PAR Signaling Research Articles

ADP-ribosylation: activation, recognition, and removal.

ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions.

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling*

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Molecular Insights into Poly(ADP-ribose) Recognition and Processing.

Poly(ADP-ribosyl)ation is a post-translational protein modification involved in the regulation of important cellular functions including DNA repair, transcription, mitosis and apoptosis. The amount of poly(ADP-ribosyl)ation (PAR) in cells reflects the balance of synthesis, mediated by the PARP protein family, and degradation, which is catalyzed by a glycohydrolase, PARG. Many of the proteins mediating PAR metabolism possess specialised high affinity PAR-binding modules that allow the efficient sensing or processing of the PAR signal. The identification of four such PAR-binding modules and the characterization of a number of proteins utilising these elements during the last decade has provided important insights into how PAR regulates different cellular activities. The macrodomain represents a unique PAR-binding module which is, in some instances, known to possess enzymatic activity on ADP-ribose derivatives (in addition to PAR-binding). The most recently discovered example for this is the PARG protein, and several available PARG structures have provided an understanding into how the PARG macrodomain evolved into a major enzyme that maintains PAR homeostasis in living cells.

Lanthanide Labeling of a Potent Protease Activated Receptor-2 Agonist for Time-Resolved Fluorescence Analysis

Protease activated receptor-2 (PAR(2)) is one of four G-protein coupled receptors (GPCRs) that can be activated by exogenous or endogenous proteases, which cleave the extracellular amino-terminus to expose a tethered ligand and subsequent G-protein signaling. Alternatively, PAR(2) can be activated by peptide or peptidomimetic ligands derived from the sequence of the natural tethered ligand. Screening of novel ligands that directly bind to PAR(2) to agonize or antagonize the receptor has been hindered by the lack of a sensitive, high-throughput, affinity binding assay. In this report, we describe the synthesis and use of a modified PAR(2) peptidomimetic agonist, 2-furoyl-LIGRLO-(diethylenetriaminepentaacetic acid)-NH(2) (2-f-LIGRLO-dtpa), designed for lanthanide-based time-resolved fluorescence screening. We first demonstrate that 2-f-LIGRLO-dtpa is a potent and specific PAR(2) agonist across a full spectrum of in vitro assays. We then show that 2-f-LIGRLO-dtpa can be utilized in an affinity binding assay to evaluate the ligand-receptor interactions between known high potency peptidomimetic agonists (2-furoyl-LIGRLO-NH(2), 2-f-LIGRLO; 2-aminothiazol-4-yl-LIGRL-NH(2), 2-at-LIGRL; 6-aminonicotinyl-LIGRL-NH(2), 6-an-LIGRL) and PAR(2). A separate N-terminal peptidomimetic modification (3-indoleacetyl-LIGRL-NH(2), 3-ia-LIGRL) that does not activate PAR(2) signaling was used as a negative control. All three peptidomimetic agonists demonstrated sigmoidal competitive binding curves, with the more potent agonists (2-f-LIGRLO and 2-at-LIGRL) displaying increased competition. In contrast, the control peptide (3-ia-LIGRL) displayed limited competition for PAR(2) binding. In summary, we have developed a europium-containing PAR(2) agonist that can be used in a highly sensitive affinity binding assay to screen novel PAR(2) ligands in a high-throughput format. This ligand can serve as a critical tool in the screening and development of PAR(2) ligands.

Signaling pathways in cell polarity.

A key function of signal transduction during cell polarization is the creation of spatially segregated regions of the cell cortex that possess different lipid and protein compositions and have distinct functions. Polarity can be initiated spontaneously or in response to signaling inputs from adjacent cells or soluble factors and is stabilized by positive-feedback loops. A conserved group of proteins, the Par proteins, plays a central role in polarity establishment and maintenance in many contexts. These proteins generate and maintain their distinct locations in cells by actively excluding one another from specific regions of the plasma membrane. The Par signaling pathway intersects with multiple other pathways that control cell growth, death, and organization.

Novel Role for Proteinase-activated Receptor 2 (PAR2) in Membrane Trafficking of Proteinase-activated Receptor 4 (PAR4)

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.

