Tyrosine Kinase-dependent Manner Research Articles

Innate immune receptors co-recognition of polysaccharides initiates multi-pathway synergistic immune response

The law and mechanism of the interaction between polysaccharides and pattern recognition receptors (PRRs) has been unclear. Herein, three glucomannans with different structures were selected to explore the universal mechanism for PRRs to recognize glucomannans. Screening results showed that the silence of TLR4 but not TLR2 severely blocked the production of inflammatory cytokines and the transduction of signal pathways. In-depth results revealed that the participation of myeloid differentiation protein 2 (MD2) and CD14 and the dimerization of the TLR4-MD2 complex were required for glucomannan-activated TLR4 signal transduction. Mannose receptor (MR) was also engaged in glucomannan-induced respiratory burst, endocytosis, and inflammatory signaling pathways in a spleen tyrosine kinase-dependent manner. The internalization of glucomannans into the cytoplasm by MR directly initiated complex intracellular signaling cascades. Finally, molecular docking characterized the binding energy and binding sites between glucomannans and multiple receptors from other perspectives. The essence of glucomannans recognized by PRRs was the non-covalent interaction of multiple receptors and the subsequent transmission of the signal cascade was triggered in a multi-channel and cooperative manner. As a result, the hypothesis that “Innate immune receptors co-recognition of polysaccharides initiates multi-pathway synergistic immune response” was proposed to outline these meaningful phenomena.

Microglia and astrocytes mediate synapse engulfment in a MER tyrosine kinase-dependent manner after traumatic brain injury.

Recent studies have shown that microglia/macrophages and astrocytes can mediate synaptic phagocytosis through the MER proto-oncokinase in developmental or stroke models, but it is unclear whether the same mechanism is also active in traumatic brain injury. In this study, we established a mouse model of traumatic brain injury and found that both microglia/macrophages and astrocytes phagocytosed synapses and expression of the MER proto-oncokinase increased 14 days after injury. Specific knockout of MER in microglia/macrophages or astrocytes markedly reduced injury volume and greatly improved neurobehavioral function. In addition, in both microglia/macrophages-specific and astrocytes-specific MER knock-out mice, the number of microglia/macrophage and astrocyte phagocytosing synapses was markedly decreased, and the total number of dendritic spines was increased. Our study suggested that MER proto-oncokinase expression in microglia/macrophages and astrocytes may play an important role in synaptic phagocytosis, and inhibiting this process could be a new strategy for treating traumatic brain injury.

Activation of EGFR-KLF4 positive feedback loop results in acquired resistance to sorafenib in hepatocellular carcinoma.

Sorafenib is the standard first-line systemic chemotherapeutic drugs for advanced hepatocellular carcinoma (HCC), but acquired resistance to sorafenib is frequently observed in clinical practice. In this study, we first produced three sorafenib resistance (SR) HCC cell lines by using two human HCC cell lines (Hep3B and Huh7) and a human primary HCC cell line. We identified that epidermal growth factor receptor (EGFR) and Kruppel-like factor 4 (KLF4) are dramatically increased in the three SR HCC cell lines. Either inhibition of tyrosine kinase activity of EGFR with Erlotinib/Icotinib or inhibition of KLF4 expression with short hairpin RNA recovered the response of three SR HCC cell lines to sorafenib, suggesting the critical roles of EGFR tyrosine kinase and KLF4 on inducing SR. Luciferase activity and chromatin immunoprecipitation assays further determined that KLF4 promoted EGFR expression through inducing its transcription by directly binding to its promoter. EGFR, conversely, could also promote KLF4 expression through inducing its transcription by binding to its promoter in a tyrosine kinase-dependent manner, suggesting that a positive feedback loop formed by EGFR and KLF4 further amplifies their effects on inducing SR. Up to now, our findings that KLF4 induces the development of SR and it cooperates with EGFR to form a positive feedback loop to amplify their SR-inducing abilities have rarely been reported. Our findings bear possible implications for the improvement of the efficacy of sorafenib in HCC.

