Tyrosine kinase Fyn promotes apoptosis after intracerebral hemorrhage in rats by activating Drp1 signaling.

Tyrosine kinase Fyn is a member of the Src kinase family, which is involved in neuroinflammation, apoptosis, and oxidative stress. Its role in intracerebral hemorrhage (ICH) is not fully understood. In this study, we found that Fyn was significantly elevated in human brain tissue after ICH. Accordingly, we investigated the role of Fyn in a rat ICH model, which was constructed by injecting blood into the right basal ganglia. In this model, Fyn expression was significantly upregulated in brain tissue adjacent to the hematoma. SiRNA-induced Fyn knockdown was neuroprotective for secondary cerebral damage, as demonstrated by reduced brain edema, suppression of the modified neurological severity score, and mitigation of blood-brain barrier permeability and neuronal damage. Fyn downregulation reduced apoptosis following ICH, as indicated by downregulation of apoptosis-related proteins AIF, Cyt.c, caspase 3, and Bax; upregulation of anti-apoptosis-related protein Bcl-2; and decreased tunnel staining. Mdivi-1, a Drp1 inhibitor, reversed Fyn overexpression induced pro-apoptosis. However, Fyn did not significantly affect inflammation-related proteins NF-κB, TNF-α, caspase 1, MPO, IL-1ß, or IL-18 after ICH. Fyn activated Drp1 signaling by phosphorylating Drp1 at serine 616, which increased apoptosis after ICH in rats. This study clarifies the relationship between Fyn, apoptosis, and inflammation following ICH and provides a new strategy for exploring the prevention and treatment of ICH. KEY MESSAGES: ICH induced an increase in Fyn expression in human and rat cerebral tissues. Knockdown of Fyn prevented cerebral damage following ICH. Inhibition of Fyn had no significant effects on inflammatory responses. However, the downregulation of Fyn exerted neuroprotective effects on apoptosis. Fyn perturbed ICH-induced cell apoptosis by interacting with and phosphorylating (Ser616) Drp1 in a rat ICH model.

LINC00152 facilitates tumorigenesis in esophageal squamous cell carcinoma via miR-153-3p/FYN axis.

Long non-coding RNAs (LncRNAs) have been found to be associated with the biological behaviors of human cancers. LINC00152 is reported as an oncogene in many kinds of malignancies. However, the functions and mechanisms of LINC00152 involved in esophageal squamous cell carcinoma (ESCC) remain elusive. Our results revealed that LINC00152 expression was up-regulated in ESCC, and correlated with advanced TNM stage, lymph node metastasis, and poor prognosis of ESCC patients. Functionally, LINC00152 knockdown suppressed proliferation, decreased colony forming ability, and induced apoptosis in ESCC cells. Mechanically, LINC00152 functioned as a competing endogenous RNA (ceRNA) to sponge miR-153-3p, thereby facilitating its downstream target FYN. Moreover, miR-153-3p-mediated tumor-suppressive effects were partly reversed following LINC00152 overexpression. Also, FYN knockdown displayed a similar anti-cancerous role in ESCC cells. Taken together, LINC00152 contributed to ESCC progression by down-regulating miR-153-3p and promoting FYN expression, uncovering a novel LINC00152/miR-153-3p/FYN regulatory pathway in ESCC.

Genetic disruption of ankyrin-G in adult mouse forebrain causes cortical synapse alteration and behavior reminiscent of bipolar disorder.

