P110α and P110δ catalytic subunits of PI3 kinase regulate lysophosphatidylcholine-induced TRPC6 externalization

Lipid oxidation products, including lysophosphatidylcholine (lysoPC) inhibit endothelial cell (EC) migration in vitro and impair EC healing of arterial injuries in vivo, in part by activating phosphatidylinositol 3-kinase (PI3K), which increases the externalization of canonical transient receptor potential 6 (TRPC6) channels and the subsequent increase in intracellular calcium. Inhibition of PI3K is a potential method to decrease TRPC6 activation and restore migration, but PI3K is involved in multiple intracellular signaling pathways and has multiple downstream effectors. The goal of this study is to identify the specific p110 catalytic subunit isoforms responsible for lysoPC-induced TRPC6 externalization to identify a target for intervention while minimizing impact on alternative signaling pathways. Down-regulation of the p110α and p110δ isoforms, but not the p110β or p110γ isoforms, with small interfering RNA significantly decreased phosphatidylinositol (3,4,5)-trisphosphate production and TRPC6 externalization, and significantly improved EC migration in the presence of lysoPC. These results identify an additional role of p110α in EC and reveal for the first time a specific role of p110δ in EC, providing a foundation for subsequent in vivo studies to investigate the impact of p110 isoform inhibition on arterial healing after injury.

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Abstract 413: Inhibition of Phospholipase A2 Preserves Endothelial Cell Migration in Presence of Lipid Oxidation Products

Circulation Research ◽

10.1161/res.127.suppl_1.413 ◽

2020 ◽

Vol 127 (Suppl_1) ◽

Author(s):

Priya Putta ◽

Andrew H Smith ◽

Pinaki Chaudhuri ◽

Linda M Graham

Keyword(s):

Arachidonic Acid ◽

Endothelial Cell ◽

Transient Receptor Potential ◽

Phospholipase A ◽

Oxidation Products ◽

Endothelial Cell Migration ◽

Enzyme Activity Assay ◽

Vascular Intervention ◽

Lipid Oxidation Products

During vascular intervention oxidized low-density lipoprotein (oxLDL) and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, which inhibits endothelial cell (EC) migration, and impedes endothelium healing. We have previously shown that lysoPC activates canonical transient receptor potential 6 (TRPC6) channel that leads to a prolonged [Ca2+]i influx, causing the inhibition in EC migration. We hypothesize that lysoPC activates phospholipase A 2 (PLA 2 ) crucial for TRPC6 activation; PLA 2 acts on cellular membranes to release arachidonic acid that opens arachidonate regulated calcium channel to release the initial calcium required to trigger TRPC6, blocking PLA 2 will prevent the opening of TRPC6 channels, preserve migration and promote endothelium healing. After incubation of bovine aortic EC with ATK or Ax048 to block PLA2G4, or with BEL, FKGK11, or FKGK18 to block PLA2G6, the effect of lysoPC-induced TRPC6 externalization and EC migration was assessed. Reversible PLA2G6 pharmacological inhibitors maximally blocked lysoPC-induced TRPC6 externalization, arachidonic acid release and preserved EC migration; exemplified with biotinylation assay, arachidonate enzyme assay, and razor scrape migration assay respectively. Immunofluorescence microscopy for TRPC6 plasma membrane translocation and PLA 2 enzyme activity assay supported these findings. To further verify the specific isoforms involved in blocking TRPC6 externalization, siRNA mediated transient knockdown studies were performed with PLA2G4A, PLA2G4C, PLA2G6A, and PLA2G6B siRNAs in EA.hy926— human endothelial cell line. PLA2G6A and not PLAG6B/4A/4C downregulation completely blocked TRPC6 externalization and preserved migration comparable to control levels. These studies confirmed the role of PLA2G6A (a calcium-independent cytosolic phospholipase A 2 group VI β isoform) in blocking lysoPC-induced TRPC6 activation and preservation of EC migration. Additional studies are in progress to confirm in vivo relevance of these findings. Our results show the potential for developing PLA 2 targeted therapies to block TRPC6 activation and promote endothelium healing, thus improving the outcomes for patients undergoing cardiovascular intervention.

