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From the Departments of Surgery* and Physiology, and the University of Maryland Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland
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Abstract |
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Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor (TNF) superfamily.5
It is a type II transmembrane protein that can be cleaved to generate an 
17-kd soluble factor with biological activity.5
Soluble TWEAK has been shown to stimulate various cellular responses when it is added to cells in culture, including cell proliferation, migration, survival, apoptosis, and differentiation.6,7
Also, TWEAK is a proinflammatory factor5,8-12
and may play a role in neuroinflammation in vivo.13
TWEAK activity is mediated via binding to Fn14, a member of the TNF receptor (TNFR) superfamily of cell surface receptors.14
The TWEAK-Fn14 signal transduction pathway is not fully understood, but it has been shown that adaptor proteins known as TNFR-associated factors can bind the Fn14 cytoplasmic tail14-16
and that TWEAK binding to Fn14 activates the nuclear factor-
B (NF-B),11,15-18
extracellular signal-regulated kinase (ERK)17
and c-Jun NH2-terminal kinase (JNK)17
signal transduction pathways. In the present study we investigated whether the TWEAK-Fn14 signaling system could be involved in ischemia-induced brain injury.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Mixed cortical cell cultures were isolated from embryonic day 15 CD1 mice and cultured as described.19 One week after plating, some of the cells were washed twice with phosphate-buffered saline (PBS), fixed in 3% formaldehyde in PBS, and then permeabilized and blocked in 0.1% saponin and 5% bovine serum albumin in PBS for 1 hour. Other cell cultures were enriched for astrocytes by maintaining them in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum, 2 mmol/L glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin for an additional 2 weeks. These cells were washed and fixed in the same manner as the 1-week-old cultures. All antibodies were diluted in the blocking buffer described above and coverslips were washed three times in between each step. Cells were incubated with either a 1:100 dilution of rabbit anti-Fn14 IgG20 in combination with a 1:500 dilution of anti-neuron-specific nuclear protein (NeuN) monoclonal antibody (Chemicon, Temecula, CA) or a 1:50 dilution of antigen-purified goat anti-TWEAK polyclonal antibody (Cell Sciences, Canton, MA) in combination with a 1:100 dilution of rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibody (DAKO, Carpinteria, CA) for 1 hour at room temperature. Goat anti-rabbit secondary antibodies conjugated to Alexa 488 (Molecular Probes, Eugene, OR) and donkey anti-mouse or anti-goat secondary antibodies conjugated to Rhodamine Red-X (Jackson ImmunoResearch, West Grove, PA) were diluted to 1:500 and applied for 1 hour at room temperature. In some cases the cells were counterstained with 4,6-diamidino-2-phenylindole (Molecular Probes) for 2 to 3 seconds. Coverslips were rinsed with PBS and mounted on glass slides with Gel/Mount (Biomedia, Foster City, CA).
Construction and Stable Transfection of the Fn14-Fc and Fc Expression Plasmids, Purification of the Fn14-Fc and Fc Proteins
The plasmid pSecTag2/Fn14-Fc was constructed and a stably transfected human embryonic kidney (HEK) 293T clonal cell line was isolated as described.17 To construct the plasmid pSecTag2/Fc, the plasmid pSecTag2/OPG-Fc (provided by M. Tondravi, National Institutes of Health, Bethesda, MD) was digested with SfiI to release the OPG cDNA insert. The DNA ends were filled in using T4 DNA polymerase and then the plasmid was self-ligated using T4 DNA ligase. DNA sequence analysis was performed to confirm the identity of the construct. This plasmid was transfected into HEK293T cells and a stably transfected cell line (a pooled population) was isolated by drug selection as described.17 The soluble Fn14-Fc and Fc proteins were purified from conditioned medium by affinity chromatography as described.17
Characterization of the Fn14-Fc and Fc Proteins Expressed in Transiently Transfected HEK293T Cells
Approximately 106 HEK293T cells (American Type Culture Collection, Manassas, VA) were grown as described17 and then transfected with 4 µg of either the pSecTag2/Fn14-Fc or pSecTag2/Fc plasmids using Lipofectamine Plus (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. At 48 hours after transfection the conditioned medium was collected and centrifuged to remove any contaminating cells. Protein A-Sepharose beads (Amersham Pharmacia, Piscataway, NJ) were added to the conditioned medium and bound proteins subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis using a 4 to 12% NuPage gradient gel (Invitrogen). Proteins were transferred to nitrocellulose membranes (Schleicher and Schuell, Keene, NH) and then visualized using Ponceau S stain (Sigma, St. Louis, MO). The membranes were blocked for 1 hour at 37°C in TBST (25 mmol/L Tris/HCl, pH 7.5, 150 mmol/L NaCl, 0.1% Tween-20) containing 5% nonfat dry milk and then incubated for 1 hour at room temperature in TBST containing 5% bovine serum albumin and a 1:500 dilution of anti-myc monoclonal antibody 9E10 (gift of Sue Robinson, University of Maryland School of Medicine, Baltimore, MD). The membranes were then washed three times with TBST, and incubated for 1 hour in TBST containing 5% nonfat dry milk and a 1:10,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA). Membranes were washed three times in TBST and bound secondary antibodies were detected using the Supersignal West Pico kit (Pierce, Rockford, IL).
