Internalized ß2-Adrenergic Receptors Oppose PLC-Dependent Hypertrophic Signaling.

BACKGROUND: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR (ß1-adrenergic receptor) and ß2-ARs (ß2-adrenergic receptor) are the 2 major subtypes of ß-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1-ARs drives detrimental cardiac remodeling while ß2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2-ARs remain unclear. METHODS: ß2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of ß2-AR signaling mechanisms in the regulation of cardiac hypertrophy. RESULTS: Here, we show that ß2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for ß2-AR-mediated phospholipase C inhibition requires internalization of ß2-AR, activation of Gi and Gßγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. CONCLUSIONS: This reveals a mechanism for ß2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.

TR(i)P Goes On: Auxiliary TRP Channel Subunits?

Transient receptor potential (TRP) cation channels are a diverse family of channels whose members play prominent roles as cellular sensors and effectors. The important role of TRP channels (and mechanosensitive piezo channels) in the complex interaction of our senses with the environment was underlined by the award of the Nobel Prize in Physiology or Medicine to 2 pioneers in this field, David Julius and Ardem Patapoutian. There are many competent and comprehensive reviews on many aspects of the TRP channels, and there is no intention to expand on them. Rather, after an introduction to the nomenclature, the molecular architecture of native TRP channel/protein complexes in vivo will be summarized using TRP channels of the canonical transient receptor potential subfamily as an example. This molecular architecture provides the basis for the signatures of native canonical transient receptor potential currents and their control by endogenous modulators and potential drugs.

Identification of phospholipase C ß downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels.

Gαq-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) depletion. When PI(4,5)P2 depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gαq-phospholipase C ß (Gαq-PLCß) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCß activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca2+ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca2+ due to Ca2+ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P2 depletion.

Identification of phospholipase C β downstream effect on transient receptor potential canonical 1/4, transient receptor potential canonical 1/5 channels

Gα(q)-coupled receptor stimulation was implied in the activation process of transient receptor potential canonical (TRPC)1/4 and TRPC1/5 heterotetrameric channels. The inactivation occurs due to phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) depletion. When PI(4,5)P₂ depletion was induced by muscarinic stimulation or inositol polyphosphate 5-phosphatase (Inp54p), however, the inactivation by muscarinic stimulation was greater compared to that by Inp54p. The aim of this study was to investigate the complete inactivation mechanism of the heteromeric channels upon Gα(q)-phospholipase C β (Gα(q)-PLCβ) activation. We evaluated the activity of heteromeric channels with electrophysiological recording in HEK293 cells expressing TRPC channels. TRPC1/4 and TRPC1/5 heteromers undergo further inhibition in PLCβ activation and calcium/protein kinase C (PKC) signaling. Nevertheless, the key factors differ. For TRPC1/4, the inactivation process was facilitated by Ca²⁺ release from the endoplasmic reticulum, and for TRPC1/5, activation of PKC was concerned mostly. We conclude that the subsequent increase in cytoplasmic Ca²⁺ due to Ca²⁺ release from the endoplasmic reticulum and activation of PKC resulted in a second phase of channel inhibition following PI(4,5)P₂ depletion.

The effect of PLCε1 gene silencing on apoptosis and invasion of esophageal carcinoma Eca109 cells and its mechanism / 肿瘤

