RESUMO
Previous studies identified the involvement of phosphoinositide-specific phospholipase C (PLC) γ1 in some events of chondrocytes. This study aims to investigate whether and how PLCγ1 modulates autophagy to execute its role in osteoarthritis (OA) progression. Rat normal or human OA chondrocytes were pretreated with IL-1ß for mimicking or sustaining OA pathological condition. Using Western blotting, immunoprecipitation, qPCR, immunofluorescence and Dimethylmethylene blue assays, and ELISA and transmission electron microscope techniques, we found that PLCγ1 inhibitor U73122 enhanced Collagen II, Aggrecan and GAG levels, accompanied with increased LC3B-II/I ratio and decreased P62 expression level, whereas autophagy inhibitor Chloroquine partially diminished its effect. Meanwhile, U73122 dissociated Beclin1 from Beclin1-IP3R-Bcl-2 complex and blocked mTOR/ULK1 axis, in which the crosstalk between PLCγ1, AMPK, Erk and Akt were involved. Additionally, by haematoxylin and eosin, Safranin O/Fast green, and immunohistochemistry staining, we observed that intra-articular injection of Ad-shPLCγ1-1/2 significantly enhanced Collagen and Aggrecan levels, accompanied with increased LC3B and decreased P62 levels in a rat OA model induced by anterior cruciate ligament transection and medial meniscus resection. Consequently, PLCγ1 inhibition-driven autophagy conferred cartilage protection against OA through promoting ECM synthesis in OA chondrocytes in vivo and in vitro, involving the crosstalk between PLCγ1, AMPK, Erk and Akt.
Assuntos
Autofagia , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/etiologia , Osteoartrite/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Autofagia/efeitos dos fármacos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Suscetibilidade a Doenças , Estrenos/farmacologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Osteoartrite/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Pirrolidinonas/farmacologia , Ratos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Chromophobe renal cell carcinoma (chRCC) accounts for approximately 5% of all renal cancers and around 30% of chRCC cases have mutations in TP53. chRCC is poorly supported by microvessels and has markably lower glucose uptake than clear cell RCC and papillary RCC. Currently, the metabolic status and mechanisms by which this tumor adapts to nutrient-poor microenvironments remain to be investigated. In this study, we performed proteome and metabolome profiling of chRCC tumors and adjacent kidney tissues and identified major metabolic alterations in chRCC tumors, including the classical Warburg effect, the downregulation of gluconeogenesis and amino acid metabolism, and the upregulation of protein degradation and endocytosis. chRCC cells depended on extracellular macromolecules as an amino acid source by activating endocytosis to sustain cell proliferation and survival. Inhibition of the phospholipase C gamma 2 (PLCG2)/inositol 1,4,5-trisphosphate (IP3)/Ca2+/protein kinase C (PKC) pathway significantly impaired the activation of endocytosis for amino acid uptakes into chRCC cells. In chRCC, whole-exome sequencing revealed that TP53 mutations were not related to expression of PLCG2 and activation of endocytosis. Our study provides novel perspectives on metabolic rewiring in chRCC and identifies the PLCG2/IP3/Ca2+/PKC axis as a potential therapeutic target in patients with chRCC. SIGNIFICANCE: This study reveals macropinocytosis as an important process utilized by chRCC to gain extracellular nutrients in a p53-independent manner.
Assuntos
Aminoácidos/metabolismo , Carcinoma de Células Renais/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Endocitose/efeitos dos fármacos , Neoplasias Renais/metabolismo , Amilorida/análogos & derivados , Amilorida/farmacologia , Compostos de Boro/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estrenos/farmacologia , Gluconeogênese , Humanos , Indóis/farmacologia , Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Inositol 1,4,5-Trifosfato/metabolismo , Neoplasias Renais/patologia , Maleimidas/farmacologia , Metaboloma , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteoma , Pirrolidinonas/farmacologiaRESUMO
Oxidative stress (OS) and neuronal apoptosis are major pathological processes after hypoxic-ischemic encephalopathy (HIE). Colony stimulating factor 1 (CSF1), binding to CSF1 receptor (CSF1R), has been shown to reduce neuronal loss after hypoxic-ischemia- (HI-) induced brain injury. In the present study, we hypothesized that CSF1 could alleviate OS-induced neuronal degeneration and apoptosis through the CSF1R/PLCG2/PKA/UCP2 signaling pathway in a rat model of HI. A total of 127 ten-day old Sprague Dawley rat pups were used. HI was induced by right common carotid artery ligation with subsequent exposure to hypoxia for 2.5 h. Exogenous recombinant human CSF1 (rh-CSF1) was administered intranasally at 1 h and 24 h after HI. The CSF1R inhibitor, BLZ945, or phospholipase C-gamma 2 (PLCG2) inhibitor, U73122, was injected intraperitoneally at 1 h before HI induction. Brain infarct volume measurement, cliff avoidance test, righting reflex test, double immunofluorescence staining, western blot assessment, 8-OHdG and MitoSOX staining, Fluoro-Jade C staining, and TUNEL staining were used. Our results indicated that the expressions of endogenous CSF1, CSF1R, p-CSF1R, p-PLCG2, p-PKA, and uncoupling protein2 (UCP2) were increased after HI. CSF1 and CSF1R were expressed in neurons and astrocytes. Rh-CSF1 treatment significantly attenuated neurological deficits, infarct volume, OS, neuronal apoptosis, and degeneration at 48 h after HI. Moreover, activation of CSF1R by rh-CSF1 significantly increased the brain tissue expressions of p-PLCG2, p-PKA, UCP2, and Bcl2/Bax ratio, but reduced the expression of cleaved caspase-3. The neuroprotective effects of rh-CSF1 were abolished by BLZ945 or U73122. These results suggested that rh-CSF1 treatment attenuated OS-induced neuronal degeneration and apoptosis after HI, at least in part, through the CSF1R/PLCG2/PKA/UCP2 signaling pathway. Rh-CSF1 may serve as therapeutic strategy against brain damage in patients with HIE.
