P2X7 receptors and Fyn kinase mediate ATP-induced oligodendrocyte progenitor cell migration.

Recruitment of oligodendrocyte precursor cells (OPCs) to the lesions is the most important event for remyelination after central nervous system (CNS) injury or in demyelinating diseases. However, the underlying molecular mechanism is not fully understood. In the present study, we found high concentrations of ATP could increase the number of migrating OPCs in vitro, while after pretreatment with oxidized ATP (a P2X7 receptor antagonist), the promotive effect was attenuated. The promotive effect of 2'(3')-O-(4-benzoylbenzoyl) adenosine 5'-triphosphate (BzATP) (a P2X7 receptor agonist) was more potent than ATP. After incubation with BzATP, the activity of Fyn, one member of the Src family of kinases, was enhanced. Moreover, the interaction between P2X7 and Fyn was identified by co-immunoprecipitation. After blocking the activity of Fyn or down-regulating the expression of Fyn, the migration of OPCs induced by BzATP was inhibited. These data indicate that P2X7 receptors/Fyn may mediate ATP-induced OPC migration under pathological conditions.

Src kinases in chondrosarcoma chemoresistance and migration: dasatinib sensitises to doxorubicin in TP53 mutant cells.

BACKGROUND: Chondrosarcomas are malignant cartilage-forming tumours of bone. Because of their resistance to conventional chemotherapy and radiotherapy, currently no treatment strategies exist for unresectable and metastatic chondrosarcoma. Previously, PI3K/AKT/GSK3ß and Src kinase pathways were shown to be activated in chondrosarcoma cell lines. Our aim was to investigate the role of these kinases in chemoresistance and migration in chondrosarcoma in relation to TP53 mutation status. METHODS: We used five conventional and three dedifferentiated chondrosarcoma cell lines and investigated the effect of PI3K/AKT/GSK3ß pathway inhibition (enzastaurin) and Src pathway inhibition (dasatinib) in chemoresistance using WST assay and live cell imaging with AnnexinV staining. Immunohistochemistry on tissue microarrays (TMAs) containing 157 cartilaginous tumours was performed for Src family members. Migration assays were performed with the RTCA xCelligence System. RESULTS: Src inhibition was found to overcome chemoresistance, to induce apoptosis and to inhibit migration. Cell lines with TP53 mutations responded better to combination therapy than wild-type cell lines (P=0.002). Tissue microarray immunohistochemistry confirmed active Src (pSrc) signalling, with Fyn being most abundantly expressed (76.1%). CONCLUSION: These results strongly indicate Src family kinases, in particular Fyn, as a potential target for the treatment of inoperable and metastatic chondrosarcomas, and to sensitise for doxorubicin especially in the presence of TP53 mutations.

Podocarpusflavone alleviated renal fibrosis in obstructive nephropathy by inhibiting Fyn/Stat3 signaling pathway.

Tubulointerstitial fibrosis is a common pathological change in end-stage renal disease. However, limited treatment methods are developed, and unexplained potential mechanisms of renal diseases are urgent problems to be solved. In the present research, we first elucidated the role of podocarpusflavone (POD), a biflavone compound, in unilateral ureteral obstruction (UUO) in rodent model which is characterized by inflammation and fibrosis. The changes in histology and immunohistochemistry were observed that POD exerted renoprotective effects by retarding the infiltration of macrophage and aberrant deposition of ɑ-SMA, Col1a1, and fibronectin. Consistent with in vivo assay, POD treatment also ameliorated the process of fibrosis in TGF-ß1-stimulated renal tubular epithelial cells and inflammation in LPS-induced RAW264.7 cells in vitro. In terms of mechanism, our results showed that treatment with POD inhibited the aggravated activation of Fyn in the UUO group, and weakened the level of phosphorylation of Stat3 which indicated that POD may alleviate the process of fibrosis by the Fyn/Stat3 signaling pathway. Furthermore, the gain of function assay by lentivirus-mediated exogenous forced expression of Fyn abrogated the therapeutic effect of the POD on renal fibrosis and inflammation. Collectively, it can be concluded that POD exerted a protective effect on renal fibrosis by mediating Fyn/Stat3 signaling pathway.

