RESUMO
BACKGROUND: Regulation of vascular permeability is critical to maintaining tissue metabolic homeostasis. VEGF (vascular endothelial growth factor) is a key stimulus of vascular permeability in acute and chronic diseases including ischemia reperfusion injury, sepsis, and cancer. Identification of novel regulators of vascular permeability would allow for the development of effective targeted therapeutics for patients with unmet medical need. METHODS: In vitro and in vivo models of VEGFA-induced vascular permeability, pathological permeability, quantitation of intracellular calcium release and cell entry, and phosphatidylinositol 4,5-bisphosphate levels were evaluated with and without modulation of PLC (phospholipase C) ß2. RESULTS: Global knock-out of PLCß2 in mice resulted in blockade of VEGFA-induced vascular permeability in vivo and transendothelial permeability in primary lung endothelial cells. Further work in an immortalized human microvascular cell line modulated with stable knockdown of PLCß2 recapitulated the observations in the mouse model and primary cell assays. Additionally, loss of PLCß2 limited both intracellular release and extracellular entry of calcium following VEGF stimulation as well as reduced basal and VEGFA-stimulated levels of phosphatidylinositol 4,5-bisphosphate compared to control cells. Finally, loss of PLCß2 in both a hyperoxia-induced lung permeability model and a cardiac ischemia:reperfusion model resulted in improved animal outcomes when compared with wild-type controls. CONCLUSIONS: The results implicate PLCß2 as a key positive regulator of VEGF-induced vascular permeability through regulation of both calcium flux and phosphatidylinositol 4,5-bisphosphate levels at the cellular level. Targeting of PLCß2 in a therapeutic setting may provide a novel approach to regulating vascular permeability in patients.
Assuntos
Permeabilidade Capilar , Fosfatidilinositol 4,5-Difosfato , Fosfolipase C beta , Mucosa Respiratória , Fator A de Crescimento do Endotélio Vascular , Animais , Cálcio/metabolismo , Permeabilidade Capilar/genética , Permeabilidade Capilar/fisiologia , Células Endoteliais/metabolismo , Humanos , Pulmão/metabolismo , Camundongos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Fosfolipase C beta/fisiologia , Mucosa Respiratória/metabolismoRESUMO
Host-pathogen interactions play an important role in defining the outcome of a disease. Recent studies have shown that the bacterial quorum sensing molecules (QSM) can interact with host cell membrane proteins, mainly G protein-coupled receptors (GPCRs), and induce innate immune responses. However, few studies have examined QSM-GPCR interactions and their influence on oral innate immune responses. In this study, we examined the role of bitter taste receptor T2R14 in sensing competence stimulating peptides (CSPs) secreted by cariogenic bacterium Streptococcus mutans and in mediating innate immune responses in gingival epithelial cells (GECs). Transcriptomic and western blot analyses identify T2R14 to be highly expressed in GECs. Our data show that only CSP-1 from S. mutans induces robust intracellular calcium mobilization compared to CSP-2 and CSP-3. By using CRISPR-Cas9, we demonstrate that CSP-1 induced calcium signaling and secretion of cytokines CXCL-8/IL-8, TNF-α, and IL-6 is mediated through T2R14 in GECs. Interestingly, the NF-kB signaling activated by CSP-1 in GECs was independent of T2R14. CSP-1-primed GECs attracted differentiated HL-60 immune cells (dHL-60) and this effect was abolished in T2R14 knock down GECs and also in cells primed with T2R14 antagonist 6-Methoxyflavone (6-MF). Our findings identify S. mutans CSP-1 as a peptide ligand for the T2R family. Our study establishes a novel host-pathogen interaction between cariogenic S. mutans CSP-1 and T2R14 in GECs leading to an innate immune response. Collectively, these findings suggest T2Rs as potential therapeutic targets to modulate innate immune responses upon oral bacterial infections.
Assuntos
Proteínas de Bactérias/fisiologia , Gengiva/imunologia , Interações Hospedeiro-Patógeno , Percepção de Quorum/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Streptococcus mutans/fisiologia , Cálcio/metabolismo , Linhagem Celular , Movimento Celular , Citocinas/biossíntese , Células Epiteliais/imunologia , Gengiva/citologia , Humanos , Imunidade Inata , NF-kappa B/fisiologia , Fosfolipase C beta/fisiologiaRESUMO
Uveal melanoma (UM) is a currently untreatable form of melanoma with a 50% mortality rate. Characterization of the essential signaling pathways driving this cancer is critical to develop target therapies. Activating mutations in the Gαq signaling pathway at the level of GNAQ, GNA11, or rarely CYSLTR2 or PLCß4 are considered alterations driving proliferation in UM and several other neoplastic disorders. Here, we systematically examined the oncogenic signaling output of various mutations recurrently identified in human tumors. We demonstrate that CYSLTR2 â GNAQ/11 â PLCß act in a linear signaling cascade that, via protein kinase C (PKC), activates in parallel the MAP-kinase and FAK/Yes-associated protein pathways. Using genetic ablation and pharmacological inhibition, we show that the PKC/RasGRP3/MAPK signaling branch is the essential component that drives the proliferation of UM. Only inhibition of the MAPK branch but not the FAK branch synergizes with inhibition of the proximal cascade, providing a blueprint for combination therapy. All oncogenic signaling could be extinguished by the novel GNAQ/11 inhibitor YM-254890, in all UM cells with driver mutation in the Gαq subunit or the upstream receptor. Our findings highlight the GNAQ/11 â PLCß â PKC â MAPK pathway as the central signaling axis to be suppressed pharmacologically to treat for neoplastic disorders with Gαq pathway mutations.
