scholarly journals Sphingosine kinase activity is required for sphingosine-mediated phospholipase D activation in C2C12 myoblasts

2004 ◽  
Vol 381 (3) ◽  
pp. 655-663 ◽  
Author(s):  
Elisabetta MEACCI ◽  
Francesca CENCETTI ◽  
Chiara DONATI ◽  
Francesca NUTI ◽  
Laura BECCIOLINI ◽  
...  
Keyword(s):  
Phospholipase D ◽  
Mechanism Of Action ◽  
Biological Effects ◽  
Regulatory Element ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Membrane Receptors ◽  
Muscle Physiology ◽  
C2c12 Myoblasts

Sphingosine (Sph) has been implicated as a modulator of membrane signal transduction systems and as a regulatory element of cardiac and skeletal muscle physiology, but little information is presently available on its precise mechanism of action. Recent studies have shown that sphingosine 1-phosphate (S1P), generated by the action of sphingosine kinase (SphK) on Sph, also possesses biological activity, acting as an intracellular messenger, as well as an extracellular ligand for specific membrane receptors. At present, however, it is not clear whether the biological effects elicited by Sph are attributable to its conversion into S1P. In the present study, we show that Sph significantly stimulated phospholipase D (PLD) activity in mouse C2C12 myoblasts via a previously unrecognized mechanism that requires the conversion of Sph into S1P and its subsequent action as extracellular ligand. Indeed, Sph-induced activation of PLD was inhibited by N,N-dimethyl-D-erythro-sphingosine (DMS), at concentrations capable of specifically inhibiting SphK. Moreover, the crucial role of SphK-derived S1P in the activation of PLD by Sph was confirmed by the observed potentiated effect of Sph in myoblasts where SphK1 was overexpressed, and the attenuated response in cells transfected with the dominant negative form of SphK1. Notably, the measurement of S1P formation in vivo by employing labelled ATP revealed that cell-associated SphK activity in the extracellular compartment largely contributed to the transformation of Sph into S1P, with the amount of SphK released into the medium being negligible. It will be important to establish whether the mechanism of action identified in the present study is implicated in the multiple biological effects elicited by Sph in muscle cells.

scholarly journals Tyrosine Phosphorylation of Eps15 Is Required for Ligand-Regulated, but Not Constitutive, Endocytosis

2000 ◽  
Vol 150 (4) ◽  
pp. 905-912 ◽  
Author(s):  
Stefano Confalonieri ◽  
Anna Elisabetta Salcini ◽  
Claudia Puri ◽  
Carlo Tacchetti ◽  
Pier Paolo Di Fiore
Keyword(s):  
Tyrosine Phosphorylation ◽  
Phosphorylation Site ◽  
Growth Factor Receptor ◽  
Differential Regulation ◽  
Dominant Negative ◽  
Membrane Receptors ◽  
Kinase Substrate ◽  
Molecular Determinant ◽  
Molecular Machinery

Membrane receptors are internalized either constitutively or upon ligand engagement. Whereas there is evidence for differential regulation of the two processes, little is known about the molecular machinery involved. Previous studies have shown that an unidentified kinase substrate is required for endocytosis of the epidermal growth factor receptor (EGFR), the prototypical ligand-inducible receptor, but not of the transferrin receptor (TfR), the prototypical constitutively internalized receptor. Eps15, an endocytic protein that is tyrosine phosphorylated by EGFR, is a candidate for such a function. Here, we show that tyrosine phosphorylation of Eps15 is necessary for internalization of the EGFR, but not of the TfR. We mapped Tyr 850 as the major in vivo tyrosine phosphorylation site of Eps15. A phosphorylation-negative mutant of Eps15 acted as a dominant negative on the internalization of the EGFR, but not of the TfR. A phosphopeptide, corresponding to the phosphorylated sequence of Eps15, inhibited EGFR endocytosis, suggesting that phosphotyrosine in Eps15 serves as a docking site for a phosphotyrosine binding protein. Thus, tyrosine phosphorylation of Eps15 represents the first molecular determinant, other than those contained in the receptors themselves, which is involved in the differential regulation of constitutive vs. regulated endocytosis.