Apoptotic cell induction of miR-10b in macrophages contributes to advanced atherosclerosis progression in ApoE-/- mice.

We previously found that miR-10b inhibits cholesterol efflux from thioglycollate-elicited mouse peritoneal macrophages through repressing ATP binding cassette transporter (ABCA1). Herein, we deciphered the mechanism underlying macrophage miR-10b expression and the role of miR-10b in atherosclerosis. MiR-10b expression was increased in the arteries with advanced but not early atherosclerotic plaques of ApoE-/- mice. Free cholesterol-induced macrophage apoptotic cells (FC-AM) promoted miR-10b expression in mouse resident peritoneal macrophages (RPM) by up-regulation of Twist1/2 in a Mer receptor tyrosine kinase dependent manner. Surprisingly, antagomiR-10b de-repressed ABCA1 in RPM engulfing FC-AM but not in RPM alone or RPM-derived foam cells; systemic delivery of antagomiR-10b enhanced reverse cholesterol transport from RPM engulfing FC-AM but not from RPM or the foam cells in ApoE-/- mice. Mechanistically, RPM engulfing FC-AM possessed sufficient miR-10b functionally repressing ABCA1 expression, whereas RPM and foam cells had little miR-10b incompetently repressing ABCA1 expression. Notably, antagomiR-10b administration reduced advanced plaque size and also enhanced plaque stability in ApoE-/-mice, which were associated with increased plaque macrophage ABCA1 expression and reduced plaque apoptosis and inflammation. However, antagomiR-10b administration did not affect early atherosclerotic plaque formation in ApoE-/- mice. These data suggest that apoptotic cell induction of miR-10b in macrophages is important in advanced atherosclerosis progression.

Functional interaction between nonreceptor tyrosine kinase c-Abl and SR-Rich protein RBM39

RBM39, also known as splicing factor HCC1.4, acts as a transcriptional coactivator for the steroid nuclear receptors JUN/AP-1, ESR1/ER-α and ESR2/ER-β. RBM39 is involved in the regulation of the transcriptional responses of these steroid nuclear receptors and promotes transcriptional initiation. In this paper, we report that RBM39 interacts with the nonreceptor tyrosine kinase c-Abl. Both the Src homology (SH) 2 and SH3 domains of c-Abl interact with RBM39. The major tyrosine phosphorylation sites on RBM39 that are phosphorylated by c-Abl are Y95 and Y99, as demonstrated by liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) and mutational analysis. c-Abl was shown boost the transcriptional coactivation activity of RBM39 for ERα and PRβ in a tyrosine kinase-dependent manner. The results suggest that mammalian c-Abl plays an important role in steroid hormone receptor-mediated transcription by regulating RBM39.

Mitochondrial events responsible for morphine's cardioprotection against ischemia/reperfusion injury

Morphine may induce cardioprotection by targeting mitochondria, but little is known about the exact mitochondrial events that mediate morphine's protection. We aimed to address the role of the mitochondrial Src tyrosine kinase in morphine's protection. Isolated rat hearts were subjected to 30min ischemia and 2h of reperfusion. Morphine was given before the onset of ischemia. Infarct size and troponin I release were measured to evaluate cardiac injury. Oxidative stress was evaluated by measuring mitochondrial protein carbonylation and mitochondrial ROS generation. HL-1 cells were subjected to simulated ischemia/reperfusion and LDH release and mitochondrial membrane potential (ΔΨm) were measured. Morphine reduced infarct size as well as cardiac troponin I release which were aborted by the selective Src tyrosine kinase inhibitors PP2 and Src-I1. Morphine also attenuated LDH release and prevented a loss of ΔΨm at reperfusion in a Src tyrosine kinase dependent manner in HL-1 cells. However, morphine failed to reduce LDH release in HL-1 cells transfected with Src siRNA. Morphine increased mitochondrial Src phosphorylation at reperfusion and this was abrogated by PP2. Morphine attenuated mitochondrial protein carbonylation and mitochondrial superoxide generation at reperfusion through Src tyrosine kinase. The inhibitory effect of morphine on the mitochondrial complex I activity was reversed by PP2. These data suggest that morphine induces cardioprotection by preventing mitochondrial oxidative stress through mitochondrial Src tyrosine kinase. Inhibition of mitochondrial complex I at reperfusion by Src tyrosine kinase may account for the prevention of mitochondrial oxidative stress by morphine.