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Role of Hyaluronan in Human Adipogenesis: Evidence from in-Vitro and in-Vivo Studies

International Journal of Molecular Sciences ◽

10.3390/ijms20112675 ◽

2019 ◽

Vol 20 (11) ◽

pp. 2675 ◽

Cited By ~ 1

Author(s):

Nicholas Wilson ◽

Robert Steadman ◽

Ilaria Muller ◽

Mohd Draman ◽

D. Aled Rees ◽

...

Keyword(s):

Stem Cell Differentiation ◽

In Vivo Studies ◽

Peroxisome Proliferator ◽

Synthesis Inhibitor ◽

Peroxisome Proliferator Activated Receptor ◽

Inhibitory Role ◽

The Impact

Hyaluronan (HA), an extra-cellular matrix glycosaminoglycan, may play a role in mesenchymal stem cell differentiation to fat but results using murine models and cell lines are conflicting. Our previous data, illustrating decreased HA production during human adipogenesis, suggested an inhibitory role. We have investigated the role of HA in adipogenesis and fat accumulation using human primary subcutaneous preadipocyte/fibroblasts (PFs, n = 12) and subjects of varying body mass index (BMI). The impact of HA on peroxisome proliferator-activated receptor gamma (PPARγ) expression was analysed following siRNA knockdown or HA synthase (HAS)1 and HAS2 overexpression. PFs were cultured in complete or adipogenic medium (ADM) with/without 4-methylumbelliferone (4-MU = HA synthesis inhibitor). Adipogenesis was evaluated using oil red O (ORO), counting adipogenic foci, and measurement of a terminal differentiation marker. Modulating HA production by HAS2 knockdown or overexpression increased (16%, p < 0.04) or decreased (30%, p = 0.01) PPARγ transcripts respectively. The inhibition of HA by 4-MU significantly enhanced ADM-induced adipogenesis with 1.52 ± 0.18- (ORO), 4.09 ± 0.63- (foci) and 2.6 ± 0.21-(marker)-fold increases compared with the controls, also increased PPARγ protein expression (40%, (p < 0.04)). In human subjects, circulating HA correlated negatively with BMI and triglycerides (r = −0.396 (p = 0.002), r = −0.269 (p = 0.038), respectively), confirming an inhibitory role of HA in human adipogenesis. Thus, enhancing HA action may provide a therapeutic target in obesity.

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Membrane translocation of TRPC6 channels and endothelial migration are regulated by calmodulin and PI3 kinase activation

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1600371113 ◽

2016 ◽

Vol 113 (8) ◽

pp. 2110-2115 ◽

Cited By ~ 22

Author(s):

Pinaki Chaudhuri ◽

Michael A. Rosenbaum ◽

Pritam Sinharoy ◽

Derek S. Damron ◽

Lutz Birnbaumer ◽

...

Keyword(s):

Cell Membrane ◽

Transient Receptor Potential ◽

Receptor Potential ◽

Oxidation Products ◽

Site Directed Mutagenesis ◽

Trpc Channels ◽

Lipid Oxidation Products ◽

Transient Receptor

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels leading to inhibition of endothelial cell (EC) migration in vitro and delayed EC healing of arterial injuries in vivo. The precise mechanism through which lysoPC activates TRPC6 channels is not known, but calmodulin (CaM) contributes to the regulation of TRPC channels. Using site-directed mutagenesis, cDNAs were generated in which Tyr99 or Tyr138 of CaM was replaced with Phe, generating mutant CaM, Phe99-CaM, or Phe138-CaM, respectively. In ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM, but not in ECs transfected with pcDNA3.1-myc-His-Phe138-CaM, the lysoPC-induced TRPC6-CaM dissociation and TRPC6 externalization was disrupted. Also, the lysoPC-induced increase in intracellular calcium concentration was inhibited in ECs transiently transfected with pcDNA3.1-myc-His-Phe99-CaM. Blocking phosphorylation of CaM at Tyr99 also reduced CaM association with the p85 subunit and subsequent activation of phosphatidylinositol 3-kinase (PI3K). This prevented the increase in phosphatidylinositol (3,4,5)-trisphosphate (PIP3) and the translocation of TRPC6 to the cell membrane and reduced the inhibition of EC migration by lysoPC. These findings suggest that lysoPC induces CaM phosphorylation at Tyr99 by a Src family kinase and that phosphorylated CaM activates PI3K to produce PIP3, which promotes TRPC6 translocation to the cell membrane.