Animal Model
Animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee. Focal cerebral ischemia was induced by permanent middle cerebral artery occlusion (MCAO) as described elsewhere.21,22 Briefly, C57BL/6J male mice, weighing 20 to 30 g, were anesthetized by intraperitoneal injection of ketamine (2 mg) and xylazine (0.2 mg). Atropine (1 mg/kg) was administered intramuscularly, and body temperature was maintained by keeping the animals on a heating pad. Brain temperature was monitored with a thermometer placed into the left masseter muscle. A U-shaped incision was made between the left ear and left eye. The skull was exposed by retraction of the temporal muscle and a small opening (1 to 2 mm in diameter) was made with a handheld drill, with saline superfusion to prevent heat injury over the middle cerebral artery (MCA) region. The meninges were removed with forceps, and the MCA was occluded by ligation with 10-0 nylon thread and transected distally to the ligation point. Finally, the temporal muscle and skin were sutured back in place. At either 0, 24, 48, or 72 hours after MCAO animals were transcardially perfused with PBS and 4% paraformaldehyde. Brains were removed and TWEAK and Fn14 expression levels were assayed as described below.
A separate group of animals was placed on a stereotaxic frame immediately after MCAO and 2 µl of either PBS, soluble Fn14-Fc decoy receptor (1 µg/µl), or soluble Fc protein (1 µg/µl) was injected intracerebroventricularly throughout a 60-second period using a Hamilton syringe. The coordinates for the injection were: bregma, –2 mm; medial-lateral, 0 mm; and dorso-ventral, 2 mm.23 The animals were allowed to recover before being returned to their cages. After 72 hours, the animals were anesthetized and perfused as described above. The brains were removed, embedded in paraffin, and coronal sections, 20 µm thick, were cut through the rostrocaudal extent of the brain. The sections were stained with hematoxylin and eosin and infarction volume was calculated by the integration of the areas of eight chosen sections and the distances between them using the NIH Scion Image Analyzer System as described.24 The rostral and caudal limits for the integration were set at the frontal and occipital poles of the cortex.25 Statistical significance between groups was evaluated by unpaired Student’s t-test for comparison between two means. Differences were considered statistically significant at a probability value of P < 0.05.
RNA Isolation and Real-Time Quantitative Reverse Transcription (RT)-Polymerase Chain Reaction (PCR) Analysis
Total RNA was isolated from mouse ipsilateral (ischemic) or contralateral (nonischemic) hemisphere brain tissue using RNA Stat-60 (Tel-Test, Friendswood, TX) according to the manufacturer’s instructions. The integrity of each RNA sample was confirmed by denaturing gel electrophoresis followed by ethidium bromide staining. One µg of each RNA sample was converted to cDNA using TaqMan reverse transcription reagents according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Each PCR reaction was performed in triplicate using an ABI Prism 7900HT sequence detector system. The reactions contained 5 µl of each cDNA, 1x TaqMan Universal PCR Master Mix, and murine TWEAK-, murine Fn14-, or rodent GAPDH-specific primers and fluorescence-labeled probes (Applied Biosystems Assay-On-Demand Products) in 100-µl total volume according to the manufacturer’s instructions (Applied Biosystems). The thermal cycling conditions comprised an initial denaturation step at 95°C for 10 minutes and 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Threshold cycle (Ct) was obtained from the PCR reaction curves and TWEAK and Fn14 mRNA levels were quantitated using the comparative Ct method with GAPDH mRNA serving as the reference. Statistical significance was evaluated as described above.
