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• W2006223811 abstract "Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B′, a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B′ protein levels accompanied by a concomitant increase in HSP70B′ extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B′, which co-localized with cell-associated oxLDL-IC. In HSP70B′-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B′ co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B′ is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B′ and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B′ resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B′, and once stimulated, HSP70B′ binds to the cell-associated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B′ in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival. Heat shock proteins (HSPs) have been implicated in the activation and survival of macrophages. This study examined the role of HSP70B′, a poorly characterized member of the HSP70 family, in response to oxidatively modified LDL (oxLDL) and immune complexes prepared with human oxLDL and purified human antibodies to oxLDL (oxLDL-IC) in monocytic and macrophage cell lines. Immunoblot analysis of cell lysates and conditioned medium from U937 cells treated with oxLDL alone revealed an increase in intracellular HSP70B′ protein levels accompanied by a concomitant increase in HSP70B′ extracellular levels. Fluorescence immunohistochemistry and confocal microscopy, however, demonstrated that oxLDL-IC stimulated the release of HSP70B′, which co-localized with cell-associated oxLDL-IC. In HSP70B′-green fluorescent protein-transfected mouse RAW 264.7 cells, oxLDL-IC-induced HSP70B′ co-localized with membrane-associated oxLDL-IC as well as the lipid moiety of internalized oxLDL-IC. Furthermore, the data demonstrated that HSP70B′ is involved in cell survival, and this effect could be mediated by sphingosine kinase 1 (SK1) activation. An examination of regularly implicated cytokines revealed a significant relationship between HSP70B′ and the release of the anti-inflammatory cytokine interleukin-10 (IL-10). Small interfering RNA knockdown of HSP70B′ resulted in a corresponding decrease in SK1 mRNA levels and SK1 phosphorylation as well as increased release of IL-10. In conclusion, these findings suggest that oxLDL-IC induce the synthesis and release of HSP70B′, and once stimulated, HSP70B′ binds to the cell-associated and internalized lipid moiety of oxLDL-IC. The data also implicate HSP70B′ in key cellular functions, such as regulation of SK1 activity and release of IL-10, which influence macrophage activation and survival. IntroductionHeat shock proteins (HSPs) 2The abbreviations used are: HSPheat shock proteinLDLlow density lipoprotein(s)oxLDLoxidized low density lipoprotein(s)oxLDL-ICoxidized low density lipoprotein immune complexesSK1sphingosine kinase 1ILinterleukinTNF-αtumor necrosis factor αIFN-γinterferon-γKLH-ICkeyhole limpet hemocyanin immune complexesDiIdioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchloratePBSphosphate-buffered salineQ-PCRquantitative PCRGFPgreen fluorescent proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolsiRNAsmall interfering RNA. belong to a group of more than 20 highly conserved stress proteins that are routinely employed by cells as cytoprotective agents against a variety of stress stimuli, including heat shock, oxidative and mechanical stress, and inflammation (1Morimoto R.I. Science. 1993; 259: 1409-1410Crossref PubMed Scopus (1212) Google Scholar, 2Xu Q. Wick G. Mol. Med. Today. 1996; 2: 372-379Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Evidence exists that HSP expression is higher at sites of atherosclerotic lesions than it is in normal tissue (3Pockley A.G. Circulation. 2002; 105: 1012-1017Crossref PubMed Scopus (216) Google Scholar, 4Roma P. Catapano A.L. Atherosclerosis. 