FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2009611959> ?p ?o ?g. }

• W2009611959 endingPage "1016" @default.
• W2009611959 startingPage "1010" @default.
• W2009611959 abstract "Epidemiological evidence suggests that cardiovascular disease is associated with osteoporosis, independent of age. Bone resorptive surface is increased in mice on a high-fat diet, and osteoclastic differentiation of bone marrow preosteoclasts is promoted by oxidized phospholipids. Because osteoclastic differentiation requires cytokines produced by osteoblasts, we hypothesized that the stimulatory mechanism of oxidized phospholipids is via induction of osteoclast-regulating cytokines in osteoblasts. To investigate the effects of oxidized phospholipids on expression of such cytokines, murine calvarial preosteoblasts, MC3T3-E1, were treated with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), an active component of oxidized lipoproteins. Results showed that ox-PAPC increased expression of interleukin-6 (IL-6) and tumor necrosis factor-α. IL-6 expression was also elevated in calvarial tissues from hyperlipidemic but not in wild-type mice. Ox-PAPC also induced IL-6 protein levels in both MC3T3-E1 and primary calvarial cells. Promoter-reporter assay analysis showed that ox-PAPC, but not PAPC, induced murine IL-6 promoter activity. Effects of ox-PAPC on IL-6 expression and the promoter activity were attenuated by H89, a PKA inhibitor. Analysis of deletion and mutant IL-6 promoter constructs suggested that CAAT/enhancer binding protein (C/EBP) partly mediates the ox-PAPC effects. Taken together, the data suggest that oxidized phospholipids induce IL-6 expression in osteoblasts in part via C/EBP. Epidemiological evidence suggests that cardiovascular disease is associated with osteoporosis, independent of age. Bone resorptive surface is increased in mice on a high-fat diet, and osteoclastic differentiation of bone marrow preosteoclasts is promoted by oxidized phospholipids. Because osteoclastic differentiation requires cytokines produced by osteoblasts, we hypothesized that the stimulatory mechanism of oxidized phospholipids is via induction of osteoclast-regulating cytokines in osteoblasts. To investigate the effects of oxidized phospholipids on expression of such cytokines, murine calvarial preosteoblasts, MC3T3-E1, were treated with oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC), an active component of oxidized lipoproteins. Results showed that ox-PAPC increased expression of interleukin-6 (IL-6) and tumor necrosis factor-α. IL-6 expression was also elevated in calvarial tissues from hyperlipidemic but not in wild-type mice. Ox-PAPC also induced IL-6 protein levels in both MC3T3-E1 and primary calvarial cells. Promoter-reporter assay analysis showed that ox-PAPC, but not PAPC, induced murine IL-6 promoter activity. Effects of ox-PAPC on IL-6 expression and the promoter activity were attenuated by H89, a PKA inhibitor. Analysis of deletion and mutant IL-6 promoter constructs suggested that CAAT/enhancer binding protein (C/EBP) partly mediates the ox-PAPC effects. Taken together, the data suggest that oxidized phospholipids induce IL-6 expression in osteoblasts in part via C/EBP. Although commonly viewed as having distinct etiologies, cardiovascular disease has been associated with osteoporosis, often coexisting in patients independent of age (1Hamerman D. Osteoporosis and atherosclerosis: biological linkages and the emergence of dual-purpose therapies.QJM. 2005; 98: 467-484Crossref PubMed Scopus (115) Google Scholar). The National Health and Nutrition Examination Survey (1988–1994) showed that an estimated 63% of osteoporotic patients exhibit hyperlipidemia (cholesterol >130 mg/dl) and epidemiological studies show that plasma LDL levels inversely correlate with bone mineral density in postmenopausal women (2Yamaguchi T. Sugimoto T. Yano S. Yamauchi M. Sowa H. Chen Q. Chihara K. Plasma lipids and osteoporosis in postmenopausal women.Endocr. J. 2002; 49: 211-217Crossref PubMed Scopus (241) Google Scholar). Postmenopausal women who have high plasma LDL levels are also more susceptible to osteopenia (3Poli A. Bruschi F. Cesana B. Rossi M. Paoletti R. Crosignani P.G. Plasma low-density lipoprotein cholesterol and bone mass densitometry in postmenopausal women.Obstet. Gynecol. 