Contribution of caveolin-1{alpha} and Akt to TNF-{alpha}-induced cell death

Koh Ono,1 Yoshitaka Iwanaga,2 Madoka Hirayama,3 Teruhisa Kawamura,4 Naoya Sowa,4 and Koji Hasegawa1

1Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto 612-8555; Departments of 2Cardiology and 3Pharmacology, Research Institute, National Cardiovascular Center, Osaka 565-8565; and 4Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan

Submitted 27 August 2003 ; accepted in final form 5 March 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-{alpha} (TNF-{alpha})-resistant lines from L929 cells. Using this approach, we discovered that caveolin-1{alpha} is required for TNF-{alpha}-induced cell death in L929 cells. The need for caveolin-1{alpha} in TNF-{alpha}-induced cell death was confirmed by the restoration of sensitivity to TNF-{alpha} after ectopic reconstitution of caveolin-1{alpha}/{beta} expression. This caveolin-1{alpha}-mutated line was also resistant to H2O2 and staurosporine, but not to lonidamine. HepG2 cells are known to lack endogenous caveolins. HepG2 cells stably transfected with caveolin-1{alpha}/{beta} were found to be much more sensitive to TNF-{alpha} than either parental cells transfected with caveolin-1{beta} or parental cells transfected with an empty vector. In contrast to its extensively documented antiapoptotic effect, the elevated activity of Akt appears to be important in sensitizing caveolin-1-expressing cells to TNF-{alpha}, since pretreatment of cells with the phosphatidylinositide 3-kinase (PI3K) inhibitor LY-294002 or wortmannin completely blocked PI3K activation and markedly improved the survival of TNF-{alpha}-treated L929 cells. The survival rates of caveolin-1{alpha}-normal and caveolin-1{alpha}-deficient L929 cells were comparable after treatment with PI3K inhibitor and TNF-{alpha}. Similar results were obtained with HepG2 cells that stably expressed caveolin-1{alpha}/{beta} or -{beta} and parental cells transfected with an empty vector. In summary, our results indicate that caveolin-1{alpha} preferentially sensitizes L929 cells to TNF-{alpha} through the activation of a PI3K/Akt signaling pathway.

caveolin; tumor necrosis factor; phosphatidylinositol 3-kinase


CAVEOLINS, A FAMILY of 22- to 24-kDa integral membrane proteins, are the principal structural components of caveolar membrane domains (26). Caveolin-1 is expressed in two isoforms, caveolin-1{alpha} and caveolin-1{beta}; the {alpha}- and {beta}-isoforms start from methionine at positions 1 and 32, respectively (27). Thus the two isoforms have in common a hydrophobic stretch of amino acids, the scaffolding domain, and the acylated COOH-terminal region, whereas the 31 NH2-terminal amino acids are found only in the {alpha}-isoform. The two isoforms have been reported to show overlapping but slightly different distributions in mammalian cells (27), but there has been no detailed study concerning their functional diversity. Although the biochemical basis is unknown, a recent study by Fujimoto et al. (9) suggests that the ratio of caveolin-1 isoforms is related to differentiation of the caveolar structure.

Although caveolin-1 is expressed in most cell types, caveolin-1 expression is downregulated or absent in oncogenically transformed NIH/3T3 cells and in human cancer cells (5, 14, 15, 25). Conversely, the overexpression of caveolin-1 blocks anchorage-independent growth of oncogenically transformed cells, indicating that caveolin-1 may act as a suppressor of oncogenic transformation. Moreover, the human caveolin-1 gene is located at a suspected tumor suppressor locus (D7S522; 7q31.1), a known fragile site (FRA7G) that is deleted in many forms of cancer (6, 7). Thus the downregulation of caveolin-1 expression and caveolar organelles may be critical for maintaining the transformed phenotype.

The potential role of caveolin-1 in cell death remains controversial. Using ceramide as a stimulus, Zundel and colleagues (36) observed that caveolin-1 expression in Rat-1 fibroblasts facilitates ceramide-induced cell death. In contrast, Timme and colleagues (31) showed that caveolin-1 suppresses c-myc-induced apoptosis in LNCaP cells. Thus contradictory evidence has been presented that caveolin-1 is both a facilitator and a suppressor of cell death in different contexts.

