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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

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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 endothelial 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-aminomethyl 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 coumarin; 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 cytotoxicity in TBBEC. TBBEC were seeded on 24-well plates and incubated overnight. Cells were treated with or without 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 experiments 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. Finally, supernatants were col- caspase 1-, 5-, 6-, and 8-like activity and for 10 min for caspase 3-like lected and a lactate dehydrogenase (LDH) assay was performed by using activity. After termination of the reaction by sudden chilling of the reaction the LDH assay kit from Roche Diagnostics. Cytotoxicity was expressed as mixture on ice, the fluorescence of the cleaved 7-amino-4-methyl-couma- percentage of total LDH release according to the following formula: per- rine (AMC) was measured using a fluorescence microplate reader (excita- centage of total ϭ (experimental release Ϫ background release) ϫ 100/ tion: 360 nm, emission: 460 nm). The activity of each caspase protein was total release. calculated from a standard curve for AMC and expressed in nanomoles of Caspase activity assay AMC cleaved per min per mg cell extract protein. Time course of caspase 1-, 3-, 5-, 6-, or 8-like activity showed linearity up to 30, 15, 40, 30, and Cells were seeded in 10-cm dishes and incubated overnight, then treated 30 min, respectively, and the dose response of cell lysates for caspase 1-, with LPS in the presence or absence of various inhibitors for various du- 3-, 5-, 6-, or 8-like activity was linear up to 20, 3, 20, 10, and 10 ␮g, rations. Then cells were scraped from the dishes with a cell scraper and respectively. 7282 LPS-INDUCED VASCULAR EC APOPTOSIS

Detection of apoptotic cells by TUNEL and annexin V/propidium iodide staining Cells were seeded onto glass chamber slides. Cells were treated with or without LPS for various time periods and cells were fixed with 3% form- aldehyde. TUNEL staining was performed by using an In Situ Cell Death Detection, POD from Roche Diagnostics according to the manufacturer’s protocol. Stained cells were observed under the microscope and Ͼ300 cells were examined for TUNEL positivity. The results are expressed as relative percentage of TUNELϩ cells, based on the total number of cells counted. For annexin V/propidium iodide staining, cells were pretreated with DMSO or inhibitors for 1 h, followed by treatment with or without LPS for various time periods. Cells were washed with PBS and incubated with Annexin V-FLUOS labeling solution (Roche Diagnostics) and analyzed using a fluorescence microscope. Statistics Data are reported as the means Ϯ SE. The statistical significance of differences between mean values was determined by Student’s t test. A value of p Ͻ 0.05 was considered significant. Results

LPS induces cell death through apoptosis in TBBEC Downloaded from FIGURE 2. Time course of TUNEL positivity (A) and PARP cleavage After stimulation with LPS, we observed cell death of TBBEC that (B) in TBBEC treated with LPS. Cells were treated with 100 ng/ml LPS for was characterized by shrinkage, appearance of apoptotic bodies, various time periods as indicated. A, TUNEL staining was performed using and nuclear condensation as early as 10 h after LPS stimulation. a commercial kit according to the manufacturer’s protocol. Results shown p Ͻ 0.01. B, PARP and cleaved PARP ,ء .Next, we determined LDH release, which detects extracellular are from triplicate experiments LDH after disintegration of the cell membrane. As shown in Fig. were detected by SDS-PAGE/Western blotting with anti-PARP Ab. Shown 1, LDH release increased over time in a linear fashion up to 24 h is one representative experiment of three independent experiments. http://www.jimmunol.org/ after stimulation with 100 ng/ml LPS. Without LPS, almost no cell damage was observed (Fig. 1A). LPS induced LDH release in a dose-dependent manner and saturation was observed for stimula- Ac-DEVD-CHO (caspase 3 inhibitor), and Z-Asp-CH2-DCB (pan- tion with Ͼ100 ng/ml LPS at 22 h of stimulation (Fig. 1B). There- caspase inhibitor) suppressed the LDH release induced by LPS. fore, we used these experimental conditions (100 ng/ml LPS for Inhibitory effects were dose dependent for up to 500 ␮⌴ Ac- 22 h in TBBEC) for all LDH release experiments. YVAD-CMK or Ac-DEVD-CHO and 250 ␮M ⌮-Asp-CH2-DCB. Next, we asked whether the cell death observed in TBBEC These results indicate that caspase activation is involved in the cell treated with LPS was apoptosis. First, we analyzed annexin V death of TBBEC and suggest that the cell death detected by LDH by guest on October 1, 2021 staining in LPS-treated TBBEC as a marker for early apoptotic release is apoptosis. events. We were able to detect annexin V-positive cells as early as Kinetics of caspase activation and subsequent induction of 1 h after LPS stimulation (Fig. 1C). Next, DNA cleavage as mea- apoptosis sured by TUNEL staining was detected as early as 4 h after LPS stimulation (Fig. 2A). Furthermore, PARP cleavage was examined Next, we tried to detect caspase activity induced by LPS in because PARP is one target of activated caspases (20–23). As TBBEC. We detected a significant increase in caspase 3-like ac- shown in Fig. 2B, PARP was cleaved at 8 h after the addition of tivity at 8 h after the addition of LPS and observed an increase in LPS and cleaved PARP accumulated over time. These results sug- activity up to 16 h (Fig. 4). In addition, caspase 6- or 8-like activity gest that LPS induces apoptosis in TBBEC. increased at 10 or 12 h, respectively, and continued to increase up To identify what signaling pathways are involved leading to cell to 16 h, but the activity was less than caspase 3-like activity (Fig. death after stimulation with LPS, we used several inhibitors of 4). No increase in caspase 3-, 6- or 8-like activity was detected signaling pathways involved in the induction of apoptosis. Herbi- without LPS stimulation (Fig. 4). In contrast, caspase 1 or caspase mycin A, a tyrosine kinase inhibitor (24), significantly suppressed 5-like activity was not detected after stimulation with LPS (Fig. 4). LDH release (Fig. 3A). We observed similar inhibitory effects with concentrations up to 20 ␮⌴ (data not shown). In contrast, Signaling pathway to induce apoptosis in TBBEC SB203580, a p38 MAPK inhibitor (25–27), PD98059, an inhibitor To clarify what molecular signaling components are involved in of MEK1 (28), and LY294002, an inhibitor of PI3K (29), failed to LPS-induced activation of caspases, we used several inhibitors. suppress LDH release induced by LPS (Fig. 3A). No inhibiting Caspase 3-like activity induced by LPS was significantly sup- effects were observed for up to 50 ␮⌴ PD98059 or LY294002 pressed by herbimycin A or LY294002, but PD98059 had no effect when cells were stimulated with LPS (data not shown). These re- (Fig. 5A). We detected significant inhibition of caspase 6-like ac- sults suggest that tyrosine kinase activation is involved in LPS- tivity after addition of herbimycin A and a strong