2003; 102: 922-926Crossref PubMed Scopus (53) Google Scholar). We and others have found that an atherogenic high-fat diet reduces bone formation rate and bone mineral density while increasing osteoclastic potential of bone marrow preosteoclasts and bone resorptive surface (4Hirasawa H. Tanaka S. Sakai A. Tsutsui M. Shimokawa H. Miyata H. Moriwaki S. Niida S. Ito M. Nakamura T. ApoE gene deficiency enhances the reduction of bone formation induced by a high-fat diet through the stimulation of p53-mediated apoptosis in osteoblastic cells.J. Bone Miner. Res. 2007; 22: 1020-1030Crossref PubMed Scopus (52) Google Scholar, 5Parhami F. Tintut Y. Beamer W.G. Gharavi N. Goodman W. Demer L.L. Atherogenic high-fat diet reduces bone mineralization in mice.J. Bone Miner. Res. 2001; 16: 182-188Crossref PubMed Scopus (230) Google Scholar, 6Tintut Y. Morony S. Demer L.L. Hyperlipidemia promotes osteoclastic potential of bone marrow cells ex vivo.Arterioscler. Thromb. Vasc. Biol. 2004; 24: e6-e10Crossref PubMed Google Scholar, 7Tintut Y. Parhami F. Tsingotjidou A. Tetradis S. Territo M. Demer L.L. 8-Isoprostaglandin E2 enhances receptor- activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway.J. Biol. Chem. 2002; 277: 14221-14226Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).High circulating plasma LDL particles accumulate in the subendothelial matrix of arteries where they have an increased susceptibility to modification, including oxidation and acetylation. These oxidized lipoproteins and phospholipids induce release of inflammatory cytokines and chemokines from endothelial cells and monocytes (8Van Lenten B.J. Wagner A.C. Navab M. Fogelman A.M. Oxidized phospholipids induce changes in hepatic paraoxonase and ApoJ but not monocyte chemoattractant protein-1 via interleukin-6.J. Biol. Chem. 2001; 276: 1923-1929Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 9Yeh M. Leitinger N. de Martin R. Onai N. Matsushima K. Vora D.K. Berliner J.A. Reddy S.T. Increased transcription of IL-8 in endothelial cells is differentially regulated by TNF-alpha and oxidized phospholipids.Arterioscler. Thromb. Vasc. Biol. 2001; 21: 1585-1591Crossref PubMed Scopus (99) Google Scholar). Al-Aly et al. (10Al-Aly Z. Shao J.S. Lai C.F. Huang E. Cai J. Behrmann A. Cheng S.L. Towler D.A. Aortic Msx2-Wnt calcification cascade is regulated by TNF-alpha-dependent signals in diabetic Ldlr−/− mice.Arterioscler. Thromb. Vasc. Biol. 2007; 27: 2589-2596Crossref PubMed Scopus (253) Google Scholar) recently showed that a high-fat diet promotes hyperlipidemia and upregulates serum tumor necrosis factor (TNF)-α in mice. Furnkranz et al. (11Furnkranz A. Schober A. Bochkov V.N. Bashtrykov P. Kronke G. Kadl A. Binder B.R. Weber C. Leitinger N. Oxidized phospholipids trigger atherogenic inflammation in murine arteries.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 633-638Crossref PubMed Scopus (121) Google Scholar) have also shown that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (ox-PAPC) induces interleukin-6 (IL-6) expression in murine carotid arteries. One active component of biologically active LDL that is known to cause inflammatory responses both in vitro and in vivo is ox-PAPC (11Furnkranz A. Schober A. Bochkov V.N. Bashtrykov P. Kronke G. Kadl A. Binder B.R. Weber C. Leitinger N. Oxidized phospholipids trigger atherogenic inflammation in murine arteries.Arterioscler. Thromb. Vasc. Biol. 2005; 25: 633-638Crossref PubMed Scopus (121) Google Scholar, 12Watson A.D. Leitinger N. Navab M. Faull K.F. Horkko S. Witztum J.L. Palinski W. Schwenke D. Salomon R.G. Sha W. et al.Structural identification by mass spectrometry of oxidized phospholipids in minimally oxidized low density lipoprotein that induce monocyte/endothelial interactions and evidence for their presence in vivo.J. Biol. Chem. 1997; 272: 13597-13607Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar).Oxidized lipids are also likely to accumulate within the skeletal bone. We previously found lipid deposits within the perivascular space of Haversian canals in osteoporotic but not in normal