1996; 127: 147-154Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Elevated HSP70 levels are associated with atherosclerotic plaques, particularly in areas with abundant activated macrophages, and seem to coincide around sites of necrosis and lipid accumulation (5Johnson A.D. Berberian P.A. Tytell M. Bond M.G. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 27-36Crossref PubMed Scopus (84) Google Scholar, 6Berberian P.A. Myers W. Tytell M. Challa V. Bond M.G. Am. J. Pathol. 1990; 136: 71-80PubMed Google Scholar).Of particular interest in the development of atherogenesis is the role of oxidatively modified LDL (oxLDL). It is established that oxLDL particles are taken up by activated macrophages, resulting in lipid accumulation (7Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1450) Google Scholar). oxLDL also triggers an immune response, initiating the production of predominantly proinflammatory IgG antibodies, which then form circulating complexes with oxLDL (oxLDL-IC) (8Virella G. Koskinen S. Krings G. Onorato J.M. Thorpe S.R. Lopes-Virella M. Clin. Immunol. 2000; 95: 135-144Crossref PubMed Scopus (55) Google Scholar, 9Virella G. Lopes-Virella M.F. Clin. Diagn. Lab. Immunol. 2003; 10: 499-505Crossref PubMed Scopus (56) Google Scholar). These immune complexes activate macrophages through the FcγRI receptor, resulting in the release of proinflammatory cytokines (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) and the associated acceleration of foam cell formation (10Virella G. Muñoz J.F. Galbraith G.M. Gissinger C. Chassereau C. Lopes-Virella M.F. Clin. Immunol. Immunopathol. 1995; 75: 179-189Crossref PubMed Scopus (57) Google Scholar, 11Griffith R.L. Virella G.T. Stevenson H.C. Lopes-Virella M.F. J. Exp. Med. 1988; 168: 1041-1059Crossref PubMed Scopus (145) Google Scholar, 12Huang Y. Jaffa A. Koskinen S. Takei A. Lopes-Virella M.F. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1600-1607Crossref PubMed Scopus (59) Google Scholar, 13Saad A.F. Virella G. Chassereau C. Boackle R.J. Lopes-Virella M.F. J. Lipid Res. 2006; 47: 1975-1983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Whereas free oxLDL have been shown to be cytotoxic to monocytic cells (14Asmis R. Begley J.G. Circ. Res. 2003; 92: e20-e29Crossref PubMed Google Scholar, 15Martinet W. Kockx M.M. Curr. Opin. Lipidol. 2001; 12: 535-541Crossref PubMed Scopus (110) Google Scholar, 16Vicca S. Massy Z.A. Hennequin C. Rihane D. Drüeke T.B. Lacour B. Free Radic. Biol. Med. 2003; 35: 603-615Crossref PubMed Scopus (26) Google Scholar), oxLDL complexed to IgG was found to promote survival (17Bianchi G. Montecucco F. Bertolotto M. Dallegri F. Ottonello L. Ann. N.Y. Acad. Sci. 2007; 1095: 209-219Crossref PubMed Scopus (10) Google Scholar, 18Hammad S.M. Taha T.A. Nareika A. Johnson K.R. Lopes-Virella M.F. Obeid L.M. Prostaglandins Other Lipid Mediat. 2006; 79: 126-140Crossref PubMed Scopus (41) Google Scholar, 19Oksjoki R. Kovanen P.T. Lindstedt K.A. Jansson B. Pentikäinen M.O. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 576-583Crossref PubMed Scopus (38) Google Scholar).Studies have demonstrated that circulating HSP60 is linked to cardiovascular disease (20Mehta T.A. Greenman J. Ettelaie C. Venkatasubramaniam A. Chetter I.C. McCollum P.T. Eur. J. Vasc. Endovasc. Surg. 2005; 29: 395-402Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, the role HSP70 family members play in the development of atherosclerotic plaques is still unclear. Increasing evidence suggests that HSPs may serve as cytokines themselves. Asea et al. (21Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar) determined that activated macrophages secrete HSP70, which then binds to CD14 on the outer membrane, triggering the production and subsequent release of proinflammatory cytokines. This finding supports earlier experiments that induced cytokine production with the addition of exogenous HSP70 (22Asea A. Kraeft S.K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1354) Google Scholar, 23Multhoff G. Mizzen L. Winchester C.C. Milner C.M. Wenk S. Eissner G. Kampinga H.H. Laumbacher B. Johnson J. Exp. Hematol. 