Levels Of Fyn Research Articles

Conformational insights into the inhibitory mechanism of phyto-compounds against Src kinase family members implicated in psoriasis

Psoriasis is a chronic immune mediated disorder of the skin. There is growing evidence that the Src family tyrosine kinases (SFK) are highly upregulated in psoriasis. The SFK are the key components of the signaling pathways triggering cell growth and differentiation in addition to the immune cascades. In the current work, the interactions between SFK and selective phyto-compounds were studied using molecular docking approach. Based on the results of docking and binding energy calculations quercetin was identified as potential lead compound. To get a deeper insight into the binding of quercetin with the SFK, a combined molecular dynamics and binding free energy calculations were performed. The binding of quercetin disrupted the intra-molecular contacts making the SFK compact except Src kinase. The MM/PBSA free energy decomposition analysis highlighted the significance of hydrophobic and polar residues which are involved in the binding of quercetin. An experimental validation was carried out against the activated forms of Fyn, Lyn and Src kinases, the top three proteins which showed high preference for quercetin. The flow cytometry analysis showed that the expression levels of Fyn, Lyn and Src kinases were dramatically increased in HaCaT cells. However, the treatment of quercetin at the concentration of 51.65 µM for 24 h markedly decreased their expression in HaCaT cells. Besides, similar results were also observed when the HaCaT cells were treated with the kinase inhibitor Ponitinib (1.43 µM) for 24 h.Communicated by Ramaswamy H. Sarma

FYN promotes mesenchymal phenotypes of basal type breast cancer cells through STAT5/NOTCH2 signaling node.

Basal type breast cancer is the most aggressive and has mesenchymal features with a high metastatic ability. However, the signaling node that determines the basal type features in breast cancer remains obscure. Here, we report that FYN among SRC family kinases is required for the maintenance of basal type breast cancer subtype. Importantly, FYN enhanced NOTCH2 activation in basal type breast cancer cells through STAT5-mediated upregulation of Jagged-1 and DLL4 NOTCH ligands, thereby contributed to mesenchymal phenotypes. In addition, we found that high levels of FYN persist in basal type breast cancer cells by a positive feedback loop between FYN and STAT5. FYN interacted directly with STAT5 and increased p-STAT5 that further acts as a transcription factor for FYN. Taken together, our findings demonstrate a pivotal role of FYN and its downstream effectors in maintaining the basal type features in breast cancer.

Fyn knockdown inhibits migration and invasion in cholangiocarcinoma through the activated AMPK/mTOR signaling pathway.

Cholangiocarcinoma (CCA) is a rare and fatal tumor. In previous decades, there has been a steady increase in the incidence and mortality rates of this tumor worldwide. Metastasis is regarded as the major factor that contributes to poor prognosis in CCA patients. Studies therefore aim to develop novel therapeutic targets to control CCA metastasis. Fyn is known to enhance expression and promote metastasis in various cancers, including pancreatic cancer, prostate cancer and colorectal cancer. However, the exact function and mechanism of Fyn in CCA metastasis remains unclear. In the present study, mRNA and protein expression levels of Fyn, AMP-activated protein kinase (AMPK), phosphorylated (p-)AMPK, mammalian target of rapamycin (mTOR) and p-mTOR were measured, using the reverse transcription-quantitative polymerase chain reaction and western blot analysis, in CCA tissues and cell lines. In addition, Transwell assays were used to determine the migratory and invasive abilities of human CCA QBC939, following transfection. In the present study, it was found that Fyn was overexpressed in CCA cell lines. Fyn knockdown inhibited CCA cell migration and invasion. Furthermore, it was demonstrated that Fyn knockdown induces phosphorylation of AMPK, inhibits downstream phosphorylation of mTOR, and activate the AMPK/mTOR signaling pathway. Compound C, an AMPK inhibitor, inhibited the AMPK/mTOR signaling pathway, and reversed the effect of Fyn knockdown on migration and invasion of CCA cells. In conclusion, the present study suggests that Fyn knockdown inhibits cell migration and invasion by regulating the AMPK/mTOR signaling pathway in CCA cell lines and that Fyn knockdown is a potential target for anti-CCA therapy.

