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B and PI 3-kinase/Akt in TNF-
-induced cytotoxicity in microvascular endothelial cells1Department of Medicine, University of Colorado at Denver and Health Sciences Center, Denver, Colorado; and 2First Affiliated Hospital of Kunming Medical College, Kunming, Yunnai, People's Republic of China
Submitted 6 February 2008 ; accepted in final form 10 July 2008
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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with the endothelium is a pivotal factor during endotoxemia. Inflammatory conditions are characterized by the activation of the transcription factor NF-
B and the expression of inflammatory mediators. Previous reports indicate that inhibition of NF-B activation during sepsis may be beneficial to the microvasculature. In addition, the phosphatidylinositol-3-kinase/Akt signaling pathway (PI3-kinase/Akt) has been shown to be cytoprotective. In this study, we examined the effect of inhibition of NF-B and PI3-kinase/Akt on cell viability, cytokine production, inducible nitric oxide synthase (iNOS) expression, and nitric oxide (NO) generation by TNF--treated cultured microvascular endothelial cells. TNF- induced significant cytotoxicity and was associated with increased inflammatory cytokines and NO and increased expression of iNOS. The NF-B inhibitor, pyrrolidine dithiocarbamate (PDTC), prevented these increases and significantly attenuated the TNF--induced cytotoxicity. TNF- also caused PI3-kinase/Akt activation, which was further increased by PDTC and prevented by the PI3-kinase inhibitor, LY294002. Inhibition of PI3-kinase/Akt also significantly potentiated TNF--mediated cytotoxicity. LY294002 treatment resulted in the appearance of increased apoptosis, compatible with the known anti-apoptotic properties of PI3-kinase/Akt. The present results therefore demonstrate a cytotoxic effect of TNF- in microvascular endothelial cells which can be attenuated by NF-B inhibition. In addition, PI3-kinase/Akt activation during TNF- exposure may represent a compensatory anti-necrotic and anti-apoptotic pathway. The cytoprotective effects of NF-B inhibition and PI3-kinase/Akt activation may have potential implications in the treatment of endotoxemia and septic shock. endotoxemia; sepsis; cell culture; PDTC; LY294002
Experimental investigations identified tumor necrosis factor- (TNF-) as a pivotal factor during endotoxemia. Endotoxemia is known to be an important modulator in the hemodynamic and inflammatory events occurring during sepsis (30). Endotoxin-related AKI is associated with dramatic increases in TNF- and soluble TNF receptor administration has been shown to significantly attenuate the AKI as assessed by glomerular filtration rate (GFR) and renal blood flow (RBF) (22). In other studies, mice without TNF receptor 1 (TNFR1–/–) were protected against AKI, as assessed by blood urea nitrogen (BUN) (12). Moreover, when kidneys from TNFR+/+ mice were transplanted into TNFR–/– mice, the recipient mice demonstrated the same rise in BUN in response to endotoxemia as control mice (12). Recent studies in our laboratory also showed that inhibition of the rise in TNF- during endotoxemia with pentolifylline affords protection against AKI (42).
Endotoxemia results in the systemic release of a cascade of proinflammatory mediators and cytokines, including TNF-, which is primarily produced by activated mononuclear leukocytes and dendritic cells (14). When endothelial cells are exposed to inflammatory cytokines such as TNF-, endothelial function can be altered or impaired. In addition, exposure to these proinflammatory mediators may cause the altered endothelial cells to produce a further burst of inflammatory mediators (19). These effects of TNF- are thought to be predominantly mediated through the TNFR1 receptor in endothelial cells (1, 11). Moreover, exposure of endothelial cells to proinflammatory cytokines can also result in the induction of inducible nitric oxide synthase (iNOS) expression, an important component in the systemic inflammatory response in sepsis (4, 40). The NO generated from iNOS is the vasoactive mediator responsible for the fall in systemic vascular resistance underlying the hypotension in septic shock (31).