cortical bone (6Tintut Y. Morony S. Demer L.L. Hyperlipidemia promotes osteoclastic potential of bone marrow cells ex vivo.Arterioscler. Thromb. Vasc. Biol. 2004; 24: e6-e10Crossref PubMed Google Scholar). In addition, we have detected oxidized lipids in the bone marrow of hyperlipidemic mice (6Tintut Y. Morony S. Demer L.L. Hyperlipidemia promotes osteoclastic potential of bone marrow cells ex vivo.Arterioscler. Thromb. Vasc. Biol. 2004; 24: e6-e10Crossref PubMed Google Scholar). Recent studies showed that osteoblasts are also capable of oxidizing LDL particles (13Brodeur M.R. Brissette L. Falstrault L. Ouellet P. Moreau R. Influence of oxidized low-density lipoproteins (LDL) on the viability of osteoblastic cells.Free Radic. Biol. Med. 2008; 44: 506-517Crossref PubMed Scopus (55) Google Scholar), suggesting a possible increased local concentration of oxidized phospholipids in bones of hyperlipidemic patients.Elevated levels of osteoclast-promoting cytokines, including receptor activator of nuclear factor kappa B ligand (RANKL), IL-6, and TNF-α have been linked to postmenopausal bone loss (14Pfeilschifter J. Koditz R. Pfohl M. Schatz H. Changes in proinflammatory cytokine activity after menopause.Endocr. Rev. 2002; 23: 90-119Crossref PubMed Scopus (706) Google Scholar). IL-6 stimulates osteoclastic differentiation and activity in bone marrow cells (15Ishimi Y. Miyaura C. Jin C.H. Akatsu T. Abe E. Nakamura Y. Yamaguchi A. Yoshiki S. Matsuda T. Hirano T. et al.IL-6 is produced by osteoblasts and induces bone resorption.J. Immunol. 1990; 145: 3297-3303PubMed Google Scholar). IL-6 also mediates parathyroid hormone-induced bone resorption in vivo (16Grey A. Mitnick M.A. Masiukiewicz U. Sun B.H. Rudikoff S. Jilka R.L. Manolagas S.C. Insogna K. A role for interleukin-6 in parathyroid hormone-induced bone resorption in vivo.Endocrinology. 1999; 140: 4683-4690Crossref PubMed Google Scholar), and has been implicated in contributing to the increased sensitivity to bone loss that is often observed in peri-menopausal or estrogen-deficient women who concurrently suffer from primary hyperthyroidism (17Insogna K. Mitnick M. Pascarella J. Nakchbandi I. Grey A. Masiukiewicz U. Role of the interleukin-6/interleukin-6 soluble receptor cytokine system in mediating increased skeletal sensitivity to parathyroid hormone in perimenopausal women.J. Bone Miner. Res. 2002; 17 (Suppl 2): N108-N116PubMed Google Scholar, 18Masiukiewicz U.S. Mitnick M. Gulanski B.I. Insogna K.L. Evidence that the IL-6/IL-6 soluble receptor cytokine system plays a role in the increased skeletal sensitivity to PTH in estrogen-deficient women.J. Clin. Endocrinol. Metab. 2002; 87: 2892-2898Crossref PubMed Scopus (0) Google Scholar). We previously found that oxidized phospholipids induced osteoclast differentiation when cocultured with osteoblasts (7Tintut Y. Parhami F. Tsingotjidou A. Tetradis S. Territo M. Demer L.L. 8-Isoprostaglandin E2 enhances receptor- activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway.J. Biol. Chem. 2002; 277: 14221-14226Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Because osteoclastic differentiation is regulated by cytokines produced by osteoblasts (19Rodan G.A. Introduction to bone biology.Bone. 1992; 13 (Suppl 1): S3-S6Crossref PubMed Scopus (217) Google Scholar), we hypothesize that oxidized phospholipids promote production of osteoclast-regulating cytokines by osteoblasts. To test the hypothesis, we investigated the effects of oxidized phospholipids on expression of osteoclastogenic cytokines in the murine calvarial cell line, MC3T3-E1. Our results showed that oxidized lipids induced mRNA expression of IL-6, and that CAAT/enhancer binding protein (C/EBP) response elements partly mediated the transcriptional regulation of IL-6 expression by oxidized phospholipids.MATERIALS AND METHODSMaterialsMC3T3-E1 cells were obtained from Riken Cell Bank (Japan). Arachidonic acid (AA) was purchased from Cayman Chemical. Ox-PAPC was prepared by auto-oxidizing PAPC (1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine; Avanti-Polar Lipids). The oxidation state was verified by liquid chromatography/ mass spectroscopy. Bioactive components of ox-PAPC, oxidized AA (ox-AA), 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5,6)-epoxyisoprostane E 2-sn-glycero-3-phosphocholine (PEIPC) were prepared as previously described (20Watson A.D. Subbanagounder G. Welsbie D.S. Faull K.F. Navab M. Jung M.E. Fogelman A.M. Berliner J.A. Structural identification of a novel pro-inflammatory epoxyisoprostane phospholipid in mildly oxidized low density lipoprotein.J. Biol. Chem. 1999; 274: 24787-24798Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). H89 was obtained from Calbiochem, and Y-27632 from Cayman Chemical.Cell cultureCalvarial preosteblasts (21Nervina J.M. Magyar C.E. Pirih F.Q. Tetradis S. PGC-1alpha is induced by parathyroid hormone and coactivates Nurr1-mediated promoter activity in osteoblasts.Bone. 2006; 39: 1018-1025Crossref PubMed Scopus (27) Google Scholar) were a kind gift from Dr. S. Tetradis (UCLA). Both calvarial preosteoblasts and MC3T3-E1 cells were cultured in α-MEM (Invitrogen) supplemented with 10% FBS, penicillin, streptomycin, and sodium pyruvate. Media was replenished every 3 to 4 days, when necessary. Short-term (6–24 h) treatment media consisted of α-MEM without ascorbic acid (Gibco BRL) supplemented with 1% FBS, whereas longer-term (72 h) treatment media consisted of α-MEM (Invitrogen) supplemented with 10% FBS.Animal tissueCalvarial tissues were harvested from 15-week-old wild-type (C57BL/6) and hyperlipidemic (Ldlr−/−) mice (Jackson Laboratory, ME), and total RNA was isolated. The animals were given a standard Purina Chow diet. The experimental protocol was approved by the Institutional Animal Care and Use Committee of the University of California, Los Angeles.RNA isolation and real-time RT-quantitative PCRTotal RNA was isolated using TRIzol reagent (Invitrogen). Real-time PCR was performed using the One-Step qRT-PCR SuperMix Kit (BioChain, Inc.) and Mx3005P (Stratagene).DNA constructsGeneration of murine IL-6 promoter luciferase reporter constructs was previously described (22Baccam M. Woo S.Y. Vinson C. Bishop G.A. CD40-mediated transcriptional regulation of the IL-6 gene in B lymphocytes: involvement of NF-kappa B, AP-1, and C/EBP.J. Immunol. 2003; 170: 3099-3108Crossref PubMed Scopus (131) Google Scholar). The constructs contain either 1277, 231, or 84 bp upstream of the transcriptional start site or a 1277 bp fragment containing mutant C/EBP response elements (-161 to -147) and are designated here as -1277 Luc, -231 Luc, -84 Luc, and mC/EBP Luc, respectively. The plasmids were verified by sequencing.Transient transfections and luciferase reporter assayPlasmid transfection was performed with Effectene transfection reagent (Qiagen), according to the manufacturer's instructions. Twenty-four h after transfection, cells were serum starved for 7 h using media containing 1% FBS and treated in the same media for 19 h. Luciferase activity (expressed as relative light units, RLU) was assayed using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase activity was normalized for transfection efficiency to Renilla luciferase.IL-6 protein levelsConfluent cells were treated for 24 h in serum-free, phenol red-free DMEM (Sigma Aldrich). Secreted IL-6 protein levels in cell media were measured using Quantikine Mouse IL-6 Immunoassay Kit (R&D Systems), following the manufacturer's protocol. Assays were performed in triplicate.Data analysisExperiments (≥ triplicate wells) were performed at least three times and data from one representative experiment are expressed as mean ± SEM. Results were compared using a two-tailed paired Student's t-test. In comparisons across more than two groups, two-way ANOVA, followed by Fisher's Protected Least Significant Difference, was performed using StatView (v4.5, Abacus). P < 0.05 is considered statistically significant.RESULTSEffects of ox-PAPC on osteoclast-regulating cytokinesTo determine whether ox-PAPC regulates expression of osteoclast-regulatory cytokines, MC3T3-E1 were treated with ox-PAPC for 6 and 72 h, and mRNA expression level was assessed by real-time RT-qPCR for IL-6, TNF-α, RANKL, and osteoprotegerin (OPG), a decoy receptor of RANKL that inhibits osteoclastic differentiation (19Rodan G.A. Introduction to bone biology.Bone. 1992; 13 (Suppl 