Genome-wide association studies have implicated the ANK3 locus in bipolar disorder, a major human psychotic illness. ANK3 encodes ankyrin-G, which organizes the neuronal axon initial segment (AIS). We generated a mouse model with conditional disruption of ANK3 in pyramidal neurons of the adult forebrain (Ank-G cKO). This resulted in the expected loss of pyramidal neuron AIS voltage-gated sodium and potassium channels. There was also dramatic loss of markers of afferent GABAergic cartridge synapses, resembling the cortical microcircuitry changes in brains from psychotic patients, and suggesting disinhibition. Expression of c-fos was increased in cortical pyramidal neurons, consistent with increased neuronal activity due to disinhibition. The mice showed robust behavioral phenotypes reminiscent of aspects of human mania, ameliorated by antimania drugs lithium and valproate. Repeated social defeat stress resulted in repeated episodes of dramatic behavioral changes from hyperactivity to "depression-like" behavior, suggestive of some aspects of human bipolar disorder. Overall, we suggest that this Ank-G cKO mouse model recapitulates some of the core features of human bipolar disorder and indicates that cortical microcircuitry alterations during adulthood may be involved in pathogenesis. The model may be useful for studying disease pathophysiology and for developing experimental therapeutics.

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-ß (TGF-ß)-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 in vivo, 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.

Regulation of Multicellular Spheroids by MAPK and FYN Kinase.

Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length ß6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, ß-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase.

Inhibitory effect of fermented Arctium lappa fruit extract on the IgE-mediated allergic response in RBL­2H3 cells.

Arctium lappa fruit has been used in traditional medicine, and it is known to exert beneficial effects, such as antioxidant, anti-inflammatory and anticancer effects. However, the effects of the Arctium lappa fruit on the allergic response remain unknown. In this study, we evaluated the anti-allergic effects of Arctium lappa fruit extract (AFE) and its fermented form (F-AFE) using immunoglobulin E (IgE)-activated RBL­2H3 cells. To investigate the anti-allergic effects of AFE or F-AFE, we examined the release of ß-hexosaminidase, a key biomarker of degranulation during an allergic reaction, and the production of pro-inflammatory mediators, such as tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2) in the cells treated with or without the above-mentioned extracts. AFE weakly inhibited the release of ß-hexosaminidase, whereas F-AFE significantly suppressed the release of ß-hexosaminidase in a dose-dependent manner. Consistently, F-AFE suppressed the production of TNF-α and PGE2 in a dose-dependent manner. F-AFE exerted an inhibitory effect on the production of ß-hexosaminidase, TNF-α and PGE2 with an IC50 value of 30.73, 46.96 and 36.27 µg/ml, respectively. Furthermore, F-AFE inhibited the phosphorylation of Lyn, Fyn and Syk, which are involved in the FcεRI signaling pathway, that of phosphoinositide phospholipase C (PLC)γ1/2 and protein kinase C (PKC)δ, which are associated with the degranulation process, as well as that of extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase 1/2 (JNK), p38 and Akt, which are associated with cytokine expression. In the late phase, F-AFE partially suppressed the phosphorylation of cytosolic phospholipase A2 (cPLA2), but not the expression of cyclooxygenase (COX)-2. To compare and identify the major components of the two extracts, we used high-performance liquid chromatography. The levels of arctigenin, one of the major compounds, were elevated 6-fold in F-AFE compared with AFE, whereas the levels of arctiin, an arctigenin glycoside, were decreased in F-AFE by approximately 57.40%. These results suggest that arctigenin plays an important role in the anti-allergic effects of F-AFE. Taken together, F-AFE containing anti-allergic phytochemicals, including arctigenin, inhibited the activation of the FcεRI receptor induced by the antigen­IgE complex. Such effects may provide further information for the development of a phytomedicine for allergic diseases.

Differential spheroid formation by oral cancer cells.

Squamous cell carcinomas (SCC) make up 96% of all oral cancers. Most laboratory SCC studies grow cells as a monolayer, which does not accurately represent the disease in vivo. We used a more relevant multicellular spheroid (MCS) model to study this disease. The SCC9ß6KDFyn cell line, which expresses full-length ß6 and a kinase dead Fyn formed the largest MCS. Cell adhesive properties are dynamic and N-cadherin was increased in the largest MCS. c-Raf mediates the survival of tumor cells and was consistently expressed both in monolayers and in the MCS by SCC9ß6D1 cells which lack the ß6 cytoplasmic tail and, do not activate Fyn. SCC9ß6KDFyn cells also express high levels of c-Raf when grown as spheroids in which Fyn suppression stimulates MCS formation. Tumor microenvironment and growth patterns modulate cell behavior and suppression of Fyn kinase may promote MCS growth.

