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The Journal of Immunology, 2002, 169: 2611-2618.
Copyright © 2002 by The American Association of Immunologists

Lipopolysaccharide Signals an Endothelial Apoptosis Pathway Through TNF Receptor-Associated Factor 6-Mediated Activation of c-Jun NH2-Terminal Kinase1

Christopher Hull*,{dagger}, Graeme McLean*,{ddagger}, Fred Wong*,{dagger}, Patrick J. Duriez*,{dagger} and Aly Karsan2,*,{dagger},{ddagger}

* Departments of Pathology and Laboratory Medicine, and Medical Biophysics, British Columbia Cancer Agency, British Columbia, Canada; and Departments of {dagger} Pathology and Laboratory Medicine and {ddagger} Experimental Medicine, University of British Columbia, British Columbia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Inflammatory mediators such as TNF and bacterial LPS do not cause significant apoptosis of endothelial cells unless the expression of cytoprotective genes is blocked. In the case of TNF, the transcription factor NF-{kappa}B conveys an important survival signal. In contrast, even though LPS can also activate NF-{kappa}B, this signal is dispensable for LPS-inducible cytoprotective activity. LPS intracellular signals are transmitted through a member of the Toll-like receptor family, TLR4. This family of receptors transduces signals through a downstream molecule, TNFR-associated factor 6 (TRAF6). In this study, we demonstrate that the C-terminal fragment of TRAF6 (TRAF6-C) inhibits LPS-induced NF-{kappa}B nuclear translocation and c-Jun NH2-terminal kinase (JNK) activation in endothelial cells. In contrast, LPS activation of p38 kinase is not inhibited by TRAF6-C. TRAF6-C also inhibits LPS-initiated endothelial apoptosis, but potentiates TNF-induced apoptosis. LPS-induced loss of mitochondrial transmembrane potential, cytochrome c release, and caspase activation are all blocked by TRAF6-C. We demonstrate that TRAF6 signals apoptosis via JNK activation, since inhibition of JNK activation using a dominant-negative mutant also inhibits apoptosis. JNK inhibition blocks caspase activation, but the reverse is not true. Hence, JNK activation lies upstream of caspase activation in response to LPS stimulation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lipopolysaccharide is a critical glycolipid component of the outer wall of Gram-negative bacteria, and many of the cellular signals activated by Gram-negative bacteria are attributed to LPS (1). Endothelial injury plays a crucial role in the pathogenesis of septic shock due to Gram-negative bacteria (2). However, at the concentrations of LPS measured in patients’ sera during sepsis, there is minimal direct toxicity of LPS on cultured human endothelial cells (3). In contrast, when the expression of new genes is inhibited using cycloheximide (CHX)3 or actinomycin D, endothelial cells become sensitized to the apoptotic signals evoked by LPS (3, 4, 5). A similar phenomenon has been described for TNF (6). While exposure to TNF does not cause endothelial apoptosis, the combination of TNF and CHX or actinomycin D does (6). In both cases, the concomitant induction of antiapoptotic genes has been shown to inhibit the apoptotic pathway that is activated (4, 6). In the case of the apoptotic pathway, however, the molecules are constitutively expressed, and simply require posttranslational activation to function.

The receptor that transduces the LPS signal has been recognized to be a member of the Toll-like receptor (TLR) family (7, 8). At least 10 members of the family have been identified to date, and TLR4 appears to be the main LPS receptor although coreceptors are most likely involved (9, 10, 11). Ligand activation of the TLRs results in recruitment of the adaptor protein, MyD88 (12). MyD88 in turn signals through a complex comprising the IL-1R-associated kinases and TNFR-associated factor 6 (TRAF6) (13, 14, 15). TRAF6 through a series of kinases transmits the LPS signal to activate NF-{kappa}B (13, 16, 17). Activation of various receptors of the inflammatory response, including the TLRs, also results in activation of c-Jun NH2-terminal kinase (JNK). However, whether activation of JNK lies downstream of TRAF6 may depend on the particular inflammatory mediator and cell type (17, 18, 19, 20).