Selective Small Molecule Inhibition of Poly(ADP-Ribose) Glycohydrolase (PARG)

The poly(ADP-ribose) (PAR) post-translational modification is essential for diverse cellular functions, including regulation of transcription, response to DNA damage, and mitosis. Cellular PAR is predominantly synthesized by the enzyme poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a critical node in the DNA damage response pathway, and multiple potent PARP-1 inhibitors have been described, some of which show considerable promise in the clinic for the treatment of certain cancers. Cellular PAR is efficiently degraded by poly(ADP-ribose) glycohydrolase (PARG), an enzyme for which no potent, readily accessible, and specific inhibitors exist. Herein we report the discovery of small molecules that effectively inhibit PARG in vitro and in cellular lysates. These potent PARG inhibitors can be produced in two chemical steps from commercial starting materials and have complete specificity for PARG over the other known PAR glycohydrolase (ADP-ribosylhydrolase 3, ARH3) and over PARP-1 and thus will be useful tools for studying the biochemistry of PAR signaling.

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

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.

Neutrophil Elastase Acts as a Biased Agonist for Proteinase-activated Receptor-2 (PAR2)

Human neutrophil proteinases (elastase, proteinase-3, and cathepsin-G) are released at sites of acute inflammation. We hypothesized that these inflammation-associated proteinases can affect cell signaling by targeting proteinase-activated receptor-2 (PAR(2)). The PAR family of G protein-coupled receptors is triggered by a unique mechanism involving the proteolytic unmasking of an N-terminal self-activating tethered ligand (TL). Proteinases can either activate PAR signaling by unmasking the TL sequence or disarm the receptor for subsequent enzyme activation by cleaving downstream from the TL sequence. We found that none of neutrophil elastase, cathepsin-G, and proteinase-3 can activate G(q)-coupled PAR(2) calcium signaling; but all of these proteinases can disarm PAR(2), releasing the N-terminal TL sequence, thereby preventing G(q)-coupled PAR(2) signaling by trypsin. Interestingly, elastase (but neither cathepsin-G nor proteinase-3) causes a TL-independent PAR(2)-mediated activation of MAPK that, unlike the canonical trypsin activation, does not involve either receptor internalization or recruitment of β-arrestin. Cleavage of synthetic peptides derived from the extracellular N terminus of PAR(2), downstream of the TL sequence, demonstrated distinct proteolytic sites for all three neutrophil-derived enzymes. We conclude that in inflammation, neutrophil proteinases can modulate PAR(2) signaling by preventing/disarming the G(q)/calcium signal pathway and, via elastase, can selectively activate the p44/42 MAPK pathway. Our data illustrate a new mode of PAR regulation that involves biased PAR(2) signaling by neutrophil elastase and a disarming/silencing effect of cathepsin-G and proteinase-3.

Roles and interactions among protease-activated receptors and P2ry12 in hemostasis and thrombosis

Toward understanding their redundancies and interactions in hemostasis and thrombosis, we examined the roles of thrombin receptors (protease-activated receptors, PARs) and the ADP receptor P2RY12 (purinergic receptor P2Y G protein-coupled 12) in human and mouse platelets ex vivo and in mouse models. Par3(-/-) and Par4(+/-) mouse platelets showed partially decreased responses to thrombin, resembling those in PAR1 antagonist-treated human platelets. P2ry12(+/-) mouse platelets showed partially decreased responses to ADP, resembling those in clopidogrel-treated human platelets. Par3(-/-) mice showed nearly complete protection against carotid artery thrombosis caused by low FeCl(3) injury. Par4(+/-) and P2ry12(+/-) mice showed partial protection. Increasing FeCl(3) injury abolished such protection; combining partial attenuation of thrombin and ADP signaling, as in Par3(-/-):P2ry12(+/-) mice, restored it. Par4(-/-) mice, which lack platelet thrombin responses, showed still better protection. Our data suggest that (i) the level of thrombin driving platelet activation and carotid thrombosis was low at low levels of arterial injury and increased along with the contribution of thrombin-independent pathways of platelet activation with increasing levels of injury; (ii) although P2ry12 acts downstream of PARs to amplify platelet responses to thrombin ex vivo, P2ry12 functioned in thrombin/PAR-independent pathways in our in vivo models; and (iii) P2ry12 signaling was more important than PAR signaling in hemostasis models; the converse was noted for arterial thrombosis models. These results make predictions being tested by ongoing human trials and suggest hypotheses for new antithrombotic strategies.