Fyn Kinase Activity and Its Role in Neurodegenerative Disease Pathology: a Potential Universal Target?

Fyn is a non-receptor tyrosine kinase belonging to the Src family of kinases (SFKs) which has been implicated in several integral functions throughout the central nervous system (CNS), including myelination and synaptic transmission. More recently, Fyn dysfunction has been associated with pathological processes observed in neurodegenerative diseases, such as multiple sclerosis (MS), Alzheimer's disease (AD) and Parkinson's disease (PD). Neurodegenerative diseases are amongst the leading cause of death and disability worldwide and, due to the ageing population, prevalence is predicted to rise in the coming years. Symptoms across neurodegenerative diseases are both debilitating and degenerative in nature and, concerningly, there are currently no disease-modifying therapies to prevent their progression. As such, it is important to identify potential new therapeutic targets. This review will outline the role of Fyn in normal/homeostatic processes, as well as degenerative/pathological mechanisms associated with neurodegenerative diseases, such as demyelination, pathological protein aggregation, neuroinflammation and cognitive dysfunction.

Hippocampal levels of phosphorylated protein kinase A (phosphor-S96) are linked to spatial memory enhancement by SGS742.

Cognitive enhancement by the GABA (B) receptor antagonist SGS742 has been well-documented, but mechanisms of action are not fully elucidated. Previous work has proposed involvement of somatostatin-14 and protein kinase C in cognitive enhancement; phospho-protein kinase A (p-PKA), fyn, and phospho-fyn are known signaling systems for spatial memory. It was the aim of the study to determine hippocampal levels of these proteins following SGS742-treatment and to correlate them with the outcome from the Morris water maze (MWM), represented by the parameter "time spent in the target quadrant" during the probe trial. OF1 mice were used for the experiments and divided into four groups: intraperitoneal SGS742 and saline solution treatment, both, tested in the MWM, and two yoked controls. Six hours following the probe trial, hippocampal protein levels were determined by immunoblotting. In the MWM, time spent in the target quadrant was significantly enhanced by SGS742 treatment. p-PKA levels were significantly increased only in the SGS742-treated group tested in the MWM as compared to saline treatment. In yoked controls, no significant differences in p-PKA levels between SGS742 and saline treatment were observed. Somatostatin-14 levels were significantly increased in both SGS742-treated groups. No statistically significant changes of other protein levels were observed. We propose that GABA (B) antagonism represented by SGS742 treatment led to cognitive enhancement involving p-PKA, because yoked controls treated with SGS742 were comparable to yoked saline-treated controls. The finding that somatostatin-14 was also induced in the SGS742-treated yoked controls points to a drug side effect, and therefore the role of somatostatin-14 for cognitive enhancement remains open.

Panaxadiol inhibits synaptic dysfunction in Alzheimer's disease and targets the Fyn protein in APP/PS1 mice and APP-SH-SY5Y cells.

AIM: Alzheimer's disease (AD), a neurodegenerative disease, is characterized by memory loss and synaptic damage. Up to now, there are limited drugs to cure or delay the state of this illness. Recently, the Fyn tyrosine kinase is implicated in AD pathology triggered by synaptic damage. Thus, Fyn inhibition may prevent or delay the AD progression. Therefore, in this paper, we investigated whether Panaxadiol could decrease synaptic damage in AD and the underlying mechanism. MAIN METHODS: The ability of learning and memory of mice has detected by Morris Water Maze. The pathological changes detected by H&E staining and Nissl staining. The percentage of cell apoptosis and the calcium concentration were detected by Flow Cytometry in vitro. The amount of synaptic protein and related proteins in the Fyn/GluN2B/CaMKIIα signaling pathway were detected by Western Blot. KEY FINDINGS: In the present article, Panaxadiol could significantly improve the ability of learning and memory of mice and reduce its synaptic dysfunction. Panaxadiol could down-regulate GluN2B's phosphorylation level by inhibition Fyn kinase activity, Subsequently, decrease Ca2+-mediated synaptic damage, reducing LDH leakage, inhibiting apoptosis in AD, resulting in facilitating the cells survival. For the underlying molecular mechanism, we used PP2 to block the Fyn/GluN2B/CaMKIIα signaling pathway. The results from WB showed that the expression of related proteins in the Fyn signaling pathway decreased with PP2 treated. SIGNIFICANCE: Our results indicate that Panaxadiol could decrease synaptic damage, which will cause AD via inhibition of the Fyn/GluN2B/CaMKIIα signaling pathway. Thus, the Panaxadiol is a best promising candidate to test as a potential therapy for AD.