Assuntos
Melanoma/genética , Oncogenes/fisiologia , Neoplasias Uveais/genética , Animais , Linhagem Celular Tumoral , Quinase 1 de Adesão Focal/fisiologia , Subunidades alfa de Proteínas de Ligação ao GTP/fisiologia , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Camundongos , Mutação , Fosfolipase C beta/fisiologia , Proteína Quinase C/fisiologia , Receptores de Leucotrienos/fisiologia , Transdução de Sinais/fisiologia , Neoplasias Uveais/patologiaRESUMO
Orexin-A is an endogenous peptide with receptors throughout the brain. According to some recent research, learning and memory are affected by the central administration of orexin; however, no study so far has investigated the long-term inhibition of the orexinergic system. The present study has evaluated the effect of pretraining administration of orexin 1 receptor (OXR1) antagonist, SB-334867, on the acquisition of memory. The Morris water maze (MWM) task was used for training and trial purposes in all groups. Memory performance was analyzed by measuring escape latency, traveled distance, and time spent in the target quadrant. Moreover, the effect of SB-334867 on phospholipase Cß3 (PLCß3) levels in the CA1 region of hippocampus slices was examined. Hippocampus slices were prepared using an immunohistochemistry (IHC) approach. SB-334867 (20 mg/kg) increased escape latency in SB-treated rats compared to SB-vehicle group (P < 0.01). SB-treated rats spent less time in the target quadrant compared to the SB-vehicle group (P < 0.001). Distance traveled in the target quadrant was significantly more in SB-treated rats compared to the SB-vehicle group (P < 0.001). Furthermore, SB-334867 decreased PLCß3 levels in the CA1 of the hippocampus (P < 0.01 and P < 0.05, respectively). Put together, our results suggest that the long-term inhibition of OXR1 plays a prominent role in spatial learning and memory, probably by attenuating PLCß3 in CA1 neurons.
Assuntos
Memória/efeitos dos fármacos , Memória/fisiologia , Fosfolipase C beta/metabolismo , Animais , Benzoxazóis/farmacologia , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Naftiridinas/farmacologia , Receptores de Orexina/metabolismo , Orexinas/metabolismo , Fosfolipase C beta/fisiologia , Ratos , Ratos Wistar , Ureia/análogos & derivados , Ureia/farmacologiaRESUMO
Sleep abnormality often accompanies the impairment of cognitive function. Both rapid eye movement (REM) and non-REM (NREM) sleep have associated with improved memory performance. However, the role of composition in NREM sleep, consisting of light and deep NREM, for memory formation is not fully understood. We investigated how the dynamics of NREM sleep states influence memory consolidation. Thalamocortical (TC) neuron-specific phospholipase C ß4 (PLCß4) knockout (KO) increased the total duration of NREM sleep, consisting of destabilized light NREM and stabilized deep NREM. Surprisingly, the longer NREM sleep did not improve memory consolidation but rather impaired it in TC-specific PLCß4 KO mice. Memory function was positively correlated with the stability of light NREM and spindle activity occurring in maintained light NREM period. Our study suggests that a single molecule, PLCß4, in TC neurons is critical for tuning the NREM sleep states and thus affects sleep-dependent memory formation.
