Activation Of Phospholipase Cγ2 Research Articles

Extracellular domain mutations of the EGF receptor differentially modulate high-affinity and low-affinity responses to EGF receptor ligands

The EGF receptor is mutated in a number of cancers. In most cases, the mutations occur in the intracellular tyrosine kinase domain. However, in glioblastomas, many of the mutations are in the extracellular ligand binding domain. To determine what changes in receptor function are induced by such extracellular domain mutations, we analyzed the binding and biological response to the seven different EGF receptor ligands in three common glioblastoma mutants--R84K, A265V, and G574V. Our data indicate that all three mutations significantly increase the binding affinity of all seven ligands. In addition, the mutations increase the potency of all ligands for stimulating receptor autophosphorylation, phospholipase Cγ, Akt, and MAP kinase activity. In all mutants, the rank order of ligand potency seen at the wild-type receptor was retained, suggesting that the receptors still discriminate among the different ligands. However, the low affinity ligands, EPR and EPG, did show larger than average enhancements of potency for stimulating Akt and MAPK, but not receptor autophosphorylation and phospholipase Cγ activation. Relative to the wild-type receptor, these changes lead to an increase in the responsiveness of these mutants to physiological concentrations of ligands and an alteration in the ratio of activation of the different pathways. This may contribute to their oncogenic potential. In the context of recent findings, our data also suggest that so-called ‘high’ affinity biological responses arise from activation by isolated receptor dimers, whereas ‘low’ affinity biological responses require clustering of receptors which occurs at higher concentrations of ligand.

Icaritin sensitizes thrombin- and TxA2-induced platelet activation and promotes hemostasis via enhancing PLCγ2-PKC signaling pathways.

Vascular injury results in uncontrollable hemorrhage in hemorrhagic diseases and excessive antithrombotic therapy. Safe and efficient hemostatic agents which can be orally administered, are urgently needed. Platelets play indispensable roles in hemostasis, but there is no drug exerting hemostatic effects through enhancing platelet function. The regulatory effects of icaritin, a natural compound isolated from Herba Epimedii, on the dense granule release, thromboxane A2 (TxA2) synthesis, α-granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by multiple agonists were investigated. The effects of icaritin on tail vein bleeding times of warfarin-treated mice were also evaluated. Furthermore, we investigated the underlying mechanisms by which icaritin exerted its pharmacological effects. Icaritin alone did not activate platelets, but significantly potentiated the dense granule release, α-granule release, activation of integrin αIIbβ3, and aggregation of platelets induced by thrombin and U46619. Icaritin also shortened tail vein bleeding times of mice treated with warfarin. In addition, phosphorylated proteome analysis, immunoblotting analysis, and pharmacological research revealed that icaritin sensitized the activation of phospholipase Cγ2 (PLCγ2)-protein kinase C (PKC) signaling pathways, which play important roles in platelet activation. Icaritin can sensitize platelet activation induced by thrombin and TxA2 through enhancing the activation of PLCγ2-PKC signaling pathways and promote hemostasis, and has potential to be developed into a novel orally deliverable therapeutic agent for hemorrhages.

Restrained glycoprotein VI-induced platelet signaling by tyrosine protein phosphatases independent of phospholipase Cγ2

The platelet collagen receptor glycoprotein VI (GPVI) signals to activation of phospholipase Cγ2 (PLCγ2) and phosphoinositide 3-kinases (PI3K), causing platelet activation and aggregation. The non-receptor Src homology tyrosine phosphatases Shp1/2 modulate GPVI signaling in partly opposite ways, both of which are targeted by the potential drug NSC87877. Effect measurements of the Shp1/2 inhibitor NSC87877 on platelet activation via GPVI using light transmission aggregometry, Ca2+ flux assay, western blotting and flow cytometry. Effect measurements of selective PI3K inhibitor TGX221. Inhibition of Shp1/2 with NSC87877 enhanced platelet aggregation induced by the GPVI agonist, collagen-related peptide (CRP). Furthermore, NSC87877 antagonized the effects of PI3Kb inhibition, but not of Btk inhibition. Both NSC87877 and TGX221 suppressed the CRP-induced phosphorylation of PLCγ2 at activation site Tyr759. These findings indicate that drug interference of the two phosphatases Shp1/2 subtly enhances GPVI-induced platelet responses via a mechanism not involving PLCγ2 activation, even upon PI3K inhibition.