Assuntos
Apoptose/efeitos dos fármacos , Hipóxia-Isquemia Encefálica/patologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Benzotiazóis/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Humanos , Fator Estimulador de Colônias de Macrófagos/genética , Fator Estimulador de Colônias de Macrófagos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Ácidos Picolínicos/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Proteína Desacopladora 2/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
Small cell lung cancer (SCLC) remains a recalcitrant disease where limited therapeutic options have not improved overall survival, and approved targeted therapies are lacking. Amplification of the tyrosine kinase receptor FGFR1 (fibroblast growth factor receptor 1) is one of the few actionable alterations found in the SCLC genome. However, efforts to develop targeted therapies for FGFR1-amplified SCLC are hindered by critical gaps in knowledge around the molecular origins and mediators of FGFR1-driven signaling as well as the physiologic impact of targeting FGFR1. Here we show that increased FGFR1 promotes tumorigenic progression in precancerous neuroendocrine cells and is required for SCLC development in vivo. Notably, Fgfr1 knockout suppressed tumor development in a mouse model lacking the retinoblastoma-like protein 2 (Rbl2) tumor suppressor gene but did not affect a model with wild-type Rbl2. In support of a functional interaction between these two genes, loss of RBL2 induced FGFR1 expression and restoration of RBL2 repressed it, suggesting a novel role for RBL2 as a regulator of FGFR1 in SCLC. Additionally, FGFR1 activated phospholipase C gamma 1 (PLCG1), whereas chemical inhibition of PLCG1 suppressed SCLC growth, implicating PLCG1 as an effector of FGFR1 signaling in SCLC. Collectively, this study uncovers mechanisms underlying FGFR1-driven SCLC that involve RBL2 upstream and PLCG1 downstream, thus providing potential biomarkers for anti-FGFR1 therapy. SIGNIFICANCE: This study identifies RBL2 and PLCG1 as critical components of amplified FGFR1 signaling in SCLC, thus representing potential targets for biomarker analysis and therapeutic development in this disease.
Assuntos
Neoplasias Pulmonares/patologia , Proteínas de Neoplasias/metabolismo , Fosfolipase C gama/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Proteína p130 Retinoblastoma-Like/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Feminino , Deleção de Genes , Genes Reguladores , Genes do Retinoblastoma , Humanos , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Células Neuroendócrinas/metabolismo , Células Neuroendócrinas/patologia , Fosfolipase C gama/antagonistas & inibidores , Lesões Pré-Cancerosas/metabolismo , Lesões Pré-Cancerosas/patologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteína p130 Retinoblastoma-Like/genética , Carcinoma de Pequenas Células do Pulmão/etiologia , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
Phosphatidylcholine-specific phospholipase Cγ1 (PLCγ1) is involved in regulating cell metabolism. However, little is known how PLCγ1 directs BMSC differentiation. Here, we investigated the role of PLCγ1 in rat BMSC differentiation into osteoblasts and chondrocytes. The results of Alizarin red and Alcian blue staining showed that PLCγ1 inhibitor U73122 significantly enhanced the mineralization capacity and proteoglycan deposition of BMSCs. The results of qPCR technique and Western blot analysis showed that long-term treatment of U73122 enhanced COL1A1 and OPG mRNA levels and Collagen 1A1, BMP2, and p-Smad1/5/9 protein levels and that short-term treatment of U73122 enhanced COL2A1 and SOX9 mRNA levels and Collagen 2, SOX9, Aggrecan, TGF-ß3, and p-Smad2/3 protein levels. Decreased p-mTOR and p-P38 contributed to enhanced osteogenic potentials of BMSCs and increased p-P38 contributed to enhanced chondrogenic potentials of BMSCs. The scaffold transplantation with U73122+BMSC was more efficacious than BMSC alone for osteochondral defect repair in a rat model. Therefore, suppressing PLCγ1 could improve the capacity to effectively use BMSCs for cell therapy of osteochondral defect.