1999; 27: 1627-1636Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar).Elevated oxLDL, a hallmark of increased risk of atherosclerosis, has been implicated as the initial factor in the HSP70-linked proinflammatory pathway of activated macrophages. Svensson et al. (24Svensson P.A. Asea A. Englund M.C. Bausero M.A. Jernås M. Wiklund O. Ohlsson B.G. Carlsson L.M. Carlsson B. Atherosclerosis. 2006; 185: 32-38Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) demonstrated that high levels of oxLDL directly up-regulate and initiate release of HSP70 in macrophages, resulting in a corresponding increase in cytokine (IL-1β and IL-12) production. Whether exposure of human macrophages to elevated levels of oxLDL-IC elicits HSP70 regulation has not been previously examined.We have recently shown that one member of the HSP70 family, HSP70B′ (also known as HSP70 protein 6; gene HSPA6), displayed a considerable increase in expression in response to oxLDL-IC but not oxLDL alone (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Furthermore, evidence was provided that IL-1β secretion is HSP70B′-dependent, suggesting a novel, oxLDL-IC-dependent proinflammatory mechanism involving the little known HSP70B′ species (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The HSP70B′ gene is unique to the human genome, probably arising after the divergence of rodents and humans (26Parsian A.J. Sheren J.E. Tao T.Y. Goswami P.C. Malyapa R. Van Rheeden R. Watson M.S. Hunt C.R. Biochim. Biophys. Acta. 2000; 1494: 201-205Crossref PubMed Google Scholar), and although HSP70B′ and HSP70 are over 80% homologous, differences are present in sequences coding for substrate-binding and activation sites, evidence of a unique cellular function (27Noonan E.J. Place R.F. Giardina C. Hightower L.E. Cell Stress Chaperones. 2007; 12: 393-402Crossref PubMed Scopus (41) Google Scholar). Until recently, sequence data and immunological reagents for HSP70B′ were unavailable, and due to the sequence homology, standard antibodies designed for the common HSP70A/B members most likely included the detection of HSP70B′. HSP70, while functionally inducible, is fairly ubiquitous at basal conditions in many different cell types, whereas HSP70B′ is strictly inducible (27Noonan E.J. Place R.F. Giardina C. Hightower L.E. Cell Stress Chaperones. 2007; 12: 393-402Crossref PubMed Scopus (41) Google Scholar, 28Leung T.K. Rajendran M.Y. Monfries C. Hall C. Lim L. Biochem. J. 1990; 267: 125-132Crossref PubMed Scopus (152) Google Scholar, 29Noonan E. Giardina C. Hightower L. Exp. Cell Res. 2008; 314: 2468-2476Crossref PubMed Scopus (32) Google Scholar). Our recent findings have led to the hypothesis that HSP70B′ expression could be induced by Fcγ-RI activation in human macrophages, resulting in increased proinflammatory cytokine release, prolonged foam cell survival, and thus the associated increased risk of atherosclerotic plaque development (18Hammad S.M. Taha T.A. Nareika A. Johnson K.R. Lopes-Virella M.F. Obeid L.M. Prostaglandins Other Lipid Mediat. 