Publications by authors named "Michael A Frohman"

102 Publications

Biallelic loss-of-function variants in PLD1 cause congenital right-sided cardiac valve defects and neonatal cardiomyopathy.

J Clin Invest 2021 03;131(5)

Feinberg School of Medicine, Northwestern University, Chicago, Illinois, USA.

Congenital heart disease is the most common type of birth defect, accounting for one-third of all congenital anomalies. Using whole-exome sequencing of 2718 patients with congenital heart disease and a search in GeneMatcher, we identified 30 patients from 21 unrelated families of different ancestries with biallelic phospholipase D1 (PLD1) variants who presented predominantly with congenital cardiac valve defects. We also associated recessive PLD1 variants with isolated neonatal cardiomyopathy. Furthermore, we established that p.I668F is a founder variant among Ashkenazi Jews (allele frequency of ~2%) and describe the phenotypic spectrum of PLD1-associated congenital heart defects. PLD1 missense variants were overrepresented in regions of the protein critical for catalytic activity, and, correspondingly, we observed a strong reduction in enzymatic activity for most of the mutant proteins in an enzymatic assay. Finally, we demonstrate that PLD1 inhibition decreased endothelial-mesenchymal transition, an established pivotal early step in valvulogenesis. In conclusion, our study provides a more detailed understanding of disease mechanisms and phenotypic expression associated with PLD1 loss of function.
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http://dx.doi.org/10.1172/JCI142148DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919725PMC
March 2021

Structure and regulation of human phospholipase D.

Adv Biol Regul 2021 01 3;79:100783. Epub 2021 Jan 3.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA. Electronic address:

Mammalian phospholipase D (PLD) generates phosphatidic acid, a dynamic lipid secondary messenger involved with a broad spectrum of cellular functions including but not limited to metabolism, migration, and exocytosis. As a promising pharmaceutical target, the biochemical properties of PLD have been well characterized. This has led to the recent crystal structures of human PLD1 and PLD2, the development of PLD specific pharmacological inhibitors, and the identification of cellular regulators of PLD. In this review, we discuss the PLD1 and PLD2 structures, PLD inhibition by small molecules, and the regulation of PLD activity by effector proteins and lipids.
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http://dx.doi.org/10.1016/j.jbior.2020.100783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8193641PMC
January 2021

Roles for Phospholipase D1 in the Tumor Microenvironment.

Adv Exp Med Biol 2020 ;1259:77-87

Center for Developmental Genetics and the Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY, USA.

The lipid-modifying signal transduction enzyme phospholipase D (PLD) has been proposed to have roles in oncogenic processes for well-on 30 years, with most of the early literature focused on potential functions for PLD in the biology of the tumor cells themselves. While such roles remain under investigation, evidence has also now been generated to support additional roles for PLD, in particular PLD1, in the tumor microenvironment, including effects on neoangiogenesis, the supply of nutrients, interactions of platelets with circulating cancer cells, the response of the immune system, and exosome biology. Here, we review these lines of investigation, accompanied by a discussion of the limitations of the existing studies and some cautionary notes regarding the study and interpretation of PLD function using model systems.
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http://dx.doi.org/10.1007/978-3-030-43093-1_5DOI Listing
July 2020

Crystal structure of human PLD1 provides insight into activation by PI(4,5)P and RhoA.

Nat Chem Biol 2020 04 16;16(4):400-407. Epub 2020 Mar 16.

Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY, USA.

The signal transduction enzyme phospholipase D1 (PLD1) hydrolyzes phosphatidylcholine to generate the lipid second-messenger phosphatidic acid, which plays roles in disease processes such as thrombosis and cancer. PLD1 is directly and synergistically regulated by protein kinase C, Arf and Rho GTPases, and the membrane lipid phosphatidylinositol-4,5-bisphosphate (PIP). Here, we present a 1.8 Å-resolution crystal structure of the human PLD1 catalytic domain, which is characterized by a globular fold with a funnel-shaped hydrophobic cavity leading to the active site. Adjacent is a PIP-binding polybasic pocket at the membrane interface that is essential for activity. The C terminus folds into and contributes part of the catalytic pocket, which harbors a phosphohistidine that mimics an intermediate stage of the catalytic cycle. Mapping of PLD1 mutations that disrupt RhoA activation identifies the RhoA-PLD1 binding interface. This structure sheds light on PLD1 regulation by lipid and protein effectors, enabling rationale inhibitor design for this well-studied therapeutic target.
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http://dx.doi.org/10.1038/s41589-020-0499-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7117805PMC
April 2020

Prospects for PLD Inhibition in Cancer and Thrombotic Disease.

Handb Exp Pharmacol 2020 ;259:79-88

Center for Developmental Genetics and the Department of Pharmacological Sciences, Stony Brook University School of Medicine, Stony Brook, NY, USA.