Src-related thrombocytopenia: a fine line between a megakaryocyte dysfunction and an immune-mediated disease

AbstractSrc-related thrombocytopenia (SRC-RT) is a rare autosomal dominant, inherited platelet disorder resulting from the p.E527K heterozygous germline gain-of-function variant of Src. To date, genetic diagnosis of the disease has only been reported in 7 patients from 3 unrelated families. The clinical features ranged from isolated thrombocytopenia to complex syndromic manifestations characterized by thrombocytopenia, bleeding, myelofibrosis, splenomegaly, and bone disease. We report a new 3-generation kindred with the Src p.E527K variant. Patients presented with rather variable platelet counts (38-139 × 109/L), mildly impaired platelet function, >15% immature platelet fraction, and with a significant proportion of large-giant platelets. Four adults from the family were diagnosed with immune thrombocytopenia (ITP) and underwent splenectomy, achieving sustained platelet counts >75 × 109/L for several years; increases in platelet counts were also observed after corticosteroid therapy. Four of 7 Src p.E527K variant carriers showed immune defects and recurrent infections. In addition, a range of neurological symptoms, from specific language impairment to epilepsy, was seen in some family members. Patient platelets exhibited constitutive Src, Bruton tyrosine kinase, and phospholipase Cγ2 activation, and after stimulating CD19 cells by crosslinking surface immunoglobulin M, phosphorylated extracellular signal-regulated kinase (ERK) was significantly increased in B cells from individuals carrying the Src p.E527K substitution. In summary, in addition to causing impaired platelet production, SRC-RT may associate immune dysregulation and increased platelet consumption. In families in whom several members are responsive to ITP-directed therapies, an underlying Src p.E527K variant should be excluded.

Phospholipase Cγ2 regulates endocannabinoid and eicosanoid networks in innate immune cells

Human genetic studies have pointed to a prominent role for innate immunity and lipid pathways in immunological and neurodegenerative disorders. Our understanding of the composition and function of immunomodulatory lipid networks in innate immune cells, however, remains incomplete. Here, we show that phospholipase Cγ2 (PLCγ2 or PLCG2)-mutations in which are associated with autoinflammatory disorders and Alzheimer's disease-serves as a principal source of diacylglycerol (DAG) pools that are converted into a cascade of bioactive endocannabinoid and eicosanoid lipids by DAG lipase (DAGL) and monoacylglycerol lipase (MGLL) enzymes in innate immune cells. We show that this lipid network is tonically stimulated by disease-relevant human mutations in PLCγ2, as well as Fc receptor activation in primary human and mouse macrophages. Genetic disruption of PLCγ2 in mouse microglia suppressed DAGL/MGLL-mediated endocannabinoid-eicosanoid cross-talk and also caused widespread transcriptional and proteomic changes, including the reorganization of immune-relevant lipid pathways reflected in reductions in DAGLB and elevations in PLA2G4A. Despite these changes, Plcg2-/- mice showed generally normal proinflammatory cytokine and chemokine responses to lipopolysaccharide treatment, instead displaying a more restricted deficit in microglial activation that included impairments in prostaglandin production and CD68 expression. Our findings enhance the understanding of PLCγ2 function in innate immune cells, delineating a role in cross-talk with endocannabinoid/eicosanoid pathways and modulation of subsets of cellular responses to inflammatory stimuli.

Overcoming Acquired Epigenetic Resistance to BTK Inhibitors.

In diffuse large B-cell lymphoma, we show that primary resistance to BTK inhibitors is due to epigenetic rather than genetic changes that circumvent the BTK blockade. We also observed this resistance mechanism in chronic lymphocytic leukemia, suggesting that epigenetic alterations may contribute more to BTK inhibitor resistance than currently thought.See related commentary by Pasqualucci, p. 555. This article is highlighted in the In This Issue feature, p. 549.

Characterization of Mice with a Platelet-Specific Deletion of the Adapter Molecule ADAP.