Previous studies have demonstrated that TLRs can initiate apoptosis through a death domain interaction of MyD88 with Fas-associated death domain protein (FADD) (5, 21). This interaction in turn initiates a caspase cascade, and ultimately cell death. While caspase inhibition is sufficient to block LPS-triggered endothelial apoptosis, it is clear that there are multiple feed-forward, amplification steps as well as inhibitory steps that are recruited following a death signal, and it is the balance of positive and negative apoptosis regulators that determines the outcome in an individual cell (5, 22). The downstream signaling events that positively regulate cell death in response to LPS have not been well investigated, particularly in endothelial cells.

In this study, we demonstrate that LPS activates both NF-{kappa}B and JNK through TRAF6 in endothelial cells. A C-terminal fragment of TRAF6 (TRAF6-C) inhibits NF-{kappa}B and c-Jun NH2-terminal kinase (JNK) activation in endothelial cells. Whereas inhibition of NF-{kappa}B sensitizes endothelial cells to TNF-induced death, NF-{kappa}B inhibition does not potentiate LPS-induced death. Notably, however, TRAF6-C is able to inhibit LPS- but not TNF-induced apoptosis. In contrast, a C-terminal fragment of TRAF2 (TRAF2-C) does not inhibit LPS-induced apoptosis. This inhibition of LPS-initiated apoptosis is due to the inhibition of JNK by TRAF6-C, since a JNK dominant-negative (JNK-APF) (23) mutant also inhibits LPS-induced apoptosis. Dominant-negative JNK is able to inhibit caspase activation, but the reverse is not true. Thus, LPS can initiate a TRAF6-JNK-mediated death pathway that is upstream of caspase activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell lines and reagents

Human microvascular endothelial cells-1 (hereafter referred to as HMEC) were provided by the Center for Disease Control and Prevention (Atlanta, GA) and were cultured in MCDB-131 medium supplemented with 10% FCS and 10 µg/ml epidermal growth factor. Flag-tagged TRAF cDNAs (TRAF2-C (aa 87–501) and TRAF6-C (aa 289–522)) were provided by Tularik (South San Francisco, CA) and Flag-I-{kappa}Bmt (I-{kappa}B{alpha}S32/36A) was a gift of D. Ballard (Vanderbilt University, Nashville, TN) (13, 24). The various constructs were cloned into the retroviral vectors: LNCX, LXSH (gift of A. D. Miller, Fred Hutchinson Cancer Research Center, Seattle, WA), or MSCVpac (gift of R. Hawley, American Red Cross, Rockville, MD). Transient transfections of the Ampho Phoenix packaging cell line (gift of G. Nolan, Stanford University, Stanford, CA) were conducted by calcium phosphate precipitation. Viral supernatants were used to transduce HMEC, and cell lines were selected in G418, hygromycin, and/or puromycin, as previously described (6). Expression of the various proteins was confirmed by immunoblotting with the anti-Flag (M5) Ab, as described (6). Polyclonal cell lines were used to avoid artifacts due to retroviral integration site.

EMSA

HMEC nuclear extracts were prepared following incubation with LPS at 100 ng/ml for 30 min. Cells were washed twice in ice-cold PBS, scraped into buffer A (10 mM HEPES-KOH, pH 7.8, 1.5 mM MgCl2, 10 mM KCl), pelleted briefly, and lysed in buffer A + 0.5% Nonidet P-40 for 10 min on ice. Nuclei were isolated by centrifugation for 10 min at 12,000 x g, then washed, repelleted, and incubated for 20 min in buffer C (50 mM HEPES-KOH, pH 7.8, 50 mM KCl, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA, 10% glycerol). Nuclear membranes were then removed by centrifuging for 10 min at 12000 x g, and the supernatant/nuclear extract was removed and frozen at -70°C until needed.

A total of 15 µg nuclear extract was diluted in 4 vol binding buffer (10 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA pH 8.0, 4% glycerol), 2 µg of poly(dIdC) (Sigma-Aldrich, St. Louis, MO), and 1 µl of double-stranded 32P-labeled oligonucleotide corresponding to the consensus NF-{kappa}B binding site, and incubated at room temperature for 20 min. This binding reaction was then loaded onto a 5% nondenaturing 0.5x Tris borate EDTA-polyacrylamide gel that had been prerun for 1 h at 80 V. Electrophoresis was conducted at 80 V for 1.5–2 h at room temperature before drying the gel and carrying out autoradiography.