Assuntos
Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Osteogênese/fisiologia , Fosfolipase C gama/antagonistas & inibidores , Alginatos , Animais , Diferenciação Celular , Células Cultivadas , Condrogênese/efeitos dos fármacos , Estrenos/farmacologia , Hidrogéis/administração & dosagem , Hidrogéis/química , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteocondrose/fisiopatologia , Osteocondrose/terapia , Osteogênese/efeitos dos fármacos , Patela/diagnóstico por imagem , Patela/patologia , Patela/cirurgia , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Pirrolidinonas/farmacologia , Ratos Sprague-Dawley , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Accumulating studies have shown that the epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in mediating cellular entry of numerous viruses. In this study, we report that bovine herpesvirus 1 (BoHV-1) productive infection in both the human lung carcinoma cell line A549 and bovine kidney (MDBK) cells leads to activation of EGFR, as demonstrated by the increased phosphorylation of EGFR at Tyr1068 (Y1068), which in turn plays important roles in virus infection. A time-of-addition assay supported that virus replication at post-entry stages was affected by the EGFR specific inhibitor Gefitinib. Interestingly, both phospholipase C-γ1 (PLC-γ1) and Akt, canonical downstream effectors of EGFR, were activated following virus infection in A549 cells, while Gefitinib could inhibit the activation of PLC-γ1 but not Akt. In addition, virus titers in A549 cells was inhibited by chemical inhibition of PLC-γ1, but not by the inhibition of Akt. However, the Akt specific inhibitor Ly294002 could significantly reduce the virus titer in MDBK cells. Taken together, our data suggest that PLC-γ1 is stimulated in part through EGFR for efficient replication in A549 cells, whereas Akt can be stimulated by virus infection independent of EGFR, and is not essential for virus productive infection, indicating that Akt modulates BoHV-1 replication in a cell type-dependent manner. This study provides novel insights on how BoHV-1 infection activates EGFR signaling transduction to facilitate virus replication.
Assuntos
Receptores ErbB/metabolismo , Herpesvirus Bovino 1/fisiologia , Transdução de Sinais , Replicação Viral , Células A549 , Animais , Bovinos , Linhagem Celular , Receptores ErbB/antagonistas & inibidores , Interações Hospedeiro-Patógeno , Humanos , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacosRESUMO
Mast cell activation through the high-affinity IgE receptor (FcεRI) plays a central role in allergic reactions. FcεRI-mediated activation triggers multiple signaling pathways leading to degranulation and synthesis of different inflammatory mediators. IgE-mediated mast cell activation can be modulated by different molecules, including several drugs. Herein, we investigated the immunomodulatory activity of the histone deacetylase inhibitor valproic acid (VPA) on IgE-mediated mast cell activation. To this end, bone marrow-derived mast cells (BMMC) were sensitized with IgE and treated with VPA followed by FcεRI cross-linking. The results indicated that VPA reduced mast cell IgE-dependent degranulation and cytokine release. VPA also induced a significant reduction in the cell surface expression of FcεRI and CD117, but not other mast cell surface molecules. Interestingly, VPA treatment inhibited the phosphorylation of PLCγ2, a key signaling molecule involved in IgE-mediated degranulation and cytokine secretion. However, VPA did not affect the phosphorylation of other key components of the FcεRI signaling pathway, such as Syk, Akt, ERK1/2, or p38. Altogether, our data demonstrate that VPA affects PLCγ2 phosphorylation, which in turn decreases IgE-mediated mast cell activation. These results suggest that VPA might be a key modulator of allergic reactions and might be a promising therapeutic candidate.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Fosfolipase C gama/antagonistas & inibidores , Receptores de IgE/efeitos dos fármacos , Ácido Valproico/farmacologia , Animais , Degranulação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Interleucina-13/metabolismo , Interleucina-6/metabolismo , Mastócitos/citologia , Camundongos , Fosfolipase C gama/fisiologia , Receptores de IgE/biossíntese , Receptores de IgE/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Our previous studies indicated that phosphoinositide specific phospholipase Cγ1 (PLCγ1) was involved in autophagy induction in colon and hepatic carcinoma cells. However, whether and how PLCγ1 regulation in human lung adenocarcinoma is linked to autophagy remains unclear. Here, we assessed the