The adhesion and degranulation-promoting adapter protein (ADAP) is expressed in T cells, NK cells, myeloid cells, and platelets. The involvement of ADAP in the regulation of receptor-mediated inside-out signaling leading to integrin activation is well characterized, especially in T cells and in platelets. Due to the fact that animal studies using conventional knockout mice are limited by the overlapping effects of the different ADAP-expressing cells, we generated conditional ADAP knockout mice (ADAPfl/fl PF4-Cretg) (PF4, platelet factor 4). We observed that loss of ADAP restricted to the megakaryocytic lineage has no impact on other hematopoietic cells even under stimulation conditions. ADAPfl/fl PF4-Cretg mice showed thrombocytopenia in combination with reduced plasma levels of PF4 and transforming growth factor β1 (TGF-β1). In vitro, platelets from these mice revealed reduced P-selectin expression, lower levels of TGF-β1 release, diminished integrin αIIbβ3 activation, and decreased fibrinogen binding after stimulation with podoplanin, the ligand of C-type lectin-like receptor 2 (CLEC-2). Furthermore, loss of ADAP was associated with impaired CLEC-2-mediated activation of phospholipase Cγ2 (PLCγ2) and extracellular signal-regulated kinase 1/2 (ERK1/2). Induction of experimental autoimmune encephalomyelitis (EAE) in mice lacking ADAP expression in platelets caused a more severe disease. In vivo administration of TGF-β1 early after T cell transfer reduced EAE severity in mice with loss of ADAP restricted to platelets. Our results reveal a regulatory function of ADAP in platelets in vitro and during autoimmune disease EAE in vivo.

Mathematical Model of Platelet Intracellular Signaling After Activation by Fucoidan

Blood platelets are the cells responsible for prevention of the blood loss. Fucoidan is a brown algae extract that is known to activate platelets via C-type lectin receptor of the second type. On the other hand, different fucoidans are now considered as perspective immunomodulators. Thus, application of fucoidan as a medicinal drug seems to be contradictory. In this work we studied activation of platelets by fucoidan in silico and in vitro. The computational model describes the behavior of the participants of the fucoidan receptor signaling cascade. The model was validated with available experimental data published earlier. In order to confirm the model predictions, the fucoidan-induced activation of platelets was assessed in flow cytometry and aggregometry experiments. The resultant model describes changes in the activity of tyrosine kinases of Syk and Sarc family and subsequent activation of phospholipase Cγ2. One of the main model prediction is a significant increase in the platelet cytosolic calcium level after the activation by fucoidan. This prediction was confirmed in the experiments. Thus, fucoidan, as a true platelet activator, cannot be applied in therapy.

α-1,3-Fucosyltransferase-VII siRNA inhibits the expression of SLex and hepatocarcinoma cell proliferation.

The increased expression of sialyl-Lewisx (SLex) epitope on the surface of tumor cells has been known for decades. However, genetic manipulation of the expression of SLex and the role of SLex in cancer cell proliferation remains to be fully elucidated. The present study suggested that the monoclonal antibody of SLex (KM93) significantly inhibited the proliferation of human hepatocarcinoma (HCC) cells. The expression levels of three sialyl-Lewis oligosaccharide antigens, SLex, SLea and dimeric SLex (SDLex), were determined on the cell surface of the MHCC97 human HCC cell line. The expression of SLex was markedly higher in MHCC97 cells than in normal liver cells. The expression of SDLex was also relatively high, however, no significant difference was observed between normal liver cells and HCC cells. The expression of SLea was only detected in trace quantities. Fucosyltransferase (FUT) is the key enzyme of the fucosylation step in the biosynthesis of sialyl-Lewis oligosaccharide antigens. Therefore, the present study investigated the expression of FUTs. It was found that the mRNA and protein expression levels of FUT7 were high in the MHCC97 HCC cell line compared with levels in normal liver cells. FUT6 was also expressed at a high level, although the difference was not statistically significant between MHCC97 cells and normal liver cells. No expression of FUT3 was detected. The results were consistent with the change insialyl-Lewis antigens. The effects of FUT7 small interfering (si)RNA transfection on the expression of FUT7, expression of SLex and MHCC97 cell proliferation were also examined. Following FUT7 siRNA transfection, the expression of FUT7 was markedly downregulated, as determined by western blot and reverse transcription-quantitative polymerase chain reaction methods. The results from flow cytometry showed that the synthesis of SLex was also inhibited, which was consistent with the downregulated expression of FUT7. MHCC97 cell proliferation was also significantly inhibited following FUT7 siRNA transfection, which was correlated with suppression of the S-phase in cell cycle progression. By using inhibitors of various signaling pathways, it was found that the knockdown of FUT7 inhibited the activation of phospholipase Cγ (PLCγ) by inhibiting the translocation and phosphorylation of PLCγ. In conclusion, the results suggested that FUT7 has animportant functional role in human HCC cell proliferation by controlling cell cycle progression via the PLCγ/extracellular signal-regulated kinase signaling pathway. The inhibition of SLex and FUT7 siRNA transfection may provide a novel therapeutic methodology to treat tumors that express SLex glycoconjugates.

