Role Of Syk Research Articles

Protein-Sequence-Based Search of Nonreceptor ITAM-Like Regions to Identify Cytosolic Syk-Recruiting Proteins.

The recruitment of the protein spleen tyrosine kinase (Syk) to membrane-bound immune receptors is an essential step in initiating an immune response mediated through the activated receptors. Syk recognizes intracellular phosphorylated regions of membrane receptors known as immunoreceptor tyrosine-based activation motifs (ITAMs) defined by a sequence with two tyrosine (Y) amino acids separated by a certain spacing of six to eight residues: YXX(I/L)X6-8YXX(I/L). Syk with doubly phosphorylated ITAM is high-affinity and negatively regulated when Syk itself becomes phosphorylated. While the role of Syk in immune signaling is well characterized, recent information affords new functionality to Syk related to cytoplasmic processes, including the clearance of stress granules and P-bodies, both formed by liquid-liquid phase separation. Little to nothing is known about the molecular interactions involving Syk in these cytoplasmic processes. Given the essential role of receptor ITAMs in recruiting and localizing Syk for immune signaling, we explore here the possibility of a similar localization mechanism occurring for cytoplasmic processes by searching sequences of proteins related to Syk cytoplasmic function for regions that resemble receptor ITAMs. Protein sequence databases were generated from a Syk-dependent phosphoproteome and from genes related to P-bodies. A search of these databases for ITAM-like sequences yielded 102 unique hits, and 33 of these were synthesized and tested experimentally for binding to Syk tandem SH2 domains. The equilibrium dissociation constants were 0.1-50 μM for 28 peptides, and binding was negatively regulated by phosphorylation for many peptides. These results identify cytoplasmic proteins with potential for regulating the localization of Syk in a phosphorylation-dependent manner to nonmembrane cellular regions.

Syk inhibitors reduce tau protein phosphorylation and oligomerization

Spleen tyrosine kinase (Syk), a non-receptor-type tyrosine kinase, has a wide range of physiological functions. A possible role of Syk in Alzheimer's disease (AD) has been proposed. We evaluated the localization of Syk in the brains of patients with AD and control participants. Human neuroblastoma M1C cells harboring wild-type tau (4R0N) were used with the tetracycline off (TetOff) induction system. In this model of neuronal tauopathy, the effects of the Syk inhibitors—BAY 61–3606 and R406—on tau phosphorylation and oligomerization were explored using several phosphorylated tau-specific antibodies and an oligomeric tau antibody, and the effects of these Syk inhibitors on autophagy were examined using western blot analyses. Moreover, the effects of the Syk inhibitor R406 were evaluated in vivo using wild-type mice. In AD brains, Syk and phosphorylated tau colocalized in the cytosol. In M1C cells, Syk protein (72 kDa) was detected using western blot analysis. Syk inhibitors decreased the expression levels of several tau phosphoepitopes including PHF-1, CP13, AT180, and AT270. Syk inhibitors also decreased the levels of caspase-cleaved tau (TauC3), a pathological tau form. Syk inhibitors increased inactivated glycogen synthase kinase 3β expression and decreased active p38 mitogen-activated protein kinase expression and demethylated protein phosphatase 2 A levels, indicating that Syk inhibitors inactivate tau kinases and activate tau phosphatases. Syk inhibitors also activated autophagy, as indicated by increased LC3II and decreased p62 levels. In vivo, the Syk inhibitor R406 decreased phosphorylated tau levels in wild-type mice. These findings suggest that Syk inhibitors offer novel therapeutic strategies for tauopathies, including AD.

