The inflammatory process involving neutrophils is critical for host defense and normal healing. However, neutrophils also play a significant role in such pathologic disorders as ischemia-reperfusion injury in the heart, emphysema, asthma, and rheumatoid arthritis. When blood flow is restored after a heart attack, neutrophils can inflict collateral damage on the inflammatory/ischemic reperfusion site and surrounding healthy tissue, because they release powerful lytic enzymes and oxygen radicals. Neutrophils arrive at the target tissue by a process called chemotaxis. The molecular mechanisms leading to chemotaxis in ischemia/reperfusion injury are not well understood. Preliminary data in our laboratory have indicated for the first time that granulocytemacrophage colony-stimulating factor (GM-CSF) is a cytokine that acts as a chemoattractant for neutrophils. GM-CSF-induced chemotaxis seems to involve the activation of the enzyme ribosomal p70S6K kinase (p70S6K). Data from our lab have also indicated that another signaling molecule, mitogen-activated protein kinase (MAPK), is active during cell migration and is spatially associated to p70S6K. Finally, we have found that there is a connection between phospholipase D (PLD), chemotaxis and p70S6K activity. This grant focuses on the molecular mechanisms underlying neutrophil chemotaxis and the crosstalk between the aforementioned molecules. Our central hypothesis is that GM-CSF-induced neutrophil chemotaxis is mediated by the rapamycin-sensitive p70S6K pathway that, upregulated by MAPK and PLD, acts upon tubulin and actin for enhanced functionality.
The Specific Aims will test the following three interrelated hypotheses, by using cellular, molecular and proteomic techniques: 1-. MAPK is an upstream regulator of S6K-induced chemotaxis in GM-CSF-stimulated neutrophils. The goal is to test if an interaction between the MAPK and p70S6K pathways, involving covalent modification by phosphorylation, leads to chemotaxis. 2-. There is a molecular crosstalk between PLD and S6K in GM-CSF-stimulated neutrophil chemotaxis. The goal is to test if PLD (PLD 1/2), and at what level (upstream or downstream), is involved in the mechanism of p70S6K activation and subsequent functional chemotaxis. 3. Alternate mechanisms of rapamycin action exist for the inhibition of GM-CSF-induced chemotaxis. The goal is to test which of the following is inhibited by rapamycin: the signaling molecules TOR (target of rapamycin) and p70S6K; nascent protein translation; or the actuator molecules actin and tubulin. Given the devastating effects inflicted by neutrophils during reperfusion, it is reasonable to act on the first event: chemotaxis. This proposal will advance our knowledge of the molecular mechanisms that govern cell movement. Inhibitors can then be designed to prevent leukocyte-mediated tissue injury that occurs during ischemia/reperfusion and in similar host-damaging inflammation pathologies.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL056653-09
Application #
7188605
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Schwartz, Lisa
Project Start
1998-04-01
Project End
2008-06-30
Budget Start
2007-03-01
Budget End
2008-06-30
Support Year
9
Fiscal Year
2007
Total Cost
$340,160
Indirect Cost
Name
Wright State University
Department
Physiology
Type
Schools of Medicine
DUNS #
047814256
City
Dayton
State
OH
Country
United States
Zip Code
45435
Gomez-Cambronero, Julian; Fite, Kristen; Miller, Taylor E (2018) How miRs and mRNA deadenylases could post-transcriptionally regulate expression of tumor-promoting protein PLD. Adv Biol Regul 68:107-119
Ganesan, Ramya; Henkels, Karen M; Wrenshall, Lucile E et al. (2018) Oxidized LDL phagocytosis during foam cell formation in atherosclerotic plaques relies on a PLD2-CD36 functional interdependence. J Leukoc Biol 103:867-883
Abdulnour, Raja-Elie E; Howrylak, Judie A; Tavares, Alexander H et al. (2018) Phospholipase D isoforms differentially regulate leukocyte responses to acute lung injury. J Leukoc Biol 103:919-932
Miller, Taylor E; Gomez-Cambronero, Julian (2017) A feedback mechanism between PLD and deadenylase PARN for the shortening of eukaryotic poly(A) mRNA tails that is deregulated in cancer cells. Biol Open 6:176-186
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
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; 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
Gomez-Cambronero, Julian (2015) FASEB Science Research Conference on phospholipid cell signaling and metabolism in inflammation and cancer. FASEB J 29:5-10
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
Henkels, Karen M; Mallets, Elizabeth R; Dennis, Patrick B et al. (2015) S6K is a morphogenic protein with a mechanism involving Filamin-A phosphorylation and phosphatidic acid binding. FASEB J 29:1299-313

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