Viability assay

To quantitate the proportion of viable cells, HMEC were seeded on 96-well plates at a density of 30,000 cells/well. On the following day, cells were incubated in TNF or LPS with or without CHX (50 µg/ml) for 14 h. Viable cell numbers were estimated by neutral red uptake, as described (25).

Mitochondrial membrane potential

To measure mitochondrial transmembrane potential, 5 x 105 cells were incubated with 3,3'-dihexyloxacarbocyanine iodide (Molecular Probes, Eugene, OR) and analyzed for fluorescence on a flow cytometer (25). Similar results were seen when the fluorescent dye, TMRE, was used. The mitochondrial uncoupler, carbonyl cyanide m-chlorophenylhydrazone (Sigma-Aldrich), was used as a positive control for the detection of mitochondrial depolarization (data not shown).

Caspase activity assay

Cleavage of the tetrapeptide substrates, IETD-p-nitroaniline (pNA) or DEVD-pNA, was performed with a colorimetric assay kit, according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). Briefly, 200 µg of whole cell lysates from HMEC cells exposed to 100 ng/ml LPS and 50 µg/ml CHX for various times were combined with 200 µM IETD-pNA or DEVD-pNA in a 96-well plate and incubated at 37°C. The release of the chromophore by active caspases was quantitated at 405 nm and normalized to untreated cell lysates.

Assessment of JNK activation

JNK activity was determined by in vitro solid-phase kinase assay, as described previously (26). Briefly, HMEC cell lines were stimulated with LPS at 100 ng/ml for various times, as indicated. Total cell lysate (300 µg) was mixed with 40 µl glutathione-agarose suspension to which 10 µg of GST-c-Jun-1–79 (gift of Kinetek Pharmaceuticals, Vancouver, Canada) was bound. The mixture was rotated at 4°C for 3 h to overnight. The kinase reaction was performed as described (26), with the following modifications. The reaction was stopped with 10 µl of 4x SDS sample buffer. Samples were boiled for 10 min and then pelleted at 10,000 x g for 20 s. A total of 35 µl of supernatant was separated by 10% SDS-PAGE to resolve phosphorylated proteins. Resolved proteins were transferred to a nitrocellulose membrane, followed by autoradiography. In parallel, 50 µg of cell lysate was subjected to 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for phospho-JNK (New England Biolabs, Beverly, MA). Membranes were stripped and reprobed with an anti-JNK Ab (Santa Cruz Biotechnology, Santa Cruz, CA) as a loading control.

Immunoblotting

Total cellular extracts were prepared from HMEC, fractionated by SDS-PAGE, electrotransferred onto nitrocellulose membranes, and detected by immunoblotting, as described (25). To separate intracellular membrane and cytosolic fractions, cells were lysed in 0.025% digitonin, and pellet (membrane) and supernatant (cytosolic) fractions were separated. Fractionation of mitochondria into the membrane fraction was confirmed in each experiment by probing with anti-cytochrome c oxidase. Equal loading of lanes was confirmed by Ponceau staining the membranes, and in some cases by stripping and reprobing blots with an anti-tubulin Ab. Anti-cytochrome c was purchased from BD PharMingen (San Diego, CA), and anti-cytochrome c oxidase from Molecular Probes (Eugene, OR). Anti-MAPK and anti-phospho-MAPK Abs were purchased from Cell Signaling Technology (Beverly, MA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF activates parallel apoptotic and antiapoptotic pathways, with the antiapoptotic pathway requiring the expression of new genes (3, 6). A major TNF-induced antiapoptotic pathway is routed through activation of NF-{kappa}B (27, 28, 29). We and others have demonstrated that similar to TNF, LPS also activates parallel death and survival pathways (3, 4, 5, 30). To unmask either the TNF- or LPS-activated apoptotic pathways in endothelial (and most other) cells, expression of cytoprotective genes must be blocked with either CHX or actinomycin D (3, 4, 5). We have previously shown in HUVECs that inhibition of NF-{kappa}B sensitizes cells to TNF-, but not LPS-induced apoptosis (31). We have also shown that LPS signals a death pathway, in part through the adaptor molecule, FADD (5). Others have demonstrated that apoptotic signaling by the TLR2 receptor is mediated by a MyD88/FADD/caspase 8 pathway (21). However, propagation and amplification of the LPS-stimulated apoptotic pathway by downstream components have not been well studied. Since the major sequelae of sepsis occur at the level of the microvasculature, we used HMEC for most of these studies.