Spleen tyrosine kinase regulates keratinocyte inflammasome activation and skin inflammation induced by UVB irradiation

UVB can induce inflammatory responses contributing to diverse skin damage. UVB-triggered inflammasome activation of human keratinocytes underlies UVB-induced skin sunburn reaction. Pleiotropic functions of spleen tyrosine kinase (Syk) have rendered it as a potential therapeutic target. In immunocytes, Syk modulates immunoreceptor signaling and NLRP3 inflammasome activation. In skin, Syk mediates EGFR signaling, regulates keratinocyte differentiation and is involved in inflammatory disorders. However, roles of Syk in UVB-induced inflammasome activation in keratinocytes remain elusive. We investigated roles of keratinocyte Syk in UVB-triggered photo-responses. Primary normal human epidermal keratinocytes (NHEKs) isolated from skin were used. Syk knockdown or Syk inhibitor R406 was applied to investigate functions of keratinocyte Syk in UVB photobiology. The possible in vivo role of Syk was evaluated by checking UVB-induced skin damage in R406-treated mice. UVB was able to induce Syk phosphorylation in NHEKs that could be regulated by reactive oxygen species (ROS) generation and EGFR. Syk knockdown or Syk inhibitor (R406) treatment reduced UVB-triggered apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) crosslinking, procaspase-1 cleavage, active IL-1β formation, and gasdermin D activation, indicating roles of Syk in UVB-triggered inflammasome activation in keratinocytes. UVB-induced production of IL-8, TNF-α, ROS, and phosphorylation of JNK and p38 were attenuated after Syk knockdown or inhibition. R406 ameliorated UVB-induced mouse skin damage, including erythema and transepidermal water loss (TEWL). Thus, Syk participated in UVB-induced inflammasome activation and inflammatory response in vitro and in vivo, suggesting potential photo-protective effects of Syk inhibition in UVB-induced skin inflammation.

P6493Syk activation is a defense mechanism in murine model of aortic dissection

Abstract Background Aortic dissection (AD) is a serious clinical condition with unknown etiology that frequently results in fatal outcome. Recent studies showed essential role of inflammatory response both in promoting AD and aortic aneurysm (AA). However, the difference of the molecular pathogenesis between AD and AA is unclear. Previously, we reported that Syk, a tyrosine kinase that regulates differentiation and activation of inflammatory cells, promotes AA formation in a mouse model. Objective In the current study, we investigated the role of Syk in AD. Methods and results A mouse AD model was created by continuous infusion of beta-aminopropionitrile (125 mg/kg/day) and angiotensin II (1,000 ng/min/kg) (BAPN+AngII), which caused AD in approximately 80% of mice within 14 days. Immunohistochemical staining for activated (phosphorylated) Syk (pSyk) revealed that Syk was inactive in normal mouse aorta, but was activated in the aortic walls after AD development. Double immunofluorescence staining for pSyk and smooth muscle alpha actin showed that Syk was active not only in the infiltrating inflammatory cells, but also in smooth muscle cells in AD tissue. Western blot analysis revealed that BAPN+AngII treatment caused Syk activation at 3 days before AD development, followed by transient suppression, and reactivation at 14 days after AD development. We examined the significance of Syk activation in AD by treating mice with fostamatinib, a specific Syk inhibitor, before and during BAPN+AngII infusion. Notably, fostamatinib-treated group developed more severe AD compared to the vehicle-treated group. The AD lesion length was 3.80±0.86 mm for vehicle group and 8.87±1.69 mm for fostamatinib group (P<0.05, n=12 for each group). In addition, fostamatinib significantly worsened the mortality of mice due to the rupture of the aorta from 0% to 42% (P<0.05, n=12 for each group). Transcriptome analysis revealed that fostamatinib suppressed both positive and negative regulators of immune response, defense response and inflammatory response. Conclusions These findings uncovered the previously unrecognized role of Syk for protecting the aortic tissue in AD pathogenesis, and suggested fundamentally different disease mechanisms of AD and AA.

