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.
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