Functions for phospholipase D1 and D2 (PLD1 and PLD2), the canonical isoforms of the PLD superfamily in mammals, have been explored using cell biological and animal disease models for two decades. PLD1 and PLD2, which are activated as a consequence of extracellular signaling events and generate the second messenger signaling lipid phosphatidic acid (PA), have been reported to play roles in settings ranging from platelet activation to the response to cardiac ischemia, viral infection, neurodegenerative disease, and cancer. Of these, the most tractable as therapeutic targets may be thrombotic disease and cancer, as will be discussed here in the context of ongoing efforts to develop small molecule PLD inhibitors.
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http://dx.doi.org/10.1007/164_2019_244DOI Listing
August 2020

Fast, volumetric live-cell imaging using high-resolution light-field microscopy.

Biomed Opt Express 2019 Jan 4;10(1):29-49. Epub 2018 Dec 4.

Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, Georgia, USA.

Visualizing diverse anatomical and functional traits that span many spatial scales with high spatio-temporal resolution provides insights into the fundamentals of living organisms. Light-field microscopy (LFM) has recently emerged as a scanning-free, scalable method that allows for high-speed, volumetric functional brain imaging. Given those promising applications at the tissue level, at its other extreme, this highly-scalable approach holds great potential for observing structures and dynamics in single-cell specimens. However, the challenge remains for current LFM to achieve a subcellular level, near-diffraction-limited 3D spatial resolution. Here, we report high-resolution LFM (HR-LFM) for live-cell imaging with a resolution of 300-700 nm in all three dimensions, an imaging depth of several micrometers, and a volume acquisition time of milliseconds. We demonstrate the technique by imaging various cellular dynamics and structures and tracking single particles. The method may advance LFM as a particularly useful tool for understanding biological systems at multiple spatio-temporal levels.
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http://dx.doi.org/10.1364/BOE.10.000029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6363205PMC
January 2019

Oxidized LDL phagocytosis during foam cell formation in atherosclerotic plaques relies on a PLD2-CD36 functional interdependence.

J Leukoc Biol 2018 05 14;103(5):867-883. Epub 2018 Apr 14.

Department of Biochemistry and Molecular Biology, Wright State University, Dayton, Ohio, USA.

The uptake of cholesterol carried by low-density lipoprotein (LDL) is tightly controlled in the body. Macrophages are not well suited to counteract the cellular consequences of excess cholesterol leading to their transformation into "foam cells," an early step in vascular plaque formation. We have uncovered and characterized a novel mechanism involving phospholipase D (PLD) in foam cell formation. Utilizing bone marrow-derived macrophages from genetically PLD deficient mice, we demonstrate that PLD2 (but not PLD1)-null macrophages cannot fully phagocytose aggregated oxidized LDL (Agg-Ox-LDL), which was phenocopied with a PLD2-selective inhibitor. We also report a role for PLD2 in coupling Agg-oxLDL phagocytosis with WASP, Grb2, and Actin. Further, the clearance of LDL particles is mediated by both CD36 and PLD2, via mutual dependence on each other. In the absence of PLD2, CD36 does not engage in Agg-Ox-LDL removal and when CD36 is blocked, PLD2 cannot form protein-protein heterocomplexes with WASP or Actin. These results translated into humans using a GEO database of microarray expression data from atheroma plaques versus normal adjacent carotid tissue and observed higher values for NFkB, PLD2 (but not PLD1), WASP, and Grb2 in the atheroma plaques. Human atherectomy specimens confirmed high presence of PLD2 (mRNA and protein) as well as phospho-WASP in diseased arteries. Thus, PLD2 interacts in macrophages with Actin, Grb2, and WASP during phagocytosis of Agg-Ox-LDL in the presence of CD36 during their transformation into "foam cells." Thus, this study provides new molecular targets to counteract vascular plaque formation and atherogenesis.
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http://dx.doi.org/10.1002/JLB.2A1017-407RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5951301PMC
May 2018

Proliferative and metastatic roles for Phospholipase D in mouse models of cancer.

Adv Biol Regul 2018 01 14;67:134-140. Epub 2017 Nov 14.