Immunohistochemistry
All immunohistochemistry was performed on 5-µm deparaffinized embedded sections. The sections were first immersed in 100% methanol/0.3% H2O2 for 30 minutes to exhaust endogenous peroxidase activity and then preincubated with either 10% goat serum (TWEAK and Fn14 staining) or 10% rabbit serum (Mac-1 staining) for 20 minutes at room temperature. Sections were then incubated with either a 1:50 dilution of rabbit anti-TWEAK IgG (gift of Timothy Zheng, Biogen Idec Inc Cambridge, MA.), a 1:10 dilution of rabbit anti-Fn14 IgG,20 a 1:100 dilution of rat anti-Mac-1 antibody M170 (gift of Li Zhang, University of Maryland School of Medicine, Baltimore, MD) or an equivalent amount of normal control rabbit or rat IgG (Sigma) for 1 hour at room temperature. After a wash with PBS, a 1:200 dilution of biotinylated anti-rabbit or anti-rat secondary antibody (Vector Laboratories, Burlingame, CA) was added for 30 minutes at room temperature. The sections were then washed in PBS, incubated with a 1:100 dilution of ABC Elite reagent (Vector Laboratories), developed with 3,3'-diaminobenzidine for 4 minutes, and counterstained with Mayer’s hematoxylin for 2 minutes.
Terminal dUTP Nick-End Labeling (TUNEL) Staining
Paraffin-embedded sections from vehicle and Fn14-Fc-treated animals (72 hours after MCAO) were prepared and TUNEL reactivity was measured using the ApopTag Plus Fluorescein In Situ Apoptosis Detection kit (Chemicon) according to the manufacturer’s instructions.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We first determined whether TWEAK and Fn14 protein expression could be detected in mouse cerebral cortex-derived neurons or astrocytes by indirect immunofluorescence analysis. In these experiments, we identified neurons by staining for the neuronal nuclear marker NeuN and astrocytes by staining for GFAP. We detected TWEAK and Fn14 expression in both neurons and astrocytes; however, TWEAK expression was significantly more pronounced in astrocytes (Figure 1; A to C)
whereas Fn14 expression was more pronounced in neurons (Figure 1; D to F)
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We then investigated TWEAK and Fn14 gene expression in the murine MCAO model of cerebral ischemia. This model produces a very reproducible area of infarct and changes in both the ischemic penumbra region of the ipsilateral hemisphere and the corresponding region of the nonischemic contralateral hemisphere can be monitored. The ischemic penumbra is defined as the region bordering the necrotic core with moderately reduced blood flow and partially preserved energy metabolism and is an area within the injured brain where significant inflammation and apoptosis occur.2,3
We first determined whether TWEAK or Fn14 mRNA expression levels were regulated in the brain after MCAO in the mouse. Ipsilateral (ischemic) or contralateral (nonischemic) hemisphere brain tissue was obtained from mice that were sacrificed either immediately after MCAO (0-hour time point) or at 24, 48, and 72 hours after MCAO and RNA was isolated. Real-time quantitative RT-PCR analysis revealed that TWEAK mRNA levels in both brain hemispheres remained relatively constant during the course of the experiment (Figure 2A)
. In contrast, we observed a statistically significant increase in Fn14 mRNA levels in the ipsilateral, ischemic hemisphere at all three time points, with peak induction at 48 hours (2.3-fold increase greater than baseline) (Figure 2B)
. An increase in Fn14 mRNA expression was also detected in the contralateral, nonischemic hemisphere at the 24-hour time point only (1.7-fold increase greater than baseline).