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr416) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.

IER3 is a crucial mediator of TAp73β-induced apoptosis in cervical cancer and confers etoposide sensitivity

Infection with high-risk human papillomaviruses (HPVs) causes cervical cancer. E6 oncoprotein, an HPV gene product, inactivates the major gatekeeper p53. In contrast, its isoform, TAp73β, has become increasingly important, as it is resistant to E6. However, the intracellular signaling mechanisms that account for TAp73β tumor suppressor activity in cervix are poorly understood. Here, we identified that IER3 is a novel target gene of TAp73β. In particular, TAp73β exclusively transactivated IER3 in cervical cancer cells, whereas p53 and TAp63 failed to do. IER3 efficiently induced apoptosis, and its knockdown promoted survival of HeLa cells. In addition, TAp73β-induced cell death, but not p53-induced cell death, was inhibited upon IER3 silencing. Moreover, etoposide, a DNA-damaging chemotherapeutics, upregulated TAp73β and IER3 in a c-Abl tyrosine kinase-dependent manner, and the etoposide chemosensitivity of HeLa cells was largely determined by TAp73β-induced IER3. Of interest, cervical carcinomas from patients express no observable levels of two proteins. Thus, our findings suggest that IER3 is a putative tumor suppressor in the cervix, and the c-Ab1/p73β/IER3 axis is a novel and crucial signaling pathway that confers etoposide chemosensitivity. Therefore, TAp73β and IER3 induction would be a valuable checkpoint for successful therapeutic intervention of cervical carcinoma patients.

Regulation of thrombomodulin expression and release in human aortic endothelial cells by cyclic strain.

Background and ObjectivesThrombomodulin (TM), an integral membrane glycoprotein expressed on the lumenal surface of vascular endothelial cells, promotes anti-coagulant and anti-inflammatory properties. Release of functional TM from the endothelium surface into plasma has also been reported. Much is still unknown however about how endothelial TM is regulated by physiologic hemodynamic forces (and particularly cyclic strain) intrinsic to endothelial-mediated vascular homeostasis.MethodsThis study employed human aortic endothelial cells (HAECs) to investigate the effects of equibiaxial cyclic strain (7.5%, 60 cycles/min, 24 hrs), and to a lesser extent, laminar shear stress (10 dynes/cm2, 24 hrs), on TM expression and release. Time-, dose- and frequency-dependency studies were performed.ResultsOur initial studies demonstrated that cyclic strain strongly downregulated TM expression in a p38- and receptor tyrosine kinase-dependent manner. This was in contrast to the upregulatory effect of shear stress. Moreover, both forces significantly upregulated TM release over a 48 hr period. With continuing focus on the cyclic strain-induced TM release, we noted both dose (0–7.5%) and frequency (0.5–2.0 Hz) dependency, with no attenuation of strain-induced TM release observed following inhibition of MAP kinases (p38, ERK-1/2), receptor tyrosine kinase, or eNOS. The concerted impact of cyclic strain and inflammatory mediators on TM release from HAECs was also investigated. In this respect, both TNFα (100 ng/ml) and ox-LDL (10–50 µg/ml) appeared to potentiate strain-induced TM release. Finally, inhibition of neither MMPs (GM6001) nor rhomboids (3,4-dichloroisocoumarin) had any effect on strain-induced TM release. However, significantly elevated levels (2.1 fold) of TM were observed in isolated microparticle fractions following 7.5% strain for 24 hrs.ConclusionsA preliminary in vitro investigation into the effects of cyclic strain on TM in HAECs is presented. Physiologic cyclic strain was observed to downregulate TM expression, whilst upregulating in a time-, dose- and frequency-dependent manner the release of TM.