Abstract 1270: Elevated levels of poly(ADP-ribose) lead to novel apoptotic signaling and the induction of caspase-independent cell death

Abstract The metabolism of poly(ADP-ribose) (PAR) is activated by DNA damage, where PAR polymerase-1 (PARP-1) transfers ADP-ribose onto nuclear proteins to form PAR and PAR glycohydrolase (PARG) catalyzes its degradation. Previous findings show that high levels of PAR lead to its presence in the cytoplasm, where it signals the release of the proapoptotic protein, apoptosis inducing factor (AIF). Our studies suggest that inhibiting PAR glycohydrolase (PARG), and thus promoting increased levels of PAR, will increase the efficacy of anti-cancer agents by promoting this alternative cell death pathway mediated by AIF. The objective of this study is to elucidate the pathway of cytoplasm PAR cell death signaling in cancer cells. We utilized MDA-MB-231 breast cancer cells to reveal the ability of AIF to induce cancer cell death following caspase inhibition. In addition, PARG null embryonic trophoblast stem cells were utilized to determine the cytoplasmic PAR signaling pattern and to purify the PAR-modified nuclear proteins released in the cytoplasm. FACS analysis demonstrated equivalent levels of cell death with or without caspases and Western blot revealed a robust release of AIF from the mitochondria following DNA damage. Following low-dose DNA-damaging agent treatment in PARG null cells, cytoplasmic PAR was detected concurrently with AIF translocation to the nucleus, as shown by Western blot and confocal immunofluorescence. In PARG null cells in the absence of stress, cytoplasmic PAR levels were maximal after 3 days as shown by cell fractionations and Western blot. The cytoplasmic fraction containing PAR was then purified using dihydroxyboronyl Bio-Rex affinity chromatography. The purified PAR-modified proteins were treated with phosphodiesterase to remove all PAR and SDS-PAGE analysis demonstrated several proteins ranging from 20-50 kDa, which together serve as the cytoplasmic signal that causes AIF release from the mitochondria. These proteins potentially reveal novel nuclear-to-mitochondrial cell signaling ligands that are necessary for the induction of the AIF pathway of caspase-independent cell death. Taken together, the results demonstrate the importance of AIF in cancer cell death and novel insight into the cell death signaling by PAR that produces AIF-mediated cell death. Therefore, ability to increase PAR levels by inhibiting PARG is potentially a unique method to induce the death of cancer cells by an alternative cell death pathway. This research was supported by funding from the American Cancer Society and the Pharmaceutical Research Manufacturers of America. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1270.

Candida albicans Releases Soluble Factors That Potentiate Cytokine Production by Human Cells through a Protease-Activated Receptor 1- and 2-Independent Pathway

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

Thrombin‐mediated thromboxane and 12‐HETE formation in human platelets is differentially regulated through PAR1 and PAR4

Thrombin‐regulated platelet activation mediates a diverse range of vascular effects which may result in thrombus formation and stroke. Thromboxane (TxB2) is thought to play a crucial role in the reinforcement of platelet activation following thrombin activation via TxB2 formation in the platelet. We demonstrate that PAR1 and PAR4 work synergistically but through unique signaling pathways following activation in order to generate both TxB2 as well as 12‐HETE. To investigate the signaling differences, we examined multiple biochemical and physiological steps involved in platelet activation including Rap1, aggregation, Ca2+ mobilization and formation of 12‐HETE and TxB2. Inhibition of PIP2 and PIP3 significantly altered PAR1‐, but not PAR4‐mediated platelet aggregation and resulted in inhibition of TxB2, but not 12‐HETE. Additionally, the time course for PAR1‐mediated TxB2 formation was faster compared to PAR4. Inhibiting PAR4 activation with YD3 only partially blocked thrombin‐mediated effects confirming the synergistic involvement of both PAR1 and PAR4 in this process. A better understanding of regulation of platelet function through PAR signaling pathways is crucial to developing a better class of anti‐platelet therapies with a lower profile for bleeding as a side effect. This study was supported in part by grants P50 HL081009 (to H.E.H.) and R00 HL089457 (to M.H.) from National Institutes of Health.

