-9 mRNA and enzymatic activity. Thrombin also increased COX2 mRNA and PGE2 in these cells. Protease-activated receptor-1 (PAR-1) was localized to amnion mesenchymal and decidual cells. PAR-1-specific inhibitors and activating peptides indicated that thrombin-induced up-regulation of MMP-9 was mediated via PAR-1. In contrast, thrombin-induced up-regulation of MMP-1 and COX-2 was mediated through Toll-like receptor-4, possibly through thrombin-induced release of soluble fetal fibronectin. In vivo, thrombin-injected pregnant mice delivered preterm. Mmp8, Mmp9, and Mmp13, and PGE2 content was increased significantly in fetal membranes from thrombin-injected animals. These results indicate that thrombin acts through multiple mechanisms to activate MMPs and PGE2 synthesis in amnion. PMID:24652285

Uncovering Mechanisms for Repair and Protection in Cold Environments Through Studies of Cold Adapted Archaea

DTIC Science & Technology

2009-12-18

Cpn60) subunits is more abundant during growth at 4°C compared to 23°C. Consistent with this, cold shock studies in thermophilic archaea, and...helicases (Mbur_0245, Mbur_1950): These enzymes may be responsible for unwinding secondary structures in messenger RNA, and a role in cold adaptation in M...limiting step, it is unsurprising that these enzymes showed higher abundance at 4ºC. ParA protein (Mbur_2141): ParA ATPases are a ubiquitous

Crumbs3 Is Essential for Proper Epithelial Development and Viability

PubMed Central

Whiteman, Eileen L.; Fan, Shuling; Harder, Jennifer L.; Walton, Katherine D.; Liu, Chia-Jen; Soofi, Abdul; Fogg, Vanessa C.; Hershenson, Marc B.; Dressler, Gregory R.; Deutsch, Gail H.; Gumucio, Deborah L.

2014-01-01

First identified in Drosophila, the Crumbs (Crb) proteins are important in epithelial polarity, apical membrane formation, and tight junction (TJ) assembly. The conserved Crb intracellular region includes a FERM (band 4.1/ezrin/radixin/moesin) binding domain (FBD) whose mammalian binding partners are not well understood and a PDZ binding motif that interacts with mammalian Pals1 (protein associated with lin seven) (also known as MPP5). Pals1 binds Patj (Pals1-associated tight-junction protein), a multi-PDZ-domain protein that associates with many tight junction proteins. The Crb complex also binds the conserved Par3/Par6/atypical protein kinase C (aPKC) polarity cassette that restricts migration of basolateral proteins through phosphorylation. Here, we describe a Crb3 knockout mouse that demonstrates extensive defects in epithelial morphogenesis. The mice die shortly after birth, with cystic kidneys and proteinaceous debris throughout the lungs. The intestines display villus fusion, apical membrane blebs, and disrupted microvilli. These intestinal defects phenocopy those of Ezrin knockout mice, and we demonstrate an interaction between Crumbs3 and ezrin. Taken together, our data indicate that Crumbs3 is crucial for epithelial morphogenesis and plays a role in linking the apical membrane to the underlying ezrin-containing cytoskeleton. PMID:24164893

Cumulative childhood risk is associated with a new measure of chronic inflammation in adulthood.

PubMed

Rasmussen, Line Jee Hartmann; Moffitt, Terrie E; Eugen-Olsen, Jesper; Belsky, Daniel W; Danese, Andrea; Harrington, HonaLee; Houts, Renate M; Poulton, Richie; Sugden, Karen; Williams, Benjamin; Caspi, Avshalom

2018-05-09

Childhood risk factors are associated with elevated inflammatory biomarkers in adulthood, but it is unknown whether these risk factors are associated with increased adult levels of the chronic inflammation marker soluble urokinase plasminogen activator receptor (suPAR). We aimed to test the hypothesis that childhood exposure to risk factors for adult disease is associated with elevated suPAR in adulthood and to compare suPAR with the oft-reported inflammatory biomarker C-reactive protein (CRP). Prospective study of a population-representative 1972-1973 birth cohort; the Dunedin Multidisciplinary Health and Development Study observed participants to age 38 years. Main childhood predictors were poor health, socioeconomic disadvantage, adverse childhood experiences (ACEs), low IQ, and poor self-control. Main adult outcomes were adulthood inflammation measured as suPAR and high-sensitivity CRP (hsCRP). Participants with available plasma samples at age 38 were included (N = 837, 50.5% male). suPAR (mean 2.40 ng/ml; SD 0.91) was positively correlated with hsCRP (r 0.15, p < .001). After controlling for sex, body mass index (BMI), and smoking, children who experienced more ACEs, lower IQ, or had poorer self-control showed elevated adult suPAR. When the five childhood risks were aggregated into a Cumulative Childhood Risk index, and controlling for sex, BMI, and smoking, Cumulative Childhood Risk was associated with higher suPAR (b 0.10; SE 0.03; p = .002). Cumulative Childhood Risk predicted elevated suPAR, after controlling for hsCRP (b 0.18; SE 0.03; p < .001). Exposure to more childhood risk factors was associated with higher suPAR levels, independent of CRP. suPAR is a useful addition to studies connecting childhood risk to adult inflammatory burden. © 2018 Association for Child and Adolescent Mental Health.

Expression of protease activated receptor-2 (PAR-2) in central airways of smokers and non-smokers

PubMed Central

Miotto, D; Hollenberg, M; Bunnett, N; Papi, A; Braccioni, F; Boschetto, P; Rea, F; Zuin, A; Geppetti, P; Saetta, M; Maestrelli, P; Fabbri, L; Mapp, C

2002-01-01

Background: Protease activated receptor-2 (PAR-2) is a transmembrane G protein coupled receptor preferentially activated by trypsin and tryptase. The protease activated receptors play an important role in most components of injury responses including cell proliferation, migration, matrix remodelling, and inflammation. Cigarette smoking causes an inflammatory process in the central airways, peripheral airways, lung parenchyma, and adventitia of pulmonary arteries. Methods: To quantify the expression of PAR-2 in the central airways of smokers and non-smokers, surgical specimens obtained from 30 subjects undergoing lung resection for localised pulmonary lesions (24 with a history of cigarette smoking and six non-smoking control subjects) were examined. Central airways were immunostained with an antiserum specific for PAR-2 and PAR-2 expression was quantified using light microscopy and image analysis. Results: PAR-2 expression was found in bronchial smooth muscle, epithelium, glands, and in the endothelium and smooth muscle of bronchial vessels. PAR-2 expression was similar in the central airways of smokers and non-smokers. When smokers were divided according to the presence of symptoms of chronic bronchitis and chronic airflow limitation, PAR-2 expression was increased in smooth muscle (median 3.8 (interquartile range 2.9–5.8) and 1.4 (1.07–3.4) respectively); glands (33.3 (18.2–43.8) and 16.2 (11.5–22.2), respectively); and bronchial vessels (54.2 (48.7–56.8) and 40.0 (36–40.4), respectively) of smokers with symptoms of chronic bronchitis with normal lung function compared with smokers with chronic airflow limitation (COPD), but the increase was statistically significant (p<0.005) only for bronchial vessels. Conclusions: PAR-2 is present in bronchial smooth muscle, glands, and bronchial vessels of both smokers and non-smokers. An increased expression of PAR-2 was found in bronchial vessels of patients with bronchitis compared with those with COPD. PMID:11828045

Compound heterozygosity of SHOX-encompassing and downstream PAR1 deletions results in Langer mesomelic dysplasia (LMD).

PubMed

Campos-Barros, Angel; Benito-Sanz, Sara; Ross, Judith L; Zinn, Andrew R; Heath, Karen E

2007-05-01

We present the clinical and molecular characteristics of a multi-generation family in which the proband presented with clinical features of Langer mesomelic dysplasia (LMD) whilst different family members had a diagnosis of Léri-Weill dyschondrosteosis (LWD) and/or pseudoachondroplasia (PSACH). In the LMD proband two different deletions were identified in the pseudoautosomal 1 region (PAR1) of the X and Y chromosomes: a SHOX-encompassing deletion inherited from his father and a downstream PAR1 deletion, which did not include SHOX, inherited from his mother. The individuals with PSACH features presented the previously described G719D mutation in the C-terminal globular domain of the cartilage oligomeric matrix protein gene (COMP). The LMD proband described here represents the first LMD case due to compound heterozygosity for deletions of the two different PAR1 regions, SHOX-encompassing and downstream from SHOX, that have been shown to be implicated in the pathogenesis of LWD and LMD.

ApoER2 and Reelin are expressed in regenerating peripheral nerve and regulate Schwann cell migration by activating the Rac1 GEF protein, Tiam1.

PubMed

Pasten, Consuelo; Cerda, Joaquín; Jausoro, Ignacio; Court, Felipe A; Cáceres, Alfredo; Marzolo, Maria-Paz

2015-11-01

ApoER2 and its ligand Reelin participate in neuronal migration during development. Upon receptor binding, Reelin induces the proteolytic processing of ApoER2 as well as the activation of signaling pathway, including small Rho GTPases. Besides its presence in the central nervous system (CNS), Reelin is also secreted by Schwann cells (SCs), the glial cells of the peripheral nervous system (PNS). Reelin deficient mice (reeler) show decreased axonal regeneration in the PNS; however neither the presence of ApoER2 nor the role of the Reelin signaling pathway in the PNS have been evaluated. Interestingly SC migration occurs during PNS development and during injury-induced regeneration and involves activation of small Rho GTPases. Thus, Reelin-ApoER2 might regulate SC migration during axon regeneration in the PNS. Here we demonstrate the presence of ApoER2 in PNS. After sciatic nerve injury Reelin was induced and its receptor ApoER2 was proteolytically processed. In vitro, SCs express both Reelin and ApoER2 and Reelin induces SC migration. To elucidate the molecular mechanism underlying Reelin-dependent SC migration, we examined the involvement of Rac1, a conspicuous small GTPase family member. FRET experiments revealed that Reelin activates Rac1 at the leading edge of SCs. In addition, Tiam1, a major Rac1-specific GEF was required for Reelin-induced SC migration. Moreover, Reelin-induced SC migration was decreased after suppression of the polarity protein PAR3, consistent with its association to Tiam1. Even more interesting, we demonstrated that PAR3 binds preferentially to the full-length cytoplasmic tail of ApoER2 corresponding to the splice-variant containing the exon 19 that encodes a proline-rich insert and that ApoER2 was required for SC migration. Our study reveals a novel function for Reelin/ApoER2 in PNS, inducing cell migration of SCs, a process relevant for PNS development and regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Abnormal Chloride Homeostasis in the Substancia Nigra Pars Reticulata Contributes to Locomotor Deficiency in a Model of Acute Liver Injury

PubMed Central

Wei, Yan-Yan; Chen, Jing; Dou, Ke-Feng; Wang, Ya-Yun

2013-01-01

Background Altered chloride homeostasis has been thought to be a risk factor for several brain disorders, while less attention has been paid to its role in liver disease. We aimed to analyze the involvement and possible mechanisms of altered chloride homeostasis of GABAergic neurons within the substantia nigra pars reticulata (SNr) in the motor deficit observed in a model of encephalopathy caused by acute liver failure, by using glutamic acid decarboxylase 67 - green fluorescent protein knock-in transgenic mice. Methods Alterations in intracellular chloride concentration in GABAergic neurons within the SNr and changes in the expression of two dominant chloride homeostasis-regulating genes, KCC2 and NKCC1, were evaluated in mice with hypolocomotion due to hepatic encephalopathy (HE). The effects of pharmacological blockade and/or activation of KCC2 and NKCC1 functions with their specific inhibitors and/or activators on the motor activity were assessed. Results In our mouse model of acute liver injury, chloride imaging indicated an increase in local intracellular chloride concentration in SNr GABAergic neurons. In addition, the mRNA and protein levels of KCC2 were reduced, particularly on neuronal cell membranes; in contrast, NKCC1 expression remained unaffected. Furthermore, blockage of KCC2 reduced motor activity in the normal mice and led to a further deteriorated hypolocomotion in HE mice. Blockade of NKCC1 was not able to normalize motor activity in mice with liver failure. Conclusion Our data suggest that altered chloride homeostasis is likely involved in the pathophysiology of hypolocomotion following HE. Drugs aimed at restoring normal chloride homeostasis would be a potential treatment for hepatic failure. PMID:23741482

Diverse roles of actin in C. elegans early embryogenesis

PubMed Central

Velarde, Nathalie; Gunsalus, Kristin C; Piano, Fabio

2007-01-01

Background The actin cytoskeleton plays critical roles in early development in Caenorhabditis elegans. To further understand the complex roles of actin in early embryogenesis we use RNAi and in vivo imaging of filamentous actin (F-actin) dynamics. Results Using RNAi, we found processes that are differentially sensitive to levels of actin during early embryogenesis. Mild actin depletion shows defects in cortical ruffling, pseudocleavage, and establishment of polarity, while more severe depletion shows defects in polar body extrusion, cytokinesis, chromosome segregation, and eventually, egg production. These defects indicate that actin is required for proper oocyte development, fertilization, and a wide range of important events during early embryogenesis, including proper chromosome segregation. In vivo visualization of the cortical actin cytoskeleton shows dynamics that parallel but are distinct from the previously described myosin dynamics. Two distinct types of actin organization are observed at the cortex. During asymmetric polarization to the anterior, or the establishment phase (Phase I), actin forms a meshwork of microfilaments and focal accumulations throughout the cortex, while during the anterior maintenance phase (Phase II) it undergoes a morphological transition to asymmetrically localized puncta. The proper asymmetric redistribution is dependent on the PAR proteins, while both asymmetric redistribution and morphological transitions are dependent upon PFN-1 and NMY-2. Just before cytokinesis, actin disappears from most of the cortex and is only found around the presumptive cytokinetic furrow. Finally, we describe dynamic actin-enriched comets in the early embryo. Conclusion During early C. elegans embryogenesis actin plays more roles and its organization is more dynamic than previously described. Morphological transitions of F-actin, from meshwork to puncta, as well as asymmetric redistribution, are regulated by the PAR proteins. Results from this study indicate new insights into the cellular and developmental roles of the actin cytoskeleton. PMID:18157918

Activation of Protease Activated Receptor 2 by Exogenous Agonist Exacerbates Early Radiation Injury in Rat Intestine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang Junru; Boerma, Marjan; Kulkarni, Ashwini

2010-07-15

Purpose: Protease-activated receptor-2 (PAR{sub 2}) is highly expressed throughout the gut and regulates the inflammatory, mitogenic, fibroproliferative, and nociceptive responses to injury. PAR{sub 2} is strikingly upregulated and exhibits increased activation in response to intestinal irradiation. We examined the mechanistic significance of radiation enteropathy development by assessing the effect of exogenous PAR{sub 2} activation. Methods and Materials: Rat small bowel was exposed to localized single-dose radiation (16.5 Gy). The PAR{sub 2} agonist (2-furoyl-LIGRLO-NH{sub 2}) or vehicle was injected intraperitoneally daily for 3 days before irradiation (before), for 7 days after irradiation (after), or both 3 days before and 7 daysmore » after irradiation (before-after). Early and delayed radiation enteropathy was assessed at 2 and 26 weeks after irradiation using quantitative histologic examination, morphometry, and immunohistochemical analysis. Results: The PAR{sub 2} agonist did not elicit changes in the unirradiated (shielded) intestine. In contrast, in the irradiated intestine procured 2 weeks after irradiation, administration of the PAR{sub 2} agonist was associated with more severe mucosal injury and increased intestinal wall thickness in all three treatment groups (p <.05) compared with the vehicle-treated controls. The PAR{sub 2} agonist also exacerbated the radiation injury score, serosal thickening, and mucosal inflammation (p <.05) in the before and before-after groups. The short-term exogenous activation of PAR{sub 2} did not affect radiation-induced intestinal injury at 26 weeks. Conclusion: The results of the present study support a role for PAR{sub 2} activation in the pathogenesis of early radiation-induced intestinal injury. Pharmacologic PAR{sub 2} antagonists might have the potential to reduce the intestinal side effects of radiotherapy and/or as countermeasures in radiologic accidents or terrorism scenarios.« less

Proteinase-Activated Receptor 2 May Drive Cancer Progression by Facilitating TGF-β Signaling.

PubMed

Ungefroren, Hendrik; Witte, David; Rauch, Bernhard H; Settmacher, Utz; Lehnert, Hendrik; Gieseler, Frank; Kaufmann, Roland

2017-11-22

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β (TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-β signaling-promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection.

