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.

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Role of Protein Kinase C<i>δ</i>-Mediated Spleen Tyrosine Kinase (Syk) Phosphorylation on Ser in the Amplification of Oral Mucosal Inflammatory Responses to <i>Porphyromonas gingivalis</i>

Journal of Biosciences and Medicines ◽

10.4236/jbm.2018.63005 ◽

2018 ◽

Vol 06 (03) ◽

pp. 70-85

Author(s):

Bronislaw L. Slomiany ◽

Amalia Slomiany

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Tyrosine Kinase ◽

Porphyromonas Gingivalis ◽

Inflammatory Responses ◽

Spleen Tyrosine Kinase ◽

Kinase C

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The Tyrosine Kinase-Binding and Proline-Rich Domains of Mutant CBL Are Essential for Leukemogenesis

Blood ◽

10.1182/blood.v126.23.2457.2457 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2457-2457

Author(s):

Scott Nadeau ◽

Wei An ◽

Gulzar Ahmad ◽

Bhopal Mohapatra ◽

Neha Zutshi ◽

...

Keyword(s):

Tyrosine Kinase ◽

Progenitor Cells ◽

Molecular Mechanisms ◽

Sh2 Domain ◽

Leukemia Cell Line ◽

Hematopoietic Stem ◽

Rich Domain ◽

The Impact ◽

Cbl Proteins

Abstract Mutations of the tyrosine kinase-directed ubiquitin ligase CBL are associated with myeloid malignancies, yet the molecular mechanisms by which this tumor suppressor becomes a dominant oncogene to initiate the leukemogenic process are unclear. In this study, we used systematic mutagenesis to delineate the importance of the various protein-protein interaction domains/motifs of a CBL mutant that is most frequently found in human leukemias, Y371H, in inducing leukemogenesis. We tested the impact of these secondary mutations on the ability of CBL-Y371H to impart hypersensitivity to cytokines and to upregulate the associated signaling pathways in the TF1 leukemia cell line model, and in mouse hematopoietic stem/progenitor cells from Cbl-null mice to mimic the lack of wildtype CBL expression in most of the mutant CBL bearing leukemia patients. The secondary mutations included: Cbl G306E, to abrogate the ability of the TKB domain of mutant Cbl to bind to activated tyrosine kinases; internal deletion of the proline-rich domain (AA 477-688) to abrogate interactions with SH3 domain containing partners; Cbl-Y700/731/774F triple phosphorylation site mutant predicted not to interact with the SH2 domain-containing partners; and Cbl-1-436 deletion construct lacking all C-terminal motifs. Analyses of stably expressed mutants in TF-1 cells for hypersensitivity to SCF1 demonstrated an essential role of an intact TKB domain and a particularly important role of the proline-rich domain. Transient retroviral expression in Cbl-null primary murine hematopoietic stem/progenitor cells confirmed these results. Deletion of the proline-rich domain led to substantially less tyrosine phosphorylation of the oncogenic mutant Cbl as well as of the c-Kit receptor upon SCF stimulation, and cells expressing this mutant lacked the sustained activation of Erk1/2 and Akt typically seen after SCF stimulation of the Cbl-Y371H-expressing cells. Together, our data provide conclusive evidence that interaction of leukemogenic mutant Cbl proteins with the upstream tyrosine kinase (via the TKB domain) and with partner proteins (via the proline-rich domain) provides a basic mechanism for gain of function phenotype of mutant Cbl proteins. Our studies suggest that identification of the leukemogenesis-critical partners of the proline-rich domain and targeting of the TKB-tyrosine kinase interface provide new therapeutic approaches against mutant Cbl-driven leukemias. Disclosures No relevant conflicts of interest to declare.

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Immune-Inflammatory Responses in Atherosclerosis: The Role of Myeloid Cells

Journal of Clinical Medicine ◽

10.3390/jcm8111798 ◽

2019 ◽

Vol 8 (11) ◽

pp. 1798 ◽

Cited By ~ 9

Author(s):

Dimitry A. Chistiakov ◽

Dmitry A. Kashirskikh ◽

Victoriya A. Khotina ◽

Andrey V. Grechko ◽

Alexander N. Orekhov

Keyword(s):