Inflammatory conditions are characterized by the activation of the ubiquitous intracellular transcription factor NF-B. The expression of inflammatory cytokines and iNOS from their respective genes is under the control of NF-B. In addition, the phosphatidylinositol-3-kinase/Akt signaling pathway (PI3-kinase/Akt) has been shown to be involved in both iNOS induction in macrophages and prevention of cytotoxicity in various cells (29, 37, 47).
In the present study, we therefore examined the effect of pharmacological inhibition of the NF-B and PI3-kinase/Akt pathways on TNF--treated cultured microvascular endothelial cells. Pyrrolidine dithiocarbamate (PDTC) and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-HCL (LY294002), potent inhibitors of NF-B and PI3-kinase/Akt activation, respectively, were utilized in these studies (9, 27).
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DMEM, FBS, Dulbecco's phosphate-buffered saline (DPBS), Trypsin-EDTA, and antibiotics for cell culture were purchased from Mediatech (Herndon, VA). Recombinant murine TNF- and recombinant murine IFN-
were purchased from Peprotech (Rocky Hill, NJ). 1400W was obtained from Cayman Chemical (Ann Arbor, MI). Q-Val-Asp(nonomethylated)-Oph [QVD-OPH] was purchased from MP Biochemicals (Aurora, OH). The vital nuclear dye bisbenzamide (Hoechst 33342) was obtained from Molecular Probes (Eugene, OR). LY294002 was purchased from EMD Chemicals (Gibbstown, NJ). Anti-IL-6 antibody was from R&D Systems (Minneapolis, MN). PDTC and all other reagents were obtained from Sigma (St. Louis, MO) unless otherwise specified.
Cell Culture
Mouse microvascular endothelial cells (MS1 cells) were purchased from American Type Culture Collection (Manassas, VA). This line expresses both Factor VIII-related antigen and VEGF receptor. Cells were grown in DMEM supplemented with 5% heat-inactivated FBS and antibiotics (100 U/l penicillin and 100 µg/ml streptomycin). Cells were kept in a humidified incubator gassed with 5% CO2-95% air at 37°C and the media were renewed two to three times weekly. Experiments were performed in six-well culture dishes when the cells became 
80% confluent. Cells were treated with 50 ng/ml recombinant murine TNF- in DMEM including serum in the presence of 1,000 U/ml recombinant murine IFN- and incubated at 37°C for 20 h. Previous studies demonstrated that the combination of TNF- and IFN- is necessary for maximal NO production in endothelial cells (26, 32, 40). Therefore, IFN- was included in the TNF--treated cells.
Cell Viability
Cell viability was assessed by percent lactate dehydrogenase (LDH) released into the medium. Incubation medium was collected and the cell pellet was dissolved in 1.5% Triton X-100 to the original volume. LDH release in the medium (supernatant) and in the cellular fraction (pellet) was measured as previously described (5, 17). The degree of cell injury was determined as the percentage of LDH released into the medium to total LDH obtained from both cellular fractions and the medium.
Apoptotic cell death was assessed at 6 h of TNF- treatment by two methods: nuclear staining with the DNA-specific dye Hoechst 33342 for evidence of nuclear condensation and fragmentation, and by immunoblotting for caspase-3 cleavage, a strong indicator of apoptosis (16). In addition, the effect of the pancaspase inhibitor, QVD-PPH, on TNF--induced cell damage was examined.
Detection of Cytokines
IL-6, IL-1β, RANTES, and IL-10 were measured in cell supernatants using the Bio-Plex cytokine assay from Bio-Rad Laboratories (Richmond, CA).
NO Production
NO production by the cells was assessed by nitrite (NO2–) accumulation in the culture supernate measured with the Griess reaction (18). An aliquot of the sample was mixed with the Griess reagent containing 1% (wt/vol) sulfanilamide, 0.1% (wt/vol) naphthylethylenediamine di-HCl, and 5% (vol/vol) phosphoric acid (85%). The mixture was incubated at room temperature for 10 min and read at 570 nm with a microplate reader. NaNO2 was used as standard.