Fyn-binding protein ADAP supports actin organization in podocytes.

The renal podocyte is central to the filtration function of the kidney that is dependent on maintaining both highly organized, branched cell structures forming foot processes, and a unique cell‐cell junction, the slit diaphragm. Our recent studies investigating the developmental formation of the slit diaphragm identified a novel claudin family tetraspannin, TM4SF10, which is a binding partner for ADAP (also known as Fyn binding protein Fyb). To investigate the role of ADAP in podocyte function in relation to Fyn and TM4SF10, we examined ADAP knockout (KO) mice and podocytes. ADAP KO mice developed glomerular pathology that began as hyalinosis and progressed to glomerulosclerosis, with aged male animals developing low levels of albuminuria. Podocyte cell lines established from the KO mice had slower attachment kinetics compared to wild‐type cells, although this did not affect the total number of attached cells nor the ability to form focal contacts. After attachment, the ADAP KO cells did not attain typical podocyte morphology, lacking the elaborate cell protrusions typical of wild‐type podocytes, with the actin cytoskeleton forming circumferential stress fibers. The absence of ADAP did not alter Fyn levels nor were there differences between KO and wild‐type podocytes in the reduction of Fyn activating phosphorylation events with puromycin aminonucleoside treatment. In the setting of endogenous TM4SF10 overexpression, the absence of ADAP altered the formation of cell‐cell contacts containing TM4SF10. These studies suggest ADAP does not alter Fyn activity in podocytes, but appears to mediate downstream effects of Fyn controlled by TM4SF10 involving actin cytoskeleton organization.

Abstract 322: Distinct pools of ShcA coupled tyrosine kinase signaling influences breast tumor heterogeneity and therapeutic responsiveness

Abstract Phospho-tyrosine (pTyr) signaling networks are frequently activated in breast cancers (BrCa) and are considered to be major oncogenic drivers of tumor progression. Therapeutic interventions, such as the tyrosine kinase (TK) inhibitor Trastuzumab, focus on targeting TK activity and downstream effectors. Although successful for early stage tumors, a subset of patients experience relapse due to intrinsic or acquired resistance. This includes activation of alternative receptor tyrosine kinase (RTK) and/or cytoplasmic TKs, many of which require recruitment of the adaptor protein, ShcA. ShcA is a key convergence point downstream of RTKs and serves to integrate multiple signal transduction pathways dysregulated in BrCa. Specifically, ShcA contains two pTyr binding motifs including an amino-terminal PTB domain and a carboxy-terminal SH2 domain which facilitate its interactions with TKs including ErbB2 and Src Family Kinases (SFK), respectively. The CH-1 domain houses three tyrosine phosphorylation sites at residues 239/240 and 317 which transduce Ras-dependent and independent signals. Using a well characterized transgenic mouse model of BrCa where ShcA can no longer engage the transforming oncogene through its PTB domain, we demonstrate that loss of PTB-driven ShcA (ShcA-PTBMut) signaling delays mammary tumor onset. However, once formed, the growth and angiogenic potential of these tumors is significantly increased relative to control mice. Increased growth potential of ShcA-PTBMut tumors is associated with the hyper-activation of the c-Src tyrosine kinase. Deletion of c-Src in ShcA-PTBMut breast tumor cells significantly delays tumor onset but is dispensable for the growth of tumors that retain an intact ShcA PTB domain. These data suggest that the ShcA PTB domain can recruit negative regulators that limit the activation of downstream tumorigenic signaling networks. Interestingly, tumors expressing ShcA-PTBMut debilitated in SH2 driven pTyr interactions (SH2Mut), are significantly delayed in tumor onset relative to ShcA-PTBMut controls. Paradoxically, deletion of c-Src in the context of ShcA-PTBMut-SH2Mut further accelerates tumor growth which is attributed to increased levels of Fyn and Lyn. These observations support the high dependence of intracellular ShcA pools on other SFK family members to retain tumorigenic potential when adapting to low levels and/or activity of c-Src. We demonstrate that uncoupling of PTB-driven ShcA signaling from upstream RTKs can potentiate ShcA signaling from intracellular pools to hyper-activate SFKs. This data is clinically relevant as c-Src is frequently hyper-activated in Trastuzumab-resistant BrCa. This is the first study to identify a tumor suppressive role of the ShcA PTB domain and to characterize an intrinsic ShcA SH2 domain-SFK dependent resistance mechanism downstream of activated RTKs in mammary tumorigenesis. Citation Format: Jacqueline R. Ha, Ryuhjin Ahn, Young Kyuen Im, Valerie Sabourin, Harvey W. Smith, Ivan Topisirovic, Tony Pawson, Peter Siegel, William J. Muller, Josie Ursini-Siegel. Distinct pools of ShcA coupled tyrosine kinase signaling influences breast tumor heterogeneity and therapeutic responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 322. doi:10.1158/1538-7445.AM2017-322