Hypoxia-inducible factors enhance glutamate signaling in cancer cells.

Signaling through glutamate receptors has been reported in human cancers, but the molecular mechanisms are not fully delineated. We report that in hepatocellular carcinoma and clear cell renal carcinoma cells, increased activity of hypoxia-inducible factors (HIFs) due to hypoxia or VHL loss-of-function, respectively, augmented release of glutamate, which was mediated by HIF-dependent expression of the SLC1A1 and SLC1A3 genes encoding glutamate transporters. In addition, HIFs coordinately regulated expression of the GRIA2 and GRIA3 genes, which encode glutamate receptors. Binding of glutamate to its receptors activated SRC family kinases and downstream pathways, which stimulated cancer cell proliferation, apoptosis resistance, migration and invasion in different cancer cell lines. Thus, coordinate regulation of glutamate transporters and receptors by HIFs was sufficient to activate key signal transduction pathways that promote cancer progression.

Transmembrane adaptor protein PAG/CBP is involved in both positive and negative regulation of mast cell signaling.

The transmembrane adaptor protein PAG/CBP (here, PAG) is expressed in multiple cell types. Tyrosine-phosphorylated PAG serves as an anchor for C-terminal SRC kinase, an inhibitor of SRC-family kinases. The role of PAG as a negative regulator of immunoreceptor signaling has been examined in several model systems, but no functions in vivo have been determined. Here, we examined the activation of bone marrow-derived mast cells (BMMCs) with PAG knockout and PAG knockdown and the corresponding controls. Our data show that PAG-deficient BMMCs exhibit impaired antigen-induced degranulation, extracellular calcium uptake, tyrosine phosphorylation of several key signaling proteins (including the high-affinity IgE receptor subunits, spleen tyrosine kinase, and phospholipase C), production of several cytokines and chemokines, and chemotaxis. The enzymatic activities of the LYN and FYN kinases were increased in nonactivated cells, suggesting the involvement of a LYN- and/or a FYN-dependent negative regulatory loop. When BMMCs from PAG-knockout mice were activated via the KIT receptor, enhanced degranulation and tyrosine phosphorylation of the receptor were observed. In vivo experiments showed that PAG is a positive regulator of passive systemic anaphylaxis. The combined data indicate that PAG can function as both a positive and a negative regulator of mast cell signaling, depending upon the signaling pathway involved.

Regulation of primary response genes in B cells.

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

Therapeutic strategies for tau mediated neurodegeneration.

Based on the amyloid hypothesis, controlling ß-amyloid protein (Aß) accumulation is supposed to suppress downstream pathological events, tau accumulation, neurodegeneration and cognitive decline. However, in recent clinical trials, Aß removal or reducing Aß production has shown limited efficacy. Moreover, while active immunisation with Aß resulted in the clearance of Aß, it did not prevent tau pathology or neurodegeneration. This prompts the concern that it might be too late to employ Aß targeting therapies once tau mediated neurodegeneration has occurred. Therefore, it is timely and very important to develop tau directed therapies. The pathomechanisms of tau mediated neurodegeneration are unclear but hyperphosphorylation, oligomerisation, fibrillisation and propagation of tau pathology have been proposed as the likely pathological processes that induce loss of function or gain of toxic function of tau, causing neurodegeneration. Here we review the strategies for tau directed treatments based on recent progress in research on tau and our understanding of the pathomechanisms of tau mediated neurodegeneration.

Enhanced antioxidant effect of prenylated polyphenols as Fyn inhibitor.

