Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

Novel Pathways RegulatingMonocyte Inflammatory ActivitiesThe focus of the research conducted in ourlaboratory is to define the mechanismsresponsible for regulating the oxidation oflipids during the activation of human monocytesand to study the role of lipids in regulatingmonocyte chemotaxis into sites of inflammation.These events likely contribute to inordinate lipidaccumulation in atherosclerosis and may mediatetissue injury in pathologic settings.Our work has defined several essentialsteps in the potentially pathologic process ofmonocyte-mediated lipid oxidation. Amongthese, we have demonstrated a critical role for thehighly reactive oxygen radical superoxide anion.Additionally, we have identified an apparentrequirement for an enzyme in the lipoxygenasefamily, enzymes that catalyze highly specificoxidation of lipids.In the course of these studies, we haveidentified a requisite role for both calcium influxand calcium release from intracellular stores; yetcalcium is not the sole required stimulus forinducing the oxidizing events. We are presentlyinvestigating the relationship between the roles ofcalcium and other signal transduction pathways inregulating monocyte-mediated lipid oxidation, inparticular, the involvement of various isoformsof protein kinase C (PKC) and phospholipase A 2(PLA 2).In this regard, we found, through the useof pharmacologic inhibitors and antisenseoligonucleotides, that PKC activity is essential foractivated monocytes to oxidize LDL lipids. Wetherefore designed studies to identify theparticular isoenzymes of PKC that participate inthis process. Isoenzymes of the cPKC family(including PKCα, PKCβI and PKCβII) wereshown to be required, and recently we have foundthat PKCα is the isoenzyme required formonocyte-mediated LDL lipid oxidation.The phospholipase A 2family of enzymesincludes an enzyme called cytosolic PLA 2(cPLA 2),believed to function in signal transduction. Inmonocytes, the activity of this enzyme isregulated by calcium. We have also examined theparticipation of this enzyme in the signaltransduction processes required for monocyteproduction of superoxide anion and oxidation ofLDL lipids. Our studies have implicated thisenzymatic pathway as an essential one and havefurther shown that cPLA 2activity is regulated byPKCα activity, thus linking these two pathways.We are pursuing studies to determine themechanisms for enzymatic regulation of thephosphorylation and translocation of thecomponents of the enzyme complex responsiblefor producing superoxide anion.The Department of Cell BiologyIn related studies, we are monitoringlipoxygenase (LO) activity during monocyteactivation and investigating the possible involvementof each of the above enzymatic pathwayson regulating LO activity and LO expression,particularly 15-LO. We were the first todemonstrate the detection of the products of thisenzyme in human atherosclerotic tissue. We areespecially interested in understanding thecytokine-mediated induction of expression ofthis enzyme. To this end, we have recentlyidentified the receptor components and immediatesignal transducing kinases involved in themonocyte response to IL-13, a cytokine that is auniquely potent inducer of 15-LO expression.Our understanding of the regulation of expressionof this enzyme may prove important forlimiting atherogenesis.Other studies in the lab are focused onunderstanding the role of phospholipases andtheir products in regulating monocyte chemotaxisin response to MCP-1. This chemokine plays acentral role in bringing monocytes into the vesselwall in atherosclerosis, and if this process isblocked, atherosclerosis can be markedlyinhibited. We have found two lipid products ofphospholipases that are required for MCP-1-induced monocyte chemotaxis and are pursuingstudies to understand how they control monocytemovement.In summary, we are studying a variety ofregulatory pathways and defining their contributionsto modulating the activity of monocytes ininflammatory responses. Our findings will suggestnew approaches for inhibiting these processes andlimiting the progression of atherosclerosis andinflammation.THE CATHCARTLABORATORYPROJECT SCIENTISTBo Xu, Ph.D.RESEARCH ASSOCIATEKevin Carnevale, M.D.POSTDOCTORAL FELLOWAshish Bhattacharjee, Ph.D.STUDENTSRyan FiccoTsung-Fu YuTECHNICIANClaudine Horton, M.S.Martha K. Cathcart, Ph.D.Cathcart, M.K., and V.A. Folcik (2000) Lipoxygenases and atherosclerosis: protection versus pathogenesis.Free Radical Biol. Med. 28:1726-1734.Carnevale, K., and M.K. Cathcart (2001) Calcium independent phospholipase A 2is required forhuman monocyte chemotaxis to monocyte chemoattractant protein 1. J. Immunol. 167:3414-3421.Roy, B., Bhattacharjee, A., Xu, B., Ford, D., Maizel, A.L., and M.K. Cathcart. (2002) IL-13 signaltransduction in human monocytes: phosphorylation of receptor components, association with Jaks,and phosphorylation/activation of Stats. J. Leuko. Biol. 72:580-9.Bey E.A., and M.K. Cathcart. (2002) Antisense oligodeoxyribonucleotides: a better way to inhibitmonocyte superoxide anion production? Methods Enzymol. 353:421-34.Zhao X., Bey, E.A., Wientjes, F.B., and M.K. Cathcart. (2002) Cytosolic phospholipase A 2(cPLA 2)regulation of human monocyte NADPH oxidase activity. cPLA 2affects translocation but not phosphorylationof p67(phox) and p47(phox). J. Biol. Chem. 277:25385-92.Xu, B., Bhattacharjee, A., Roy, B., Xu, H.M., Anthony, D., Frank, D.A., Feldman, G.M., and M.K.Cathcart (2003) Interleukin-13 induction of 15-lipoxygenase gene expression requires p38 mitogenactivatedprotein kinase-mediated serine 727 phosphorylation of Stat1 and Stat3. Mol. Cell. Biol.23:3918-3928.Carnevale, K.A., and M.K. Cathcart (2003) Protein kinase Cb is required for human monocytechemotaxis to MCP-1. J. Biol. Chem. 278:25317-25322.69