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and invivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

The HLA-B*35 allele modulates ER stress, inflammation and proliferation in PBMCs from Limited Cutaneous Systemic Sclerosis patients.

IntroductionHLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics.MethodsPBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression.ResultsER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs.ConclusionsOur study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-015-0881-1) contains supplementary material, which is available to authorized users.

Postsynaptic density protein 95-regulated NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in levodopa-induced dyskinesia rat models.

ContextAbnormality in interactions between N-methyl-d-aspartate (NMDA) receptor and its signaling molecules occurs in the lesioned striatum in Parkinson’s disease (PD) and levodopa-induced dyskinesia (LID). It was reported that Fyn-mediated NR2B tyrosine phosphorylation, can enhance NMDA receptor function. Postsynaptic density protein 95 (PSD-95), one of the synapse-associated proteins, regulates interactions between receptor and downstream-signaling molecules. In light of the relationship between PSD-95, NR2B, and Fyn kinases, does PSD-95 contribute to the overactivity of NMDA receptor function induced by dopaminergic treatment? To further prove the possibility, the effects of regulating the PSD-95 expression on the augmented NR2B tyrosine phosphorylation and on the interactions of Fyn and NR2B in LID rat models were evaluated.MethodsIn the present study, parkinsonian rat models were established by injecting 6-hydroxydopamine. Subsequently, valid PD rats were treated with levodopa (50 mg/kg/day with benserazide 12.5 mg/kg/day, twice daily) intraperitoneally for 22 days to create LID rat models. Then, the effect of pretreatment with an intrastriatal injection of the PSD-95mRNA antisense oligonucleotides (PSD-95 ASO) on the rotational response to levodopa challenge was assessed. The effects of pretreatment with an intrastriatal injection of PSD-95 ASO on the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B in the LID rat models were detected by immunoblotting and immunoprecipitation.ResultsLevodopa administration twice daily for 22 days to parkinsonian rats shortened the rotational duration and increased the peak turning responses. The altered rotational responses were attenuated by PSD-95 ASO pretreatment. Meanwhile, PSD-95 ASO pretreatment decreased the level of PSD-95 protein expression and reduced both the augmented NR2B tyrosine phosphorylation and interactions of Fyn with NR2B triggered during the levodopa administration in the lesioned striatum of PD rats. However, the protein levels of Fyn and NR2B showed no difference under the above conditions.ConclusionThe data demonstrate that the inhibition of PSD-95 protein expression suppressed the interactions of Fyn with NR2B and NR2B tyrosine phosphorylation and subsequently downregulated NMDA receptor overactivation, thus providing benefit for the therapy of LID. Therefore, PSD-95 is important for overactivity of NMDA receptor function due to facilitating NR2B tyrosine phosphorylation dependent on Fyn kinase by regulating interactions of Fyn with NR2B under the pathological conditions of LID.