2006; 79: 126-140Crossref PubMed Scopus (41) Google Scholar, 25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Herein, we show that HSP70B′ was induced and released in response to oxLDL-IC and that HSP70B′ could bind to the cell-associated and internalized lipid moiety of oxLDL-IC. The presented data also implicated HSP70B′ in cellular functions that underlie the ability of oxLDL-IC to promote prolonged activation of foam cells, such as regulation of sphingosine kinase 1 (SK1) activity and release of IL-10.DISCUSSIONIn this study, we examined the role of HSP70B′, a poorly characterized member of the heat shock protein 70 family, in foam cell activation and survival induced by oxLDL-IC. HSP70B′ appears to play a unique and complex role in macrophage activation associated with oxLDL-IC signaling. The results showed for the first time that human monocytic cells respond to oxLDL-IC by stimulating and secreting HSP70B′. The results also showed that HSP70B′ appears to become associated with the intracellular as well as membrane-associated oxLDL-IC lipid moiety. Furthermore, the data demonstrated that HSP70B′ is involved in cell survival, and this effect could be mediated by SK1 activation. The regulation of the anti-inflammatory cytokine IL-10 by HSP70B′ provides further evidence of the involvement of the little known member of the HSP70 family, HSP70B′, in foam cell functionality. Investigating the significance of HSPs, not only as classical chaperones and stress proteins, but as complex signaling molecules is of increasing importance in understanding the mechanisms involved in the activity of foam cells, a hallmark of atherosclerotic plaques.In a recent study, we identified 83 genes as being similarly regulated by oxLDL-IC in the human leukemic monocyte lymphoma U937 cell line (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Among the up-regulated genes, HSP70B′ showed the highest increase in expression. Despite the substantial increase of HSP70B′ at the gene level in response to oxLDL-IC, the fate of the protein could not be determined using immunoblot analysis (Fig. 1). The lack of HSP70B′ protein suggested aborted translation, improper folding, and/or complexing to an undetermined molecule(s) concealing the antibody binding sites. Due to cross-linking of the Fcγ receptors with immune complexes (oxLDL-IC and KLH-IC), we believe that the HSP70B′ protein and possibly other stimulated proteins, such as SK1 (18Hammad S.M. Taha T.A. Nareika A. Johnson K.R. Lopes-Virella M.F. Obeid L.M. Prostaglandins Other Lipid Mediat. 2006; 79: 126-140Crossref PubMed Scopus (41) Google Scholar, 37Hengst J.A. Guilford J.M. Fox T.E. Wang X. Conroy E.J. Yun J.K. Arch. Biochem. Biophys. 2009; 492: 62-73Crossref PubMed Scopus (41) Google Scholar), become part of lipid rafts and/or the detergent-resistant membrane fraction. To extract the cell membrane-associated HSP70B′, which is demonstrated clearly in the confocal microscopy images, we used several different detergents and extraction buffers (38Shogomori H. Brown D.A. Biol. Chem. 2003; 384: 1259-1263Crossref PubMed Scopus (170) Google Scholar) (data not shown). These did not improve the extractability of HSP70B′, from cells induced by immune complexes beyond what is shown in Fig. 1D. Interestingly, we were able to detect more HSP70B′ protein in response to KLH-IC than oxLDL-IC (Fig. 1D), which reflects appropriately the higher gene expression of HSP70B′ in response to KLH-IC compared with oxLDL-IC (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Apparently, the protein expression of HSP70B′ in response to oxLDL alone is mainly post-translational because it reached a plateau at 6 h post-treatment.HSP70 has been shown to form complexes with a number of intracellular proteins (39Davidoff A.M. Iglehart J.D. Marks J.R. Proc. Natl. Acad. Sci. U.S.A. 1992; 89: 3439-3442Crossref PubMed Scopus (312) Google Scholar), and more recently HSPs have been shown to bind to intracellular lipids (29Noonan E. Giardina C. Hightower L. Exp. Cell Res. 2008; 314: 2468-2476Crossref PubMed Scopus (32) Google Scholar, 40Osterloh A. Kalinke U. Weiss S. Fleischer B. Breloer M. J. Biol. Chem. 2007; 282: 4669-4680Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 41Tsan M.F. Gao B. Cell. Mol. Immunol. 