1): S3-S6Crossref PubMed Scopus (217) Google Scholar). As shown in Fig. 1A, ox-PAPC significantly induced IL-6 at both 6 and 72 h, whereas it only increased TNF-α at 72 h. OPG and RANKL expressions were not significantly affected by ox-PAPC (Fig. 1A). RANKL expression was not detectable at 72 h in these cells. Because hyperlipidemic mice have elevated levels of oxidized phospholipids, we compared the in vivo cytokine profile of calvarial tissue from hyperlipidemic and wild-type mice with our in vitro results. We found that IL-6 expression, but not RANKL, OPG, or TNF-α, was elevated in the hyperlipidemic Ldlr−/− mice compared with wild-type mice (Fig. 1B). Results from IL-6 ELISA showed that treatment of MC3T3-E1 cells with ox-PAPC also induced IL-6 protein levels in a dose-dependent manner (Fig. 1C).Intracellular mechanism mediating ox-PAPC effectsBecause IL-6 expression is induced both in vitro and in vivo, we further investigated the mechanism of IL-6 induction by ox-PAPC. To investigate whether IL-6 induction was at the level of transcriptional activation, we assessed IL-6 promoter activity using -1277 Luc, which contains luciferase reporter gene under the transcriptional control of the IL-6 promoter (1277 bp upstream of the transcriptional start site). As shown in Fig. 2A, ox-PAPC induced IL-6 promoter activity in a dose-dependent manner. The IL-6 promoter activity was also induced by ox-AA but not unoxidized PAPC (Fig. 2B), confirming that the oxidized arachidonic side chain is the active component. As shown previously (23Bordin L. Priante G. Musacchio E. Giunco S. Tibaldi E. Clari G. Baggio B. Arachidonic acid-induced IL-6 expression is mediated by PKC alpha activation in osteoblastic cells.Biochemistry. 2003; 42: 4485-4491Crossref PubMed Scopus (47) Google Scholar), AA (40 µg/ml) also induced IL-6 promoter activity (control: 0.63 ± 0.32 vs. AA: 31.96 ± 6.83, P < 0.05). Interestingly, IL-6 induction was not induced by POVPC, PGPC, or PEIPC, some of the active components of ox-PAPC (Fig. 2C). In primary calvarial osteoblasts, ox-PAPC also induced both IL-6 protein levels and promoter activity (Fig. 3A, B).Fig. 2Effects of ox-PAPC on IL-6 promoter activity. Luciferase activity assay (expressed as relative light units, RLU) of MC3T3-E1 cells transfected with -1277 Luc and treated with (A) ox-PAPC at various concentrations, as indicated (B) with control vehicle, PAPC, ox-PAPC, or ox-AA (all at 40 µg/ml) (C) with ox-PAPC or its components at 10 or 40 µg/ml, as indicated. Firefly luciferase activity was normalized to Renilla luciferase for transfection efficiency. ∗P < 0.005, ∗∗P < 0.0001 (vs. control), N.S. statistically nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3Effects of ox-PAPC in primary calvarial cells. A: IL-6 immunoassay (ELISA) of culture media from primary calvarial cells treated with control vehicle or ox-PAPC at 40 or 60 µg/ml. B: Luciferase activity assay of primary calvarial cells transfected with -1277 Luc and treated with control vehicle or ox-PAPC (40 µg/ml). ∗P < 0.005 (vs. control).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Because the protein kinase A pathway mediates effects of oxidized lipoproteins and ox-PAPC (7Tintut Y. Parhami F. Tsingotjidou A. Tetradis S. Territo M. Demer L.L. 8-Isoprostaglandin E2 enhances receptor- activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway.J. Biol. Chem. 2002; 277: 14221-14226Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Birukov K.G. Leitinger N. Bochkov V.N. Garcia J.G. Signal transduction pathways activated in human pulmonary endothelial cells by OxPAPC, a bioactive component of oxidized lipoproteins.Microvasc. Res. 2004; 67: 18-28Crossref PubMed Scopus (56) Google Scholar, 25Cole A.L. Subbanagounder G. Mukhopadhyay S. Berliner J.A. Vora D.K. Oxidized phospholipid-induced endothelial cell/monocyte interaction is mediated by a cAMP-dependent R-Ras/PI3-kinase pathway.Arterioscler. Thromb. Vasc. Biol. 2003; 23: 1384-1390Crossref PubMed Scopus (119) Google Scholar, 26Parhami F. Fang Z.T. Fogelman A.M. Andalibi A. Territo M.C. Berliner J.A. Minimally modified low density lipoprotein-induced inflammatory responses in endothelial cells are mediated by cyclic adenosine monophosphate.J. Clin. Invest. 