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Integration of TRPC6 and NADPH oxidase activation in lysophosphatidylcholine-induced TRPC5 externalization

AJP Cell Physiology ◽

10.1152/ajpcell.00028.2017 ◽

2017 ◽

Vol 313 (5) ◽

pp. C541-C555 ◽

Cited By ~ 9

Author(s):

Pinaki Chaudhuri ◽

Michael A. Rosenbaum ◽

Lutz Birnbaumer ◽

Linda M. Graham

Keyword(s):

Reactive Oxygen Species ◽

Nadph Oxidase ◽

Transient Receptor Potential ◽

Reactive Oxygen ◽

Oxidation Products ◽

Oxygen Species ◽

Subsequent Increase ◽

Erk Activation ◽

Lipid Oxidation Products ◽

Nadph Oxidase Activation

Lipid oxidation products, including lysophosphatidylcholine (lysoPC), activate canonical transient receptor potential 6 (TRPC6) channels, and the subsequent increase in intracellular Ca2+ leads to TRPC5 activation. The goal of this study is to elucidate the steps in the pathway between TRPC6 activation and TRPC5 externalization. Following TRPC6 activation by lysoPC, extracellular regulated kinase (ERK) is phosphorylated. This leads to phosphorylation of p47phox and subsequent NADPH oxidase activation with increased production of reactive oxygen species. ERK activation requires TRPC6 opening and influx of Ca2+ as evidenced by the failure of lysoPC to induce ERK phosphorylation in TRPC6−/− endothelial cells. ERK siRNA blocks the lysoPC-induced activation of NADPH oxidase, demonstrating that ERK activation is upstream of NADPH oxidase. The reactive oxygen species produced by NADPH oxidase promote myosin light chain kinase (MLCK) activation with phosphorylation of MLC and TRPC5 externalization. Downregulation of ERK, NADPH oxidase, or MLCK with the relevant siRNA prevents TRPC5 externalization. Blocking MLCK activation prevents the prolonged rise in intracellular calcium levels and preserves endothelial migration in the presence of lysoPC.

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Murine roseolovirus does not accelerate amyloid-β pathology and human roseoloviruses are not over-represented in Alzheimer disease brains

Molecular Neurodegeneration ◽

10.1186/s13024-021-00514-8 ◽

2022 ◽

Vol 17 (1) ◽

Author(s):

Tarin M. Bigley ◽

Monica Xiong ◽

Muhammad Ali ◽

Yun Chen ◽

Chao Wang ◽

...

Keyword(s):