We focused our attention on TRAF6, as this molecule appears to integrate many of the LPS-generated signals (13, 16, 17). A C-terminal fragment of TRAF6 (TRAF6-C) has been shown to act as a dominant-negative molecule to inhibit NF-{kappa}B activation by the IL-1R and TLRs (13, 16). Flag epitope-tagged TRAF6-C and TRAF2-C HMEC lines were constructed to study the role of TRAF6 in HMEC (Fig. 1GoA). We confirmed that similar to HUVECs (16), TRAF6-C was able to block LPS-induced NF-{kappa}B signaling in HMEC, as measured by nuclear translocation of the NF-{kappa}B complex (Fig. 1GoB). A C-terminal fragment of TRAF2, TRAF2-C, did not block LPS- or TNF-induced NF-{kappa}B nuclear translocation. However, TRAF2-C was able to inhibit TNF-induced JNK activation (data not shown), indicating that it was able to function in a dominant-negative fashion.



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  		FIGURE 1. A transdominant inhibitor of TRAF6, TRAF6-C, inhibits NF-{kappa}B and JNK activation in HMEC treated with LPS. A, The expression of Flag-tagged TRAF2-C and TRAF6-C in transduced HMEC lines was verified by immunoblotting of total cell extracts using the M5 anti-Flag mAb. B, EMSA was performed on nuclear extracts from HMEC cells transduced with TRAF2-C, TRAF6-C, or the vector alone. Cells were left unstimulated, or stimulated with TNF (10 ng/ml) or LPS (100 ng/ml) for 30 min before harvesting nuclear extracts. C, HMEC-TRAF6-C or vector control cells were stimulated with LPS (100 ng/ml) for various times, as shown, and assessed for JNK activity by in vitro kinase assay and JNK phosphorylation by immunoblotting. Equal loading was confirmed by reprobing the membrane with an Ab against total JNK. Results shown are a single experiment representative of three independent experiments showing similar findings.

 
There is conflicting evidence as to whether TLR-mediated JNK activation lies upstream or downstream of TRAF6 (17, 18, 19, 20). This process may be cell type and/or stimulus dependent. To determine whether JNK activation lies downstream of TRAF6 in the LPS signaling pathway in HMEC, we performed phospho-JNK immunoblots as well as in vitro kinase assays using GST-c-jun as a substrate. These results demonstrate that TRAF6-C inhibits LPS-induced JNK activation, thus placing JNK activation downstream of TRAF6 in endothelial cells (Fig. 1GoC). Although the block in JNK activation does not appear complete, this is most likely due to the fact that we used polyclonal cell lines, with variable expression of TRAF6-C, to avoid integration artifacts.

Since TRAF6-C appeared to act as a dominant-negative molecule for at least two apoptosis-related pathways in HMEC, we tested whether inhibition of these signals would result in LPS-induced apoptosis in the absence of CHX. Fig. 2GoA demonstrates that TRAF6-C did not sensitize HMEC to either LPS- or TNF-induced apoptosis. In contrast, using microvascular endothelial cells transduced with a superrepressor I-{kappa}B (I-{kappa}Bmt) to block NF-{kappa}B nuclear translocation, we demonstrate that NF-{kappa}B activation is critical for TNF-, but not LPS-induced death (Fig. 2GoB).



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  		FIGURE 2. TRAF6-C does not sensitize HMEC to LPS-induced death. A, Determination of HMEC-TRAF2-C and HMEC-TRAF6-C viability following TNF or LPS stimulation. Neutral red incorporation was measured after cells were treated for 14 h with TNF (10 ng/ml) or LPS (100 ng/ml), and viability was normalized to untreated cells. B, Determination of HMEC-I-{kappa}Bmt viability following TNF or LPS stimulation. Neutral red incorporation was measured after cells were treated for 14 h with TNF (10 ng/ml) or LPS (100 ng/ml), and viability was normalized to untreated cells. Data shown are the mean ± SD of a single experiment done in triplicate and are representative of at least three independent experiments.