1993; 92: 471-478Crossref PubMed Scopus (207) Google Scholar) as well as IL-6 gene expression in epithelial cells (27Hershko D.D. Robb B.W. Luo G. Hasselgren P.O. Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 283: R1140-R1148Crossref PubMed Scopus (80) Google Scholar), we tested the effects of a PKA inhibitor, H89, on ox-PAPC induction of IL-6. As shown in Fig. 4A, H89 attenuated basal IL-6 expression, as reported previously (28Chen C. Koh A.J. Datta N.S. Zhang J. Keller E.T. Xiao G. Franceschi R.T. D'Silva N.J. McCauley L.K. Impact of the mitogen-activated protein kinase pathway on parathyroid hormone-related protein actions in osteoblasts.J. Biol. Chem. 2004; 279: 29121-29129Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). In addition, H89 attenuated ox-PAPC-induced IL-6 mRNA expression. Consistent with this finding, H89 also inhibited IL-6 promoter activity (Fig. 4B). Because rho-associated kinase II (ROCK-II) has been shown to be inhibited by H89 (29Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors.Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (0) Google Scholar) and also shown to mediate parathyroid hormone-induced IL-6 promoter activity (30Radeff J.M. Nagy Z. Stern P.H. Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C alpha translocation and IL-6 promoter activity in osteoblastic cells.J. Bone Miner. Res. 2004; 19: 1882-1891Crossref PubMed Scopus (47) Google Scholar), we tested the effect of Y-27632, which inhibits 87% of ROCK-II activity but only 9% of PKA activity (29Davies S.P. Reddy H. Caivano M. Cohen P. Specificity and mechanism of action of some commonly used protein kinase inhibitors.Biochem. J. 2000; 351: 95-105Crossref PubMed Scopus (0) Google Scholar). As shown in Fig. 4C, Y-27632 did not attenuate ox-PAPC induction of IL-6 promoter activity, suggesting that ROCK-II does not mediate ox-PAPC effects. Western blot analyses also showed that ox-PAPC did not activate two other kinases that are also inhibited by H89, mitogen- and stress-activated protein kinase 2 or p70 ribosomal protein s6 kinase 1 (data not shown).Fig. 4Effect of H89 on ox-PAPC induced IL-6. A: RT-qPCR analysis of total RNA isolated from MC3T3-E1 cells treated with vehicle, ox-PAPC (40 µg/ml) and/or H89 (10 µM), as indicated. B: Luciferase activity assay of MC3T3-E1 cells transfected with -1277 Luc and treated with vehicle, ox-PAPC (40 µg/ml) and/or H89 (10 µM), as indicated. C: Luciferase activity assay of MC3T3-E1 cells transfected with -1277 Luc and treated with vehicle, ox-PAPC (40 µg/ml), and/or Y-27632 (10 µM) as indicated. ∗P < 0.001 (vs. control), N.S. statistically nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Multiple transcription factors, including activator protein-1 (AP-1) and C/EBP, have been reported to regulate IL-6 promoter activity in several cell types (22Baccam M. Woo S.Y. Vinson C. Bishop G.A. CD40-mediated transcriptional regulation of the IL-6 gene in B lymphocytes: involvement of NF-kappa B, AP-1, and C/EBP.J. Immunol. 2003; 170: 3099-3108Crossref PubMed Scopus (131) Google Scholar, 27Hershko D.D. Robb B.W. Luo G. Hasselgren P.O. Multiple transcription factors regulating the IL-6 gene are activated by cAMP in cultured Caco-2 cells.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2002; 283: R1140-R1148Crossref PubMed Scopus (80) Google Scholar, 31Motomura T. Kasayama S. Takagi M. Kurebayashi S. Matsui H. Hirose T. Miyashita Y. Yamauchi-Takihara K. Yamamoto T. Okada S. et al.Increased interleukin-6 production in mouse osteoblastic MC3T3–E1 cells expressing activating mutant of the stimulatory G protein.J. Bone Miner. Res. 1998; 13: 1084-1091Crossref PubMed Scopus (40) Google Scholar). Therefore, we tested whether AP-1 and C/EBP mediate ox-PAPC effects. To delineate the involvement of AP-1 sites, we used two deletion constructs. As shown in Fig. 5A, ox-PAPC retained induction of IL-6 in the -231 Luc construct where upstream AP-1 response element (-277 to -271) was deleted. In contrast, ox-PAPC effect was attenuated in the -84 Luc construct containing downstream AP-1 response elements (-61 to -55), indicating that AP-1 response elements do not mediate ox-PAPC induction