protein expression of PLCγ1 in human lung adenocarcinoma tissue using immunohistochemistry assay and the relationship between PLCG1 and autophagy in The Cancer Genome Atlas Network (TCGA) using Spearman correlation analysis and GSEA software. Furthermore, the interaction between PLCγ1 and autophagy-related signal molecules was investigated in human lung adenocarcinoma A549 cells treated with different inhibitors or transduction with lentivirus-mediated PLCγ1 gene short-hairpin RNA (shRNA) vectors using MTT, clonogenicity, Transwell migration, RT-PCR, Caspase-3, mitochondrial transmembrane potential, and western blotting assays, as well as transmission electron microscope technique. Additionally, the effect of shRNA/PLCγ1 alone or combined with autophagic activator Lithium Chloride (LiCl) on tumor growth and metastasis was measured using immunohistochemistry and assays in A549 xenograft nude mouse model. The results showed that increased PLCγ1 expression occurred frequently in human lung adenocarcinoma tissue with higher grades of T in TNM staging classification. PLCγ1 significantly enriched in autophagic process and regulation, which negatively regulating autophagy was enriched in higher expression of PLCγ1. PLCγ1 inhibition partially reduced cell proliferation and migration of A549 cells, with an increased autophagic flux involving alterations of AMPKα, mTOR, and ERK levels. However, PLCγ1 inhibition-driven autophagy led to cell death without depending on Caspase-3 and RIP1. Additionally, the abrogation of PLCγ1 signaling by shRNA and combination with autophagic activator LiCl could efficaciously suppress tumor growth and metastasis in A549 xenograft nude mice, in combination with a decrease in P62 level. These findings collectively suggest that reduction of cell proliferation and migration by PLCγ1 inhibition could be partially attributed to PLCγ1 inhibition-driven autophagic cell death (ACD). It highlights the potential role of a combination between targeting PLCγ1 and autophagy pathway in anti-tumor therapy, which may be an efficacious new strategy to overcome the autophagy addition of tumor and acquired resistance to current therapy.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Autofagia/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Fosfatidilinositóis/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Células A549 , Animais , Antineoplásicos/farmacologia , Apoptose , Morte Celular Autofágica , Linhagem Celular Tumoral , Proliferação de Células , Dano ao DNA , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genoma Humano , Células HEK293 , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Transplante de Neoplasias , Fosfolipase C gama/metabolismo , RNA Interferente Pequeno/metabolismoRESUMO
Nepetin derived from the flowers of Inula japonica, Inulae flos, has been reported to exert several biological activities, including anti-inflammatory responses. In this study, we evaluated the anti-allergic property of nepetin with its molecular mechanisms in bone marrow-derived mast cells (BMMC) and mice. In this in vitro study, we investigated the inhibitory effects of nepetin on degranulation and generation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in IgE/antigen (Ag)-stimulated BMMC. The effect of nepetin on passive cutaneous anaphylaxis (PCA) reaction was also studied in mice. Nepetin reduced degranulation and LTC4 generation in BMMC. The IgE/Ag-mediated signaling pathway demonstrated that nepetin suppressed intracellular Ca2+ level and activation of PLCγ1 and cPLA2. However, MAPKs were not affected by nepetin in BMMC. In addition, nepetin treatment reduced PGD2 production and suppressed cyclooxygenase-2 protein expression via the inhibition of the Akt and nuclear factor-κB signaling pathways. With respect to the local allergic response in vivo, oral administration of nepetin suppressed mast cell-dependent PCA reaction in a dose-dependent manner. The results of this study suggest that nepetin might have an anti-allergic potential related to mast cell-mediated inflammatory diseases.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Produtos Biológicos/farmacologia , Flavonas/farmacologia , Inula/química , Leucotrieno C4/antagonistas & inibidores , Prostaglandina D2/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/química , Anti-Inflamatórios não Esteroides/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonas/química , Flavonas/isolamento & purificação , Leucotrieno C4/metabolismo , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Prostaglandina D2/metabolismo , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
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.
