ß1,6 GlcNAc branches-modified protein tyrosine phosphatase Mu attenuates its tyrosine phosphatase activity and promotes glioma cell migration through PLCγ-PKC pathways.

The metastatic potential of malignant tumor has been shown to be correlated with the increased expression of tri- and tetra-antennary ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) N-glycans. In this study, We found that GnT-V expression was negatively correlated with receptor protein tyrosine phosphatase type µ(RPTPµ) in human glioma tissues. To study whether RPTPµ is a novel substance of GnT-V which further affect RPTPµ's downstream dephosphorylation function, we preform lentiviral infection with GnT-V gene to construct stably transfected GnT-V glial cell lines. We found RPTPµ undergone severer cleavage in GnT-V transfected glioma cells compare to Mock cells. RPTPµ intracellular domain fragments increased while ß1,6-GlcNAc-branched N-glycans increased, in consistent with the decrease of RPTPµ's catalytic activity. The results showed that abnormal glycosylation could decrease the phosphorylation activity of PTP µ, and affect PLCγ-PKC pathways. Both protease inhibitor Furin and N-glycan biosynthesis inhibitor swainsonine could decrease cell mobility in GnT-V-U87 transfectants and other glioma cell lines. All results above suggest increased post-translational modification of RPTPµ N-glycans by GnT-V attenuates its tyrosine phosphatase activity and promotes glioma cell migration through PLCγ-PKC pathways, and that the ß1,6-GlcNAc-branched N-glycans of RPTPµ play a crucial role in glioma invasivity.

7,8-dihydroxyflavone enhanced cholinergic contraction of rat gastric smooth muscle via augmenting muscarinic M3 receptor expression.

Although 7,8-dihydroxyflavone (7,8-DHF), a synthetic agonist specific for tyrosine kinase receptor B (TrkB), has been reported to promote intestinal dynamics, its effect on gastric dynamics has not been studied as yet. In this study, we explored how 7,8-DHF affected the carbachol (CCh)-induced contraction of rat gastric muscle by way of measuring the contractile tension of muscular strips. We found that although 7,8-DHF did not directly cause contraction of gastric muscle, it enhanced CCh-induced, instead of substance P- or high K+ -induced, contraction. The enhancing role of 7,8-DHF was partially blocked by ANA-12, a blocker specific for TrkB the activation of which in the gastric strips was evidenced by its phosphorylation. Although 7,8-DHF alone did not activate : phospholipase C (PLC)-γ in gastric muscle, CCh did, and importantly, the combined treatment with CCh + 7,8-DHF activated more PLC-γ. U73122, an antagonist to PLC-γ blocked both the CCh-induced and the 7,8-DHF-enhanced/CCh-induced contraction by ~30%. To pursue how 7,8-DHF could augment CCh-activated PLC-γ phosphorylation, we first examined the effect of 7,8-DHF on the expression of muscarinic receptors in gastric muscle and found that 7,8-DHF specifically increased M3 but not M2 receptor expression possibly through TrkB/Akt (protein kinase B) pathway because the Akt antagonist, LY294002 significantly suppressed the 7,8-DHF-augmemted M3 expression and completely blocked the 7,8-DHF-enhanced cholinergic contraction. Supporting the result, Akt phosphorylation in the gastric muscle was enhanced by 7,8-DHF treatment. The in vivo experiment showed that orally fed 7,8-DHF increased gastric emptying rate. The results imply a possibility that 7,8-DHF may be developed into a drug in the future for enhancing gastric dynamics.

Roles and Signaling Pathways of Des-γ-Carboxyprothrombin in the Progression of Hepatocellular Carcinoma.

