Platelet 12-lipoxygenase activation via glycoprotein VI - Involvement of multiple signaling pathways in agonist control of H(P)ETE synthesis

Lipoxygenases (LOX) contribute to vascular disease and inflammation through generation of bioactive lipids, including 12-hydro(pero)xyeicosatetraenoic acid (12-H(P)ETE). The physiological mechanisms that acutely control LOX product generation in mammalian cells are uncharacterized. Human platelets that contain a 12-LOX isoform (p12-LOX) were used to define pathways that activate H( P) ETE synthesis in the vasculature. Collagen and collagen-related peptide (CRP) (1 to 10 mug/mL) acutely induced platelet 12-H(P)ETE synthesis. This implicated the collagen receptor glycoprotein VI ( GPVI), which signals via the immunoreceptor-based activatory motif (ITAM)-containing FcRgamma chain. Conversely, thrombin only activated at high concentrations (> 0.2 U/mL), whereas U46619 and ADP alone were ineffective. Collagen or CRP-stimulated 12-H( P) ETE generation was inhibited by staurosporine, PP2, wortmannin, BAPTA/AM, EGTA, and L-655238, implicating src-tyrosine kinases, PI3-kinase, Ca2+ mobilization, and p12-LOX translocation. In contrast, protein kinase C (PKC) inhibition potentiated 12-H( P) ETE generation. Finally, activation of the immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing platelet endothelial cell adhesion molecule (PECAM-1) inhibited p12-LOX product generation. This study characterizes a receptor-dependent pathway for 12-H(P) ETE synthesis via the collagen receptor GPVI, which is negatively regulated by PECAM-1 and PKC, and demonstrates a novel link between immune receptor signaling and lipid mediator generation in the vasculature.

N-acetylgalactosamine kinase: a naturally promiscuous small molecule kinase

N-acetylgalactosamine kinase is a member of the GHMP family of small molecule kinases which catalyses the ATP-dependent phosphorylation of N-acetylgalactosamine. It is highly similar in structure and sequence to galactokinase. Alteration of galactokinase at a key tyrosine residue (Tyr-379 in the human enzyme) has been shown to dramatically enhance the substrate range of this enzyme. Here, we investigated the substrate specificity of the wild type N-acetylgalactosamine kinase and demonstrated that it can also catalyse the phosphorylation of N-acetylglucosamine and N-acetylmannosamine. In human N-acetylgalactosamine kinase, the equivalent residue to Tyr-379 in galactokinase is Phe-444. Alteration of this residue did not result in dramatic changes to the specificity of the enzyme. The more relaxed substrate specificity of N-acetylgalactosamine kinase, compared to galactokinase, can be explained by the greater flexibility of a glycine rich loop in the active site of the enzyme. These results suggest that N-acetylgalactosamine kinase is a potential biocatalyst for the phosphorylation of N-acetyl sugars. However, it is unlikely that it will be possible to further broaden the substrate range by alteration of Phe-444.

Environmental, lifestyle, and familial/ethnic factors associated with myeloproliferative neoplasms

Myeloproliferative neoplasms (MPNs) are characterized by overproduction of mature functional blood cells and are often associated with an acquired genetic mutation of Janus Kinase 2V617F. The etiology of MPNs remains unknown. The aim of this article was to review and collate all known published data investigating environmental and lifestyle factors associated with MPNs. Medline, Embase, PubMed, Cochrane, and Web of Science were systematically searched using terms for MPNs and observational study designs to identify studies investigating the risk factors for MPNs published before March 2010. Of 9,156 articles identified, 19 met the selection criteria. Although the studies exhibited heterogeneity, in case definitions, study design, and risk factors investigated, several themes emerged. A strong association was found with Jewish descent, and with a family history of MPNs. Autoimmune conditions, specifically Crohn's disease, were more common in patients with MPNs. Certain occupational groups were significantly associated with MPNs including occupations with potential exposure to benzene and/or petroleum. Blood donation was associated with an increased risk of polycythemia vera specifically. The vast heterogeneity in studies identified as part of this review suggests that large scale systematic assessment of etiological factors associated with MPNs is warranted. (C) 2011 Wiley-Liss, Inc.