Polyphenols have antioxidant effects. In view of the diverse biological activities of prenylated natural products, this study investigated whether polyphenols with prenyl residues have improved antioxidant and cytoprotective activity against oxidative stress, and explored the underlying basis for this effect. A set of structurally related polyphenols exhibited varying degrees of antioxidant effect in HepG2 cells, as evidenced by increases in cell viability against oxidative injury; kazinol E and C with three prenyls had greater potency than other kazinols having fewer prenyl chains. Polyphenols without prenyl (tupichinol C and resveratrol) showed weaker potency. Treatment with kazinol E diminished H(2)O(2) production and enabled cells to protect the mitochondria, as indicated by the inhibition of mitochondrial fragmentation, mitochondrial permeability transition, and cytochrome c release. Moreover, kazinol E activated LKB1 by its phosphorylation and cytoplasmic translocation, contributing to the protection of mitochondria via AMPK. In vitro or in a cell-based assay, tyrosine phosphorylation of Fyn was prohibited by kazinol E, which led to LKB1 activation, as shown by the experiments using Fyn over-expression construct or siRNA. SU6656, a known Fyn inhibitor, had a similar effect. Moreover, oxidative stress facilitated Fyn phosphorylation with repression of AMPKα and GSK3ß phosphorylation, which was abolished by kazinol E treatment. The role of Fyn inhibition by kazinol E in AMPK-mediated protection of the cell viability and mitochondrial function was strengthened by ectopically expressed Fyn's reversal of these effects. In conclusion, kazinols as multi-prenylated polyphenols possess increased antioxidant and cytoprotective activity, which depends on the activation of LKB1-AMPK pathway downstream of Fyn inhibition.

Heterogeneous expression of Src tyrosine kinases Lyn, Fyn and Syk in classical Hodgkin lymphoma: prognostic implications.

The aim of this study was to determine the prognostic significance of the expression of Lyn, Fyn and Syk in Hodgkin lymphoma and its correlation with Epstein-Barr virus (EBV) infection. With this in mind, 96 patients with classical Hodgkin lymphoma were immunohistochemically evaluated for Lyn, Fyn and Syk expression in Hodgkin and Reed-Sternberg cells, and the results were correlated with the presence of EBV and patient outcomes. These three kinases were heterogeneously expressed in classical Hodgkin lymphoma cases. As there are no cut-offs established for these antibodies, they were introduced as continuous variables in the model. Statistical analysis showed that the expression of Syk and Fyn was significantly associated with shorter failure-free survival. Syk and Fyn may be useful to predict at diagnosis the treatment response of patients with classical Hodgkin lymphoma. There was a significant association between EBV infection and Lyn expression (p < 0.05). Overexpression of Syk and the availability of Syk inhibitors suggest that this molecule might be a therapeutic strategy worthy of development for cases expressing this molecule.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells.

Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl2) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl2-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl2-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

Regulation of peripheral myelination by Src-like kinases.

Fyn, a nonreceptor Src-like tyrosine kinase (SLK), plays an important role in oligodendrocyte differentiation and myelination in the brain. However, its role in myelination of peripheral nerves remains undefined. Here we report that selective inhibitors of SLKs (PP2 and SU6656) caused a dose-dependent decrease in the accumulation of several myelin proteins, including myelin basic protein (MBP), protein zero (P0) and myelin-associated glycoprotein (MAG) in rat Schwann cell-dorsal root ganglion neuron (SC-DRGN) co-cultures. Interestingly, SLK inhibition was insufficient to completely abrogate myelin synthesis, as removal of PP2 after several days of treatment permitted a partial recovery of myelin proteins expression. Furthermore, fewer and shorter myelinated segments formed in the continuous presence of PP2, although the myelin formed was normally compacted. PP2 also decreased the number of SCs expressing Krox-20, a master-regulatory transcription factor expressed by myelinating SCs, by 50%. These results were corroborated by selective knockdown of Fyn and Lyn kinases using siRNA. Extracellular matrix is important to SC differentiation and peripheral myelination. Using phospho-specific antibodies, we showed that addition of extracellular matrix extracts to SC-DRGN co-cultures resulted in the activation of ERK, Akt and p38 MAPK, three protein kinases involved in SC proliferation, differentiation and peripheral myelination. PP2 blocked the phosphorylation of all three kinases. Our results support a role for SLKs in the initiation of peripheral myelination via the activation of p38, Akt and ERK, which regulate Krox-20 expression and peripheral myelination.