of IL-6 promoter activity. However, the -84 Luc construct lacks the C/EBP response element (-161 to -147), suggesting that C/EBP partly mediates the effects of ox-PAPC. Consistent with this hypothesis, ox-PAPC effects were attenuated partially in the construct containing mutations in the C/EBP response element in the full-length IL-6 promoter (mC/EBP Luc) (Fig. 5B). The incomplete attenuation suggests the involvement of other transcription factors in mediating ox-PAPC effects.Fig. 5Effect of ox-PAPC on IL-6 promoter constructs. A: Luciferase activity assay of MC3T3-E1 cells transfected with full-length or deleted constructs (as shown in the schematic diagram) and treated with ox-PAPC (40 µg/ml). B: Luciferase activity assay of MC3T3-E1 cells transfected with the full-length construct or the construct containing mutation in the C/EBP response elements and treated with ox-PAPC (40 µg/ml). ∗P < 0.001, N.S. statistically nonsignificant.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONWe previously found that bone marrow cells from hyperlipidemic (Ldlr−/−) mice have increased osteoclastic potential (6Tintut Y. Morony S. Demer L.L. Hyperlipidemia promotes osteoclastic potential of bone marrow cells ex vivo.Arterioscler. Thromb. Vasc. Biol. 2004; 24: e6-e10Crossref PubMed Google Scholar), and that oxidized phospholipids promote osteoclastic differentiation both directly and also indirectly through osteoblasts (7Tintut Y. Parhami F. Tsingotjidou A. Tetradis S. Territo M. Demer L.L. 8-Isoprostaglandin E2 enhances receptor- activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway.J. Biol. Chem. 2002; 277: 14221-14226Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Because osteoblasts are principal physiological regulators of osteoclastic differentiation, we investigated the mechanism of lipid-mediated osteoclast induction through osteoblasts. The data suggest that oxidized phospholipids promote osteoblastic production of the osteoclastogenic cytokines IL-6 and TNF-α. The induction of IL-6 expression is also evident in the calvarial tissues of hyperlipidemic mice, suggesting that local cytokine expression may be increased. The present results also showed that oxidized lipids did not alter expression of RANKL and OPG, the main regulators of osteoclast differentiation (19Rodan G.A. Introduction to bone biology.Bone. 1992; 13 (Suppl 1): S3-S6Crossref PubMed Scopus (217) Google Scholar). Lack of RANKL induction and/or OPG inhibition by oxidized phospholipids both in vitro and in tissue suggests that hyperlipidemia/oxidized phospholipid regulation of osteoclastogenesis via osteoblasts may be secondary to their direct effects on preosteoclasts, as we showed previously (7Tintut Y. Parhami F. Tsingotjidou A. Tetradis S. Territo M. Demer L.L. 8-Isoprostaglandin E2 enhances receptor- activated NFkappa B ligand (RANKL)-dependent osteoclastic potential of marrow hematopoietic precursors via the cAMP pathway.J. Biol. Chem. 2002; 277: 14221-14226Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar).We previously found that oxidized phospholipids have differential effects on vascular and bone cells (32Parhami F. Morrow A.D. Balucan J. Leitinger N. Watson A.D. Tintut Y. Berliner J.A. Demer L.L. Lipid oxidation products have opposite effects on calcifying vascular cell and bone cell differentiation. A possible ex" @default.
• W2009611959 created "2016-06-24" @default.
• W2009611959 creator A5028525233 @default.
• W2009611959 creator A5038345311 @default.
• W2009611959 creator A5038851818 @default.
• W2009611959 creator A5057604637 @default.
• W2009611959 creator A5059995837 @default.
• W2009611959 creator A5062873812 @default.
• W2009611959 creator A5090431009 @default.
• W2009611959 date "2010-05-01" @default.
• W2009611959 modified "2023-09-27" @default.
• W2009611959 title "Regulation of interleukin-6 expression in osteoblasts by oxidized phospholipids" @default.
• W2009611959 cites W1820445151 @default.
• W2009611959 cites W1855737037 @default.
• W2009611959 cites W1978437735 @default.
• W2009611959 cites W1983424760 @default.
• W2009611959 cites W1986596274 @default.
• W2009611959 cites W1994103933 @default.
• W2009611959 cites W2013553103 @default.