Extraction of Nuclear Proteins
Nuclear proteins were extracted using the Nuclear Extract Kit from Active Motif (Carlsbad, CA). Cells were grown to confluence in a 100-mm tissue culture dish. Briefly, the cells were collected in ice-cold PBS in the presence of phosphatase inhibitors and centrifuged for 5 min at 500 rpm in a centrifuge precooled at 4°C. The cells were swollen in hypotonic buffer for 15 min on ice. Cytoplasmic proteins were released by the addition of detergent and centrifugation for 30 s at 14,000 g at 4°C. After collection of the cytoplasmic fraction, the nuclei are lysed and the nuclear proteins are solubilized in lysis buffer in the presence of protease inhibitors for 30 min on ice on a rocking platform. After centrifugation for 10 min at 14,000 g at 4°C, the supernatant (nuclear fraction) is collected and stored at –80°C.
Western Blot Analyses
Western blot analyses were performed as previously described (42) with some modifications. Protein extracts (20–50 µg) prepared from cell cultures were separated on NuPAGE 10% Bis-Tris gels (Invitrogen Life Technologies, Carlsbad, CA) and electrophoretically transferred to Immun-Blot PVDE membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat dry milk or with 4–5% bovine serum albumin (BSA) in Tris-buffered saline (TBS) containing 0.1% Tween. After being blocked, the membranes were incubated overnight at 4°C with antibodies to one of the following compounds: iNOS (anti-iNOS, 1:1,000), endothelial nitric oxide synthase (anti-eNOS, 1:1,000; Upstate Biotechnology, Billerica, MA), phospho-NF-B p65 (anti-pNF-B p65, 1:1,000), NF-B p65 (anti-NF-B p65, 1:1,000), phospho-38MAP kinase, p38 MAP kinase, phospho-p44/p42 MAP kinase, p44/p42 MAP kinase, phospho-SAPK/JNK, SAPK/JNK kinase, phospho-IB- (ser 32) kinase, IB- (ser 32), phospho-Akt (Ser473) (193H12), Akt (Ser473) (193H12; Cell Signaling Technology, Danvers, MA), and caspase-3 (Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then washed with TBS containing 0.1% Tween and incubated for 1 h at room temperature with a 1:3,000 dilution of rabbit anti-IgG-horseradish peroxidase conjugate (Pierce Laboratories, Rockford, IL). Bands were visualized using chemiluminescence (Amersham, Arlington Heights, IL) on a Versa Doc Imaging System (Bio-Rad). Densitometric results were reported as integrated values (area x density of band) and expressed as a percentage compared with the mean value in control groups (100%). Blots shown in RESULTS are representative of the results obtained from all samples. Densitometry as shown in RESULTS reflects means ± SE densitometry of all samples.
Measurement of NF-B DNA-Binding Activity
DNA-binding activity of NF-B was measured by a sensitive ELISA-based assay using a TransAM NF-B kit (Active Motif). Briefly, cultured MS1 cells were scraped and centrifuged for 10 min at 1,500 rpm at 4°C. The pellet was resuspended in 100 µl of lysis buffer and the lysate was centrifuged for 20 min at 15,000 rpm at 4°C. Supernatants (cell extract; 5 µg) from each sample were incubated in 96-well plates coated with NF-B consensus double-strand oligonucleotide sequence (5'-AGTTGAGGGGACTTTCCCAGGC-3'; Active Motif) for 1 h, and then with the supplied primary NF-B antibody (1:500) for 1 h, and subsequently with secondary peroxidase-conjugated antibody (1:1,000) for 1 h at room temperature. The optical density was read at 450 nm.
Luciferase Reporter Gene Assay
Cells were plated in 60-mm dishes at 600,000/dish and incubated overnight. Promoter transfections were performed using 1 µg pNF-B-Luc plasmids and CMV-β-galactosidase (β-gal) by using Lipofectamine reagent (Invitrogen) as previously described (44). After 5-h incubation, transfected cells were returned to growth medium for overnight incubation. Cells were then treated with different agents for 24 h and harvested in reporter lysis buffer (Promega, Madison, WI). Total DNA content was matched for each sample using the appropriate empty vector, and luciferase was normalized to β-galactosidase.