Proteinase-Activated Receptor 2 May Drive Cancer Progression by Facilitating TGF-β Signaling

PubMed Central

Ungefroren, Hendrik; Witte, David; Settmacher, Utz; Lehnert, Hendrik; Kaufmann, Roland

2017-01-01

The G protein-coupled receptor proteinase-activated receptor 2 (PAR2) has been implicated in various aspects of cellular physiology including inflammation, obesity and cancer. In cancer, it usually acts as a driver of cancer progression in various tumor types by promoting invasion and metastasis in response to activation by serine proteinases. Recently, we discovered another mode through which PAR2 may enhance tumorigenesis: crosstalk with transforming growth factor-β (TGF-β) signaling to promote TGF-β1-induced cell migration/invasion and invasion-associated gene expression in ductal pancreatic adenocarcinoma (PDAC) cells. In this chapter, we review what is known about the cellular TGF-β responses and signaling pathways affected by PAR2 expression, the signaling activities of PAR2 required for promoting TGF-β signaling, and the potential molecular mechanism(s) that underlie(s) the TGF-β signaling–promoting effect. Since PAR2 is activated through various serine proteinases and biased agonists, it may couple TGF-β signaling to a diverse range of other physiological processes that may or may not predispose cells to cancer development such as local inflammation, systemic coagulation and pathogen infection. PMID:29165389

Characterization of In Vitro Release of Neurochemicals from the Intermediate Area of the Rat Thoracic Spinal Cord: Regulation by Coexisting Neurochemical and Presynaptic Autoreceptors

DTIC Science & Technology

1995-06-16

university of the Health Sciences ABSTRACT serotonin (5-HT), substance P (SP), neurokinin A (NKA) and thyrotropin-releasing hormone (TRH) coexist in the...intermediolateral cell column IML pars funiculars IML pars principal is immunoreactivity mean arterial pressure neurokinin A neurokinin B nucleus tractus...thyrotropin-releasing hormone (TRH) and neurokinin A (NKA) coexist in these IML-projecting neurons. We propose that each of these colocalized

Actinic cheilitis: epithelial expression of COX-2 and its association with mast cell tryptase and PAR-2.

PubMed

Rojas, I Gina; Martínez, Alejandra; Brethauer, Ursula; Grez, Patricia; Yefi, Roger; Luza, Sandra; Marchesani, Francisco J

2009-03-01

Cyclooxygenase-2 (COX-2) is overexpressed in various types of human malignancies, including oral cancers. Recent studies have shown that mast cell-derived protease tryptase can induce COX-2 expression by the cleavage of proteinase-activated receptor-2 (PAR-2). Actinic cheilitis (AC) is a premalignant form of lip cancer characterized by an increased density of tryptase-positive mast cells. To investigate the possible contribution of tryptase to COX-2 overexpression during early lip carcinogenesis, normal lip (n=24) and AC (n=45) biopsies were processed for COX-2, PAR-2 and tryptase detection, using RT-PCR and immunohistochemistry. Expression scores were obtained for each marker and tested for statistical significance using Mann-Whitney and Spearmann's correlation tests as well as multivariate logistic regression analysis. Increased epithelial co-expression of COX-2 and PAR-2, as well as, elevated subepithelial density of tryptase-positive mast cells were found in AC as compared to normal lip (P<0.001). COX-2 overexpression was found to be a significant predictor of AC (P<0.034, forward stepwise, Wald), and to be correlated with both tryptase-positive mast cells and PAR-2 expression (P<0.01). The results suggest that epithelial COX-2 overexpression is a key event in AC, which is associated with increased tryptase-positive mast cells and PAR-2. Therefore, tryptase may contribute to COX-2 up-regulation by epithelial PAR-2 activation during early lip carcinogenesis.

Low-concentration hydrogen peroxide can upregulate keratinocyte intracellular calcium and PAR-2 expression in a human keratinocyte-melanocyte co-culture system.

PubMed

Li, Jian; Tang, Lu-Yan; Fu, Wen-Wen; Yuan, Jin; Sheng, You-Yu; Yang, Qin-Ping

2016-12-01

Hydrogen peroxide (H 2 O 2 ) may have a biphasic effect on melanin synthesis and melanosome transfer. High H 2 O 2 concentrations are involved in impaired melanosome transfer in vitiligo. However, low H 2 O 2 concentration promotes the beneficial proliferation and migration of melanocytes. The aim of this study was to explore low H 2 O 2 and its mechanism in melanosome transfer, protease-activated receptor-2 (PAR-2) expression and calcium balance. Melanosomes were fluorescein-labeled for clear visualization of their transfer. The expression of protease-activated receptor-2 (PAR-2) in keratinocytes was determined by western blot analysis. Flow cytometry was employed to evaluate the effects of H 2 O 2 on calcium levels in keratinocytes. Fluorescence microscopy showed the upregulation of melanosome transfer into keratinocytes following 0.3 mM H 2 O 2 treatment in the co-cultures rather than in the untreated control groups, which was associated with higher expression of PAR-2 protein and increased calcium concentration. The addition of a PAR-2 antagonist inhibited the positive activity of H 2 O 2 and calcium flow in keratinocytes. When calcium flow was blocked by a calcium chelator, the addition of H 2 O 2 did not increase the PAR-2 expression level in keratinocytes, therefore, inhibiting dendrite formation and melanosome transfer. Low H 2 O 2 concentration promotes melanosome transfer with increased PAR-2 expression level and calcium concentration in keratinocytes. In addition, the interaction between melanocytes and keratinocytes is more beneficial to enhance calcium levels in keratinocytes which mediate melanin transfer. Moreover, low H 2 O 2 concentration promotes dendrite formation, in which extracellular calcium and Par-2 were involved.

Allergic sensitization enhances anion current responsiveness of murine trachea to PAR-2 activation.

PubMed

Rievaj, Juraj; Davidson, Courtney; Nadeem, Ahmed; Hollenberg, Morley; Duszyk, Marek; Vliagoftis, Harissios

2012-03-01

Protease-activated receptor 2 (PAR-2) is a G protein-coupled receptor possibly involved in the pathogenesis of asthma. PAR-2 also modulates ion transport in cultured epithelial cells, but these effects in native airways are controversial. The influence of allergic inflammation on PAR-2-induced changes in ion transport has received little attention. Here, we studied immediate changes in transepithelial short circuit current (I (sc)) induced by PAR-2 activation in the tracheas of naive and allergic mice. Activation of PAR-2 with an apically added activation peptide (AP) induced a small increase in I (sc), while a much larger increase was observed following basolateral AP addition. In ovalbumin-sensitized and -challenged animals used as a model of allergic airway inflammation, the effect of basolateral AP addition was enhanced. Responses to basolateral AP in both naive and allergic mice were not decreased by blocking sodium absorption with amiloride or CFTR function with CFTR(inh)172 but were reduced by the cyclooxygenase inhibitor indomethacin and largely blocked (>80%) by niflumic acid, a calcium-activated chloride channels' (CaCC) blocker. Allergic mice also showed an enhanced response to ATP and thapsigargin. There was no change in mRNA expression of Par-2 or of the chloride channels Ano1 (Tmem16a) and Bestrophin 2 in tracheas from allergic mice, while mRNA levels of Bestrophin 1 were increased. In conclusion, basolateral PAR-2 activation in the mouse airways led to increased anion secretion through apical CaCC, which was more pronounced in allergic animals. This could be a protective mechanism aimed at clearing allergens and defending against mucus plugging.

Function of Platelet-Induced Epithelial Attachment at Titanium Surfaces Inhibits Microbial Colonization.

PubMed

Maeno, M; Lee, C; Kim, D M; Da Silva, J; Nagai, S; Sugawara, S; Nara, Y; Kihara, H; Nagai, M

2017-06-01

The aim of this study was to evaluate the barrier function of platelet-induced epithelial sheets on titanium surfaces. The lack of functional peri-implant epithelial sealing with basal lamina (BL) attachment at the interface of the implant and the adjacent epithelium allows for bacterial invasion, which may lead to peri-implantitis. Although various approaches have been reported to combat bacterial infection by surface modifications to titanium, none of these have been successful in a clinical application. In our previous study, surface modification with protease-activated receptor 4-activating peptide (PAR4-AP), which induced platelet activation and aggregation, was successful in demonstrating epithelial attachment via BL and epithelial sheet formation on the titanium surface. We hypothesized that the platelet-induced epithelial sheet on PAR4-AP-modified titanium surfaces would reduce bacterial attachment, penetration, and invasion. Titanium surface was modified with PAR4-AP and incubated with platelet-rich plasma (PRP). The aggregated platelets released collagen IV, a critical BL component, onto the PAR4-AP-modified titanium surface. Then, human gingival epithelial cells were seeded on the modified titanium surface and formed epithelial sheets. Green fluorescent protein (GFP)-expressing Escherichia coli was cultured onto PAR4-AP-modified titanium with and without epithelial sheet formation. While Escherichia coli accumulated densely onto the PAR4-AP titanium lacking epithelial sheet, few Escherichia coli were observed on the epithelial sheet on the PAR4-AP surface. No bacterial invasion into the interface of the epithelial sheet and the titanium surface was observed. These in vitro results indicate the efficacy of a platelet-induced epithelial barrier that functions to prevent bacterial attachment, penetration, and invasion on PAR4-AP-modified titanium.

3D view to tumor suppression: Lkb1, polarity and the arrest of oncogenic c-Myc.

PubMed

Partanen, Johanna I; Nieminen, Anni I; Klefstrom, Juha

2009-03-01

Machiavelli wrote, in his famous political treatise Il Principe, about disrupting organization by planting seeds of dissension or by eliminating necessary support elements. Tumor cells do exactly that by disrupting the organized architecture of epithelial cell layers during progression from contained benign tumor to full-blown invasive cancer. However, it is still unclear whether tumor cells primarily break free by activating oncogenes powerful enough to cause chaos or by eliminating tumor suppressor genes guarding the order of the epithelial organization. Studies in Drosophila have exposed genes that encode key regulators of the epithelial apicobasal polarity and which, upon inactivation, cause disorganization of the epithelial layers and promote unscheduled cell proliferation. These polarity regulator/tumor suppressor proteins, which include products of neoplastic tumor suppressor genes (nTSGs), are carefully positioned in polarized epithelial cells to maintain the order of epithelial structures and to impose a restraint on cell proliferation. In this review, we have explored the presence and prevalence of somatic mutations in the human counterparts of Drosophila polarity regulator/tumor suppressor genes across the human cancers. The screen points out LKB1, which is a causal genetic lesion in Peutz-Jeghers cancer syndrome, a gene mutated in certain sporadic cancers and a human homologue of the fly polarity gene par-4. We review the evidence linking Lkb1 protein to polarity regulation in the scope of our recent results suggesting a coupled role for Lkb1 as an architect of organized acinar structures and a suppressor of oncogenic c-Myc. We finally present models to explain how Lkb1-dependent formation of epithelial architecture is coupled to suppression of normal and oncogene-induced proliferation.

Insights from spectroscopic and in-silico techniques for the exploitation of biomolecular interactions between Human serum albumin and Paromomycin.

PubMed

Raza, Muslim; Jiang, Yang; Wei, Yun; Ahmad, Aftab; Khan, Ajmal; Qipeng, Yuan

2017-09-01

The study of molecular interactions of drug-protein are extremely important from the biological aspect in all living organisms, and therefore such type of investigation hold a tremendous significance in rational drug design and discovery. In the present study, the molecular interactions between paromomycin (PAR) and human serum albumin (HSA) have been studied by different biophysical techniques and validated by in-silico approaches. The results obtained from Ultraviolet-visible spectroscopy (UV) and Fourier transform infrared spectroscopy (FT-IR) demonstrated a remarkable change upon the complexation of PAR with HSA. Circular Dichroism (CD), Dynamic Light Scattering (DLS) and Resonance Rayleigh scattering (RRS) results revealed a significant secondary structure alteration and reduction of hydrodynamic radii upon the conjugation of PAR with HSA. The fluorescence spectroscopy results also apparently revealed the static quenching mechanism. The number of binding sites, binding constants, and Gibbs free energy values were calculated to illustrate the nature of intermolecular interactions. Similarly, the in-silico docking and molecular dynamics simulation clearly explain the theoretical basis of the binding mechanism of PAR with HSA. The experimental and docking approaches suggested that PAR binds to the hydrophobic cavity site I of HSA. The finding of present investigation will provide binding insight of PAR and associated alterations in the stability and conformation of HSA. Copyright © 2017 Elsevier B.V. All rights reserved.

Polycystin-1 Binds Par3/aPKC and Controls Convergent Extension During Renal Tubular Morphogenesis

PubMed Central

Castelli, Maddalena; Boca, Manila; Chiaravalli, Marco; Ramalingam, Harini; Rowe, Isaline; Distefano, Gianfranco; Carroll, Thomas; Boletta, Alessandra

2013-01-01

Several organs, including lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintanance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1 (PC-1), a large receptor of unknown function. Here we demonstrate that PC-1 plays an essential role in establishment of correct tubular diameter during nephron development. PC-1 associates with Par3 favoring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a program of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis and in renal cyst formation. Our data define PC-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis. PMID:24153433

Anti-Urokinase Receptor Antisense Oligonucleotide (uPAR-aODN) to Prevent and Cure Long-Term Space Exploration-Related Retinal Pathological Angiogenesis

NASA Astrophysics Data System (ADS)

Lazzarano, Stefano; Lulli, Matteo; Fibbi, Gabriella; Margheri, Francesca; Papucci, Laura; Serrati, Simona; Witort, Ewa; Chilla, Anastasia; Lapucci, Andrea; Donnini, Martino; Quaglierini, Paolo; Romiti, Alice; Specogna, Rebecca; Del Rosso, Mario; Capaccioli, Sergio

2008-06-01

Angiogenesis underlies a variety of physiological processes and its possible deregulation during long term space exploration needs to be investigated. Angiogenesis is a multistep process of new blood capillary formation, where degradation of the extracellular matrix (ECM) by proteolytic enzymes, including uPA (urokinase plasminogen activator) and opening the way to migration of endothelial cells (EC), is critical. Plasminogen activation system regulates angiogenesis by both uPA-driven ECM degradation and uPA receptor (uPAR). Microgravity and low dose irradiations promote tissue neoangiogeenesis and neovascularization is often common occurence in ophthalmologic pathologies. We have designed and patented the uPAR antisense oligonucleotide (aODN) and evaluated its antiangiogenetic activity by EC cellular migration and capillary morphogenesis assays. The uPAR aODN treatment caused a 75% inhibition of human microvascular EC migration and a complete inhibition of capillary morphogenesis, suggesting its therapeutic application to prevent neoangiogenesis-related ophthalmologic pathologies during space exploration.

Polycystin-1 binds Par3/aPKC and controls convergent extension during renal tubular morphogenesis

NASA Astrophysics Data System (ADS)

Castelli, Maddalena; Boca, Manila; Chiaravalli, Marco; Ramalingam, Harini; Rowe, Isaline; Distefano, Gianfranco; Carroll, Thomas; Boletta, Alessandra

2013-10-01

Several organs, including the lungs and kidneys, are formed by epithelial tubes whose proper morphogenesis ensures correct function. This is best exemplified by the kidney, where defective establishment or maintenance of tubular diameter results in polycystic kidney disease, a common genetic disorder. Most polycystic kidney disease cases result from loss-of-function mutations in the PKD1 gene, encoding Polycystin-1, a large receptor of unknown function. Here we demonstrate that PC-1 has an essential role in the establishment of correct tubular diameter during nephron development. Polycystin-1 associates with Par3 favouring the assembly of a pro-polarizing Par3/aPKC complex and it regulates a programme of cell polarity important for oriented cell migration and for a convergent extension-like process during tubular morphogenesis. Par3 inactivation in the developing kidney results in defective convergent extension and tubular morphogenesis, and in renal cyst formation. Our data define Polycystin-1 as central to cell polarization and to epithelial tube morphogenesis and homeostasis.

The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation.

PubMed

Umbarger, Mark A; Toro, Esteban; Wright, Matthew A; Porreca, Gregory J; Baù, Davide; Hong, Sun-Hae; Fero, Michael J; Zhu, Lihua J; Marti-Renom, Marc A; McAdams, Harley H; Shapiro, Lucy; Dekker, Job; Church, George M

2011-10-21

We have determined the three-dimensional (3D) architecture of the Caulobacter crescentus genome by combining genome-wide chromatin interaction detection, live-cell imaging, and computational modeling. Using chromosome conformation capture carbon copy (5C), we derive ~13 kb resolution 3D models of the Caulobacter genome. The resulting models illustrate that the genome is ellipsoidal with periodically arranged arms. The parS sites, a pair of short contiguous sequence elements known to be involved in chromosome segregation, are positioned at one pole, where they anchor the chromosome to the cell and contribute to the formation of a compact chromatin conformation. Repositioning these elements resulted in rotations of the chromosome that changed the subcellular positions of most genes. Such rotations did not lead to large-scale changes in gene expression, indicating that genome folding does not strongly affect gene regulation. Collectively, our data suggest that genome folding is globally dictated by the parS sites and chromosome segregation. Copyright © 2011 Elsevier Inc. All rights reserved.

Expression of protease-activated receptor-2 (PAR-2) is related to advanced clinical stage and adverse prognosis in ovarian clear cell carcinoma.