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and TWEAK immunoreactivity was much lower in the corresponding region of the nonischemic contralateral hemisphere (Figure 3, B and D)
. Fn14 immunoreactivity was also detected in the ipsilateral hemisphere and again staining was most intense in the ischemic penumbra at 48 hours after MCAO (Figure 4, A and C)
. Fn14 immunoreactivity was significantly lower in the corresponding region of the contralateral hemisphere (Figure 4, B and D)
. Staining of the ischemic penumbra region was not detected when control rabbit IgG was used instead of anti-TWEAK or anti-Fn14 IgG as the primary immunological reagent (data not shown).
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We next investigated whether TWEAK-Fn14 interactions played a role in cerebral ischemia-mediated brain injury using a soluble Fn14-Fc decoy receptor. This protein consists of the extracellular ligand-binding domain of murine Fn14 fused to the Fc portion and hinge region of the mouse IgG1 heavy chain17
(Figure 5, A and B)
. We have previously shown that Fn14-Fc can bind TWEAK with high affinity and inhibit TWEAK-stimulated cellular responses in vitro.17,26
For the present studies, we also constructed an additional plasmid that expresses only the Fc portion and hinge region of the mouse IgG1 heavy chain (Figure 5A)
. Initial transient transfection experiments demonstrated that the Fc protein was the appropriate apparent molecular mass and was efficiently secreted from cells (Figure 5C)
. This Fc protein, which does not bind TWEAK (data not shown), was purified from the conditioned medium of stably transfected cells in the same manner as the Fn14-Fc decoy receptor.17
PBS vehicle, the soluble Fn14-Fc decoy receptor, and the soluble Fc protein were each administered via intracerebroventricular injection immediately after MCAO. Mice were sacrificed 72 hours later and the volume of the ischemic area was calculated. Fn14-Fc decoy receptor administration resulted in a 37% reduction in infarct volume; in contrast, the Fc protein had no detectable effect (Figure 6)
.
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Early after the onset of cerebral ischemia there is activation of microglial cells and a strong inflammatory reaction in the area surrounding the necrotic core (ischemic penumbra),2-4,27,28
followed by the appearance of apoptotic cell death in the same area.2,29
Because TWEAK appears to be a proinflammatory cytokine,5,8-12
we postulated that the Fn14-Fc decoy receptor could be acting, at least in part, by inhibiting TWEAK-stimulated brain inflammation. To test this hypothesis, PBS vehicle and the soluble Fn14-Fc decoy receptor were each administered via intracerebroventricular injection immediately after MCAO. Mice were sacrificed 72 hours later and tissue sections were prepared. Immunohistochemistry was then performed using an antibody that recognizes the integrin Mac-1 (
Mß2), a marker for microglial activation.30
TUNEL staining was also performed to examine apoptotic cell death. We found that Fn14-Fc treatment decreased both ischemia-induced microglial cell activation (Figure 7)
and cellular apoptosis (Figure 8
) in the area of the ischemic penumbra.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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We then determined whether TWEAK and/or Fn14 gene expression was regulated in response to focal cerebral ischemia using a mouse MCAO model and both real-time quantitative RT-PCR and immunohistochemical assays. TWEAK mRNA was detected at similar levels in both brain hemispheres at all time points after MCAO; nevertheless, an elevated amount of TWEAK protein was found in the ischemic penumbra region of the ipsilateral hemisphere using an immunohistochemical approach. There are several possible explanations for this apparent discrepancy. First, TWEAK mRNA expression may be rapidly induced in the ischemic penumbra region and then return to basal levels by 24 hours, the first time point we examined using the real-time RT-PCR assay. If TWEAK is a relatively stable protein, then increased TWEAK immunoreactivity might be apparent at 24, 48, and 72 hours after MCAO. Second, the tissue specimen used for RNA isolation represented the entire ipsilateral hemisphere and contained multiple cell types, including endothelial cells, which express relatively high levels of TWEAK (data not shown). This could make it difficult to detect any changes in TWEAK mRNA expression that may only occur in the neurons or glia residing in the ischemic penumbra. Third, it is possible that TWEAK mRNA levels do not change at all in response to cerebral ischemia, but ischemia itself promotes either the redistribution of TWEAK or a conformational change in TWEAK in such a manner that antibody recognition is increased.