Alzheimer Disease ◽

Rna Sequencing ◽

Amyloid Β ◽

Plaque Formation ◽

In Vivo Studies ◽

Sequencing Analysis ◽

The Impact

Abstract Background The role of viral infection in Alzheimer Disease (AD) pathogenesis is an area of great interest in recent years. Several studies have suggested an association between the human roseoloviruses, HHV-6 and HHV-7, and AD. Amyloid-β (Aβ) plaques are a hallmark neuropathological finding of AD and were recently proposed to have an antimicrobial function in response to infection. Identifying a causative and mechanistic role of human roseoloviruses in AD has been confounded by limitations in performing in vivo studies. Recent -omics based approaches have demonstrated conflicting associations between human roseoloviruses and AD. Murine roseolovirus (MRV) is a natural murine pathogen that is highly-related to the human roseoloviruses, providing an opportunity to perform well-controlled studies of the impact of roseolovirus on Aβ deposition. Methods We utilized the 5XFAD mouse model to test whether MRV induces Aβ deposition in vivo. We also evaluated viral load and neuropathogenesis of MRV infection. To evaluate Aβ interaction with MRV, we performed electron microscopy. RNA-sequencing of a cohort of AD brains compared to control was used to investigate the association between human roseolovirus and AD. Results We found that 5XFAD mice were susceptible to MRV infection and developed neuroinflammation. Moreover, we demonstrated that Aβ interacts with viral particles in vitro and, subsequent to this interaction, can disrupt infection. Despite this, neither peripheral nor brain infection with MRV increased or accelerated Aβ plaque formation. Moreover, −omics based approaches have demonstrated conflicting associations between human roseoloviruses and AD. Our RNA-sequencing analysis of a cohort of AD brains compared to controls did not show an association between roseolovirus infection and AD. Conclusion Although MRV does infect the brain and cause transient neuroinflammation, our data do not support a role for murine or human roseoloviruses in the development of Aβ plaque formation and AD.

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Aberrant Patterns of Sensory-Evoked Activity in the Olfactory Bulb of LRRK2 Knockout Mice

Cells ◽

10.3390/cells10113212 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3212

Author(s):

Andrea Maset ◽

Marco Albanesi ◽

Antonio di Soccio ◽

Martina Canova ◽

Marco dal Maschio ◽

...

Keyword(s):

Olfactory Bulb ◽

Intracellular Signaling ◽

System Level ◽

Strong Impact ◽

Lrrk2 Gene ◽

Evoked Activity ◽

Sensory Evoked ◽

The Impact

The LRRK2 gene is the major genetic determinant of familiar Parkinson’s disease (PD). Leucine-rich repeat kinase 2 (LRRK2) is a multidomain protein involved in several intracellular signaling pathways. A wealth of evidence indicates that LRRK2 is enriched at the presynaptic compartment where it regulates vesicle trafficking and neurotransmitter release. However, whether the role of LRRK2 affects neuronal networks dynamic at systems level remains unknown. Addressing this question is critical to unravel the impact of LRRK2 on brain function. Here, combining behavioral tests, electrophysiological recordings, and functional imaging, we investigated neuronal network dynamics, in vivo, in the olfactory bulb of mice carrying a null mutation in LRRK2 gene (LRRK2 knockout, LRRK2 KO, mice). We found that LRRK2 KO mice exhibit olfactory behavioral deficits. At the circuit level, the lack of LRRK2 expression results in altered gamma rhythms and odorant-evoked activity with significant impairments, while the spontaneous activity exhibited limited alterations. Overall, our data in the olfactory bulb suggest that the multifaced role of LRRK2 has a strong impact at system level when the network is engaged in active sensory processing.

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The Role of Activator Protein-1 (AP-1) Family Members in CD30-Positive Lymphomas

Cancers ◽

10.3390/cancers10040093 ◽

2018 ◽

Vol 10 (4) ◽

pp. 93 ◽

Cited By ~ 28

Author(s):

Ines Garces de los Fayos Alonso ◽

Huan-Chang Liang ◽

Suzanne Turner ◽

Sabine Lagger ◽

Olaf Merkel ◽

...

Keyword(s):

Transcriptional Response ◽

Large Cell ◽

In Vivo Studies ◽

Activator Protein 1 ◽

Activator Protein ◽

Cell Type Specific ◽

The Impact

The Activator Protein-1 (AP-1) transcription factor (TF) family, composed of a variety of members including c-JUN, c-FOS and ATF, is involved in mediating many biological processes such as proliferation, differentiation and cell death. Since their discovery, the role of AP-1 TFs in cancer development has been extensively analysed. Multiple in vitro and in vivo studies have highlighted the complexity of these TFs, mainly due to their cell-type specific homo- or hetero-dimerization resulting in diverse transcriptional response profiles. However, as a result of the increasing knowledge of the role of AP-1 TFs in disease, these TFs are being recognized as promising therapeutic targets for various malignancies. In this review, we focus on the impact of deregulated expression of AP-1 TFs in CD30-positive lymphomas including Classical Hodgkin Lymphoma and Anaplastic Large Cell Lymphoma.