Des-γ-carboxyprothrombin (DCP), an abnormal prothrombin produced in human hepatocellular carcinoma (HCC), plays crucial roles in the progression of HCC. DCP binding to cellular mesenchymal-epithelial transition factor (c-Met) is an initial event and consequently stimulates HCC through the increase of c-Met-Janus kinase 1- signal transducers and activators of transcription pathways. DCP stimulates HCC invasion through activation of matrix metalloproteinase via upregulation of extracellular signal-regulated kinase-mitogen-activated protein kinase (MAPK) pathway. DCP stimulates HCC angiogenesis through activation of the DCP-kinase insert domain receptor-phospholipaseC-γ-MAPK pathway. Identification of these pathways is important for designing the therapeutic strategy for HCC.

Gentiana lutea exerts anti-atherosclerotic effects by preventing endothelial inflammation and smooth muscle cell migration.

BACKGROUND AND AIMS: Studies suggest that Gentiana lutea (GL), and its component isovitexin, may exhibit anti-atherosclerotic properties. In this study we sought to investigate the protective mechanism of GL aqueous root extract and isovitexin on endothelial inflammation, smooth muscle cell migation, and on the onset and progression of atherosclerosis in streptozotocin (STZ)-induced diabetic rats. METHODS AND RESULTS: Our results show that both GL extract and isovitexin, block leukocyte adhesion and generation of reactive oxygen species in human umbilical vein endothelial cells (HUVECs) and rat aortic smooth muscle cells (RASMCs), following TNF-alpha and platelet derived growth factor-BB (PDGF-BB) challenges respectively. Both the extract and isovitexin blocked TNF-α induced expression of ICAM-1 and VCAM-1 in HUVECs. PDGF-BB induced migration of RASMCs and phospholipase C-γ activation, were also abrogated by GL extract and isovitexin. Fura-2 based ratiometric measurements demonstrated that, both the extact, and isovitexin, inhibit PDGF-BB mediated intracellular calcium rise in RASMCs. Supplementation of regular diet with 2% GL root powder for STZ rats, reduced total cholesterol in blood. Oil Red O staining demonstrated decreased lipid accumulation in aortic wall of diabetic animals upon treatment with GL. Medial thickness and deposition of collagen in the aortic segment of diabetic rats were also reduced upon supplementation. Immunohistochemistry demonstrated reduced expression of vascular cell adhesion molecule-1 (VCAM-1), inducible nitric oxide synthase (iNOS), and vascular endothelial cadherin (VE-cadherin) in aortic segments of diabetic rats following GL treatment. CONCLUSIONS: Thus, our results support that GL root extract/powder and isovitexin exhibit anti-atherosclerotic activities.

Spiraeoside inhibits mast cells activation and IgE-mediated allergic responses by suppressing phospholipase C-γ-mediated signaling.

Mast cells are responsible for IgE-mediated allergic responses through the secretion of various inflammatory cytokines and mediators. Therefore, the pharmacological regulation of mast cell activation is an important goal in the development of novel anti-allergic drugs. In this study, we found that spiraeoside (SP) inhibits mast cell activation and allergic responses in vivo. SP dose-dependently inhibited the degranulation induced by IgE-antigen (Ag) stimulation in RBL-2H3 mast cells without cytotoxic effects. At the molecular level, SP reduced the Ag-induced phosphorylation and subsequent activation of phospholipase C-γ2 (PLC-γ2). Moreover, SP inhibited the phosphorylation of spleen tyrosine kinase (Syk), linker for activation of T cells (LAT), and downstream MAPKs, such as ERK1/2, p38, and JNK, eventually attenuating expression of TNF-α and IL-4. Finally, we found that SP significantly inhibited IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. Taken together, our results strongly suggest that SP suppresses IgE-mediated mast cell activation and allergic responses by inhibiting Lyn-induced PLC-γ2/MAPK signaling in mast cells.

Mesenchymal stem cell secreted platelet derived growth factor exerts a pro-migratory effect on resident Cardiac Atrial appendage Stem Cells.