Epidermal growth factor receptors containing a single tyrosine in their C-terminal tail bind different effector molecules and are signaling-competent

The EGF receptor is a classic receptor tyrosine kinase. It contains nine tyrosines in its C-terminal tail, many of which are phosphorylated and bind proteins containing SH2 or phosphotyrosine-binding (PTB) domains. To determine how many and which tyrosines are required to enable EGF receptor-mediated signaling, we generated a series of EGF receptors that contained only one tyrosine in their C-terminal tail. Assays of the signaling capabilities of these single-Tyr EGF receptors indicated that they can activate a range of downstream signaling pathways, including MAP kinase and Akt. The ability of the single-Tyr receptors to signal correlated with their ability to bind Gab1 (Grb2-associated binding protein 1). However, Tyr-992 appeared to be almost uniquely required to observe activation of phospholipase Cγ. These results demonstrate that multiply phosphorylated receptors are not required to support most EGF-stimulated signaling but identify Tyr-992 and its binding partners as a unique node within the network. We also studied the binding of the isolated SH2 domain of Grb2 (growth factor receptor-bound protein 2) and the isolated PTB domain of Shc (SHC adaptor protein) to the EGF receptor. Although these adapter proteins bound readily to wild-type EGF receptor, they bound poorly to the single-Tyr EGF receptors, even those that bound full-length Grb2 and Shc well. This suggests that in addition to pTyr-directed associations, secondary interactions between the tail and regions of the adapter proteins outside of the SH2/PTB domains are important for stabilizing the binding of Grb2 and Shc to the single-Tyr EGF receptors.

Association of C-Type Lectin Mincle with Fc\u03b5RI\u03b2\u03b3 Subunits Leads to Functional Activation of RBL-2H3 Cells through Syk

Macrophage-inducible C-type lectin (Mincle) interacts with the γ-subunit of high-affinity IgE receptor (FcεRIγ) and activates Syk by recognizing its specific ligand, trehalose-6,6′-dimycolate, a glycolipid produced by Mycobacterium tuberculosis. It has been suggested that mast cells participate in the immune defense against pathogenic microbes including M. tuberculosis, although the functions are still uncertain. In this study, we examined the Mincle-mediated signaling pathway and cellular responses using RBL-2H3 cells. Mincle formed a protein complex with not only FcεRIγ but also FcεRIβ in a stable cell line expressing myc-tagged Mincle. In addition, engagement of Mincle increased the levels of protein tyrosine phosphorylation and ERK phosphorylation. A pull-down assay demonstrated that cross-linking of Mincle induced binding of FcεRIβγ subunits to the Src homology 2 domain of Syk. Pharmacological and genetic studies indicated that activation of Syk was critical for Mincle-mediated activation of phospholipase Cγ2, leading to the activation of ERK and nuclear factor of activated T cells. Moreover, engagement of Mincle efficiently induced up-regulation of characteristic mast cell genes in addition to degranulation. Taken together, our present results suggest that mast cells contribute to Mincle-mediated immunity through Syk activation triggered by association with the FcεRIβγ complex.

Peroxisome proliferator-activated receptor-β/δ modulates mast cell phenotype.

The peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) is known to have multiple anti-inflammatory effects, typically observed in endothelial cells, macrophages, T cells and B cells. Despite the fact that mast cells are important mediators of inflammation, to date, the role of PPARβ/δ in mast cells has not been examined. Hence, the present study examined the hypothesis that PPARβ/δ modulates mast cell phenotype. Bone-marrow-derived mast cells (BMMCs) and peritoneal mast cells from Pparβ/δ+/+ mice expressed higher levels of high-affinity IgE receptor (FcεRI) compared with Pparβ/δ-/- mice. BMMCs from Pparβ/δ+/+ mice also exhibited dense granules, associated with higher expression of enzymes and proteases compared with Pparβ/δ-/- mice. Resting BMMCs from Pparβ/δ+/+ mice secreted lower levels of inflammatory cytokines, associated with the altered activation of phospholipase Cγ1 and extracellular signal-regulated kinases compared with Pparβ/δ-/- mice. Moreover, the production of cytokines by mast cells induced by various stimuli was highly dependent on PPARβ/δ expression. This study demonstrates that PPARβ/δ is an important regulator of mast cell phenotype.

