PLCγ Inhibitor Research Articles

Abstract P2-07-01: Kidins220 (kinase D interacting substrate 220) has a connection with neutrotrophic related factors, BDNF and NGF, in human breast cancer

Abstract Background: Kidins220 (Kinase D Interacting Substrate 220) also known as ARMS (ankyrin repeat-rich membrane spanning) is a transmembrane protein that plays a significant role in neuronal development. It acts as the common downstream effector of neuronal signalling regulated by neurotrophin family including nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF). Expression abnormalities of Kidins220 have been identified in certain solid tumours, but the role played remains controversial. The present study aimed to examine the correlation between Kidins220 and BDNF, factor known to be aberrantly expressed in breast cancer cells (1,2), together with NGF and NGF receptor (BGFR). The involvement of Kidins220 in the chain event of cells response to BDNF, in particular the role played by phospholipase-gamma (PLC- γ) were also been determined. Method: Human breast cancer tissues were collected immediately after surgery. The transcription levels of Kidins220 and the neurotrophic factors and their receptors were determined using quantitative transcript analyses. The correlations between Kidins220 with BDNF, NGF, and NGFR were analysed against the clinical and pathological parameters in the clinical breast cancer cohort. Kidins220 knockdown cell models, using MDA MB-231 and MCF-7 cells, were created by employing anti-Kidins220 shRNA lentiviral technologies. Cell matrix attachment, cellular migration and cell growth were determined in breast cancer cells treated with BDNF and PLC- γ specific inhibitors and analysed using an automated cell analyser (ECIS) and crystal violet method, respectively. Result: The analyses of the clinical cohort demonstrated reduced expression of NGF and NGFR in high grade and node positive breast tumours, which is in contrast to the the reported pattern seen with BDNF expression. Furthermore, the expressions of NGF and NGFR were reduced in patients with poor prognosis and clinical outcomes. Interestingly, Kidins220 was found to have a significant negative correlation with BDNF (r=-0.429, p<0.001), but positive correlation with NGF (r=0.269, p<0.01) and NGFR (r=0.211, p<0.01). Knockdown of Kidins220 increased cellular migration in MDA-MB-231 and MCF-7 breast cells. In vitro, breast cancer cells responded to BDNF and exhibited changes in cellular migration, after knocking down Kidins220 in the cells. Loss of Kidins220 also resulted in cells which were more responsive to BDNF treatment when compared with the controls. Moreover, breast cancer cells exhibited a marked response to PLC-γ inhibitor, in the presence of BDNF, this was particularly so when they lost Kidins220. Conclusion: Our observations suggest that Kidins220 is involved in the metastasis of breast cancer, and it is a potential regulator in the response chain to neurotrophic factors including BDNF. Furthermore, it down-regulates the cells in response to BDNF, which is connected to PLC-γ.

Effect and mechanisms of sacral nerve stimulation on visceral hypersensitivity mediated by nerve growth factor.

To investigate the efficacy of sacral nerve stimulation (SNS) on nerve growth factor (NGF) mediated visceral sensitivity in normal rat and visceral hypersensitivity model rats. 120 male newborn rats were randomly divided into 6 groups: group A was normal model group; group B ~ F were all sensitized with acetic acid enema and grouped again. Group c2 was given NGF antagonist, d2 group was given NGF agonist, e2 group was given PI3K inhibitor, and f2 group was given PLC‐γ inhibitor. After treatment, the expression of NGF, TrKA, PI3K, AKT, PLC‐γ, NF‐κB, TRPV1, pTRPV1 and intracellular Ca2+ content were detected. The expression of protein TRPV1 and pTRPV1 was increased, and Ca2+ was increased in the visceral hypersensitive group. NGF, TrKA in NGF antagonist group, PI3K, AKT, NF‐κB in PI3K inhibitor group, PLC‐γ in PLC‐γ inhibitor group were all almost not expressed. The relative expression of NGF, TrKA, PI3K, AKT, PLC‐γ and NF‐κB in NGF antagonist group was lower than that in visceral hypersensitivity group and NGF activator group (P < .01). The relative expression of NGF, TrKA, PI3K and AKT mRNA in NGF antagonist group was lower than that in the normal model group (P < .01). There was no significant difference in the relative expression of PLC‐γ and NF‐κB mRNA (P > .05). The expression level of MAPK, ERK1 and ERK2 in visceral hypersensitivity group was higher than that in PI3K inhibitor group and PLC‐γ inhibitor group. The normal group Ca2+ curve was flat, and the NGF agonist group had the highest Ca2+ curve peak. Calcium concentration in visceral hypersensitivity group was higher than that in PI3K inhibitor group and that in PLC‐γ inhibitor group was higher than that in NGF antagonist group. The binding of TrkA receptor to NGF activates the MAPK/ERK pathway, the PI3K/Akt pathway and the PLC‐γ pathway, causing changes in the fluidity of intracellular and extracellular Ca2+, resulting in increased sensitivity of visceral tissues and organs.