Mesenchymal stem cells (MSCs) modulate cardiac healing after myocardial injury through the release of paracrine factors, but the exact mechanisms are still unknown. One possible mechanism is through mobilization of endogenous cardiac stem cells (CSCs). This study aimed to test the pro-migratory effect of MSC conditioned medium (MSC-CM) on endogenous CSCs from human cardiac tissue. By using a three-dimensional collagen assay, we found that MSC-CM improved migration of cells from human cardiac tissue. Cell counts, perimeter and area measurements were utilized to quantify migration effects. To examine whether resident stem cells were among the migrating cells, specific stem cell properties were investigated. The migrating cells displayed strong similarities with resident Cardiac Atrial appendage Stem Cells (CASCs), including a clonogenic potential of ~21.5% and expression of pluripotency associated genes like Oct-4, Nanog, c-Myc and Klf-4. Similar to CASCs, migrating cells demonstrated high aldehyde dehydrogenase activity and were able to differentiate towards cardiomyocytes. Receptor tyrosine kinase analysis and collagen assays performed with recombinant platelet derived growth factor (PDGF)-AA and Imatinib Mesylate, a PDGF receptor inhibitor, suggested a role for the PDGF-AA/PDGF receptor α axis in enhancing the migration process of CASCs. In conclusion, our findings demonstrate that factors present in MSC-CM improve migration of resident stem cells from human cardiac tissue. These data open doors towards future therapies in which MSC secreted factors, like PDGF-AA, can be utilized to enhance the recruitment of CASCs towards the site of myocardial injury.

PLC-γ-Ca2+ pathway regulates axonal TrkB endocytosis and is required for long-distance propagation of BDNF signaling.

Brain-derived neurotrophic factor (BDNF) and its tropomyosin receptor kinase B (TrkB) are important signaling proteins that regulate dendritic growth and maintenance in the central nervous system (CNS). After binding of BDNF, TrkB is endocytosed into endosomes and continues signaling within the cell soma, dendrites, and axon. In previous studies, we showed that BDNF signaling initiated in axons triggers long-distance signaling, inducing dendritic arborization in a CREB-dependent manner in cell bodies, processes that depend on axonal dynein and TrkB activities. The binding of BDNF to TrkB triggers the activation of different signaling pathways, including the ERK, PLC-γ and PI3K-mTOR pathways, to induce dendritic growth and synaptic plasticity. How TrkB downstream pathways regulate long-distance signaling is unclear. Here, we studied the role of PLC-γ-Ca2+ in BDNF-induced long-distance signaling using compartmentalized microfluidic cultures. We found that dendritic branching and CREB phosphorylation induced by axonal BDNF stimulation require the activation of PLC-γ in the axons of cortical neurons. Locally, in axons, BDNF increases PLC-γ phosphorylation and induces intracellular Ca2+ waves in a PLC-γ-dependent manner. In parallel, we observed that BDNF-containing signaling endosomes transport to the cell body was dependent on PLC-γ activity and intracellular Ca2+ stores. Furthermore, the activity of PLC-γ is required for BDNF-dependent TrkB endocytosis, suggesting a role for the TrkB/PLC-γ signaling pathway in axonal signaling endosome formation.

PLC-γ directly binds activated c-Src, which is necessary for carbachol-mediated inhibition of NHE3 activity in Caco-2/BBe cells.