trend of inhibi- induced cell death in TBBEC. To confirm that herbimycin A in- tion after LY294002 (Fig. 5B), but caspase 8-like activity was not hibits apoptosis in LPS-treated TBBEC, we analyzed annexin V inhibited by any of the inhibitors used in Fig. 5 (data not shown). binding in TBBEC treated with LPS and herbimycin A. Herbimy- These results suggest that LPS activates caspases through tyrosine cin A inhibited the apoptosis as measured by annexin V binding of kinases and PI3K, which ultimately leads to LDH release. LPS-treated TBBEC at 2 h while PD98059 did not inhibit apopto- It has been reported that JNK plays an important role in induc- sis in LPS-treated TBBEC (Fig. 3B). Similar results were obtained tion of apoptosis (12, 13). Moreover, our data showed involvement at1and4hofLPSstimulation (data not shown). of tyrosine kinase, which is upstream of MAPK, but neither p38 Next, we performed experiments with several caspase inhibi- nor ERK1/2 seem to be involved in LPS-induced apoptosis in tors. As shown in Fig. 3, Ac-YVAD-CHO (caspase 1 inhibitor), TBBEC (Figs. 3 and 5). First, we performed experiments with the The Journal of Immunology 7283

FIGURE 3. Effects of various inhibitors on LPS-induced cytotoxicity in TBBEC. Cells were pretreated with 0.5% DMSO, 250 ␮M Ac-YVAD- CHO, Ac-DEVD-CHO, Z-Asp-CH2- DCB, 13.3 ␮M SB203580, 5 ␮M herbimycin A, 25 ␮M PD98059, or 5 ␮M LY294002 for 1 h and then treated with or without 100 ng/ml LPS for 22 h. Cytotoxicity was as- sayed by a LDH release assay (A). Re- sults are means Ϯ SE for at least three ;p Ͻ 0.02 ,ء .independent experiments .p Ͻ 0.001 ,ءءء p Ͻ 0.01; and ,ءء Cells were pretreated with 0.5% Downloaded from DMSO, 5 ␮M herbimycin A, or 25 ␮M PD98059 for 1 h and then treated with or without 100 ng/ml LPS for 2 h. Apoptosis was detected by annexin V-ﬂuorescein and propidium iodide staining. Representative photo- http://www.jimmunol.org/ graphs of three independent experiments are shown (B). by guest on October 1, 2021

JNK inhibitor SP600125. As shown in Fig. 6, SP600125 suppressed cytotoxicity induced by LPS in a dose-dependent manner. Next, we analyzed the effect of SP600125 on LPS-induced caspase activation. Forty micromolar SP600125 inhibited caspase 3- and 6-like activity signiﬁcantly but not completely (Fig. 7). As shown in Fig. 5, LY294002 suppressed activation of caspases 3 and 6, suggesting that both PI3K and JNK signaling pathways are involved in the induction of apoptosis in TBBEC. Actually, the combination of SP600125 with LY294002 inhibited LPS-induced caspase 3 and 6 activities completely (Fig. 7). These results suggest that LPS induces apoptosis by engaging PI3K and JNK signaling pathways through tyrosine kinase in TBBEC.

Kinetics of mitogen-activated proteins and caspase 3 activation in HMEC In contrast to TBBEC, LPS does not induce apoptosis in HMEC (7). However, in the presence of the protein synthesis inhibitor CHX, LPS induces apoptosis in HMEC or macrophages (7). More- over, CHX itself does not induce apoptosis at the concentration used to inhibit de novo protein synthesis (30), suggesting that the LPS signaling pathway is involved in induction of apoptosis. To investigate why HMEC are protected from LPS-induced apoptosis, we analyzed activation of MAPK in response to LPS in the pres- FIGURE 4. Time course of induction of caspase-like activities in TB- ence or absence of CHX in HMEC. As shown in Fig. 8, phosphor- BEC treated with LPS. Cells were treated with (Œ, F, and f) or without (‚, E, and Ⅺ) 100 ng/ml LPS for various time periods as labeled on the ylation of JNK was observed 2 h after the addition of LPS and abscissa. After incubation, cells were lysed with ICE extraction buffer and CHX, and this activation was sustained for6hinthecase of caspase-like activities were measured in ICE standard buffer as described combined LPS and CHX treatment. Moreover, activation of in Materials and Methods. The results are means Ϯ SE from four inde- caspase 3, a typical feature of apoptosis, occurred at 4 h and was p Ͻ 0.05. sustained for at least 6 h (Fig. 8). These results show that ,ء .pendent experiments 7284 LPS-INDUCED VASCULAR EC APOPTOSIS Downloaded from http://www.jimmunol.org/