2004; 1: 274-279PubMed Google Scholar). Our data suggest that oxLDL-IC on the surface of the cells become associated with secreted HSP70B′ (FIGURE 2, FIGURE 3). This association may inhibit the presumed autocrine effect of HSP70B′ on cell signaling.The differential trafficking of oxLDL and oxLDL-IC probably plays a role in the action of HSP70B′. Differences in trafficking could be due to differences in receptor binding, uptake, and delivery to lysosomes and/or to lysosomal and post-lysosomal processing. Several studies have previously shown that the lipid and protein moieties are metabolized in lysosomes within hours after internalization of oxLDL (42Li W. Yuan X.M. Brunk U.T. Free Radic. Res. 1998; 29: 389-398Crossref PubMed Scopus (57) Google Scholar, 43Mander E.L. Dean R.T. Stanley K.K. Jessup W. Biochim. Biophys. Acta. 1994; 1212: 80-92Crossref PubMed Scopus (51) Google Scholar, 44Yancey P.G. Jerome W.G. J. Lipid Res. 1998; 39: 1349-1361Abstract Full Text Full Text PDF PubMed Google Scholar, 45Brown A.J. Mander E.L. Gelissen I.C. Kritharides L. Dean R.T. Jessup W. J. Lipid Res. 2000; 41: 226-237Abstract Full Text Full Text PDF PubMed Google Scholar). Live cell imaging shown in Fig. 3A demonstrated internalization of oxLDL-IC and co-localization with HSP70B′-GFP at 4 h post-treatment. This novel finding suggests that induced HSP70B′ could bind to cytoplasmic oxLDL-IC but not to oxLDL in the lysosomal compartment.There have been several reports implicating HSPs in survival of immune cells (46Kusher D.I. Ware C.F. Gooding L.R. J. Immunol. 1990; 145: 2925-2931PubMed Google Scholar, 47Williams R.S. Thomas J.A. Fina M. German Z. Benjamin I.J. J. Clin. Investig. 1993; 92: 503-508Crossref PubMed Scopus (152) Google Scholar, 48Hirvonen M.R. Brüne B. Lapetina E.G. Biochem. J. 1996; 315: 845-849Crossref PubMed Scopus (61) Google Scholar), mostly suggesting a protective function against inflammation and other types of stressors. Our data suggest that inducible HSP70B′ may have an inhibitory effect on macrophage cell growth and survival. For example, cells overexpressing HSP70B′ showed significantly decreased cell proliferation and knockdown of HSP70B′-attenuated oxLDL-induced cell death compared with controls.A mechanism regularly implicated in mediating prosurvival and inflammatory responses to macrophage cell survival is regulation of SK1, an enzyme that is responsible for the generation of the signaling molecule sphingosine 1-phosphate (49Taha T.A. Hannun Y.A. Obeid L.M. J. Biochem. Mol. Biol. 2006; 39: 113-131Crossref PubMed Google Scholar). We have recently shown that SK1 mRNA levels increased in response to oxLDL-IC in U937 cells (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and we also showed that oxLDL-IC prompt the release of SK1 into the medium, suggesting the generation of sphingosine 1-phosphate extracellularly (18Hammad S.M. Taha T.A. Nareika A. Johnson K.R. Lopes-Virella M.F. Obeid L.M. Prostaglandins Other Lipid Mediat. 2006; 79: 126-140Crossref PubMed Scopus (41) Google Scholar). It has recently been reported that overexpression of HSP70 in RAW 264.7 macrophages resulted in both increased SK1 protein and mRNA levels, contributing to the partial reversal of cell death caused by the combination of LPS and SK1 inhibitor (50Ding X.Z. Feng X.R. Borschel R.H. Nikolich M.P. Feng J. Li Y.S. Hoover D.L. Prostaglandins Other Lipid Mediat. 2010; (in press)PubMed Google Scholar). In this study, we show that knockdown of HSP70B′ reduced the expression of SK1 by half in response to oxLDL-IC, suggesting that HSP70B′ is an upstream mediator of SK1. Furthermore, phosphorylated SK1 is significantly reduced in U937 cells transfected with HSP70B′ siRNA compared with control cells treated with oxLDL. These data are the first to suggest an interaction between HSP70B′ and SK1 and could significantly influence further research to define SK1 effects mediating macrophage activation and survival.The anti-inflammatory cytokine, IL-10, has been shown to be cytoprotective in macrophages (51Arai T. Hiromatsu K. Nishimura H. Kimura Y. Kobayashi N. Ishida H. Nimura Y. Yoshikai Y. Biochem. Biophys. Res. Commun. 