Syk/PECAM-1 Signaling Pathway Mediates Low Shear Stress-Induced Inflammatory in Atherosclerosis

Although blood flow shear stress has been demonstrated to alter the physiology of vascular endothelial cells, little is known about the role of Syk in PECAM-1 signaling pathway exposed to low shear stress (LSS). Methods: In vitro, primary human umbilical vein endothelial cells (HUVECs) were stimulated with parallel plate flow chamber system for 12h under normal shear stress (NSS, 15dyne/cm2), LSS (5dyne/cm2) and high shear stress (HSS, 25dyne/cm2), respectively, followed by inflammatory response analysis. In vivo, atherosclerotic animal models of rat and rabbit were created by feeding with high cholesterol diets. The animals were applied with LSS stimulation by constricting left carotid artery with a blunted needle (0.8mm in diameter). Small molecule R406, a well-demonstrated Syk inhibitor, was applied to the atherosclerotic animal models as well as HUVEC cells. Results: LSS influenced carotid atherosclerosis in the LSS model of rabbits; LSS promoted expression of PECAM-1 and Syk in LSS-treated atherosclerotic rat model; LSS influenced the morphology of abdominal aortic endothelial cells in rats; LSS increased the expression of pPECAM-1, Syk and NF-κB in HUVECs, and inhibition of Syk attenuated LSSinduced inflammatory response. Conclusion: Activation of spleen tyrosine kinase (Syk)/platelet endothelial cell adhesion molecule-1(PECAM-1) signaling pathway was accompanied with inflammatory response induced by LSS stimulation both in vitro and in vivo. This work demonstrated the important role Syk/PECAM1/NF-κB signaling pathway plays in LSS-induced inflammatory response. Funding Statement: This work was supported by Pudong New Area Health Bureau of Shanghai (grant no.PWZbr2017-09), and the National Natural Science Foundation of China (grant no.81401428, 81571693 and 81871361). Declaration of Interests: The authors have declared that no competing interest exists. Ethics Approval Statement: Human umbilical vein from normal full-term newborns was obtained with informed consent and approved in accordance with the procedures of the Medical Ethics Committee of Shanghai East Hospital Affiliated to Tongji University (China).

The role of Syk in peripheral T cells

The aim of this study was to understand how Syk affects peripheral T cell function. T cells from Syk−/− chimeric mice and DR1 Sykfl/fl CD4cre conditional mice gave strong CD3-induced Th1, Th2, and Th17 cytokine responses. However, an altered peptide ligand (APL) of human CII (256–276) with two substitutions (F263N, E266D), also called A12, elicited only Th2 cytokine responses from Sykfl/fl T cells but not Sykfl/fl-CD4cre T cells. Western blots revealed a marked increase in the phosphorylation of Syk, JNK and p38 upon A12/DR1 activation in WT or Sykfl/fl T cells but not in Sykfl/flCD4-cre cells. We demonstrate that Syk is required for the APL- induction of suppressive cytokines. Chemical Syk inhibitors blocked activation of GATA-3 by peptide A12/DR1. In conclusion, this study provides novel insights into the role that Syk plays in directing T cell activity, and may shape therapeutic approaches for autoimmune diseases.

The Syk Tyrosine Kinase Is Required for Skin Inflammation in an In Vivo Mouse Model of Epidermolysis Bullosa Acquisita

The inflammatory form of epidermolysis bullosa acquisita is caused by autoantibodies against type VII collagen (C7), a component of the dermal-epidermal junction. We have previously shown that myeloid Src family kinases mediate skin inflammation triggered by anti-C7 antibodies. Here we identify the Syk tyrosine kinase as a critical component of autoantibody-induced skin inflammation downstream of Src family kinases. Immobilized C7–anti-C7 immune complexes triggered neutrophil activation and Syk phosphorylation in a Src family kinase-dependent manner. Bone marrow chimeric mice lacking Syk in their hematopoietic compartment were completely protected from skin inflammation triggered by anti-C7 antibodies despite normal circulating anti-C7 levels. Syk deficiency abrogated the accumulation of CXCL2, IL-1β, and leukotriene B4 at the site of inflammation and resulted in defective in vivo neutrophil recruitment. Syk–/– neutrophils had a normal intrinsic migratory capacity but failed to release CXCL2 or leukotriene B4 upon activation by immobilized C7–anti-C7 immune complexes, indicating a role for Syk in the amplification of the inflammation process. These results identify Syk as a critical component of skin inflammation in a mouse model of epidermolysis bullosa acquisita and as a potential therapeutic target in epidermolysis bullosa acquisita and other mechanistically related inflammatory skin diseases such as bullous pemphigoid.