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Magnesium Chloride is an Effective Therapeutic Agent for Prostate Cancer

Functional Foods in Health and Disease ◽

10.31989/ffhd.v8i1.368 ◽

2018 ◽

Vol 8 (1) ◽

pp. 62 ◽

Cited By ~ 2

Author(s):

Julianna Maria Santos ◽

Fazle Hussain

Keyword(s):

Prostate Cancer ◽

Magnesium Chloride ◽

Epithelial Mesenchymal Transition ◽

Chromatin Condensation ◽

Myosin Ii ◽

In Vivo Studies ◽

Migration Speed ◽

Mesenchymal Transition

Background: Reduced levels of magnesium can cause several diseases and increase cancer risk. Motivated by magnesium chloride’s (MgCl2) non-toxicity, physiological importance, and beneficial clinical applications, we studied its action mechanism and possible mechanical, molecular, and physiological effects in prostate cancer with different metastatic potentials.Methods: We examined the effects of MgCl2, after 24 and 48 hours, on apoptosis, cell migration, expression of epithelial mesenchymal transition (EMT) markers, and V-H+-ATPase, myosin II (NMII) and the transcription factor NF Kappa B (NFkB) expressions.Results: MgCl2 induces apoptosis, and significantly decreases migration speed in cancer cells with different metastatic potentials. MgCl2 reduces the expression of V-H+-ATPase and myosin II that facilitates invasion and metastasis, suppresses the expression of vimentin and increases expression of E-cadherin, suggesting a role of MgCl2 in reversing the EMT. MgCl2 also significantly increases the chromatin condensation and decreases NFkB expression.Conclusions: These results suggest a promising preventive and therapeutic role of MgCl2 for prostate cancer. Further studies should explore extending MgCl2 therapy to in vivo studies and other cancer types.Keywords: Magnesium chloride, prostate cancer, migration speed, V-H+-ATPase, and EMT.

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Nanocurcumin: A Double-Edged Sword for Microcancers

Current Pharmaceutical Design ◽

10.2174/1381612826666201118100045 ◽

2020 ◽

Vol 26 (45) ◽

pp. 5783-5792

Author(s):

Kholood Abid Janjua ◽

Adeeb Shehzad ◽

Raheem Shahzad ◽

Salman Ul Islam ◽

Mazhar Ul Islam

Keyword(s):

Intracellular Signaling ◽

Dosage Forms ◽

The Body ◽

In Vivo Studies ◽

Mrna Levels ◽

Anticancer Activities ◽

Road Map ◽

Anticancer Effects

There is compelling evidence that drug molecules isolated from natural sources are hindered by low systemic bioavailability, poor absorption, and rapid elimination from the human body. Novel approaches are urgently needed that could enhance the retention time as well as the efficacy of natural products in the body. Among the various adopted approaches to meet this ever-increasing demand, nanoformulations show the most fascinating way of improving the bioavailability of dietary phytochemicals through modifying their pharmacokinetics and pharmacodynamics. Curcumin, a yellowish pigment isolated from dried ground rhizomes of turmeric, exhibits tremendous pharmacological effects, including anticancer activities. Several in vitro and in vivo studies have shown that curcumin mediates anticancer effects through the modulation (upregulation and/or downregulations) of several intracellular signaling pathways both at protein and mRNA levels. Scientists have introduced multiple modern techniques and novel dosage forms for enhancing the delivery, bioavailability, and efficacy of curcumin in the treatment of various malignancies. These novel dosage forms include nanoparticles, liposomes, micelles, phospholipids, and curcumin-encapsulated polymer nanoparticles. Nanocurcumin has shown improved anticancer effects compared to conventional curcumin formulations. This review discusses the underlying molecular mechanism of various nanoformulations of curcumin for the treatment of different cancers. We hope that this study will make a road map for preclinical and clinical investigations of cancer and recommend nano curcumin as a drug of choice for cancer therapy.

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