FIGURE 7. Effects of the combination of LY294002 (LY) and SP600125 (SP) on LPS-induced activation of caspase 3 (A) and caspase 6 (B) in TBBEC. Cells were pretreated with 40 ␮M SP600125 plus 5 ␮M LY294002 for 1 h and cultured with 100 ng/ml LPS for 14 h. Then caspase 3 and caspase 6 activities were measured as described in Materials and FIGURE 5. Effects of various inhibitors on LPS-induced activation of Methods. The results are means Ϯ SE from three independent experiments. .p Ͻ 0.0001 ,ءءءء p Ͻ 0.001; and ,ءءء ;p Ͻ 0.01 ,ءء ;p Ͻ 0.02 ,ء ,caspase 3 (A) and caspase 6 (B). Cells were treated with 0.5% DMSO 5 ␮M LY294002, 25 ␮M PD98059, or 5 ␮M herbimycin A for 1 h by guest on October 1, 2021 followed by incubation for 14 h without or with LPS. The results shown p Ͻ 0.05; might be involved in the induction of apoptosis in HMEC. To ,ء .are means Ϯ SE from four independent experiments Ͻ ءءء Ͻ ءء , p 0.02; and , p 0.01. further investigate whether differences in the JNK pathway activation can fully account for these differences in susceptibility phosphorylation of JNK precedes cleavage of caspase 3 in HMEC treated with LPS and CHX. On the other hand, activation of p38, ERK1/2, or AKT showed no correlation with the activation of caspase 3 (Fig. 8). These results suggest that the JNK pathway

FIGURE 6. Effect of SP600125 on LPS-induced cytotoxicity in TB- FIGURE 8. Time course of LPS-induced activation of caspase 3 and BEC. Cells were preincubated with various concentration of SP600125 for MAPK in HMEC. HMEC were treated with or without 100 ng/ml LPS and 1 h and then treated with 100 ng/ml LPS for 22 h. LDH was measured in with or without 40 ␮g/ml CHX for the indicated times. Western blotting the supernatants. The results are means Ϯ SE from four independent ex- experiments were performed as described in Materials and Methods. One .p Ͻ 0.02. representative experiment of three independent experiments is shown ,ء .periments The Journal of Immunology 7285

through JNK and PI3K. Furthermore, time-course experiments of expression of FLIP, an inhibitor of caspase 8 (9), did not show any difference up to 12 h after the addition of LPS in TBBEC (data not shown), suggesting that caspase 8 is not important for LPS-induced TBBEC apoptosis. These results suggest that any contribu- tion of FLIP to LPS-induced apoptosis is minimal. Our study showed that the PI3K pathway is involved in the activation of caspase 3 or 6. The PI3K signaling pathway, which also includes AKT, is part of a survival signaling pathway (34, 35). One mechanism is that AKT phosphorylates apoptosis signal-regulating kinase 1 and inhibits proapoptotic function in intestinal epithelial cells (34). We observed that AKT is phosphorylated in response to LPS plus CXH in HMEC, conditions that lead to ap- FIGURE 9. Effect of JNK inhibition on LPS-induced activation of JNK optosis in these EC. This is consistent with previous observations and caspase 3 in the presence of CHX. Cells were pretreated with 25 ␮M that LPS induces both pro- and antiapoptotic pathways in EC (36– SP600125 for 1 h and stimulated with LPS, CHX, or LPS plus CHX for 39). However, it is still