We used retrovirus insertion-mediated random mutagenesis to generate tumor necrosis factor-{alpha} (TNF-{alpha})-resistant lines from L929 cells. Our results indicate that 1) caveolin-1{alpha} is required for TNF-{alpha}-induced cell death in L929 cells, 2) the elevated activity of Akt appears to be important for sensitizing caveolin-1{alpha}-expressing cells to TNF-{alpha}, and 3) caveolin-1{alpha} preferentially sensitizes L929 cells to TNF-{alpha} through the activation of a phosphatidylinositide 3-kinase (PI3K)/Akt signaling pathway.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell lines, reagents, and antibodies. All cell lines were obtained from the American Type Culture Collection (Rockville, MD) and were cultured under the recommended conditions. TNF-{alpha} sensitivity was examined in L929 murine fibrosarcoma cells, which exhibited a spontaneous survival rate of <1 in 106 after 48 h of exposure to TNF-{alpha} at 100 ng/ml. Stable TNF-{alpha}-resistant cell lines derived from L929 cells were established either by retroviral infection or by plasmid transfection with the Lipofect Amine PLUS reagent (GIBCO-BRL). Stably transfected TNF-{alpha}-resistant clones were selected in normal growth medium with the addition of G-418 (1 mg/ml; GIBCO-BRL). Stably transfected HepG2 cells were selected in normal growth medium with the addition of G-418 (1 mg/ml). Cyclohexamide (10 µg/ml; Sigma) was added simultaneously when HepG2 cells were treated with TNF-{alpha}. Expression vectors encoding human caveolin-1{alpha}/{beta} and caveolin-1{beta} were kindly provided by Dr. Toyoshi Fujimoto and Dr. Hiroshi Kogo. The expression vector encoding human caveolin-1{alpha}/{beta} expresses both caveolin-1{alpha} and -{beta} since it contains sequence for Met-32. The anti-caveolin-1 antibodies used were as follows: rabbit polyclonal anti-caveolin-1 antibody (sc-894; Santa Cruz Biotechnology) and mouse monoclonal anti-caveolin-1 antibody (Clone-2297; Transduction Laboratories).

Retroviral vector construction. The pDisrup retroviral vector was constructed based on the Moloney murine leukemia virus retroviral vector pLNCX backbone, as described previously (24).

Retroviral production and cell infection. pDisrup recombinant retrovirus was generated in Phoenix Amphotropic producer cells using the calcium phosphate method of transfection (21). Viruses were produced at 32°C, and virus-containing medium was collected 24 h posttransfection and filtered through a 0.45-µm filter. L929 cells were plated in six-well plates at a density of 5 x 105 cells/well. One round of retroviral infection was performed by replacing medium with 2 ml pDisrup virus (containing 4 µg Polybrene/ml), followed by centrifugation of the six-well plates at 2,500 rpm for 30 min at 32°C.

3'-Rapid amplification of cDNA ends. The portion of the endogenous gene that was fused with the neo gene was amplified by the 3'-rapid amplification of cDNA ends (RACE) technique, as described previously (24).

Western blot analysis. Cell lysates were prepared as described and subjected to SDS-PAGE, followed by standard Western blotting procedures. For analysis of Akt, the blots were probed with anti-phospho-Akt or anti-Akt (1:1,000; Cell Signaling Technology), followed by horseradish peroxidase-conjugated anti-rabbit IgG (1:10,000; Bio-Rad). For analysis of GSK-3{beta} phosphorylation, 0.5 mg lysate was immunoprecipitated by using 1 µg anti-GSK-3{beta} (Transduction Laboratory) and probed with anti-phospho-GSK-3{beta} (1:1,000; Cell Signaling Technology) or anti-glycogen synthase kinase (GSK)-3{beta} (1:2,500), followed by horseradish peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG, respectively. The probed protein was then visualized by the enhanced chemiluminescence system (Amersham).

Akt kinase assay. Akt kinase activity was measured using GSK-3 fusion protein as a substrate, as described previously (29).

Cell viability assay using propidium iodide staining and forward-angle light scattering. The integrity of the plasma membrane was assessed by the ability of cells to exclude propidium iodide (PI; Sigma). Cells were trypsinized, collected by centrifugation, washed one time with PBS, and resuspended in PBS containing 1 µg/ml PI. The levels of PI incorporation were quantified by flow cytometry on a FACScan flow cytometer. Cell size was evaluated by forward-angle light scatting. PI-negative cells of normal size were considered live cells.

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The absorbance of the untreated cells was considered 100% viability, and the results were expressed as the percent viability with comparison to untreated cells.

