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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.
Dept. of Cell Biology & Molecular Medicine
Friday, June 26, 2009
MSB G-609, 2:00 p.m.
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 PLCâĎ inhibitor U73122, in the presence of AR agonists, promoted macrophage VEGF expression, while simultaneously down-regulating TNFâĐ. Suppression of PLCâĎ2 but not PLCâĎ1 using siRNA increased VEGF expression in response to AR agonists. In contrast to U73122, this depletion did not suppress TNFâĐ expression. VEGF expression by PLCâĎ2-/- macrophages was also stimulated by AR agonists. Involvement of A2ARs in PLCâĎ2 íVdependent VEGF up-regulation was shown using A2AR-/- macrophages. Since PLCâĎ2 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 PLCâĎ1 and âĎ2 protein and mRNA in macrophages. To determine the mechanism of this suppression, we cloned the PLCâĎ2 promoter into a reporter plasmid and transfected RAW264.7 cells. LPS did not induce luciferase expression in these cells, suggesting that PLCâĎ2 suppression by LPS is not transcriptionally regulated. This was confirmed in macrophages by nuclear run-on assay. PLCâĎ2 and âĎ1 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 PLCâĎ1 and âĎ2 expression in vivo, we used a mouse model of sub-lethal endotoxemia. LPS (1.4 âŢg/g body weight i.p.) rapidly suppressed PLCâĎ1 and âĎ2 mRNA and protein expression in spleen, liver and lungs. Finally, LPS-induced suppression of PLCâĎ1 and âĎ2 expression both in vitro and in vivo was shown to be MyD88-dependent.
In summary, PLCâĎ2 plays an important role in the M1 to M2-like switch induced by the synergistic action of LPS with A2AR agonists. LPS suppresses PLCâĎ2 expression, mediating the up-regulation of VEGF expression by A2ARs. PLCú]2 signaling may be a target for modulating macrophage phenotype and thus inflammation and angiogenesis.