Tumor-promoting and pro-angiogenic effects of roxarsone via VEGFR2/PLCγ/PKC signaling

Roxarsone is an organoarsenic feed additive used in livestock and poultry production that is released into the environment, where it poses a risk to human health. It is known to have a tumor-promoting effect that is brought about by pro-angiogenic factors such as vascular endothelial growth factor (VEGF) and it receptors (VEGFR). However, little information is available about the other signaling molecules that could be involved. This study aims to investigate the role of PLCγ/PKC signaling in roxarsone-induced angiogenesis in a mouse B16-F10 melanoma xenograft model and rat vascular endothelial cells (ECs). Results showed treatment with 5 mg/kg and 25 mg/kg roxarsone resulted in an obvious increase in the weight and volume of B16-F10 xenografts and PLCγ/PKC phosphorylation in a dose-dependent manner in C57BL/6 mice. SU5416, a VEGFR2 inhibitor, significantly attenuated the tumor growth induced by roxarsone. Further, 1.0 μmol/L roxarsone treatment in rat ECs was observed to significantly increase the optical density rate in the MTT assay, the number of BrdU-positive cells in the proliferation assay, the migration distance in the scratch test, and the number of meshes formed in the tube formation assay. In addition, treatment with 1.0 μmol/L roxarsone was associated with significantly higher phosphorylation of PLCγ/PKC than the control treatment. U73122, a PLCγ inhibitor, was found significantly to combat the effects of 1.0 μmol/L roxarsone on the ECs. Roxarsone is capable of promoting the growth of mouse B16-F10 xenografts and tube formation in vascular ECs. Moreover, VEGFR2/PLCγ/PKC signaling may play a regulatory role in in vivo and in vitro roxarsone-induced angiogenesis.

The inhibition of PLCγ1 protects chondrocytes against osteoarthritis, implicating its binding to Akt.

Previous studies have addressed the involvement of phosphoinositide-specifc phospholipase γ1 (PLCγ1) and protein kinase B (PKB/Akt) in osteoarthritis (OA) pathogenesis, but it is not ascertained the possibility of them to be potential targets for OA therapy. Here, through local intra-articular injection of PLCγ or Akt inhibitor in a rat OA model induced by anterior cruciate ligament transaction plus medial meniscus resection, the architecture of chondrocyte and matrix organization of articular cartilage were observed using histopathological assays and Aggrecan, Col2, PLCγ1, and Akt levels were detected using immunohistochemistry assays. By treatment of Akt or PLCγ inhibitor and transfection of different PLCγ1- or Akt-expressing vectors in rat OA model chondrocytes, Aggrecan, Col2, PLCγ1, p-PLCγ1, Akt, and p-Akt levels were detected using western blotting analysis. The binding between PLCγ1 and Akt was assessed with co-immunoprecipitation assays in human OA chondrocytes. These results showed that PLCγ inhibition protected chondrocytes against OA, but Akt inhibition did not dramatically aggravate OA progression. There were mutual antagonism and binding between PLCγ1 and Akt that could be regulated by their phosphorylation levels. Consequently, the data reveal that the inhibition of PLCγ1 may provide an attractive therapeutic target for OA therapy, implicating its binding to Akt.

Dual roles of protein tyrosine phosphatase kappa in coordinating angiogenesis induced by pro-angiogenic factors.