RGS protein regulation and signaling diversity of protease‐activated receptors (PAR1 and PAR2)

PAR signaling must be tightly controlled since these G protein coupled receptors are proteolytically cleaved and thereby irreversibly activated. Regulators of G protein signaling (RGS) proteins provide a potential mechanism for such regulation since their conserved RGS domains give them the capacity to act as GTPase‐activating proteins and thereby inhibit G protein signaling. Little is known about RGS regulation of PAR signaling but our data using purified proteins and intact cells suggest that RGS2 and RGS4 but not RGS1 or RGS16, directly bind to and selectively regulate PAR1 and PAR2 signaling. Adding to the complexity of these studies, PAR1 and PAR2 have both been shown to couple to Gi, Gq, and G12. Our data shows that PAR1 and PAR2 activation of Gq (and potentially other G proteins) can be blocked by RGS2 and RGS4 but not by RGS1 or RGS16. Using selective G protein inhibitors and receptor mutants, ongoing studies will determine whether RGS regulation of PARs is receptor‐dependent, G protein‐ dependent or both. A detailed understanding of PAR signaling will provide insight into how these promiscuous receptors activate multiple G proteins and how their divergent signaling pathways are regulated. These studies will define PAR signaling mechanisms that underlie their roles in cellular processes such as inflammation, gliosis, and hyperalgesia. Support: R01NS037112 and R01NS049195 (JRH), A.H.A. 0715465B (KLM).

Advances in Neuronal Cell Death 2007

In a stroke lesion, there is a core of necrotic cell death surrounded by a zone of tissue at risk, termed the penumbra. In the penumbra, there is usually less severe tissue damage. Programmed cell death (PCD) pathways have been documented to be present in the penumbra. While many studies have used the term apoptosis as equivalent to PCD, the absence of complete biochemical and morphological characteristics of apoptosis, coupled with the ineffectiveness of caspase inhibitors, indicates that there are other important cell death pathways activated during stroke. A few caspase-independent cell death pathways have been identified and there are likely more to be described. Stroke injury leads to the activation of a cell death program, dependent on poly(ADP-ribose) polymerase-1 (PARP1) activation1 and culminating in apoptosis inducing factor (AIF) mediated cell death.2 Poly(ADP-ribose) (PAR) polymer is the death signal in this pathway and Thanatos is the Greek personification of death and mortality, hence, the name parthanatos. In this form of cell death that occurs in many different organ systems during ischemia-reperfusion injury, PAR polymer translocates from the nucleus to the cytoplasm and mitochondria. This leads to release of AIF from the mitochondria, an effect that is inhibited by poly(ADP-ribose) glycohydrolase (PARG), an enzyme that degrades PAR polymer.3 After cerebral focal ischemia reperfusion injury, mice with reduced PARG levels had higher infarct volumes than controls. On the other hand, mice overexpressing PARG had significantly lower infarct volumes. Combined, the current data indicate that PAR is a powerful death signal. What is not yet understood are the mechanisms of PAR translocation out of the nucleus and PAR-mediated AIF release from the mitochondria. Parthanatos is caspase-independent and is biochemically and morphologically distinct from apoptosis.4 Regulation of PAR signaling may be a useful therapeutic intervention to reduce stroke-mediated damage. Additional …

Hematopoeitic lineage cell-specific protein-1 (HS1) regulates PAR-mediated ERK activation and thromboxane generation in platelets

Thrombin-induced platelet activation leads to tyrosine phosphorylation of hematopoietic lineage cell-specific protein-1 (HS1), a 75 kDa adapter protein expressed exclusively in cells of hematopoietic lineage. We have shown HS1 to be a functionally important signaling molecule downstream of PAR-4 and GPVI collagen receptor. We have thus begun to elucidate PAR signaling pathway of HS1 phosphorylation, and its functional implications. PAR-1 and PAR-4 activating peptides (SFLLRN and AYPGKF, respectively) induced HS1 phosphorylation in a Gq-dependent manner as shown by incubation with the Gq inhibitor, YM254890. Consistently, HS1 phosphorylation was abolished in platelets from Gq deficient mice upon AYPGKF stimulation. Treatment with ADP receptor antagonists did not affect HS1 phosphorylation. Pretreatment of platelets with Src kinase inhibitors abolished HS1 phosphorylation. Further Syk activation, as measured by tyrosine phosphorylation of Syk (residues 525/526), in response to PAR activation was abolished in the presence of Src inhibitors. HS1 null mice show inhibition of PAR-mediated thromboxane A2 generation compared to wild type littermates. Phosphorylation of Erk, a key signaling molecule in thromboxane generation, was also diminished in HS1 null mice platelets. Based on these findings, we conclude that tyrosine phosphorylation of HS1 occurs downstream of both PAR-1 and PAR-4. HS1 phosphorylation is a Gq mediated response regulated by Src kinases. Thus, HS1 may mediate PAR-induced thromboxane generation through regulation of Erk phosphorylation.