Terminal deoxynucleotidyl trasnferase-mediated dUTP nick end labeling staining. Cells were treated with TNF-{alpha} or vehicle. At 16 h postexposure, the cells were harvested and centrifuged (1,500 rpm for 5 min). The pellets were fixed with 1x ORTHO Permeafix (Ortho Diagnostics) at room temperature for 40 min. After being washed with PBS containing 1% BSA, cells were stained by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method with the Apop Tag Direct Fluorescein kit (Oncor). Positive control is the result of Jurkat cells treated with anti-Fas antibody (MBL) or vehicle for 8 h.

RT-PCR. cDNA was synthesized by a SuperScript preamplification system from total RNA extracted with Tri-Zol reagent (GIBCO-BRL). PCR was performed using forward and reverse primers synthesized according to published cDNA sequences of caveolin-1, -2, and -3, and products were electrophoresed on 2% agarose gels and stained by ethidium bromide.

Construction of plasmids that contained DNA templates for the synthesis of small interfering RNAs. We selected two 19-nt target sequences that were flanked in the mRNA with amino acid at the 5'-end to construct plasmids for the synthesis of small interfering RNA (siRNA) for caveolin-1. They were 5'-GGCCAGCTTCACCACCTTC-3' for pSUPER caveolin-1.1 and 5'-GCCCAACAACAAGGCCATG-3' for pSUPER caveolin-1.2. A control vector (pSUPER Control) was constructed using a 19-nt sequence (5'-GCGCGCTTTGTAGGATTCG-3') with no significant homology to any mammalian gene sequence and therefore serves as a nonsilencing control. The pSUPER RNAi System (Oligoengine) was used to construct plasmid for the synthesis of siRNA under the control of the H1 promoter.

Statistical analysis. Results are expressed as means ± SE. Comparisons between groups were analyzed by t-test (2-sided) or ANOVA for experiments with more than two subgroups. A P value of <0.05 was considered significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Caveolin-1{alpha} was disrupted by pDisrup insertion. We used retrovirus insertion-mediated mutagenesis in L929 fibroblasts coupled with TNF-{alpha} treatment to select TNF-{alpha}-resistant cell lines that were generated by the mutagenesis. There is no poly(A) signal after the neo gene in this retroviral vector, but a splicing donor instead. Therefore, the neo gene was fused to the sequence of the exon that was at the 3'-end of the viral insertion site. The identities of the disrupted genes in various TNF-{alpha}-resistant cell lines were determined by 3'-RACE of the fused neo mRNA.

Caveolin-1 is expressed in two isoforms, caveolin-1{alpha} and caveolin-1{beta}; the {alpha}- and {beta}-isoforms start from methionine at positions 1 and 32, respectively. The gene disrupted in one of the TNF-{alpha}-resistant cell lines was identified as caveolin-1{alpha}. Caveolin-1 isoforms are encoded by distinct mRNAs (13) and the location at which pDisrup was inserted is indicated in Fig. 1A. A partial sequence of the fused gene product generated by retroviral insertion in this line (called Caveolin-1{alpha}mut) is shown in Fig. 1B. Western blotting analysis using a monoclonal antibody that recognized caveolin-1{alpha} and -{beta} (Clone-2297) showed that there was a reduction in caveolin-1{alpha} immunoreactivity in the Caveolin-1{alpha}mut cell line compared with parental L929 cells. No difference was observed in caveolin-1{beta} immunoreactivity (Fig. 1C). This is consistent with the prediction that the caveolin-1{alpha} protein level was reduced in Caveolin-1{alpha}mut cells.



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Fig. 1. Identification of caveolin-1{alpha} as a gene required for tumor necrosis factor (TNF)-{alpha}-induced L929 cell death. A: schematic representation of mouse caveolin-1 mRNAs with the gene structure. The diagram shows the location at which pDisrup is inserted. AA, amino acid; RACE, rapid amplification of cDNA ends. B: mRNA sequence at the junction between neo and caveolin-1{alpha}. The amino acid sequence at the COOH-terminus of neo is shown beneath the mRNA sequence. The splicing donor is shown in lowercase letters. The number in parentheses shows the beginning of the disrupted caveolin-1{alpha} gene relative to the start codon (as +1). C: amount of caveolin-1{alpha} protein is reduced in caveolin-1{alpha} mutant cells (Caveolin-1{alpha}mut). An antibody that recognizes caveolin-1{alpha} and -{beta} was used in Western blotting analysis on membrane protein samples from Caveolin-1{alpha}mut and parental wild-type L929 cells. D: total RNA was prepared from Caveolin-1{alpha}mut and one other clonal cell line that had viral insertions in other loci. neo mRNA levels were analyzed by Northern blot using a 32P-labeled double-stranded neo probe. A single neo fusion mRNA was detected in Caveolin-1{alpha}mut cells, as indicated by the triangle. *Transcripts driven by the 5'-long terminal repeat, which was detected in two lines by the neo probe because it contains the antisense sequence of neo.