A potential role may be played by receptor-type protein tyrosine phosphatase kappa (PTPRK) in angiogenesis due to its critical function in coordinating intracellular signal transduction from various receptors reliant on tyrosine phosphorylation. In the present study, we investigated the involvement of PTPRK in the cellular functions of vascular endothelial cells (HECV) and its role in angiogenesis using in vitro assays and a PTPRK knockdown vascular endothelial cell model. PTPRK knockdown in HECV cells (HECVPTPRKkd) resulted in a decrease of cell proliferation and cell-matrix adhesion; however, increased cell spreading and motility were seen. Reduced focal adhesion kinase (FAK) and paxillin protein levels were seen in the PTPRK knockdown cells which may contribute to the inhibitory effect on adhesion. HECVPTPRKkd cells were more responsive to the treatment of fibroblast growth factor (FGF) in their migration compared with the untreated control and cells treated with VEGF. Moreover, elevated c-Src and Akt1 were seen in the PTPRK knockdown cells. The FGF-promoted cell migration was remarkably suppressed by an addition of PLCγ inhibitor compared with other small inhibitors. Knockdown of PTPRK suppressed the ability of HECV cells to form tubules and also impaired the tubule formation that was induced by FGF and conditioned medium of cancer cells. Taken together, it suggests that PTPRK plays dual roles in coordinating angiogenesis. It plays a positive role in cell proliferation, adhesion and tubule formation, but suppresses cell migration, in particular, the FGF-promoted migration. PTPRK bears potential to be targeted for the prevention of tumour associated angiogenesis.

Brain-derived neurotrophic factor promotes vesicular glutamate transporter 3 expression and neurite outgrowth of dorsal root ganglion neurons through the activation of the transcription factors Etv4 and Etv5.

Brain-derived neurotrophic factor (BDNF) is critical for sensory neuron survival and is necessary for vesicular glutamate transporter 3 (VGLUT3) expression. Whether the transcription factors Etv4 and Etv5 are involved in these BDNF-induced effects remains unclear. In the present study, primary cultured dorsal root ganglion (DRG) neurons were used to test the link between BDNF and transcription factors Etv4 and Etv5 on VGLUT3 expression and neurite outgrowth. BDNF promoted the mRNA and protein expression of Etv4 and Etv5 in DRG neurons. These effects were blocked by extracellular signal-regulated protein kinase 1/2 (ERK1/2) inhibitor PD98059 but not phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or phospholipase C-γ (PLC-γ) inhibitor U73122. Etv4 siRNA and Etv5 siRNA effectively blocked the VGLUT3 expression and neurite elongation induced by BNDF. The overexpression of Etv4 or Etv5 potentiated the effects of BNDF-induced neurite elongation and growth-associated protein 43 (GAP-43), medium neurofilament (NF-M), and light neurofilament (NF-L) expression while these effects could be inhibited by Etv4 and Etv5 siRNA. These data imply that Etv4 and Etv5 are essential transcription factors in modulating BDNF/TrkB signaling-mediated VGLUT3 expression and neurite outgrowth. BDNF, through the ERK1/2 signaling pathway, activates Etv4 and Etv5 to initiate GAP-43 expression, promote neurofilament (NF) protein expression, induce neurite outgrowth, and mediate VGLUT3 expression for neuronal function improvement. The biological effects initiated by BDNF/TrkB signaling linked to E26 transformation-specific (ETS) transcription factors are important to elucidate neuronal differentiation, axonal regeneration, and repair in various pathological states.

Definition of a Bidirectional Activity-Dependent Pathway Involving BDNF and Narp.

One of the cardinal features of neural development and adult plasticity is the contribution of activity-dependent signaling pathways. However, the interrelationships between different activity-dependent genes are not well understood. The immediate early gene neuronal-activity-regulated pentraxin (NPTX2 or Narp) encodes a protein that has been associated with excitatory synaptogenesis, AMPA receptor aggregation, and the onset of critical periods. Here, we show that Narp is a direct transcriptional target of brain-derived neurotrophic factor (BDNF), another highly regulated activity-dependent gene involved in synaptic plasticity. Unexpectedly, Narp is bidirectionally regulated by BDNF. Acute BDNF withdrawal results in downregulation of Narp, whereas transcription of Narp is greatly enhanced by BDNF. Furthermore, our results show that BDNF directly regulates Narp to mediate glutamatergic transmission and mossy fiber plasticity. Hence, Narp serves as a significant epistatic target of BDNF to regulate synaptic plasticity during periods of dynamic activity.