Expression of B-cell-associated genes in peripheral blood mononuclear cells of patients with symptomatic pulmonary embolism.

The aim of the present study was to identify differentially expressed B‑cell‑associated genes in peripheral blood mononuclear cells and investigate the gene expression characteristics of the different stages of B‑cell activation. A total of 20 patients with pulmonary embolisms (PE) and 20 age‑ and gender‑matched controls were enrolled in the present study. Human complementary DNA microarray analysis was used in order to detect the differential expression of B‑cell‑associated genes between the PE and control groups. Messenger (m)RNA expression was detected for 82 genes involved in B‑cell activation. The results showed that PE patients exhibited significantly increased expression levels of the B‑cell receptor genes LYN, CD22, SYK, BTK, PTPRC and NFAM1, whereas expression levels of FYN, FCRL4 and LAX1 were significantly decreased compared to those of the control group. Expression levels of T‑cell‑dependent B‑cell‑activation genes, including EMR2, TNFSF9, CD86, ICOSLG, CD37 and CD97, were significantly upregulated in PE patients, whereas SPN mRNA expression was significantly downregulated compared with those of the control group. LILRA1 and TLR9 T cell‑independent B‑cell activation mRNAs were significantly upregulated in PE patients compared with those of the control group. In addition, the expression levels of B‑cell‑activation regulator genes, including CR1, LILRB4 and VAV1, were significantly increased, whereas SLAMF7 expression levels were significantly decreased in PE patients compared with those of the control group. Furthermore, the expression levels of B‑cell‑activation‑associated cytokine genes demonstrated a significant upregulation of LTA and IL10 and downregulation of L1A, IFNA5, IFNA6, IFNA8, IFNA14, IL2, IL13 and IFNG in PE patients compared to those of the control group. In conclusion, the differential gene expression at different stages of B‑cell activation between healthy controls and PE patients indicated that B‑cell function was reduced or disorganized in patients with symptomatic PE.

CD36 and Fyn Kinase Mediate Malaria-Induced Lung Endothelial Barrier Dysfunction in Mice Infected with Plasmodium berghei

Severe malaria can trigger acute lung injury characterized by pulmonary edema resulting from increased endothelial permeability. However, the mechanism through which lung fluid conductance is altered during malaria remains unclear. To define the role that the scavenger receptor CD36 may play in mediating this response, C57BL/6J (WT) and CD36−/− mice were infected with P. berghei ANKA and monitored for changes in pulmonary endothelial barrier function employing an isolated perfused lung system. WT lungs demonstrated a >10-fold increase in two measures of paracellular fluid conductance and a decrease in the albumin reflection coefficient (σalb) compared to control lungs indicating a loss of barrier function. In contrast, malaria-infected CD36−/− mice had near normal fluid conductance but a similar reduction in σalb. In WT mice, lung sequestered iRBCs demonstrated production of reactive oxygen species (ROS). To determine whether knockout of CD36 could protect against ROS-induced endothelial barrier dysfunction, mouse lung microvascular endothelial monolayers (MLMVEC) from WT and CD36−/− mice were exposed to H2O2. Unlike WT monolayers, which showed dose-dependent decreases in transendothelial electrical resistance (TER) from H2O2 indicating loss of barrier function, CD36−/− MLMVEC demonstrated dose-dependent increases in TER. The differences between responses in WT and CD36−/− endothelial cells correlated with important differences in the intracellular compartmentalization of the CD36-associated Fyn kinase. Malaria infection increased total lung Fyn levels in CD36−/− lungs compared to WT, but this increase was due to elevated production of the inactive form of Fyn further suggesting a dysregulation of Fyn-mediated signaling. The importance of Fyn in CD36-dependent endothelial signaling was confirmed using in vitro Fyn knockdown as well as Fyn−/− mice, which were also protected from H2O2- and malaria-induced lung endothelial leak, respectively. Our results demonstrate that CD36 and Fyn kinase are critical mediators of the increased lung endothelial fluid conductance caused by malaria infection.