1995; 213: 600-607Crossref PubMed Scopus (65) Google Scholar) and induced by the addition of exogenous HSP70 in synovial cells (52Detanico T. Rodrigues L. Sabritto A.C. Keisermann M. Bauer M.E. Zwickey H. Bonorino C. Clin. Exp. Immunol. 2004; 135: 336-342Crossref PubMed Scopus (50) Google Scholar, 53Luo X. Zuo X. Zhang B. Song L. Wei X. Zhou Y. Xiao X. Cell Stress Chaperones. 2008; 13: 365-373Crossref PubMed Scopus (36) Google Scholar), suggesting that HSPs play an active role in suppressing inflammation by the regulation of IL-10. Here we provide evidence that secretion of IL-10 may be regulated by HSP70B′.Although HSPs are classically viewed as intracellular proteins, we provided evidence that HSP70B′ is induced and secreted in response to cell activation by oxLDL-IC. This is in agreement with previous reports demonstrating that HSPs may be released by viable cells under inflammatory stress (3Pockley A.G. Circulation. 2002; 105: 1012-1017Crossref PubMed Scopus (216) Google Scholar, 22Asea A. Kraeft S.K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1354) Google Scholar). Recently, it has been shown that HSP70 can be secreted from cells via exosomes (54Clayton A. Turkes A. Navabi H. Mason M.D. Tabi Z. J. Cell Sci. 2005; 118: 3631-3638Crossref PubMed Scopus (343) Google Scholar, 55Lancaster G.I. Febbraio M.A. J. Biol. Chem. 2005; 280: 23349-23355Abstract Full Text Full Text PDF PubMed Scopus (439) Google Scholar). Moreover, it has been shown that in stressed cells, HSPs may be inserted into the plasma membrane before release into the extracellular environment in membrane-associated structures (56Vega V.L. Rodríguez-Silva M. Frey T. Gehrmann M. Diaz J.C. Steinem C. Multhoff G. Arispe N. De Maio A. J. Immunol. 2008; 180: 4299-4307Crossref PubMed Scopus (320) Google Scholar). The molecular mechanisms that regulate the mobilization of secretory vesicles and secretion of mediators from inflammatory cells, including macrophages, are still obscure (57Medzhitov R. Nature. 2008; 454: 428-435Crossref PubMed Scopus (3733) Google Scholar, 58Logan M.R. Odemuyiwa S.O. Moqbel R. J. Allergy Clin. Immunol. 2003; 111: 923-932Abstract Full Text Full Text PDF PubMed Google Scholar).In conclusion, our current data suggest that HSP70B′ is induced and released by macrophages stimulated by oxLDL-IC and that once released into the extracellular space, HSP70B′ immediately associates with oxLDL-IC. We also provided evidence that a variety of cellular responses, including regulation of SK1 and release of the anti-inflammatory prosurvival cytokine IL-10, could be mediated by HSP70B′. Our findings contribute to the understanding of foam cell survival and activation and may advance efforts to reveal additional therapeutic targets that could mediate the stabilization of vulnerable atherosclerotic plaques. IntroductionHeat shock proteins (HSPs) 2The abbreviations used are: HSPheat shock proteinLDLlow density lipoprotein(s)oxLDLoxidized low density lipoprotein(s)oxLDL-ICoxidized low density lipoprotein immune complexesSK1sphingosine kinase 1ILinterleukinTNF-αtumor necrosis factor αIFN-γinterferon-γKLH-ICkeyhole limpet hemocyanin immune complexesDiIdioctadecyl-3,3,3′,3-tetramethylindocarbocyanine perchloratePBSphosphate-buffered salineQ-PCRquantitative PCRGFPgreen fluorescent proteinBisTris2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diolsiRNAsmall interfering RNA. belong to a group of more than 20 highly conserved stress proteins that are routinely employed by cells as cytoprotective agents against a variety of stress stimuli, including heat shock, oxidative and mechanical stress, and inflammation (1Morimoto R.I. Science. 