PubMed

Aman, Murasaki; Ohishi, Yoshihiro; Imamura, Hiroko; Shinozaki, Tomoko; Yasutake, Nobuko; Kato, Kiyoko; Oda, Yoshinao

2017-06-01

Recent studies demonstrated that protease-activated receptor-2 (PAR-2) correlates with tumor progression in various tissues. On the other hand, oxidative stress arising from endometriosis has been considered a cause of carcinogenesis in ovarian clear cell carcinoma (OCCC). We previously demonstrated that oxidative stress up-regulates PAR-2 expression, and we conducted the present study to investigate the PAR-2 expression and its relation to clinicopathological factors and oxidative stress in OCCC. We performed an immunohistochemical evaluation in 95 cases of OCCC. For the evaluation of oxidative stress markers, 31 cases of ovarian endometrioid carcinoma (OEC) were also examined. No significant differences in the expression of cyclooxygenase-2 and inducible nitric oxide synthase were observed between OCCC and OEC. Sixty-two percent of the OCCC cases showed high 8-hydroxydeoxyguanosine expression, whereas all of the OEC cases showed almost negative immunoreactivities. The presence of endometriosis did not affect the expression of these oxidative stress markers or prognosis. High PAR-2 expression was observed in 20% (14/71) of the early International Federation of Gynecology and Obstetrics (FIGO) stage cases and 58% (14/24) of the advanced FIGO stage cases. High PAR-2 expression was significantly correlated with advanced FIGO stage and shorter overall survival. We found no correlations between PAR-2 expression and oxidative stress in OCCC. Our results suggest that PAR-2 plays an important role in the progression of OCCC. The expression of 8-hydroxydeoxyguanosine is a characteristic finding of OCCC, indicating that the injury of DNA by oxidative stress may be involved in the carcinogenesis of OCCC. Copyright © 2017 Elsevier Inc. All rights reserved.

Protease-Activated Receptor 1 Inhibition by SCH79797 Attenuates Left Ventricular Remodeling and Profibrotic Activities of Cardiac Fibroblasts

PubMed Central

Sonin, Dmitry L.; Wakatsuki, Tetsuro; Routhu, Kasi V.; Harmann, Leanne M.; Petersen, Matthew; Meyer, Jennifer; Strande, Jennifer L.

2013-01-01

Purpose Fibroblast activity promotes adverse left ventricular (LV) remodeling that underlies the development of ischemic cardiomyopathy. Transforming growth factor-β (TGF-β) is a potent stimulus for fibrosis, and the extracellular signal-regulated kinases(ERK) 1/2 pathway also contributes to the fibrotic response. The thrombin receptor, protease-activated receptor 1 (PAR1), has been shown to play an important role in the excessive fibrosis in different tissues. The aim of this study was to investigate the influence of a PAR1 inhibitor, SCH79797, on cardiac fibrosis, tissue stiffness and postinfarction remodeling, and effects of PAR1 inhibition on thrombin-induced TGF-β and (ERK) 1/2 activities in cardiac fibroblasts. Methods We used a rat model of myocardial ischemia–reperfusion injury, isolated cardiac fibroblasts, and 3-dimensional (3D) cardiac tissue models fabricated to ascertain the contribution of PAR1 activation on cardiac fibrosis and LV remodeling. Results The PAR1 inhibitor attenuated LV dilation and improved LV systolic function of the reperfused myocardium at 28 days. This improvement was associated with a nonsignificant decrease in scar size (%LV) from 23 ± % in the control group (n = 10) to 16% ± 5.5% in the treated group (n = 9; P = .052). In the short term, the PAR1 inhibitor did not rescue infarct size or LV systolic function after 3 days. The PAR1 inhibition abolished thrombin-mediated ERK1/2 phosphorylation, TGF-β and type I procollagen production, matrix metalloproteinase-2/9 activation, myofibroblasts transformation in vitro, and abrogated the remodeling of 3D tissues induced by chronic thrombin treatment. Conclusion These studies suggest PAR1 inhibition initiated after ischemic injury attenuates adverse LV remodeling through late-stage antifibrotic events. PMID:23598708

Diagnostic value of decoy receptor 3 combined with procalcitonin and soluble urokinase-type plasminogen activator receptor for sepsis.

PubMed

Zhao, Jing-Jing; Lou, Xiao-Li; Chen, Hong-Wei; Zhu, Feng-Ting; Hou, Yan-Qiang

2018-01-01

The levels of decoy receptor 3 (DcR3), soluble urokinase type plasminogen activator receptor (suPAR) and procalcitonin (PCT) are significantly increased in sepsis. We investigated the diagnostic value of DcR3 combined with suPAR and PCT in sepsis. Patients with sepsis, non-infectious systemic inflammatory response comprehensive syndrome (SIRS) and healthy controls were recruited according to the diagnostic standard. We measured DcR3, suPAR, PCT, interleukin-6 (IL-6) and C-reactive protein (CRP), and the diagnostic value was evaluated by receiver operating characteristics (ROC) curves. In our analysis, serum DcR3, suPAR and PCT levels of the sepsis group were significantly higher than those of the SIRS and control groups. However, IL-6, CRP and WBC showed no significant difference between the SIRS group and the sepsis group. The serum DcR3 level was positively correlated with the serum suPAR level ( r  = 0.37, p  = 0.0022) and PCT level ( r  = 0.37, p  = 0.0021). Using DcR3, suPAR and PCT to distinguish SIRS from sepsis, the area under the curve (AUC) values were 0.892, 0.778 and 0.692. When DcR3, suPAR and PCT combined were used for diagnosis of sepsis, the AUC was 0.933, at a cut-off point of 0.342. This combination improved the sensitivity and specificity of diagnosis of sepsis, suggesting that use of the combination of three indexes enhanced the efficiency of sepsis diagnosis.

Exploiting Amino Acid Composition for Predicting Protein-Protein Interactions

PubMed Central

Roy, Sushmita; Martinez, Diego; Platero, Harriett; Lane, Terran; Werner-Washburne, Margaret

2009-01-01

Background Computational prediction of protein interactions typically use protein domains as classifier features because they capture conserved information of interaction surfaces. However, approaches relying on domains as features cannot be applied to proteins without any domain information. In this paper, we explore the contribution of pure amino acid composition (AAC) for protein interaction prediction. This simple feature, which is based on normalized counts of single or pairs of amino acids, is applicable to proteins from any sequenced organism and can be used to compensate for the lack of domain information. Results AAC performed at par with protein interaction prediction based on domains on three yeast protein interaction datasets. Similar behavior was obtained using different classifiers, indicating that our results are a function of features and not of classifiers. In addition to yeast datasets, AAC performed comparably on worm and fly datasets. Prediction of interactions for the entire yeast proteome identified a large number of novel interactions, the majority of which co-localized or participated in the same processes. Our high confidence interaction network included both well-studied and uncharacterized proteins. Proteins with known function were involved in actin assembly and cell budding. Uncharacterized proteins interacted with proteins involved in reproduction and cell budding, thus providing putative biological roles for the uncharacterized proteins. Conclusion AAC is a simple, yet powerful feature for predicting protein interactions, and can be used alone or in conjunction with protein domains to predict new and validate existing interactions. More importantly, AAC alone performs at par with existing, but more complex, features indicating the presence of sequence-level information that is predictive of interaction, but which is not necessarily restricted to domains. PMID:19936254

Affinity Pulldown of Biotinylated RNA for Detection of Protein-RNA Complexes.

PubMed

Panda, Amaresh C; Martindale, Jennifer L; Gorospe, Myriam

2016-12-20

RNA-binding proteins (RBPs) have recently emerged as crucial players in the regulation of gene expression. The interactions of RBPs with target mRNAs control the levels of gene products by altering different regulatory steps, including pre-mRNA splicing and maturation, nuclear mRNA export, and mRNA stability and translation (Glisovic et al. , 2008). There are several methodologies available today to identify RNAs bound to specific RBPs; some detect only recombinant molecules in vitro , others detect recombinant and endogenous molecules, while others detect only endogenous molecules. Examples include systematic evolution of ligands by exponential enrichment (SELEX), biotinylated RNA pulldown assay, RNA immunoprecipitation (RIP) assay, electrophoretic mobility shift assay (EMSA), RNA footprinting analysis, and various UV crosslinking and immunoprecipitation (CLIP) methods such as CLIP, PAR-CLIP, and iCLIP (Popova et al. , 2015). Here, we describe a simple and informative method to study and identify the RNA region of interaction between an RBP and its target transcript (Panda et al. , 2014 and 2016). Its reproducibility and ease of use make this protocol a fast and useful method to identify interactions between RBPs and specific RNAs.

Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP

PubMed Central

Hafner, Markus; Landthaler, Markus; Burger, Lukas; Khorshid, Mohsen; Hausser, Jean; Berninger, Philipp; Rothballer, Andrea; Ascano, Manuel; Jungkamp, Anna-Carina; Munschauer, Mathias; Ulrich, Alexander; Wardle, Greg S.; Dewell, Scott; Zavolan, Mihaela; Tuschl, Thomas

2010-01-01

Summary RNA transcripts are subject to post-transcriptional gene regulation involving hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) expressed in a cell-type dependent fashion. We developed a cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs. The crosslinked sites are revealed by thymidine to cytidine transitions in the cDNAs prepared from immunopurified RNPs of 4-thiouridine-treated cells. We determined the binding sites and regulatory consequences for several intensely studied RBPs and miRNPs, including PUM2, QKI, IGF2BP1-3, AGO/EIF2C1-4 and TNRC6A-C. Our study revealed that these factors bind thousands of sites containing defined sequence motifs and have distinct preferences for exonic versus intronic or coding versus untranslated transcript regions. The precise mapping of binding sites across the transcriptome will be critical to the interpretation of the rapidly emerging data on genetic variation between individuals and how these variations contribute to complex genetic diseases. PMID:20371350

Circadian and feeding cues integrate to drive rhythms of physiology in Drosophila insulin-producing cells.

PubMed

Barber, Annika F; Erion, Renske; Holmes, Todd C; Sehgal, Amita

2016-12-01

Circadian clocks regulate much of behavior and physiology, but the mechanisms by which they do so remain poorly understood. While cyclic gene expression is thought to underlie metabolic rhythms, little is known about cycles in cellular physiology. We found that Drosophila insulin-producing cells (IPCs), which are located in the pars intercerebralis and lack an autonomous circadian clock, are functionally connected to the central circadian clock circuit via DN1 neurons. Insulin mediates circadian output by regulating the rhythmic expression of a metabolic gene (sxe2) in the fat body. Patch clamp electrophysiology reveals that IPCs display circadian clock-regulated daily rhythms in firing event frequency and bursting proportion under light:dark conditions. The activity of IPCs and the rhythmic expression of sxe2 are additionally regulated by feeding, as demonstrated by night feeding-induced changes in IPC firing characteristics and sxe2 levels in the fat body. These findings indicate circuit-level regulation of metabolism by clock cells in Drosophila and support a role for the pars intercerebralis in integrating circadian control of behavior and physiology. © 2016 Barber et al.; Published by Cold Spring Harbor Laboratory Press.

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin

PubMed Central

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun

2016-01-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2. Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. PMID:27534820

Covalent ISG15 conjugation positively regulates the ubiquitin E3 ligase activity of parkin.

PubMed

Im, Eunju; Yoo, Lang; Hyun, Minju; Shin, Woo Hyun; Chung, Kwang Chul

2016-08-01

Parkinson's disease (PD) is characterized by selective loss of dopaminergic neurons in the pars compacta of the substantia nigra and accumulation of ubiquitinated proteins in aggregates called Lewy bodies. Several mutated genes have been found in familial PD patients, including SNCA (α-synuclein), PARK2 (parkin), PINK1, PARK7 (DJ-1), LRRK2 and ATP13A2 Many pathogenic mutations of PARK2, which encodes the ubiquitin E3 ligase parkin, result in loss of function, leading to accumulation of parkin substrates and consequently contributing to dopaminergic cell death. ISG15 is a member of the ubiquitin-like modifier family and is induced by stimulation with type I interferons. Similar to ubiquitin and ubiquitination, covalent conjugation of ISG15 to target proteins (ISGylation) regulates their biochemical properties. In this study, we identified parkin as a novel target of ISGylation specifically mediated by the ISG15-E3 ligase HERC5. In addition, we identified two ISGylation sites, Lys-349 and Lys-369, in the in-between-ring domain of parkin. ISGylation of these sites promotes parkin's ubiquitin E3 ligase activity by suppressing the intramolecular interaction that maintains its autoinhibited conformation and increases its cytoprotective effect. In conclusion, covalent ISG15 conjugation is a novel mode of modulating parkin activity, and alteration in this pathway may be associated with PD pathogenesis. © 2016 The Authors.

The transcription factor DBP affects circadian sleep consolidation and rhythmic EEG activity.

PubMed

Franken, P; Lopez-Molina, L; Marcacci, L; Schibler, U; Tafti, M

2000-01-15

Albumin D-binding protein (DBP) is a PAR leucine zipper transcription factor that is expressed according to a robust circadian rhythm in the suprachiasmatic nuclei, harboring the circadian master clock, and in most peripheral tissues. Mice lacking DBP display a shorter circadian period in locomotor activity and are less active. Thus, although DBP is not essential for circadian rhythm generation, it does modulate important clock outputs. We studied the role of DBP in the circadian and homeostatic aspects of sleep regulation by comparing DBP deficient mice (dbp-/-) with their isogenic controls (dbp+/+) under light-dark (LD) and constant-dark (DD) baseline conditions, as well as after sleep loss. Whereas total sleep duration was similar in both genotypes, the amplitude of the circadian modulation of sleep time, as well as the consolidation of sleep episodes, was reduced in dbp-/- under both LD and DD conditions. Quantitative EEG analysis demonstrated a marked reduction in the amplitude of the sleep-wake-dependent changes in slow-wave sleep delta power and an increase in hippocampal theta peak frequency in dbp-/- mice. The sleep deprivation-induced compensatory rebound of EEG delta power was similar in both genotypes. In contrast, the rebound in paradoxical sleep was significant in dbp+/+ mice only. It is concluded that the transcriptional regulatory protein DBP modulates circadian and homeostatic aspects of sleep regulation.

The Ras-related Protein, Rap1A, Mediates Thrombin-stimulated, Integrin-dependent Glioblastoma Cell Proliferation and Tumor Growth*

PubMed Central

Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A.; Stupack, Dwayne G.; Brown, Joan Heller

2014-01-01

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. PMID:24790104

Kid cleaves specific mRNAs at UUACU sites to rescue the copy number of plasmid R1

PubMed Central

Pimentel, Belén; Madine, Mark A; de la Cueva-Méndez, Guillermo

2005-01-01

Stability and copy number of extra-chromosomal elements are tightly regulated in prokaryotes and eukaryotes. Toxin Kid and antitoxin Kis are the components of the parD stability system of prokaryotic plasmid R1 and they can also function in eukaryotes. In bacteria, Kid was thought to become active only in cells that lose plasmid R1 and to cleave exclusively host mRNAs at UA(A/C/U) trinucleotide sites to eliminate plasmid-free cells. Instead, we demonstrate here that Kid becomes active in plasmid-containing cells when plasmid copy number decreases, cleaving not only host- but also a specific plasmid-encoded mRNA at the longer and more specific target sequence UUACU. This specific cleavage by Kid inhibits bacterial growth and, at the same time, helps to restore the plasmid copy number. Kid targets a plasmid RNA that encodes a repressor of the synthesis of an R1 replication protein, resulting in increased plasmid DNA replication. This mechanism resembles that employed by some human herpesviruses to regulate viral amplification during infection. PMID:16163387

Granulocyte-macrophage colony-stimulating factor primes interleukin-13 production by macrophages via protease-activated receptor-2.

PubMed

Aoki, Manabu; Yamaguchi, Rui; Yamamoto, Takatoshi; Ishimaru, Yasuji; Ono, Tomomichi; Sakamoto, Arisa; Narahara, Shinji; Sugiuchi, Hiroyuki; Hirose, Eiji; Yamaguchi, Yasuo

2015-04-01

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation. Copyright © 2015 Elsevier Inc. All rights reserved.

The Asymmetric Cell Division Regulators Par3, Scribble and Pins/Gpsm2 Are Not Essential for Erythroid Development or Enucleation

PubMed Central

Wölwer, Christina B.; Gödde, Nathan; Pase, Luke B.; Elsum, Imogen A.; Lim, Krystle Y. B.; Sacirbegovic, Faruk; Walkley, Carl R.; Ellis, Sarah; Ohno, Shigeo; Matsuzaki, Fumio; Russell, Sarah M.; Humbert, Patrick O.