Abstract 2864: Regulation and targeting of Fyn in cutaneous squamous cell carcinoma

Abstract Over 700,000 cases of cutaneous squamous cell carcinoma (cSCC) occur in the United States every year. Despite this high rate of diagnosis no therapies currently exist that target the molecular mechanism of tumorigenesis. Between 20 and 60% of these tumors contain mutated Ras; however targeting Ras with therapeutics has been unsuccessful in clinical trials. Expression of active H-Ras (G12V) in HaCaT cells (immortalized, non-transformed keratinocytes) results in upregulated expression of Fyn, a Src family kinase that is active in many malignancies and is involved in the elevated invasion/migration of Ras-transformed HaCaT cells. The specific objectives of this project include exploring the mechanism through which Ras activation increases Fyn levels and determining how high levels of Fyn promote cancer cell migration and survival. Using RT-qPCR we verified that Fyn expression is selectively elevated in cells with increased Ras activity, but levels of other Src family kinases (eg Src, Lyn, Yes, Blk, Lck) are not altered. To explore the transcriptional regulation of Fyn in Ras-transformed HaCaT cells we subcloned segments (−2 kb to −50 bp) of the human Fyn promoter in front of firefly luciferase and transfected the reporter constructs into HaCaT and HaCaT-Ras cells (transformed by constitutively active H-Ras). We identified a region −100 to −50 base pairs upstream of the transcription start site that is necessary for Fyn upregulation in HaCaT-Ras cells. The −80 to −65 region of the Fyn promoter contains putative GATA-1, GATA-2, GATA-3, CP2 or TATA transcription factor binding sites. We have also targeted Fyn in Ras-transformed cells using the Src family kinase inhibitor Dasatinib. Already used to treat chronic myelogenous leukemia and Philadelphia chromosome positive acute lymphoblastic leukemia, Dasatinib is also in clinical trials to treat solid tumors. Treatment with varying concentrations of Dasatinib did not reveal greater changes in cell viability, cell cycle progression, apoptosis or autophagy in transformed HaCaT-Ras cells as compared to HaCaT cells. However, upon Dasatinib exposure HaCaT-Ras cells change dramatically in morphology, enhancing cell-cell contacts and reverting from a stellate appearance to the rounded morphology of untransformed HaCaT cells. Due to previous work verifying Fyn's role in promoting the invasion and migration of transformed keratinocytes, we propose that Fyn inhibition in keratinocytes with increased Ras activity will restore cell to cell contacts and provide a potential therapeutic target in the treatment of cSCCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2864. doi:1538-7445.AM2012-2864

Human vascular endothelial cells reduce sphingosylphosphorylcholine-induced smooth muscle cell contraction in co-culture system through integrin β4 and Fyn

In vascular strips, the adjacent endothelial cells modulate the contraction of vascular smooth muscle cells (VSMCs) induced by sphingosylphosphorylcholine (SPC) through nitric oxide (NO). The aim of this study was to elucidate the mechanisms by which vascular endothelial cells (VECs) reduce the SPC-induced contraction of VSMCs in a co-culture system. Human umbilical VECs and VSMCs were co-cultured. The VECs were transfected with integrin β4- or Fyn-specific siRNA. The areas of VSMCs that are involved in cell contractility were quantified using the Leica confocal software and collagen contractility assay. The production of NO in VECs was measured in the cell supernatants using NO Detection Kit. The levels of integrin β4 and Fyn in VECs and the levels of Rho kinase (ROCK) in VSMC were detected using immunofluorescence assays or Western blots. Co-culture with VECs reduced the contraction of VSMCs induced by SPC (30 μmol/L). The down-regulation of integrin β4 or Fyn in VECs by the specific siRNA (20 nmol/L) was able to counteract the effects of VECs on the SPC-induced VSMC contractions. Furthermore, the integrin β4-specific siRNA (20 and 40 nmol/L) significantly reduced the level of Fyn protein and the production of NO in VECs, while increased the level of ROCK in VSMCs that had been stimulated by SPC. The VECs reduced the SPC-induced contraction of VSMCs in the co-culture system through integrin β4 and Fyn proteins. In this process, NO may be the factor downstream of integrin β4 in VECs, while ROCK may be the key protein regulating the contraction of VSMCs.