Expression of Fyn kinase modulates EMT in oral cancer cells.

Oral squamous cell carcinoma (SCC) is an aggressive tumor with a poor 5-year survival rate. Oral SCC can undergo epithelial to mesenchymal transition (EMT). We previously showed that the epithelial integrin alphavbeta6 complexes with Fyn kinase in oral SCC to promote EMT. Using immunofluorescence microscopy and Western blotting, we evaluated whether the expression of specific markers of EMT were influenced by modulating serum concentration (ie. growth factors). The SCC cultures were grown under contrasting levels of serum. In low serum (1%), Fyn promoted EMT; whereas suppression of Fyn kinase promoted the epithelial phenotype. However, when the SCC cells were grown in 10% serum, activation of Fyn had the reverse effect. Lastly, cell migration was evaluated under low serum conditions (1% FBS). Activation of Fyn promoted SCC cell migration and its suppression thwarted SCC migration toward FN. These results indicate that the activation of Fyn kinase as well as local growth factor concentration modulate EMT in oral SCC.

Expression of full-length and truncated Fyn tyrosine kinase transcripts and encoded proteins during spermatogenesis and localization during acrosome biogenesis and fertilization.

We report that full-length and truncated transcripts of Fyn tyrosine protein kinase are expressed during testicular development. Truncated Fyn (tr-Fyn) transcripts encode a 24 kDa protein with a N-terminal (NT) domain, a complete Src homology (SH) 3 domain and an incomplete SH2 domain. The kinase domain is missing in tr-Fyn. In contrast, full-length Fyn transcripts encode a 59-55 kDa protein. Fractionated spermatids by centrifugal elutriation express tr-Fyn transcripts and protein, but not full-length Fyn transcripts and protein. Neither full-length Fyn nor tr-Fyn transcripts and encoded proteins are detected in elutriated pachytene spermatocytes. Sertoli cells express full-length and truncated Fyn throughout testicular development. In contrast, sperm contain full-length Fyn transcripts and protein but not the truncated form. tr-Fyn protein is visualized at the cytosolic side of Golgi membranes, derived proacrosomal vesicles, along the outer acrosome membrane and the inner acrosome membrane-acroplaxome complex anchoring the acrosome to the spermatid nuclear envelope. Fyn and phosphotyrosine immunoreactivity coexist in the tail of capacitated sperm. During fertilization, the Fyn-containing acroplaxome seen in the egg-bound and egg-fused sperm is no longer detected upon decondensation of the sperm nucleus. tr-Fyn expands the catalog of truncated tyrosine protein kinases expressed during spermiogenesis. We suggest that the NT and SH3 domains of tr-Fyn may recruit adaptor and effector proteins, in particular GTPase activating proteins, required for acrosome-acroplaxome biogenesis, acroplaxome F-actin dynamics and Sertoli cell function. During fertilization, full-length Fyn in the acroplaxome may contribute to a transient local signaling burst during the early events of sperm-egg interaction.

SRC family kinases are required for limb trajectory selection by spinal motor axons.

Signal relay by guidance receptors at the axonal growth cone is a process essential for the assembly of a functional nervous system. We investigated the in vivo function of Src family kinases (SFKs) as growth cone guidance signaling intermediates in the context of spinal lateral motor column (LMC) motor axon projection toward the ventral or dorsal limb mesenchyme. Using in situ mRNA detection we determined that Src and Fyn are expressed in LMC motor neurons of chick and mouse embryos at the time of limb trajectory selection. Inhibition of SFK activity by C-terminal Src kinase (Csk) overexpression in chick LMC axons using in ovo electroporation resulted in LMC axons selecting the inappropriate dorsoventral trajectory within the limb mesenchyme, with medial LMC axon projecting into the dorsal and ventral limb nerve with apparently random incidence. We also detected LMC axon trajectory choice errors in Src mutant mice demonstrating a nonredundant role for Src in motor axon guidance in agreement with gain and loss of Src function in chick LMC neurons which led to the redirection of LMC axons. Finally, Csk-mediated SFK inhibition attenuated the retargeting of LMC axons caused by EphA or EphB over-expression, implying the participation of SFKs in Eph-mediated LMC motor axon guidance. In summary, our findings demonstrate that SFKs are essential for motor axon guidance and suggest that they play an important role in relaying ephrin:Eph signals that mediate the selection of motor axon trajectory in the limb.