The Graduate Program in Molecular and Cellular Pharmacology, The Medical Scientist Training Program, and the Department of Pharmacological Sciences, Stony Brook University, New York, 11794, USA. Electronic address:

Phospholipase D (PLD) activity has been proposed to facilitate multiple steps in cancer progression including growth, metabolism, angiogenesis, and mobility. The canonical enzymes PLD1 and PLD2 enact their diverse effects through hydrolyzing the membrane lipid phosphatidylcholine to generate the second messenger and signaling lipid phosphatidic acid (PA). However, the widespread expression of PLD1 and PLD2 in normal tissues and the additional distinct enzymatic mechanisms through which PA can be generated have produced uncertainty regarding the optimal settings in which PLD inhibition might ameliorate cancer. Recent studies in mouse model systems have demonstrated that inhibition or elimination of PLD activity reduces tumor growth and metastasis. One mechanism proposed for this outcome involves proliferative signaling mediated by receptor tyrosine kinases (RTK) and G protein-coupled receptors (GPCR), which is attenuated when downstream PLD signal propagation is suppressed. The reduced proliferative signaling has been reported to be compounded by dysfunctional energetic metabolism in the tumor cells under conditions of nutrient deprivation. Moreover, cancer cells lacking PLD activity display inefficiencies across multiple steps of the metastatic cascade, limiting the tumor's lethal spread. Using PLD isoform knockout mice, recent studies have reported on the net effects of inhibition and ablation in multiple cancer models through examining the role of PLD in the non-tumor cells comprising the stroma and microenvironment. The promising results of such in vivo studies, combined with the apparent low toxicity of highly-specific and potent inhibitors, highlights PLD as an attractive target for therapeutic inhibition in cancer. We discuss here the array of anti-tumor effects produced by PLD inhibition and ablation in cancer models with a focus on animal studies.
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http://dx.doi.org/10.1016/j.jbior.2017.11.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910061PMC
January 2018

Phospholipase D2 loss results in increased blood pressure via inhibition of the endothelial nitric oxide synthase pathway.

Sci Rep 2017 08 22;7(1):9112. Epub 2017 Aug 22.

Department of Pharmacological Sciences, Stony Brook University, New York, USA.

The Phospholipase D (PLD) superfamily is linked to neurological disease, cancer, and fertility, and a recent report correlated a potential loss-of-function PLD2 polymorphism with hypotension. Surprisingly, PLD2 mice exhibit elevated blood pressure accompanied by associated changes in cardiac performance and molecular markers, but do not have findings consistent with the metabolic syndrome. Instead, expression of endothelial nitric oxide synthase (eNOS), which generates the potent vasodilator nitric oxide (NO), is decreased. An eNOS inhibitor phenocopied PLD2 loss and had no further effect on PLD2 mice, confirming the functional relationship. Using a human endothelial cell line, PLD2 loss of function was shown to lower intracellular free cholesterol, causing upregulation of HMG Co-A reductase, the rate-limiting enzyme in cholesterol synthesis. HMG Co-A reductase negatively regulates eNOS, and the PLD2-deficiency phenotype of decreased eNOS expression and activity could be rescued by cholesterol supplementation and HMG Co-A reductase inhibition. Together, these findings identify a novel pathway through which the lipid signaling enzyme PLD2 regulates blood pressure, creating implications for on-going therapeutic development of PLD small molecule inhibitors. Finally, we show that the human PLD2 polymorphism does not trigger eNOS loss, but rather creates another effect, suggesting altered functioning for the allele.
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http://dx.doi.org/10.1038/s41598-017-09852-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5567230PMC
August 2017

Coincident Phosphatidic Acid Interaction Restrains Drp1 in Mitochondrial Division.

Mol Cell 2016 09;63(6):1034-43

Department of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA. Electronic address:

Mitochondria divide to control their size, distribution, turnover, and function. Dynamin-related protein 1 (Drp1) is a critical mechanochemical GTPase that drives constriction during mitochondrial division. It is generally believed that mitochondrial division is regulated during recruitment of Drp1 to mitochondria and its oligomerization into a division apparatus. Here, we report an unforeseen mechanism that regulates mitochondrial division by coincident interactions of Drp1 with the head group and acyl chains of phospholipids. Drp1 recognizes the head group of phosphatidic acid (PA) and two saturated acyl chains of another phospholipid by penetrating into the hydrophobic core of the membrane. The dual phospholipid interactions restrain Drp1 via inhibition of oligomerization-stimulated GTP hydrolysis that promotes membrane constriction. Moreover, a PA-producing phospholipase, MitoPLD, binds Drp1, creating a PA-rich microenvironment in the vicinity of a division apparatus. Thus, PA controls the activation of Drp1 after the formation of the division apparatus.
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http://dx.doi.org/10.1016/j.molcel.2016.08.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5028122PMC
September 2016

The DNA Damage Transducer RNF8 Facilitates Cancer Chemoresistance and Progression through Twist Activation.

Mol Cell 2016 09 8;63(6):1021-33. Epub 2016 Sep 8.