Abstract 384: The Prothrombotic Profile of BCR-ABL Inhibitors Ponatinib, Nilotinib and Imatinib

Background: Thrombotic complications observed in patients treated with the tyrosine kinase inhibitor ponatinib resulted in the temporary suspension of the drug by the FDA in late 2013. Ponatinib is a pan-BCR-ABL inhibitor designed for treatment of chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Ponatinib has been shown to inhibit a subset of the class III/IV family of receptor tyrosine kinases. The pathogenic mechanism underlying the prothrombotic phenotype associated with ponatinib remains ill-defined. Hypothesis: We tested the hypothesis that BCR-ABL inhibitors regulate platelet activation, spreading, and aggregation. Methods & Results: Our results show that treatment of platelets with ponatinib (1 μM) abrogated platelet spreading on fibrinogen (50 μg/ml) or collagen (100 μg/ml). Both nilotinib and imatinib inhibited platelet spreading, although not to the same extent as ponatinib at equimolar concentrations. We next examined the effects of BCR-ABL inhibitors on platelet activation and aggregation in response to the GPVI-agonist, CRP, which mediates platelet activation in a receptor tyrosine kinase-dependent manner. Intriguingly, our results show that ponatinib abrogated platelet aggregation in response to CRP (1 μg/ml), whereas equimolar concentrations of nilotinib or imatinib had minimal effects on CRP-induced platelet aggregation. Conclusions: While tyrosine kinase inhibitors are generally associated with bleeding diathesis, thrombotic complications have been observed in patients treated with ponatinib. Our results indicate that ponatinib, nilotinib, and imatinib inhibit platelet spreading and aggregation, with ponatinib having the most significant effect, suggesting that ponatinib may act as a platelet antagonist. Our future work will be focused on identifying platelet proteins affected by the BCR-ABL inhibitors as well as the pathogenic mechanisms underlying the prothrombotic phenotype associated with ponatinib.

A novel cysteine-rich neurotrophic factor in Aplysia facilitates growth, MAPK activation, and long-term synaptic facilitation

Neurotrophins are critically involved in developmental processes such as neuronal cell survival, growth, and differentiation, as well as in adult synaptic plasticity contributing to learning and memory. Our previous studies examining neurotrophins and memory formation in Aplysia showed that a TrkB ligand is required for MAPK activation, long-term synaptic facilitation (LTF), and long-term memory (LTM) for sensitization. These studies indicate that neurotrophin-like molecules in Aplysia can act as key elements in a functionally conserved TrkB signaling pathway. Here we report that we have cloned and characterized a novel neurotrophic factor, Aplysia cysteine-rich neurotrophic factor (apCRNF), which shares classical structural and functional characteristics with mammalian neurotrophins. We show that apCRNF (1) is highly enriched in the CNS, (2) enhances neurite elongation and branching, (3) interacts with mammalian TrkB and p75NTR, (4) is released from Aplysia CNS in an activity-dependent fashion, (5) facilitates MAPK activation in a tyrosine kinase dependent manner in response to sensitizing stimuli, and (6) facilitates the induction of LTF. These results show that apCRNF is a native neurotrophic factor in Aplysia that can engage the molecular and synaptic mechanisms underlying memory formation.

Caspase-8 as a regulator of tumor cell motility.

