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Hyaluronan (HA) is the major glycosaminoglycan in the extracellular matrix. During inflammation there is an increased breakdown of HA, resulting in the accumulation of low molecular weight (LMW) HA and activation of monocytes and macrophages. Eicosanoids, derived from cytosolic phospholipase A2 group IVA (cPLA2α) activation, are potent lipid mediators also attributed to acute and chronic inflammation. The aim of this study was to determine the effect of LMW HA on cPLA2α activation, arachidonic acid (AA) release and subsequent eicosanoid production, and to examine the receptors and downstream mechanisms involved in these processes in monocytes and differently polarized macrophages. LMW HA was a potent stimulant of AA release in a time and dose-dependent manner, induced cPLA2α, ERK 1/2, p38 and JNK phosphorylation, as well as activated COX2 expression and PGE2 production in primary human monocytes, murine RAW264.7 and wild-type bone-marrow derived macrophages. Specific cPLA2α inhibitor blocked HA-induced AA release and PGE2 production in all of these cells. Using CD44, TLR4, TLR2, MyD88, Rhamm or Stabilin-2 siRNA-transfected macrophages and monocytes we found that AA release, cPLA2α, ERK 1/2, p38 and JNK phosphorylation, COX2 expression and PGE2 production were activated by LMW HA through a TLR4/MyD88 pathway. Likewise, PGE2 production and COX2 expression were blocked in Tlr4-/- and MyD88-/-mice, but not in Cd44-/- mice, after LMW HA stimulation. Moreover, we demonstrated that LMW HA activated M1 macrophage phenotype with the unique cPLA2α/COX2high and COX1/ALOX15/ALOX5/LTAH4low gene and PGE2/PGD2/15-HETEhigh and LXA4low eicosanoid profile. These findings reveal a novel link between HA-mediated inflammation and lipid metabolism.
Cyclooxygenase (COX) pathway; Eicosanoid; Extracellular matrix; Hyaluronan; Inflammation; Monocytes; Prostaglandin; Toll-like receptors (TLR); cPLA2; macrophage polarization
...Murine and human COX2 Page 4. Hyaluronan activates cPLA2α and eicosanoid production 4 antibodies were obtained from Cayman (Ann Arbor, MI). Monoclonal horseradish peroxidase (HRP)-conjugated beta actin antibody was obtained from GenScript (Piscataway, NJ)....
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