Leptin increases TNF-α expression and production through phospholipase D1 in Raw 264.7 cells.

Epidemiological evidence suggests that obesity is associated with inflammation of the respiratory tract and the pathogenesis of asthma. The purpose of this study was to examine the role of phospholipase D1 (PLD1) in leptin-induced expression of the proinflammatory cytokine, tumor necrosis factor (TNF)-α, and to suggest a molecular link between obesity and respiratory tract inflammation. We investigated whether leptin, a typical adipocytokine, plays a role in the expression of TNF-α through increased PLD1 activity in Raw 264.7. Leptin enhanced the activity of PLD1 through activation of PLCγ and Src, while PLD1 siRNA decreased the leptin-induced expression and production of TNF-α. Leptin-induced PLD activation was also inhibited by a PLCγ inhibitor (PAO) and Src kinase inhibitor (PP2), indicating that PLCγ and Src kinase are upstream activators of PLD1. Down-regulation of PLD1 also completely blocked activation of p70S6K, an activator of JNK. Leptin-induced expression of TNF-α was also prevented by inhibition of p70S6K and JNK. Taken together, these results indicate that PLD1 acts as an important regulator of leptin-induced expression of TNF-α by participating in the PLCγ/Src/PLD1/PA/p70S6K/JNK pathway.

Phospholipase D1 increases Bcl-2 expression during neuronal differentiation of rat neural stem cells.

We studied the possible role of phospholipase D1 (PLD1) in the neuronal differentiation, including neurite formation of neural stem cells. PLD1 protein and PLD activity increased during neuronal differentiation. Bcl-2 also increased. Downregulation of PLD1 by transfection with PLD1 siRNA or a dominant-negative form of PLD1 (DN-PLD1) inhibited both neurite outgrowth and Bcl-2 expression. PLD activity was dramatically reduced by a PLCγ (phospholipase Cγ) inhibitor (U73122), a Ca(2+)chelator (BAPTA-AM), and a PKCα (protein kinase Cα) inhibitor (RO320432). Furthermore, treatment with arachidonic acid (AA) which is generated by the action of PLA2 (phospholipase A2) on phosphatidic acid (a PLD1 product), increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, indicating that PLA2 is involved in the differentiation process resulting from PLD1 activation. PGE2 (prostaglandin E2), a cyclooxygenase product of AA, also increased during neuronal differentiation. Moreover, treatment with PGE2 increased the phosphorylation of p38 MAPK and CREB, as well as Bcl-2 expression, and this effect was inhibited by a PKA inhibitor (Rp-cAMP). As expected, inhibition of p38 MAPK resulted in loss of CREB activity, and when CREB activity was blocked with CREB siRNA, Bcl-2 production also decreased. We also showed that the EP4 receptor was required for the PKA/p38MAPK/CREB/Bcl-2 pathway. Taken together, these observations indicate that PLD1 is activated by PLCγ/PKCα signaling and stimulate Bcl-2 expression through PLA2/Cox2/EP4/PKA/p38MAPK/CREB during neuronal differentiation of rat neural stem cells.

A General Network Pharmacodynamic Model–Based Design Pipeline for Customized Cancer Therapy Applied to the VEGFR Pathway

A unified approach to optimize multidrug chemotherapy using a pharmacokinetic (PK)/enhanced pharmacodynamic model was developed using the vascular endothelial growth factor receptor (VEGFR) signaling system. The base VEGFR network model, characterized by ligand–receptor interactions, enzyme recruitment (Grb2-Sos, phospholipase C γ (PLCγ), and phosphoinositide-3 kinase (PI3K)), and downstream mitogen-activated protein kinase and Akt cascade activation, was linked to a sunitinib (VEGFR inhibitor) PK model and underwent Sobol sensitivity analysis that revealed potential sunitinib-enhancing mechanisms. Drugs targeting these mechanisms (a VEGF inhibitor, a PI3K inhibitor, a PLCγ inhibitor, and a mitogen-activated protein kinase inhibitor) and sunitinib were input to optimization-based control analyses to design multidrug regimens that maintained 80% pERK and pAkt inhibition for 28 days while minimizing drug dose. The resultant combination regimens contained both continuous and discontinuous schedules, mostly at low doses, and were altered by oncogenic mutations. This pipeline of computational analyses demonstrates how model-based methods can capture the complexities of drug action, tailor cancer chemotherapy, and empower personalized medicine.