Inverse regulation of plasticity-related immediate early genes by calcineurin in hippocampal neurons.

In the mammalian hippocampus, changes in the expression of immediate early genes (IEGs) is thought to contribute to long term plastic changes in neurons brought about by learning tasks and high frequency stimulation of synapses. The phosphatase calcineurin has emerged as an important negative regulator of hippocampus-dependent learning and long term potentiation. Here we investigated the possibility that the constraining action of calcineurin on hippocampal plasticity is mediated in part by regulation of gene expression through negative control of transcription factors, such as cAMP-response element (CRE)-binding protein (CREB). We assessed the effect of calcineurin inhibitors on CREB activation by neuronal activity and show that calcineurin activity is in fact required for CREB-mediated gene expression. However, inhibition of calcineurin had disparate effects on the transcriptional induction of CREB-dependent IEGs. We find that the IEG c-fos is unaffected by suppression of calcineurin activity, the plasticity-related genes Egr1/Zif268 and Egr2/Krox-20 are up-regulated, and genes encoding the orphan nuclear hormone receptors Nor1 and Nur77 are down-regulated. We further show that the up-regulation of particular IEGs is probably due to the presence of serum response elements (SREs) in their promoters, because SRE-mediated gene expression is enhanced by calcineurin blockers. Moreover, expression of the c-fos gene, which is unaffected by calcineurin inhibitors, could be down-regulated by mutating the SRE. Conversely, SRE-mediated c-fos induction in the absence of a functional CRE was enhanced by calcineurin inhibitors. Our experiments thus implicate calcineurin as a negative regulator of SRE-dependent neuronal genes.

Fyn kinase is a direct molecular target of delphinidin for the inhibition of cyclooxygenase-2 expression induced by tumor necrosis factor-alpha.

Tumor necrosis factor (TNF)-alpha-mediated cyclooxygenase (COX)-2 expression plays key roles in inflammation and tumorigenesis, particularly skin carcinogenesis, and hence targeting the TNF-alpha-mediated signaling pathway might be a promising strategy for developing chemopreventive agents against skin cancer and other skin disorders. Here we report that Fyn kinase - one of the members of the nonreceptor protein tyrosine kinase family - is involved in TNF-alpha-induced COX-2 expression, and that delphinidin - a major anthocyanidin present in red wine and berries - inhibits these effects by directly inhibiting Fyn kinase activity. Delphinidin strongly inhibited TNF-alpha-induced COX-2 expression in JB6 P+ mouse epidermal (JB6 P+) cells, whereas two other major phenolic compounds (resveratrol and gallic acid) did not exert significant inhibitory effects. Delphinidin inhibited the TNF-alpha-induced phosphorylations of JNK, p38 MAP kinase, Akt, p90RSK, MSK1, and ERK, and subsequently blocked the activation of the eukaryotic transcription factors AP-1 and NF-kappaB. Kinase and pull-down assay data revealed that delphinidin inhibited Fyn kinase activity and directly bound with Fyn kinase noncompetitively with ATP. By using PP2 (a commercial inhibitor of Fyn kinase) and siRNA-Fyn, we confirmed that Fyn kinase is involved in TNF-alpha-induced COX-2 expression mainly by down-regulating JNK in JB6 P+ cells. Together these findings suggest that the targeted inhibition of Fyn kinase activity and COX-2 expression by delphinidin contributes to the chemopreventive potential of red wine and berries.