1993; 259: 1409-1410Crossref PubMed Scopus (1212) Google Scholar, 2Xu Q. Wick G. Mol. Med. Today. 1996; 2: 372-379Abstract Full Text PDF PubMed Scopus (141) Google Scholar). Evidence exists that HSP expression is higher at sites of atherosclerotic lesions than it is in normal tissue (3Pockley A.G. Circulation. 2002; 105: 1012-1017Crossref PubMed Scopus (216) Google Scholar, 4Roma P. Catapano A.L. Atherosclerosis. 1996; 127: 147-154Abstract Full Text PDF PubMed Scopus (37) Google Scholar). Elevated HSP70 levels are associated with atherosclerotic plaques, particularly in areas with abundant activated macrophages, and seem to coincide around sites of necrosis and lipid accumulation (5Johnson A.D. Berberian P.A. Tytell M. Bond M.G. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 27-36Crossref PubMed Scopus (84) Google Scholar, 6Berberian P.A. Myers W. Tytell M. Challa V. Bond M.G. Am. J. Pathol. 1990; 136: 71-80PubMed Google Scholar).Of particular interest in the development of atherogenesis is the role of oxidatively modified LDL (oxLDL). It is established that oxLDL particles are taken up by activated macrophages, resulting in lipid accumulation (7Steinberg D. J. Biol. Chem. 1997; 272: 20963-20966Abstract Full Text Full Text PDF PubMed Scopus (1450) Google Scholar). oxLDL also triggers an immune response, initiating the production of predominantly proinflammatory IgG antibodies, which then form circulating complexes with oxLDL (oxLDL-IC) (8Virella G. Koskinen S. Krings G. Onorato J.M. Thorpe S.R. Lopes-Virella M. Clin. Immunol. 2000; 95: 135-144Crossref PubMed Scopus (55) Google Scholar, 9Virella G. Lopes-Virella M.F. Clin. Diagn. Lab. Immunol. 2003; 10: 499-505Crossref PubMed Scopus (56) Google Scholar). These immune complexes activate macrophages through the FcγRI receptor, resulting in the release of proinflammatory cytokines (interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α)) and the associated acceleration of foam cell formation (10Virella G. Muñoz J.F. Galbraith G.M. Gissinger C. Chassereau C. Lopes-Virella M.F. Clin. Immunol. Immunopathol. 1995; 75: 179-189Crossref PubMed Scopus (57) Google Scholar, 11Griffith R.L. Virella G.T. Stevenson H.C. Lopes-Virella M.F. J. Exp. Med. 1988; 168: 1041-1059Crossref PubMed Scopus (145) Google Scholar, 12Huang Y. Jaffa A. Koskinen S. Takei A. Lopes-Virella M.F. Arterioscler. Thromb. Vasc. Biol. 1999; 19: 1600-1607Crossref PubMed Scopus (59) Google Scholar, 13Saad A.F. Virella G. Chassereau C. Boackle R.J. Lopes-Virella M.F. J. Lipid Res. 2006; 47: 1975-1983Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Whereas free oxLDL have been shown to be cytotoxic to monocytic cells (14Asmis R. Begley J.G. Circ. Res. 2003; 92: e20-e29Crossref PubMed Google Scholar, 15Martinet W. Kockx M.M. Curr. Opin. Lipidol. 2001; 12: 535-541Crossref PubMed Scopus (110) Google Scholar, 16Vicca S. Massy Z.A. Hennequin C. Rihane D. Drüeke T.B. Lacour B. Free Radic. Biol. 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However, the role HSP70 family members play in the development of atherosclerotic plaques is still unclear. Increasing evidence suggests that HSPs may serve as cytokines themselves. Asea et al. (21Asea A. Rehli M. Kabingu E. Boch J.A. Bare O. Auron P.E. Stevenson M.A. Calderwood S.K. J. Biol. Chem. 2002; 277: 15028-15034Abstract Full Text Full Text PDF PubMed Scopus (1243) Google Scholar) determined that activated macrophages secrete HSP70, which then binds to CD14 on the outer membrane, triggering the production and subsequent release of proinflammatory cytokines. This finding supports earlier experiments that induced cytokine production with the addition of exogenous HSP70 (22Asea A. Kraeft S.K. Kurt-Jones E.A. Stevenson M.A. Chen L.B. Finberg R.W. Koo G.C. Calderwood S.K. Nat. Med. 2000; 6: 435-442Crossref PubMed Scopus (1354) Google Scholar, 23Multhoff G. Mizzen L. Winchester C.C. Milner C.M. Wenk S. Eissner G. Kampinga H.H. Laumbacher B. Johnson J. Exp. Hematol. 