2017-01-01

Erythroid enucleation is the process by which the future red blood cell disposes of its nucleus prior to entering the blood stream. This key event during red blood cell development has been likened to an asymmetric cell division (ACD), by which the enucleating erythroblast divides into two very different daughter cells of alternate molecular composition, a nucleated cell that will be removed by associated macrophages, and the reticulocyte that will mature to the definitive erythrocyte. Here we investigated gene expression of members of the Par, Scribble and Pins/Gpsm2 asymmetric cell division complexes in erythroid cells, and functionally tested their role in erythroid enucleation in vivo and ex vivo. Despite their roles in regulating ACD in other contexts, we found that these polarity regulators are not essential for erythroid enucleation, nor for erythroid development in vivo. Together our results put into question a role for cell polarity and asymmetric cell division in erythroid enucleation. PMID:28095473

76 FR 68747 - Hess Corporation

Federal Register 2010, 2011, 2012, 2013, 2014

2011-11-07

... Respondent to make a minor adjustment to two phase angle regulators (PAR) owned by Public Service Electric... contacts for the Respondent as listed on the Commission's list of Corporate Officials. Any person desiring...

ssHMM: extracting intuitive sequence-structure motifs from high-throughput RNA-binding protein data

PubMed Central

Krestel, Ralf; Ohler, Uwe; Vingron, Martin; Marsico, Annalisa

2017-01-01

Abstract RNA-binding proteins (RBPs) play an important role in RNA post-transcriptional regulation and recognize target RNAs via sequence-structure motifs. The extent to which RNA structure influences protein binding in the presence or absence of a sequence motif is still poorly understood. Existing RNA motif finders either take the structure of the RNA only partially into account, or employ models which are not directly interpretable as sequence-structure motifs. We developed ssHMM, an RNA motif finder based on a hidden Markov model (HMM) and Gibbs sampling which fully captures the relationship between RNA sequence and secondary structure preference of a given RBP. Compared to previous methods which output separate logos for sequence and structure, it directly produces a combined sequence-structure motif when trained on a large set of sequences. ssHMM’s model is visualized intuitively as a graph and facilitates biological interpretation. ssHMM can be used to find novel bona fide sequence-structure motifs of uncharacterized RBPs, such as the one presented here for the YY1 protein. ssHMM reaches a high motif recovery rate on synthetic data, it recovers known RBP motifs from CLIP-Seq data, and scales linearly on the input size, being considerably faster than MEMERIS and RNAcontext on large datasets while being on par with GraphProt. It is freely available on Github and as a Docker image. PMID:28977546

Differences in PAR-2 activating potential by king crab (Paralithodes camtschaticus), salmon (Salmo salar), and bovine (Bos taurus) trypsin.

PubMed

Larsen, Anett K; Kristiansen, Kurt; Sylte, Ingebrigt; Seternes, Ole-Morten; Bang, Berit E

2013-07-20

Salmon trypsin is shown to increase secretion of the pro-inflammatory cytokine interleukin (IL)-8 from human airway epithelial cells through activation of PAR-2. Secretion of IL-8 induced by king crab trypsin is observed in a different concentration range compared to salmon trypsin, and seems to be only partially related to PAR-2 activation. This report aim to identify differences in the molecular structure of king crab trypsin (Paralithodes camtschaticus) compared to salmon (Salmo salar) and bovine trypsin (Bos taurus) that might influence the ability to activate protease-activated receptor-2 (PAR-2). During purification king crab trypsin displayed stronger binding capacity to the anionic column used in fast protein liquid chromatography compared to fish trypsins, and was identified as a slightly bigger molecule. Measurements of enzymatic activity yielded no obvious differences between the trypsins tested. Molecular modelling showed that king crab trypsin has a large area with strong negative electrostatic potential compared to the smaller negative areas in bovine and salmon trypsins. Bovine and salmon trypsins also displayed areas with strong positive electrostatic potential, a feature lacking in the king crab trypsin. Furthermore we have identified 3 divergent positions (Asp196, Arg244, and Tyr247) located near the substrate binding pocket of king crab trypsin that might affect the binding and cleavage of PAR-2. These preliminary results indicate that electrostatic interactions could be of importance in binding, cleavage and subsequent activation of PAR-2.

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia.

PubMed

Schoergenhofer, Christian; Schwameis, Michael; Gelbenegger, Georg; Buchtele, Nina; Thaler, Barbara; Mussbacher, Marion; Schabbauer, Gernot; Wojta, Johann; Jilma-Stohlawetz, Petra; Jilma, Bernd

2018-06-04

The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11-49%), thrombin-anti-thrombin concentrations by 22% (-3 to 46%) and plasmin-anti-plasmin levels by 38% (23-53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (-11-71%), 50% (15-79%) and 23% (16-38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26-51%) and soluble E-selectin concentrations by 30% (25-38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia. Schattauer GmbH Stuttgart.

Soluble urokinase plasminogen activator receptor level is an independent predictor of the presence and severity of coronary artery disease and of future adverse events.

PubMed

Eapen, Danny J; Manocha, Pankaj; Ghasemzadeh, Nima; Ghasemzedah, Nima; Patel, Riyaz S; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc-Anh; Pielak, Tomasz; Thorball, Christian W; Velegraki, Aristea; Kremastinos, Dimitrios T; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A

2014-10-23

Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD, is unknown. We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (≥50% stenosis in ≥1 coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C-reactive protein levels. Elevated suPAR levels were independently associated with the presence of CAD (P<0.0001) and its severity (P<0.0001). A plasma suPAR level ≥3.5 ng/mL (cutoff by Youden's index) predicted future risk of MI (hazard ratio [HR]=3.2; P<0.0001), cardiac death (HR=2.62; P<0.0001), and the combined endpoint of death and MI (HR=1.9; P<0.0001), even after adjustment of covariates. The C-statistic for a model based on traditional risk factors was improved from 0.72 to 0.74 (P=0.008) with the addition of suPAR. Elevated levels of plasma suPAR are associated with the presence and severity of CAD and are independent predictors of death and MI in patients with suspected or known CAD. © 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Soluble Urokinase Plasminogen Activator Receptor Level Is an Independent Predictor of the Presence and Severity of Coronary Artery Disease and of Future Adverse Events

PubMed Central

Eapen, Danny J.; Manocha, Pankaj; Ghasemzedah, Nima; Patel, Riyaz S.; Al Kassem, Hatem; Hammadah, Muhammad; Veledar, Emir; Le, Ngoc‐Anh; Pielak, Tomasz; Thorball, Christian W.; Velegraki, Aristea; Kremastinos, Dimitrios T.; Lerakis, Stamatios; Sperling, Laurence; Quyyumi, Arshed A.

2014-01-01

Introduction Soluble urokinase plasminogen activator receptor (suPAR) is an emerging inflammatory and immune biomarker. Whether suPAR level predicts the presence and the severity of coronary artery disease (CAD), and of incident death and myocardial infarction (MI) in subjects with suspected CAD, is unknown. Methods and Results We measured plasma suPAR levels in 3367 subjects (67% with CAD) recruited in the Emory Cardiovascular Biobank and followed them for adverse cardiovascular (CV) outcomes of death and MI over a mean 2.1±1.1 years. Presence of angiographic CAD (≥50% stenosis in ≥1 coronary artery) and its severity were quantitated using the Gensini score. Cox's proportional hazard survival and discrimination analyses were performed with models adjusted for established CV risk factors and C‐reactive protein levels. Elevated suPAR levels were independently associated with the presence of CAD (P<0.0001) and its severity (P<0.0001). A plasma suPAR level ≥3.5 ng/mL (cutoff by Youden's index) predicted future risk of MI (hazard ratio [HR]=3.2; P<0.0001), cardiac death (HR=2.62; P<0.0001), and the combined endpoint of death and MI (HR=1.9; P<0.0001), even after adjustment of covariates. The C‐statistic for a model based on traditional risk factors was improved from 0.72 to 0.74 (P=0.008) with the addition of suPAR. Conclusion Elevated levels of plasma suPAR are associated with the presence and severity of CAD and are independent predictors of death and MI in patients with suspected or known CAD. PMID:25341887

Differential Dynamics of Platelet Contact and Spreading

PubMed Central

Lee, Dooyoung; Fong, Karen P.; King, Michael R.; Brass, Lawrence F.; Hammer, Daniel A.

2012-01-01

Platelet spreading is critical for hemostatic plug formation and thrombosis. However, the detailed dynamics of platelet spreading as a function of receptor-ligand adhesive interactions has not been thoroughly investigated. Using reflection interference contrast microscopy, we found that both adhesive interactions and PAR4 activation affect the dynamics of platelet membrane contact formation during spreading. The initial growth of close contact area during spreading was controlled by the combination of different immobilized ligands or PAR4 activation on fibrinogen, whereas the growth of the total area of spreading was independent of adhesion type and PAR4 signaling. We found that filopodia extend to their maximal length and then contract over time; and that filopodial protrusion and expansion were affected by PAR4 signaling. Upon PAR4 activation, the integrin αIIbβ3 mediated close contact to fibrinogen substrata and led to the formation of ringlike patterns in the platelet contact zone. A systematic study of platelet spreading of GPVI-, α2-, or β3-deficient platelets on collagen or fibrinogen suggests the integrin α2 is indispensable for spreading on collagen. The platelet collagen receptors GPVI and α2 regulate integrin αIIbβ3-mediated platelet spreading on fibrinogen. This work elucidates quantitatively how receptor-ligand adhesion and biochemical signals synergistically control platelet spreading. PMID:22325269

C. elegans STRADalpha and SAD cooperatively regulate neuronal polarity and synaptic organization.

PubMed

Kim, Joanne S M; Hung, Wesley; Narbonne, Patrick; Roy, Richard; Zhen, Mei

2010-01-01

Neurons are polarized cells with morphologically and functionally distinct axons and dendrites. The SAD kinases are crucial for establishing the axon-dendrite identity across species. Previous studies suggest that a tumour suppressor kinase, LKB1, in the presence of a pseudokinase, STRADalpha, initiates axonal differentiation and growth through activating the SAD kinases in vertebrate neurons. STRADalpha was implicated in the localization, stabilization and activation of LKB1 in various cell culture studies. Its in vivo functions, however, have not been examined. In our present study, we analyzed the neuronal phenotypes of the first loss-of-function mutants for STRADalpha and examined their genetic interactions with LKB1 and SAD in C. elegans. Unexpectedly, only the C. elegans STRADalpha, STRD-1, functions exclusively through the SAD kinase, SAD-1, to regulate neuronal polarity and synaptic organization. Moreover, STRD-1 tightly associates with SAD-1 to coordinate its synaptic localizations. By contrast, the C. elegans LKB1, PAR-4, also functions in an additional genetic pathway independently of SAD-1 and STRD-1 to regulate neuronal polarity. We propose that STRD-1 establishes neuronal polarity and organizes synaptic proteins in a complex with the SAD-1 kinase. Our findings suggest that instead of a single, linear genetic pathway, STRADalpha and LKB1 regulate neuronal development through multiple effectors that are shared in some cellular contexts but distinct in others.

Proteinase-activated receptor 2 (PAR-2) in gastrointestinal and pancreatic pathophysiology, inflammation and neoplasia.

PubMed

Søreide, Kjetil

2008-08-01

Of all the body systems, the gastrointestinal (GI) tract is the most exposed to proteinases. Proteolytic activity must thus be tightly regulated in the face of diverse environmental challenges, because unrestrained or excessive proteolysis leads to pathological GI conditions. The protease-activated receptor-2 (PAR-2) is expressed in numerous cell types within the GI tract, suggesting both multiple functions and numerous modes of receptor activation. Although best known as a pancreatic digestive enzyme, trypsin has also been found in other tissues and various cancers. Of interest, trypsin and PAR-2 act together in an autocrine loop that promotes proliferation, invasion and metastasis in neoplasia through various mechanisms. Trypsin and PAR-2 seem to act both directly and indirectly through activation of other proteinase cascades, including metalloproteinases. PAR-2 activation can participate in inflammatory reactions, be protective to mucosal surfaces, send or inhibit nociceptive messages, modify gut motility or secretory functions, and stimulate cell proliferation and motility. Several studies point to a role for the PARs in disease processes of the GI tract and pancreas ranging from inflammatory bowel disease, symptoms associated with irritable bowel syndrome, pain in pancreatitis, development of colon and other GI cancers, and even infectious colitis. Proteinases should not only be considered from the traditional view as digestive or degradative enzymes in the gut, but additionally as signalling molecules that actively participate in the spectrum of physiology and diseased states of the GI tract.

Late-onset Parkinsonism in NFκB/c-Rel-deficient mice

PubMed Central

Baiguera, Cristina; Alghisi, Manuela; Pinna, Annalisa; Bellucci, Arianna; De Luca, Maria Antonietta; Frau, Lucia; Morelli, Micaela; Ingrassia, Rosaria; Benarese, Marina; Porrini, Vanessa; Pellitteri, Michele; Bertini, Giuseppe; Fabene, Paolo Francesco; Sigala, Sandra; Spillantini, Maria Grazia; Liou, Hsiou-Chi; Spano, Pier Franco

2012-01-01

Activation of the nuclear factor κB/c-Rel can increase neuronal resilience to pathological noxae by regulating the expression of pro-survival manganese superoxide dismutase (MnSOD, now known as SOD2) and Bcl-xL genes. We show here that c-Rel-deficient (c-rel−/−) mice developed a Parkinson’s disease-like neuropathology with ageing. At 18 months of age, c-rel−/− mice exhibited a significant loss of dopaminergic neurons in the substantia nigra pars compacta, as assessed by tyrosine hydroxylase-immunoreactivity and Nissl staining. Nigral degeneration was accompanied by a significant loss of dopaminergic terminals and a significant reduction of dopamine and homovanillic acid levels in the striatum. Mice deficient of the c-Rel factor exhibited a marked immunoreactivity for fibrillary α-synuclein in the substantia nigra pars compacta as well as increased expression of divalent metal transporter 1 (DMT1) and iron staining in both the substantia nigra pars compacta and striatum. Aged c-rel−/− mouse brain were characterized by increased microglial reactivity in the basal ganglia, but no astrocytic reaction. In addition, c-rel−/− mice showed age-dependent deficits in locomotor and total activity and various gait-related deficits during a catwalk analysis that were reminiscent of bradykinesia and muscle rigidity. Both locomotor and gait-related deficits recovered in c-rel−/− mice treated with l-3,4-dihydroxyphenylalanine. These data suggest that c-Rel may act as a regulator of the substantia nigra pars compacta resilience to ageing and that aged c-rel−/− mice may be a suitable model of Parkinson’s disease. PMID:22915735

Thiol-redox signaling, dopaminergic cell death, and Parkinson's disease.

PubMed

Garcia-Garcia, Aracely; Zavala-Flores, Laura; Rodriguez-Rocha, Humberto; Franco, Rodrigo

2012-12-15

Parkinson's disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta, which has been widely associated with oxidative stress. However, the mechanisms by which redox signaling regulates cell death progression remain elusive. Early studies demonstrated that depletion of glutathione (GSH), the most abundant low-molecular-weight thiol and major antioxidant defense in cells, is one of the earliest biochemical events associated with PD, prompting researchers to determine the role of oxidative stress in dopaminergic cell death. Since then, the concept of oxidative stress has evolved into redox signaling, and its complexity is highlighted by the discovery of a variety of thiol-based redox-dependent processes regulating not only oxidative damage, but also the activation of a myriad of signaling/enzymatic mechanisms. GSH and GSH-based antioxidant systems are important regulators of neurodegeneration associated with PD. In addition, thiol-based redox systems, such as peroxiredoxins, thioredoxins, metallothioneins, methionine sulfoxide reductases, transcription factors, as well as oxidative modifications in protein thiols (cysteines), including cysteine hydroxylation, glutathionylation, and nitrosylation, have been demonstrated to regulate dopaminergic cell loss. In this review, we summarize major advances in the understanding of the role of thiol-redox signaling in dopaminergic cell death in experimental PD. Future research is still required to clearly understand how integrated thiol-redox signaling regulates the activation of the cell death machinery, and the knowledge generated should open new avenues for the design of novel therapeutic approaches against PD.

The relationship between levels of plasma-soluble urokinase plasminogen activator receptor (suPAR) and presence of migraine attack and aura.

PubMed

Yılmaz, Nigar; Yılmaz, Mustafa; Sirin, Burcu; Yılmaztekin, Sureyya; Kutlu, Gülnihal

2017-10-01

Migraine is one of the most common types of pain associated with sterile inflammatory conditions. Soluble urokinase plasminogen activator receptor (suPAR) is a potential novel inflammatory marker. We aim to determine the association between serum values of suPAR, procalcitonin, fibrinogen, and high-sensitivity C-reactive protein (hs-CRP) and migraine disease characteristics. The study involved a total of 60 migraine patients (33 patients in the interictal period, 27 patients in the attack period) and 30 healthy individuals. The serum values of suPAR were found to be significantly higher in migraine patients in the attack period than in migraine patients in the interictal period, and in healthy individuals (p < .01 for both). In addition, levels of suPAR were determined to be higher in migraine with aura patients than in migraine without aura patients. When we subdivided migraine patients according to frequency of attack (attacks/month), significant differences were found between the suPAR and procalcitonin levels (measured during the attack period) of those in the frequent-attack group (4-5 or more) versus those in the less frequent attack group (less than 4). Serum levels of procalcitonin were shown to be significantly higher in migraine patients during the attack period compared with migraine patients in the interictal period and in control subjects (p = .001 for both). Significant differences were found between plasma levels of fibrinogen in migraine patients versus control subjects (p < .01). No statistically significant difference was found between levels of hs-CRP in migraine patients versus the control group. These findings may show that presenting a high level of suPAR in migraine patients with attack and aura results to predisposition to occurring on the symptoms and that high levels of suPAR, procalcitonin and fibrinogen in patients with migraine result in neurogenic inflammation during migraine headaches.

Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

PubMed

Khedr, M E M S; Abdelmotelb, A M; Pender, S L F; Zhou, X; Walls, A F

2018-05-01

Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. We have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH 2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2. © 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.

Productive infection of HUVEC by HHV-8 is associated with changes compatible with angiogenic transformations.

PubMed

Foglieni, C; Scabini, S; Belloni, D; Broccolo, F; Lusso, P; Malnati, M S; Ferrero, E

2005-01-01

Kaposi's Sarcoma (KS) is an angioproliferative disease associated with human herpesvirus 8 (HHV-8) infection. We have characterized the morphologic and phenotypic modifications of HUVEC in a model of productive HHV-8 infection. HHV-8 replication was associated with ultra-structural changes, flattened soma and a loss of marginal folds and intercellular contacts, and morphologic features, spindle cell conversion and cordon-like structures formation. Phenotypic changes observed on cordon-like structures included partial loss and redistribution of CD31/PECAM-1 and VE-cadherin, uPAR up-regulation and de novo expression of CD13/APN. Such changes demonstrate the induction, in HUVEC, of an angiogenic profile. Most of these findings are directly linked to HHV-8-encoded proteins expression, suggesting that HHV-8 itself may participate to the initial steps of the angiogenic transformation in KS.

Feeding a higher plane of nutrition and providing exogenous estrogen increases mammary gland development in Holstein heifer calves.

PubMed

Geiger, A J; Parsons, C L M; Akers, R M

2016-09-01

Feeding heifers a higher plane of nutrition postweaning but before puberty can negatively affect mammary gland development and future milk yield. However, enhanced nutrition preweaning may promote development and future production. Our objectives were to determine the effects of enhanced feeding preweaning and exogenous estrogen immediately postweaning on mammary gland development and the composition of the mammary parenchyma (PAR) and mammary fat pad (MFP). Thirty-six Holstein heifer calves (<1 wk old) were reared on 1 of 2 dietary treatments for 8 wk: (1) a restricted milk replacer fed at 0.45 kg/d (R; 20% crude protein, 20% fat), or (2) an enhanced milk replacer fed at 1.13 kg/d (EH; 28% crude protein, 25% fat). Upon weaning, calves from each diet (n=6) were given either a placebo or estrogen implant for 2 wk, creating 4 treatments: R, R + estrogen (R-E2), EH, and EH + estrogen (EH-E2). Calves were housed individually with ad libitum access to water. Starter feeding began at wk 5 and was balanced between treatments. Udders were evaluated by palpation and physical measurements weekly. Subsets of calves were killed at weaning (n=6 per diet) and at the conclusion of the trial (n=6 per treatment). Udders were removed, dissected, and weighed. At wk 8, EH calves had longer front and rear teats. Providing estrogen to EH calves increased the length of rear teats during wk 9 and 10. Enhanced-fed calves had 5.2-fold more trimmed mammary gland mass than R calves. Providing estrogen to EH calves further increased mammary gland weight. Masses of PAR and MFP were markedly greater for EH calves than for R calves (e.g., 7.3-fold greater PAR tissue). Estrogen increased the mass of both PAR and MFP in EH calves. Feeding a higher plane of nutrition increased total protein, DNA, and fat in the MFP and total protein and DNA in the PAR. Dual-energy x-ray absorptiometry estimates of mammary fat mass were highly correlated with biochemical analyses of fat content. From histological study, we observed that the degree of expansion of epithelium into the adjacent stromal tissue and the complexity of ductal development were minimal in R, increased in EH, and increased by estrogen in both dietary treatments. Results provide compelling evidence that preweaning nutrition and estrogen administration immediately postweaning markedly increase mammary gland development in dairy calves. Cellular and molecular mechanisms responsible for these differences are currently under study. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

[Atp6ap2/ (Pro) renin Receptor is Required for Laminar Formation during Retinal Development in Mice].

PubMed

Kanda, Atsuhiro

2015-11-01

(Pro) renin receptor [(P) RR], a key molecule for tissue renin-angiotensin system, was originally identified as Atp6ap2, an accessory subunit for vacuolar H(+)-ATPase that is a multi-subunit proton pump involved in fundamental cellular physiology. In this study, to elucidate the physiological functions of Atp6ap2/ (P) RR during retinal development in mammals, we used Cre-LoxP system to generate photoreceptor-specific conditional knock-out (CKO) mice, and revealed a critical role of Atp6ap2/(P) RR in photoreceptor development. Deletion of photoreceptor Atp6ap2/ (P) RR did not affect retinal cell differentiation, but led to laminar disorganization in the photoreceptor layer with dysfunction of photoreceptors. Cell adhesion and polarity molecules, all of which were co-localized with Atp6ap2 at the apical edge of the developing retina, were dispersed together with mislocalization of retinal progenitors apart from the apical surface in Atp6ap2 conditional knockout mice. Among these molecules, co-immunoprecipitation using retinal homogenates and Atp6ap2/(P) RR-transfected cells showed that Atp6ap2/(P) RR interacted with partitioning defective 3 homolog (Par3) protein, known to play a pivotal role in planar cell polarity in the Par-atypical protein kinase C system. Atp6ap2 interacted with Par3 protein that plays a pivotal role in planar cell polarity. Our data provide a novel function of Atp6ap2 required as a cell polarity determinant for retinal laminar formation.

Elevated fecal peptidase D at onset of colitis in Galphai2-/- mice, a mouse model of IBD.

PubMed

Bergemalm, Daniel; Kruse, Robert; Sapnara, Maria; Halfvarson, Jonas; Hörnquist, Elisabeth Hultgren

2017-01-01

The identification of novel fecal biomarkers in inflammatory bowel disease (IBD) is hampered by the complexity of the human fecal proteome. On the other hand, in experimental mouse models there is probably less variation. We investigated the fecal protein content in mice to identify possible biomarkers and pathogenic mechanisms. Fecal samples were collected at onset of inflammation in Galphai2-/- mice, a well-described spontaneous model of chronic colitis, and from healthy littermates. The fecal proteome was analyzed by two-dimensional electrophoresis and quantitative mass spectrometry and results were then validated in a new cohort of mice. As a potential top marker of disease, peptidase D was found at a higher ratio in Galphai2-/- mouse feces relative to controls (fold change 27; p = 0.019). Other proteins found to be enriched in Gαi2-/- mice were mainly pancreatic proteases, and proteins from plasma and blood cells. A tendency of increased calprotectin, subunit S100-A8, was also observed (fold change 21; p = 0.058). Proteases are potential activators of inflammation in the gastrointestinal tract through their interaction with the proteinase-activated receptor 2 (PAR2). Accordingly, the level of PAR2 was found to be elevated in both the colon and the pancreas of Galphai2-/- mice at different stages of disease. These findings identify peptidase D, an ubiquitously expressed intracellular peptidase, as a potential novel marker of colitis. The elevated levels of fecal proteases may be involved in the pathogenesis of colitis and contribute to the clinical phenotype, possibly by activation of intestinal PAR2.

Cadmium exposure is associated with soluble urokinase plasminogen activator receptor, a circulating marker of inflammation and future cardiovascular disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fagerberg, Björn, E-mail: bjorn.fagerberg@wlab.gu.

Background: Diet and smoking are the main sources of cadmium exposure in the general population. Cadmium increases the risk of cardiovascular diseases, and experimental studies show that it induces inflammation. Blood cadmium levels are associated with macrophages in human atherosclerotic plaques. Soluble urokinase-type plasminogen activator receptor (suPAR) is an emerging biomarker for cardiovascular events related to inflammation and atherosclerotic plaques. The aim was to examine whether blood cadmium levels are associated with circulating suPAR and other markers of inflammation. Methods: A population sample of 4648 Swedish middle-aged women and men was examined cross-sectionally in 1991–1994. Plasma suPAR was assessed bymore » ELISA, leukocytes were measured by standard methods, and blood cadmium was analysed by inductively coupled plasma mass spectrometry. Prevalent cardiovascular disease, ultrasound-assessed carotid plaque occurrence, and several possible confounding factors were recorded. Results: After full adjustment for risk factors and confounding variables, a 3-fold increase in blood cadmium was associated with an 10.9% increase in suPAR concentration (p<0.001). In never-smokers, a 3-fold increase in blood cadmium was associated with a 3.7% increase in suPAR concentration (p<0.01) after full adjustment. Blood cadmium was not associated with C-reactive protein, white blood cell count and Lp-PLA2 but with neutrophil/lymphocyte ratio in one of two statistical models. Conclusions: Exposure to cadmium was associated with increased plasma suPAR in the general population, independently of smoking and cardiovascular disease. These results imply that cadmium is a possible cause for raised levels of this inflammatory marker. - Highlights: • Cadmium is a toxic proinflammatory, proatherosclerotic metal. • Soluble urokinase-type plasminogen activator receptor (suPAR) in plasma is a promising proinflammatory marker of atherosclerosis. • Blood cadmium and plasma suPAR were measured in a cohort of 4648 Swedish men and women. • Blood cadmium was positively associated with plasma suPAR, also in never smokers.« less

MicroRNA-146 inhibits thrombin-induced NF-κB activation and subsequent inflammatory responses in human retinal endothelial cells.

PubMed

Cowan, Colleen; Muraleedharan, Chithra K; O'Donnell, James J; Singh, Pawan K; Lum, Hazel; Kumar, Ashok; Xu, Shunbin

2014-07-01

Nuclear factor-κB (NF-κB), a key regulator of immune and inflammatory responses, plays important roles in diabetes-induced microvascular complications including diabetic retinopathy (DR). Thrombin activates NF-κB through protease-activated receptor (PAR)-1, a member of the G-protein-coupled receptor (GPCR) superfamily, and contributes to DR. The current study is to uncover the roles of microRNA (miRNA) in thrombin-induced NF-κB activation and retinal endothelial functions. Target prediction was performed using the TargetScan algorithm. Predicted target was experimentally validated by luciferase reporter assays. Human retinal endothelial cells (HRECs) were transfected with miRNA mimics or antimiRs and treated with thrombin. Expression levels of miR-146 and related protein-coding genes were analyzed by quantitative (q)RT-PCR. Functional changes of HRECs were analyzed by leukocyte adhesion assays. We identified that caspase-recruitment domain (CARD)-containing protein 10 (CARD10), an essential scaffold/adaptor protein of GPCR-mediated NF-κB activation pathway, is a direct target of miR-146. Thrombin treatment resulted in NF-κB-dependent upregulation of miR-146 in HRECs; while transfection of miR-146 mimics resulted in significant downregulation of CARD10 and prevented thrombin-induced NF-κB activation, suggest that a negative feedback regulation of miR-146 on thrombin-induced NF-κB through targeting CARD10. Furthermore, overexpression of miR-146 prevented thrombin-induced increased leukocyte adhesion to HRECs. We uncovered a novel negative feedback regulatory mechanism on thrombin-induced GPCR-mediated NF-κB activation by miR-146. In combination with the negative feedback regulation of miR-146 on the IL-1R/toll-like receptor (TLR)-mediated NF-κB activation in RECs that we reported previously, our results underscore a pivotal, negative regulatory role of miR-146 on multiple NF-κB activation pathways and related inflammatory processes in DR. Copyright 2014 The Association for Research in Vision and Ophthalmology, Inc.

Immunoglobulin A with protease activity secreted in human milk activates PAR-2 receptors, of intestinal epithelial cells HT-29, and promotes beta-defensin-2 expression.

PubMed

Barrera, G J; Portillo, R; Mijares, A; Rocafull, M A; del Castillo, J R; Thomas, L E

2009-03-24

Secretory antibodies of the immunoglobulin A (sIgA) class constitute the first line of antigen-specific immune protection against pathogens and other antigens at mucosal surfaces. Although initially perceived as potentially deleterious, catalytic antibodies have been proposed to participate in the removal of metabolic wastes and in protection against infection. Here we show that the presence of sIgA endowed with serine protease-like hydrolytic activity in milk strongly correlates with PAR-2 activation in human intestinal epithelial cells. F(ab')(2) fragments of sIgA activated the epithelial cells in culture to produce beta-defensin-2 (hBD2). Intracellular Ca(2+) mobilization was induced by treatment with (1) sIgA-F(ab')(2) fragments; (2) trypsin, a recognized PAR-2 agonist; or (3) a synthetic PAR-2 agonist peptide (SLIGKV). The co-treatment with a synthetic PAR-2 antagonist peptide (FSLLRY) and sIgA-F(ab')(2) fragments eliminates the latter's effect; nevertheless, cells were not refractory to subsequent stimulation with sIgA-F(ab')(2) fragments. Both the induction of hBD-2 expression in epithelial cells and the increase in intracellular [Ca(2+)] stimulated by sIgA-F(ab')(2) fragments were inhibited by treatment with serine protease inhibitors or pertussis toxin (PTX). These findings suggest that catalytic antibodies can activate intestinal epithelial cells through G-protein-coupled PAR-2, and could actively participate in the immune system of breastfed babies inducing the production of peptides related to innate defense, such as defensins.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy

NASA Astrophysics Data System (ADS)

Hou, Guangjin; Lu, Xingyu; Vega, Alexander J.; Polenova, Tatyana

2014-09-01

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear 1H-X (X = 13C, 15N, 31P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the 1H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the 1H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from 1H chemical shift anisotropy, while keeping the 1H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [15N]-N-acetyl-valine and [U-13C,15N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate 1H-15N dipolar couplings in the context of 3D experiments is presented on U-13C,15N-enriched dynein light chain protein LC8.

p38α phosphorylates serine 258 within the cytoplasmic domain of tissue factor and prevents its incorporation into cell-derived microparticles.

PubMed

Ettelaie, Camille; Elkeeb, Azza M; Maraveyas, Anthony; Collier, Mary Elizabeth W

2013-03-01

We previously showed that the phosphorylation of Ser253 within the cytoplasmic domain of human tissue factor (TF) initiates the incorporation and release of this protein into cell-derived microparticles. Furthermore, subsequent phosphorylation of Ser258 terminates this process. However, the identity of the kinase responsible for the phosphorylation of Ser258 and mode of action of this enzyme remain unknown. In this study, p38α was identified as the proline-directed kinase capable of phosphorylating Ser258 specifically, and without any detectable activity towards Ser253. Furthermore, using synthetic peptides, it was shown that the Km for the reaction decreased by approximately 10 fold on substitution of Ser253 with phospho-Ser253. Either inhibition of p38 using SB202190 or knockdown of p38α expression in coronary artery endothelial cells overexpressing wild-type TF, resulted in decreased phosphorylation of Ser258, following activation of cells with PAR2-agonist peptide (PAR2-AP). In agreement with our previous data, inhibition of phosphorylation of this residue maintained the release of TF. Activation of PAR2 in cells transfected to overexpress TF, resulted in two separate peaks of p38 activity at approximately 40 and 120 min post-activation. Furthermore, overexpression of Ala253-substituted TF enhanced the second p38 activation peak. However, the second peak was absent in cells devoid of TF or in cells overexpressing the Asp253-substituted TF. Our data clearly identifies p38α as a kinase capable of phosphorylating Ser258 within the cytoplasmic domain of TF. Moreover, it appears that the presence of TF within the cells regulates the late activation of p38 and consequently the termination of TF release into microparticles. Copyright © 2012 Elsevier B.V. All rights reserved.

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei.

PubMed

Schlesinger, Mariana; Vilchez Larrea, Salomé C; Haikarainen, Teemu; Narwal, Mohit; Venkannagari, Harikanth; Flawiá, Mirtha M; Lehtiö, Lari; Fernández Villamil, Silvia H

2016-03-23

Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H2O2, was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H2O2 treatment when compared to their wild type counterparts. PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems.

Interactions of Kid-Kis toxin-antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid-Kis oligomers.

PubMed

Monti, Maria C; Hernández-Arriaga, Ana M; Kamphuis, Monique B; López-Villarejo, Juan; Heck, Albert J R; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H H

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin-antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid-kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid-Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2-Kis2-Kid2-Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2-Kis2-Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2-Kis2)n complexes repress the kid-kis operon.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling.

PubMed

Bai, Zhiyong; Grant, Barth D

2015-03-24

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1-positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42-associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling.

A TOCA/CDC-42/PAR/WAVE functional module required for retrograde endocytic recycling

PubMed Central

Bai, Zhiyong; Grant, Barth D.