Ethanol-mediated long-lasting adaptations of the NR2B-containing NMDA receptors in the dorsomedial striatum

We recently found that ethanol-induced long-term facilitation (LTF) of NMDAR activity is mediated by NR2B-NMDARs and is observed in the dorsomedial striatum (DMS) but not in the dorsolateral striatum (DLS).9 We also showed that repeated administration of ethanol causes a long-lasting increase in NMDAR activity in the DMS, resulting from ethanol-mediated Fyn phosphorylation of NR2B subunits.9 In this addendum, we report that the different sensitivity of NMDARs to ethanol between the DMS and DLS is not attributed to the abundance of synaptic NR2B-NMDARs or differences in Fyn levels. We further show that LTF is specific for NR2B-, but not NR2A-NMDARs, and that the duration of the in vivo ethanol-mediated increase in NMDAR activity is associated with the period of ethanol exposure, but not with alteration in NR1 or NR2A protein levels. Together, these results suggest that upregulation of NR2B-NMDAR activity by ethanol is selective and that ethanol's effect on NMDAR activity is gradual and cumulative.

Roles of the Src Tyrosine Kinases Lck and Fyn in Regulating γδTCR Signal Strength

Lck and Fyn, members of the Src family of tyrosine kinases, are key components of the αβTCR-coupled signaling pathway. While it is generally accepted that both Lck and Fyn positively regulate signal transduction by the αβTCR, recent studies have shown that Lck and Fyn have distinct functions in this signaling pathway, with Lck being a positive regulator and Fyn being a negative regulator of αβTCR signal transduction. To determine whether Lck and Fyn also differentially regulate γδTCR signal transduction, we analyzed γδ T cell development and function in mice with reduced Lck or Fyn expression levels. We found that reducing Lck or Fyn levels altered the strength of the γδTCR signaling response, with low levels of Lck weakening γδTCR signal strength and low levels of Fyn augmenting γδTCR signal strength. These alterations in γδTCR signal strength had profound effects not only on αβ/γδ lineage choice, but also on γδ thymocyte maturation and γδ T cell effector function. These results indicate that the cellular levels of Lck and Fyn play a role in regulating the strength of the γδTCR signaling response at different stages in the life of the γδ T cell.

Srcasm Inhibits Fyn-Induced Cutaneous Carcinogenesis with Modulation of Notch1 and p53

Src family tyrosine kinases (SFK) regulate cell proliferation, and increased SFK activity is common in human carcinomas, including cutaneous squamous cell carcinomas (SCC) and its precursors. The elevated SFK activity in cutaneous SCCs was modeled using K14-Fyn Y528F transgenic mice, which spontaneously form punctate keratotic lesions, scaly plaques, and large tumors resembling actinic keratoses, SCC in situ, and SCCs, respectively. Lesional tissue showed increased levels of activated SFKs, PDK1, STAT3, and ERK1/2, whereas Notch1/NICD protein and transcript levels were decreased. p53 levels also were decreased in SCC in situ and SCCs. Increasing Srcasm levels using a K14-Fyn Y528F/K14-Srcasm double transgenic model markedly inhibited cutaneous neoplasia. In contrast, increased expression of a nonphosphorylatable Srcasm mutant maintained the neoplastic phenotype. Increasing Srcasm levels decreased levels of Fyn, activated SFKs, ERK1/2, PDK1, and phospho-STAT3, and increased Notch1/NICD and p53 levels. Analysis of human specimens revealed that levels of Fyn and activated SFKs were elevated in SCCs compared with adjacent nonlesional epidermis. In addition, Notch1 and Srcasm protein and transcript levels were decreased in human SCCs compared with nonlesional epidermis. Therefore, the SCCs produced by the Fyn Y528F mice resemble their human counterparts at the molecular level. K14-Fyn Y528F mice represent a robust model of cutaneous carcinogenesis that manifests precancerous lesions and SCCs resembling human disease. The Fyn/Srcasm signaling nexus modulates activity of STAT3, PDK1, ERK1/2, Notch1, and p53. Further study of Fyn and Srcasm should provide insights into the mechanisms regulating keratinocyte proliferation and skin carcinogenesis.