Targeting Fyn Kinase in Alzheimer's Disease.

The past decade has brought tremendous progress in unraveling the pathophysiology of Alzheimer's disease (AD). While increasingly sophisticated immunotherapy targeting soluble and aggregated brain amyloid-beta (Aß) continues to dominate clinical research in AD, a deeper understanding of Aß physiology has led to the recognition of distinct neuronal signaling pathways linking Aß to synaptotoxicity and neurodegeneration and to new targets for therapeutic intervention. Identifying specific signaling pathways involving Aß has allowed for the development of more precise therapeutic interventions targeting the most relevant molecular mechanisms leading to AD. In this review, I highlight the discovery of cellular prion protein as a high-affinity receptor for Aß oligomers, and the downstream signaling pathway elucidated to date, converging on nonreceptor tyrosine kinase Fyn. I discuss preclinical studies targeting Fyn as a therapeutic intervention in AD and our recent experience with the safety, tolerability, and cerebrospinal fluid penetration of the Src family kinase inhibitor saracatinib in patients with AD. Fyn is an attractive target for AD therapeutics, not only based on its activation by Aß via cellular prion protein but also due to its known interaction with tau, uniquely linking the two key pathologies in AD. Fyn is also a challenging target, with broad expression throughout the body and significant homology with other members of the Src family kinases, which may lead to unintended off-target effects. A phase 2a proof-of-concept clinical trial in patients with AD is currently under way, providing critical first data on the potential effectiveness of targeting Fyn in AD.

A new mechanism of resistance to ABL1 tyrosine kinase inhibitors in a BCR-ABL1-positive cell line.

Tyrosine kinase inhibitors (TKI) constitute the frontline treatment for chronic myeloid leukemia patients. Dasatinib, a second-generation TKI, was developed to overcome TKI resistances. However, dasatinib resistances are also described but remain less characterized. To mimic in vivo acquired dasatinib resistance, the BCR-ABL1-positive cell line K562 was transiently treated with a pharmacological concentration of dasatinib, for a short time in the presence of stem cell factor. A dasatinib resistant counterpart (K562 RES) was developed. Investigation of resistance mechanisms using kinase substrate arrays revealed that FYN was overactivated in K562 RES. The FYN inhibitor KX2-391 cooperated with dasatinib to block K562 RES proliferation. Cell tracking experiments showed that activated FYN support cell proliferation independently of BCR-ABL1 in K562 RES cells. Moreover, the MEK-ERK pathway was found hyper-phosphorylated in K562 RES cells even in the presence of dasatinib. Actually, ERK1/2 activity supported viability in K562 RES only in the absence of BCR-ABL1 activity. Finally, BCR-ABL1 and MEK inhibitor combination was sufficient to induce cell death even in non-proliferating resistant cells. Considering the conditions used to generate this dasatinib resistant cell line, such a resistance mechanism could be found in dasatinib treated patients. Consequently, it is valuable to know that inhibition of the MEK-ERK1/2 axis can overcome this resistance.

Muscarinic acetylcholine receptors mediate oligodendrocyte progenitor survival through Src-like tyrosine kinases and PI3K/Akt pathways.