Assuntos
Colo/metabolismo , Gânglios Espinais/metabolismo , Fator de Crescimento Neural/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Animais , Cálcio/metabolismo , Colo/citologia , Colo/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , NF-kappa B/metabolismo , Fator de Crescimento Neural/agonistas , Fator de Crescimento Neural/antagonistas & inibidores , Fator de Crescimento Neural/genética , Fosfatidilinositol 3-Quinases/genética , Inibidores de Fosfoinositídeo-3 Quinase/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor trkA/genética , Receptor trkA/metabolismo , Sacro/inervação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Canais de Cátion TRPV/metabolismoRESUMO
The enzyme phospholipase C gamma 1 (PLCγ1) has been identified as a potential drug target of interest for various pathological conditions such as immune disorders, systemic lupus erythematosus, and cancers. Targeting its SH3 domain has been recognized as an efficient pharmacological approach for drug discovery against PLCγ1. Therefore, for the first time, a combination of various biophysical methods has been employed to shed light on the atomistic interactions between PLCγ1 and its known binding partners. Indeed, molecular modeling of PLCγ1 with SLP76 peptide and with previously reported inhibitors (ritonavir, anethole, daunorubicin, diflunisal, and rosiglitazone) facilitated the identification of the common critical residues (Gln805, Arg806, Asp808, Glu809, Asp825, Gly827, and Trp828) as well as the quantification of their interaction through binding energies calculations. These features are in agreement with previous experimental data. Such an in depth biophysical analysis of each complex provides an opportunity to identify new inhibitors through pharmacophore mapping, molecular docking and MD simulations. From such a systematic procedure, a total of seven compounds emerged as promising inhibitors, all characterized by a strong binding with PLCγ1 and a comparable or higher binding affinity to ritonavir (∆Gbind < -25 kcal/mol), one of the most potent inhibitor reported till now.
Assuntos
Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Peptídeos/química , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/química , Inibidores Enzimáticos/metabolismo , Humanos , Peptídeos/metabolismo , Fosfolipase C gama/metabolismo , Ligação Proteica , Domínios ProteicosRESUMO
Calcium (Ca2+) signaling within the cell nucleus regulates specific cellular events such as gene transcription and cell proliferation. Nuclear and cytosolic Ca2+ levels can be independently regulated, and nuclear translocation of receptor tyrosine kinases (RTKs) is one way to locally activate signaling cascades within the nucleus. Nuclear RTKs, including the epidermal growth factor receptor (EGFR), are important for processes such as transcriptional regulation, DNA-damage repair, and cancer therapy resistance. RTKs can hydrolyze phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) within the nucleus, leading to Ca2+ release from the nucleoplasmic reticulum by inositol 1,4,5-trisphosphate receptors. PI(4,5)P2 hydrolysis is mediated by phospholipase C (PLC). However, it is unknown which nuclear PLC isoform is triggered by EGFR. Here, using subcellular fractionation, immunoblotting and fluorescence, siRNA-based gene knockdowns, and FRET-based biosensor reporter assays, we investigated the role of PLCδ4 in epidermal growth factor (EGF)-induced nuclear Ca2+ signaling and downstream events. We found that EGF-induced Ca2+ signals are inhibited when translocation of EGFR is impaired. Nuclear Ca2+ signals also were reduced by selectively buffering inositol 1,4,5-trisphosphate (InsP3) within the nucleus. EGF induced hydrolysis of nuclear PI(4,5)P2 by the intranuclear PLCδ4, rather than by PLCγ1. Moreover, protein kinase C, a downstream target of EGF, was active in the nucleus of stimulated cells. Furthermore, PLCδ4 and InsP3 modulated cell cycle progression by regulating the expression of cyclins A and B1. These results provide evidence that EGF-induced nuclear signaling is mediated by nuclear PLCδ4 and suggest new therapeutic targets to modulate the proliferative effects of this growth factor.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Núcleo Celular/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Fosfolipase C delta/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cadeias Pesadas de Clatrina/antagonistas & inibidores , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Ciclina A/metabolismo , Ciclina B1/metabolismo , Receptores ErbB/metabolismo , Humanos , Hidrólise , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C delta/antagonistas & inibidores , Fosfolipase C delta/genética , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Proteína Quinase C/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
Recently, several plexins and semaphorins have been associated with osteoclastogenesis, a vital process for bone remodeling. Plexin-A2 is implicated in bone homeostasis, however, whether it plays a role in osteoclastogenesis and the underlying mechanism remain unknown. We show that plexin-A2 expression is upregulated during RANKL-induced osteoclastogenesis. In addition, the soluble Sema6A fused with IgG1 Fc region (Fc-Sema6A) interacts with plexin-A2 from cell lysates of osteoclasts, suggesting that plexin-A2 acts as a receptor of Sema6A in osteoclasts. Moreover, Sema6A treatment stimulates RANKL-induced osteoclastogenesis, and this effect is abolished when plexin-A2 is neutralized, which illustrates an indispensable role of plexin-A2 in mediating Sema6A effect on osteoclastogenesis. Mechanistically, Sema6A-plexin-A2 axis enhances RANKL-induced activation of PLCγ as well as downstream target NFATc1, one master transcriptional factor of osteoclastogenesis. Lastly, inhibition of PLCγ by pharmacological inhibitor U73122 abrogates Sema6A-stimulated NFATc1 activation and RANKL-induced osteoclastogenesis, thus demonstrating that the PLCγ-mediated NFATc1 activation accounts for the promotive role of Sema6A-plexin-A2 axis in RANKL-induced osteoclastogenesis. Taken together, this study uncovers a novel role of Sema6A and plexin-A2 in osteoclastogenesis, and also offers them as possible therapeutic targets in the intervention of osteolytic diseases.
