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"SUPPRESSION OF PHOSPHOLIPASE-C2 PLAYS A ROLE IN THE SYNERGISTIC SWITCH OF MACROPHAGES FROM AN INFLAMMATORY TO AN ANGIOGENIC PHENOTYPE BY TOLL-LIKE RECEPTOR-4 AND ADENOSINE A2A RECEPTOR AGONISTS"

by
Stan Grinberg
Cell Biology & Molecular Medicine

B.S. 1997, York College, CUNY
M.S. 1999, NYU Graduate School of Arts & Science

Thesis Advisor: S. Joseph Leibovich, Ph.D.
Professor
Dept. of Cell Biology & Molecular Medicine

Friday, June 26, 2009
MSB G-609, 2:00 p.m.


Abstract

Macrophages produce several cytokines and growth factors that are critical for inflammation and wound healing. Quiescent macrophages produce low levels of these factors, but respond to micro-environmental signals to regulate their expression. Macrophages can be switched from a pro-inflammatory (M1) to a pro-angiogenic (M2-like) phenotype by the synergistic action of TLR agonists with adenosine A2A receptor (A2AR) agonists. The PLC inhibitor U73122, in the presence of AR agonists, promoted macrophage VEGF expression, while simultaneously down-regulating TNF. Suppression of PLC2 but not PLC1 using siRNA increased VEGF expression in response to AR agonists. In contrast to U73122, this depletion did not suppress TNF expression. VEGF expression by PLC2-/- macrophages was also stimulated by AR agonists. Involvement of A2ARs in PLC2 Vdependent VEGF up-regulation was shown using A2AR-/- macrophages. Since PLC2 suppression by U73122 or siRNA mediates A2AR-dependent VEGF expression, the effects of LPS (endotoxin, TLR4 agonist) on expression of PLC isoforms was examined. LPS rapidly suppressed PLC1 and 2 protein and mRNA in macrophages. To determine the mechanism of this suppression, we cloned the PLC2 promoter into a reporter plasmid and transfected RAW264.7 cells. LPS did not induce luciferase expression in these cells, suggesting that PLC2 suppression by LPS is not transcriptionally regulated. This was confirmed in macrophages by nuclear run-on assay. PLC2 and 1 mRNA half-life was decreased by LPS (t1/2 = 2.5 h) compared to untreated controls (t1/2 = >12 h), indicating that LPS destabilizes these mRNAs. To investigate whether LPS down-regulates PLC1 and 2 expression in vivo, we used a mouse model of sub-lethal endotoxemia. LPS (1.4 g/g body weight i.p.) rapidly suppressed PLC1 and 2 mRNA and protein expression in spleen, liver and lungs. Finally, LPS-induced suppression of PLC1 and 2 expression both in vitro and in vivo was shown to be MyD88-dependent.
In summary, PLC2 plays an important role in the M1 to M2-like switch induced by the synergistic action of LPS with A2AR agonists. LPS suppresses PLC2 expression, mediating the up-regulation of VEGF expression by A2ARs. PLC]2 signaling may be a target for modulating macrophage phenotype and thus inflammation and angiogenesis.


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