Assuntos
Consolidação da Memória/fisiologia , Transtornos da Memória/enzimologia , Proteínas do Tecido Nervoso/fisiologia , Fosfolipase C beta/fisiologia , Fases do Sono/fisiologia , Tálamo/enzimologia , Animais , Córtex Cerebral/enzimologia , Condicionamento Clássico/fisiologia , Ritmo Delta/fisiologia , Eletroencefalografia , Eletromiografia , Éxons/genética , Comportamento Exploratório , Medo/fisiologia , Masculino , Transtornos da Memória/fisiopatologia , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Neurônios/enzimologia , Fosfolipase C beta/deficiência , Reconhecimento Psicológico , Deleção de Sequência , Sono de Ondas Lentas/fisiologia , Fatores de TempoRESUMO
GSK-3 and PLCbeta enzymes are responsible for the regulation of several signalling pathways related to many cellular functions. In hematopoietic cells, GSK-3 deficiency is correlated with an MDS-like phenotype and with leukemogenesis, showing a prognostic potential in AML cells. GSK-3 interacts with Wnt or MAPK signalling, but it is also linked to PI3K/Akt/mTOR pathways to regulate cell proliferation and apoptosis of hematopoietic stem cell progenitors. PLCbeta enzymes are involved in cell cycle progression of hematopoietic, MDS/AML and immune cells, through activation of PKC or calcium signalling. Of note, a PLCbeta1/PKCalpha pathway is modulated during MDS pathogenesis, with a specific involvement of the inositides localized in the nucleus. Here we focus on GSK-3 and PLCbeta signalling, describing the many evidences that underline the pivotal role of both GSK-3 and PLCbeta-dependent pathways in MDS/AML, their association with therapy and their possible interactions.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Leucemia Mieloide Aguda/enzimologia , Síndromes Mielodisplásicas/enzimologia , Fosfolipase C beta/metabolismo , Transdução de Sinais , Quinase 3 da Glicogênio Sintase/antagonistas & inibidores , Quinase 3 da Glicogênio Sintase/fisiologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Síndromes Mielodisplásicas/tratamento farmacológico , Síndromes Mielodisplásicas/genética , Síndromes Mielodisplásicas/metabolismo , Fosfolipase C beta/fisiologiaRESUMO
Enhanced contractility of airway smooth muscle (ASM) is a major pathophysiological characteristic of asthma. Expanding the therapeutic armamentarium beyond ß-agonists that target ASM hypercontractility would substantially improve treatment options. Recent studies have identified naturally occurring phytochemicals as candidates for acute ASM relaxation. Several flavonoids were evaluated for their ability to acutely relax human and murine ASM ex vivo and murine airways in vivo and were evaluated for their ability to inhibit procontractile signaling pathways in human ASM (hASM) cells. Two members of the flavonol subfamily, galangin and fisetin, significantly relaxed acetylcholine-precontracted murine tracheal rings ex vivo (n = 4 and n = 5, respectively, P < 0.001). Galangin and fisetin also relaxed acetylcholine-precontracted hASM strips ex vivo (n = 6-8, P < 0.001). Functional respiratory in vivo murine studies demonstrated that inhaled galangin attenuated the increase in lung resistance induced by inhaled methacholine (n = 6, P < 0.01). Both flavonols, galangin and fisetin, significantly inhibited purified phosphodiesterase-4 (PDE4) (n = 7, P < 0.05; n = 7, P < 0.05, respectively), and PLCß enzymes (n = 6, P < 0.001 and n = 6, P < 0.001, respectively) attenuated procontractile Gq agonists' increase in intracellular calcium (n = 11, P < 0.001), acetylcholine-induced increases in inositol phosphates, and CPI-17 phosphorylation (n = 9, P < 0.01) in hASM cells. The prorelaxant effect retained in these structurally similar flavonols provides a novel pharmacological method for dual inhibition of PLCß and PDE4 and therefore may serve as a potential treatment option for acute ASM constriction.
Assuntos
Flavonoides/farmacologia , Relaxamento Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Fosfolipase C beta/antagonistas & inibidores , Animais , Aorta/efeitos dos fármacos , Aorta/fisiopatologia , Asma/tratamento farmacológico , Broncoconstrição/efeitos dos fármacos , Sinalização do Cálcio , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/química , Avaliação Pré-Clínica de Medicamentos , Flavonoides/química , Flavonóis , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Masculino , Camundongos , Contração Muscular , Músculo Liso/fisiologia , Músculo Liso/fisiopatologia , Inibidores da Fosfodiesterase 4/química , Inibidores da Fosfodiesterase 4/farmacologia , Fosfolipase C beta/fisiologiaRESUMO
Hematopoietic stem/progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment and are retained there by the interaction of membrane lipid raft-associated receptors, such as the α-chemokine receptor CXCR4 and the α4ß1-integrin (VLA-4, very late antigen 4 receptor) receptor, with their respective specific ligands, stromal-derived factor 1 and vascular cell adhesion molecule 1, expressed in BM stem cell niches. The integrity of the lipid rafts containing these receptors is maintained by the glycolipid glycosylphosphatidylinositol anchor (GPI-A). It has been reported that a cleavage fragment of the fifth component of the activated complement cascade, C5a, has an important role in mobilizing HSPCs into the peripheral blood (PB) by (i) inducing degranulation of BM-residing granulocytes and (ii) promoting their egress from the BM into the PB so that they permeabilize the endothelial barrier for subsequent egress of HSPCs. We report here that hematopoietic cell-specific phospholipase C-ß2 (PLC-ß2) has a crucial role in pharmacological mobilization of HSPCs. On the one hand, when released during degranulation of granulocytes, it digests GPI-A, thereby disrupting membrane lipid rafts and impairing retention of HSPCs in BM niches. On the other hand, it is an intracellular enzyme required for degranulation of granulocytes and their egress from BM. In support of this dual role, we demonstrate that PLC-ß2-knockout mice are poor mobilizers and provide, for the first time, evidence for the involvement of this lipolytic enzyme in the mobilization of HSPCs.