Overexpression of sphingosine kinase 1 is an oncogenic event in erythroleukemic progression

Blood ◽  
2005 ◽  
Vol 106 (5) ◽  
pp. 1808-1816 ◽  
Author(s):  
Erwan Le Scolan ◽  
Dimitri Pchejetski ◽  
Yoshiko Banno ◽  
Nicole Denis ◽  
Patrick Mayeux ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Molecular Mechanisms ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Recurrent Event ◽  
Dominant Negative Mutant ◽  
Apoptosis Resistance ◽  
Kinase Gene ◽  
Extracellular Signal

Abstract The erythroleukemia developed by spi-1/PU.1-transgenic mice is a model of multistage oncogenic process. Isolation of tumor cells representing discrete stages of leukemic progression enables the dissection of some of the critical events required for malignant transformation. To elucidate the molecular mechanisms of multistage leukemogenesis, we developed a microarray transcriptome analysis of nontumorigenic (HS1) and tumorigenic (HS2) proerythroblasts from spi-1-transgenic mice. The data show that transcriptional up-regulation of the sphingosine kinase gene (SPHK1) is a recurrent event associated with the tumorigenic phenotype of these transgenic proerythroblasts. SPHK1 is an enzyme of the metabolism of sphingolipids, which are essential in several biologic processes, including cell proliferation and apoptosis. HS1 erythroleukemic cells engineered to overexpress the SPHK1 protein exhibited growth proliferative advantage, increased clonogenicity, and resistance to apoptosis in reduced serum level by a mechanism involving activation of the extracellular signal-related kinases 1/2 (ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. In addition, SPHK1-overexpressing HS1 cells acquired tumorigenicity when engrafted in vivo. Finally, enforced expression of a dominant-negative mutant of SPHK1 in HS2 tumorigenic cells or treatment with a pharmacologic inhibitor reduced both cell growth and apoptosis resistance. Altogether, these data suggest that overexpression of the sphingosine kinase may represent an oncogenic event during the multistep progression of an erythroleukemia. (Blood. 2005;106:1808-1816)

scholarly journals Basal and angiopoietin-1–mediated endothelial permeability is regulated by sphingosine kinase-1

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3489-3497 ◽  
Author(s):  
Xiaochun Li ◽  
Milena Stankovic ◽  
Claudine S. Bonder ◽  
Christopher N. Hahn ◽  
Michelle Parsons ◽  
...  
Keyword(s):  
Endothelial Permeability ◽  
Sphingosine Kinase ◽  
Dominant Negative ◽  
Sphingosine Kinase 1 ◽  
Sphingosine 1 Phosphate ◽  
Functional States ◽  
Interfering Rna ◽  
Angiopoietin 1

Abstract Endothelial cells (ECs) regulate the barrier function of blood vessels. Here we show that basal and angiopoietin-1 (Ang-1)–regulated control of EC permeability is mediated by 2 different functional states of sphingosine kinase-1 (SK-1). Mice depleted of SK-1 have increased vascular leakiness, whereas mice transgenic for SK-1 in ECs show attenuation of leakiness. Furthermore, Ang-1 rapidly and transiently stimulates SK-1 activity and phosphorylation, and induces an increase in intracellular sphingosine-1-phosphate (S1P) concentration. Overexpression of SK-1 resulted in inhibition of permeability similar to that seen for Ang-1, whereas knockdown of SK-1 by small interfering RNA blocked Ang-1-mediated inhibition of permeability. Transfection with SKS225A, a nonphosphorylatable mutant of SK-1, inhibited basal leakiness, and both SKS225A and a dominant-negative SK-1 mutant removed the capacity of Ang-1 to inhibit permeability. These effects were independent of extracellular S1P as knockdown or inhibition of S1P1, S1P2, or S1P3, did not affect the Ang-1 response. Thus, SK-1 levels in ECs powerfully regulate basal permeability in vitro and in vivo. In addition, the Ang-1–induced inhibition of leakiness is mediated through activation of SK-1, defining a new signaling pathway in the Ang-1 regulation of permeability.

