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Cells and Human Dermal Microvessel Endothelial Cells: the Role of JNK This Information Is Current As of October 1, 2021 Lipopolysaccharide-Induced Apoptosis in Transformed Bovine Brain Endothelial Cells and Human Dermal Microvessel Endothelial Cells: The Role of JNK This information is current as of October 1, 2021. Hisae Karahashi, Kathrin S. Michelsen and Moshe Arditi J Immunol 2009; 182:7280-7286; ; doi: 10.4049/jimmunol.0801376 http://www.jimmunol.org/content/182/11/7280 Downloaded from References This article cites 45 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/182/11/7280.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2009 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Lipopolysaccharide-Induced Apoptosis in Transformed Bovine Brain Endothelial Cells and Human Dermal Microvessel Endothelial Cells: The Role of JNK1 Hisae Karahashi,*† Kathrin S. Michelsen,*‡ and Moshe Arditi2* Stimulation of transformed bovine brain endothelial cells (TBBEC) with LPS leads to apoptosis while human microvessel endo- thelial cells (HMEC) need the presence of cycloheximide (CHX) with LPS to induce apoptosis. To investigate the molecular mechanism of LPS-induced apoptosis in HMEC or TBBEC, we analyzed the involvement of MAPK and PI3K in TBBEC and HMEC. LPS-induced apoptosis in TBBEC was hallmarked by the activation of caspase 3, caspase 6, and caspase 8 after the stimulation of LPS, followed by poly(ADP-ribose) polymerase cleavage and lactate dehydrogenase release. We also observed DNA cleavage determined by TUNEL staining in TBBEC treated with LPS. Herbimycin A, a tyrosine kinase inhibitor, and SP600125, a JNK inhibitor, suppressed the activation of caspases and lactate dehydrogenase release. Moreover, a PI3K inhibitor (LY294002) Downloaded from suppressed activation of caspases and combined treatment with both SP600125 and LY294002 completely inhibited the activation of caspases. These results suggest that the JNK signaling pathway through the tyrosine kinase and PI3K pathways is involved in the induction of apoptosis in LPS-treated TBBEC. On the other hand, we observed sustained JNK activation in HMEC treated with LPS and CHX, and neither ERK1/2 nor AKT were activated. The addition of SP600125 suppressed phosphorylation of JNK and the activation of caspase 3 in HMEC treated with LPS and CHX. These results suggest that JNK plays an important role in the induction of apoptosis in endothelial cells. The Journal of Immunology, 2009, 182: 7280–7286. http://www.jimmunol.org/ ipopolysaccharide is a major component of Gram-nega- EC (TBBEC), LPS itself induces cell death (8, 9). Mediators in tive bacteria and is known as a potent activator of proin- both the MAPK and PI3K signaling pathways have been reported L flammatory responses in various types of cells (1–3). LPS to play important roles in regulating apoptosis (10). JNK is a can induce cell death indirectly via the secretion of TNF-␣ or NO MAPK that responds to LPS by inducing inflammation in some in macrophages or endothelial cells (EC)3 (4–6). In contrast, LPS cell types (11). Phosphorylation of JNK triggers UV-induced ap- can directly induce cell death when mRNA or protein synthesis is optosis (12) and apoptosis of EC (13). On the other hand, inhibi- inhibited (7). This suggests that the LPS may inhibit de novo tion of JNK phosphorylation can also induce apoptosis in EC (14). mRNA or protein synthesis of antiapoptotic proteins and thus These reports suggest a relationship between JNK and induction of by guest on October 1, 2021 cause cell death of EC or macrophages. apoptosis. LPS induces apoptosis in human dermal microvessel EC To determine the molecular bases underlying differences in the (HMEC) only in the presence of protein synthesis inhibitors such response to LPS in these two types of EC (TBBEC vs HMEC), we as cycloheximide (CHX). However, in transformed bovine brain investigated signaling by the MAPK and PI3K pathways after LPS stimulation. In this study, we show that the cell death induced by LPS in TBBEC is apoptotic and is followed by cell necrosis. More- *Division of Pediatrics Infectious Diseases and Immunology, Immunobiology Re- over, we determined that the JNK signaling pathway through ty- search Institute, †Women’s Cancer Research Institute, and ‡Inflammatory Bowel Dis- rosine kinase and PI3K pathways plays an important role in the ease Center, Cedars-Sinai Medical Center, David Geffen School of Medicine, Uni- versity of California Los Angeles, CA 90048 induction of apoptosis in TBBEC. In contrast, in HMEC, LPS does not induce phosphorylation of JNK, while CHX forced sustained Received