Suppressors of cytokine signalling (SOCS): putative modulators of cytokine bioactivity in health and disease

Cytokines are important modulators of homeostatic processes such as development, haematopoiesis and host defence, A recently identified family of proteins, the supressors of cytokine signalling (SOCS) act as negative regulators of the key cytokine-activated signalling pathway, the Janus kinase/signal transducers and activators of transcription (JAK/STAT) cascade, In the current review, the discovery, structural features, regulation of expression, mechanisms of JAK/STAT inhibition and putative role in health and disease of the SOCS family are discussed.

Activation of extracellular signal-regulated kinase in proliferative glomerulonephritis in rats

Multiple extracellular mitogens are involved in the pathogenesis of proliferative forms of glomerulonephritis (GN), In vitro studies demonstrate the pivotal role of extracellular signal-regulated kinase (ERK) in the regulation of cellular proliferation in response to extracellular mitogens. In this study, we examined whether this kinase, as a convergence point of mitogenic stimuli, is activated in proliferative GN in vivo, Two different proliferative forms of anti-glomerular basal membrane (GEM) GN in rats were induced and whole cortical tissue as well as isolated glomeruli examined using kinase activity assays and Western blot analysis, Administration of rabbit anti-rat GEM serum to rats, preimmunized with rabbit IgG, induced an accelerated crescentic anti-GEM GN. A significant increase in cortical, and more dramatically glomerular ERK activity was detected at 1, 3, and 7 d after induction of GN, Immunization of Wistar-Kyoto rats with bovine GEM also induced a crescentic anti-GBM GN with an increase of renal cortical ERK activity after 4, 6, and 8 wk, ERK is phosphorylated and activated by the MAP kinase/ERK kinase (MEK), We detected a significant increase in the expression of glomerular MEK in the accelerated form of anti-GEM CN, providing a possible mechanism of long-term activation of ERK in this disease model, In contrast to ERK, activation of stress-activated protein kinase was only detectable at early stages of proliferative GN, indicating these related kinases to serve distinct roles in the pathogenesis of GN, Our observations point to ERK as a putative mediator of the proliferative response to immune injury in GN and suggest that upregulation of MEK is involved in the long-term regulation of ERK in vivo.

Heat shock protein 90 inhibition is cytotoxic to primary AML cells expressing mutant FLT3 and results in altered downstream signalling

Activating mutations of the FMS-like tyrosine kinase 3 gene (FLT3) occur in approximately one-third of patients with acute myeloid leukaemia (AML) and predict for a poor outcome. Heat shock protein 90 (Hsp90) is a molecular chaperone that is frequently used by cancer cells to stabilise mutant oncoproteins. Mutant FLT3 is chaperoned by Hsp90 in primary AML blasts whereas unmutated FLT3 is not, making Hsp90 inhibitors potentially useful therapeutically. The present study showed that inhibition of Hsp90 by 17-allylamino-17-demethoxygeldanamycin (17-AAG) was cytotoxic to primary AML cells expressing mutant FLT3. Inhibition of Hsp90 results in altered downstream signalling effects in primary AML cells with disruption of Janus kinase-signal transducer and activator of transcription (JAK-STAT), mitogen-activated protein kinase and phosphatidylinositol 3/AKT signalling pathways. Co-treatment of blasts with 17-AAG and cytarabine resulted in a synergistic or additive effect in approximately 50% of AML cases tested. Our results confirm that Hsp90 is a valid molecular target in the therapy of AML. Inhibition of Hsp90 in parallel with conventional AML therapies may have particular benefit in those patients with the poor prognostic FLT3 mutant disease.

Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation

In mammals, the ATM (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response. The checkpoint functions of ATR and ATM are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2. In mammalian cells, evidence has been presented that Chk1 is devoted to the ATR signaling pathway and is modified by ATR in response to replication inhibition and UV-induced damage, whereas Chk2 functions primarily through ATM in response to ionizing radiation (IR), suggesting that Chk2 and Chk1 might have evolved to channel the DNA damage signal from ATM and ATR, respectively. We demonstrate here that the ATR-Chk1 and ATM-Chk2 pathways are not parallel branches of the DNA damage response pathway but instead show a high degree of cross-talk and connectivity. ATM does in fact signal to Chk1 in response to IR. Phosphorylation of Chk1 on Ser-317 in response to IR is ATM-dependent. We also show that functional NBS1 is required for phosphorylation of Chk1, indicating that NBS1 might facilitate the access of Chk1 to ATM at the sites of DNA damage. Abrogation of Chk1 expression by RNA interference resulted in defects in IR-induced S and G(2)/M phase checkpoints; however, the overexpression of phosphorylation site mutant (S317A, S345A or S317A/S345A double mutant) Chk1 failed to interfere with these checkpoints. Surprisingly, the kinase-dead Chk1 (D130A) also failed to abrogate the S and G(2) checkpoint through any obvious dominant negative effect toward endogenous Chk1. Therefore, further studies will be required to assess the contribution made by phosphorylation events to Chk1 regulation. Overall, the data presented in the study challenge the model in which Chk1 only functions downstream from ATR and indicate that ATM does signal to Chk1. In addition, this study also demonstrates that Chk1 is essential for IR-induced inhibition of DNA synthesis and the G(2)/M checkpoint.

Myeloid Angiogenic Cells Act as Alternative M2 Macrophages and Modulate Angiogenesis through Interleukin-8

Endothelial progenitor cells (EPCs) promote angiogenesis, and clinical trials have shown such cell therapy to be feasible for treating ischemic disease. However, clinical outcomes have been contradictory owing to the diverse range of EPC types used. We recently characterized two EPC subtypes, and identified outgrowth endothelial cells as the only EPC type with true progenitor and endothelial characteristics. By contrast, myeloid angiogenic cells (MACs) were shown to be monocytic cells without endothelial characteristics despite being widely described as "EPCs." In the current study we demonstrated that although MACs do not become endothelial cells or directly incorporate into a microvascular network, they can significantly induce endothelial tube formation in vitro and vascular repair in vivo. MAC-derived interleukin-8 (IL-8) was identified as a key paracrine factor, and blockade of IL-8 but not vascular endothelial growth factor (VEGF) prevented MAC-induced angiogenesis. Extracellular IL-8 transactivates VEGFR2 and induces phosphorylation of extracellular signal-regulated kinases. Further transcriptomic and immunophenotypic analysis indicates that MACs represent alternative activated M2 macrophages. Our findings demonstrate an unequivocal role for MACs in angiogenesis, which is linked to paracrine release of cytokines such as IL-8. We also show, for the first time, the true identity of these cells as alternative M2 macrophages with proangiogenic, antiinflammatory and pro-tissue-repair properties.

Klebsiella pneumoniae subverts the activation of inflammatory responses in a NOD1-dependent manner

Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Subversion of inflammation is essential for pathogen survival during infection. Evidence indicates that K. pneumoniae infections are characterized by lacking an early inflammatory response although the molecular bases are currently unknown. Here we unveil a novel strategy employed by a pathogen to counteract the activation of inflammatory responses. K. pneumoniae attenuates pro-inflammatory mediators-induced IL-8 secretion. Klebsiella antagonizes the activation of NF-?B via the deubiquitinase CYLD and blocks the phosphorylation of mitogen-activated protein kinases (MAPKs) via the MAPK phosphatase MKP-1. Our studies demonstrate that K. pneumoniae has evolved the capacity to manipulate host systems dedicated to control the immune balance. To exert this anti-inflammatory effect, Klebsiella engages NOD1. In NOD1 knock-down cells, Klebsiella neither induces the expression of CYLD and MKP-1 nor blocks the activation of NF-?B and MAPKs. Klebsiella inhibits Rac1 activation; and inhibition of Rac1 activity triggers a NOD1-mediated CYLD and MKP-1 expression which in turn attenuates IL-1ß-induced IL-8 secretion. A capsule (CPS) mutant does not attenuate the inflammatory response. However, purified CPS neither reduces IL-1ß-induced IL-8 secretion nor induces the expression of CYLD and MKP-1 thereby indicating that CPS is necessary but not sufficient to attenuate inflammation.