Inhibition of spleen tyrosine kinase attenuates IgE-mediated airway contraction and mediator release in human precision cut lung slices.

Asthma presents as a heterogeneous syndrome characterized by airway obstruction, inflammation and hyper-reactivity (AHR). Spleen tyrosine kinase (Syk) mediates allergen-induced mast cell degranulation, a central component of allergen-induced inflammation and AHR. However, the role of Syk in IgE-mediated constriction of human small airways remains unknown. In this study, we addressed whether selective inhibition of Syk attenuates IgE-mediated constriction and mast cell mediator release in human small airways. Human precision cut lung slices (hPCLS) ex vivo derived from non-asthmatic donors were incubated overnight with human IgE, dexamethasone, montelukast, antihistamines or a selective Syk inhibitor (SYKi). High-affinity IgE receptor (FcεRI) activation by anti-IgE cross-linking was performed, and constriction and mediator release measured. Airway constriction was normalized to that induced by maximal carbachol stimulation. Syk expression (determined by qPCR and immunoblot) was also evaluated in human primary airway smooth muscle (HASM) cells to determine whether Syk directly modulates HASM function. While dexamethasone had little effect on FcεR-mediated contraction, montelukast or antihistamines partially attenuated the response. SYKi abolished anti-IgE-mediated contraction and suppressed the release of mast cell or basophil mediators from the IgE-treated hPCLS. In contrast, SYKi had little effect on the non-allergic contraction induced by carbachol. Syk mRNA and protein were undetectable in HASM cells. A selective Syk inhibitor, but not corticosteroids, abolished FcεR-mediated contraction in human small airways ex vivo. The mechanism involved FcεRI receptor activation on mast cells or basophils that degranulate causing airway constriction, rather than direct actions on HASM.

Regulatory roles of Syk in epidermal keratinocyte differentiation

Regulatory roles of Syk in epidermal keratinocyte differentiation

The role of Syk in cutaneous lupus erythematosus.

The role of Syk in cutaneous lupus erythematosus.

Cholesterol crystals activate Syk and PI3 kinase in human macrophages and dendritic cells

Background and aimsCholesterol crystals are a key component of atherosclerotic lesions where they promote pro-inflammatory cytokine production and plaque destabilization. Antagonists of inflammatory mediators and agents that dissolve or prevent the formation of cholesterol crystals are being explored as potential therapeutics for atherothrombosis. We sought to identify signalling molecules activated following exposure of immune cells to cholesterol crystals with the view to identifying novel therapeutic targets. MethodsHuman macrophages and dendritic cells (DC) were exposed to cholesterol crystals and activation of signalling molecules was assessed by immunoblotting. The role of Syk and PI3K in crystal-induced interleukin (IL)-1 production was determined by ELISA using specific kinase inhibitors. Real-time PCR was employed to examine the role of Syk/PI3K in cholesterol crystal-induced expression of S100 proteins and MMPs. ResultsExposure of human macrophages and DC to cholesterol crystals induced robust activation of Syk and PI3K within 2–5 min. Pharmacological inhibition of Syk/PI3K reduced crystal-induced IL-1α/β production by approximately 80%. Activation of the downstream MAP kinases, MEK and ERK, was suppressed following inhibition of Syk and PI3K. Finally, inhibition of both Syk and PI3K significantly reduced cholesterol crystal-induced S100A8 and MMP1 gene expression by >70% while inhibition of PI3K also reduced S100A12 expression. ConclusionCholesterol crystals activate specific cell signalling pathways which drive the production of inflammatory cytokines and degradative enzymes known to contribute to disease initiation and progression. These molecular events are dependent on activation of Syk and PI3K, hence, they represent potential therapeutic targets for the treatment of cholesterol crystal-related pathologies.