for publication April 29, 2008. Accepted for publication April 1, 2009. phosphorylation of JNK in LPS-treated HMEC. Inhibition of JNK The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance phosphorylation suppressed the activation of caspase 3, one feature with 18 U.S.C. Section 1734 solely to indicate this fact. of apoptosis. These results suggest that the JNK pathway is com- 1 This work was supported by National Institutes of Health Grants AI 067995, AI mon in apoptosis of TBBEC and HMEC. However, in HMEC LPS 058128, and HL66436 (to M.A.). alone does not induce JNK phosphorylation and subsequent 2 Address correspondence and reprint requests to Dr. Moshe Arditi, Division of Pe- apoptosis. diatrics Infectious Diseases and Immunology, Immunobiology Research Institute, Ce- dars-Sinai Medical Center, 8700 Beverly Boulevard, Room 4220, Los Angeles, CA 90048. E-mail address: [email protected] Materials and Methods 3 Abbreviations used in this paper: EC, endothelial cell; HMEC, human microvessel Cell culture EC; TBBEC, transformed bovine brain EC; Ac-DEVD-AMC, acetyl-DEVD-amino- methyl coumarin; Ac-DEVD-CHO, acetyl-DEVD-aldehyde; Ac-VEID-AMC, acetyl- HMEC were cultured as described previously (15, 16). TBBEC were ob- VEID-aminomethyl coumarin; Ac-YVAD-AMC, acetyl-YVAD-aminomethyl cou- tained from K. S. Kim (Johns Hopkins Hospital, Baltimore, MD) and iso- marin; Ac-YVAD-CMK, acetyl-YVAD-chloromethyl ketone; AMC, 7-amino-4- lation and purity were described extensively earlier (17) and maintained in methyl coumarin; CHX, cycloheximide; LDH, lactate dehydrogenase; PARP, F12/DMEM (Invitrogen) supplemented with 10% FBS, antibiotic/antimy- poly(ADP-ribose) polymerase; Z-Asp-CH2-DCB, carbobenzoxy-L-aspart-1-yl-[(2,6- cotic, and EC growth supplement (Upstate Biotechnology). dichlorobenzoyl)oxy]methane; Ac-IETD-AMC, acetyl-IETD- aminomethyl couma- rin; Ac-WEHD-AMC, acetyl-WEHD- aminomethyl coumarin; ICE, IL-1␤-convert- Reagents and Abs ing enzyme. LY294002, SB203580, PD98059, Ac-YVAD-AMC, AC-DEVD-AMC, Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00 AC-WEHD-AMC, and AC-VEID-AMC were obtained from Calbiochem. www.jimmunol.org/cgi/doi/10.4049/jimmunol.0801376 The Journal of Immunology 7281 FIGURE 1. Time course and dose dependency of LPS-induced cytotox- icity in TBBEC. TBBEC were seeded on 24-well plates and incubated over- night. Cells were treated with or with- out LPS and incubated for various time points (A) or treated with various concentrations of LPS for 22 h (B). A Downloaded from LDH release assay was performed as described in Materials and Methods. The results are means Ϯ SE for three .p Ͻ 0.05 ,ء .independent experiments TBBEC were seeded on coverslips and incubated overnight. Cells were treated with or without LPS and incu- http://www.jimmunol.org/ bated for 1, 2, or 4 h and stained with annexin V-fluorescein and propidium iodide (C). Representative photo- graphs of three independent experi- ments are shown. ctrl, Control. by guest on October 1, 2021 AC-IETD-AMC was purchased from Sigma-Aldrich. Highly purified phe- lysed in IL-1␤-converting enzyme (ICE) extraction buffer (19) (50 mM ␮ nol-water extracted protein-free Escherichia coli K235 LPS was obtained KCl, 5 mM EGTA, 2 mM MgCl2, 1 mM DTT, 20 M cytochalasin B, 1 from Dr. S. N. Vogel (Uniformed Services University, Bethesda, MD). The mM PMSF, 1 ␮g/ml leupeptin, 1 ␮g/ml pepstatin A, 50 ␮g/ml antipain, purity of this LPS preparation has been previously demonstrated (18). AC- and 10 ␮g/ml chymopapain in 50 mM PIPES-NaOH, pH 7.0) and soni- YVAD-CMK, AC-DEVD-CHO, and Z-Asp-CH2-DCB were from Onco- cated briefly on ice using a sonicator. Crude extracts were centrifuged at gene. Anti-FLIP Ab was obtained from Upstate Biotechnology. Anti-poly- 2000 rpm for 5 min at 4°C and subsequent dilution with 100 ␮l of the (ADP-ribose) polymerase (PARP) Ab was purchased from Santa Cruz reaction buffer (ICE standard buffer) (19), which consisted of 10% sucrose, Biotechnology. Anti-cleaved caspase 3 Ab was from Cell Signaling. 0.1% CHAPS, 10 mM DTT, and 0.1 mg/ml OVA in 100 mM HEPES- KOH (pH 7.5). The reaction was started by the addition of the substrate, Cytotoxicity assay i.e., 200 ␮M Ac-YVAD-AMC for caspase 1-like, 200 ␮M Ac-WEHD- AMC for caspase 5-like, 200 ␮M Ac-DEVD-AMC for caspase 3-like, 200 Cells were seeded at 1.5 ϫ 105 cells/well onto 24-well plates, incubated ␮M Ac-VEID-AMC for caspase 6-like, or 200 ␮M Ac-IETD-AMC for overnight, and treated with or without LPS in the presence or absence of caspase 8-like activity, followed by incubation at 37°C for 20 min for various inhibitors for various durations.
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