Fyn and CD70 Expression in CD4+ T Cells from Patients with Systemic Lupus Erythematosus

CD4+ T cells from patients with systemic lupus erythematosus (SLE) display defective function that contributes to abnormal activation of B cells and autoantibody production. We compared the transcript and protein levels of Fyn and CD70 in CD4+ T cells from patients with SLE (n = 41) and healthy individuals (n = 34). The CD4+ T cells were isolated by positive biomagnetic separation technique. The quantitative analysis of messenger RNA was performed by reverse transcription and real-time quantitative PCR. The protein contents in the CD4+ T cells were determined by Western blotting analysis. We observed significantly higher levels of Fyn (p = 0.03) and CD70 (p = 0.029) transcripts in SLE CD4+ T cells than in controls. There was a significant increase in CD70 protein levels (p < 0.0001), but not Fyn protein levels (p = 0.081) in CD4+ T cells from patients with SLE compared to healthy individuals. In the group with high disease activity [SLE Disease Activity Index (SLEDAI) >/= 9], we observed a significantly higher Fyn protein content than in controls (p = 0.030). There was no correlation between Fyn and CD70 protein levels in SLE CD4+ T cells and disease activity as expressed in the SLEDAI scale. We confirmed previous observations of higher expression of CD70 in CD4+ T cells from patients with SLE. Our findings suggest that increased Fyn protein content in CD4+ T cells can be associated with high SLE disease activity.

Expression patterns of Src-family tyrosine kinases during Xenopus laevis development

Src family tyrosine kinases (SFKs) play important roles in cell morphology, differentiation, motility and proliferation. Elevated expression and/or specific activity of Src kinases are characteristic for several types of human cancer. However, little information is available about the role and spatio-temporal expression of SFKs in early embryonic development. In this study we characterized, in Xenopus laevis, the expression patterns of five SFK genes src, fyn, yes, lyn and laloo as well as of the csk gene, a negative regulator of SFKs, using RT-qPCR and in situ hybridisation. We found that transcripts of all SFKs and csk were already detectable in one-cell embryos and their levels similarly oscillated during subsequent development. First, after stage 8, the levels of SFK and csk mRNAs began to decrease, reached a minimum between stages 10 and 28 and increased again. In the later stages (33-45), the levels of fyn, yes and csk mRNAs returned to approximately maternal ones, whereas the src, laloo and lyn mRNA transcripts exceeded, up to about 3.5-6-fold, their maternal levels. In situ hybridisation analysis located the SFK and csk transcripts in the animal hemisphere of Xenopus embryos. Subsequent gastrula stages showed signals in ectodermal cells, mid-neurula stage embryos at neural folds, and the tailbud stages showed strong signals in the brain and neural tube. RT-qPCR concentration profiling along the animal-vegetal axis proved in blastula and gastrula the preferential localisation of yes, src, lyn and csk transcripts towards the animal pole in a gradient-like manner. In contrast, laloo and fyn displayed a vegetal pole preference.