Dissection of Host Cell Signal Transduction during Acinetobacter baumannii – Triggered Inflammatory Response

Infected airway epithelial cells up-regulate the expression of chemokines, chiefly IL-8, and antimicrobial molecules including ß-defensins (BD). Acinetobacter baumannii is a cause of hospital-acquired pneumonia. We examined whether A. baumannii induced the expressions of IL-8 and BD2 by airway epithelial cells and the receptors implicated in bacterial detection. A549 and human primary airway cells released IL-8 upon infection. A. baumannii-infected cells also increased the expression of BD2 which killed A. baummannii strains. IL-8 induction was via NF-B and mitogen-activated kinases p38 and p44/42-dependent pathways. A. baumannii engaged Toll-like receptor (TLR) 2 and TLR4 pathways and A549 cells could use soluble CD14 as TLRs co-receptor. A. baumannii lipopolysaccharide stimulated IL-8 release by A549 cells and sCD14 facilitated the recognition of the lipopolysaccharide. Mass spectrometry analysis revealed that A. baumannii lipid A structure matches those with endotoxic potential. These results demonstrate that airway epithelial cells produce mediators important for A. baumannii clearance. © 2010 March et al.

Klebsiella pneumoniae targets an EGF receptor-dependent pathway to subvert inflammation

The NF-kB transcriptional factor plays a key role governing the activation of immune responses. Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that K. pneumoniae infections are characterized by lacking an early in?ammatory response. Recently, we have demonstrated that Klebsiella antagonizes the activation of NF-kB via the deubiquitinase CYLD. In this work, by applying a high-throughput siRNA gain-of-function screen interrogating the human kinome, we identi?ed 17 kinases that when targeted by siRNA restored IL-1b-dependent NF-kB translocation in infected cells. Further characterization revealed that K. pneumoniae activates an EGF receptor (EGFR)- phosphatidylinositol 3-OH kinase (PI3K)–AKT–PAK4–ERK–GSK3b signalling pathway to attenuate the cytokine-dependent nuclear translocation of NF-kB. Our data also revealed that CYLD is a downstream effector of K. pneumoniae-induced EGFR–
PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. Our efforts to identify the bacterial factor(s) responsible for EGFR activation demonstrate that a capsule (CPS) mutant did not activate EGFR hence
suggesting that CPS could mediate the activation of EGFR. Supporting this notion, puri?ed CPS did activate EGFR as well as the EGFR-dependent PI3K–AKT–PAK4–ERK–GSK3b signalling pathway. CPS-mediated EGFR activation was dependent on a TLR4–MyD88–c-SRC-dependent pathway. Several promising drugs have been developed to antagonize this cascade. We propose that agents targeting this signalling pathway might provide selective alternatives for the management of K. pneumoniae pneumonias.

Protection of corneal endothelium from irrigation damage:A comparison of sodium hyaluronate and hydroxypropylmethyl-cellulose

Purpose: To compare the endothelial protection of sodium hyaluronate and hydroxypropylmethylcellulose against endothelial damage induced by irrigation. Methods: An in vitro assay with freshly excised porcine eyes was developed using the Janus green photometry technique. Irrigation and aspiration technique was standardised. Forty pairs of porcine eyes were used. One randomly chosen eye was filled with sodium hyaluronate (SH) and the other with hydroxypropylmethylcellulose (HPMC). Irrigation and aspiration was carried out with balanced salt solution for 5 min. Twenty additional pairs of porcine eyes served as controls. Student's t-test was used for statistical analysis. Results: Both viscoelastic agents protected the endothelium as compared with controls. The endothelial protection, determined with the Janus green photometric technique, was significantly greater with HPMC than with SH. Conclusions: Viscoelastic agents are effective in protecting the endothelium from irrigation damage in porcine eyes in vitro. HPMC provided greater protection than SH in this particular model.