Elevated levels of intracellular Ca(2+) ([Ca(2+)]i) inhibit Na(+)/H(+) exchanger 3 (NHE3) activity in the intact intestine. We previously demonstrated that PLC-γ directly binds NHE3, an interaction that is necessary for [Ca(2+)]i inhibition of NHE3 activity, and that PLC-γ Src homology 2 (SH2) domains may scaffold Ca(2+) signaling proteins necessary for regulation of NHE3 activity. [Ca(2+)]i regulation of NHE3 activity is also c-Src dependent; however, the mechanism by which c-Src is involved is undetermined. We hypothesized that the SH2 domains of PLC-γ might link c-Src to NHE3-containing complexes to mediate [Ca(2+)]i inhibition of NHE3 activity. In Caco-2/BBe cells, carbachol (CCh) decreased NHE3 activity by ∼40%, an effect abolished with the c-Src inhibitor PP2. CCh treatment increased the amount of active c-Src as early as 1 min through increased Y(416) phosphorylation. Coimmunoprecipitation demonstrated that c-Src associated with PLC-γ, but not NHE3, under basal conditions, an interaction that increased rapidly after CCh treatment and occurred before the dissociation of PLC-γ and NHE3 that occurred 10 min after CCh treatment. Finally, direct binding to c-Src only occurred through the PLC-γ SH2 domains, an interaction that was prevented by blocking the PLC-γ SH2 domain. This study demonstrated that c-Src 1) activity is necessary for [Ca(2+)]i inhibition of NHE3 activity, 2) activation occurs rapidly (∼1 min) after CCh treatment, 3) directly binds PLC-γ SH2 domains and associates dynamically with PLC-γ under elevated [Ca(2+)]i conditions, and 4) does not directly bind NHE3. Under elevated [Ca(2+)]i conditions, PLC-γ scaffolds c-Src into NHE3-containing multiprotein complexes before dissociation of PLC-γ from NHE3 and subsequent endocytosis of NHE3.

FGF2 Prevents Sunitinib-Induced Cardiotoxicity in Zebrafish and Cardiomyoblast H9c2 Cells.

Sunitinib is used extensively in the treatment of metastatic renal cell carcinoma and imatinib-resistant gastrointestinal stromal tumors. However, the undesirable cardiotoxic effects of sunitinib, such as congestive heart failure and hypertension, limit its use in the clinical setting. As multiple receptor tyrosine kinases are inhibited by sunitinib, it raises a question as to which target mediates sunitinib-induced cardiotoxicity. Here, we reported that the injection of fibroblast growth factor 2 (FGF2) mRNA into one- to two-cell stage embryos protected against sunitinib-induced cardiotoxicity in zebrafish. In addition, FGF2 significantly prevented sunitinib-induced cardiotoxicity in cardiomyoblast H9c2 cells, possibly via activating the PLC-γ/c-Raf/CREB pathway. Importantly, FGF2 did not compromise the antitumor activity of sunitinib in Caki-1 and OS-RC-2 renal cell carcinoma cells. Molecular docking simulations further revealed an interaction between the tyrosine kinase domain of FGF receptor 1 (FGFR1) and sunitinib. Taken together, our results clearly demonstrated that FGF2 inhibition plays an important role in sunitinib-induced cardiotoxicity both in vitro and in vivo. This study also provided a basis for further research on sunitinib-induced cardiotoxicity and may allow rational design of new sunitinib derivatives with fewer or weak cardiotoxic effects.

Expression of breast cancer metastasis suppressor-1, BRMS-1, in human breast cancer and the biological impact of BRMS-1 on the migration of breast cancer cells.

UNLABELLED: Breast cancer metastasis suppressor-1 (BRMS1) is a candidate metastasis-suppressing gene and has been shown to potentially inhibit tumor progression without blocking the growth of orthotopic tumors, in different tumor types including non-small cell lung cancer, ovarian, melanoma and breast cancers. MATERIALS AND METHODS: BRMS-1 gene transcript was quantified in breast cancer sample tissues and analyzed against histological and clinical patient outcome. Human breast cancer cell lines, MDA MB-231 and MCF-7 were used to genetically-modify the expression of BRMS-1 and test for biological responses following BRMS-1 modifications. Key candidate signal pathways, influenced by BRMS-1 were also explored. RESULTS: BRMS1 was present in MDA MB-231 and MCF-7 cell lines. Using anti-BRMS1 transgenes, we knocked-down the transcripts of BRMS1 in both cells at the mRNA and protein levels. Knockdown of BRMS1 gave both cells a faster cell growth rate, rapid pace of cellular migration and invasion, compared to respective wild-type and control cells (p<0.05). Blocking phospholipase-Cγ (PLCγ) had a significant influence on the BRMS-1-induced cell migration. Finally, significantly low levels of BRMS1 were observed in patients with high-grade tumors (p=0.12), in patients with distant metastasis (p=0.05) and those who died of breast cancer (p=0.0037). In addition, patients with low levels of BRMS1 had a significantly shorter overall survival (p=0.035). CONCLUSION: BRMS-1 is aberrantly expressed in human breast cancer and is inversely-correlated with disease progression and patient survival. This is likely to be occurring via its influence on invasion and migration of breast cancer cells.