The caspases are a family of ubiquitously expressed cysteine proteases best known for their roles in programmed cell death. However, caspases play a number of other roles in vertebrates. In the case of caspase-8, loss of expression is an embryonic lethal phenotype, and caspase-8 plays roles in suppressing cellular necrosis, promoting differentiation and immune signaling, regulating autophagy, and promoting cellular migration. Apoptosis and migration require localization of caspase-8 in the periphery of the cells, where caspase-8 acts as part of distinct biosensory complexes that either promote migration in appropriate cellular microenvironments, or cell death in inappropriate settings. In the cellular periphery, caspase-8 interacts with components of the focal adhesion complex in a tyrosine-kinase dependent manner, promoting both cell migration in vitro and metastasis in vivo. Mechanistically, caspase-8 interacts with components of both focal adhesions and early endosomes, enhancing focal adhesion turnover and promoting rapid integrin recycling to the cell surface. Clinically, this suggests that the expression of caspase-8 may not always be a positive prognostic sign, and that the role of caspase-8 in cancer progression is likely context-dependent.

BRCA1-Down-Regulation In Chronic Myeloid Leukemia (CML) Occurs Via The Deubiquitinase / Tumor Suppressor BRCA1-Associated Protein 1 (BAP1)

BCR-ABL has been shown to lead to a genetic instability in leukemic cells either directly by inducing oxidative stress or indirectly by compromising DNA repair mechanisms. BRCA1 is a major DNA repair gene as it promotes homologous recombination and plays thereby a critical role for preserving genomic integrity. We have previously reported that BCR-ABL down-regulates BRCA1 protein using a post-transcriptional mechanism (Deutsch et al, Blood 2003). The precise mechanism of this down regulation had not been established so far. BAP1 (BRCA1 associated protein-1) is a tumor suppressor gene that encodes a nuclear ubiquitin carboxy-terminal hydrolase, which interacts with BRCA1 protein and with many other cell cycle regulators. BAP1 is mutated in hereditary cancers and the overexpression of a deleted form of BAP1 has been shown to lead to a myelodysplastic syndrome in mice (Dey et al, Science 2012). In a gene profiling analysis of the human UT7 cells expressing BCR-ABL, we have discovered that the expression of BAP-1 is down-regulated as compared to parental UT7 cells. Using qRT-PCR and Western blotting analyses, we have confirmed the reduction of BAP1 transcript and protein levels in UT7 cells expressing BCR-ABL. This occurs in a tyrosine kinase dependent manner as exposure to Imatinib reverted BCR-ABL-associated BAP1 down-regulation. To determine the effects of BAP1 complementation in leukemic cells, we have transfected UT7-BCR-ABL cells with a BAP1 expression vector. The enforced expression of BAP1 in BCR-ABL expressing cells restored BRCA1 protein levels without affecting its mRNA level. As BAP1 is a deubiquitinase, we wondered whether there was an increased ubiquitination of BRCA1 in BCR-ABL expressing cells, due to a BAP1 deficiency. In the UT7-BCR-ABL model, we have performed immunoprecipitation of BRCA1 followed by Western blotting using anti-ubiquitin antibodies. These experiments revealed that BRCA1 was highly ubiquitinylated in BCR-ABL-expressing cells as compared to parental UT7 cells, explaining potentially its down-regulation in CML at the protein level via a proteasome-related mechanism. We next wished to validate these findings in primary human CML samples using qRT-PCR. In a cohort of newly diagnosed chronic phase CML patients before any therapy (n= 21) blood mRNA levels of BAP1 were significantly reduced ( p=0.0032, Mann Whitney Test ) as compared to normal controls (n= 8). Thus, our report reveals for the first time loss of BAP1 expression as a mechanism of the down-regulation of the DNA repair protein BRCA1 in CML. In addition to its contribution to genetic instability, BAP1 could be directly involved in the pathophysiology of CML due to its interactions with epigenetic factors such as polycomb proteins. The molecular mechanisms of BAP1 downregulation in BCR-ABL-expressing leukemic cells is under investigation. Disclosures:Guilhot:Novartis, BMS, Ariad, Pfizer: Honoraria. Turhan:BMS, Novartis: Honoraria, Research Funding.