 
Northern blot analysis using a neo probe indicated that there is a single viral insertion in the Caveolin-1{alpha}mut line (Fig. 1D). The size of the neo-caveolin-1{alpha} fusion mRNA was consistent with the predicted length.

Disruption of caveolin-1{alpha} gene in L929 confers resistance to TNF-{alpha}-induced cell death. As shown in Fig. 2, A and B, the parental cells and caveolin-1{alpha}-reconstituted cells (expressing wild-type caveolin-1{alpha}/{beta}) were highly sensitive to TNF-{alpha}-induced cell death. In contrast, the Caveolin-1{alpha}mut and vector-transfected cell lines were significantly more resistant to TNF-{alpha}-induced cytotoxicity than to the parental and reconstituted cell lines (P < 0.05, n = 6). Thus the resistance to TNF-{alpha}-induced cell death observed in the Caveolin-1{alpha}mut cell line is the result of the reduced level of caveolin-1{alpha} expression in these cells. Figure 2C shows that the cell death observed in L929 cells is necrosis. Although more than half of the cells are dead 16 h after TNF treatment, the percentage of TUNEL-positive cells is low. Positive control is the result of the Jurkat cells treated with anti-Fas antibody for 8 h.



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Fig. 2. TNF-{alpha} resistance in Caveolin-1{alpha}mut cells. Wild-type parental L929 cells, Caveolin-1{alpha}mut cells, reconstituted cells, and vector control cells were treated with TNF-{alpha} for different periods of time. A: cell viability was measured by propidium iodide (PI) staining. *P < 0.05 vs. reconstituted and wild-type L929 (n = 6). B: cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. *P < 0.05 vs. reconstituted and wild-type L929 (n = 6). C: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining of the L929 and Caveolin-1{alpha}mut cell line 16 h after treatment with TNF or vehicle. Positive control is the result of the Jurkat cells treated with anti-Fas antibody (MBL), as described in MATERIALS AND METHODS.

 
Caveolin-1{alpha}mut line is selectively resistant to some death stimuli. To establish whether Caveolin-1{alpha}mut is selectively resistant to different death triggers, we examined its sensitivity to several death stimuli. As shown in Fig. 3, Caveolin-1{alpha}mut was resistant to TNF-{alpha}-, H2O2-, and staurosporine-induced cell death. In contrast, Caveolin-1{alpha}mut was still sensitive to lonidamine-induced cell killing. The different sensitivities of Caveolin-1{alpha}mut cells to different death stimuli support the idea that there are multiple pathways for cell death and demonstrate that caveolin-1{alpha} mutation-mediated TNF-{alpha} resistance is not the result of a general promotion of cell survival but rather to an impairment of the death pathway used by TNF-{alpha}.



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Fig. 3. Sensitivity of Caveolin-1{alpha}mut cells to certain death stimuli was decreased. Wild-type parental L929 cells, Caveolin-1{alpha}mut cells, reconstituted cells, and vector control cells were treated with different concentrations of TNF-{alpha}, lonidamine, H2O2, and staurosporine for 24, 48, 8, and 24 h, respectively. *P < 0.05 vs. reconstituted and wild-type L929 (n = 6).

 
LY-294002 can abolish the differences in TNF-{alpha} sensitivity between parental and Caveolin-1{alpha}mut cells. The effect of the PI3K inhibitor LY-294002 on TNF-{alpha}-induced cell death was compared in cells expressing normal levels of caveolin-1{alpha} (parental and reconstituted cells) and cells deficient in caveolin-1{alpha} (Caveolin-1{alpha}mut and vector-transfected Caveolin-1{alpha}mut; Fig. 4). When the samples were treated with TNF-{alpha} alone, parental and reconstituted cells had an average survival rate of <25%, whereas Caveolin-1{alpha}mut and vector-transfected Caveolin-1{alpha}mut cells had a survival rate of {approx}50% (P < 0.05, n = 4). Thus the survival rate in Caveolin-1{alpha}mut cells was nearly two times that in caveolin-1{alpha}-normal cells. However, when the cells were treated with both LY-294002 and TNF-{alpha}, the survival rates of caveolin-1{alpha}-normal and caveolin-1{alpha}-deficient lines were both {approx}70%. Similar results were obtained with wortmannin as a PI3K inhibitor.