Abstract IA38: Identification of targetable vulnerabilities in lymphoma.

Abstract Diffuse large B- cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma in adults and a clinically and genetically heterogeneous disorder. With current therapy, over 60% of patients with DLBCL can be cured; however, the remaining patients succumb to their disease. DLBCL largely originates in germinal center (GC) B cells, which have high growth rates and increased genomic instability. As a consequence, DLBCLs exhibit multiple low frequency genetic alterations including chromosomal translocations, somatic mutations and copy number alterations (CNAs). We have used several complementary comprehensive strategies to characterize the molecular heterogeneity of DLBCL and identify associated rational therapeutic targets. Using a combination of high-density – SNP arrays, whole exome sequencing, gene expression profiling and pathway analyses, we have comprehensively defined CNAs, candidate driver genes and perturbed signaling pathways in large series of newly diagnosed DLBCLs. The approach revealed a complementary set of CNAs and somatic mutations that decreased p53 activity and perturbed cell cycle regulation. The genetic signature of p53 deficiency and cell cycle deregulation was highly predictive for unfavorable outcome and potentially amenable to targeted therapy. We previously applied consensus clustering methods to the transcriptional profiles of primary DLBCLs and identified 3 highly reproducible tumor groups: BCR (B-cell receptor), OxPhos (oxidative phosphorylation) and HR (host response). “BCR” DLBCLs have increased expression of multiple components of the BCR signaling pathway and increased reliance on BCR-mediated survival signals. We recently characterized distinct BCR/SYK/PI3K-dependent survival pathways in DLBCLs with high or low baseline NF-κB activity and defined specific survival mechanisms in BCR-dependent NF-κB low DLBCLs. In these studies, SYK/PI3K/AKT-dependent cholesterol biosynthesis was found to be a feed-forward mechanism of preserving the integrity of BCRs in lipid rafts in DLBCLs with low- or high-baseline NF-κB. Consistent with the roles of SYK and PI3K/AKT as important mediators of BCR survival signals in primary DLBCLs, SYK amplification and PTEN deletion were identified as selective and largely mutually exclusive genetic alterations in “BCR”-type tumors. These studies suggest potential strategies for specifically targeting BCR-dependent survival signals in defined subtypes of DLBCL. In additional studies, we found that predominant fuel utilization pathways in BCR and OxPhos DLBCLs differ. Whereas BCR DLBCLs primarily utilize aerobic glycolysis, OxPhos DLBCLs largely depend upon fatty acid oxidation and exhibit selective sensitivity to targeted inhibition of this fuel utilization pathway. These findings further define molecular subsets of DLBCLs with distinct metabolic features that are linked to the functional state of upstream BCR signaling components. Citation Format: Margaret Shipp. Identification of targetable vulnerabilities in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr IA38.

Signal Transduction of Zymosan and Spleen Tyrosine Kinase

Zymosan is a component produced by polysaccharide which is obtained from cell wall of Saccharomyces cerevisiae. Zymosan consists of cross-linked polysaccharides such as 1,3-s glucan, 1,6-s glucan, and ?-mannan. Zymosan plays a role in inflammmatory diseases such as fungal sepsis, non-septic shock, multiple organ dysfunction syndrome, acute peritonitis, irritable bowel syndrome and it is used in related experimental models. Toll-like receptor 2, dectin-1, mannose, and compleman receptors mediate zymosan’s effects on immune system cells such as monocytes, macrophages, and dendritic cells. Spleen tyrosine kinase (Syk) is a cytosolic, intracellular, 72-kDa protein tyrosine kinase. It is demonstrated that Syk is expressed in hematopoietic cells such as mast cells, neutrophils, macrophages, B cells, leukocytes, and platelets and in non-hematopoietic cells such as epithelial, fibroblast, neuronal cells, and vascular endothelial cells. Spleen tyrosine kinase is a critical kinase that mediates cellular responses through dectin-1 receptor by zymosan. In this review, the role of syk in is evaluated in the signal transduction of zymosan.