Assuntos
Medula Óssea/enzimologia , Complemento C5a/metabolismo , Granulócitos/metabolismo , Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Microdomínios da Membrana , Fosfolipase C beta/fisiologia , Animais , Apoptose , Adesão Celular , Movimento Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Granulócitos/citologia , Células-Tronco Hematopoéticas/citologia , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de SinaisRESUMO
Intestinal dual oxidase (DUOX) activation is the first line of host defense against enteric infection in Drosophila. DUOX enzymatic activity is mainly controlled by phospholipase C-ß (PLCß)-dependent calcium mobilization, whereas DUOX gene expression is mainly controlled by the MEKK1-p38 mitogen-activated protein kinase pathway. Furthermore, bacterial-derived uracil molecules act as ligands for DUOX activation. However, our current understanding of uracil-induced signal transduction pathways remain incomplete. We have recently found that uracil stimulates Hedgehog signaling, which in turn upregulates cadherin99C (Cad99C) expression in enterocytes. Cad99C molecules, along with PLCß and protein kinase C, induce the formation of signaling endosomes that facilitate intracellular calcium mobilization for DUOX activity. These observations illustrate the complexity of signaling cascades in uracil-induced signaling pathways. Here, we further demonstrated the role of lipid raft formation and calmodulin-dependent protein kinase-II on endosome formation and calcium mobilization, respectively. Moreover, we will provide a brief discussion on two different models for uracil recognition and uracil-induced DUOX activation in Drosophila enterocytes.
Assuntos
Drosophila/imunologia , Modelos Imunológicos , NADPH Oxidases/fisiologia , Uracila/farmacologia , Animais , Caderinas/metabolismo , Cálcio/metabolismo , Resistência à Doença , Drosophila/genética , Drosophila/metabolismo , Enterócitos/efeitos dos fármacos , Enterócitos/imunologia , Regulação da Expressão Gênica , Proteínas Hedgehog/metabolismo , Sistema de Sinalização das MAP Quinases , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/fisiologia , NADPH Oxidases/metabolismo , Fosfolipase C beta/metabolismo , Fosfolipase C beta/fisiologia , Transdução de Sinais , Transcriptoma/efeitos dos fármacos , Uracila/fisiologiaRESUMO
BACKGROUND: G proteins are known to modulate various growth signals and are implicated in the regulation of tumorigenesis. The tumor suppressor Fhit is a newly identified interaction partner of Gq proteins that typically stimulate the phospholipase C pathway. Activated Gαq subunits have been shown to interact directly with Fhit, up-regulate Fhit expression and enhance its suppressive effect on cell growth and migration. Other signaling molecules may be involved in modulating Gαq/Fhit interaction. METHODS: To test the relationship of PLCß with the interaction between Gαq and Fhit, co-immunoprecipication assay was performed on HEK293 cells co-transfected with different combinations of Flag-Fhit, Gα16, Gα16QL, pcDNA3 vector, and PLCß isoforms. Possible associations of Fhit with other effectors of Gαq were also demonstrated by co-immunoprecipitation. The regions of Gαq for Fhit interaction and PLCß stimulation were further evaluated by inositol phosphates accumulation assay using a series of Gα16/z chimeras with discrete regions of Gα16 replaced by those of Gαz. RESULTS: PLCß1, 2 and 3 interacted with Fhit regardless of the expression of Gαq. Expression of PLCß increased the affinities of Fhit for both wild-type and activated Gαq. Swapping of the Fhit-interacting α2-ß4 region of Gαq with Gαi eliminated the association of Gαq with Fhit without affecting the ability of the mutant to stimulate PLCß. Other effectors of Gαq including RGS2 and p63RhoGEF were unable to interact with Fhit. CONCLUSIONS: PLCß may participate in the regulation of Fhit by Gq in a unique way. PLCß interacts with Fhit and increases the interaction between Gαq and Fhit. The Gαq/PLCß/Fhit complex formation points to a novel signaling pathway that may negatively regulate tumor cell growth.