 
Although the differences were not significant, as there is only minimal death of LPS-stimulated HMEC, the data in Fig. 2GoA suggested that TRAF6-C may be providing a protective effect against LPS-induced apoptosis. Since NF-{kappa}B does not appear to mediate a critical survival signal in response to LPS (31), and JNK has been reported in different systems to either promote or inhibit apoptosis (32, 33, 34, 35, 36, 37, 38), we tested whether TRAF6-C would inhibit apoptosis induced by LPS and CHX (to magnify the apoptotic response). As seen in Fig. 3GoA, TRAF6-C, but not TRAF2C, was able to protect HMEC from LPS/CHX-induced death. Surprisingly, both TRAF6-C and TRAF2-C sensitized HMEC to TNF/CHX-induced death (Fig. 3GoB). The differential effect of the TRAF6-C construct in response to LPS and TNF suggests that TRAF6 signaling in endothelial cells is stimulus dependent. TRAF2-C has previously been shown to sensitize other cell types to TNF-induced death (39). As a further control, we initiated apoptosis of HMEC-TRAF6-C, HMEC-TRAF2-C, and vector-transduced HMEC with different doses of ceramide (0–50 µM). All three lines underwent cell death to similar degrees, suggesting that the LPS-specific TRAF6-C protective effect is truly stimulus dependent (Fig. 3GoC).



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  		FIGURE 3. TRAF6-C protects against LPS- but not TNF-induced endothelial death. HMEC lines were treated with LPS and CHX (A; 50 µg/ml), TNF and CHX (B; 50 µg/ml), or C2 ceramide (C) for 14 h. Cell survival was measured by neutral red incorporation and normalized to cells treated with CHX alone (A and B) or to vehicle-treated cells (C). Data shown are the mean ± SD of a single experiment done in triplicate and are representative of at least four independent experiments.

 
To determine whether LPS signaling in the presence of CHX is similar to LPS alone, HMEC were stimulated with CHX or LPS + CHX. As seen in Fig. 4GoA, CHX alone did not activate either JNK or p38 in HMEC. Fig. 4GoB shows a similar time course of JNK activation with LPS + CHX as seen with LPS alone (Fig. 1GoC). JNK activation by LPS + CHX is inhibited by TRAF6-C, as seen when cells were stimulated with LPS alone (Fig. 1GoC). Additionally, LPS + CHX activation of p38 kinase, another potential proapoptotic kinase, was not inhibited by TRAF6-C (Fig. 4GoB), and mimicked the temporal patterns of activation seen with LPS stimulation alone (data not shown).



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  		FIGURE 4. TRAF6-C inhibits LPS/CHX-induced JNK activation, but not p38 kinase activation. HMEC lines were treated with CHX (50 µg/ml) only (A), or LPS (100 ng/ml) and CHX (50 µg/ml) (B) for various times, and cell lysates were immunoblotted to detect expression of phospho- or total JNK and phospho- or total p38 kinase.

 
We also wanted to determine whether the TRAF6-C effects on JNK activation and cell survival were reproducible in primary endothelial cells. To this end, we generated bovine aortic endothelial cells (BAEC) that overexpressed TRAF6-C. Similar to HMEC, enforced expression of TRAF6-C inhibited JNK activation (Fig. 5GoA) and BAEC death (Fig. 5GoB) in response to LPS + CHX. Similar results were seen using HUVECs (data not shown). Because BAEC will undergo apoptosis in response to LPS in the absence of CHX, we tested whether TRAF6-C would protect these cells from death in response to stimulation by LPS alone. Fig. 5GoC demonstrates that inhibition of TRAF6-dependent signals by TRAF6-C prevents LPS-induced endothelial death.



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  		FIGURE 5. TRAF6-C inhibits LPS-induced JNK activation and apoptosis in primary endothelial cells. BAEC were transduced with TRAF6-C or a control vector. Following stimulation with LPS (100 ng/ml) and CHX (50 µg/ml), cell lysates were assessed for JNK activity by in vitro kinase assay (A), and cell survival by neutral red uptake at 14 h (B). The effect of TRAF6-C on BAEC survival was also tested following stimulation with LPS alone (C). Results shown are a single experiment representative of two independent experiments showing similar findings.

 
The above findings suggested that LPS may be inducing an apoptotic pathway via TRAF6-mediated JNK activation. Analysis of DNA fragmentation using propidium iodide staining and quantitation of the hypodiploid region by flow cytometry confirmed that TRAF6-C blocked LPS + CHX-initiated DNA fragmentation (data not shown). Since JNK-mediated apoptosis has been reported to act through a mitochondrial pathway (40, 41), we tested whether TRAF6-C blocks mitochondrial changes of apoptosis. Fig. 6GoA demonstrates that TRAF6-C attenuated loss of mitochondrial transmembrane potential in response to LPS + CHX. TRAF6-C also inhibited release of cytochrome c from the mitochondria to the cytosol (Fig. 6GoB).