Inhibition of DNA methyltransferase activity upregulates Fyn tyrosine kinase expression in Hut-78 T-lymphoma cells

Tyrosine kinase Fyn, the expression of which is epigenetically regulated, has been proposed to be a tumour suppressor gene. A frequent deletion at the 6q chromosomal region encoding the Fyn gene in lymphomas has been reported. Therefore, we assessed the impact of 5-Aza-2′-deoxycytidine (5-dAzaC), a DNA methyltransferase (DNMTs) inhibitor on Fyn expression in Hut-78 T-lymphoma cells. Using reverse transcription, real-time quantitative PCR (RQ-PCR), and western blot analyses, we found that 5-dAzaC significantly increased transcript and protein levels of Fyn in Hut-78 T-lymphoma cells. However, bisulfite sequencing revealed that Hut-78 T-lymphoma cells cultured in the absence of 5-dAzaC contained unmethylated cytosine in the cytosine-guanosine dinucleotide island of the Fyn promoter. Our results suggest that the DNMTs activity may have an indirect influence on the expression of Fyn without altering the methylation level of its promoter in Hut-78 T-lymphoma cells.

Fyn-mediated Phosphorylation of NR2B Tyr-1336 Controls Calpain-mediated NR2B Cleavage in Neurons and Heterologous Systems

Cleavage of the intracellular carboxyl terminus of the N-methyl-d-aspartate (NMDA) receptor 2 subunit (NR2) by calpain regulates NMDA receptor function and localization. Here, we show that Fyn-mediated phosphorylation of NR2B controls calpain-mediated NR2B cleavage. In cultured neurons, calpain-mediated NR2B cleavage is significantly attenuated by blocking NR2B phosphorylation of Tyr-1336, but not Tyr-1472, via inhibition of Src family kinase activity or decreasing Fyn levels by small interfering RNA. In HEK cells, mutation of Tyr-1336 eliminates the potentiating effect of Fyn on calpain-mediated NR2B cleavage. The potentiation of NR2B cleavage by Fyn is limited to cell surface receptors and is associated with calpain translocation to plasma membranes during NMDA receptor activation. Finally, reducing full-length NR2B by calpain does not decrease extrasynaptic NMDA receptor function, and truncated NR1/2B receptors similar to those generated by calpain have electrophysiological properties matching those of wild-type receptors. Thus, the Fyn-controlled regulation of NMDA receptor cleavage by calpain may play critical roles in controlling NMDA receptor properties during synaptic plasticity and excitotoxicity.

Effects of chronic multiple stress on learning and memory and the expression of Fyn, BDNF, TrkB in the hippocampus of rats

The effect of chronic stress on cognitive functions has been one of the hot topics in neuroscience. But there has been much controversy over its mechanism. The aim of this study was to investigate the effects of chronic multiple stress on spatial learning and memory as well as the expression of Fyn, BDNF and TrkB in the hippocampus of rats. Adult rats were randomly divided into control and chronic multiple stressed groups. Rats in the multiple stressed group were irregularly and alternatively exposed to situations of vertical revolution, sleep expropriation and restraint lasting for 6 weeks, 6 hours per day with night illumination for 6 weeks. Before and after the period of chronic multiple stresses, the performance of spatial learning and memory of all rats was measured using the Morris Water Maze (MWM). The expression of Fyn, BDNF and TrkB proteins in the hippocampus was assayed by Western blotting and immunohistochemical methods. The levels of Fyn and TrkB mRNAs in the hippocampus of rats were detected by RT-PCR technique. The escape latency in the control group and the stressed group were 15.63 and 8.27 seconds respectively. The performance of spatial learning and memory of rats was increased in chronic multiple stressed group (P < 0.05). The levels of Fyn, BDNF and TrkB proteins in the stressed group were higher than those of the control group (P < 0.05). The results of immunoreactivity showed that Fyn was present in the CA3 region of the hippocampus and BDNF positive particles were distributed in the nuclei of CA1 and CA3 pyramidal cells as well as DG granular cells. Quantitative analysis indicated that level of Fyn mRNA was also upregulated in the hippocampus of the stressed group (P < 0.05). Chronic multiple stress can enhance spatial learning and memory function of rats. The expression of Fyn, BDNF and TrkB proteins and the level of Fyn mRNA are increased in the stessed rat hippocampus. These suggest that Fyn and BDNF/TrkB signal transduction pathways may participate in the process of the enhanced learning and memory during chronic multiple stress.