Assuntos
Hidrolases Anidrido Ácido/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Fosfolipase C beta/metabolismo , Western Blotting , Células HEK293 , Humanos , Imunoprecipitação , Fosfolipase C beta/fisiologia , Ligação Proteica , Transdução de Sinais/fisiologia , Regulação para CimaRESUMO
The G protein-coupled estrogen receptor (GPER) mediates both the genomic and nongenomic effects of estrogen and has been implicated in breast cancer development. Here, we compared GPER expression in cancerous tissue and adjacent normal tissue in patients with invasive ductal carcinoma (IDC) of the breast and determined that GPER is highly upregulated in cancerous cells. Additionally, our studies revealed that GPER stimulation activates yes-associated protein 1 (YAP) and transcriptional coactivator with a PDZ-binding domain (TAZ), 2 homologous transcription coactivators and key effectors of the Hippo tumor suppressor pathway, via the Gαq-11, PLCß/PKC, and Rho/ROCK signaling pathways. TAZ was required for GPER-induced gene transcription, breast cancer cell proliferation and migration, and tumor growth. Moreover, TAZ expression positively correlated with GPER expression in human IDC specimens. Together, our results suggest that the Hippo/YAP/TAZ pathway is a key downstream signaling branch of GPER and plays a critical role in breast tumorigenesis.
Assuntos
Neoplasias da Mama/fisiopatologia , Carcinoma Ductal de Mama/fisiopatologia , Estrogênios/fisiologia , Proteínas de Neoplasias/fisiologia , Neoplasias Hormônio-Dependentes/fisiopatologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/metabolismo , Divisão Celular , Movimento Celular , Transformação Celular Neoplásica , Estrogênios/farmacologia , Feminino , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/fisiologia , Regulação Neoplásica da Expressão Gênica , Via de Sinalização Hippo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Hormônio-Dependentes/genética , Neoplasias Hormônio-Dependentes/metabolismo , Fosfolipase C beta/fisiologia , Fosfoproteínas/fisiologia , Fosforilação , Proteína Quinase C/fisiologia , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/análise , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Receptores de Estrogênio/efeitos dos fármacos , Receptores Acoplados a Proteínas G/efeitos dos fármacos , Serina-Treonina Quinase 3 , Fatores de Transcrição/fisiologia , Transcrição Gênica , Proteínas Supressoras de Tumor/análise , Proteínas de Sinalização YAP , Quinases Associadas a rho/fisiologiaRESUMO
Drosophila phototransduction is a model system for the ubiquitous phosphoinositide signaling. In complete darkness, spontaneous unitary current events (dark bumps) are produced by spontaneous single Gqα activation, while single-photon responses (quantum bumps) arise from synchronous activation of several Gqα molecules. We have recently shown that most of the spontaneous single Gqα activations do not produce dark bumps, because of a critical phospholipase Cß (PLCß) activity level required for bump generation. Surpassing the threshold of channel activation depends on both PLCß activity and cellular [Ca(2+)], which participates in light excitation via a still unclear mechanism. We show here that in IP3 receptor (IP3R)-deficient photoreceptors, both light-activated Ca(2+) release from internal stores and light sensitivity were strongly attenuated. This was further verified by Ca(2+) store depletion, linking Ca(2+) release to light excitation. In IP3R-deficient photoreceptors, dark bumps were virtually absent and the quantum-bump rate was reduced, indicating that Ca(2+) release from internal stores is necessary to reach the critical level of PLCß catalytic activity and the cellular [Ca(2+)] required for excitation. Combination of IP3R knockdown with reduced PLCß catalytic activity resulted in highly suppressed light responses that were partially rescued by cellular Ca(2+) elevation, showing a functional cooperation between IP3R and PLCß via released Ca(2+). These findings suggest that in contrast to the current dogma that Ca(2+) release via IP3R does not participate in light excitation, we show that released Ca(2+) plays a critical role in light excitation. The positive feedback between PLCß and IP3R found here may represent a common feature of the inositol-lipid signaling.
Assuntos
Drosophila/fisiologia , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Fosfolipase C beta/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Animais , Animais Geneticamente Modificados , Sinalização do Cálcio/fisiologia , Eletrorretinografia , Hipóxia/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Luz , Masculino , Técnicas de Patch-Clamp , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Interferência de RNARESUMO
We previously observed that the human bitter taste receptor T2R38 is an important component of upper respiratory innate defense because it detects acyl homoserine lactone (AHL) quorum-sensing molecules secreted by Gram-negative bacteria. T2R38 activation in human sinonasal epithelial cells stimulates calcium and NO signals that increase mucociliary clearance, the major physical respiratory defense against inhaled pathogens. While mice do not have a clear T2R38 ortholog, they do have bitter taste receptors capable of responding to T2R38 agonists, suggesting that T2R-mediated innate immune mechanisms may be conserved in mice. We examined whether AHLs activate calcium and NO signaling in mouse nasal epithelial cells, and utilized pharmacology, as well as cells from knockout mice lacking important components of canonical taste signal transduction pathways, to determine if AHL-stimulated responses require taste signaling molecules. We found that AHLs stimulate calcium-dependent NO production that increases mucociliary clearance and thus likely serves an innate immune role against Gram-negative bacteria. These responses require PLCß2 and TRPM5 taste signaling components, but not α-gustducin. These data suggest the mouse may be a useful model for further studies of T2R-mediated innate immunity.