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA; Stony Brook Cancer Center, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address:

Twist has been shown to cause treatment failure, cancer progression, and cancer-related death. However, strategies that directly target Twist are not yet conceivable. Here we reveal that K63-linked ubiquitination is a crucial regulatory mechanism for Twist activation. Through an E3 ligase screen and biochemical studies, we unexpectedly identified that RNF8 functions as a direct Twist activator by triggering K63-linked ubiquitination of Twist. RNF8-promoted Twist ubiquitination is required for Twist localization to the nucleus for subsequent EMT and CSC functions, thereby conferring chemoresistance. Our histological analyses showed that RNF8 expression is upregulated and correlated with disease progression, EMT features, and poor patient survival in breast cancer. Moreover, RNF8 regulates cancer cell migration and invasion and cancer metastasis, recapitulating the effect of Twist. Together, our findings reveal a previously unrecognized tumor-promoting function of RNF8 and provide evidence that targeting RNF8 is an appealing strategy to tackle tumor aggressiveness and treatment resistance.
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http://dx.doi.org/10.1016/j.molcel.2016.08.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5026628PMC
September 2016

Genetic and Stress-Induced Loss of NG2 Glia Triggers Emergence of Depressive-like Behaviors through Reduced Secretion of FGF2.

Neuron 2015 Dec 20;88(5):941-956. Epub 2015 Nov 20.

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY 11794, USA. Electronic address:

NG2-expressing glia (NG2 glia) are a uniformly distributed and mitotically active pool of cells in the central nervous system (CNS). In addition to serving as progenitors of myelinating oligodendrocytes, NG2 glia might also fulfill physiological roles in CNS homeostasis, although the mechanistic nature of such roles remains unclear. Here, we report that ablation of NG2 glia in the prefrontal cortex (PFC) of the adult brain causes deficits in excitatory glutamatergic neurotransmission and astrocytic extracellular glutamate uptake and induces depressive-like behaviors in mice. We show in parallel that chronic social stress causes NG2 glia density to decrease in areas critical to Major Depressive Disorder (MDD) pathophysiology at the time of symptom emergence in stress-susceptible mice. Finally, we demonstrate that loss of NG2 glial secretion of fibroblast growth factor 2 (FGF2) suffices to induce the same behavioral deficits. Our findings outline a pathway and role for NG2 glia in CNS homeostasis and mood disorders.
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http://dx.doi.org/10.1016/j.neuron.2015.10.046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5354631PMC
December 2015

Physiological and pathophysiological roles for phospholipase D.

J Lipid Res 2015 Dec 29;56(12):2229-37. Epub 2015 Apr 29.

Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY

Individual members of the mammalian phospholipase D (PLD) superfamily undertake roles that extend from generating the second messenger signaling lipid, phosphatidic acid, through hydrolysis of the membrane phospholipid, phosphatidylcholine, to functioning as an endonuclease to generate small RNAs and facilitating membrane vesicle trafficking through seemingly nonenzymatic mechanisms. With recent advances in genome-wide association studies, RNA interference screens, next-generation sequencing approaches, and phenotypic analyses of knockout mice, roles for PLD family members are being uncovered in autoimmune, infectious neurodegenerative, and cardiovascular disease, as well as in cancer. Some of these disease settings pose opportunities for small molecule inhibitory therapeutics, which are currently in development.
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http://dx.doi.org/10.1194/jlr.R059220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4655994PMC
December 2015

Krüppel-like factor 6 regulates mitochondrial function in the kidney.

J Clin Invest 2015 Mar 17;125(3):1347-61. Epub 2015 Feb 17.

Maintenance of mitochondrial structure and function is critical for preventing podocyte apoptosis and eventual glomerulosclerosis in the kidney; however, the transcription factors that regulate mitochondrial function in podocyte injury remain to be identified. Here, we identified Krüppel-like factor 6 (KLF6), a zinc finger domain transcription factor, as an essential regulator of mitochondrial function in podocyte apoptosis. We observed that podocyte-specific deletion of Klf6 increased the susceptibility of a resistant mouse strain to adriamycin-induced (ADR-induced) focal segmental glomerulosclerosis (FSGS). KLF6 expression was induced early in response to ADR in mice and cultured human podocytes, and prevented mitochondrial dysfunction and activation of intrinsic apoptotic pathways in these podocytes. Promoter analysis and chromatin immunoprecipitation studies revealed that putative KLF6 transcriptional binding sites are present in the promoter of the mitochondrial cytochrome c oxidase assembly gene (SCO2), which is critical for preventing cytochrome c release and activation of the intrinsic apoptotic pathway. Additionally, KLF6 expression was reduced in podocytes from HIV-1 transgenic mice as well as in renal biopsies from patients with HIV-associated nephropathy (HIVAN) and FSGS. Together, these findings indicate that KLF6-dependent regulation of the cytochrome c oxidase assembly gene is critical for maintaining mitochondrial function and preventing podocyte apoptosis.
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http://dx.doi.org/10.1172/JCI77084DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4362257PMC
March 2015

The phospholipase D superfamily as therapeutic targets.