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  		FIGURE 6. TRAF6-C inhibits the mitochondrial pathway of LPS-induced apoptosis. A, HMEC lines were treated with LPS (100 ng/ml) and CHX (50 µg/ml) for the times indicated before incubation with the lipophilic dye, 3,3'-dihexyloxacarbocyanine iodide. Loss of mitochondrial transmembrane potential ({Delta}{Psi}m) is indicated by a decrease in fluorescence, as measured by flow cytometry. The net proportion of cells with low {Delta}{Psi}m is shown. B, Cytosolic extracts from vector only- or TRAF6-C-transduced HMEC were immunoblotted to detect cytochrome c release from mitochondria. Results shown represent a single experiment of at least three independent experiments.

 
Release of cytochrome c results in caspase 3 activation through the oligomerization of Apaf1/caspase 9 (42). Caspase 8 has also been implicated in TLR-induced apoptosis (21). We thus tested for activated caspase 3 and caspase 8 in lysates from control cells or cells expressing TRAF6-C, using chromogenic caspase substrates. TRAF6-C blocked both DEVDase activity (caspase 3/7) (Fig. 7GoA) and IETDase activity (caspase 8) (Fig. 7GoB). While these substrates are not entirely specific for individual caspases (43), our results suggest that caspase 8 activity is inhibited to a greater extent than caspase 3, by TRAF6-C.



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  		FIGURE 7. TRAF6-C inhibits caspase 3/7 and caspase 8 activation induced by LPS. HMEC lines were treated with LPS (100 ng/ml) and CHX (50 µg/ml) for the times indicated before assaying for DEVD-pNA (A), or IETD-pNA cleavage activity. Results represent fold increase in activity over CHX only-treated cells, and show the mean ± SEM of three independent experiments.

 
Activation of JNK has been reported to have no effect on apoptosis, an antiapoptotic effect, or a proapoptotic effect depending on the stimulus and cell type, and whether JNK activation is transient or sustained (32, 33, 34, 35, 36, 37, 38, 44, 45). We thus tested directly whether JNK was involved in LPS-induced apoptosis. HMEC were transduced with a dominant-negative JNK in which the phosphorylation site, Thr-Pro-Tyr, is mutated to Ala-Pro-Phe (JNK-APF) (23). Expression of the JNK construct was demonstrated by immunoblotting (data not shown), and JNK-inhibitory activity was confirmed by in vitro kinase assay (Fig. 8GoA). JNK-APF did not inhibit LPS-induced extracellular signal-regulated kinase 1/2 or p38 MAPK activation (Fig. 8GoB). Fig. 8GoC demonstrates that inhibition of JNK inhibits LPS + CHX-initiated endothelial death. TRAF6-C and JNK-APF inhibit LPS-induced HMEC apoptosis to similar degrees (Fig. 8GoD), suggesting that JNK activation may be the major contributor to the TRAF6-dependent LPS-induced apoptotic pathway.



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  		FIGURE 8. JNK mediates the TRAF6-C-induced apoptotic pathway stimulated by LPS. After stimulation with LPS (100 ng/ml) and CHX (50 µg/ml) for the times shown, HMEC transduced with the empty vector or dominant-negative JNK-APF were tested for JNK activity by in vitro kinase assay (A), or extracellular signal-regulated kinase 1/2 and p38 kinase activity by immunoblotting with specific anti-phosphokinase Abs (B). C, HMEC lines were treated with LPS and CHX (50 µg/ml) for 14 h. Cell survival was measured by neutral red incorporation and normalized to cells treated with CHX alone. D, HMEC transduced with empty vector, TRAF6-C, or JNK-APF were treated with LPS and CHX (50 µg/ml) for 14 h. Cell survival was measured by neutral red incorporation and normalized to cells treated with CHX alone. Data shown for C and D are the mean ± SD of a single experiment done in triplicate and are representative of at least three independent experiments.