scholarly journals Effect ofOenothera odorataRoot Extract on Microgravity and Disuse-Induced Muscle Atrophy

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Yong-Hyeon Lee ◽  
Dong-Hyun Seo ◽  
Ji-Hyung Park ◽  
Kazuya Kabayama ◽  
Joerg Opitz ◽  
...  
Keyword(s):  
Muscle Atrophy ◽  
Biological Effects ◽  
Microgravity Environment ◽  
Root Extract ◽  
Oxygen Species ◽  
C2c12 Myoblasts ◽  
Antioxidant Function ◽  
Positive Effect

Muscle atrophy, a reduction of muscle mass, strength, and volume, results from reduced muscle use and plays a key role in various muscular diseases. In the microgravity environment of space especially, muscle atrophy is induced by muscle inactivity. Exposure to microgravity induces muscle atrophy through several biological effects, including associations with reactive oxygen species (ROS). This study used 3D-clinostat to investigate muscle atrophy caused by oxidative stressin vitro, and sciatic denervation was used to investigate muscle atrophyin vivo. We assessed the effect ofOenothera odorataroot extract (EVP) on muscle atrophy. EVP helped recover cell viability in C2C12 myoblasts exposed to microgravity for 24 h and delayed muscle atrophy in sciatic denervated mice. However, the expressions of HSP70, SOD1, and ceramide in microgravity-exposed C2C12 myoblasts and in sciatic denervated mice were either decreased or completely inhibited. These results suggested that EVP can be expected to have a positive effect on muscle atrophy by disuse and microgravity. In addition, EVP helped characterize the antioxidant function in muscle atrophy.

Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells

2001 ◽  
Vol 281 (2) ◽  
pp. L435-L449 ◽  
Author(s):  
Viswanathan Natarajan ◽  
William M. Scribner ◽  
Andrew J. Morris ◽  
Shukla Roy ◽  
Suryanarayana Vepa ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
P38 Mapk ◽  
Phospholipase D ◽  
Tyrosine Phosphatase ◽  
P38 Map Kinase ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Mapk Activity

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.

INTERACTION OF DECAVANADATE WITH INTERFACES AND BIOLOGICAL MODEL MEMBRANE SYSTEMS: CHARACTERIZATION OF SOFT OXOMETALATE SYSTEMS

2014 ◽  
Vol 02 (01) ◽  
pp. 1440007 ◽  
Author(s):  
NUTTAPORN SAMART ◽  
JESSICA SAEGER ◽  
KENNETH J. HALLER ◽  
MANUEL AURELIANO ◽  
DEBBIE C. CRANS
Keyword(s):  
Coordination Compounds ◽  
Structural Model ◽  
Noncovalent Interactions ◽  
Biological Effects ◽  
Cellular Systems ◽  
Membrane Receptors ◽  
Model Systems ◽  
Membrane Model ◽  
Coulombic Interactions