The function of muscarinic acetylcholine receptors expressed in oligodendrocytes and in myelin has remained largely undetermined. Here we present evidence that incubation of oligodendrocyte progenitors, deprived of growth factor, with the acetylcholine analog carbachol significantly reduced cell death by apoptosis and blocked caspase-3 cleavage. This protective effect was reversed by atropine, a muscarinic acetylcholine receptor antagonist, as well as by specific inhibitors of intracellular signaling molecules, including phosphatidylinositol 3-kinase (Wortmannin and LY294002), Akt (Akt inhibitor III) and Src-like tyrosine kinases (PP2), but not by the mitogen-activated protein kinase kinase inhibitor, PD98059. Activation of Akt by carbachol was antagonized by atropine and inhibited by LY294002 and PP2. The Src-like tyrosine kinase inhibitor, PP2, also reduced carbachol stimulation of extracellular signal-regulated kinases 1/2 and cAMP-response element binding protein in a dose-dependent manner. Furthermore, carbachol increased tyrosine-phosphorylation of Fyn, a member of the Src-like tyrosine kinases. These results indicate that muscarinic acetylcholine receptors play an important role in oligodendrocyte progenitor survival through transduction pathways involving activation of Src-like tyrosine kinases and phosphatidylinositol 3-kinase/Akt.

Phosphotyrosine enrichment identifies focal adhesion kinase and other tyrosine kinases for targeting in canine hemangiosarcoma.

Canine hemangiosarcoma (HSA) is an endothelial cell malignancy driven, in part, by activating mutations in receptor and non-receptor tyrosine kinases. Proteomics, Western blots and a tyrosine kinase inhibitor were used to elucidate activating mechanisms in HSA cell lines. Phosphotyrosine peptides from focal adhesion kinase (FAK) STAT3, Lyn, Fyn and other signal transduction kinases were identified by mass spectrometry. FAK was constitutively activated at tyrosine 397, the autophosphorylation site, and this was reversible with high concentrations of a FAK inhibitor. FAK inhibitor-14 suppressed migration and phosphorylation of FAK tyrosine 397 and tyrosines 576/577 and was cytotoxic to HSA cells suggesting FAK signalling may be an important contributor to canine HSA survival.

A co-culture system reveals the involvement of intercellular pathways as mediators of the lutropin receptor (LHR)-stimulated ERK1/2 phosphorylation in Leydig cells.

Co-cultures of lutropin receptor (LHR) positive and negative Leydig cells were used to test the hypothesis that the LHR provokes phosphorylation of the extracellular regulated kinases (ERK1/2) using intracellular and intercellular pathways. Addition of hCG to MA-10 cells (LHR positive) stimulates phosphorylation of the EGF receptor (EGFR) and ERK1/2 whereas addition of hCG to I-10 cells (LHR negative) does not. Addition of hCG to co-cultures of MA-10 and I-10 cells rapidly stimulates the phosphorylation of the EGFR and ERK1/2 in I-10 cells, however. Transfection of interfering constructs shows that the LHR-mediated activation of Fyn in MA-10 cells is necessary for the phosphorylation of the EGFR and ERK1/2 in I-10 cells. This pathway can also be demonstrated in MA-10 cells but the phosphorylation of ERK1/2 in MA-10 cells also involves a second pathway mediated by protein kinase A (PKA). We propose that the LHR-mediated stimulation of the ERK1/2 cascade in Leydig cells depends on two independent pathways. One is intracellular and is mediated by PKA. The second is mediated by Fyn and it involves the release of soluble factors that act to phosphorylate the EGFR in an autocrine/paracrine fashion.

Restoration of FcRgamma/Fyn signaling repairs central nervous system demyelination.