unknown how PI3K and JNK signaling 6 h. Western blotting was performed as described. pathways cooperate in the induction of caspase activity in LPS-treated EC. Furthermore, we demonstrate here that JNK phosphorylation is between HMEC and TBBEC, we examined phosphorylation of a common signaling pathway inducing apoptosis in TBBEC and Downloaded from JNK in response to LPS in primary human coronary aortic EC HMEC treated with LPS. Although, LPS directly induced apopto- (HCAEC). Consistent with our findings in HMEC, we observed sis in TBBEC, in HMEC LPS alone was unable to induce JNK that LPS does only induce minute phosphorylation of JNK in phosphorylation, but the presence of inhibitors of de novo protein HCAEC. However, treatment of HCAEC with LPS and CHX leads synthesis induces JNK phosphorylation followed by caspase 3 to an increase of JNK phosphorylation (data not shown). cleavage and apoptosis. Inhibition of JNK activation efficiently blocked caspase 3 cleavage and apoptosis in TBBEC and HMEC. http://www.jimmunol.org/ The role of JNK in induction of apoptosis in HMEC treated LPS activates proapoptotic and antiapoptotic pathways (7). In with LPS and CHX in HMEC HMEC, LPS induced apoptosis in the presence of either CHX or To verify the involvement of JNK in LPS/CHX-induced apoptosis actinomycin D (7). This indicates that de novo mRNA or protein in HMEC, we used an inhibitor of JNK. As shown in Fig. 9, 25 synthesis induced by LPS is necessary for protection from apo- ␮M SP600125 suppressed phosphorylation of JNK completely, ptosis, suggesting that LPS itself induces antiapoptotic effects. We but had no effect on JNK protein expression. Cleaved caspase 3 have previously demonstrated that the addition of CHX after Ͼ30 induced by addition of LPS and CHX was also suppressed by the min of LPS stimulation blocked LPS in macrophages (30), sup- addition of SP600125 compared with DMSO controls (Fig. 9), porting the notion that early de novo protein synthesis induced by by guest on October 1, 2021 suggesting that the JNK pathway is involved in the induction of LPS has antiapoptotic effects. Previous studies report that FLIP apoptosis in HMEC by LPS and CHX. In addition, it has been inhibits apoptosis in HMEC and, accordingly, CHX decreases reported that FLIP is induced by LPS itself but CHX suppresses FLIP expression and thereby promotes apoptosis in HMEC (9). this expression. Since FLIP functions as inhibitor of caspase 8 However, we did not observe any differences in FLIP protein ex- activation, we determined whether FLIP expression is altered in pression in LPS-treated HMEC, CHX-treated HMEC or LPS plus LPS- and CHX-treated cells (8). We did not observe a correlation CHX-treated HMEC (Fig. 9). These results suggest that other mol- between JNK activation and FLIP expression (Fig. 9). These data ecule(s) are also important for survival of HMEC-treated suggest that Flip is not involved in induction of apoptosis induced with LPS. by LPS and CHX. Recently, phosphorylation of JNK has been reported to be involved in apoptosis induction (40). However, the role of JNK dur- Discussion ing apoptosis is controversial, with some reports demonstrating In this study, we characterized LPS-induced apoptosis in bovine that JNK is proapoptotic (40), while others demonstrated protec- brain EC (TBBEC). LPS-induced signaling pathways leading to tion by JNK (41). Apoptosis signal-regulating kinase 1 was re- apoptosis in TBBEC included tyrosine kinase, JNK, and PI3K ac- ported as an associated molecule involved in sustained JNK acti- tivation, but not ERK1/2 or p38 activation. In TBBEC treated with vation (42). We show here sustained phosphorylation of JNK