Assuntos
Fatores de Transcrição NFATC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Osteogênese/fisiologia , Fosfolipase C gama/metabolismo , Ligante RANK/metabolismo , Receptores de Superfície Celular/metabolismo , Semaforinas/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Estrenos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/farmacologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Inibidores de Fosfodiesterase/farmacologia , Fosfolipase C gama/antagonistas & inibidores , Pirrolidinonas/farmacologia , Semaforinas/farmacologiaRESUMO
Objective- Venous malformations (VMs) arise from developmental defects of the vasculature and are characterized by massively enlarged and tortuous venous channels. VMs grow commensurately leading to deformity, obstruction of vital structures, bleeding, and pain. Most VMs are associated with the activating mutation L914F in the endothelial cell (EC) tyrosine kinase receptor TIE2. Therapeutic options for VM are limited and ineffective while therapy with the mammalian target of rapamycin inhibitor rapamycin shows moderate efficacy. Here, we investigated novel therapeutic targets promoting VM regression. Approach and Results- We performed an unbiased screen of Food and Drug Administration-approved drugs in human umbilical vein ECs expressing the TIE2-L914F mutation (HUVEC-TIE2-L914F). Three ABL (Abelson) kinase inhibitors prevented cell proliferation of HUVEC-TIE2-L914F. Moreover, c-ABL, common target of these inhibitors, was highly phosphorylated in HUVEC-TIE2-L914F and VM patient-derived ECs with activating TIE2 mutations. Knockdown of c-ABL/ARG in HUVEC-TIE2-L914F reduced cell proliferation and vascularity of murine VM. Combination treatment with the ABL kinase inhibitor ponatinib and rapamycin caused VM regression in a xenograft model based on injection of HUVEC-TIE2-L914F. A reduced dose of this drug combination was effective in this VM murine model with minimal side effects. The drug combination was antiproliferative, enhanced cell apoptosis and vascular channel regression both in vivo and in a 3-dimensional fibrin gel assay. Conclusions- This is the first report of a combination therapy with ponatinib and rapamycin promoting regression of VM. Mechanistically, the drug combination enhanced AKT inhibition compared with single drug treatment and reduced PLCγ (phospholipase C) and ERK (extracellular signal-regulated kinase) activity.
Assuntos
Imidazóis/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Piridazinas/uso terapêutico , Sirolimo/uso terapêutico , Malformações Vasculares/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Células Cultivadas , Quimiotaxia , Avaliação Pré-Clínica de Medicamentos , Quimioterapia Combinada , Xenoenxertos , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Imidazóis/administração & dosagem , Imidazóis/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Camundongos , Camundongos Nus , Mutação de Sentido Incorreto , Fosfolipase C gama/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Piridazinas/administração & dosagem , Piridazinas/farmacologia , Receptor TIE-2/genética , Transdução de Sinais/efeitos dos fármacos , Sirolimo/administração & dosagem , Sirolimo/farmacologia , Malformações Vasculares/patologiaRESUMO
Nerve growth factor elicits signaling outcomes by interacting with both its high-affinity receptor, TrkA, and its low-affinity receptor, p75NTR. Although these two receptors can regulate distinct cellular outcomes, they both activate the extracellular-signal-regulated kinase pathway upon nerve growth factor stimulation. To delineate TrkA subcircuits in PC12 cell differentiation, we developed an optogenetic system whereby light was used to specifically activate TrkA signaling in the absence of nerve growth factor. By using tyrosine mutants of the optogenetic TrkA in combination with pathway-specific pharmacological inhibition, we find that Y490 and Y785 each contributes to PC12 cell differentiation through the extracellular-signal-regulated kinase pathway in an additive manner. Optogenetic activation of TrkA eliminates the confounding effect of p75NTR and other potential off-target effects of the ligand. This approach can be generalized for the mechanistic study of other receptor-mediated signaling pathways.