Objective: To investigate the effect of phospholipase C epsilon 1 (PLCε1) gene silencing on apoptosis and invasion of esophageal carcinoma Eca109 cells and its possible mechanism. Methods: The recombinant vectors targeting short hairpin RNA (shRNA) of PLCε1 (pGenesil-1-PLCε1-shRNA, pGenesil-1-PLCε1-shRNA2 and pGenesil-1-PLCε1-shRNA3) were transfected into Eca109cells by cationic liposome method and to screen out the expression vector with best interference effect. The apoptosis rate and invasive ability of Eca109 cells 48 h after transfection were determined by flow cytometry (FCM) and Transwell chamber method, respectively. After PLCε1 gene silencing, the expression levels of factorassociated suicide (Fas), Fas ligand (FasL), CD44, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) mRNAs were detected by semi-quantitative reverse transcriptionpolymerase chain reaction (RT-PCR). Results: The pGenesil-1-PLCε1-shRNA2 had the best interference effect on PLCε1 gene. The apoptosis rate of Eca109 cells 48 h after transfection with PLCε1-shRNA2 was (30.27±5.13)%, which was significantly higher than that of the cells transfected with pGenesil-1-NC-shRNA (NC group) [(22.06±4.47)%, P < 0.05]. The number of Eca109 cells across the polycarbonate membrane of Transwell chamber in the NC group and PLCε1-shRNA2 group 48 h after transfection were 82.00±2.00 and 62.67±3.06, respectively. The number of Eca109 cells across the polycarbonate membrane in PLCε1-shRNA2 group was lower than that of the NC group (P < 0.05). The expression level of Fas mRNA in Eca109 cells of PLCε1-shRNA2 group was significantly up-regulated as compared with that of the NC group (P < 0.05), while the expression levels of FasL, MMP-9 and VEGF mRNAs in PLCε1 - shRNA2 group were significantly down-regulated (all P < 0.05). There was no significant difference in CD44 mRNA expression level between the NC group and PLCε1-shRNA2 group. Conclusion: Silencing PLCε1 gene might promote the apoptosis of Eca109 cells by up-regulating the expression of Fas and down-regulating the expression of FasL, and it can also suppress the invasive ability of Eca109 cells by down-regulating the expressions of MMP-9 and VEGF.

Syringaresinol causes vasorelaxation by elevating nitric oxide production through the phosphorylation and dimerization of endothelial nitric oxide synthase

Nitric oxide (NO) produced by endothelial NO synthase (eNOS) plays an important role in vascular functions, including vasorelaxation. We here investigated the pharmacological effect of the natural product syringaresinol on vascular relaxation and eNOS-mediated NO production as well as its underlying biochemical mechanism in endothelial cells. Treatment of aortic rings from wild type, but not eNOS-/- mice, with syringaresinol induced endothelium-dependent relaxation, which was abolished by addition of the NOS inhibitor NG-monomethyl-L-arginine. Treatment of human endothelial cells and mouse aortic rings with syringaresinol increased NO production, which was correlated with eNOS phosphorylation via the activation of Akt and AMP kinase (AMPK) as well as elevation of intracellular Ca2+ levels. A phospholipase C (PLC) inhibitor blocked the increases in intracellular Ca2+ levels, AMPK-dependent eNOS phosphorylation, and NO production, but not Akt activation, in syringaresinol-treated endothelial cells. Syringaresinol-induced AMPK activation was inhibited by co-treatment with PLC inhibitor, Ca2+ chelator, calmodulin antagonist, and CaMKKbeta siRNA. This compound also increased eNOS dimerization, which was inhibited by a PLC inhibitor and a Ca2+-chelator. The chemicals that inhibit eNOS phosphorylation and dimerization attenuated vasorelaxation and cGMP production. These results suggest that syringaresinol induces vasorelaxation by enhancing NO production in endothelial cells via two distinct mechanisms, phosphatidylinositol 3-kinase/Akt- and PLC/Ca2+/CaMKKbeta-dependent eNOS phosphorylation and Ca2+-dependent eNOS dimerization.

PLCE1 over-expression inhibits migration of colon cancer SW620 cells and induces their apoptosis / 肿瘤