 
Activation of JNK has been placed either upstream or downstream of caspase activation, again depending on the model studied (34, 46, 47). To place JNK in the apoptotic cascade initiated by LPS in endothelial cells, we first tested whether JNK-APF could block caspase activation. Similar to TRAF6-C (Fig. 7Go), the dominant-negative JNK blocked both DEVDase activity (caspase 3/7) and IETDase activity (caspase 8) following 8 h of stimulation by LPS and CHX (Fig. 9GoA). In contrast, caspase inhibition, using a broad-spectrum caspase inhibitor, zVAD-fmk, did not affect activation of JNK by LPS/CHX (Fig. 9GoB). As we have previously shown, the concentration of zVAD-fmk used (50 µM) was sufficient to abrogate LPS-induced cell death in this model (Fig. 9GoC) (5). Hence, JNK activation in response to LPS lies downstream of TRAF6, but upstream of caspases.



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  		FIGURE 9. Caspases are activated downstream of JNK following LPS stimulation. A, HMEC lines were treated with LPS (100 ng/ml) and CHX (50 µg/ml) for the times indicated before assaying for DEVD-pNA or IETD-pNA cleavage activity. Results represent fold increase in activity over CHX only-treated cells, and show the results of one of two independent experiments. B, HMEC were stimulated with LPS (100 ng/ml) and CHX (50 µg/ml) for various times, as shown following pretreatment (30 min) with zVAD-fmk (50 µM) or DMSO (vehicle), and assessed for JNK activity by immunoblotting with an anti-phospho-JNK Ab. C, HMEC were stimulated with LPS and CHX (50 µg/ml) following pretreatment with zVAD-fmk (50 µM) or DMSO (vehicle), and assessed for cell survival by neutral red incorporation. Results shown are a single experiment representative of two independent experiments showing similar findings.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Endotoxic or septic shock is a potentially lethal complication of systemic infection by Gram-negative bacteria (1, 48). Endotoxin or LPS, a glycolipid present on the outer wall of Gram-negative bacteria, is responsible for many of the sequelae of Gram-negative sepsis (1, 48). Death in sepsis is due to circulatory collapse and multiple organ failure (1, 48). The integrity of the endothelium is crucial during sepsis, and endothelial death and loss of its barrier function may play a key role in the pathogenesis of septic shock (49). LPS induces endothelial damage in vivo, and microvascular injury occurs in numerous tissues during sepsis (2, 50, 51, 52). Endothelial damage precedes tissue damage during endotoxemia, suggesting that loss of vascular integrity may play a role in the parenchymal tissue damage and the multiorgan failure seen in sepsis (2).

A number of cytokines are released by LPS-activated inflammatory cells in vivo, and it has been argued that the sequelae of sepsis are mediated by these other cytokines (1, 2, 30). However, it is also clear that LPS has direct effects on the endothelium (53, 54, 55, 56, 57). As with TNF, in most studies LPS does not induce significant death of human endothelial cells unless new gene expression is blocked (3, 6). Since LPS-initiated endothelial death occurs mainly in the presence of CHX in vitro, this model cannot directly be applied to in vivo findings during Gram-negative sepsis. However, there is evidence to suggest that the LPS-survival pathway in vivo may fail due to inhibition by other inflammatory mediators such as IFN-{gamma}, or that the death pathway overwhelms the survival pathway by synergism with other cytokines or physical factors during sepsis (30, 58, 59).

Because TLR4 was only recently identified as the LPS receptor, signals evoked by LPS stimulation have not been well studied. LPS stimulation of endothelial cells has been shown to up-regulate various antiapoptotic factors such as A1, A20, and XIAP (4, 60). In parallel and similar to the TNFR1, apoptotic signals are also initiated by TLRs through the death domain adaptor protein, FADD (5, 21). However, in contrast to death that is initiated by TNF, LPS-induced endothelial apoptosis is not completely blocked by a dominant-negative FADD (5). Thus, other important apoptotic pathways may be functioning independently of FADD-caspase 8 activation. Our findings presented in this study suggest that a TRAF6-JNK pathway routes an important alternate proapoptotic pathway in endothelial cells responding to LPS.