T Cells ◽

Immune Cells ◽

Inflammatory Responses ◽

Oxidized Ldl ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Cell Types ◽

Foam Cells ◽

Intracellular Lipid Accumulation

Inflammation plays a key role in the initiation and progression of atherosclerosis and can be caused by multiple agents, including increased concentration of circulating low-density lipoprotein (LDL) cholesterol. Areas of the arterial wall affected by atherosclerosis are enriched with lymphocytes and dendritic cells (DCs). Atherosclerotic plaques contain a variety of proinflammatory immune cells, such as macrophages, DCs, T cells, natural killer cells, neutrophils and others. Intracellular lipid accumulation in atherosclerotic plaque leads to formation of so-called foam cells, the cytoplasm of which is filled with lipid droplets. According to current understanding, these cells can also derive from the immune cells that engulf lipids by means of phagocytosis. Macrophages play a crucial role in the initial stages of atherogenesis by engulfing oxidized LDL (oxLDL) in the intima that leads to their transformation to foam cells. Dying macrophages inside the plaque form a necrotic core that further aggravates the lesion. Proinflammatory DCs prime differentiation of naïve T cells to proinflammatory Th1 and Th17 subsets. In this review, we discuss the roles of cell types of myeloid origin in atherosclerosis-associated inflammation.

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Oxidized LDL Disrupts Metabolism and Inhibits Macrophage Survival by Activating a miR-9/Drp1/Mitochondrial Fission Signaling Pathway

Oxidative Medicine and Cellular Longevity ◽

10.1155/2020/8848930 ◽

2020 ◽

Vol 2020 ◽

pp. 1-16

Author(s):

Ting Xin ◽

Chengzhi Lu ◽

Jing Zhang ◽

Jiaxin Wen ◽

Shuangbin Yan ◽

...

Keyword(s):

Inflammatory Responses ◽

Cholesterol Metabolism ◽

Oxidized Ldl ◽

Mitochondrial Fission ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

The Novel ◽

Macrophage Viability

Mitochondrial dysfunction is associated with macrophage damage, but the role of mitochondrial fission in macrophage cholesterol metabolism is not fully understood. In this study, we explored the influences of miR-9 and mitochondrial fission on macrophage viability and cholesterol metabolism. Macrophages were incubated with oxidized low-density lipoprotein (ox-LDL) in vitro, after which mitochondrial fission, cell viability, and cholesterol metabolism were examined using qPCR, ELISAs, and immunofluorescence. ox-LDL treatment significantly increased Drp1-associated mitochondrial fission. Transfection of Drp1 siRNA significantly reduced cell death, attenuated oxidative stress, and inhibited inflammatory responses in ox-LDL-treated macrophages. Interestingly, inhibition of Drp1-related mitochondrial fission also improved cholesterol metabolism by balancing the transcription of cholesterol influx/efflux enzymes. We also found that miR-9 was downregulated in ox-LDL-treated macrophages, and administration of a miR-9 mimic decreased Drp1 transcription and mitochondrial fission, as well as its effects. These results indicate that signaling via the novel miR-9/Drp1/mitochondrial fission axis is a key determinant of macrophage viability and cholesterol metabolism.

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The Role of the Endothelium in Premature Atherosclerosis: Molecular Mechanisms

Current Medicinal Chemistry ◽

10.2174/0929867326666190911141951 ◽

2020 ◽

Vol 27 (7) ◽

pp. 1041-1051 ◽

Cited By ~ 1

Author(s):

Michael Spartalis ◽

Eleftherios Spartalis ◽

Antonios Athanasiou ◽

Stavroula A. Paschou ◽

Christos Kontogiannis ◽

...

Keyword(s):

Molecular Mechanisms ◽

Low Density Lipoprotein ◽

Critical Role ◽

Density Lipoprotein ◽

Number Of Genes ◽

Causes Of Mortality ◽

Artery Disease ◽

Genetic And Environmental Factors ◽

Made In

Atherosclerotic disease is still one of the leading causes of mortality. Atherosclerosis is a complex progressive and systematic artery disease that involves the intima of the large and middle artery vessels. The inflammation has a key role in the pathophysiological process of the disease and the infiltration of the intima from monocytes, macrophages and T-lymphocytes combined with endothelial dysfunction and accumulated oxidized low-density lipoprotein (LDL) are the main findings of atherogenesis. The development of atherosclerosis involves multiple genetic and environmental factors. Although a large number of genes, genetic polymorphisms, and susceptible loci have been identified in chromosomal regions associated with atherosclerosis, it is the epigenetic process that regulates the chromosomal organization and genetic expression that plays a critical role in the pathogenesis of atherosclerosis. Despite the positive progress made in understanding the pathogenesis of atherosclerosis, the knowledge about the disease remains scarce.