Fibroblast growth factor 23 signaling in hippocampal cells: impact on neuronal morphology and synaptic density

Endocrine fibroblast growth factor 23 (FGF23) is predominantly secreted by osteocytes and facilitates renal phosphate excretion. However, FGF23 is also present in cerebrospinal fluid. In chronic kidney disease, FGF23 serum levels are excessively elevated and associated with learning and memory deficits. Structural plasticity of the hippocampus such as formation of new synapses or an altered dendritic arborization comprises a cellular and morphological correlate of memory formation. Therefore, we hypothesize that FGF23 alters hippocampal neuron morphology and synapses. To address this, we prepared primary murine hippocampal cultures and incubated them with recombinant FGF23 alone or together with a soluble isoform of its co-receptor α-Klotho. Neuronal expression of a fluorescent reporter allowed for a detailed evaluation of the neuronal morphology by Sholl analysis. Additionally, we evaluated synaptic density, identified by stainings, for synaptic markers. We show an enhanced number of primary neurites combined with a reduced arborization, resulting in a less complex morphology of neurons treated with FGF23. Moreover, FGF23 enhances the synaptic density in a FGF-receptor (FGF-R) dependent manner. Finally, we addressed the corresponding signaling events downstream of FGF-R employing a combination of western blots and quantitative immunofluorescence. Interestingly, FGF23 induces phospholipase Cγ activity in primary hippocampal neurons. Co-application of soluble α-Klotho leads to activation of the Akt-pathway and modifies FGF23-impact on neuronal morphology and synaptic density. Compared with other FGFs, this alternative signaling pattern is a possible reason for differential effects of FGF23 on hippocampal neurons and may thereby contribute to learning and memory deficits in chronic kidney disease patients. In this study, we show that fibroblast growth factor 23 inhibits neuronal ramification and enhances the synaptic density in primary hippocampal cultures accompanied by phospholipase Cγ-activation. Co-application of the co-receptor α-Klotho leads to an Akt-activation and further modifies neuronal morphology and number of synapses. Those effects provide a mechanistic basis for memory deficits in patients suffering from chronic kidney disease (CKD) characterized by excessively elevated FGF23 levels as well as memory deficits.

MicroRNA-204 suppresses epileptiform discharges through regulating TrkB-ERK1/2-CREB signaling in cultured hippocampal neurons

MicroRNAs (miRs) have been increasingly recognized as post-transcriptional regulators involved in the pathogenesis of epilepsy. Extensive evidence suggests that inhibition of tropomyosin related kinase type B (TrkB) signaling limits epileptogenesis. It has been reported that miR-204 directly targeted and downregulated TrkB protein in a variety of cancers to suppress tumorigenesis, but its effect on epileptogenesis is unknown. Here we showed that miR-204 was downregulated in cultured hippocampal neurons following status epilepticus (SE) induced by Mg2+-free treatment. Overexpression of miR-204 using a transfection technique negatively regulated the expression of TrkB mRNA. Insight into the downstream signaling pathway activated by TrkB may provide valuable clues to the underlying mechanisms of epileptogenesis. Biochemical measures revealed activation of phospholipase Cγ1 (PLCγ1), extracellular signal-regulated kinase1/2 (ERK1/2) and cAMP response-element binding protein (CREB) signaling molecules in the Mg2+-free model in hippocampal neurons, of which ERK1/2-CREB but not PLCγ1 signaling pathway was able to be inhibited by miR-204. Epileptiform discharges assessed using whole-cell current–clamp techniques were suppressed by miR-204 in cultured hippocampus neurons of SE. These results suggest that miR-204 exerts an antiepileptogenic effect in the Mg2+-free model in hippocampal neurons through regulating TrkB and downstream ERK1/2-CREB signaling pathway.

Functional interplay between ganglioside GM1 and cross-linking galectin-1 induces axon-like neuritogenesis via integrin-based signaling and TRPC5-dependent Ca²⁺ influx.