The effect of a thieno[2,3-b]pyridine PLC-γ inhibitor on the proliferation, morphology, migration and cell cycle of breast cancer cells

3-Amino-N-(3-chlorophenyl)-5-oxo-5,6,7,8-tetrahydrothieno[2,3-b]quinoline-2-carboxamide (compound 1) is a putative phosphoinositide specific-phospholipase C-γ (PLC-γ) enzyme inhibitor. This enzyme is a plausible anticancer target linked to cell motility, important for the invasion and dissemination of tumour cells. In this work it is shown that IC50 values of compound 1 are in the low nanomolar range against a host of breast cancer cell lines as a consequence of anti-proliferative activity. These results correlate well with previously published results (Feng et al., Eur. Med. Chem., 54, 2012, 463–469) on tumour cell viability confirming the efficacy of compound 1. Flow cytometry experiments revealed that compound 1 arrests the cell cycle in the G2/M phases. Furthermore, the morphology and cell migration for the MDA-MB-231 breast cancer cell line are severely affected by administration of compound 1, which fits the hypothesis of PLC-γ inhibition. Finally, a detailed docking study against PLC reveals that the side chains of the amino acids His356, Glu341, Arg549 and Lys438 are involved in hydrogen bonding with ligand 1 as well as a lipophilic pocket is occupied by the phenyl moiety. The results presented in this study are particularly interesting because compound 1 affects triple-negative breast cancer cells, which are difficult to treat in the clinic and are in a dire need for an effective targeted therapy. We believe that compound 1 and its thieno[2,3-b]pyridine derivatives demonstrate that such a therapy can be developed.

The expression of vesicular glutamate transporter 3 and vesicular monoamine transporter 2 induced by brain-derived neurotrophic factor in dorsal root ganglion neurons in vitro

The vesicular glutamate transporter 3 (VGLUT3) and the vesicular monoamine transporter 2 (VMAT2) are expressed in dorsal root ganglion (DRG) neurons and play an important role in packing the neurotransmitter into synaptic vesicles. Brain-derived neurotrophic factor (BDNF) is one of the most profound known regulators of survival in the developing peripheral nervous system (PNS). Whether BDNF regulates the expression of VGLUT3 and VMAT2 in DRG neurons is still unclear. In the present study, primary cultured rat DRG neurons were used to evaluate the effects of BDNF on VGLUT3 and VMAT2 expression. The signaling pathways of the extracellular signal-regulated protein kinase 1/2 (ERK1/2), the phosphatidylinositol 3-kinase (PI3K)/Akt, and the phospholipase C-gamma (PLC-γ) involved in these effects were also determined. DRG neurons at 48h post-culture were incubated with BDNF and/or ERK1/2 inhibitor PD98059, PI3K inhibitor LY294002, and PLC-γ inhibitor U73122 for an additional 24h. After that, the neurite growth and growth-associated protein 43 (GAP-43) expressions after different doses of BDNF treatment were determined by immunofluorescent labeling. The expression of mRNA and protein of VGLUT3 and VMAT2 in different experimental conditions was assessed by real-time PCR, immunoblotting, and immunofluorescent labeling, respectively. The results showed that BDNF exposure promoted neurite growth and GAP-43 expression in DRG neurons in a dose-dependent manner. BDNF induced VGLUT3 upregulation through activation of PLC-γ signaling pathway. Although BDNF administration did not elevate the levels of VMAT2, the block of the PI3K/Akt or PLC-γ signaling pathways could inhibit VMAT2 expression in DRG neurons in the presence of BDNF. The knockdown of VGLUT3 or VMAT2 gene by siRNA did not affect the BDNF's effects on GAP-43 upregulation and neurite growth. The upregulation of VGLUT3 induced by BDNF might be that BDNF improved neuronal outgrowth status by promoting GAP-43 expression to stimulate neurite elongation. The contribution of distinct VGLUT3 and VMAT2 transporter expression induced by BDNF might be one of the mechanisms that BDNF regulates neuropathic pain. These data imply that BDNF signaling system might be a potential target on modifying distinct transporter-mediated biological effects of primary sensory neurons.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study, we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.