Trends Pharmacol Sci 2015 Mar 3;36(3):137-44. Epub 2015 Feb 3.

Department of Pharmacological Sciences and the Center for Developmental Genetics, 438 Centers for Molecular Medicine, Stony Brook University, Stony Brook, NY 11794-5140, USA. Electronic address:

The phospholipase D (PLD) lipid-signaling enzyme superfamily has long been studied for its roles in cell communication and a wide range of cell biological processes. With the advent of loss-of-function genetic mouse models that have revealed that PLD1 and PLD2 ablation is overtly tolerable, small-molecule PLD1/2 inhibitors that do not cause unacceptable clinical toxicity, a PLD2 polymorphism that has been linked to altered physiology, and growing delineation of processes that are subtly altered in mice lacking PLD1/2 activity, the stage is being set for assessment of PLD1/2 inhibition for therapeutic purposes. Based on findings to date, PLD1/2 inhibition may be of more utility in acute rather than chronic settings, although this generalization will depend on the specific risks and benefits in each disease setting.
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http://dx.doi.org/10.1016/j.tips.2015.01.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355084PMC
March 2015

Role of mitochondrial lipids in guiding fission and fusion.

J Mol Med (Berl) 2015 Mar 5;93(3):263-9. Epub 2014 Dec 5.

Center for Developmental Genetics, Stony Brook University, Stony Brook, NY, 11794, USA,

Clinically important links have been established between mitochondrial function and cardiac physiology and disease in the context of signaling mechanisms, energy production, and muscle cell development. The proteins and processes that drive mitochondrial fusion and fission are now known to have emergent functions in intracellular calcium homeostasis, apoptosis, vascular smooth muscle cell proliferation, myofibril organization, and Notch-driven cell differentiation, all key issues in cardiac disease. Moreover, decreasing fission may confer protection against ischemic heart disease, particularly in the setting of obesity, diabetes, and heart failure. The importance of lipids in controlling mitochondrial fission and fusion is increasingly becoming appreciated. Roles for the bulk and signaling lipids cardiolipin, phosphatidylethanolamine, phosphatidic acid, diacylglycerol, and lysophosphatidic acid and the enzymes that synthesize or metabolize them in the control of mitochondrial shape and function are reviewed here. A number of diseases have been linked to loss-of-function alleles for a subset of the enzymes, emphasizing the importance of the lipid environment in this context.
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http://dx.doi.org/10.1007/s00109-014-1237-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4334719PMC
March 2015

Phospholipase D1 facilitates second-phase myoblast fusion and skeletal muscle regeneration.

Mol Biol Cell 2015 Feb 26;26(3):506-17. Epub 2014 Nov 26.

The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, Changchun 130021, China Division of Genetics and Genomics, Boston Children's Hospital, Boston, MA 02115

Myoblast differentiation and fusion is a well-orchestrated multistep process that is essential for skeletal muscle development and regeneration. Phospholipase D1 (PLD1) has been implicated in the initiation of myoblast differentiation in vitro. However, whether PLD1 plays additional roles in myoblast fusion and exerts a function in myogenesis in vivo remains unknown. Here we show that PLD1 expression is up-regulated in myogenic cells during muscle regeneration after cardiotoxin injury and that genetic ablation of PLD1 results in delayed myofiber regeneration. Myoblasts derived from PLD1-null mice or treated with PLD1-specific inhibitor are unable to form mature myotubes, indicating defects in second-phase myoblast fusion. Concomitantly, the PLD1 product phosphatidic acid is transiently detected on the plasma membrane of differentiating myocytes, and its production is inhibited by PLD1 knockdown. Exogenous lysophosphatidylcholine, a key membrane lipid for fusion pore formation, partially rescues fusion defect resulting from PLD1 inhibition. Thus these studies demonstrate a role for PLD1 in myoblast fusion during myogenesis in which PLD1 facilitates the fusion of mononuclear myocytes with nascent myotubes.
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http://dx.doi.org/10.1091/mbc.E14-03-0802DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4310741PMC
February 2015

Aquaporin-3 re-expression induces differentiation in a phospholipase D2-dependent manner in aquaporin-3-knockout mouse keratinocytes.

J Invest Dermatol 2015 Feb 18;135(2):499-507. Epub 2014 Sep 18.