Objective: To investigate the effects of phospholipase C epsilon-1 (PLCE1 ) over-expression on migration, cell cycle and apoptosis of human colon cancer cells. Methods: The SW620 cells overexpressing PLCE1 were constructed through lipofection. Three groups were designed as follows: parent group (without transfection), control group (transfected with empty plasmid containing green fluorescent protein) and experimental group (transfected with pcDNA-DEST53-PLCE1 plasmid). The expression levels of PLCE1 mRNA and protein in SW620 cells were detected by real-time fluorogenic quantitative-PCR (RFQ-PCR) and Western blotting, respectively. The effect of PLCE 1 over-expression on migation ability of SW620 cells was detected by Transwell chamber assay. The cell cycle distribution and apoptosis rate of SW620 cells were detected by flow cytometry (FCM). The apoptosis was analyzed by using DNA ladder method. Results: The migration ability of SW620 colon cancer cells was inhibited by over-expression of PLCE 1. The numbers of migrated cells in the parent, control and experimental groups were 32.60±2.42, 32.20±3.25 and 8.80±1.72, respectively, and the difference among three groups was significant (P < 0.01). The over-expression of PLCE1 prolonged phase G1 and induced apoptosis. Conclusion: PLCE1 over-expression can inhibit the migration ability of colon cancer cells and induce their apoptosis. PLCE1 over-expression can reduce the malignant degree of colon cancer cells, and this gene may be a new antioncogene related to colon cancer. Copyright© 2011 by TUMOR.

Genetic diversity of phospholipase C-encoding genes among 102 clinical isolates of Mycobacterium tuberculosis / 中华传染病杂志

Objective To explore and analyze genetic mutations of phospholipase C (PLC)-encoding genes plcABCD among clinical isolates of Mycobacterium tuberculosis and to provide scientific basis for understanding virulence and pathogenesis of Mycobacterium tuberculosis. Methods A total of 102 isolates from patients with active tuberculosis were identified. Bacterial DNA was extracted. All plcABCD genes were amplified by polymerase chain reaction (PCR) and checked for deletions or mutations by agarose gel electrophoresis. Results According to presence or absence of plcABCD genes, 102 isolates were divided into 13 genotypes. Thirty-one (30.39%) isolates were plc wild genotype with all plcABCD genes; 71(69. 61%) isolates were plc mutant type with different plc gene deletions. Of 71 plc mutant isolates, 48 missed only one of four plc genes, 14 had deletions of 2 plc genes, 8 were triple mutants and 1 was quadruple mutant. There were 61 (59. 80%) isolates with plcD gene mutation, while the mutation rates of plcA, plcB and plcC genes were lower, which were 15. 69%(16/102), 9. 80%(10/102) and 16. 67%(17/ 102), respectively. Conclusion This study shows great diversity in plcABCD genes among clinical isolates of Mycobacterium tuberculosis, with the most common of plcD.

Negative feedback regulation of Wnt signaling by Gbetagamma-mediated reduction of Dishevelled

Wnt signaling is known to be important for diverse embryonic and post-natal cellular events and be regulated by the proteins Dishevelled and Axin. Although Dishevelled is activated by Wnt and involved in signal transduction, it is not clear how Dishevelled-mediated signaling is turned off. We report that guanine nucleotide binding protein beta 2 (Gnb2; Gbeta2) bound to Axin and Gbeta2 inhibited Wnt mediated reporter activity. The inhibition involved reduction of the level of Dishevelled, and the Gbeta2gamma2 mediated reduction of Dishevelled was countered by increased expression of Axin. Consistent with these effects in HEK293T cells, injection of Gbeta2gamma2 into Xenopus embryos inhibited the formation of secondary axes induced either by XWnt8 or Dishevelled, but not by beta-catenin. The DEP domain of Dishevelled is necessary for both interaction with Gbeta2gamma2 and subsequent degradation of Dishevelled via the lysosomal pathway. Signaling induced by Gbeta2gamma2 is required because a mutant of Gbeta2, Gbeta2 (W332A) with lower signaling activity, had reduced ability to downregulate the level of Dishevelled. Activation of Wnt signaling by either of two methods, increased Frizzled signaling or transient transfection of Wnt, also led to increased degradation of Dishevelled and the induced Dishevelled loss is dependent on Gbeta1 and Gbeta2. Other studies with agents that interfere with PLC action and calcium signaling suggested that loss of Dishevelled is mediated through the following pathway: Wnt/Frizzled-->Gbetagamma-->PLC-->Ca+2/PKC signaling. Together the evidence suggests a novel negative feedback mechanism in which Gbeta2gamma2 inhibits Wnt signaling by degradation of Dishevelled.