Adiponectin Induces Dendritic Cell Activation via PLCγ/JNK/NF-κB Pathways, Leading to Th1 and Th17 Polarization

Adiponectin (APN) is a crucial regulator for many inflammatory processes, but its effect on Th cell-mediated responses has not been fully understood. Thus, we investigated the immune-modulatory effects of APN on dendritic cells (DCs) controlling Th cell polarization. APN induced maturation and activation of DCs, as demonstrated by the increased expression of MHC class II, costimulatory molecules in both mouse and human DCs, and it significantly enhanced production of proinflammatory cytokines. APN triggered degradation of IκB proteins, nuclear translocation of NF-κB p65 subunit, and phosphorylation of MAPKs in DCs. Pretreatment with a phospholipase C (PLC)γ inhibitor and a JNK inhibitor suppressed IL-12 production and NF-κB binding activity. Additionally, PLCγ inhibitor downregulated phosphorylation of JNK, indicating that PLCγ and JNK may be upstream molecules of NF-κB. Importantly, APN-treated DCs significantly induced both Th1 and Th17 responses in allogeneic CD4(+) T cells. The addition of a neutralizing anti-IL-12 mAb to the cocultures abolished the secretion of IFN-γ, whereas the blockage of IL-23 and IL-1β suppressed APN-induced IL-17 production. Immunization of mice with OVA-pulsed, APN-treated DCs efficiently led to Ag-specific Th1 and Th17 cell responses. Taken together, these results demonstrated that APN effectively induced activation of DCs through PLCγ/JNK/NF-κB-signaling pathways, leading to enhanced Th1 and Th17 responses.

Activation of phospholipase‐Cγ and protein kinase C signal pathways helps the survival of spinal motoneurons injured by root avulsion

The signaling transduction processes involved in avulsion-induced motoneuron (MN) death have not been elucidated. Using the brachial plexus root avulsion rat model, we showed that avulsion-activated phosphorylation of phospholipase-Cγ (PLCγ) and protein kinase C (PKC) occurred in injured spinal MNs within 72 h of injury. Moreover, some MNs positive for PLCγ and PKC are also positive for avulsion-induced neuronal nitric oxide synthase (nNOS). Inhibition of PLCγ/PKC signal pathway, either with PLCγ inhibitor, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione, or with PLCγ siRNA augmented avulsion-induced MN death. 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione also inhibited PKC phosphorylation and exacerbated avulsion-induced reductions in the nNOS protein level in injured spinal segments. Moreover, activation of PLCγ/PKC signal pathway with PKC activator, phorbol-12-myristate-13-acetate, decreased avulsion-induced MN death. The temporal profile of PLCγ/PKC signaling appears to be crucial for the survival of spinal MNs after root avulsion. Our data suggest that PLCγ mediates, while PKC and nNOS are associated with, the avulsion-induced MN death in brachial plexus root avulsion.

Phospholipase D1 mediates bFGF-induced Bcl-2 expression leading to neurite outgrowth in H19-7 cells

The purpose of the present study was to investigate the role of PLD (phospholipase D) in bFGF (basic fibroblast growth factor)-induced Bcl-2 expression and to examine whether overexpressed Bcl-2 influences neurite outgrowth in immortalized hippocampal progenitor cells (H19-7 cells). We found that Bcl-2 expression was maximally induced by bFGF within 24h, and that this effect was reduced by inhibiting PLD1 expression with PLD1 small interfering RNA or by overexpressing DN (dominant-negative)-PLD1, whereas PLD1 overexpression markedly induced Bcl-2 expression. bFGF treatment activated Ras, Src, PI3K (phosphoinositide 3-kinase), PLCγ (phospholipase Cγ) and PKCα (protein kinase Cα). Among these molecules, Src and PKCα were not required for Bcl-2 expression. PLD activity was decreased by Ras, PI3K or PLCγ inhibitor, suggesting that PLD1 activation occurred through Ras, PI3K or PLCγ. We found that Ras was the most upstream molecule among these proteins, followed by the PI3K/PLCγ pathway, indicating that bFGF-induced PLD activation took place through the Ras/PI3K/PLCγ pathway. Furthermore, PLD1 was required for activation of JNK (c-Jun N-terminal kinase), which led to activation of STAT3 (signal transducer and activator of transcription 3) and finally Bcl-2 expression. When Bcl-2 was overexpressed, neurite outgrowth was stimulated along with induction of neurotrophic factors such as brain-derived neurotrophic factor and neurotrophin 4/5. In conclusion, PLD1 acts as a downstream effector of bFGF/Ras/PI3K/PLCγ signalling and regulates Bcl-2 expression through JNK/STAT3, which leads to neurite outgrowth in H19-7 cells.