Human platelets generate phospholipid-esterified prostaglandins via cyclooxygenase-1 that are inhibited by low dose aspirin supplementation

Oxidized phospholipids (oxPLs) generated nonenzymatically display pleiotropic biological actions in inflammation. Their generation by cellular cyclooxygenases (COXs) is currently unknown. To determine whether platelets generate prostaglandin (PG)-containing oxPLs, then characterize their structures and mechanisms of formation, we applied precursor scanning-tandem mass spectrometry to lipid extracts of agonist-activated human platelets. Thrombin, collagen, or ionophore activation stimulated generation of families of PGs comprising PGE2 and D2 attached to four phosphatidylethanolamine (PE) phospholipids (16:0p/, 18:1p/, 18:0p/, and 18:0a/). They formed within 2 to 5 min of activation in a calcium, phospholipase C, p38 MAP kinases, MEK1, cPLA2, and src tyrosine kinase-dependent manner (28.1 ± 2.3 pg/2 × 108 platelets). Unlike free PGs, they remained cell associated, suggesting an autocrine mode of action. Their generation was inhibited by in vivo aspirin supplementation (75 mg/day) or in vitro COX-1 blockade. Inhibitors of fatty acyl reesterification blocked generation significantly, while purified COX-1 was unable to directly oxidize PE in vitro. This indicates that they form in platelets via rapid esterification of COX-1 derived PGE2/D2 into PE. In summary, COX-1 in human platelets acutely mediates membrane phospholipid oxidation via formation of PG-esterified PLs in response to pathophysiological agonists.

Osteoarthritis-associated basic calcium phosphate crystals induce IL-1β, IL-18 and S100A8 production in a tyrosine kinase dependent manner

s / Osteoarthritis and Cartilage 20 (2012) S54–S296 S233 Th2, Th17, Treg) and activation markers. T-cell cytokine secretion (IL-2, IL4, IL-6, IL-10, IL-17A, INF-g, TNF) in SF was measured by cytometric bead array (CBA) analysis. Results: Analysis of PB did not show any significant difference within the OA patient groups and in comparison to controls. In SM a distinct infiltration of CD4+ T-cells was present in all three OA groups, which significantly increasedwith OA stage (p<0.05). Further, CD4+ T-cell subsets in SM and PB samples were analysed. Whereas in PB only a small proportion of CD4+ T-cells were activated and stained positive for the specific subsets, the majority of SM T-cells were differentiated into the T-cell subsets and this increasedwith OA stage (Figure 1). The highest increase wasmeasured for the inflammatory T cell subsets Th1 and Th17, which significantly altered the Th1/Th2 and Th17/Treg balance. Further analysis of activation markers displayed an activated phenotype of CD4+ T-cells in the SM (CD69+, CD45RO+, CD45RA-, CD62L-) but not in PB. CBA analysis confirmed a significant increase of all T-cell cytokines in the SF of OA groups 2 and 3 when compared to group 1 (p<0.05). Between group 2 and 3, pro-inflammatory Th1 and Th17 cytokines (TNF and IL-17) were further increased and anti-inflammatory Th2 and Treg cytokines (IL-4, IL-10) decreased in group 3. IL-2, IL-6 and INF-g were slightly higher in group 2. Conclusions: In contrast to SM, no differences were observed between the PB samples of OA patients and controls, showing that the relevant role of inflammatory cells in OA remains a local process. The infiltration of OA synoviumwith inflammatory cells is not solely a phenomenon of late stage OA. In fact, CD4+ T-cells are already present and activated in the affected joints of early OA stages. T-cell infiltration and polarisation correlates with OA disease stages and shifts the Th1/Th2 and Th17/Treg balance towards an inflammatory T-cell phenotype. Thus, onset and progression of OA is accompanied by an increasing influx of CD4+ T-cells into the SM, their activation, cytokine production and polarisation. These results underline the relevance of T-cells in the inflammatory process of OA, not only in latestage disease but also in early OA stages. In “classical” inflammatory joint diseases such as rheumatoid arthritis we know that T-cell derived inflammation contributes to cartilage degeneration and joint destruction. The role of these cells in OA needs to be further studied. Nevertheless our current results suggest that T-cell activation and polarisation may play a role in both the initiation and progression of OA, and as such provide a potential therapeutic target.