3,4-Methylenedioxy-β-nitrostyrene Inhibits NLRP3 Inflammasome Activation by Blocking Assembly of the Inflammasome

The NLRP3 inflammasome is a critical component of the innate immune system. NLRP3 activation is induced by diverse stimuli associated with bacterial infection or tissue damage, but its inappropriate activation is involved in the pathogenesis of inherited and acquired inflammatory diseases. However, the mechanism by which NLRP3 is activated remains poorly understood. In this study, we explored the role of kinases in NLRP3 inflammasome activation by screening a kinase inhibitor library and identified 3,4-methylenedioxy-β-nitrostyrene (MNS) as an inhibitor for NLRP3 inflammasome activation. Notably, MNS did not affect the activation of the NLRC4 or AIM2 (absent in melanoma 2) inflammasome. Mechanistically, MNS specifically prevented NLRP3-mediated ASC speck formation and oligomerization without blocking potassium efflux induced by NLRP3 agonists. Surprisingly, Syk kinase, the reported target of MNS, did not mediate the inhibitory activity of MNS on NLRP3 inflammasome activation. We also found that the nitrovinyl group of MNS is essential for the inhibitory activity of MNS. Immunoprecipitation, mass spectrometry, and mutation studies suggest that both the nucleotide binding oligomerization domain and the leucine-rich repeat domain of NLRP3 were the intracellular targets of MNS. Administration of MNS also inhibited NLRP3 ATPase activity in vitro, suggesting that MNS blocks the NLRP3 inflammasome by directly targeting NLRP3 or NLRP3-associated complexes. These studies identified a novel chemical probe for studying the molecular mechanism of NLRP3 inflammasome activation which may advance the development of novel strategies to treat diseases associated with abnormal activation of NLRP3 inflammasome.

Study of the expression and function of Syk and VEGF-C in the development of non-small cell lung cancer

To explore the role of Syk and VEGF-C in the development of NSCLC. Transfered these genes(eukaryotic expression vector pcDNA3.1-VEGF-C and pLNCX-syk)into A549 cells with the liposomes. Tested the expression of VEGF-C mRNA and Syk mRNA with RT-PCR.Investigated the cell invasion assay with transwell chamber in vitro. Analysis the expression of VEGF-C proteins in A549 cells and detect Syk and VEGF-C proteins of 81 NSCLC tissue samples with immunohistochemical staining. Higher expression of VEGF-C was revealed in VEGF-C-construct-transfected A549 cell than that in controls through RT-PCR (P < 0.05) and immunohistochemistry(P < 0.01).RT-PCR also revealed that Syk expression was higher in Syk-construct-transfected cells than in controls (P < 0.05). The cell invading experiments showed that there was more invaded cells in both transfected terms than in controls (P < 0.05). The expression of Syk protein in NSCLC tissue were significantly lower than that in the normal lung tissue (P < 0.05), while the expression of VEGF-C protein in NSCLC tissue were significantly higher than that in the normal lung tissue(P < 0.05). The expression of Syk and VEGF-C has a negative correlationship (r = -1.000, P = 0.019). The expression of Syk and VEGF-C has a negative correlationship in NSCLC tissue, VEGF-C-construct-transfected A549 cells are more aggressive than Syk-construct-transfected cells. And they may cooperated with each other in the development of NSCLC.