Charlie Norwood VA Medical Center, Augusta, Georgia, USA; Department of Physiology, Georgia Regents University, Augusta, Georgia, USA; Section of Dermatology, Department of Medicine, Georgia Regents University, Augusta, Georgia, USA. Electronic address:

Aquaporin-3 (AQP3) is a water and glycerol channel expressed in epidermal keratinocytes. Despite many studies, controversy remains about the role of AQP3 in keratinocyte differentiation. Previously, our laboratory has shown co-localization of AQP3 and phospholipase D2 (PLD2) in caveolin-rich membrane microdomains. We hypothesized that AQP3 transports glycerol and "funnels" this primary alcohol to PLD2 to form a pro-differentiative signal, such that the action of AQP3 to induce differentiation should require PLD2. To test this idea, we re-expressed AQP3 in mouse keratinocytes derived from AQP3-knockout mice. The re-expression of AQP3, which increased [3H]glycerol uptake, also induced mRNA and protein expression of epidermal differentiation markers such as keratin 1, keratin 10, and loricrin, with or without the induction of differentiation by an elevated extracellular calcium concentration. Re-expression of AQP3 had no effect on the expression of the proliferation markers keratin 5 and cyclin D1. Furthermore, a selective inhibitor of PLD2, CAY10594, and a lipase-dead (LD) PLD2 mutant, but not a LD PLD1 mutant, significantly inhibited AQP3 re-expression-induced differentiation marker expression with calcium elevation, suggesting a role for PLD2 in this process. Thus, our results indicate that AQP3 has a pro-differentiative role in epidermal keratinocytes and that PLD2 activity is necessary for this effect.
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http://dx.doi.org/10.1038/jid.2014.412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7278525PMC
February 2015

Trans-regulation of oligodendrocyte myelination by neurons through small GTPase Arf6-regulated secretion of fibroblast growth factor-2.

Nat Commun 2014 Aug 21;5:4744. Epub 2014 Aug 21.

Faculty of Medicine, Department of Physiological Chemistry, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan.

The small G protein ADP-ribosylation factor 6 (Arf6) plays important roles in a wide variety of membrane dynamics-based cellular events such as neurite outgrowth and spine formation in vitro. However, little is known about physiological function of Arf6 in vivo. Here we generate conditional knockout mice lacking Arf6 in neurons, oligodendrocytes, or both cell lineages, and unexpectedly find that Arf6 expression in neurons, but not in oligodendrocytes, is crucial for oligodendrocyte myelination in the hippocampal fimbria and the corpus callosum during development, and that this is through the regulation of secretion of fibroblast growth factor-2, a guidance factor for migration of oligodendrocyte precursor cells (OPCs). These results suggest that Arf6 in neurons plays an important role in OPC migration through regulation of FGF-2 secretion during neuronal development.
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http://dx.doi.org/10.1038/ncomms5744DOI Listing
August 2014

Cellular and physiological roles for phospholipase D1 in cancer.

J Biol Chem 2014 Aug 2;289(33):22567-22574. Epub 2014 Jul 2.

Center for Developmental Genetics and the Department of Pharmacological Sciences, Stony Brook University, Stony Brook, New York 11794. Electronic address:

Phospholipase D enzymes have long been proposed to play multiple cell biological roles in cancer. With the generation of phospholipase D1 (PLD1)-deficient mice and the development of small molecule PLD-specific inhibitors, in vivo roles for PLD1 in cancer are now being defined, both in the tumor cells and in the tumor environment. We review here tools now used to explore in vivo roles for PLD1 in cancer and summarize recent findings regarding functions in angiogenesis and metastasis.
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http://dx.doi.org/10.1074/jbc.R114.576876DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4132764PMC
August 2014

Phospholipase D activity underlies very-low-density lipoprotein (VLDL)-induced aldosterone production in adrenal glomerulosa cells.

Endocrinology 2014 Sep 23;155(9):3550-60. Epub 2014 Jun 23.

Charlie Norwood VA Medical Center (V.C., W.B.B.), Augusta, Georgia 30904; Department of Physiology (Y.-Y.T., W.E.R., Z.P., V.C., W.B.B.), Medical College of Georgia at Georgia Regents University, Augusta, Georgia 30912; and Department of Pharmacology and Center for Developmental Genetics (M.A.F.), Stony Brook University, Stony Brook, New York 11794.

Aldosterone is the mineralocorticoid responsible for sodium retention, thus increased blood volume and pressure. Excessive production of aldosterone results in high blood pressure as well as renal disease, stroke, and visual loss via both direct effects and effects on blood pressure. Weight gain is often associated with increased blood pressure, but it remains unclear how obesity increases blood pressure. Obese patients typically have higher lipoprotein levels; moreover, some studies have suggested that aldosterone levels are also elevated and represent a link between obesity and hypertension. Very-low-density lipoprotein (VLDL) functions to transport triglycerides from the liver to peripheral tissues. Although previous studies have demonstrated that VLDL can stimulate aldosterone production, the mechanisms underlying this effect are largely unclear. Here we show for the first time that phospholipase D (PLD) is involved in VLDL-induced aldosterone production in both a human adrenocortical cell line (HAC15) and primary cultures of bovine zona glomerulosa cells. Our data also reveal that PLD mediates steroidogenic acute regulatory (StAR) protein and aldosterone synthase (CYP11B2) expression via increasing the phosphorylation (activation) of their regulatory transcription factors. Finally, by using selective PLD inhibitors, our studies suggest that both PLD1 and PLD2 isoforms play an important role in VLDL-induced aldosterone production.
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http://dx.doi.org/10.1210/en.2014-1159DOI Listing
September 2014

Regulation of mitochondrial morphology by lipids.