Decavanadate is a polyoxometalate consisting of 10 octahedral vanadium centers, which has been found to exert biological effects and has been observed in vivo. Biological activity implies that a material is taken up into a cell or that the material interacts with membrane receptors. Because of the large size and the high molecular charge, it is nontrivial to anticipate how such a large anion interacts with membranes and whether it will be taken up by cells. Therefore, it becomes important to investigate how the anion interacts with membranes and membrane model systems. Since ion pairing is important for the interaction of this large complex with any membrane interface system, we investigate both the nature of Coulombic and neutral noncovalent interactions with membrane model interface systems and cellular systems. Specifically, we used microemulsions as model systems, and in the specific phase diagram regime where reverse micelles form. We find that, there is a large difference in the interaction with different interfaces, and that charge can have an important role. The negatively charged interface repels the anion, whereas a positive interface attracts the anion. However, the interface with neutral surfactant head groups also is found to repel the decavanadate. This result demonstrates that the discrete charge Coulombic interactions are not the only forces in effect, and that the interactions are at least to a first approximation dictated by the interface charge and not by the counterions in the system. Alternative forces include van der Waals attraction, pH of the water pool, and field and surface effects. Because biological membranes have differently charged ligands, it is not clear which interface systems provide the best analogy with cell surfaces. However, surface charge may affect the compounds and facilitate the interactions that could be important. For example, a positively charged surface could potentially facilitate hydrolysis and sequential abstraction of one or two vanadium atoms at a time from decavanadate. Recently, decavanadate was used as a structural model for the V2O5material. Negatively charged interfaces have also been found to accelerate compound hydrolysis or in other ways alter reactions in compounds near the interface. Lipid-like interfaces potentially contribute to processing of coordination compounds. Decavanadate has been found to interact with proteins and insulin enhancing effects have been reported. Interactions with coordination compounds and the mechanisms of interactions should continue to be investigated because such systems may reveal the mode of interaction of these compounds.

Resveratrol attenuates C5a‐induced inflammatory responses in vitro and in vivo by inhibiting phospholipase D and sphingosine kinase activities

2009 ◽  
Vol 23 (8) ◽  
pp. 2412-2424 ◽  
Author(s):  
Priya D. A. Issuree ◽  
Peter N. Pushparaj ◽  
Shazib Pervaiz ◽  
Alirio J. Melendez
Keyword(s):  
Phospholipase D ◽  
Inflammatory Responses ◽  
Sphingosine Kinase

scholarly journals Regulation of SREBP1c Gene Expression in Skeletal Muscle: Role of Retinoid X Receptor/Liver X Receptor and Forkhead-O1 Transcription Factor

Endocrinology ◽  
2008 ◽  
Vol 149 (5) ◽  
pp. 2293-2305 ◽  
Author(s):  
Yasutomi Kamei ◽  
Shinji Miura ◽  
Takayoshi Suganami ◽  
Fumiko Akaike ◽  
Sayaka Kanai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Transcription Factor ◽  
Transgenic Mice ◽  
Regulatory Element ◽  
Liver X Receptor ◽  
Retinoid X Receptor ◽  
Dominant Negative

Sterol regulatory element binding protein 1c (SREBP1c) is a master regulator of lipogenic gene expression in liver and adipose tissue, where its expression is regulated by a heterodimer of nuclear receptor-type transcription factors retinoid X receptor-α (RXRα) and liver X receptor-α (LXRα). Despite the potential importance of SREBP1c in skeletal muscle, little is known about the regulation of SREBP1c in that setting. Here we report that gene expression of RXRγ is markedly decreased by fasting and is restored by refeeding in mouse skeletal muscle, in parallel with changes in gene expression of SREBP1c. RXRγ or RXRα, together with LXRα, activate the SREBP1c promoter in vitro. Moreover, transgenic mice overexpressing RXRγ specifically in skeletal muscle showed increased gene expression of SREBP1c with increased triglyceride content in their skeletal muscles. In contrast, transgenic mice overexpressing the dominant-negative form of RXRγ showed decreased SREBP1c gene expression. The expression of Forkhead-O1 transcription factor (FOXO1), which can suppress the function of multiple nuclear receptors, is negatively correlated to that of SREBP1c in skeletal muscle during nutritional change. Moreover, transgenic mice overexpressing FOXO1 specifically in skeletal muscle exhibited decreased gene expression of both RXRγ and SREBP1c. In addition, FOXO1 suppressed RXRα/LXRα-mediated SREBP1c promoter activity in vitro. These findings provide in vivo and in vitro evidence that RXR/LXR up-regulates SREBP1c gene expression and that FOXO1 antagonizes this effect of RXR/LXR in skeletal muscle.

scholarly journals miR-378a-3p Participates in Metformin’s Mechanism of Action on C2C12 Cells under Hyperglycemia