Identification of Direct Tyrosine Kinase Substrates Based on Protein Kinase Assay-Linked Phosphoproteomics

Protein kinases are implicated in multiple diseases such as cancer, diabetes, cardiovascular diseases, and central nervous system disorders. Identification of kinase substrates is critical to dissecting signaling pathways and to understanding disease pathologies. However, methods and techniques used to identify bona fide kinase substrates have remained elusive. Here we describe a proteomic strategy suitable for identifying kinase specificity and direct substrates in high throughput. This approach includes an in vitro kinase assay-based substrate screening and an endogenous kinase dependent phosphorylation profiling. In the in vitro kinase reaction route, a pool of formerly phosphorylated proteins is directly extracted from whole cell extracts, dephosphorylated by phosphatase treatment, after which the kinase of interest is added. Quantitative proteomics identifies the rephosphorylated proteins as direct substrates in vitro. In parallel, the in vivo quantitative phosphoproteomics is performed in which cells are treated with or without the kinase inhibitor. Together, proteins phosphorylated in vitro overlapping with the kinase-dependent phosphoproteome in vivo represents the physiological direct substrates in high confidence. The protein kinase assay-linked phosphoproteomics was applied to identify 25 candidate substrates of the protein-tyrosine kinase SYK, including a number of known substrates and many novel substrates in human B cells. These shed light on possible new roles for SYK in multiple important signaling pathways. The results demonstrate that this integrated proteomic approach can provide an efficient strategy to screen direct substrates for protein tyrosine kinases.

Abstract 189: The Role of Spleen Tyrosine Kinase, Syk, in CD36-Toll Like Receptor Cooperative Signaling in Atherosclerosis

Atherosclerosis is characterized by chronic sterile inflammation of the artery wall in which cells of the monocyte lineage accumulate in response to the deposition of low density lipoprotein (LDL). We previously established that recognition of oxidized LDL (oxLDL) by CD36 triggers assembly of a novel Toll-like receptor heterodimer composed of TLR4 and TLR6. Here we set out to understand the molecular mechanisms of CD36/TLR4/TLR6 activation and establish how it triggers downstream signals that lead to the expression of the pro-inflammatory mediators that have been directly implicated in the deleterious effects of oxLDL and atherosclerosis progression. By confocal microscopy we demonstrate that oxLDL induces CD36, TLR4 and TLR6 co-localization in intracellular compartments, but not on the cell surface of macrophages. Notably, inhibition of oxLDL endocytosis (with Dynasore) or lysosomal maturation (with Bafilomycin A or NH4Cl) blocks CD36-TLR4-TLR6 complex formation and oxLDL-induced cytokine responses in macrophages. These data indicate that both ligand internalization and lysosomal acidification are required for assembly of a functional CD36/TLR4/TLR6 signaling complex. Notably, CD36 contains a hemi-ITIM motif in the C-terminus that is reported to interact with the spleen tyrosine kinase Syk through its SH2 domain. As Syk has recently been implicated in the trafficking of CD14 and TLR4 to the endosome in response to LPS, we investigated the role of this kinase in CD36/TLR4/TLR6 signaling. We find that Syk is required for CD36 internalization and TLR4/TLR6 heterodimerization. Using a pharmacological inhibitor, we show that inhibition of Syk activity blocks oxLDL-induced TLR4-TLR6 co-precipitation and abrogates macrophage expression of both MyD88- (IL-1b, CXCL1) and TRIF-dependent (CCL5) cytokines/chemokines. Together, our data are consistent with a key role for Syk in the trafficking of CD36 and oxLDL to the lysosome, where it coordinates the assembly of a functional TLR4-TLR6 heterodimer to initiate signaling. This model highlights the importance of CD36 as a co-receptor that orchestrates TLR4-TLR6 trafficking and assembly to initiate the detrimental inflammatory responses that promote the progression of atherosclerosis.