LPS, caspase 3 was activated first, followed by caspase 6 and followed by sustained caspase 3 cleavage and HMEC apoptosis caspase 8. We did not observe caspase 1- or caspase 5-like pro- (Fig. 8). LPS induces transient activation of JNK (43, 44) followed tease activity (Fig. 4). These data are in contrast to our experiments by NF-␬B activation, which regulates cellular survival. In HMEC- using the caspase 1 inhibitor Ac-YVAD-CMK. Ac-YVAD-CMK treated with LPS plus CHX, we detected sustained degradation of efficiently suppressed LPS-induced cytotoxicity in TBBEC. Ac- I␬B␣ and NF-␬B activity (data not shown) during progression of YVAD-CMK inhibits both caspase 1-like and caspase 3-like ac- apoptosis. Sustained NF-␬B activation can lead to inflammation- tivity, which explains the discrepancy between our data derived induced tissue damage or malignancy (45), which might suggest from inhibitor studies and direct measurements of caspase 1 ac- that perturbation of NF-␬B regulation affects cell survival. tivity (31). These results show that activation of caspase 3 is not During sepsis, endotoxin released from bacteria affects EC func- dependent on caspase 1 or caspase 8 in LPS-induced TBBEC ap- tion and leads to induction of cytokines, adhesion molecules, and optosis, despite the fact that caspase 1 or 8 is upstream of caspase NO, which enhances sepsis by promoting further inflammation, 3 (32, 33). As shown in Figs. 5 and 6, herbimycin A, SP600125, coagulation, and a decrease of blood pressure (9). In addition, en- or LY294002 suppressed LPS induction of caspase 3 or 6, but did dotoxin induces apoptosis of vessel EC. In this study, we elucidate not inhibit caspase 8 (data not shown). These results suggest that further the mechanism and molecular signaling by which LPS in- activation of caspase 3 and caspase 6 are dependent on signaling duces cytotoxicity in vascular EC. 7286 LPS-INDUCED VASCULAR EC APOPTOSIS

Acknowledgments 23. Lazebnik, Y. A., S. H. Kaufmann, S. Desnoyers, G. G. Poirier, and W. C. Earnshaw. 1994. Cleavage of poly(ADP-ribose) polymerase by a protein- We thank Wenxuan Zhang for excellent technical assistance. ase with properties like ICE. Nature 371: 346–347. 24. Obinata, A., Y. Akimoto, H. Hirano, and H. Endo. 1991. Stimulation by Bt2cAMP of epidermal mucous metaplasia in retinol-pretreated chick embryonic Disclosures cultured skin, and its inhibition by herbimycin A, an inhibitor for protein-tyrosine The authors have no financial conflict of interest. kinase. Exp. Cell Res. 193: 36–44. 25. Jiang, Y., C. Chen, Z. Li, W. Guo, J. A. Gegner, S. Lin, and J. Han. 1996. Characterization of the structure and function of a new mitogen-activated protein References kinase (p38␤). J. Biol. Chem. 271: 17920–17926. 1. Matic, M., and S. R. Simon. 1991. Tumor necrosis factor release from lipopo- 26. Kramer, R. M., E. F. Roberts, S. L. Um, A. G. Borsch-Haubold, S. P. Watson, lysaccharide-stimulated human monocytes: lipopolysaccharide tolerance in vitro. M. J. Fisher, and J. A. Jakubowski. 1996. p38 mitogen-activated protein kinase Cytokine 3: 576–583. phosphorylates cytosolic phospholipase A2 (cPLA2) in thrombin-stimulated 2. Morrison, D. C., and J. L. Ryan. 1979. Bacterial endotoxins and host immune platelets: evidence that proline-directed phosphorylation is not required for mo- responses. Adv. Immunol. 28: 293–450. bilization of arachidonic acid by cPLA2. J. Biol. Chem. 271: 27723–27729. 3. Tanabe, Y., C. Kohchi, N. Kitahara-Tanabe, D. Mizuno, and G. Soma. 1998. 