2015-01-01

Endosome-to-Golgi transport is required for the function of many key membrane proteins and lipids, including signaling receptors, small-molecule transporters, and adhesion proteins. The retromer complex is well-known for its role in cargo sorting and vesicle budding from early endosomes, in most cases leading to cargo fusion with the trans-Golgi network (TGN). Transport from recycling endosomes to the TGN has also been reported, but much less is understood about the molecules that mediate this transport step. Here we provide evidence that the F-BAR domain proteins TOCA-1 and TOCA-2 (Transducer of Cdc42 dependent actin assembly), the small GTPase CDC-42 (Cell division control protein 42), associated polarity proteins PAR-6 (Partitioning defective 6) and PKC-3/atypical protein kinase C, and the WAVE actin nucleation complex mediate the transport of MIG-14/Wls and TGN-38/TGN38 cargo proteins from the recycling endosome to the TGN in Caenorhabditis elegans. Our results indicate that CDC-42, the TOCA proteins, and the WAVE component WVE-1 are enriched on RME-1–positive recycling endosomes in the intestine, unlike retromer components that act on early endosomes. Furthermore, we find that retrograde cargo TGN-38 is trapped in early endosomes after depletion of SNX-3 (a retromer component) but is mainly trapped in recycling endosomes after depletion of CDC-42, indicating that the CDC-42–associated complex functions after retromer in a distinct organelle. Thus, we identify a group of interacting proteins that mediate retrograde recycling, and link these proteins to a poorly understood trafficking step, recycling endosome-to-Golgi transport. We also provide evidence for the physiological importance of this pathway in WNT signaling. PMID:25775511

Immunohistochemistry of connexin 43 throughout anterior pituitary gland in a transgenic rat with green fluorescent protein-expressing folliculo-stellate cells.

PubMed

Horiguchi, Kotaro; Fujiwara, Ken; Kouki, Tom; Kikuchi, Motoshi; Yashiro, Takashi

2008-12-01

Folliculo-stellate (FS) cells in the anterior pituitary gland have been speculated to possess multifunctional properties. Because gap junctions (GJ) have been identified between FS cells, FS cells may be interconnected electrophysiologically by GJ and serve as signal transmission networks to modulate hormone release in the anterior pituitary gland. But whether GJ are localized among FS cells from the pars tuberalis through the pars distalis is unclear. The S100b-GFP transgenic rat has recently been generated, which expresses green fluorescent protein (GFP) specifically in FS cells in the anterior pituitary. This model is expected to be a powerful tool for studies of FS cells. The purpose of the present paper was therefore to examine the localization of GJ on connexin 43 immunohistochemistry throughout the anterior pituitary gland of S100b-GFP rats under confocal laser microscopy. The localization patterns of FS cells was also observed in primary culture of anterior pituitary cells and the question of whether GJ between FS cells are reconstructed in vitro was investigated. In vivo studies showed that GJ were present specifically between FS cells from the pars tuberalis to the pars distalis in the anterior pituitary gland. The appearance of FS cells was distinguished into two types, with localization of GJ differing between types. In vitro, it was observed for the first time that FS cells in primary culture could be categorized into two types. In vivo localization of GJ between FS cells was reconstructed in vitro. These morphological observations are consistent with the hypothesis that FS cells form an electrophysiological network throughout the anterior pituitary for signal transmission.

The Ras-related protein, Rap1A, mediates thrombin-stimulated, integrin-dependent glioblastoma cell proliferation and tumor growth.

PubMed

Sayyah, Jacqueline; Bartakova, Alena; Nogal, Nekeisha; Quilliam, Lawrence A; Stupack, Dwayne G; Brown, Joan Heller

2014-06-20

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1 integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1 integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1 integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Metabolic learning and memory formation by the brain influence systemic metabolic homeostasis.

PubMed

Zhang, Yumin; Liu, Gang; Yan, Jingqi; Zhang, Yalin; Li, Bo; Cai, Dongsheng

2015-04-07

Metabolic homeostasis is regulated by the brain, but whether this regulation involves learning and memory of metabolic information remains unexplored. Here we use a calorie-based, taste-independent learning/memory paradigm to show that Drosophila form metabolic memories that help in balancing food choice with caloric intake; however, this metabolic learning or memory is lost under chronic high-calorie feeding. We show that loss of individual learning/memory-regulating genes causes a metabolic learning defect, leading to elevated trehalose and lipid levels. Importantly, this function of metabolic learning requires not only the mushroom body but also the hypothalamus-like pars intercerebralis, while NF-κB activation in the pars intercerebralis mimics chronic overnutrition in that it causes metabolic learning impairment and disorders. Finally, we evaluate this concept of metabolic learning/memory in mice, suggesting that the hypothalamus is involved in a form of nutritional learning and memory, which is critical for determining resistance or susceptibility to obesity. In conclusion, our data indicate that the brain, and potentially the hypothalamus, direct metabolic learning and the formation of memories, which contribute to the control of systemic metabolic homeostasis.

Baicalin protects against thrombin induced cell injury in SH-SY5Y cells

PubMed Central

Ju, Xiao-Ning; Mu, Wei-Na; Liu, Yuan-Tao; Wang, Mei-Hong; Kong, Feng; Sun, Chao; Zhou, Qing-Bo

2015-01-01

Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. PMID:26823714

The Allergen Der p3 from House Dust Mite Stimulates Store-Operated Ca2+ Channels and Mast Cell Migration through PAR4 Receptors.

PubMed

Lin, Yu-Ping; Nelson, Charmaine; Kramer, Holger; Parekh, Anant B

2018-04-19

The house dust mite is the principal source of perennial aeroallergens in man. How these allergens activate innate and adaptive immunity is unclear, and therefore, there are no therapies targeting mite allergens. Here, we show that house dust mite extract activates store-operated Ca 2+ channels, a common signaling module in numerous cell types in the lung. Activation of channel pore-forming Orai1 subunits by mite extract requires gating by STIM1 proteins. Although mite extract stimulates both protease-activated receptor type 2 (PAR2) and PAR4 receptors, Ca 2+ influx is more tightly coupled to the PAR4 pathway. We identify a major role for the serine protease allergen Der p3 in stimulating Orai1 channels and show that a therapy involving sub-maximal inhibition of both Der p3 and Orai1 channels suppresses mast cell activation to house dust mite. Our results reveal Der p3 as an important aeroallergen that activates Ca 2+ channels and suggest a therapeutic strategy for treating mite-induced asthma. Copyright © 2018 Elsevier Inc. All rights reserved.

Enhancement of cutaneous wound healing by Dsg2 augmentation of uPAR secretion.

PubMed

Cooper, Felicia; Overmiller, Andrew M; Loder, Anthony; Brennan-Crispi, Donna M; McGuinn, Kathleen P; Marous, Molly R; Freeman, Theresa A; Riobo-Del Galdo, Natalia A; Siracusa, Linda D; Wahl, James K; Mahoney, Mỹ G

2018-05-09

In addition to playing a role in adhesion, desmoglein 2 (Dsg2) is an important regulator of growth and survival signaling pathways, cell proliferation, migration and invasion, and oncogenesis. While low-level Dsg2 expression is observed in basal keratinocytes and is downregulated in non-healing venous ulcers, overexpression has been observed in both melanomas and non-melanoma malignancies. Here, we show that transgenic mice overexpressing Dsg2 in basal keratinocytes primed the activation of mitogenic pathways, but did not induce dramatic epidermal changes or susceptibility to chemical-induced tumor development. Interestingly, acceleration of full-thickness wound closure and increased wound-adjacent keratinocyte proliferation was observed in these mice. As epidermal cytokines and their receptors play critical roles in wound healing, Dsg2-induced secretome alterations were assessed with an antibody profiler array and revealed increased release and proteolytic processing of the urokinase-type plasminogen activator receptor (uPAR). Dsg2 induced uPAR expression in the skin of transgenic compared to wild-type mice. Wound healing further enhanced uPAR in both epidermis and dermis with concomitant increase in the pro-healing laminin-332, a major component of the basement membrane zone, in transgenic mice. This study demonstrates that Dsg2 induces epidermal activation of various signaling cascades and accelerates cutaneous wound healing, in part, through uPAR-related signaling cascades. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

TRPV3 Channel in Keratinocytes in Scars with Post-Burn Pruritus

PubMed Central

Park, Chun Wook; Kim, Hyun Ji; Choi, Yong Won; Chung, Bo Young; Woo, So-Youn; Song, Dong-Keun; Kim, Hye One

2017-01-01

Post-burn pruritus is a common and distressing sequela of burn scars. Empirical antipruritic treatments usually fail to have a satisfactory outcome because of their limited selectivity and possible side effects. Therefore, novel drug targets need to be identified. Here, we aimed to investigate the possible role of protease-activated receptor 2 (PAR2) and transient receptor potential vanniloid 3 (TRPV3), along with the relation of TRPV3 to thymic stromal lymphopoietin (TSLP). Specimens from normal (unscarred) or burn-scarred (with or without pruritus) tissue were obtained from burn patients for this study. In each sample, the keratinocytes were isolated and cultured, and the intracellular Ca2+ level at the time of stimulation of each factor was quantified and the interaction was screened. PAR2 function was reduced by antagonism of TRPV3. Inhibiting protein kinase A (PKA) and protein kinase C (PKC) reduced TRPV3 function. TSLP mRNA and protein, and TSLPR protein expressions, increased in scars with post-burn pruritus, compared to scars without it or to normal tissues. In addition, TRPV1 or TRPV3 activation induced increased TSLP expression. Conclusively, TRPV3 may contribute to pruritus in burn scars through TSLP, and can be considered a potential therapeutic target for post-burn pruritus. PMID:29140280

Dopaminergic Neuron-Specific Deletion of p53 Gene Attenuates Methamphetamine Neurotoxicity.

PubMed

Lu, Tao; Kim, Paul P; Greig, Nigel H; Luo, Yu

2017-08-01

p53 plays an essential role in the regulation of cell death in dopaminergic (DA) neurons and its activation has been implicated in the neurotoxic effects of methamphetamine (MA). However, how p53 mediates MA neurotoxicity remains largely unknown. In this study, we examined the effect of DA-specific p53 gene deletion in DAT-p53KO mice. Whereas in vivo MA binge exposure reduced locomotor activity in wild-type (WT) mice, this was significantly attenuated in DAT-p53KO mice and associated with significant differences in the levels of the p53 target genes BAX and p21 between WT and DAT-p53KO. Notably, DA-specific deletion of p53 provided protection of substantia nigra pars reticulata (SNpr) tyrosine hydroxylase (TH) positive fibers following binge MA, with DAT-p53KO mice having less decline of TH protein levels in striatum versus WT mice. Whereas DAT-p53KO mice demonstrated a consistently higher density of TH fibers in striatum compared to WT mice at 10 days after MA exposure, DA neuron counts within the substantia nigra pars compacta (SNpc) were similar. Finally, supportive of these results, administration of a p53-specific inhibitor (PFT-α) provided a similarly protective effect on MA binge-induced behavioral deficits. Neither DA specific p53 deletion nor p53 pharmacological inhibition affected hyperthermia induced by MA binge. These findings demonstrate a specific contribution of p53 activation in behavioral deficits and DA neuronal terminal loss by MA binge exposure.

Bufo arenarum egg jelly coat: purification and characterization of two highly glycosylated proteins.

PubMed Central

Arranz, S E; Albertali, I E; Cabada, M O

1997-01-01

Egg jelly coats from Bufo arenarum are formed by components secreted along the oviduct. These secretion products overlay the oocytes as they transit along the different oviductal portions. In this study, we have isolated two highly glycosylated proteins of the jelly coat, which are secreted almost all the way along the oviduct. Both glycoproteins [designated as highly glycosylated protein (HGP) and low-molecular-mass highly glycosylated protein (L-HGP)] were purified to homogeneity, from the secretion of the caudal oviduct portion, by CsCl density gradient ultracentrifugation. HGP is a high-molecular-mass protein with mucin-like characteristics: high viscosity, a high content of serine and threonine, about 70% carbohydrate by weight, and a protease-resistant domain. Cleavage of disulphide bridges with reducing agents resulted in the release of a single subunit (300000 Da). L-HGP is also a disulphide-cross-linked protein with lower apparent monomeric molecular mass, in the range 100-120 kDa and containing 50% carbohydrate by weight. HGP contains galactose, fucose, N-acetylgalactosamine and sialic acid, but no mannose, suggesting the presence of O-linked oligosaccharides exclusively. The secretion ratio of HGP increases from cephalic (16% of total protein in pars preconvoluta) to caudal (40% of total protein in pars convoluta) oviductal portions. It appears to be the major structural component of the jelly coat. Our purification data suggest that HGP is non-covalently linked to the other egg jelly proteins. Polyclonal antiserum to each purified glycoprotein from secretion was raised in rabbits and used to localize both glycoproteins in the different oviductal portions, total egg jelly and the aqueous medium where oocyte strings were incubated. HGP forms a stable fibre matrix around the oocyte. L-HGP is present in the jelly coat and is released into the incubation medium. PMID:9173897

Multiple PAR and E4BP4 bZIP transcription factors in zebrafish: diverse spatial and temporal expression patterns.

PubMed

Ben-Moshe, Zohar; Vatine, Gad; Alon, Shahar; Tovin, Adi; Mracek, Philipp; Foulkes, Nicholas S; Gothilf, Yoav

2010-09-01

Circadian rhythms of physiology and behavior are generated by an autonomous circadian oscillator that is synchronized daily with the environment, mainly by light input. The PAR subfamily of transcriptional activators and the related E4BP4 repressor belonging to the basic leucine zipper (bZIP) family are clock-controlled genes that are suggested to mediate downstream circadian clock processes and to feedback onto the core oscillator. Here, the authors report the characterization of these genes in the zebrafish, an increasingly important model in the field of chronobiology. Five novel PAR and six novel e4bp4 zebrafish homolog genes were identified using bioinformatic tools and their coding sequences were cloned. Based on their evolutionary relationships, these genes were annotated as ztef2, zhlf1 and zhlf2, zdbp1 and zdbp2, and ze4bp4-1 to -6. The spatial and temporal mRNA expression pattern of each of these factors was characterized in zebrafish embryos in the context of a functional circadian clock and regulation by light. Nine of the factors exhibited augmented and rhythmic expression in the pineal gland, a central clock organ in zebrafish. Moreover, these genes were found to be regulated, to variable extents, by the circadian clock and/or by light. Differential expression patterns of multiple paralogs in zebrafish suggest multiple roles for these factors within the vertebrate circadian clock. This study, in the genetically accessible zebrafish model, lays the foundation for further research regarding the involvement and specific roles of PAR and E4BP4 transcription factors in the vertebrate circadian clock mechanism.

Sex differences in associations of arginine vasopressin and oxytocin with resting-state functional brain connectivity.

PubMed

Rubin, Leah H; Yao, Li; Keedy, Sarah K; Reilly, James L; Bishop, Jeffrey R; Carter, C Sue; Pournajafi-Nazarloo, Hossein; Drogos, Lauren L; Tamminga, Carol A; Pearlson, Godfrey D; Keshavan, Matcheri S; Clementz, Brett A; Hill, Scot K; Liao, Wei; Ji, Gong-Jun; Lui, Su; Sweeney, John A

2017-01-02

Oxytocin (OT) and arginine vasopressin (AVP) exert robust and sexually dimorphic influences on cognition and emotion. How these hormones regulate relevant functional brain systems is not well understood. OT and AVP serum concentrations were assayed in 60 healthy individuals (36 women). Brain functional networks assessed with resting-state functional magnetic resonance imaging (rs-fMRI) were constructed with graph theory-based approaches that characterize brain networks as connected nodes. Sex differences were demonstrated in rs-fMRI. Men showed higher nodal degree (connectedness) and efficiency (information propagation capacity) in left inferior frontal gyrus (IFG) and bilateral superior temporal gyrus (STG) and higher nodal degree in left rolandic operculum. Women showed higher nodal betweenness (being part of paths between nodes) in right putamen and left inferior parietal gyrus (IPG). Higher hormone levels were associated with less intrinsic connectivity. In men, higher AVP was associated with lower nodal degree and efficiency in left IFG (pars orbitalis) and left STG and less efficiency in left IFG (pars triangularis). In women, higher AVP was associated with lower betweenness in left IPG, and higher OT was associated with lower nodal degree in left IFG (pars orbitalis). Hormones differentially correlate with brain networks that are important for emotion processing and cognition in men and women. AVP in men and OT in women may regulate orbital frontal cortex connectivity, which is important in emotion processing. Hormone associations with STG and pars triangularis in men and parietal cortex in women may account for well-established sex differences in verbal and visuospatial abilities, respectively. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Improved antimicrobial activity of DU-6859a, a new fluoroquinolone, against quinolone-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates with alterations in GyrA and ParC proteins.