Axon‐like neuritogenesis in neuroblastoma (NG108‐15) cells and primary cerebellar granular neurons is furthered by the presence of ganglioside GM1. We describe here that galectin‐1 (Gal‐1), a homobivalent endogenous lectin, is an effector by cross‐linking the ganglioside and its associated glycoprotein α5β1‐integrin. The thereby triggered signaling cascade involves autophosphorylation of focal adhesion kinase and activation of phospholipase Cγ and phosphoinositide‐3 kinase. This leads to a transient increase in the intracellular Ca2+ concentration by opening of TRPC5 channels, which belong to the signal transduction‐gated cation channels. Controls with GM1‐defective cells (NG‐CR72 and neurons from ganglio‐series KO mice) were retarded in axonal growth, underscoring the relevance of GM1 as functional counterreceptor for Gal‐1. The lectin's presence was detected in the NG108‐15 cells, suggesting an autocrine mechanism of action, and in astrocytes in situ. Gal‐1, as cross‐linking lectin, can thus translate metabolic conversion of ganglioside GD1a to GM1 by neuraminidase action into axon growth. Galectin‐1 (Gal‐1) was shown an effector of axonogenesis in cerebellar granule neurons (CGNs) and NG108‐15 cells by cross‐linking GM1 ganglioside and its associated glycoprotein α5β1‐integrin. The resulting signaling led to a transient increase in intracellular Ca2+ by opening TRPC5 channels. CGNs deficient in GM1 showed retarded axonogenesis, underscoring the relevance of GM1 as functional counterreceptor for Gal‐1 in this process. This Gal‐1/GM1‐induced signaling was manifest only at the earliest, initiating stage of axon development.

Orai3 Surface Accumulation and Calcium Entry Evoked by Vascular Endothelial Growth Factor.

Vascular endothelial growth factor (VEGF) acts, in part, by triggering calcium ion (Ca(2+)) entry. Here, we sought understanding of a Synta66-resistant Ca(2+) entry pathway activated by VEGF. Measurement of intracellular Ca(2+) in human umbilical vein endothelial cells detected a Synta66-resistant component of VEGF-activated Ca(2+) entry that occurred within 2 minutes after VEGF exposure. Knockdown of the channel-forming protein Orai3 suppressed this Ca(2+) entry. Similar effects occurred in 3 further types of human endothelial cell. Orai3 knockdown was inhibitory for VEGF-dependent endothelial tube formation in Matrigel in vitro and in vivo in the mouse. Unexpectedly, immunofluorescence and biotinylation experiments showed that Orai3 was not at the surface membrane unless VEGF was applied, after which it accumulated in the membrane within 2 minutes. The signaling pathway coupling VEGF to the effect on Orai3 involved activation of phospholipase Cγ1, Ca(2+) release, cytosolic group IV phospholipase A2α, arachidonic acid production, and, in part, microsomal glutathione S-transferase 2, an enzyme which catalyses the formation of leukotriene C4 from arachidonic acid. Shear stress reduced microsomal glutathione S-transferase 2 expression while inducing expression of leukotriene C4 synthase, suggesting reciprocal regulation of leukotriene C4-synthesizing enzymes and greater role of microsomal glutathione S-transferase 2 in low shear stress. VEGF signaling via arachidonic acid and arachidonic acid metabolism causes Orai3 to accumulate at the cell surface to mediate Ca(2+) entry and downstream endothelial cell remodeling.

Transient Receptor Potential Channel 1 Deficiency Impairs Host Defense and Proinflammatory Responses to Bacterial Infection by Regulating Protein Kinase Cα Signaling.

Transient receptor potential channel 1 (TRPC1) is a nonselective cation channel that is required for Ca(2+) homeostasis necessary for cellular functions. However, whether TRPC1 is involved in infectious disease remains unknown. Here, we report a novel function for TRPC1 in host defense against Gram-negative bacteria. TRPC1(-/-) mice exhibited decreased survival, severe lung injury, and systemic bacterial dissemination upon infection. Furthermore, silencing of TRPC1 showed decreased Ca(2+) entry, reduced proinflammatory cytokines, and lowered bacterial clearance. Importantly, TRPC1 functioned as an endogenous Ca(2+) entry channel critical for proinflammatory cytokine production in both alveolar macrophages and epithelial cells. We further identified that bacterium-mediated activation of TRPC1 was dependent on Toll-like receptor 4 (TLR4), which induced endoplasmic reticulum (ER) store depletion. After activation of phospholipase Cγ (PLC-γ), TRPC1 mediated Ca(2+) entry and triggered protein kinase Cα (PKCα) activity to facilitate nuclear translocation of NF-κB/Jun N-terminal protein kinase (JNK) and augment the proinflammatory response, leading to tissue damage and eventually mortality. These findings reveal that TRPC1 is required for host defense against bacterial infections through the TLR4-TRPC1-PKCα signaling circuit.