27. Lee, J. C., J. T. Laydon, P. C. McDonnell, T. F. Gallagher, S. Kumar, D. Green, Involvement of 26-kDa membrane-bound tumour necrosis factor precursor in D. McNulty, M. J. Blumenthal, J. R. Heys, S. W. Landvatter, et al. 1994. A bidirectional feedback regulation on 17-kDa tumour necrosis factor production protein kinase involved in the regulation of inflammatory cytokine biosynthesis. after stimulation by lipopolysaccharide. Cytokine 10: 82–92. Nature 372: 739–746. 4. Albina, J. E., S. Cui, R. B. Mateo, and J. S. Reichner. 1993. Nitric oxide-mediated 28. Dudley, D. T., L. Pang, S. J. Decker, A. J. Bridges, and A. R. Saltiel. 1995. A apoptosis in murine peritoneal macrophages. J. Immunol. 150: 5080–5085. synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc. Natl. 5. Sarih, M., V. Souvannavong, and A. Adam. 1993. Nitric oxide synthase induces Acad. Sci. USA 92: 7686–7689. macrophage death by apoptosis. Biochem. Biophys. Res. Commun. 191: 503–508. 29. Avruch, J. 1998. Insulin signal transduction through protein kinase cascades. 6. Woods, K. M., and S. K. Chapes. 1993. Three distinct cell phenotypes of in- Mol. Cell. Biochem. 182: 31–48. duced-TNF cytotoxicity and their relationship to apoptosis. J. Leukocyte Biol. 53: 30. Amano, F., and H. Karahashi. 1996. A cytotoxic effect of lipopolysaccharide on 37–44. a macrophage-like cell line, J774.1, in the presence of cycloheximide. 7. Pohlman, T. H., and J. M. Harlan. 1989. Human endothelial cell response to J. Endotoxin Res. 3: 415–423. Downloaded from lipopolysaccharide, interleukin-1, and tumor necrosis factor is regulated by pro- 31. Karahashi, H., and F. Amano. 2000. Changes of caspase activities involved in tein synthesis. Cell Immunol. 119: 41–52. apoptosis of a macrophage-like cell line J774.1/JA-4 treated with lipopolysac- 8. Bannerman, D. D., M. Sathyamoorthy, and S. E. Goldblum. 1998. Bacterial li- charide (LPS) and cycloheximide. Biol. Pharm. Bull. 23: 140–144. popolysaccharide disrupts endothelial monolayer integrity and survival signaling 32. Enari, M., R. V. Talanian, W. W. Wong, and S. Nagata. 1996. Sequential acti- events through caspase cleavage of adherens junction proteins. J. Biol. Chem. vation of ICE-like and CPP32-like proteases during Fas-mediated apoptosis. Na- 273: 35371–35380. ture 380: 723–726. 9. Bannerman, D. D., J. C. Tupper, W. A. Ricketts, C. F. Bennett, R. K. Winn, and 33. Muzio, M., G. S. Salvesen, and V. M. Dixit. 1997. FLICE induced apoptosis in

J. M. Harlan. 2001. A constitutive cytoprotective pathway protects endothelial a cell-free system. Cleavage of caspase zymogens. J. Biol. Chem. 272: http://www.jimmunol.org/ cells from lipopolysaccharide-induced apoptosis. J. Biol. Chem. 276: 2952–2956. 14924–14932. 34. Kim, A. H., G. Khursigara, X. Sun, T. F. Franke, and M. V. Chao. 2001. AKT 10. Frey, E. A., and B. B. Finlay. 1998. Lipopolysaccharide induces apoptosis in a phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. bovine endothelial cell line via a soluble CD14 dependent pathway. Microb. Mol. Cell. Biol. 21: 893–901. Pathog. 24: 101–109. 35. Zhang, H. M., J. N. Rao, X. Guo, L. Liu, T. Zou, D. J. Turner, and J. Y. Wang. 11. Rui, Y., X. Liu, N. Li, Y. Jiang, G. Chen, X. Gao, and J. Wang. 2007. PECAM-1 2004. AKT kinase activation blocks apoptosis in intestinal epithelial cells by ligation negatively regulates TLR4 signaling in macrophages. J. Immunol. 179: inhibiting caspase-3 after polyamine depletion. J. Biol. Chem. 279: 7344–7351. 22539–22547. 12. Lai, Y., D. Xin, J. Bai, and Y. Na. 2007. The important anti-apoptotic role and 36. Hu, X., E. Yee, J. M. Harlan, F. Wong, and A. Karsan. 1998. Lipoplysaccharide its regulation mechanism of PTTG1 in UV-induced apoptosis. J. Biochem. Mol. induces the antiapoptotic molecules, A1 and A2, microvascular endothelial cells. Biol. 40: 966–972. Blood 92: 2759–2765.