Statistical Analyses
All data were expressed as means ± SE. Comparisons between experimental groups were performed by ANOVA followed by the Tukey's multiple range test. P < 0.05 was considered to be significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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on Cell Viability
The effect of TNF- on cell viability in MS1 cells was assessed by percent LDH release, a measure of membrane integrity (necrosis). As shown in Fig. 1, incubation with TNF- for 20 h significantly increased the release of LDH compared with that in the control group. Moreover, the TNF--induced increase in LDH release at 20 h was not found to be secondary to apoptotic cell death, since no significant changes in nuclear staining or caspase-3 cleavage were observed with TNF- treatment at 6 h. In addition, TNF--induced cell damage was unaffected by the pancaspase inhibitor, Q-VD-OPH (not shown).
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B activation. The TNF--induced increase in LDH release was markedly attenuated by PDTC, in a concentration-dependent fashion (Fig. 1). PDTC had no effect on the release of LDH of control cells.
Effect of TNF- on Cytokine Production
IL-6 and IL-1β are critical proinflammatory cytokines in the pathogenesis of sepsis (7). The role of RANTES is less clear in sepsis but has been shown to be increased in response to TNF- in myofibroblasts (2). We also measured the anti-inflammatory cytokine, IL-10. As shown in Fig. 2, A–D, TNF- induced a significant increase in IL-1β, IL-6, RANTES, and IL-10. Since NF-B activation is critical for the maximal expression of many cytokines involved in the inflammatory process, we also examined the effect of PDTC on the TNF--induced increase in these cytokines. As shown in Fig. 2, A-C, 500 µM PDTC significantly reduced the release of IL-1β, IL-6, and RANTES in the TNF--stimulated cells. On the other hand, PDTC did not inhibit the increase in IL-10 in the TNF--stimulated cells (Fig. 2D), thus supporting a delayed and possibly distinct pathway of its secretion (39).
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on NO Production
The induction of iNOS is an important component in the systemic inflammatory response to sepsis. The NO generated from iNOS is the vasoactive mediator responsible for the fall in blood pressure in septic shock (31). Therefore, we measured the effect of TNF- on NO production after 20-h incubation. NO was assessed by the measurement of nitrite in the supernatant using the Griess reaction. As shown in Fig. 3, TNF- treatment resulted in a large increase in the nitrite concentration of the media. In addition, since activation of the NF-B pathway has been shown to be critical in the induction of iNOS (46), we examined the effect of PDTC on TNF--induced nitrite production. As shown in Fig. 3A, nitrite accumulation in the media was significantly suppressed by PDTC in a concentration-dependent fashion. PDTC alone had no effect on the basal level of NO formation. Moreover, the addition of 1400W (5 µM), an iNOS-specific inhibitor, completely prevented the TNF--induced increase in nitrite, verifying the source of TNF--stimulated NO formation as iNOS (Fig. 3B).
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on iNOS Protein Expression
As shown in Fig. 4A, expression of the iNOS protein was barely detectable in unstimulated cells, but markedly increased 20 h after TNF- treatment. To confirm whether the inhibition of NO production observed with PDTC in the previous experiments was due to less enzymatic activity or decreased protein expression of iNOS, we examined the effect of PDTC on iNOS protein expression. Treatment with PDTC attenuated iNOS protein expression in a concentration-dependent fashion. PDTC alone had no effect on iNOS protein expression. Figure 4B shows that the expression of endothelial NOS (eNOS) protein was unaffected by exposure to TNF- or PDTC.
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on Activation of NF-B
The effect of TNF- on activation of NF-B was assessed by three different methods.
ELISA.
Nuclear extracts were prepared using the Active Motif Nuclear Extract kit and NF-B binding was quantitated using the TransAM NF-B Chemiluminescence kit from Active Motif. As shown in Fig. 5A, cells exposed to TNF- demonstrated an increase in NF-B DNA-binding capacity. Treatment with PDTC prevented the TNF--induced stimulation of NF-B activity.