BDNF-induced local protein synthesis and synaptic plasticity.

Brain-derived neurotrophic factor (BDNF) is an important regulator of synaptic transmission and long-term potentiation (LTP) in the hippocampus and in other brain regions, playing a role in the formation of certain forms of memory. The effects of BDNF in LTP are mediated by TrkB (tropomyosin-related kinase B) receptors, which are known to be coupled to the activation of the Ras/ERK, phosphatidylinositol 3-kinase/Akt and phospholipase C-γ (PLC-γ) pathways. The role of BDNF in LTP is best studied in the hippocampus, where the neurotrophin acts at pre- and post-synaptic levels. Recent studies have shown that BDNF regulates the transport of mRNAs along dendrites and their translation at the synapse, by modulating the initiation and elongation phases of protein synthesis, and by acting on specific miRNAs. Furthermore, the effect of BDNF on transcription regulation may further contribute to long-term changes in the synaptic proteome. In this review we discuss the recent progress in understanding the mechanisms contributing to the short- and long-term regulation of the synaptic proteome by BDNF, and the role in synaptic plasticity, which is likely to influence learning and memory formation. This article is part of the Special Issue entitled 'BDNF Regulation of Synaptic Structure, Function, and Plasticity'.

Chloride channel 4 is required for nerve growth factor-induced TrkA signaling and neurite outgrowth in PC12 cells and cortical neurons.

We have previously shown that chloride ion flux plays an important role in receptor tyrosine kinase A (TrkA)-mediated signaling pathway during nerve growth factor (NGF)-induced differentiation in pheochromocytoma (PC12) cells. Here we found out that chloride channel 4 (CLC-4) is responsible for the NGF-induced neurite outgrowth in neuronal cells. Using a patch-clamp technique, we found that NGF treatment increased anionic conductance in PC12 cells, an effect which was blocked by transfection of siRNA of CLC-4. Also, the NGF-induced TrkA phosphorylation and subsequent Akt/moesin phosphorylation was suppressed in the CLC-4 knock down cells. Moreover, CLC-4 knock down also suppressed the neurite outgrowth in response to long-term treatment of NGF in PC12 cells and in primary cortical neurons. In summary, our results suggest that CLC-4 is an important mediator of the TrkA-mediated signaling pathway and thus, the NGF-induced differentiation of neuronal cells.

The natural killer cell cytotoxic function is modulated by HIV-1 accessory proteins.

Natural killer (NK) cells' major role in the control of viruses is to eliminate established infected cells. The capacity of NK cells to kill virus-infected cells is dependent on the interactions between ligands on the infected cell and receptors on the NK cell surface. Because of the importance of ligand-receptor interactions in modulating the NK cell cytotoxic response, HIV has developed strategies to regulate various NK cell ligands making the infected cell surprisingly refractory to NK cell lysis. This is perplexing because the HIV-1 accessory protein Vpr induces expression of ligands for the NK cell activating receptor, NKG2D. In addition, the accessory protein Nef removes the inhibitory ligands HLA-A and -B. The reason for the ineffective killing by NK cells despite the strong potential to eliminate infected cells is due to HIV-1 Vpu's ability to down modulate the co-activation ligand, NTB-A, from the cell surface. Down modulation of NTB-A prevents efficient NK cell degranulation. This review will focus on the mechanisms through which the HIV-1 accessory proteins modulate their respective ligands, and its implication for NK cell killing of HIV-infected cells.