• W2009611959 cites W2026162137 @default.
• W2009611959 cites W2028497030 @default.
• W2009611959 cites W2036857341 @default.
• W2009611959 cites W2038720983 @default.
• W2009611959 cites W2052151860 @default.
• W2009611959 cites W2054881581 @default.
• W2009611959 cites W2059950148 @default.
• W2009611959 cites W2060551064 @default.
• W2009611959 cites W2060817141 @default.
• W2009611959 cites W2065947716 @default.
• W2009611959 cites W2072353574 @default.
• W2009611959 cites W2104719053 @default.
• W2009611959 cites W2122252604 @default.
• W2009611959 cites W2127524709 @default.
• W2009611959 cites W2130149021 @default.
• W2009611959 cites W2133889308 @default.
• W2009611959 cites W2137511741 @default.
• W2009611959 cites W2142259273 @default.
• W2009611959 cites W2146675158 @default.
• W2009611959 cites W2151027438 @default.
• W2009611959 cites W2156227990 @default.
• W2009611959 cites W2158487294 @default.
• W2009611959 cites W2262311617 @default.
• W2009611959 cites W4247904703 @default.
• W2009611959 doi "https://doi.org/10.1194/jlr.m001099" @default.
• W2009611959 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/2853427" @default.
• W2009611959 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19965598" @default.
• W2009611959 hasPublicationYear "2010" @default.
• W2009611959 type Work @default.
• W2009611959 sameAs 2009611959 @default.
• W2009611959 citedByCount "23" @default.
• W2009611959 countsByYear W20096119592012 @default.
• W2009611959 countsByYear W20096119592013 @default.
• W2009611959 countsByYear W20096119592014 @default.
• W2009611959 countsByYear W20096119592016 @default.
• W2009611959 countsByYear W20096119592018 @default.
• W2009611959 countsByYear W20096119592019 @default.
• W2009611959 countsByYear W20096119592021 @default.
• W2009611959 countsByYear W20096119592022 @default.
• W2009611959 countsByYear W20096119592023 @default.
• W2009611959 crossrefType "journal-article" @default.
• W2009611959 hasAuthorship W2009611959A5028525233 @default.
• W2009611959 hasAuthorship W2009611959A5038345311 @default.
• W2009611959 hasAuthorship W2009611959A5038851818 @default.
• W2009611959 hasAuthorship W2009611959A5057604637 @default.
• W2009611959 hasAuthorship W2009611959A5059995837 @default.
• W2009611959 hasAuthorship W2009611959A5062873812 @default.
• W2009611959 hasAuthorship W2009611959A5090431009 @default.
• W2009611959 hasBestOaLocation W20096119591 @default.
• W2009611959 hasConcept C126322002 @default.
• W2009611959 hasConcept C134018914 @default.
• W2009611959 hasConcept C185592680 @default.
• W2009611959 hasConcept C202751555 @default.
• W2009611959 hasConcept C203014093 @default.
• W2009611959 hasConcept C2778260815 @default.
• W2009611959 hasConcept C2778690821 @default.
• W2009611959 hasConcept C3020625565 @default.
• W2009611959 hasConcept C55493867 @default.
• W2009611959 hasConcept C71924100 @default.
• W2009611959 hasConcept C74172505 @default.
• W2009611959 hasConcept C86803240 @default.
• W2009611959 hasConcept C95444343 @default.
• W2009611959 hasConceptScore W2009611959C126322002 @default.
• W2009611959 hasConceptScore W2009611959C134018914 @default.
• W2009611959 hasConceptScore W2009611959C185592680 @default.
• W2009611959 hasConceptScore W2009611959C202751555 @default.
• W2009611959 hasConceptScore W2009611959C203014093 @default.
• W2009611959 hasConceptScore W2009611959C2778260815 @default.
• W2009611959 hasConceptScore W2009611959C2778690821 @default.
• W2009611959 hasConceptScore W2009611959C3020625565 @default.
• W2009611959 hasConceptScore W2009611959C55493867 @default.
• W2009611959 hasConceptScore W2009611959C71924100 @default.
• W2009611959 hasConceptScore W2009611959C74172505 @default.
• W2009611959 hasConceptScore W2009611959C86803240 @default.
• W2009611959 hasConceptScore W2009611959C95444343 @default.
• W2009611959 hasIssue "5" @default.
• W2009611959 hasLocation W20096119591 @default.
• W2009611959 hasLocation W20096119592 @default.
• W2009611959 hasLocation W20096119593 @default.

• next