Suppression of Spleen Tyrosine Kinase Attenuated Obliterative Bronchiolitis Associated Lymphoid Neogenesis after Lung Transplantation

Purpose Lymphoid neogenesis, also known as tertiary lymphoid tissues, has been demonstrated as an important role in development of chronic rejection represented by obliterative bronchiolitis (OB) in human and mouse studies. Spleen tyrosine kinase (Syk) is an immunoregulatory protein regarding proliferation and receptor signaling in B cells. The study purpose was to examine the role of Syk in alloimmune-related lymphoid neogenesis in OB. Methods and Materials Using a mouse intrapulmonary tracheal transplantation (IPTT) model, BALB/c tracheae were transplanted into the left lung of C57BL/6 mice or conditional Syk knockout mice on day 0, and then C57BL/6 mice were received the treatment with Syk inhibitor (30mg/kg) or vehicle control orally once a day. Mice were sacrificed on day 28 after IPTT. Results H&E staining revealed the lumen of transplanted trachea to be open and the lymphoid aggregates smaller in Syk knock-out mice and mice treated with Syk inhibitor compared to control. Quantification of the graft occlusion (mean±SEM) revealed significant attenuation in mice treated with Syk inhibitor compared to control (19.1±4.6 vs 63.2±7.0 vs 97.4±0.8(%), p Conclusions The treatment with Syk inhibitor and Syk deletion not only abrogated development of OB but also attenuated formation of lymphoid neogenesis through impairment of B cell maturation and proliferation. Inhibition of Syk is a novel potential strategy to control chronic rejection associated with lymphoid neogenesis after organ transplantation.

Dextran sulphate induces fibrinogen receptor activation through a novel Syk-independent PI-3 kinase-mediated tyrosine kinase pathway in platelets

In our attempt to find a physiological agonist that activates PAR3 receptors, we screened several coagulation proteases using PAR4 null platelets. We observed that FXIIa and heat inactivated FXIIa, but not FXII, caused platelet aggregation. We have identified a contaminant activating factor in FXIIa preparation as dextran sulfate (DxS), which caused aggregation of both human and mouse platelets. DxS-induced platelet aggregation was unaffected by YM254890, a Gq inhibitor, but abolished by pan-Src family kinase (SFK) inhibitor PP2, suggesting a role for SFKs in this pathway. However, DxS-induced platelet aggregation was unaffected in FcRγ-chain null murine platelets, ruling out the possibility of glycoprotein VI-mediated events. More interesting, OXSI-2 and Go6976, two structurally unrelated inhibitors shown to affect Syk, had only a partial effect on DxS-induced PAC-1 binding. DxS-induced platelet aggregation and intracellular calcium increases were abolished by the pan PI-3 kinase inhibitor LY294002, or an isoform-specific PI-3 kinase β inhibitor TGX-221. Pretreatment of platelets with Syk inhibitors or ADP receptor antagonists had little effect on Akt phosphorylation following DxS stimulation. These results, for the first time, establish a novel tyrosine kinase pathway in platelets that causes fibrinogen receptor activation in a PI-3 kinase-dependent manner without a crucial role for Syk.

A Peptide-Based Biosensor Assay To Detect Intracellular Syk Kinase Activation and Inhibition

Spleen tyrosine kinase (Syk) has been implicated in a number of pathologies including cancer and rheumatoid arthritis and thus has been pursued as a novel therapeutic target. Because of the complex relationship between Syk's auto- and other internal phosphorylation sites, scaffolding proteins, enzymatic activation state and sites of phosphorylation on its known substrates, the role of Syk's activity in these diseases has not been completely clear. To approach such analyses, we developed a Syk-specific artificial peptide biosensor (SAStide) to use in a cell-based assay for direct detection of intracellular Syk activity and inhibition in response to physiologically relevant stimuli in both laboratory cell lines and primary splenic B cells. This peptide contains a sequence derived from known Syk substrate preference motifs linked to a cell permeable peptide, resulting in a biosensor that is phosphorylated in live cells in a Syk-dependent manner, thus serving as a reporter of Syk catalytic activity in intact cells. Because the assay is compatible with live, primary cells and can report pharmacodynamics for drug action on an intended target, this methodology could be used to facilitate a better understanding of Syk's function and the effect of its inhibition in disease.