PubMed Central

Deguchi, T; Yasuda, M; KawamuraT; Nakano, M; Ozeki, S; Kanematsu, E; Nishino, Y; Kawada, Y

1997-01-01

MICs of DU-6859a, a novel fluoroquinolone, for 18 Klebsiella pneumoniae isolates and 21 Enterobacter cloacae isolates with altered GyrA or altered GyrA and ParC ranged from < or =0.025 to 6.25 microg/ml and from 0.1 to 3.13 microg/ml, respectively. Based on the MICs at which 90% of the isolates were inhibited for these strains of K. pneumoniae and E. cloacae, DU-6859a exhibited 16- to 256-fold-greater activity than currently available fluoroquinolones. PMID:9371364

Improved antimicrobial activity of DU-6859a, a new fluoroquinolone, against quinolone-resistant Klebsiella pneumoniae and Enterobacter cloacae isolates with alterations in GyrA and ParC proteins.

PubMed

Deguchi, T; Yasuda, M; KawamuraT; Nakano, M; Ozeki, S; Kanematsu, E; Nishino, Y; Kawada, Y

1997-11-01

MICs of DU-6859a, a novel fluoroquinolone, for 18 Klebsiella pneumoniae isolates and 21 Enterobacter cloacae isolates with altered GyrA or altered GyrA and ParC ranged from < or =0.025 to 6.25 microg/ml and from 0.1 to 3.13 microg/ml, respectively. Based on the MICs at which 90% of the isolates were inhibited for these strains of K. pneumoniae and E. cloacae, DU-6859a exhibited 16- to 256-fold-greater activity than currently available fluoroquinolones.

Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

PubMed

Uno, Tomohide; Furutani, Masayuki; Sakamoto, Katsuhiko; Uno, Yuichi; Kanamaru, Kengo; Mizoguchi, Akira; Hiragaki, Susumu; Takeda, Makio

2017-09-01

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori. © 2017 Wiley Periodicals, Inc.

Different mechanisms are involved in the antibody mediated inhibition of ligand binding to the urokinase receptor: a study based on biosensor technology.

PubMed

List, K; Høyer-Hansen, G; Rønne, E; Danø, K; Behrendt, N

1999-01-01

Certain monoclonal antibodies are capable of inhibiting the biological binding reactions of their target proteins. At the molecular level, this type of effect may be brought about by completely different mechanisms, such as competition for common binding determinants, steric hindrance or interference with conformational properties of the receptor critical for ligand binding. This distinction is central when employing the antibodies as tools in the elucidation of the structure-function relationship of the protein in question. We have studied the effect of monoclonal antibodies against the urokinase plasminogen activator receptor (uPAR), a protein located on the surface of various types of malignant and normal cells which is involved in the direction of proteolytic degradation reactions in the extracellular matrix. We show that surface plasmon resonance/biomolecular interaction analysis (BIA) can be employed as a highly useful tool to characterize the inhibitory mechanism of specific antagonist antibodies. Two inhibitory antibodies against uPAR, mAb R3 and mAb R5, were shown to exhibit competitive and non-competitive inhibition, respectively, of ligand binding to the receptor. The former antibody efficiently blocked the receptor against subsequent ligand binding but was unable to promote the dissociation of a preformed receptor-ligand complex. The latter antibody was capable of binding the preformed complex, forming a transient trimolecular assembly, and promoting the dissociation of the uPA/uPAR complex. The continuous recording of binding and dissociation, obtained in BIA, is central in characterizing these phenomena. The identification of a non-competitive inhibitory mechanism against this receptor reveals the presence of a determinant which influences the binding properties of a remote site in the molecular structure and which could be an important target for a putative synthetic antagonist.

Proteases in agricultural dust induce lung inflammation through PAR-1 and PAR-2 activation.

PubMed

Romberger, Debra J; Heires, Art J; Nordgren, Tara M; Souder, Chelsea P; West, William; Liu, Xiang-de; Poole, Jill A; Toews, Myron L; Wyatt, Todd A

2015-08-15

Workers exposed to aerosolized dust present in concentrated animal feeding operations (CAFOs) are susceptible to inflammatory lung diseases, such as chronic obstructive pulmonary disease. Extracts of dust collected from hog CAFOs [hog dust extract (HDE)] are potent stimulators of lung inflammatory responses in several model systems. The observation that HDE contains active proteases prompted the present study, which evaluated the role of CAFO dust proteases in lung inflammatory processes and tested whether protease-activated receptors (PARs) are involved in the signaling pathway for these events. We hypothesized that the damaging proinflammatory effect of HDE is due, in part, to the proteolytic activation of PARs, and inhibiting the proteases in HDE or disrupting PAR activation would attenuate HDE-mediated inflammatory indexes in bronchial epithelial cells (BECs), in mouse lung slices in vitro, and in a murine in vivo exposure model. Human BECs and mouse lung slice cultures stimulated with 5% HDE released significantly more of each of the cytokines measured (IL-6, IL-8, TNF-α, keratinocyte-derived chemokine/CXC chemokine ligand 1, and macrophage inflammatory protein-2/CXC chemokine ligand 2) than controls, and these effects were markedly diminished by protease inhibition. Inhibition of PARs also blunted the HDE-induced cytokine release from BECs. In addition, protease depletion inhibited HDE-induced BEC intracellular PKCα and PKCε activation. C57BL/6J mice administered 12.5% HDE intranasally, either once or daily for 3 wk, exhibited increased total cellular and neutrophil influx, bronchial alveolar fluid inflammatory cytokines, lung histopathology, and inflammatory scores compared with mice receiving protease-depleted HDE. These data suggest that proteases in dust from CAFOs are important mediators of lung inflammation, and these proteases and their receptors may provide novel targets for therapeutic intervention in CAFO dust-induced airways disease.

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Guangjin, E-mail: hou@udel.edu, E-mail: tpolenov@udel.edu; Lu, Xingyu, E-mail: luxingyu@udel.edu, E-mail: lexvega@comcast.net; Vega, Alexander J., E-mail: luxingyu@udel.edu, E-mail: lexvega@comcast.net

2014-09-14

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear {sup 1}H-X (X = {sup 13}C, {sup 15}N, {sup 31}P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the {sup 1}H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the {sup 1}H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from {supmore » 1}H chemical shift anisotropy, while keeping the {sup 1}H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [{sup 15}N]-N-acetyl-valine and [U-{sup 13}C,{sup 15}N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate {sup 1}H-{sup 15}N dipolar couplings in the context of 3D experiments is presented on U-{sup 13}C,{sup 15}N-enriched dynein light chain protein LC8.« less

Astragaloside Alleviates Hepatic Fibrosis Function via PAR2 Signaling Pathway in Diabetic Rats.

PubMed

Wang, Zhenchang; Li, Quanqiang; Xiang, Mingpeng; Zhang, Fengying; Wei, Dongyu; Wen, Zhixi; Zhou, Ying

2017-01-01

Astragaloside (AGS) extracted from radix astragalin (Huangqi) has been considered to be beneficial to liver diseases. In this study, we examined the role played by AGS in alleviating hepatic fibrosis function via protease-activated receptor-2 (PAR2) mechanisms. We hypothesized that AGS affects PAR2 signaling pathway thereby improving hepatic function in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). We further hypothesized that AGS attenuates impaired hepatic function evoked by CCl4 to a greater degree in diabetic animals. ELISA and Western Blot analysis were used to examine PAR2 signaling pathway in diabetic CCl4-rats and non-diabetic CCl4-rats. AGS inhibited the protein expression of PAR2 and its downstream pathway PKA and PKCɛ in CCl4-rats. Notably, the effects of AGS were greater in CCl4-rats with diabetes. AGS also significantly attenuated the CCl4-induced upregulations of pro-inflammatory cytokines, namely interleukin-1β, interleukin-6 and tumor necrosis factor-α accompanied with decreases of collagenic parameters such as hexadecenoic acid, laminin and hydroxyproline. Additionally, AGS improved the CCl4-induced exaggerations of liver index and functions including alanine aminotransferase, aspartate aminotransferase. Moreover, TGF-β1, a marker of hepatic fibrosis, was increased in CCl4-rats and AGS inhibited increases in TGF-β1 induced by CCl4. AGS alleviates hepatic fibrosis by inhibiting PAR2 signaling expression and its effects are largely enhanced in diabetic animals. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of hepatic fibrosis; and results of our study are likely to shed light on strategies for application of AGS because it has potentially greater therapeutic effectiveness for hepatic fibrosis in diabetes. © 2017 The Author(s)Published by S. Karger AG, Basel.

Serine proteases, inhibitors and receptors in renal fibrosis

PubMed Central

Eddy, Allison A.

2011-01-01

Summary Chronic kidney disease (CKD) is estimated to affect one in eight adults. Their kidney function progressively deteriorates as inflammatory and fibrotic processes damage nephrons. New therapies to prevent renal functional decline must build on basic research studies that identify critical cellular and molecular mediators. Plasminogen activator inhibitor-1 (PAI-1), a potent fibrosis-promoting glycoprotein, is one promising candidate. Absent from normal kidneys, PAI-1 is frequently expressed in injured kidneys. Studies in genetically engineered mice have demonstrated its potency as a pro-fibrotic molecule. Somewhat surprising, its ability to inhibit serine protease activity does not appear to be its primary pro-fibrotic effect in CKD. Both tissue-type plasminogen activator and plasminogen deficiency significantly reduced renal fibrosis severity after ureteral obstruction, while genetic urokinase (uPA) deficiency had no effect. PAI-1 expression is associated with enhanced recruitment of key cellular effectors of renal fibrosis – interstitial macrophages and myofibroblasts. The ability of PAI-1 to promote cell migration involves interactions with the low-density lipoprotein receptor-associate protein-1 and also complex interactions with uPA bound to its receptor (uPAR) and several leukocyte and matrix integrins that associate with uPAR as co-receptors. uPAR is expressed by several cell types in damaged kidneys, and studies in uPAR-deficient mice have shown that its serves a protective role. uPAR mediates additional anti-fibrotic effects - it interacts with specific co-receptors to degrade PAI-1 and extracellular collagens, and soluble uPAR has leukocyte chemoattractant properties. Molecular pathways activated by serine proteases and their inhibitor, PAI-1, are promising targets for future anti-fibrotic therapeutic agents. PMID:19350108

cAMP-secretion coupling is impaired in diabetic GK/Par rat β-cells: a defect counteracted by GLP-1.

PubMed

Dolz, Manuel; Movassat, Jamileh; Bailbé, Danielle; Le Stunff, Hervé; Giroix, Marie-Hélène; Fradet, Magali; Kergoat, Micheline; Portha, Bernard

2011-11-01

cAMP-raising agents with glucagon-like peptide-1 (GLP-1) as the first in class, exhibit multiple actions that are beneficial for the treatment of type 2 diabetic (T2D) patients, including improvement of glucose-induced insulin secretion (GIIS). To gain additional insight into the role of cAMP in the disturbed stimulus-secretion coupling within the diabetic β-cell, we examined more thoroughly the relationship between changes in islet cAMP concentration and insulin release in the GK/Par rat model of T2D. Basal cAMP content in GK/Par islets was significantly higher, whereas their basal insulin release was not significantly different from that of Wistar (W) islets. Even in the presence of IBMX or GLP-1, their insulin release did not significantly change despite further enhanced cAMP accumulation in both cases. The high basal cAMP level most likely reflects an increased cAMP generation in GK/Par compared with W islets since 1) forskolin dose-dependently induced an exaggerated cAMP accumulation; 2) adenylyl cyclase (AC)2, AC3, and G(s)α proteins were overexpressed; 3) IBMX-activated cAMP accumulation was less efficient and PDE-3B and PDE-1C mRNA were decreased. Moreover, the GK/Par insulin release apparatus appears less sensitive to cAMP, since GK/Par islets released less insulin at submaximal cAMP levels and required five times more cAMP to reach a maximal secretion rate no longer different from W. GLP-1 was able to reactivate GK/Par insulin secretion so that GIIS became indistinguishable from that of W. The exaggerated cAMP production is instrumental, since GLP-1-induced GIIS reactivation was lost in the presence the AC blocker 2',5'-dideoxyadenosine. This GLP-1 effect takes place in the absence of any improvement of the [Ca(2+)](i) response and correlates with activation of the cAMP-dependent PKA-dependent pathway.

Circadian Clock Regulates Response to Pesticides in Drosophila via Conserved Pdp1 Pathway

PubMed Central

Beaver, Laura Michelle; Hooven, Louisa Ada; Butcher, Shawn Michael; Krishnan, Natraj; Sherman, Katherine Alice; Chow, Eileen Shin-Yeu; Giebultowicz, Jadwiga Maria

2010-01-01

Daily rhythms generated by the circadian clock regulate many life functions, including responses to xenobiotic compounds. In Drosophila melanogaster, the circadian clock consists of positive elements encoded by cycle (cyc) and Clock (Clk) and negative elements encoded by period (per) and timeless (tim) genes. The ϵ-isoform of the PAR-domain protein 1 (Pdp1ε) transcription factor is controlled by positive clock elements and regulates daily locomotor activity rhythms. Pdp1 target genes have not been identified, and its involvement in other clock output pathways is not known. Mammalian orthologs of Pdp1 have been implicated in the regulation of xenobiotic metabolism; therefore, we asked whether Pdp1 has a similar role in the fly. Using pesticides as model toxicants, we determined that disruption of Pdp1ε increased pesticide-induced mortality in flies. Flies deficient for cyc also showed increased mortality, while disruption of per and tim had no effect. Day/night and Pdp1-dependent differences in the expression of xenobiotic-metabolizing enzymes Cyp6a2, Cyp6g1, and α-Esterase-7 were observed and likely contribute to impaired detoxification. DHR96, a homolog of constitutive androstane receptor and pregnane X receptor, is involved in pesticide response, and DHR96 expression decreased when Pdp1 was suppressed. Taken together, our data uncover a pathway from the positive arm of the circadian clock through Pdp1 to detoxification effector genes, demonstrating a conserved role of the circadian system in modulating xenobiotic toxicity. PMID:20348229

Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease.

PubMed

Stockley, Jacqueline; Nisar, Shaista P; Leo, Vincenzo C; Sabi, Essa; Cunningham, Margaret R; Eikenboom, Jeroen C; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C; Watson, Steve P; Mundell, Stuart J; Daly, Martina E

2015-01-01

The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious, though neither of them affected PAR4 receptor expression. A third single nucleotide variation in F2RL3 (c.65 C>A; p.T22N) was co-inherited with a synonymous single nucleotide variation in TBXA2R (c.6680 C>T, p.S218 =). Expression and signalling of the p.T22N PAR4 variant was similar to wild-type, while the TBXA2R variation introduced a cryptic splice site that was predicted to cause premature termination of protein translation. The enrichment of single nucleotide variations in G protein-coupled receptor genes among type 1 von Willebrand disease patients supports the view of type 1 von Willebrand disease as a polygenic disorder.

Simultaneous determination of paracetamol and methocarbamol in human plasma By HPLC using UV detection with time programming: application to pharmacokinetic study.

PubMed

Helmy, S A; El-Bedaiwy, H M

2014-07-01

A combination of methocarbamol (MET) and paracetamol (PAR) is a widely used treatment approach. It provides complementary modes of action for treatment of pain associated with muscle spasm. The aim of this work was to develop and validate a new sensitive and reproducible isocratic reversed phase HPLC-UV detection method for simultaneous determination of MET and PAR in human plasma for the routine use in a therapeutic drug monitoring and pharmacokinetic laboratories. A simple HPLC assay was developed and validated for the simultaneous determination of the above-mentioned drugs in small samples of human plasma (0.25 mL). After protein precipitation with methanol, satisfactory separation was achieved on a Hypersil® BDS C18 column (250 mm × 4.6 mm, 5 μm) using a mobile phase comprising 20 mM sodium dihydrogen phosphate buffer (pH=3) and methanol at a ratio of 80:20, v/v; the elution was isocratic at ambient temperature with a flow rate of 1.2 ml/min. The UV detector was programmed at 254 nm for 7.0 min to measure PAR and IS and at 272 nm for the subsequent 3 min to measure MET. Linearity was demonstrated over the concentration range from 0.02 to 20 µg/ml (mean R(2) = 0.9998, n = 10). The observed within- and between-day assay precision ranged from 1.11 to 9.4 and 2.46 to 10.0% for PAR and MET, respectively; whereas, accuracy varied between 95.2-101% and 93.9-102.2% for PAR and MET, respectively. Mean drug recovery was 99.8 for PAR and 99.0% for MET. PAR and MET were stable in frozen plasma over a period of 3 months at -80 °C. The validated method was applied successfully to a bioequivalence study of PAR/MET (500/400 mg) fixed dose combination tablet in healthy volunteers (n=24). © Georg Thieme Verlag KG Stuttgart · New York.

Choroid plexus epithelial cells express the adhesion protein P-cadherin at cell-cell contacts and syntaxin-4 in the luminal membrane domain.