TRAF6 integrates signals from many cytokine receptors, including receptor activator of NF-{kappa}B, CD40, and the receptors for IL-1, IL-18, and IL-17 (61, 62, 63). However, TRAF6 plays different roles when activated by different receptors and in different cell types (19). For example, TRAF6 is required for NF-{kappa}B, but not JNK activation in response to receptor activator of NF-{kappa}B signaling (20). In contrast, TRAF6 is essential for IL-17-stimulated activation of JNK and NF-{kappa}B (63). Our results show that both TRAF6-C and TRAF2-C sensitize endothelial cells to TNF-induced death without affecting NF-{kappa}B-activation. It has been demonstrated that TRAF2 is not critical for TNF-induced NF-{kappa}B activation, but that TRAF2 may propagate an NF-{kappa}B-independent cytoprotective signal (39, 64, 65, 66). That TRAF6-C also potentiates TNF-mediated apoptosis is unexpected in light of the fact that it promotes an antiapoptotic response following LPS stimulation in the same cell type. Nevertheless, these results clearly demonstrate that TRAF6 can mediate alternate signaling pathways in the same cell type depending on the type of stimulus.

A previous report has implicated TRAF6 in signaling apoptotic responses in the CNS, but the stimuli or pathways involved were not identified (67). In our endothelial model, the TRAF6-mediated apoptotic signal is, at least in part, transmitted through JNK. Others have shown that JNK is able to phosphorylate and inactivate Bcl-xL and Bcl-2 (41, 68). Given that the major antiapoptotic function of Bcl-2 family members resides at the mitochondria, abrogation of Bcl-2/Bcl-xL function may explain the requirement of JNK for release of cytochrome c from the mitochondria, and induction of apoptosis in at least one model (25, 40). Although JNK activation is not dependent on caspase activation in our model, inhibition of JNK attenuates activation of caspases by LPS. The findings from this study taken with previous results indicating that caspase inhibition can completely inhibit LPS-initiated death (5) suggest that the downstream apoptotic property of JNK is mediated by caspases following changes at the mitochondria.

It is of interest that TRAF6-C delays caspase 8 activation (IETDase activity) to a greater extent than caspase 3 activation (DEVDase activity). This suggests that even though caspase 8 activation may be initiated at the plasma membrane following oligomerization by FADD, the majority of caspase 8 cleavage and activation occurs downstream of caspase 3. We have shown previously that following stimulation of HMEC by TNF, caspase 8 cleavage is detected primarily after caspase 3 cleavage (25). This again suggests that while the initial death signal may involve FADD-caspase 8, mitochondria appear to act as important integrators to amplify and propagate the death signal, in response to both LPS and TNF in endothelial cells. Because caspase activation is not required for JNK activation and a FADD dominant-negative construct can block caspase activation (21), our findings would suggest that JNK activation is not downstream of FADD oligomerization, and these two signals operate independently. Alternatively, FADD clustering may lie downstream of JNK activation, as has been shown in Jurkat cells (69). Further studies are required to define whether FADD and JNK lie on serial or parallel pathways.

The central role of the endothelium in mediating inflammatory responses is well recognized. Disruption of the endothelial barrier during sepsis is most likely deleterious for the organism. The data presented in this study implicate a TRAF6-JNK pathway in mediating a mitochondrial-regulated endothelial apoptotic pathway in response to LPS. Current studies are directed toward understanding the role that this pathway plays in an in vivo model of sepsis.


   Acknowledgments
 
We thank Z. Cao, D. Goeddel, and M. Rothe at Tularik for providing TRAF cDNAs.


   Footnotes
 
1 This work was funded by grants to A.K. from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of British Columbia and the Yukon. G.M. was funded by a studentship from the Michael Smith Foundation for Health Research. P.J.D. was funded by a postdoctoral fellowship from the Heart and Stroke Foundation of Canada. A.K. is a clinician-scientist of the Canadian Institutes of Health Research and a scholar of the Michael Smith Foundation for Health Research. Back

2 Address correspondence and reprint requests to Dr. Aly Karsan, Department of Medical Biophysics, British Columbia Cancer Research Center, Vancouver, British Columbia, Canada V5Z 1L3. E-mail address: akarsan{at}bccancer.bc.ca Back

3 Abbreviations used in this paper: CHX, cycloheximide; BAEC, bovine aortic endothelial cell; FADD, Fas-associated death domain protein; HMEC, human microvascular endothelial cell; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; pNA, p-nitroaniline; TLR, Toll-like receptor; TRAF, TNFR-associated factor. Back

Received for publication May 31, 2001. Accepted for publication June 27, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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