Assuntos
Diferenciação Celular , Optogenética , Proteínas Tirosina Quinases/metabolismo , Animais , Diferenciação Celular/efeitos da radiação , Membrana Celular/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Luz , Fator de Crescimento Neural/metabolismo , Células PC12 , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Proteínas Tirosina Quinases/genética , Ratos , Receptor trkA/metabolismo , Transdução de Sinais/efeitos da radiaçãoRESUMO
The classical mast cells degranulation pathway is mediated by FcεRI aggregation and varies in strength among subjects. Dehydroandrographolide (DA) is one of principal components of Andrographis paniculata (Burm.f.) Nees (family: Acanthaceae) and considered the main contributors of its therapeutic properties, such as anti-tumor. In this study, inhibition of IgE-mediated anaphylactic reactions and anti-inflammatory potential of DA were investigated. The anti-anaphylactic activity of DA was investigated using skin swelling and extravasation assays in vivo and mast cell degranulation assay in vitro. The release of cytokines was measured using ELISA kits. Human Phospho-Kinase Array kit and western blotting were used to explore the related molecular signaling pathways. DA inhibited IgE-mediated mast cell activation, including degranulation and release of cytokines in vitro. Moreover, DA reduced the degree of swelling and Evans blue exudation of mice paw in a dose-dependent manner by inhibiting mast cell degranulation. DA obviously reduced the concentrations of histamine, TNF-α, MCP-1, IL-8, IL-13, and IL-4 in mice serum and inhibited IgE-mediated anaphylactic reactions as a potential P-PLCγ inhibitor. Our study reveals that DA can inhibit allergic responses in vivo and in vitro, and it may be regarded as a novel P-PLCγ inhibitor for preventing mast cell-immediate and delayed allergic diseases.
Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Diterpenos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Imunoglobulina E/imunologia , Mastócitos/efeitos dos fármacos , Anafilaxia/imunologia , Anafilaxia/metabolismo , Animais , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Humanos , Masculino , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos Endogâmicos C57BL , Ovalbumina , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismoRESUMO
The endoplasmic reticulum stress sensor, the unfolded protein response (UPR), regulates intracellular protein homeostasis. While transient activation of the reactive UPR by unfolded protein is protective, prolonged and sustained activation of the reactive UPR triggers CHOP-mediated apoptosis. In the recently characterized, evolutionarily conserved anticipatory UPR, mitogenic hormones and other effectors pre-activate the UPR; how strong and sustained activation of the anticipatory UPR induces cell death was unknown. To characterize this cell death pathway, we used BHPI, a small molecule that activates the anticipatory UPR through estrogen receptor α (ERα) and induces death of ERα+ cancer cells. We show that sustained activation of the anticipatory UPR by BHPI kills cells by inducing depletion of intracellular ATP, resulting in classical necrosis phenotypes, including plasma membrane disruption and leakage of intracellular contents. Unlike reactive UPR activation, BHPI-induced hyperactivation of the anticipatory UPR does not induce apoptosis or sustained autophagy. BHPI does not induce CHOP protein or PARP cleavage, and two pan-caspase inhibitors, or Bcl2 overexpression, have no effect on BHPI-induced cell death. Moreover, BHPI does not increase expression of autophagy markers, or work through recently identified programmed-necrosis pathways, such as necroptosis. Opening of endoplasmic reticulum IP3R calcium channels stimulates cell swelling, cPLA2 activation, and arachidonic acid release. Notably, cPLA2 activation requires ATP depletion. Importantly, blocking rapid cell swelling or production of arachidonic acid does not prevent necrotic cell death. Rapid cell death is upstream of PERK activation and protein synthesis inhibition, and results from strong and sustained activation of early steps in the anticipatory UPR. Supporting a central role for ATP depletion, reversing ATP depletion blocks rapid cell death, and the onset of necrotic cell death is correlated with ATP depletion. Necrotic cell death initiated by strong and sustained activation of the anticipatory UPR is a newly discovered role of the UPR.
Assuntos
Apoptose , Resposta a Proteínas não Dobradas , Trifosfato de Adenosina/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Ácido Araquidônico/metabolismo , Canais de Cálcio/metabolismo , Linhagem Celular , Tamanho Celular/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Receptor alfa de Estrogênio/antagonistas & inibidores , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Humanos , Necrose , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/genética , Fosfolipase C gama/metabolismo , Fosfolipases A2 Citosólicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacos , eIF-2 Quinase/metabolismoRESUMO
BACKGROUND/AIMS: HSP70/HSP90-organizing protein (HOP) is an adaptor protein that mediates heat shock protein 70 (HSP70) and HSP90 folding. HOP can be secreted by cancer cells and promote malignant cell growth in an autocrine manner. Here, we studied its role in gastric cancer (GC). METHODS: HOP mRNA and protein levels were detected by quantitative real-time PCR and western blotting, respectively, and enzyme-linked immunosorbent assay was used to determine the serum levels. Immunohistochemistry was performed to analyze HOP expression in 117 GC tissues and 32 adjacent normal tissues. The Cell Counting Kit-8 cell viability assay, flow cytometry, and western blotting were used to analyze the effects of HOP on cell proliferation and apoptosis, and the potential underlying mechanisms. RESULTS: HOP mRNA and protein levels were significantly higher in GC tissues than in normal tissues in our medical center (P< 0.001) and in The Cancer Genome Atlas database (P< 0.001). GC patients had higher serum levels of HOP than age-matched healthy controls (P< 0.001); however, once tumors were removed, serum levels significantly decreased (P< 0.01). In human GC tissues, increased HOP expression was associated with tumor progression and poor survival. Notably, autocrine HOP promoted cell proliferation through the phospholipase Cγ1-extracellular signal-regulated kinase 1/2-dependent pathway, and inhibited cell apoptosis by regulating the activities of caspase 9, caspase 3, and B-cell lymphoma 2. Blocking extracellular HOP with neutralizing antibody reduced proliferation and enhanced fluorouracil-induced apoptosis of GC cells. CONCLUSIONS: Our findings demonstrate that HOP is an important molecular marker and prognostic factor for GC, and functionally contributes to tumor cell growth and survival. These results provide a rationale for considering HOP as a potential therapeutic target and chemosensitizer in GC.