Biofactors 2014 Jul-Aug;40(4):419-24. Epub 2014 Apr 26.

Center for Developmental Genetics, Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, USA.

Although great progress has been made in identifying key protein factors that regulate mitochondrial morphology through mediating fission and fusion, signaling lipids are increasingly being recognized as important in the process as well. We review here roles that have been proposed for the signaling and bulk lipids cardiolipin, phosphatidic acid, lysophosphatidic acid, diacylglycerol, and phosphatidylethanolamine and the enzymes that generate or catabolize them in the regulation of mitochondrial morphology in yeast and mammals. Mutations in some of these enzymes are causal in a number of disease settings, highlighting the significance of controlling the lipid environment in this setting.
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http://dx.doi.org/10.1002/biof.1169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4146713PMC
May 2015

Phosphatidic acid (PA)-preferring phospholipase A1 regulates mitochondrial dynamics.

J Biol Chem 2014 Apr 5;289(16):11497-11511. Epub 2014 Mar 5.

School of Life Sciences, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan,. Electronic address:

Recent studies have suggested that phosphatidic acid (PA), a cone-shaped phospholipid that can generate negative curvature of lipid membranes, participates in mitochondrial fusion. However, precise mechanisms underling the production and consumption of PA on the mitochondrial surface are not fully understood. Phosphatidic acid-preferring phospholipase A1 (PA-PLA1)/DDHD1 is the first identified intracellular phospholipase A1 and preferentially hydrolyzes PA in vitro. Its cellular and physiological functions have not been elucidated. In this study, we show that PA-PLA1 regulates mitochondrial dynamics. PA-PLA1, when ectopically expressed in HeLa cells, induced mitochondrial fragmentation, whereas its depletion caused mitochondrial elongation. The effects of PA-PLA1 on mitochondrial morphology appear to counteract those of MitoPLD, a mitochondrion-localized phospholipase D that produces PA from cardiolipin. Consistent with high levels of expression of PA-PLA1 in testis, PA-PLA1 knock-out mice have a defect in sperm formation. In PA-PLA1-deficient sperm, the mitochondrial structure is disorganized, and an abnormal gap structure exists between the middle and principal pieces. A flagellum is bent at that position, leading to a loss of motility. Our results suggest a possible mechanism of PA regulation of the mitochondrial membrane and demonstrate an in vivo function of PA-PLA1 in the organization of mitochondria during spermiogenesis.
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http://dx.doi.org/10.1074/jbc.M113.531921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4036285PMC
April 2014

IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling.

Nat Commun 2013 ;4:2333

Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria 3052, Australia.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5'RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.
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http://dx.doi.org/10.1038/ncomms3333DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3778833PMC
April 2014

Pharmacological inhibition of phospholipase D protects mice from occlusive thrombus formation and ischemic stroke--brief report.

Arterioscler Thromb Vasc Biol 2013 Sep 18;33(9):2212-7. Epub 2013 Jul 18.

University Hospital Würzburg, Rudolf Virchow Center, DFG Research Center for Experimental Biomedicine, University of Würzburg, Würzburg, Germany.

Objective: We recently showed that mice lacking the lipid signaling enzyme phospholipase (PL) D1 or both PLD isoforms (PLD1 and PLD2) were protected from pathological thrombus formation and ischemic stroke, whereas hemostasis was not impaired in these animals. We sought to assess whether pharmacological inhibition of PLD activity affects hemostasis, thrombosis, and thrombo-inflammatory brain infarction in mice.

Approach And Results: Treatment of platelets with the reversible, small molecule PLD inhibitor, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), led to a specific blockade of PLD activity that was associated with reduced α-granule release and integrin activation. Mice that received FIPI at a dose of 3 mg/kg displayed reduced occlusive thrombus formation upon chemical injury of carotid arteries or mesenterial arterioles. Similarly, FIPI-treated mice had smaller infarct sizes and significantly better motor and neurological function 24 hours after transient middle cerebral artery occlusion. This protective effect was not associated with major intracerebral hemorrhage or prolonged tail bleeding times.

Conclusions: These results provide the first evidence that pharmacological PLD inhibition might provide a safe therapeutic strategy to prevent arterial thrombosis and ischemic stroke.
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http://dx.doi.org/10.1161/ATVBAHA.113.302030DOI Listing
September 2013

Deficiencies of the lipid-signaling enzymes phospholipase D1 and D2 alter cytoskeletal organization, macrophage phagocytosis, and cytokine-stimulated neutrophil recruitment.