2021 ◽  
Vol 22 (2) ◽  
pp. 541
Author(s):  
Ivo F. Machado ◽  
João S. Teodoro ◽  
Ana C. Castela ◽  
Carlos M. Palmeira ◽  
Anabela P. Rolo
Keyword(s):  
Mechanism Of Action ◽  
Metabolic Diseases ◽  
Biological Effects ◽  
C2c12 Cells ◽  
Alternative Mechanism ◽  
C2c12 Myoblasts ◽  
Atp Production ◽  
Selective Degradation ◽  
Autophagic Process

Metformin is the most used biguanide drug for the treatment of type 2 diabetes mellitus. Despite being mostly known for its hepatic anti-gluconeogenic effect, it is also known to modulate microRNAs (miRNAs, miRs) associated with metabolic diseases. The latter mechanism could be relevant for better understanding metformin’s mechanisms underlying its biological effects. In the current work, we found that metformin increases miR-378a-3p expression (p < 0.002) in C2C12 myoblasts previously exposed to hyperglycemic conditions. While the inhibition of miR-378a-3p was shown to impair metformin’s effect in ATP production, PEPCK activity and the expression of Tfam. Finally, mitophagy, an autophagic process responsible for the selective degradation of mitochondria, was found to be induced by miR-378a-3p (p < 0.04). miR-378a-3p stimulated mitophagy through a process independent of sestrin-2 (SESN2), a stress-responsible protein that has been recently demonstrated to positively modulate mitophagy. Our findings provide novel insights into an alternative mechanism of action of metformin involving miR-378a-3, which can be used in the future for the development of improved therapeutic strategies against metabolic diseases.

scholarly journals Exploring the Mechanism of Action of Herbal Medicine (Gan-Mai-Da-Zao Decoction) for Poststroke Depression Based on Network Pharmacology and Molecular Docking

2021 ◽  
Vol 2021 ◽  
pp. 1-14
Author(s):  
Zhicong Ding ◽  
Fangfang Xu ◽  
Qidi Sun ◽  
Bin Li ◽  
Nengxing Liang ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Mechanism Of Action ◽  
Biological Effects ◽  
Network Pharmacology ◽  
Integrated Analysis ◽  
Poststroke Depression ◽  
Active Ingredients ◽  
Active Components ◽  
Composite Target

Background. Poststroke depression (PSD) is the most common and serious neuropsychiatric complication occurring after cerebrovascular accidents, seriously endangering human health while also imposing a heavy burden on society. Nevertheless, it is difficult to control disease progression. Gan-Mai-Da-Zao Decoction (GMDZD) is effective for PSD, but its mechanism of action in PSD is unknown. In this study, we explored the mechanism of action of GMDZD in PSD treatment using network pharmacology and molecular docking. Material and methods. We obtained the active components of all drugs and their targets from the public database TCMSP and published articles. Then, we collected PSD-related targets from the GeneCards and OMIM databases. Cytoscape 3.8.2 was applied to construct PPI and composite target disease networks. In parallel, the DAVID database was used to perform GO and KEGG enrichment analyses to determine the biological processes enriched in the treatment-related drugs in vivo. Finally, molecular docking was used to verify the association between the main active ingredients and their targets. Results. The network pharmacological analysis of GMDZD in PSD revealed 107 active ingredients with important biological effects, including quercetin, luteolin, kaempferol, naringenin, and isorhamnetin. In total, 203 potential targets for the treatment of this disease were screened, including STAT3, JUN, TNF, TPT53, AKT1, and EGFR. These drugs are widely enriched in a series of signaling pathways, such as TNF, HIF-1, and toll-like receptor. Moreover, molecular docking analysis showed that the core active components were tightly bound to their core targets, further confirming their anti-PSD effects. Conclusion. This prospective study was based on the integrated analysis of large data using network pharmacology technology to explore the feasibility of GMDZD for PSD treatment that was successfully validated by molecular docking. It reflects the multicomponent and multitarget characteristics of Chinese medicine and, more importantly, brings hope for the clinical treatment of PSD.

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