Influence of interleukin-8 (IL-8) and IL-8 receptors on the migration of human keratinocytes, the role of PLC-γ and potential clinical implications

Interleukin (IL)-8 is a pro-inflammatory cytokine that has a direct effect on immune cells, including polymorphonuclear cells. Keratinocytes are a rich source of IL-8. However, there is little knowledge on the role of IL-8 in clinical wound healing and the direct biological effect of IL-8 on keratinocytes. In this study, the effect of recombinant human IL-8 (rhIL-8) on migration and adhesion was tested using HaCaT keratinocytes as a cell model. The cell functions were evaluated using impedance cell sensing. The expression of IL-8 receptor (IL-8R) transcripts in human skin and wounds (acute and chronic) was assessed using real-time transcript analysis. rhIL-8 significantly increased the migration of keratinocytes (3.5±0.3 for cells treated with IL-8 vs. 2.7±0.6 for controls; p=0.029). It is interesting to note that treatment of keratinocytes with IL-8 resulted in a marked shift in the responsive frequencies. IL-8 only resulted in a marginal increase in cell adhesion, which was particularly noticeable at high frequencies. The PLC-γ inhibitor completely eradicated the action of IL-8 on the migration of HaCaT cells. Using real time PCR, it was found that chronic wounds had significantly lower levels of the B form of the IL-8R (IL-8RB) (p=0.045) and marginally lower levels of the A form, IL-8RA, in comparison with acute wounds. Therefore, IL-8 has a direct and profound stimulatory effect on the migration of human keratinocytes, which is likely to occur via the PLC-γ pathway. Together with a reduced level of IL-8Rs in difficult-healing wounds, IL-8 has a clear prognostic and therapeutic value in wound healing.

Anti-proliferative actions of 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone in vascular smooth muscle cells

Naphthoquinone derivatives have been reported to possess various pharmacological activities, such as antiplatelet, anticancer, antifungal, and antiviral properties. In this study, we investigated the effects of a newly-synthesized naphthoquinone derivative, 2-decylamino-5,8-dimethoxy-1,4-naphthoquinone (2-decylamino-DMNQ), on VSMC proliferation and examined the molecular basis of the underlying mechanism. In a dose-dependent manner, 2-decylamino-DMNQ inhibited PDGF-stimulated VSMC proliferation with no apparent cytotoxic effect. While 2-decylamino-DMNQ did not affect PDGF-Rβ or Akt, it did inhibit the phosphorylation of Erk1/2 and PLCγ1 induced by PDGF. Moreover, 2-decylamino-DMNQ suppressed DNA synthesis through the arrest of cell cycle progression at the G0/G1 phase, including the suppression of pRb phosphorylation and a decrease in PCNA expression, which was related to the downregulation of cell cycle regulatory factors, such as cyclin D1/E and CDK 2/4. It was demonstrated that both U0126, an Erk1/2 inhibitor, and U73122, a PLCγ inhibitor, increased the proportion of cells in the G0/G1 phase of the cell cycle. Thus, these results suggest that 2-decylamino DMNQ has an inhibitory effect on PDGF-induced VSMC proliferation and the mechanism of this action is through cell cycle arrest at the G0/G1 phase. This may be a useful tool for studying interventions for vascular restenosis in coronary revascularization procedures and stent implantation.