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B-luciferase construct along with CMV-β-gal to normalize for transfection efficiency. Cells were then treated with TNF- and/or PDTC for 20 h. As shown in Fig. 5B, TNF- stimulation caused a significant increase in NF-B-dependent luciferase activity which was significantly reduced by cotreatment with PDTC. PDTC alone had no effect on basal NF-B transcriptional gene activity.
Degradation of cytoplasmic IB.
Translocation of NF-B to the nucleus is proceeded by the phosphorylation, ubiquitination, and proteolytic degradation of IB. Therefore, we also assessed activation of NF-B by measuring the degradation of IB by Western blot. As shown in Fig. 5C, cells exposed to TNF- showed significant degradation of cytosolic IB, indicating increased NF-B activity. Cotreatment with PDTC attenuated the TNF--induced degradation of IB, indicating an inhibition of the TNF--induced NF-B activity. Moreover, PDTC alone had no effect on degradation of IB. Thus, the finding of nuclear translocation of NF-B in the first method and induction of NF-B DNA-binding activity in the second method correlated with IB degradation observed in the third method. These results indicate that TNF- activates NF-B in MS1 cells by causing IB degradation, which is prevented by PDTC (500 µM).
Effect of NF-B Inhibition on MAPKs
Mitogen-activated protein (MAP) kinases play a critical role in the regulation of cell growth and differentiation and in the control of cellular responses to cytokines and various stresses. To investigate whether the cytoprotective effect of PDTC in TNF--treated cells involved the MAP kinase pathway, we examined the levels of phosphorylated p38, phosphorylated ERK 1/2(p-p42/p44), and phosphorylated SAPK/JNK MAP kinase in TNF--treated MS1 cells using Western blot analyses. As shown in Fig. 6, TNF- treatment induced strong increases in the levels of phosphorylated p38 and phosphorylated JNK, but PDTC treatment had no effect. Therefore, although these MAP kinase pathways may play a role in the effects of TNF-, the PDTC did not affect their activation.
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on PI3-kinase/Akt Pathway
The PI3-kinase represents another major signaling transducer involved in the regulation of cell proliferation, survival, metabolism, cytoskeleton reorganization, and membrane trafficking (15). It also may have a role in iNOS gene expression (29). Akt represents an important signaling molecule downstream of PI3-kinase. Therefore, we performed Western blot analyses to determine the effect of TNF- treatment on this pathway. As shown in Fig. 7A, phosphorylated Akt was increased in cells treated with TNF- and further increased by PDTC (500 µM). The amount of nonphosphorylated Akt was unaffected by TNF- or PDTC treatment. In addition, as shown in Fig. 7B, treatment of cells with LY294002, a specific inhibitor of PI3-kinase, prevented the increase in TNF--induced phosphorylated Akt. The amount of nonphosphorylated Akt was unaffected by TNF- or LY294002 treatment.
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Since the PI3-kinase/Akt pathway has been reported to be upstream of iNOS gene expression (29), we examined the effect of inhibition of this pathway with LY294002 on NO production and iNOS protein expression in TNF--treated cells. As shown in Fig. 8A, LY294002 treatment did not reduce NO production in TNF--treated MS1 cells. In Fig. 8B, it is shown that the increase in iNOS protein expression in TNF--treated cells was also unaffected by LY294002. In addition, Fig. 9 shows that inhibition of the PI3-kinase/Akt pathway in TNF--treated cells had no effect on the activation of NF-B as measured by IB degradation.