We found that Fn14 mRNA levels transiently increased in both the ipsilateral and contralateral hemispheres. In the ipsilateral, ischemic hemisphere, an increase in Fn14 mRNA levels was first detected at 24 hours, peak expression was at 48 hours, and the level was still greater than baseline at 72 hours after MCAO. In contrast, in the contralateral, nonischemic hemisphere, increased Fn14 mRNA expression was noted at only one time point (24 hours). The increase we observe in this hemisphere may reflect cellular stress because of the surgical procedure. The molecular mechanism responsible for increased Fn14 mRNA (and protein) expression in the ipsilateral hemisphere ischemic penumbra after MCAO is not known. Because the penumbra is an area of moderate hypoxia,31 Fn14 may be a hypoxia-inducible factor-1 target gene,32 but this possibility has not yet been investigated. However, we have shown that several growth factors can induce Fn14 gene expression in vitro.14,17,20,33 Two of these growth factors, FGF-234 and VEGF-A,31 are expressed at elevated levels in the ischemic border after focal cerebral ischemia and thus one or both could play a role in Fn14 gene up-regulation after MCAO.
Finally, we investigated whether TWEAK-Fn14 signaling within the brain could contribute to focal ischemic injury by blocking the interaction between endogenously-expressed TWEAK and Fn14 using a previously characterized soluble Fn14-Fc decoy receptor.17,26 Intracerebroventricular injection of this protein immediately after MCAO significantly reduced infarct volume. This result implies that TWEAK is acting as a neurotoxic factor in the ischemic brain. We propose that this could occur via two alternative mechanisms. First, TWEAK may be directly stimulating apoptosis. Although there are reports that TWEAK treatment of certain human tumor cell lines can induce apoptosis,35 this does not appear to be its major biological activity.6 Indeed, of particular relevance to this study, TWEAK treatment of rat PC12 cells,36 human glioma cells,26 human astrocytes,8 or mouse astrocytes13 does not cause cell death. Second, TWEAK may be indirectly stimulating apoptosis by contributing to the focal ischemia-associated inflammatory response.2-4,27,28 This is the mechanism that we favor at the present time because TWEAK treatment of several different cell types, including astrocytes, has been shown to stimulate the expression of various proinflammatory molecules.5,8-12 Our results demonstrating that Fn14-Fc decoy receptor treatment results in a significant decrease in microglial cell activation and apoptotic cell death in the area of the ischemic penumbra support this hypothesis.
It has been reported that TWEAK binding to Fn14 activates the NF-B signal transduction pathway,11,15-18
and this pathway is a key regulator of numerous inflammatory response genes.37
TWEAK regulation of NF-B function is of particular interest in the present context because this transcription factor is activated during ischemic brain injury and contributes to neuronal cell death.38,39
It should be noted that TWEAK also activates the ERK and JNK signal transduction pathways.17
TWEAK-stimulated ERK and JNK pathway activation in the brain may also be involved in TWEAK neurotoxicity in consideration of recent studies demonstrating that specific inhibitors of these pathways reduce infarct volume in rodent models of focal cerebral ischemia.40-43
In summary, we have shown that TWEAK and Fn14 are present at elevated levels in the ischemic penumbra in a murine model of stroke. We hypothesize that TWEAK binding to Fn14 receptors promotes the activation of signal transduction pathways, the secretion of proinflammatory and proapoptotic cytokines, and cell death (Figure 9)
. Our findings suggest that the TWEAK-Fn14 signaling pathway might be a potential target for therapeutic strategies aimed at promoting neuronal cell survival during acute cerebral ischemia.
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Acknowledgements |
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Footnotes |
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Supported in part by the National Institutes of Health (grants NS-02223 to M.Y., HL-55374 and HL-55747 to D.A.L., and HL-39727 to J.A.W.).
Accepted for publication October 7, 2004.
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