Requirement for PLCγ2 in IL-3 and GM-CSF-stimulated MEK/ERK phosphorylation in murine and human hematopoietic stem/progenitor cells

Even though the involvement of intracellular Ca(2+) Ca(i)(2+) in hematopoiesis has been previously demonstrated, the relationship between Ca(i)(2+) signaling and cytokine-induced intracellular pathways remains poorly understood. Herein, the molecular mechanisms integrating Ca(2+) signaling with the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in primary murine and human hematopoietic stem/progenitor cells stimulated by IL-3 and GM-CSF were studied. Our results demonstrated that IL-3 and GM-CSF stimulation induced increased inositol 1,4,5-trisphosphate (IP(3) ) levels and Ca(i)(2+) release in murine and human hematopoietic stem/progenitor cells. In addition, Ca(i)(2+) signaling inhibitors, such as inositol 1,4,5-trisphosphate receptor antagonist (2-APB), PKC inhibitor (GF109203), and CaMKII inhibitor (KN-62), blocked phosphorylation of MEK activated by IL-3 and GM-CSF, suggesting the participation of Ca(2+) -dependent kinases in MEK activation. In addition, we identify phospholipase Cγ2 (PLCγ2) as a PLCγ responsible for the induction of Ca(2+) release by IL-3 and GM-CSF in hematopoietic stem/progenitor cells. Furthermore, the PLCγ inhibitor U73122 significantly reduced the numbers of granulocyte-macrophage colony-forming units after cytokine stimulation. Similar results were obtained in both murine and human hematopoietic stem/progenitor cells. Taken together, these data indicate a role for PLCγ2 and Ca(2+) signaling through the modulation of MEK in both murine and human hematopoietic stem/progenitor cells.

Kaposi's Sarcoma-Associated Herpesvirus-Induced Angiogenin Plays Roles in Latency via the Phospholipase Cγ Pathway: Blocking Angiogenin Inhibits Latent Gene Expression and Induces the Lytic Cycle

ABSTRACT During de novo infection of human dermal microvascular endothelial cells (HMVEC-d), Kaposi's sarcoma-associated herpesvirus (KSHV) induced the multifunctional angiogenin (ANG) protein, which entered the nuclei and nucleoli of infected cells and stimulated 45S rRNA gene transcription, proliferation, and tube formation, which were inhibited by blocking ANG nuclear translocation with the antibiotic neomycin (S. Sadagopan et al., J. Virol. 83: 3342-3364, 2009). ANG was induced by KSHV latency protein LANA-1 (open reading frame 73 [ORF73]). Here we examined the presence and functions of ANG in KSHV-positive (KSHV + ) primary effusion lymphoma (PEL/BCBL) cells. Significant ANG gene expression and secretion were observed in KSHV + (BCBL-1 and BC-3) and KSHV + and Epstein-Barr virus-positive (KSHV + EBV + ) (JSC-1) PEL cells and in BJAB-KSHV cells but not in EBV − KSHV − lymphoma cells (Akata, Loukes, Ramos, and BJAB), EBV + lymphoma cells (Akata-EBV and Raji), and cells from an EBV + lymphoblastoid cell line, thus suggesting a specific association of ANG in KSHV biology. Inhibition of nuclear translocation of ANG resulted in reduced BCBL-1 and TIVE-LTC (latently infected endothelial) cell survival and proliferation, while EBV − and EBV + Akata cells were unaffected. Blocking nuclear transport of ANG inhibited latent ORF73 gene expression and increased lytic switch ORF50 gene expression, both during de novo infection and in latently infected cells. A greater quantity of infectious KSHV was detected in the supernatants of neomycin-treated BCBL-1 cells than 12- O -tetradecanoylphorbol-13-acetate (TPA)-treated cells. Neomycin treatment and ANG silencing inhibited phospholipase Cγ (PLC-γ) and AKT phosphorylation, and in contrast, ANG induced ORF73 expression and PLC-γ and AKT phosphorylation. Further studies provided evidence that blockage of PLC-γ activation by neomycin appears to be mediating the inhibition of latent gene expression, since treatment with the conventional PLC-γ inhibitor U73122 also showed similar results. Silencing of ANG also resulted in reduced cell survival, reduced ORF73 gene expression, and lytic gene activation in BCBL-1 and TIVE-LTC cells and during de novo infection. Taken together, these studies suggest that KSHV has evolved to exploit ANG for its advantage via a so-far-unexplored PLC-γ pathway for maintaining its latency.