Thrombin‐mediated hepatocellular carcinoma cell migration: Cooperative action via proteinase‐activated receptors 1 and 4

Proteinase-activated receptor-1 (PAR(1)), a thrombin receptor and the prototype of a newly discovered G-protein-coupled receptor subfamily, plays an important role in tumor development and progression. In this study, we documented the expression of the thrombin receptors PAR(1), PAR(3), and PAR(4) in permanent hepatocellular carcinoma (HCC) cell lines and primary HCC cell cultures. Stimulation of HCC cells with thrombin and the PAR(1)-selective activating peptide, TFLLRN-NH(2), increased transmembrane migration across a collagen barrier. This effect was blocked by the PAR(1) antagonist SCH 79797, confirming that the PAR(1) thrombin receptor subtype is involved in regulating hepatoma cell migration. In addition, the PAR(4)-selective agonist, AYPGKF-NH(2), also stimulated HCC cell migration whilst the PAR(4) antagonist, trans-cinnamoyl-YPGKF-NH(2), attenuated the effect of thrombin on HCC cell migration. PAR(1)- and PAR(4)-triggered HCC cell migration was blocked by inhibiting a number of key mediators of signal transduction, including G proteins of the G(i)/G(o) family, matrix metalloproteinases, ERK/MAPKinase, cyclic AMP-dependent protein kinase, Src tyrosine kinase, and the EGF receptor kinase. Our data point to a cooperative PAR(1)/PAR(4) signaling network that contributes to thrombin-mediated tumor cell migration. We suggest that a combined inhibition of coagulation cascade serine proteinases, the two PARs and their complex signaling pathways may provide a new strategy for treating hepatocellular carcinoma.

Tissue factor and PAR signaling in tumor progression

Tumor development depends on multiple reciprocal interactions of tumor cells with the host cell compartment. Tumor cells initiate TF-dependent crosstalks with the tumor microenvironment by releasing procoagulant microparticles, soluble cytokines and angiogenic growth factors. Conversely, the hemostatic system in the host compartment provides multiple circuits that regulate tumor growth and sustain angiogenesis. A combination of experimental models of spontaneous and transplanted tumor development and metastasis start to delineate the role of TF in tumor progression and identified potential therapeutic approaches to target the TF pathway.

Condensin I Interacts with the PARP-1-XRCC1 Complex and Functions in DNA Single-Strand Break Repair

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.

Flow Perturbation Is Linked to Endothelial PAR Signaling

The vascular endothelium integrates a variety of humoral stimuli and biophysical forces to maintain patent microvascular and macrovacular beds. At the interface between the blood and vessel wall, endothelial cells communicate in both directions by the release of growth factors, cytokines, and bioactive mediators to maintain vascular homeostasis. Endothelial dysfunction is central for acute systemic inflammatory syndromes including sepsis and chronic inflammatory disorders such as atherosclerosis. Early atherosclerotic lesions preferentially develop at sites where laminar flow patterns are disturbed, and endothelial cells sense changes in biomechanical forces that result from flow perturbation. Exposure to shear forces rapidly activates G proteins, ion fluxes, and mechanosensing receptors, including integrins and a complex involving PECAM-1, VEGFR-2, and vascular endothelial cell cadherin.1 Activation of downstream signaling pathways results in cytoskeletal reorganization and a flow-adapted endothelial phenotype.2 Endothelial cells under laminar flow have an atheroprotected phenotype, but perturbation of laminar flow patterns induces a complex transcriptional response that favors the induction of adhesion molecules, proinflammatory cytokines, and procoagulant genes. See page 1185 Dekker et al profiled the repertoire of laminar flow-regulated genes in endothelial cells and identified lung Krueppel-like factor (LKLF or KLF-2) that was selectively induced on flow adaptation and suppressed by the inflammatory mediator tumor necrosis factor (TNF)α.3 KLF2 was indeed tonically expressed in vivo in noninflamed large vessel endothelial cells. KLFs are zinc finger transcription factors similar to Sp1 and Egr-1 families that act as transcriptional repressors or activators by high affinity binding to a CACCC promoter/enhancer element. KLF2 was originally identified …