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* Departments of Pathology and Laboratory Medicine, and Medical Biophysics, British Columbia Cancer Agency, British Columbia, Canada; and Departments of
Pathology and Laboratory Medicine and
Experimental Medicine, University of British Columbia, British Columbia, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B conveys an important survival signal. In contrast, even
though LPS can also activate NF-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-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 |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-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-B and
JNK through TRAF6 in endothelial cells. A C-terminal fragment of TRAF6
(TRAF6-C) inhibits NF-B and c-Jun NH2-terminal
kinase (JNK) activation in endothelial cells. Whereas inhibition of
NF-B sensitizes endothelial cells to TNF-induced death, NF-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.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-Bmt (I-B
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-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).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-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-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. 1
A). We confirmed that similar
to HUVECs (16), TRAF6-C was able to block LPS-induced
NF-B signaling in HMEC, as measured by nuclear translocation of the
NF-B complex (Fig. 1B). A C-terminal fragment of TRAF2,
TRAF2-C, did not block LPS- or TNF-induced NF-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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C). 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. 2A
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-B (I-Bmt) to block
NF-B nuclear translocation, we demonstrate that NF-B activation
is critical for TNF-, but not LPS-induced death (Fig. 2B).
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A suggested
that TRAF6-C may be providing a protective effect against LPS-induced
apoptosis. Since NF-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. 3A, 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. 3B). 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. 3C).
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A, CHX alone did not activate
either JNK or p38 in HMEC. Fig. 4B shows a similar time
course of JNK activation with LPS + CHX as seen with LPS alone (Fig. 1C). JNK activation by LPS + CHX is inhibited by TRAF6-C, as
seen when cells were stimulated with LPS alone (Fig. 1C).
Additionally, LPS + CHX activation of p38 kinase, another potential
proapoptotic kinase, was not inhibited by TRAF6-C (Fig. 4B),
and mimicked the temporal patterns of activation seen with LPS
stimulation alone (data not shown).
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A) and BAEC death (Fig. 5B) 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. 5C demonstrates that inhibition of TRAF6-dependent
signals by TRAF6-C prevents LPS-induced endothelial death.
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A
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. 6B).
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A) and
IETDase activity (caspase 8) (Fig. 7B). 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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A).
JNK-APF did not inhibit LPS-induced extracellular signal-regulated
kinase 1/2 or p38 MAPK activation (Fig. 8B). Fig. 8C 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. 8D),
suggesting that JNK activation may be the major contributor to the
TRAF6-dependent LPS-induced apoptotic pathway.
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), 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. 9A). In contrast, caspase
inhibition, using a broad-spectrum caspase inhibitor, zVAD-fmk, did not
affect activation of JNK by LPS/CHX (Fig. 9B). 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. 9C) (5). Hence, JNK activation in response to
LPS lies downstream of TRAF6, but upstream of caspases.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-
, 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-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-B, but not
JNK activation in response to receptor activator of NF-B signaling
(20). In contrast, TRAF6 is essential for IL-17-stimulated
activation of JNK and NF-B (63). Our results show that
both TRAF6-C and TRAF2-C sensitize endothelial cells to TNF-induced
death without affecting NF-B-activation. It has been demonstrated
that TRAF2 is not critical for TNF-induced NF-B activation, but that
TRAF2 may propagate an NF-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.
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Acknowledgments |
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Footnotes |
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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
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.
Received for publication May 31, 2001. Accepted for publication June 27, 2002.
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 J. E. Kirby Anthrax Lethal Toxin Induces Human Endothelial Cell Apoptosis Infect. Immun., January 1, 2004; 72(1): 430 - 439. [Abstract] [Full Text] [PDF]
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M. Alikhani, Z. Alikhani, H. He, R. Liu, B. I. Popek, and D. T. Graves Lipopolysaccharides Indirectly Stimulate Apoptosis and Global Induction of Apoptotic Genes in Fibroblasts J. Biol. Chem., December 26, 2003; 278(52): 52901 - 52908. [Abstract] [Full Text] [PDF]
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 M. Comalada, J. Xaus, A. F. Valledor, C. Lopez-Lopez, D. J. Pennington, and A. Celada PKC{epsilon} is involved in JNK activation that mediates LPS-induced TNF-{alpha}, which induces apoptosis in macrophages Am J Physiol Cell Physiol, November 1, 2003; 285(5): C1235 - C1245. [Abstract] [Full Text] [PDF]
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I. Pollet, C. J. Opina, C. Zimmerman, K. G. Leong, F. Wong, and A. Karsan Bacterial lipopolysaccharide directly induces angiogenesis through TRAF6-mediated activation of NF-{kappa}B and c-Jun N-terminal kinase Blood, September 1, 2003; 102(5): 1740 - 1742. [Abstract] [Full Text] [PDF]
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I. Sabroe, R. C. Read, M. K. B. Whyte, D. H. Dockrell, S. N. Vogel, and S. K. Dower Toll-Like Receptors in Health and Disease: Complex Questions Remain J. Immunol., August 15, 2003; 171(4): 1630 - 1635. [Full Text] [PDF]
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 D. D. Bannerman and S. E. Goldblum Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis Am J Physiol Lung Cell Mol Physiol, June 1, 2003; 284(6): L899 - L914. [Abstract] [Full Text] [PDF]
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m) is
indicated by a decrease in fluorescence, as measured by flow cytometry.
The net proportion of cells with low 