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-Induced Cytotoxicity
We also examined the effect of LY294002 on cell viability. As shown in Fig. 10A, treatment with LY294002 (10 µM) increased the release of LDH in TNF--treated cells. LY294002 alone did not significantly affect cell viability of untreated cells. These results suggest that activation of the PI3-kinase/Akt pathway in TNF--treated cells may be a compensatory cytoprotective mechanism, since blocking of the pathway potentiates the cell death. This finding is compatible with reports of a protective effect of the PI3-kinase/Akt pathway in other cell types (6, 25, 37). In this regard, although no evidence of apoptosis was initially observed in this model of TNF--induced cytotoxicity in MS1 cells, subsequent cotreatment with LY294002 resulted in a significant increase in apoptosis as indicated by caspase-3 activation/cleavage (Fig. 10B) and nuclear condensation (not shown) at 6 h (20). This is compatible with the well-known anti-apoptotic properties of Akt activation.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(12, 22). Previous studies showed that the integrity of the endothelium is important in attenuating the AKI during endotoxemia. Specifically, eNOS knockout mice (41) and cyclooxygenase inhibition in mice (43) increased susceptibility to endotoxin-related AKI. The present paper was undertaken to examine the mechanisms whereby TNF- causes endothelial damage.
In the present studies, therefore, we examined the effect of TNF- in cultured microvascular endothelial cells. As assessed by LDH release, cell viability was greatly reduced after 20-h exposure to TNF-. No detectable changes in nuclear staining or caspase-3 cleavage were observed, and the effect of TNF- was insensitive to caspase inhibition. Therefore, in these experiments, the TNF--induced loss of cell viability did not occur secondary to apoptotic cell death. In addition, this TNF--induced cytotoxicity was accompanied by increased levels of the proinflammatory cytokines, IL-1β, IL-6, and RANTES, as well as the anti-inflammatory cytokine, IL-10. Nitrite production due to the induction of iNOS was also significantly increased with TNF- exposure. TNF- exposure had no effect on eNOS expression.
TNF- also resulted in activation of NF-B, the ubiquitous transcription factor necessary for the expression of many proinflammatory cytokines as well as iNOS. In unstimulated cells, NF-B exists in the cytoplasm complexed with its inhibitory subunit IB. When cells are exposed to extracellular stimuli such as bacterial lipopolysaccharides (LPS) or TNF-, IB undergoes phosphorylation and ubiquitination allowing proteosomal degradation. Free NF-B is then released from the complex and translocates into the nucleus where it activates transcription of target genes (21). In our studies, NF-B activation was verified by three different methods: nuclear translocation of NF-B, induction of NF-B DNA-binding activity, and IB degradation.
PDTC is a potent inhibitor of NF-B activation. Inhibition of NF-B activation by PDTC has been demonstrated to inhibit cytokine production induced by inflammatory mediators in human umbilical vein endothelial cells (28). In addition, in vivo studies demonstrated reduced LPS-induced microvascular injury in multiple organs by inhibition of NF-B activation with PDTC (24). Moreover, PDTC administration prevented induction of iNOS expression as well as the systemic hypotension in a rat model of septic shock via inhibition of NF-B (23). Based on these reports, we examined the effect of PDTC inhibition of NF-B activity on our TNF--treated endothelial cells. We found TNF--stimulated NF-B activity to be significantly attenuated by PDTC. This inhibition of NF-B activity was associated with a significant reduction in TNF--induced iNOS expression, NO generation, and proinflammatory cytokines, IL-1β, IL-6, and RANTES. The TNF--induced increase in the anti-inflammatory cytokine, IL-10, was unchanged by PDTC. Inhibition of NF-B activity with PDTC also resulted in a significant reduction in TNF--induced cytotoxicity in a concentration-dependent manner. Moreover, this cytoprotective effect of PDTC was not due to changes in activation of p38 or JNK MAP kinases.
The mechanism of action of PDTC in our studies is not clear. PDTC and other dithiocarbamates are members of a group of antioxidants which inhibit the activation of NF-B (36, 38). It has been reported that NF-B activity is controlled by the intracellular redox state (13, 33). It is possible that the antioxidant properties of PDTC may result in inhibition of reactive oxygen species that activate the upstream kinase, IB kinase, causing the degradation of IB and nuclear translocation of NF-B (10). In this regard, we found that PDTC attenuated TNF--induced IB degradation, indicating that PDTC acted upstream of this event in our studies. On the other hand, other reports demonstrated that the antioxidant properties of PDTC are not required for its inhibition of NF-B (8, 45).
