Blood leukocytes migrate towards the sites of inflammation (a beneficial effect) or towards compromised tissues after an ischemic attack (a deleterious effect), such as in the case of ischemia/reperfusion injury (I/RI) in the heart. In I/R, when the supply of blood is restored to the affected area, neutrophils cause damage to healthy tissue via the massive release of reactive oxygen species (ROS). We have discovered that the enzyme Phospholipase D2 (PLD2) is a chemoattractant for neutrophils. In addition to this, we present three major lines of evidence that have allowed us to propose three Specific Aims. (1) First, novel data in our lab has revealed an unexpected GEF activity that exists in the lipase PLD2. Since the target, Rac2 Rho GTPase is responsible for cell movement PLD2 might be a major GEF mediating chemotaxis. Because GEFs are not constitutively active but are kept under tight regulation, we propose in AIM 1 to investigate the regulation of PLD2 novel GEF activity. The hypothesis is that PLD2 GEF activity is regulated by tyrosine phosphorylation and by interaction with PDZ domain proteins, which lead to PX and PH domains of PLD2 and phosphatidic acid (PA) combined action to ensure a GTP/GDP exchange activity. The candidate kinases are VEGFR2 and JAK3, while the PDZ-Domain proteins are MUPP1 and Mda9/Syntenin. (2) Second, we present preliminary evidence of PLD2 as the center of a protein network during cell signaling. We propose in AIM 2 to study a new pathway through which PLD2 and 14-3-3 interact to prevent chemotaxis in ischemia reperfusion injury conditions via GDF-15. We will study the molecular interactions involving PLD2, Rac2, WASp, and the adaptors Grb2 and 14-3-3 through biochemical, genetic (Rac2-/-, WASp-/- and PLD2-/- KO mice) and fluorescence microscopy experiments. Through visualization of protein complex formation in real time by FRET, we will quantify the spatial organization of these signaling molecules during chemotaxis and in I/RI-like conditions. (3) Third, PLD inhibitors protect the heart from injury caused by ischemia/reperfusion in a Murine myocardial/reperfusion injury model, placing PLD at the center of this disease.
In AIM 3 we hypothesize that PLD from leukocytes that infiltrate an ischemia/infarct area will have a deleterious effect on the heart conducive to the exacerbation of the injury in vivo. We will test this hypothesis in wild type and PLD2- /- KO mice, as well as in osmotic pump-implanted mice with specific PLD inhibitors. We will also ascertain the PLD regulatory mechanisms of signal transduction that operate in vivo. We expect to demonstrate that depriving PLD confers myocardial protection, with therapeutic potential in conjunction with currently used thrombolytic therapy. This grant will uncover targets that can be exploited pharmacologically to diminish the harmful presence of neutrophils in inflammation-mediated heart injury. If we can avoid the untimely presence of these cells in ischemia, heart failure after myocardial infarction would then be diminished.

Public Health Relevance

Blood leukocytes migrate towards sites of inflammation (a beneficial effect) or towards compromised tissue after an ischemic attack (a deleterious effect) such as in ischemia/reperfusion injury (I/RI). In I/RI, when the supply of blood is restored to an ischemic area, neutrophil leukocytes swarm the healthy tissue and cause damage mediated by Phospholipase D (PLD) and cell chemotaxis. In this project we will use biochemical, cellular assays and animal models to identify PLD-mediated movement mechanisms. Our long-term goal is to identify new signaling pathways that can be targeted with inhibitors to ameliorate the tissue damage inflicted during I/RI using a Murine Myocardial Ischemia- Reperfusion model.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL056653-14A1
Application #
8441684
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Schwartz, Lisa
Project Start
1998-04-01
Project End
2017-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
14
Fiscal Year
2013
Total Cost
$374,564
Indirect Cost
$115,907
Name
Wright State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047814256
City
Dayton
State
OH
Country
United States
Zip Code
45435
Ganesan, Ramya; Mallets, Elizabeth; Gomez-Cambronero, Julian (2016) The transcription factors Slug (SNAI2) and Snail (SNAI1) regulate phospholipase D (PLD) promoter in opposite ways towards cancer cell invasion. Mol Oncol 10:663-76
Fite, Kristen; Gomez-Cambronero, Julian (2016) Down-regulation of MicroRNAs (MiRs) 203, 887, 3619 and 182 Prevents Vimentin-triggered, Phospholipase D (PLD)-mediated Cancer Cell Invasion. J Biol Chem 291:719-30
Fite, Kristen; Elkhadragy, Lobna; Gomez-Cambronero, Julian (2016) A Repertoire of MicroRNAs Regulates Cancer Cell Starvation by Targeting Phospholipase D in a Feedback Loop That Operates Maximally in Cancer Cells. Mol Cell Biol 36:1078-89
Mahankali, Madhu; Henkels, Karen M; Speranza, Francis et al. (2015) A non-mitotic role for Aurora kinase A as a direct activator of cell migration upon interaction with PLD, FAK and Src. J Cell Sci 128:516-26
Mahankali, Madhu; Farkaly, Terry; Bedi, Shimpi et al. (2015) Phosphatidic Acid (PA) can Displace PPARα/LXRα Binding to The EGFR Promoter Causing its Transrepression in Luminal Cancer Cells. Sci Rep 5:15379
Hatton, Nathaniel; Lintz, Erin; Mahankali, Madhu et al. (2015) Phosphatidic Acid Increases Epidermal Growth Factor Receptor Expression by Stabilizing mRNA Decay and by Inhibiting Lysosomal and Proteasomal Degradation of the Internalized Receptor. Mol Cell Biol 35:3131-44
Mahankali, Madhu; Alter, Gerald; Gomez-Cambronero, Julian (2015) Mechanism of enzymatic reaction and protein-protein interactions of PLD from a 3D structural model. Cell Signal 27:69-81
Ganesan, Ramya; Mahankali, Madhu; Alter, Gerald et al. (2015) Two sites of action for PLD2 inhibitors: The enzyme catalytic center and an allosteric, phosphoinositide biding pocket. Biochim Biophys Acta 1851:261-72
Gomez-Cambronero, Julian (2015) FASEB Science Research Conference on phospholipid cell signaling and metabolism in inflammation and cancer. FASEB J 29:5-10
Kotha, Poornima L N; Sharma, Priyanka; Kolawole, Abimbola O et al. (2015) Adenovirus entry from the apical surface of polarized epithelia is facilitated by the host innate immune response. PLoS Pathog 11:e1004696

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