An antinociceptive role for substance P in acid-induced chronic muscle pain

Release of substance P (SP) from nociceptive nerve fibers and activation of its receptor neurokinin 1 (NK1) are important effectors in the transmission of pain signals. Nonetheless, the role of SP in muscle pain remains unknown. Here we show that a single i.m. acid injection in mice lacking SP signaling by deletion of the tachykinin precursor 1 (Tac1) gene or coadministration of NK1 receptor antagonists produces long-lasting hyperalgesia rather than the transient hyperalgesia seen in control animals. The inhibitory effect of SP was found exclusively in neurons expressing acid-sensing ion channel 3, where SP enhances M-channel-like potassium currents through the NK1 receptor in a G protein-independent but tyrosine kinase-dependent manner. Furthermore, the SP signaling could alter action potential thresholds and modulate the expression of TTX-resistant sodium currents in medium-sized muscle nociceptors. Thus, i.m. SP mediates an unconventional NK1 receptor signal pathway to inhibit acid activation in muscle nociceptors, resulting in an unexpected antinociceptive effect against chronic mechanical hyperalgesia, here induced by repeated i.m. acid injection.

Alternatively activated macrophage-derived RELM-α is a negative regulator of type 2 inflammation in the lung

Differentiation and recruitment of alternatively activated macrophages (AAMacs) are hallmarks of several inflammatory conditions associated with infection, allergy, diabetes, and cancer. AAMacs are defined by the expression of Arginase 1, chitinase-like molecules, and resistin-like molecule (RELM) α/FIZZ1; however, the influence of these molecules on the development, progression, or resolution of inflammatory diseases is unknown. We describe the generation of RELM-α–deficient (Retnla−/−) mice and use a model of T helper type 2 (Th2) cytokine-dependent lung inflammation to identify an immunoregulatory role for RELM-α. After challenge with Schistosoma mansoni (Sm) eggs, Retnla−/− mice developed exacerbated lung inflammation compared with their wild-type counterparts, characterized by excessive pulmonary vascularization, increased size of egg-induced granulomas, and elevated fibrosis. Associated with increased disease severity, Sm egg–challenged Retnla−/− mice exhibited elevated expression of pathogen-specific CD4+ T cell–derived Th2 cytokines. Consistent with immunoregulatory properties, recombinant RELM-α could bind to macrophages and effector CD4+ Th2 cells and inhibited Th2 cytokine production in a Bruton's tyrosine kinase–dependent manner. Additionally, Retnla−/− AAMacs promoted exaggerated antigen-specific Th2 cell differentiation. Collectively, these data identify a previously unrecognized role for AAMac-derived RELM-α in limiting the pathogenesis of Th2 cytokine-mediated pulmonary inflammation, in part through the regulation of CD4+ T cell responses.

The Dynamics of the Rapsyn Scaffolding Protein at Individual Acetylcholine Receptor Clusters