Assuntos
Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Neoplasias Gástricas/patologia , Apoptose/efeitos dos fármacos , Comunicação Autócrina/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Bases de Dados Genéticas , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Fluoruracila/farmacologia , Proteínas de Choque Térmico HSP70/sangue , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP90/sangue , Proteínas de Choque Térmico HSP90/genética , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidadeRESUMO
We investigated the effects of recombinant adenovirus vectors that overexpress or silence PLCγ2 on the expression of this gene during hepatocyte proliferation. Hepatocytes were isolated, identified by immunofluorescent cytochemical staining and infected by previously constructed Ad-PLCγ2 and Ad-PLCγ2 siRNA1, siRNA2 and siRNA3. Green fluorescent protein (GFP) expression was observed by fluorescence microscopy. Infection percentage was calculated by flow cytometry. mRNA and protein levels of PLCγ2 were detected by quantitative reverse transcription-PCR (qRT-PCR) and western blotting, respectively. The viability of the infected hepatocytes was measured by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. We found that nearly 97% of cells were positive for the hepatocyte marker, CK18. After infection of Ad-PLCγ2 and Ad-PLCγ2 siRNA, more than 99% of hepatocytes expressed GFP significantly, and mRNA and protein expression of PLCγ2 was up-regulated significantly in Ad-PLCγ2 infected hepatocytes, but down-regulated in Ad-PLCγ2 siRNA2 infected cells. The cell proliferation rate decreased in PLCγ2-overexpressing cells, while the rate increased in PLCγ2-silencing cells. We verified that recombinant Ad-PLCγ2 and Ad-PLCγ2 siRNA2 were constructed successfully. These two recombinant vectors promoted or decreased the expression of PLCγ2 in rat hepatocytes and affected the cell proliferation rate, which provides a useful tool for further investigation of the role of PLCγ2 in hepatocyte apoptosis.
Assuntos
Regulação da Expressão Gênica/genética , Hepatócitos/metabolismo , Fosfolipase C gama/genética , RNA Interferente Pequeno/genética , Adenoviridae/genética , Animais , Sobrevivência Celular/genética , Vetores Genéticos/genética , Fosfolipase C gama/antagonistas & inibidores , Fosfolipase C gama/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Mutations in the gene encoding phospholipase C-γ2 (PLCγ2) have been shown to be associated with resistance to targeted therapy of chronic lymphocytic leukemia (CLL) with the Bruton's tyrosine kinase inhibitor ibrutinib. The fact that two of these mutations, R665W and L845F, imparted upon PLCγ2 an â¼2-3-fold ibrutinib-insensitive increase in the concentration of cytosolic Ca2+ following ligation of the B cell antigen receptor (BCR) led to the assumption that the two mutants exhibit constitutively enhanced intrinsic activity. Here, we show that the two PLCγ2 mutants are strikingly hypersensitive to activation by Rac2 such that even wild-type Rac2 suffices to activate the mutant enzymes upon its introduction into intact cells. Enhanced "basal" activity of PLCγ2 in intact cells is shown using the pharmacologic Rac inhibitor EHT 1864 and the PLCγ2F897Q mutation mediating Rac resistance to be caused by Rac-stimulated rather than by constitutively enhanced PLCγ2 activity. We suggest that R665W and L845F be referred to as allomorphic rather than hypermorphic mutations of PLCG2 Rerouting of the transmembrane signals emanating from BCR and converging on PLCγ2 through Rac in ibrutinib-resistant CLL cells may provide novel drug treatment strategies to overcome ibrutinib resistance mediated by PLCG2 mutations or to prevent its development in ibrutinib-treated CLL patients.