1999; 27: 1627-1636Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar).Elevated oxLDL, a hallmark of increased risk of atherosclerosis, has been implicated as the initial factor in the HSP70-linked proinflammatory pathway of activated macrophages. Svensson et al. (24Svensson P.A. Asea A. Englund M.C. Bausero M.A. Jernås M. Wiklund O. Ohlsson B.G. Carlsson L.M. Carlsson B. Atherosclerosis. 2006; 185: 32-38Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) demonstrated that high levels of oxLDL directly up-regulate and initiate release of HSP70 in macrophages, resulting in a corresponding increase in cytokine (IL-1β and IL-12) production. Whether exposure of human macrophages to elevated levels of oxLDL-IC elicits HSP70 regulation has not been previously examined.We have recently shown that one member of the HSP70 family, HSP70B′ (also known as HSP70 protein 6; gene HSPA6), displayed a considerable increase in expression in response to oxLDL-IC but not oxLDL alone (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Furthermore, evidence was provided that IL-1β secretion is HSP70B′-dependent, suggesting a novel, oxLDL-IC-dependent proinflammatory mechanism involving the little known HSP70B′ species (25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The HSP70B′ gene is unique to the human genome, probably arising after the divergence of rodents and humans (26Parsian A.J. Sheren J.E. Tao T.Y. Goswami P.C. Malyapa R. Van Rheeden R. Watson M.S. Hunt C.R. Biochim. Biophys. Acta. 2000; 1494: 201-205Crossref PubMed Google Scholar), and although HSP70B′ and HSP70 are over 80% homologous, differences are present in sequences coding for substrate-binding and activation sites, evidence of a unique cellular function (27Noonan E.J. Place R.F. Giardina C. Hightower L.E. Cell Stress Chaperones. 2007; 12: 393-402Crossref PubMed Scopus (41) Google Scholar). Until recently, sequence data and immunological reagents for HSP70B′ were unavailable, and due to the sequence homology, standard antibodies designed for the common HSP70A/B members most likely included the detection of HSP70B′. HSP70, while functionally inducible, is fairly ubiquitous at basal conditions in many different cell types, whereas HSP70B′ is strictly inducible (27Noonan E.J. Place R.F. Giardina C. Hightower L.E. Cell Stress Chaperones. 2007; 12: 393-402Crossref PubMed Scopus (41) Google Scholar, 28Leung T.K. Rajendran M.Y. Monfries C. Hall C. Lim L. Biochem. J. 1990; 267: 125-132Crossref PubMed Scopus (152) Google Scholar, 29Noonan E. Giardina C. Hightower L. Exp. Cell Res. 2008; 314: 2468-2476Crossref PubMed Scopus (32) Google Scholar). Our recent findings have led to the hypothesis that HSP70B′ expression could be induced by Fcγ-RI activation in human macrophages, resulting in increased proinflammatory cytokine release, prolonged foam cell survival, and thus the associated increased risk of atherosclerotic plaque development (18Hammad S.M. Taha T.A. Nareika A. Johnson K.R. Lopes-Virella M.F. Obeid L.M. Prostaglandins Other Lipid Mediat. 2006; 79: 126-140Crossref PubMed Scopus (41) Google Scholar, 25Hammad S.M. Twal W.O. Barth J.L. Smith K.J. Saad A.F. Virella G. Argraves W.S. Lopes-Virella M.F. Atherosclerosis. 2009; 202: 394-404Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Herein, we show that HSP70B′ was induced and released in response to oxLDL-IC and that HSP70B′ could bind to the cell-associated and internalized lipid moiety of oxLDL-IC. The presented data also implicated HSP70B′ in cellular functions that underlie the ability of oxLDL-IC to promote prolonged activation of foam cells, such as regulation of sphingosine kinase 1 (SK1) activity and release of IL-10." @default.
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