Rapsyn, a cytoplasmic receptor-associated protein, is required for the clustering of acetylcholine receptors (AChRs). Although AChR dynamics have been extensively studied, little is known about the dynamics of rapsyn. Here, we used a rapsyn-green fluorescent protein (GFP) fusion protein and quantitative fluorescent imaging to study the dynamics of rapsyn in transfected C2C12 myotubes. First, we found that rapsyn-GFP expression at clusters did not alter AChR aggregation, function, or turnover. Quantification of rapsyn immunofluorescence indicated that the expression of rapsyn-GFP proteins at clusters does not increase the overall rapsyn density compared with untransfected myotube clusters. Using time lapse imaging and fluorescence recovery after photobleaching, we demonstrated that the recovery of rapsyn-GFP fluorescence at clusters was very fast, with a halftime of about approximately 1.5 h (approximately 3 times faster than AChRs). Inhibition of protein kinase C significantly altered receptor insertion, but it had no effect on rapsyn insertion. When cells were treated with the broad spectrum kinase inhibitor staurosporine, receptor insertion was decreased even further. However, inhibition of protein kinase A had no effect on insertion of either rapsyn or receptors. Finally, when cells were treated with neural agrin, rapsyn and AChRs were both directed away from preexisting clusters and accumulated together in new small clusters. These results demonstrate the remarkable dynamism of rapsyn, which may underlie the stability and maintenance of the postsynaptic scaffold and suggest that the insertion of different postsynaptic proteins may be operating independently.

Expression of CCL9/MIP-1γ is repressed by BCR/ABL and its restoration suppresses in vivo leukemogenesis of 32D-BCR/ABL cells

Transformation of hematopoietic cells by the BCR/ABL oncogene is caused by perturbation of signal transduction pathways leading to altered patterns of gene expression and activity. By oligonucleotide microarray hybridization of polysomal RNA of untreated and STI571-treated 32D-BCR/ABL cells, we identified the beta-chemokine CCL9 as a gene regulated by BCR/ABL in a tyrosine kinase-dependent manner. BCR/ABL repressed CCL9 expression at the transcriptional level by mechanisms involving suppression of p38 MAP kinase, and modulation of the activity of CDP/cut and C/EBPalpha, two transcription regulators of myeloid differentiation. However, repression of C/EBP-dependent transcription did not prevent the induction of CCL9 expression by STI571, suggesting that C/EBPalpha is involved in maintaining rather than in inducing CCL9 expression. Restoration of CCL9 expression in 32D-BCR/ABL cells had no effect on the in vitro proliferation of these cells, but reduced their leukemogenic potential in vivo, possibly by recruitment of CD3-positive immune cells. Together, these findings suggest that downregulation of chemokine expression may be involved in BCR/ABL-dependent leukemogenesis by altering the relationship between transformed cells and the microenvironment.

Differential Roles of Vascular Endothelial Growth Factor Receptor-1 and Receptor-2 in Angiogenesis

Vascular endothelial growth factor (VEGF)-A, a major regulator for angiogenesis, binds and activates two tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/Flk-1). These receptors regulate physiological as well as pathological angiogenesis. VEGFR2 has strong tyrosine kinase activity, and transduces the major signals for angiogenesis. However, unlike other representative tyrosine kinase receptors which use the Ras pathway, VEGFR2 mostly uses the Phospholipase-Cgamma-Protein kinase-C pathway to activate MAP-kinase and DNA synthesis. VEGFR2 is a direct signal transducer for pathological angiogenesis including cancer and diabetic retinopathy, thus, VEGFR2 itself and the signaling appear to be critical targets for the suppression of these diseases. VEGFR1 plays dual role, a negative role in angiogenesis in the embryo most likely by trapping VEGF-A, and a positive role in adulthood in a tyrosine kinase-dependent manner. VEGFR1 is expressed not only in endothelial cells but also in macrophage-lineage cells, and promotes tumor growth, metastasis, and inflammation. Furthermore, a soluble form of VEGFR1 was found to be present at abnormally high levels in the serum of preeclampsia patients, and induces proteinurea and renal dysfunction. Therefore, VEGFR1 is also an important target in the treatment of human diseases. Recently, the VEGFR2-specific ligand VEGF-E (Orf-VEGF) was extensively characterized. Interestingly, the activation of VEGFR2 via VEGF-E in vivo results in a strong angiogenic response in mice with minor side effects such as inflammation compared with VEGF-A, suggesting VEGF-E to be a novel material for pro-angiogenic therapy.