Editorial: Cell and molecular signaling, and transport pathways involved in growth factor control of synaptic development and function.

Since the discovery of nerve growth factor (NGF) more than a half century ago (Levi-Montalcini and Cohen, 1960), the prototypic neurotrophin family has included brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4). Neurotrophins bind to the Trk family of receptors, as well as the p75 receptor, to activate multiple intracellular signaling cascades (reviewed by Reichardt, 2006). BDNF receptor tropomyosin receptor kinase B (TrkB) signaling has been extensively studied for its roles in the central nervous system (CNS) ranging from cell survival, axonal and dendritic growth and synapse formation. The pathway mediates long-lasting activity-modulated synaptic changes on excitatory and inhibitory neurons and plays critical roles in circuit development and maintenance. In addition to BDNF, many studies have identified other “growth” or signaling factors in the CNS that play important roles in the development, maintenance, and control of synaptic and circuit function. However, details of the intracellular signaling systems downstream of these events are frequently unexplored. In this Research Topic, we have collected original studies and review articles that present cellular and molecular mechanisms concerning activity-dependent synapse formation and their implications for behavior and brain disorders.

AXL Mediates Resistance to PI3Kα Inhibition by Activating the EGFR/PKC/mTOR Axis in Head and Neck and Esophageal Squamous Cell Carcinomas

Phosphoinositide-3-kinase (PI3K)-α inhibitors have shown clinical activity in squamous cell carcinomas (SCCs) of head and neck (H&N) bearing PIK3CA mutations or amplification. Studying models of therapeutic resistance, we have observed that SCC cells that become refractory to PI3Kα inhibition maintain PI3K-independent activation of the mammalian target of rapamycin (mTOR). This persistent mTOR activation is mediated by the tyrosine kinase receptor AXL. AXL is overexpressed in resistant tumors from both laboratory models and patients treated with the PI3Kα inhibitor BYL719. AXL dimerizes with and phosphorylates epidermal growth factor receptor (EGFR), resulting in activation of phospholipase Cγ (PLCγ)-protein kinase C (PKC), which, in turn, activates mTOR. Combined treatment with PI3Kα and either EGFR, AXL, or PKC inhibitors reverts this resistance.

Mechanism of Rac1-induced amplification in gastric mucosal phospholipase Cγ2 activation in response to Helicobacter pylori: modulatory effect of ghrelin.

Membrane recruitment followed by targeted phosphorylation of specific Tyr and Ser residues and the interaction with Rac GTPases are the crucial parts of an elaborate mechanism of PLCγ2 activation essential for its role in linking the specific receptor responses to a variety of hormones and bacterial endotoxins with the intended intracellular targets. Here, we explored the involvement of Rac in mediation of PLCγ2 activation associated with gastric mucosal inflammatory responses to H. pylori LPS and the hormone, ghrelin. We show that stimulation of gastric mucosal cells with the LPS leads to the membrane translocation of Rac1 as well as PLCγ2, while the effect of ghrelin is manifested by elevation in the membrane PLCγ2 activation and suppression in Rac1 translocation. However, blocking the LPS-induced Rac1 translocation, while detrimental to the PLCγ2 activation, has no effect on its membrane translocation. We reveal further that PLCγ2, localized in the membrane in association with Rac1 following the LPS stimulation, exhibits a marked increase in phosphorylation on Ser, while the modulatory effect of ghrelin, manifested by a drop in Rac1 translocation, is associated with a distinct decrease in PLCγ2 phosphorylation on Ser. Thus, the results suggest that H. pylori-elicited increase in gastric mucosal PLCγ2 phosphorylation on Ser serves as an essential platform for Rac1 colocalization and amplification in PLCγ2 activation.