13. Ho, F. M., W. W. Lin, B. C. Chen, C. M. Chao, C. R. Yang, L. Y. Lin, C. C. Lai, 37. Choi, K. B., F. Wong, J. M. Harlan, P. M. Chaudhary, L. Hood, and A. Karsan. by guest on October 1, 2021 S. H. Liu, and C. S. Liau. 2006. High glucose-induced apoptosis in human vas- 1998. Lipoplysaccharide mediates endothelial apoptosis by a FADD-dependent cular endothelial cells is mediated through NF␬B and prevented by PI3K/AKT/ pathway. J. Biol. Chem. 273: 20185–20188. eNOS pathway. Cell. Signal. 18: 391–399. 38. Hull, C., G. McLean, F. Wong, P. J., Duriez, and A. Karsan. 2002. Lipoplysac- 14. Kirby, J. E. 2004. Anthrax lethal toxin induces human endothelial cell apoptosis. charide signals an endothelial apoptosis pathway through TNF receptor-associ- Infect. Immun. 72: 430–439. ated factor 6-mediated activation of c-Jun NH2-terminal kinase. J. Immunol. 169: 15. Ades, E. W., F. J. Candal, R. A. Swerlick, V. G. George, S. Summers, 2611–2618 D. C. Bosse, and T. J. Lawley. 1992. HMEC-1: establishment of an immortalized 39. Wong, F., C. Hull, R. Zhande, J. Law, and A. Karsan. 2004. Lipopolysaccharide human microvascular endothelial cell line. J. Invest. Dermatol. 99: 683–690. initiates a TRAF 6-mediated endothelial survival signal. Blood 103: 4520–4526. 16. Zhang, F. X., C. J. Kirschning, R. Mancinelli, X. P. Xu, Y. Jin, E. Faure, 40. Kasper, B., E. Brandt, S. Brandau, and F. Petersen. 2007. Platelet factor 4 (CXC A. Mantovani, M. Rothe, M. Muzio, and M. Arditi. 1999. Bacterial lipopolysac- chemokine ligand 4) differentially regulates respiratory burst, survival, and cy- charide activates nuclear factor-␬B through interleukin-1 signaling mediators in tokine expression of human monocytes by using distinct signaling pathways. cultured human dermal endothelial cells and mononuclear phagocytes. J. Biol. J. Immunol. 179: 2584–2591. Chem. 274: 7611–7614. 41. Yang, S., M. Lim, L. K. Pham, S. E. Kendall, H. Reddi, D. C. Altieri, and 17. Stins, M. F., N. V. Prasadarao, J. Zhou, M. Arditi, and K. S. Kim. 1997. Bovine P. Roy-Burman. 2006. Bone morphogenetic protein 7 protects prostate cancer brain microvascular endothelial cells transfected with SV40-large T antigen: de- cells from stress-induced apoptosis via both smad and c-jun NH2-terminal kinase velopment of an immortalized cell line to study pathophysiology of CNS disease. pathways. Cancer Res. 66: 4285–4290. In Vitro Cell Dev. Biol. Anim. 33: 243–247. 42. Tobiume, K., A. Matsuzawa, T. Takahashi, H. Nishitoh, K. Morita, K. Takeda, 18. Hirschfeld, M., Y. Ma, J. H. Weis, S. N. Vogel, and J. J. Weis. 2000. Cutting O. Minowa, K. Miyazono, T. Noda, and H. Ichijo. 2001. ASK1 is required for edge: repurification of lipopolysaccharide eliminates signaling through both hu- sustained activation of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2: man and murine Toll-like receptor 2. J. Immunol. 165: 618–622. 222–228. 19. Enari, M., A. Hase, and S. Nagata. 1995. Apoptosis by a cytosolic extract from 43. Rui, Y., X. Liu, N. Li, Y. Jiang, G. Chen, X. Cao, and J. Wang. 2007. PECAM-1 Fas-activated cells. EMBO J. 14: 5201–5208. ligation negatively regulates TLR4 signaling in macrophages. J. Immunol. 179: 20. Althaus, F. R., and C. Richter. 1987. ADP-ribosylation of proteins: enzymology 7344–7351. and biological significance. Mol. Biol. Biochem. Biophys. 37: 1–237. 44. Qin, H., K. L. Roberts, S. A. Niyongere, Y. Cong, C. O. Elson, and 21. de Murcia, G., A. Huletsky, and G. G. Poirier. 1988. Modulation of chromatin E. N. Benveniste. 2007. Molecular mechanism of lipopolysaccharide-induced structure by poly(ADP-ribosyl)ation. Biochem. Cell Biol. 66: 626–635. SOC-3 gene expression in macrophages and microglia. J. Immunol. 179: 22. Kaufmann, S. H., S. Desnoyers, Y. Ottaviano, N. E. Davidson, and G. G. Poirier. 5966–5976. 1993. Specific proteolytic cleavage of poly(ADP-ribose) polymerase: an early 45. Karin, M., and F. R. Greten. 2005. NF␬B: linking inflammation and immunity to marker of chemotherapy-induced apoptosis. Cancer Res. 53: 3976–3985. cancer development and progression. Nat. Rev. Immunol. 5: 749–759.