Disruption of myelin causes severe neurological diseases. An understanding of the mechanisms that control myelination and remyelination is needed to develop therapeutic strategies for demyelinating diseases such as multiple sclerosis (MS). Our previous finding indicating the critical involvement of the gamma chain of immunogloblin Fc receptors (FcRgamma) and Fyn signaling in oligodendrocyte differentiaion and myelination demands a fundamental revision of the strategies used for MS therapy, because antigen-antibody complexes in MS patients may induce the direct dysregulation of myelination process as well as the inflammatory destruction of myelin sheath. Here we show that the FcRgamma/Fyn signaling cascade is critically involved in cuprizone-induced demyelination/remyelination, with no lymphocytic response. The levels of phosphorylated myelin basic proteins (p-MBPs), especially the 21.5-kDa isoform, but not the levels of total MBPs, decreased markedly during demyelination induced by aging, cuprizone treatment, and double knockout of FcRgamma/Fyn genes. We also showed that the recovery from demyelination in cuprizone-treated and aged mice is achieved after administration of the herbal medicine Ninjin'yoeito, an effective therapy targeting the FcRgamma/Fyn-Rho (Rac1)-MAPK (P38 MAPK)-p-MBPs signaling cascade. These results suggest that the restoration of FcRgamma/Fyn signaling represents a new approach for the treatment of demyelinating diseases.

TNF-alpha increases tyrosine phosphorylation of vascular endothelial cadherin and opens the paracellular pathway through fyn activation in human lung endothelia.

Tumor necrosis factor (TNF)-alpha is a key mediator of sepsis-associated multiorgan failure, including the acute respiratory distress syndrome. We examined the role of protein tyrosine phosphorylation in TNF-alpha-induced pulmonary vascular permeability. Postconfluent human lung microvascular and pulmonary artery endothelial cell (EC) monolayers exposed to human recombinant TNF-alpha displayed a dose- and time-dependent increase in transendothelial [(14)C]albumin flux in the absence of EC injury. TNF-alpha also increased tyrosine phosphorylation of EC proteins, and several substrates were identified as the zonula adherens proteins vascular endothelial (VE)-cadherin, and beta-catenin, gamma-catenin, and p120 catenin (p120(ctn)). Prior protein tyrosine kinase (PTK) inhibition protected against the TNF-alpha effect. TNF-alpha activated multiple PTKs, including src family PTKs. Prior PTK inhibition with the src-selective agents PP1 and PP2 each protected against approximately 60% of the TNF-alpha-induced increment in [(14)C]albumin flux. PP2 also blocked TNF-alpha-induced tyrosine phosphorylation of VE-cadherin, gamma-catenin, and p120(ctn). To identify which src family kinase(s) was required for TNF-alpha-induced vascular permeability, small interfering RNA (siRNA) targeting each of the three src family PTKs expressed in human EC, c-src, fyn, and yes, were introduced into the barrier function assay. Only fyn siRNA protected against the TNF-alpha effect, whereas the c-src and yes siRNAs did not. These combined data suggest that TNF-alpha regulates the pulmonary vascular endothelial paracellular pathway, in part, through fyn activation.

Influence of mizolastine on antigen-induced activation of signalling pathways in murine mast cells.

BACKGROUND: There is accumulating evidence that some antihistamines can interrupt intermediate signalling events that regulate cell function. The effect of mizolastine on both the generation and release process of many cytokines in mast cells further implies that the inhibition by mizolastine may target signalling pathways. AIM: To observe the influence of mizolastine on antigen-induced activation of signalling pathways in murine mast cells. METHODS: Western blot analysis and enzyme assay were performed. Immunoblots were prepared from whole cell lysates and probed with antibodies against Fyn, Akt, ERK, p38, phospho-Fyn, phospho-Akt, phospho-ERK and phospho-p38, respectively. RESULTS: Our study showed that signalling molecules such as IP3, Fyn, p38 and ERK were enhanced when mast cells were stimulated by antigen, and that this was not inhibited by treatment with mizolastine. Mizolastine at concentrations from 10(-9) to 10(-5) mol/L could inhibit activation of the PI3K kinase downstream signalling molecule Akt to antigen stimulation. The study also demonstrated that mizolastine exerted inhibitory ability on protein kinase C (PKC) activation in a dose-dependent manner. CONCLUSION: PKC-mediated phosphorylation of Akt can be blocked by mizolastine. There may be a PKC-independent pathway effectively activating MAPK pathways in mast cells in response to antigen induction, which cannot be affected by mizolastine.