Arabidopsis Rab-E GTPases exhibit a novel interaction with a plasma-membrane phosphatidylinositol-4-phosphate 5-kinase

Rab GTPases of the Arabidopsis Rab-E subclass are related to mammalian Rab8 and are implicated in membrane trafficking from the Golgi to the plasma membrane. Using a yeast two-hybrid assay, Arabidopsis phosphatidylinositol-4-phosphate 5-kinase 2 (PtdIns(4)P 5-kinase 2; also known as PIP5K2), was shown to interact with all five members of the Rab-E subclass but not with other Rab subclasses residing at the Golgi or trans-Golgi network. Interactions in yeast and in vitro were strongest with RAB-E1d[Q74L] and weakest with the RAB-E1d[S29N] suggesting that PIP5K2 interacts with the GTP-bound form. PIP5K2 exhibited kinase activity towards phosphatidylinositol phosphates with a free 5-hydroxyl group, consistent with PtdIns(4)P 5-kinase activity and this activity was stimulated by Rab binding. Rab-E proteins interacted with PIP5K2 via its membrane occupancy and recognition nexus (MORN) domain which is missing from animal and fungal PtdIns(4)P 5-kinases. In plant cells, GFP:PIP5K2 accumulated at the plasma membrane and caused YFP:RAB-E1d to relocate there from its usual position at the Golgi. GFP:PIP5K2 was rapidly turned over by proteasomal activity in planta, and overexpression of YFP:PIP5K2 caused pleiotropic growth abnormalities in transgenic Arabidopsis. We propose that plant cells exhibit a novel interaction in which PIP5K2 binds GTP-bound Rab-E proteins, which may stimulate temporally or spatially localized PtdIns(4,5)P(2) production at the plasma membrane.

Phosphorylation of plant actin-depolymerising factor by calmodulin-like domain protein kinase

actin-depolymerising factor (ADF)/cofilin group of proteins are stimulus-responsive actin-severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser-6 but the kinase responsible is unknown [Smertenko et al,, Plant J. 14 (1998) 187-193]. We have partially purified the ADF kinase(s) and found it to be calcium-regulated and inhibited by N-(6-aminohesyl)-[H-3]5-chloro-1-naphthalenesulphonamide. Immunoblotting reveals that calmodulin-like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti-CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation, These data strongly suggest that plant ADP is phosphorylation by CDPK(s), a class of protein kinases unique to plants and protozoa. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Molecular mapping the presence of druggable targets in preinvasive and precursor breast lesions:a comprehensive review of biomarkers related to therapeutic interventions

The analysis of clinical breast samples using biomarkers is integral to current breast cancer management. Currently, a limited number of targeted therapies are standard of care in breast cancer treatment. However, these targeted therapies are only suitable for a subset of patients and resistance may occur. Strategies to prevent the occurrence of invasive lesions are required to reduce the morbidity and mortality associated with the development of cancer. In theory, application of targeted therapies to pre-invasive lesions will prevent their progression to invasive lesions with full malignant potential. The diagnostic challenge for pathologists is to make interpretative decisions on early detected pre-invasive lesions. Overall, only a small proportion of these pre-invasive lesions will progress to invasive carcinoma and morphological assessment is an imprecise and subjective means to differentiate histologically identical lesions with varying malignant potential. Therefore differential biomarker analysis in pre-invasive lesions may prevent overtreatment with surgery and provide a predictive indicator of response to therapy. There follows a review of established and emerging potential druggable targets in pre-invasive lesions and correlation with lesion morphology.