PubMed

Christensen, Inga Baasch; Mogensen, Esben Nees; Damkier, Helle Hasager; Praetorius, Jeppe

2018-05-01

The choroid plexus epithelial cells (CPECs) belong to a small group of polarized cells, where the Na + -K + -ATPase is expressed in the luminal membrane. The basic polarity of the cells is, therefore, still debated. We investigated the subcellular distribution of an array of proteins known to play fundamental roles either in establishing and maintaining basic cell polarity or in the polarized delivery and recycling of plasma membrane proteins. Immunofluorescence histochemical analysis was applied to determine the subcellular localization of apical and basolateral membrane determinants. Mass spectrometry analysis of CPECs isolated by fluorescence-activated cell sorting was applied to determine the expression of specific forms of the proteins. CPECs mainly express the cell-adhesive P-cadherin, which is localized to the lateral membranes. Proteins belonging to the Crumbs and partitioning defective (Par) protein complexes were all localized to the luminal membrane domain. Par-1 and the Scribble complex were localized to the basolateral membrane domain. Lethal(2) giant larvae homolog 2 (Lgl2) labeling was preferentially observed in the luminal membrane domain. Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) was immunolocalized to the basolateral membrane domain, while phosphatidylinositol 4,5-bisphosphate (PIP 2 ) staining was most prominent in the luminal membrane domain along with the PIP 3 phosphatase, Pten. The apical target-SNARE syntaxin-3 and the basolateral target-SNARE syntaxin-4 were both localized to the apical membrane domain in CPECs, which lack cellular expression of the clathrin adaptor protein AP-1B for basolateral protein recycling. In conclusion, the CPECs are conventionally polarized, but express P-cadherin at cell-cell contacts, and Lgl2 and syntaxin-4 in the luminal plasma membrane domain.

Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks

PubMed Central

Cheng, Qiao; Barboule, Nadia; Frit, Philippe; Gomez, Dennis; Bombarde, Oriane; Couderc, Bettina; Ren, Guo-Sheng; Salles, Bernard; Calsou, Patrick

2011-01-01

In mammalian cells, the main pathway for DNA double-strand breaks (DSBs) repair is classical non-homologous end joining (C-NHEJ). An alternative or back-up NHEJ (B-NHEJ) pathway has emerged which operates preferentially under C-NHEJ defective conditions. Although B-NHEJ appears particularly relevant to genomic instability associated with cancer, its components and regulation are still largely unknown. To get insights into this pathway, we have knocked-down Ku, the main contributor to C-NHEJ. Thus, models of human cell lines have been engineered in which the expression of Ku70/80 heterodimer can be significantly lowered by the conditional induction of a shRNA against Ku70. On Ku reduction in cells, resulting NHEJ competent protein extracts showed a shift from C- to B-NHEJ that could be reversed by addition of purified Ku protein. Using a cellular fractionation protocol after treatment with a strong DSBs inducer followed by western blotting or immunostaining, we established that, among C-NHEJ factors, Ku is the main counteracting factor against mobilization of PARP1 and the MRN complex to damaged chromatin. In addition, Ku limits PAR synthesis and single-stranded DNA production in response to DSBs. These data support the involvement of PARP1 and the MRN proteins in the B-NHEJ route for the repair of DNA DSBs. PMID:21880593

The Chromobacterium violaceum ArsR Arsenite Repressor Exerts Tighter Control on Its Cognate Promoter Than the Escherichia coli System

PubMed Central

Arruda, Letícia M.; Monteiro, Lummy M. O.; Silva-Rocha, Rafael

2016-01-01

Environmental bacteria are endowed with several regulatory systems that have potential applications in biotechnology. In this report, we characterize the arsenic biosensing features of the ars response system from Chromobacterium violaceum in the heterologous host Escherichia coli. We show that the native Pars/arsR system of C. violaceum outperforms the chromosomal ars copy of E. coli when exposed to micromolar concentrations of arsenite. To understand the molecular basis of this phenomenon, we analyzed the interaction between ArsR regulators and their promoter target sites as well as induction of the system at saturating concentrations of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum has stronger binding affinity for its target promoter than the regulator from E. coli does. Additionally, arsenite induction experiments at saturating regulator concentration demonstrates that although the Pars/arsR system from E. coli displays a gradual response to increasing concentration of the inducer, the system from C. violaceum has a steeper response with a stronger promoter induction after a given arsenite threshold. Taken together, these data demonstrate the characterization of a novel arsenic response element from an environmental bacterium with potentially enhanced performance that could be further explored for the construction of an arsenic biosensor. PMID:27917165

The Chromobacterium violaceum ArsR Arsenite Repressor Exerts Tighter Control on Its Cognate Promoter Than the Escherichia coli System.

PubMed

Arruda, Letícia M; Monteiro, Lummy M O; Silva-Rocha, Rafael

2016-01-01

Environmental bacteria are endowed with several regulatory systems that have potential applications in biotechnology. In this report, we characterize the arsenic biosensing features of the ars response system from Chromobacterium violaceum in the heterologous host Escherichia coli . We show that the native Pars/arsR system of C. violaceum outperforms the chromosomal ars copy of E. coli when exposed to micromolar concentrations of arsenite. To understand the molecular basis of this phenomenon, we analyzed the interaction between ArsR regulators and their promoter target sites as well as induction of the system at saturating concentrations of the regulators. In vivo titration experiments indicate that ArsR from C. violaceum has stronger binding affinity for its target promoter than the regulator from E. coli does. Additionally, arsenite induction experiments at saturating regulator concentration demonstrates that although the Pars/arsR system from E. coli displays a gradual response to increasing concentration of the inducer, the system from C. violaceum has a steeper response with a stronger promoter induction after a given arsenite threshold. Taken together, these data demonstrate the characterization of a novel arsenic response element from an environmental bacterium with potentially enhanced performance that could be further explored for the construction of an arsenic biosensor.

Interactions of Kid–Kis toxin–antitoxin complexes with the parD operator-promoter region of plasmid R1 are piloted by the Kis antitoxin and tuned by the stoichiometry of Kid–Kis oligomers

PubMed Central

Monti, Maria C.; Hernández-Arriaga, Ana M.; Kamphuis, Monique B.; López-Villarejo, Juan; Heck, Albert J. R.; Boelens, Rolf; Díaz-Orejas, Ramón; van den Heuvel, Robert H. H.

2007-01-01

The parD operon of Escherichia coli plasmid R1 encodes a toxin–antitoxin system, which is involved in plasmid stabilization. The toxin Kid inhibits cell growth by RNA degradation and its action is neutralized by the formation of a tight complex with the antitoxin Kis. A fascinating but poorly understood aspect of the kid–kis system is its autoregulation at the transcriptional level. Using macromolecular (tandem) mass spectrometry and DNA binding assays, we here demonstrate that Kis pilots the interaction of the Kid–Kis complex in the parD regulatory region and that two discrete Kis-binding regions are present on parD. The data clearly show that only when the Kis concentration equals or exceeds the Kid concentration a strong cooperative effect exists between strong DNA binding and Kid2–Kis2–Kid2–Kis2 complex formation. We propose a model in which transcriptional repression of the parD operon is tuned by the relative molar ratio of the antitoxin and toxin proteins in solution. When the concentration of the toxin exceeds that of the antitoxin tight Kid2–Kis2–Kid2 complexes are formed, which only neutralize the lethal activity of Kid. Upon increasing the Kis concentration, (Kid2–Kis2)n complexes repress the kid–kis operon. PMID:17317682

The novel gene tank, a tumor suppressor homolog, regulates ethanol sensitivity in Drosophila.

PubMed

Devineni, Anita V; Eddison, Mark; Heberlein, Ulrike

2013-05-08

In both mammalian and insect models of ethanol intoxication, high doses of ethanol induce motor impairment and eventually sedation. Sensitivity to the sedative effects of ethanol is inversely correlated with risk for alcoholism. However, the genes regulating ethanol sensitivity are largely unknown. Based on a previous genetic screen in Drosophila for ethanol sedation mutants, we identified a novel gene, tank (CG15626), the homolog of the mammalian tumor suppressor EI24/PIG8, which has a strong role in regulating ethanol sedation sensitivity. Genetic and behavioral analyses revealed that tank acts in the adult nervous system to promote ethanol sensitivity. We localized the function of tank in regulating ethanol sensitivity to neurons within the pars intercerebralis that have not been implicated previously in ethanol responses. We show that acutely manipulating the activity of all tank-expressing neurons, or of pars intercerebralis neurons in particular, alters ethanol sensitivity in a sexually dimorphic manner, since neuronal activation enhanced ethanol sedation in males, but not females. Finally, we provide anatomical evidence that tank-expressing neurons form likely synaptic connections with neurons expressing the neural sex determination factor fruitless (fru), which have been implicated recently in the regulation of ethanol sensitivity. We suggest that a functional interaction with fru neurons, many of which are sexually dimorphic, may account for the sex-specific effect induced by activating tank neurons. Overall, we have characterized a novel gene and corresponding set of neurons that regulate ethanol sensitivity in Drosophila.

The Novel Gene tank, a Tumor Suppressor Homolog, Regulates Ethanol Sensitivity in Drosophila

PubMed Central

Eddison, Mark; Heberlein, Ulrike

2013-01-01

In both mammalian and insect models of ethanol intoxication, high doses of ethanol induce motor impairment and eventually sedation. Sensitivity to the sedative effects of ethanol is inversely correlated with risk for alcoholism. However, the genes regulating ethanol sensitivity are largely unknown. Based on a previous genetic screen in Drosophila for ethanol sedation mutants, we identified a novel gene, tank (CG15626), the homolog of the mammalian tumor suppressor EI24/PIG8, which has a strong role in regulating ethanol sedation sensitivity. Genetic and behavioral analyses revealed that tank acts in the adult nervous system to promote ethanol sensitivity. We localized the function of tank in regulating ethanol sensitivity to neurons within the pars intercerebralis that have not been implicated previously in ethanol responses. We show that acutely manipulating the activity of all tank-expressing neurons, or of pars intercerebralis neurons in particular, alters ethanol sensitivity in a sexually dimorphic manner, since neuronal activation enhanced ethanol sedation in males, but not females. Finally, we provide anatomical evidence that tank-expressing neurons form likely synaptic connections with neurons expressing the neural sex determination factor fruitless (fru), which have been implicated recently in the regulation of ethanol sensitivity. We suggest that a functional interaction with fru neurons, many of which are sexually dimorphic, may account for the sex-specific effect induced by activating tank neurons. Overall, we have characterized a novel gene and corresponding set of neurons that regulate ethanol sensitivity in Drosophila. PMID:23658154

Modeles numeriques de la stimulation optique de neurones assistee par nanoparticules plasmoniques

NASA Astrophysics Data System (ADS)

Le Hir, Nicolas

La stimulation de neurones par laser emerge depuis plusieurs annees comme une alternative aux techniques plus traditionnelles de stimulation artificielle. Contrairement a celles-ci, la stimulation lumineuse ne necessite pas d'interagir directement avec le tissu organique, comme c'est le cas pour une stimulation par electrodes, et ne necessite pas de manipulation genetique comme c'est le cas pour les methodes optogenetiques. Plus recemment, la stimulation lumineuse de neurones assistee par nanoparticules a emerge comme un complement a la stimulation simplement lumineuse. L'utilisation de nanoparticules complementaires permet d'augmenter la precision spatiale du procede et de diminuer la fluence necessaire pour observer le phenomene. Ceci vient des proprietes d'interaction entre les nanoparticules et le faisceau laser, comme par exemple les proprietes d'absorption des nanoparticules. Deux phenomenes princpaux sont observes. Dans certains cas, il s'agit d'une depolarisation de la membrane, ou d'un potentiel d'action. Dans d'autres experiences, un influx de calcium vers l'interieur du neurone est detecte par une augmentation de la fluorescence d'une proteine sensible a la concentration calcique. Certaines stimulations sont globales, c'est a dire qu'une perturbation se propage a l'ensemble du neurone : c'est le cas d'un potentiel d'action. D'autres sont, au contraire, locales et ne se propagent pas a l'ensemble de la cellule. Si une stimulation lumineuse globale est rendue possible par des techniques relativement bien maitrisees a l'heure actuelle, comme l'optogenetique, une stimulation uniquement locale est plus difficile a realiser. Or, il semblerait que les methodes de stimulation lumineuse assistees par nanoparticules puissent, dans certaines conditions, offrir cette possibilite. Cela serait d'une grande aide pour conduire de nouvelles etudes sur le fonctionnement des neurones, en offrant de nouvelles possibilites experimentales en complement des possibilites actuelles. Cependant, le mecanisme physique a l'origine de la stimulation lumineuse de neurones, ainsi que celui a l'orgine de la stimulation lumineuse assistee par nanoparticules, n'est a ce jour pas totalement compris. Des hypotheses ont ete formulees concernant ce mecanisme : il pourrait etre photothermique, photomecanique, ou encore photochimique. Il se pourrait egalement que plusieurs mecanismes soient a l'oeuvre conjointement, etant donne la variete des observations. La litterature ne converge pas a ce sujet et l'existence d'un mecanisme commun aux differentes situations n'a pas ete demontree.

UV-B Radiation Induces Macrophage Migration Inhibitory Factor–Mediated Melanogenesis through Activation of Protease-Activated Receptor-2 and Stem Cell Factor in Keratinocytes

PubMed Central

Enomoto, Akiko; Yoshihisa, Yoko; Yamakoshi, Takako; Ur Rehman, Mati; Norisugi, Osamu; Hara, Hiroshi; Matsunaga, Kenji; Makino, Teruhiko; Nishihira, Jun; Shimizu, Tadamichi

2011-01-01

UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation. PMID:21281800

Kallikrein-related peptidase 8 is expressed in myocardium and induces cardiac hypertrophy

PubMed Central

Cao, Buqing; Yu, Qing; Zhao, Wei; Tang, Zhiping; Cong, Binghai; Du, Jiankui; Lu, Jianqiang; Zhu, Xiaoyan; Ni, Xin

2016-01-01

The tissue kallikrein-related peptidase family (KLK) is a group of trypsin- and chymotrypsin-like serine proteases that share a similar homology to parent tissue kallikrein (KLK1). KLK1 is identified in heart and has anti-hypertrophic effects. However, whether other KLK family members play a role in regulating cardiac function remains unknown. In the present study, we demonstrated for the first time that KLK8 was expressed in myocardium. KLK8 expression was upregulated in left ventricle of cardiac hypertrophy models. Both intra-cardiac adenovirus-mediated and transgenic-mediated KLK8 overexpression led to cardiac hypertrophy in vivo. In primary neonatal rat cardiomyocytes, KLK8 knockdown inhibited phenylephrine (PE)-induced cardiomyocyte hypertrophy, whereas KLK8 overexpression promoted cardiomyocyte hypertrophy via a serine protease activity-dependent but kinin receptor-independent pathway. KLK8 overexpression increased epidermal growth factor (EGF) production, which was blocked by the inhibitors of serine protease. EGF receptor (EGFR) antagonist and EGFR knockdown reversed the hypertrophy induced by KLK8 overexpression. KLK8-induced cardiomyocyte hypertrophy was also significantly decreased by blocking the protease-activated receptor 1 (PAR1) or PAR2 pathway. Our data suggest that KLK8 may promote cardiomyocyte hypertrophy through EGF signaling- and PARs-dependent but a kinin receptor-independent pathway. It is implied that different KLK family members can subtly regulate cardiac function and remodeling. PMID:26823023

Phloretin suppresses thrombin-mediated leukocyte-platelet-endothelial interactions.

PubMed

Kim, Min Soo; Park, Sin-Hye; Han, Seon-Young; Kim, Yun-Ho; Lee, Eun-Jung; Yoon Park, Jung Han; Kang, Young-Hee

2014-04-01

Thrombin playing a pivotal role in coagulation cascade may influence the onset and progression of atherosclerosis as a pro-inflammatory mediator. This study investigated whether phloretin found in apple tree leaves, severed a linkage between thrombosis and atherosclerosis by thrombin. Human endothelial cells were pre-treated with 1-20 μM phloretin and stimulated with 10 U/mL thrombin. Phloretin attenuated adhesion of THP-1 monocytes and platelets to thrombin-inflamed endothelial cells with concurrent inhibition of protease-activated receptor (PAR-1) induction. The thrombin induction of endothelial CD40, endothelial integrin β3 and P-selectin, and monocytic CD40L was dampened by phloretin. Additionally, phloretin inhibited monocyte secretion of MCP-1, IL-6 and IL-8 responsible for pro-inflammatory activity of thrombin inducing endothelial CD40. The monocyte COX-2 induction and PGE2 secretion due to thrombin were down-regulated by phloretin, deterring endothelial CD40 expression. Thrombin promoted production of PAI-1 and tissue factor in monocytes was attenuated by phloretin through blocking PAR-1 and CD40. Thrombin up-regulated the induction of endothelial connective tissue growth factor independent of PAR-1 activation, which was reversed by phloretin. Phloretin disturbed tethering and stable adhesion of monocytes and platelets onto endothelium during increased thrombosis by thrombin. Phloretin would be a potent agent preventing thrombosis and atherosclerosis. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.