Assuntos
Imunidade Inata/imunologia , Mucosa Nasal/imunologia , Pseudomonas aeruginosa/química , Percepção de Quorum , Transdução de Sinais/efeitos dos fármacos , Paladar/efeitos dos fármacos , Acil-Butirolactonas/farmacologia , Animais , Camundongos , Camundongos Knockout , Óxido Nítrico/fisiologia , Fosfolipase C beta/genética , Fosfolipase C beta/fisiologia , Transdução de Sinais/genética , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/fisiologia , Paladar/genética , Transducina/genéticaRESUMO
Phospholipase C (PLC) enzymes convert phosphatidylinositol-4,5-bisphosphate into the second messengers diacylglycerol and inositol-1,4,5-triphosphate. The production of these molecules promotes the release of intracellular calcium and activation of protein kinase C, which results in profound cellular changes. The PLCß subfamily is of particular interest given its prominent role in cardiovascular and neuronal signaling and its regulation by G protein-coupled receptors, as PLCß is the canonical downstream target of the heterotrimeric G protein Gαq. However, this is not the only mechanism regulating PLCß activity. Extensive structural and biochemical evidence has revealed regulatory roles for autoinhibitory elements within PLCß, Gßγ, small molecular weight G proteins, and the lipid membrane itself. Such complex regulation highlights the central role that this enzyme plays in cell signaling. A better understanding of the molecular mechanisms underlying the control of its activity will greatly facilitate the search for selective small molecule modulators of PLCß.
Assuntos
Fosfolipase C beta/química , Fosfolipase C beta/fisiologia , Animais , Humanos , Isoenzimas/química , Isoenzimas/fisiologia , Isoformas de Proteínas/química , Isoformas de Proteínas/fisiologiaRESUMO
AIM: To determine the effects of BN52021 on platelet-activating factor receptor (PAFR) signaling molecules under lipopolysaccharide (LPS)-induced inflammatory conditions in MS1 cells. METHODS: MS1 cells (a mouse pancreatic islet endothelial cell line) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine and 100 µg/mL penicillin/streptomycin in 5% CO2 at 37â °C. After growth to confluency in media, the cells were processed for subsequent studies. The MS1 cells received 0, 0.1, 1 and 10 µg/mL LPS in this experiment. The viability/proliferation of the cells induced by LPS was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Apoptosis and necrosis of the cells under the inflammatory condition described previously were observed using Hoechst 33342-propidium iodide staining. Adenylate cyclase (AC), phospholipase A2 (PLA2), phospholipase Cß (PLCß), protein tyrosine kinase (PTK), G protein-coupled receptor kinases (GRK) and p38-mitogen-activated protein kinase (p38 MAPK) mRNA in the PAFR signaling pathway were measured by real-time polymerase chain reaction. The protein expression level of phosphorylated AC (p-AC), phosphorylated PLA2 (p-PLA2), phosphorylated PTK (p-PTK), phosphorylated p38 MAPK (p-p38 MAPK), PLCß and GRK was measured using Western blotting analysis. RESULTS: The activity of MS1 cells incubated with different concentrations of LPS for 6 h decreased significantly in the 1 µg/mL LPS group (0.49 ± 0.10 vs 0.67 ± 0.13, P < 0.05) and 10 µg/mL LPS group (0.44 ± 0.10 vs 0.67 ± 0.13, P < 0.001), but not in 0.1 µg/mL group. When the incubation time was extended to 12 h (0.33 ± 0.05, 0.32 ± 0.03 and 0.25 ± 0.03 vs 0.69 ± 0.01) and 24 h (0.31 ± 0.01, 0.29 ± 0.03 and 0.25 ± 0.01 vs 0.63 ± 0.01), MS1 cell activity decreased in all LPS concentration groups compared with the blank control (P < 0.001). BN52021 significantly improved the cell activity when its concentration reached 50 µmol/L compared with the group that received LPS treatment alone, which was consistent with the results obtained from fluorescence staining. The mRNAs levels of AC (4.02 ± 0.14 vs 1.00 ± 0.13), GRK (2.63 ± 0.03 vs 1.00 ± 0.12), p38 MAPK (3.87 ± 0.07 vs 1.00 ± 0.17), PLA2 (3.31 ± 0.12 vs 1.00 ± 0.12), PLCß (2.09 ± 0.08 vs 1.00 ± 0.06) and PTK (1.85 ± 0.07 vs 1.00 ± 0.11) were up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated mRNAs including AC (2.35 ± 0.13 vs 3.87 ± 0.08), GRK (1.17 ± 0.14 vs 2.65 ± 0.12), p38 MAPK (1.48 ± 0.18 vs 4.30 ± 0.07), PLCß (1.69 ± 0.10 vs 2.41 ± 0.13) and PLA2 (1.87 ± 0.11 vs 2.96 ± 0.08) were significantly suppressed by BN52021 except for that of PTK. The level of p-AC (1.11 ± 0.12 vs 0.65 ± 0.08), GRK (0.83 ± 0.07 vs 0.50 ± 0.03), PLCß (0.83 ± 0.16 vs 0.50 ± 0.10) and p-p38 MAPK (0.74 ± 0.10 vs 0.38 ± 0.05) was up-regulated after LPS stimulation as compared with the blank control (P < 0.05). The up-regulated proteins, including p-AC (0.65 ± 0.15 vs 1.06 ± 0.14), GRK (0.47 ± 0.10 vs 0.80 ± 0.06), PLCß (0.47 ± 0.04 vs 0.80 ± 0.19) and p-p38 MAPK (0.30 ± 0.10 vs 0.97 ± 0.05), was significantly suppressed by BN52021, but p-PLA2 and p-PTK protein level were not suppressed. CONCLUSION: BN52021 could effectively inhibit LPS-induced inflammation by down-regulating the mRNA and protein levels of AC, GRK, p38 MAPK, PLA2 and PLCß in the PAFR signaling pathway.
Assuntos
Células Endoteliais/fisiologia , Fibrinolíticos/farmacologia , Ginkgolídeos/farmacologia , Inflamação/fisiopatologia , Ilhotas Pancreáticas/fisiopatologia , Lactonas/farmacologia , Fator de Ativação de Plaquetas/fisiologia , Transdução de Sinais/efeitos dos fármacos , Adenilil Ciclases/efeitos dos fármacos , Adenilil Ciclases/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Fibrinolíticos/uso terapêutico , Quinases de Receptores Acoplados a Proteína G/efeitos dos fármacos , Quinases de Receptores Acoplados a Proteína G/fisiologia , Ginkgolídeos/uso terapêutico , Inflamação/induzido quimicamente , Inflamação/prevenção & controle , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/patologia , Lactonas/uso terapêutico , Lipopolissacarídeos/efeitos adversos , Camundongos , Fosfolipase C beta/efeitos dos fármacos , Fosfolipase C beta/fisiologia , Fosfolipases A2/efeitos dos fármacos , Fosfolipases A2/fisiologia , Fator de Ativação de Plaquetas/efeitos dos fármacos , Proteínas Tirosina Quinases/efeitos dos fármacos , Proteínas Tirosina Quinases/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Mutation of tub gene in mice induces obesity, suggesting that tub could be an important regulator of energy balance. In the current study, we investigated whether insulin, leptin, and obesity can modulate Tub in vivo in hypothalamic nuclei, and we investigated possible consequences on energy balance, neuropeptide expression, and hepatic glucose metabolism. Food intake, metabolic characteristics, signaling proteins, and neuropeptide expression were measured in response to fasting and refeeding, intracerebroventricular insulin and leptin, and Tub antisense oligonucleotide (ASO). Tub tyrosine phosphorylation (Tub-p-tyr) is modulated by nutritional status. Tub is a substrate of insulin receptor tyrosine kinase (IRTK) and leptin receptor (LEPR)-Janus kinase 2 (JAK2) in hypothalamic nuclei. After leptin or insulin stimulation, Tub translocates to the nucleus. Inhibition of Tub expression in hypothalamus by ASO increased food intake, fasting blood glucose, and hepatic glucose output, decreased O(2) consumption, and blunted the effect of insulin or leptin on proopiomelanocortin, thyroid-releasing hormone, melanin-concentrating hormone, and orexin expression. In hypothalamus of mice administered a high-fat diet, there is a reduction in leptin and insulin-induced Tub-p-tyr and nuclear translocation, which is reversed by reducing protein tyrosine phosphatase 1B expression. These results indicate that Tub has a key role in the control of insulin and leptin effects on food intake, and the modulation of Tub may contribute to insulin and leptin resistance in DIO mice.