PLoS One 2013 28;8(1):e55325. Epub 2013 Jan 28.

Department of Pharmacology, Stony Brook University, Stony Brook, New York, United States of America.

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0055325PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557251PMC
July 2013

A C-Terminal Transmembrane Anchor Targets the Nuage-Localized Spermatogenic Protein Gasz to the Mitochondrial Surface.

ISRN Cell Biol 2013 ;2013

Department of Pharmacology and Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5140, USA.

Mitochondria, normally tubular and distributed throughout the cell, are instead found in spermatocytes in perinuclear clusters in close association with nuage, an amorphous organelle composed of RNA and RNA-processing proteins that generate piRNAs. piRNAs are a form of RNAi required for transposon suppression and ultimately fertility. MitoPLD, another protein required for piRNA production, is anchored to the mitochondrial surface, suggesting that the nuage, also known as intermitochondrial cement, needs to be juxtaposed there to bring MitoPLD into proximity with the remainder of the piRNA-generating machinery. However, the mechanism underlying the juxtaposition is unknown. Gasz, a multidomain protein of known function found in the nuage in vertebrates, is required for piRNA production and interacts with other nuage proteins involved in this pathway. Unexpectedly, we observed that Gasz, in nonspermatogenic mammalian cells lines, localizes to mitochondria and does so through a previously unrecognized conserved C-terminal mitochondrial targeting sequence. Moreover, in this setting, Gasz is able to recruit some of the normally nuage-localized proteins to the mitochondrial surface. Taken together, these findings suggest that Gasz is a nuage-localized protein in spermatocytes that facilitates anchoring of the nuage to the mitochondrial surface where piRNA generation takes place as a collaboration between nuage and mitochondrial-surface proteins.
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http://dx.doi.org/10.1155/2013/707930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4240317PMC
January 2013

Key roles for the lipid signaling enzyme phospholipase d1 in the tumor microenvironment during tumor angiogenesis and metastasis.

Sci Signal 2012 Nov 6;5(249):ra79. Epub 2012 Nov 6.

Department of Pharmacological Sciences and Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5140, USA.

Angiogenesis inhibitors, which target tumor cells, confer only short-term benefits on tumor growth. We report that ablation of the lipid signaling enzyme phospholipase D1 (PLD1) in the tumor environment compromised the neovascularization and growth of tumors. PLD1 deficiency suppressed the activation of Akt and mitogen-activated protein kinase signaling pathways by vascular endothelial growth factor in vascular endothelial cells, resulting in decreased integrin-dependent cell adhesion to, and migration on, extracellular matrices, as well as reduced tumor angiogenesis in a xenograft model. In addition, mice lacking PLD1 incurred fewer lung metastases than did wild-type mice. Bone marrow transplantation and binding studies identified a platelet-derived mechanism involving decreased tumor cell-platelet interactions, in part because of impaired activation of αIIbβ3 integrin in platelets, which decreased the seeding of tumor cells into the lung parenchyma. Treatment with a small-molecule inhibitor of PLD1 phenocopied PLD1 deficiency, efficiently suppressing both tumor growth and metastasis in mice. These findings reveal that PLD1 in the tumor environment promotes tumor growth and metastasis and, taken together with previous reports on the roles of PLD in tumor cell-intrinsic adaptations to stress, suggest the potential use of PLD inhibitors as cancer therapeutics.
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http://dx.doi.org/10.1126/scisignal.2003257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3721670PMC
November 2012

Mitochondria: signaling with phosphatidic acid.

Int J Biochem Cell Biol 2012 Aug 15;44(8):1346-50. Epub 2012 May 15.

Molecular and Cellular Biology Graduate Program and the Department of Pharmacology & Center for Developmental Genetics, Stony Brook University, Stony Brook, NY 11794-5140, USA.

Mitochondria, once viewed as functioning relatively autonomously in the cell, have increasingly been recognized to be involved in numerous signaling networks that impact on a wide range of cell biological processes. In addition to the many types of proteins that mediate these pathways, the importance of signaling functions regulated via lipids and lipid second messengers generated on the mitochondrial surface is also becoming well appreciated. We focus here on phosphatidic acid, a lipid second messenger produced via several different pathways that can in turn stimulate the formation of multiple other bioactive lipids. Taken together, fascinating roles for phosphatidic acid and the connected lipids in mitochondrial function and interaction with other organelles are being uncovered. These pathways present new opportunities for the development of therapeutic approaches relevant to reproduction, metabolism, and neurodegenerative disease.
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http://dx.doi.org/10.1016/j.biocel.2012.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3380155PMC
August 2012
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