Signaling by TNF is an active process in which cell death receptors are stimulated to induce apoptosis. Other signaling pathways act in the opposite direction to promote cell survival by inhibiting apoptosis. Therefore, most cells are resistant to TNF--induced apoptosis (47). Our data are consistent with this observation, since we found no significant evidence of apoptosis in the TNF--treated endothelial cells. One of the major anti-apoptotic intracellular signaling pathways responsible for promoting cell survival is initiated by the enzyme PI3-kinase. PI3-kinase phosphorylates the membrane phospholipid PIP2 to form PIP3, which activates the protein-serine/threonine kinase Akt. Akt then phosphorylates a number of proteins that regulate apoptosis (15, 37). For example, Akt can phosphorylate the proapoptotic Bcl-2 family member BAD. This creates a binding site for 14-3-3 (a group of highly expressed adaptor proteins). This bound form of BAD is then unable to inhibit the activity of the survival proteins Bcl-2 or Bcl-XL (15).
The PI3-kinase/Akt pathway may also be important in protecting cells from nonapoptotic, necrotic cell death (6, 37). We therefore examined the effect of TNF- treatment on PI3-kinase/Akt activation in our microvascular endothelial cell culture. The results demonstrated that TNF- induced significant PI3-kinase/Akt activation. As discussed above, this could explain the lack of any observable apoptosis in these TNF--treated cells. In addition, PI3-kinase/Akt activity was further increased with PDTC. This may indicate an anti-necrotic effect of PI3-kinase/Akt activation and explain in part the cytoprotective effect of PDTC.
Treatment of the cells with the PI3-kinase inhibitor, LY294002, completely prevented the TNF--induced activation. However, iNOS expression and NO production were unaffected by LY294002. Finally, inhibition of the PI3-kinase/Akt pathway with LY294002 resulted in increased cytotoxicity in the TNF--treated cells. This LY294002-induced increase in cytotoxicity was associated with a significant increase in apoptosis as assessed by caspase-3 activation and the increased presence of nuclear condensation. Taken together, the effects of PDTC and LY294002 on PI3-kinase/Akt activity and cell viability in these TNF--treated cells suggest that this pathway provides cytoprotective effects through both anti-necrotic and anti-apoptotic properties.
In conclusion, the present results demonstrate a cytotoxic effect of TNF- in microvascular endothelial cells which can be attenuated by NF-B inhibition. In addition, PI3-kinase/Akt activation during TNF- exposure may represent a compensatory anti-necrotic and anti-apoptotic pathway. To our knowledge, these results are the first to demonstrate NF-B-dependent nonapoptotic necrotic cell death in microvascular endothelial cells. Moreover, these results indicate that NF-B-induced PI3-kinase/Akt activation acts in opposition to the NF-B-induced apoptotic pathway in these cells. Therefore, the cytoprotective effects of NF-B inhibition and PI3-kinase/Akt activation may have potential implications in the treatment of endotoxemia and septic shock.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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-induced RANTES gene expression in human colonic subepithelial myofibroblasts. J Immunol 169: 1683–1687, 2002.. Am J Physiol Heart Circ Physiol 268: H1293–H1303, 1995.B, a ubiquitous transcription factor in the initiation of diseases. Clin Chem 45: 7–17, 1999.B in human endothelial cells. J Biol Chem 275: 15458–15465, 2000.B and AP-1 transcription factors activity. Blood 88: 3482–3490, 1996.: central factors in the altered cytokine network of uremia–the good, the bad, and the ugly. Kidney Int 67: 1216–1233, 2005.[CrossRef][Web of Science][Medline] and IFN-. J Immunol 151: 3283–3291, 1993.[Abstract]This article has been cited by other articles:
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  Y. Zhang, B.-S. Herbert, G. Rajashekhar, D. A. Ingram, M. C. Yoder, M. Clauss, and J. Rehman Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-{alpha} via the p38 mitogen-activated protein kinase pathway FASEB J, May 1, 2009; 23(5): 1358 - 1365. [Abstract] [Full Text] [PDF]
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P = NS vs. control.