Assuntos
Hipotálamo/fisiologia , Insulina/farmacologia , Leptina/farmacologia , Proteínas/fisiologia , Transdução de Sinais/fisiologia , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal , Animais , Jejum , Janus Quinase 2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos Antissenso/farmacologia , Fosfolipase C beta/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 1/fisiologia , Proteínas/antagonistas & inibidoresRESUMO
Myelodysplastic syndromes (MDS), clonal hematopoietic stem-cell disorders mainly affecting older adult patients, show ineffective hematopoiesis in one or more of the lineages of the bone marrow. A number of MDS progresses to acute myeloid leukemia (AML) with the involvement of genetic and epigenetic mechanisms affecting PI-PLC ß1. The molecular mechanisms underlying the MDS evolution to AML are still unclear, even though it is now clear that the nuclear signaling elicited by PI-PLC ß1, Cyclin D3, and Akt plays an important role in the control of the balance between cell cycle progression and apoptosis in both normal and pathologic conditions. Moreover, a correlation between other PI-PLCs, such as PI-PLC ß3, kinases and phosphatases has been postulated in MDS pathogenesis. Here, we review the findings hinting at the role of nuclear lipid signaling pathways in MDS, which could become promising therapeutic targets.
Assuntos
Núcleo Celular/enzimologia , Síndromes Mielodisplásicas/etiologia , Fosfatidilinositóis/metabolismo , Fosfolipase C beta/fisiologia , Epigenômica , Humanos , Transdução de Sinais/fisiologiaRESUMO
Circadian clocks of most organisms are synchronized with the 24-hour solar day by the changes of light and dark. In Drosophila, both the visual photoreceptors in the compound eyes as well as the blue-light photoreceptor Cryptochrome expressed within the brain clock neurons contribute to this clock synchronization. A specialized photoreceptive structure located between the retina and the optic lobes, the Hofbauer-Buchner (H-B) eyelet, projects to the clock neurons in the brain and also participates in light synchronization. The compound eye photoreceptors and the H-B eyelet contain Rhodopsin photopigments, which activate the canonical invertebrate phototransduction cascade after being excited by light. We show here that 2 of the photopigments present in these photoreceptors, Rhodopsin 5 (Rh5) and Rhodopsin 6 (Rh6), contribute to light synchronization in a mutant (norpA(P41) ) that disrupts canonical phototransduction due to the absence of Phospholipase C-ß (PLC-ß). We reveal that norpA(P41) is a true loss-of-function allele, resulting in a truncated PLC-ß protein that lacks the catalytic domain. Light reception mediated by Rh5 and Rh6 must therefore utilize either a different (nonretinal) PLC-ß enzyme or alternative signaling mechanisms, at least in terms of clock-relevant photoreception. This novel signaling mode may distinguish Rhodopsin-mediated irradiance detection from image-forming vision in Drosophila.
Assuntos
Relógios Biológicos/fisiologia , Proteínas de Drosophila/fisiologia , Fosfolipase C beta/fisiologia , Rodopsina/fisiologia , Animais , Criptocromos/fisiologia , Drosophila melanogaster , MasculinoRESUMO
Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca ( 2+) influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP 2, or mediate the Ca ( 2+) -dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca ( 2+ ) following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.
Assuntos
Drosophila/genética , Células Fotorreceptoras de Invertebrados/citologia , Degeneração Retiniana/genética , Animais , Autofagia , Cálcio/metabolismo , Modelos Animais de Doenças , Drosophila/citologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Humanos , Modelos Biológicos , Fosfolipase C beta/genética , Fosfolipase C beta/metabolismo , Fosfolipase C beta/fisiologia , Células Fotorreceptoras de Invertebrados/metabolismo , Células Fotorreceptoras de Invertebrados/patologia , Dobramento de Proteína , Degeneração Retiniana/patologia , Rodopsina/genética , Rodopsina/metabolismo , Rodopsina/fisiologia , Transdução de SinaisRESUMO
Constitutive activation of the transcription factor Stat5 in hematopoietic stem/progenitor cells leads to various hematopoietic malignancies including myeloproliferative neoplasm (MPN). Our recent study found that phospholipase C (PLC)-ß3 is a novel tumor suppressor involved in MPN, lymphoma and other tumors. Stat5 activity is negatively regulated by the SH2 domain-containing protein phosphatase SHP-1 in a PLC-ß3-dependent manner. PLC-ß3 can form the multimolecular SPS complex together with SHP-1 and Stat5. The close physical proximity of SHP-1 and Stat5 brought about by interacting with the C-terminal segment of PLC-ß3 (PLC-ß3-CT) accelerates SHP-1-mediated dephosphorylation of Stat5. Here we identify the minimal sequences within PLC-ß3-CT required for its tumor suppressor function. Two of the three Stat5-binding noncontiguous regions, one of which also binds SHP-1, substantially inhibited in vitro proliferation of Ba/F3 cells. Surprisingly, an 11-residue Stat5-binding peptide (residues 988-998) suppressed Stat5 activity in Ba/F3 cells and in vivo proliferation and myeloid differentiation of hematopoietic stem/progenitor cells. Therefore, this study further defines PLC-ß3-CT as the Stat5- and SHP-1-binding domain by identifying minimal functional sequences of PLC-ß3 for its tumor suppressor function and implies their potential utility in the control of hematopoietic malignancies.