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Classic and Novel Signaling Pathways Involved in Cancer: Targeting the NF-κB and Syk Signaling Pathways

Current Stem Cell Research & Therapy ◽

10.2174/1574888x13666180723104340 ◽

2019 ◽

Vol 14 (3) ◽

pp. 219-225 ◽

Cited By ~ 6

Author(s):

Cong Tang ◽

Guodong Zhu

Keyword(s):

Cancer Therapy ◽

Tyrosine Kinase ◽

Signaling Pathways ◽

Molecular Mechanisms ◽

Therapeutic Potential ◽

Cell Receptor ◽

Transmembrane Receptors ◽

Biological Responses ◽

Different Types

The nuclear factor kappa B (NF-κB) consists of a family of transcription factors involved in the regulation of a wide variety of biological responses. Growing evidence support that NF-κB plays a major role in oncogenesis as well as its well-known function in the regulation of immune responses and inflammation. Therefore, we made a review of the diverse molecular mechanisms by which the NF-κB pathway is constitutively activated in different types of human cancers and the potential role of various oncogenic genes regulated by this transcription factor in cancer development and progression. We also discussed various pharmacological approaches employed to target the deregulated NF-κB signaling pathway and their possible therapeutic potential in cancer therapy. Moreover, Syk (Spleen tyrosine kinase), non-receptor tyrosine kinase which mediates signal transduction downstream of a variety of transmembrane receptors including classical immune-receptors like the B-cell receptor (BCR), which can also activate the inflammasome and NF-κB-mediated transcription of chemokines and cytokines in the presence of pathogens would be discussed as well. The highlight of this review article is to summarize the classic and novel signaling pathways involved in NF-κB and Syk signaling and then raise some possibilities for cancer therapy.

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TRIM31 facilitates K27-linked polyubiquitination of SYK to regulate antifungal immunity

Signal Transduction and Targeted Therapy ◽

10.1038/s41392-021-00711-3 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Xueer Wang ◽

Honghai Zhang ◽

Zhugui Shao ◽

Wanxin Zhuang ◽

Chao Sui ◽

...

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Fungal Pathogen ◽

Essential Role ◽

Molecular Mechanisms ◽

Systemic Infection ◽

Lectin Receptors ◽

Innate And Adaptive Immunity ◽

Cytokines And Chemokines

AbstractSpleen tyrosine kinase (SYK) is a non-receptor tyrosine kinase, which plays an essential role in both innate and adaptive immunity. However, the key molecular mechanisms that regulate SYK activity are poorly understood. Here we identified the E3 ligase TRIM31 as a crucial regulator of SYK activation. We found that TRIM31 interacted with SYK and catalyzed K27-linked polyubiquitination at Lys375 and Lys517 of SYK. This K27-linked polyubiquitination of SYK promoted its plasma membrane translocation and binding with the C-type lectin receptors (CLRs), and also prevented the interaction with the phosphatase SHP-1. Therefore, deficiency of Trim31 in bone marrow-derived dendritic cells (BMDCs) and macrophages (BMDMs) dampened SYK-mediated signaling and inhibited the secretion of proinflammatory cytokines and chemokines against the fungal pathogen Candida albicans infection. Trim31−/− mice were also more sensitive to C. albicans systemic infection than Trim31+/+ mice and exhibited reduced Th1 and Th17 responses. Overall, our study uncovered the pivotal role of TRIM31-mediated K27-linked polyubiquitination on SYK activation and highlighted the significance of TRIM31 in anti-C. albicans immunity.

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Spleen Tyrosine Kinase Inhibition Modulates p53 Activity

Journal of Cell Death ◽

10.1177/1179066017731564 ◽

2017 ◽

Vol 10 ◽

pp. 117906601773156 ◽

Cited By ~ 2

Author(s):

Mohammad Althubiti

Keyword(s):

Tyrosine Kinase ◽

Survival Rates ◽

Lymphocytic Leukemia ◽

Spleen Tyrosine Kinase ◽

Ht1080 Cell ◽

P53 Expression ◽

Chemical Inhibitors ◽

Dependent Mechanism

Spleen tyrosine kinase (SYK) is a cytoplasmic enzyme that promotes survival and proliferation of B cells. SYK inhibition has shown promising results in the treatment of arthritis and chronic lymphocytic leukemia (CLL). However, in other context, it has been shown that SYK overexpression in epithelial cancer cells induced senescence in p53-dependent mechanism, which underscored its antineoplastic activity in vitro. Here, we show that SYK was induced in response of DNA damage in parallel with p53 levels. In addition, using chemical inhibitors of SYK reduced p53 levels in HCT116 and HT1080 cell lines, which underlines the role of SYK inhibition on p53 activity. Furthermore, SYK inhibition modulated the cell growth, which resulted in a decreasing in cell death. Interestingly, SYK expression showed a positive prognosis in patients with solid tumors in correlations with their survival rates, as expected negative correlation was seen between SYK expression and survival rate of patients with CLL. In conclusion, these findings demonstrate that SYK inhibition modulates p53 expression and activity in HCT116 and HT1080 cells. Reconsidering using of SYK inhibitors in clinical setting in the future should be evaluated carefully in accordance with these findings to prevent the formation of secondary malignancies.

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