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Fig. 4. LY-294002 can abolish the differences in TNF-{alpha} sensitivity between parental and Caveolin-1{alpha}mut cells. Parental L929, Caveolin-1{alpha}mut, reconstituted, and vector cells were treated with TNF-{alpha} in the presence or absence of LY-294002, and cell viability was determined 24 h later. *P < 0.05 vs. reconstituted and wild-type L929 (n = 4).

 
TNF-{alpha}-triggered activation of Akt in L929 and Caveolin-1{alpha}mut cells. Because the survival rates of caveolin-1{alpha}-normal and caveolin-1{alpha}-deficient lines were comparable after treatment with PI3K inhibitor and TNF-{alpha}, we examined the Akt signaling pathway after these cell lines were exposed to TNF-{alpha}. A significant activation of Akt was observed in parental L929 cells, whereas TNF-{alpha} caused slight time-dependent Akt activation in Caveolin-1{alpha}mut cells. We also evaluated the phosphorylation status of GSK-3{beta}, a known substrate of Akt. The phosphorylation of GSK-3 correlated directly with the Akt phosphorylation status, supporting the view that the Akt pathway is functionally active (Fig. 5A). As shown in Fig. 5B, and consistent with the Akt phosphorylation levels shown in Fig. 5A, Akt kinase activity was significantly higher in parental L929 cells than in Caveolin-1{alpha}mut cells after treatment with TNF-{alpha}. The levels of phosphate and tensin homolog (PTEN; which catalyzes the reverse reaction as PI3K, thereby leading to Akt dephosphorylation) were not affected by either TNF-{alpha} treatment or caveolin mutation (Fig. 5A). It is thus unlikely that differences in PTEN expression play a significant role in the elevation of Akt phosphorylation induced by caveolin expression.



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Fig. 5. Changes in Akt signaling pathways after L929 and Caveolin-1{alpha}mut cells were exposed to TNF-{alpha}. A: parental L929 and Caveolin-1{alpha}mut cells were treated with TNF-{alpha} for the indicated times, and Western blot analysis was carried out to assess the levels of phosphorylated (p)-Akt, Akt, p-glycogen synthase kinase (GSK)-3{beta}, GSK-3{beta}, and phosphate and tensin homolog (PTEN). B: parental L929 and Caveolin-1{alpha}mut cells were harvested 1 h after TNF-{alpha} treatment, and Akt was immunoprecipitated from lysates with anti-Akt antibody. Akt kinase activity was assessed by using GSK-3 as a substrate.

 
Effect of caveolin-1{alpha} gene deficiency on other signaling pathways. The effect of caveolin-1{alpha} deficiency was also tested on other signaling pathways, including Ras, extracellular signal-regulated kinase (ERK) 1/2, and NF-{kappa}B. As shown in Fig. 6 and in agreement with previous reports, Ras and ERK phosphorylation was significantly enhanced in Caveolin-1{alpha}mut cells, whereas we observed no change in the activation of NF-{kappa}B.



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Fig. 6. Changes in Ras, extracellular signal-regulated kinase (ERK), and NF-{kappa}B signaling pathways after L929 and Caveolin-1{alpha}mut cells were exposed to TNF-{alpha}. Parental L929 and Caveolin-1{alpha}mut cells were treated with TNF-{alpha} for the indicated times, and Western blot analysis was carried out to assess the levels of p-Ras, Ras, pERK1/2, ERK1/2, pNF-{kappa}B, and NF-{kappa}B.

 
HepG2 cells that stably express caveolin-1{alpha}/{beta} are sensitive to TNF-{alpha}-induced cell death. To further elucidate the difference between the two isoforms, HepG2 cells, which do not express caveolins endogenously (9), were transfected with cDNA of either the {alpha}/{beta}-isoform or {beta}-isoform of human caveolin-1, or empty vector. The lack of caveolins in HepG2 cells was confirmed by RT-PCR (Fig. 7A) and Western blotting (data not shown). HepG2 cells that had been stably transfected with caveolin-1{alpha}/{beta} were much more sensitive to TNF-{alpha} than either caveolin-1{beta}-transfected parental cells or parental cells transfected with an empty vector (P < 0.05, n = 6; Fig. 7B). However, when these cell lines were treated with both LY-294002 and TNF-{alpha}, the survival rates of cells that stably expressed caveolin-1{alpha}/{beta} or -{beta} or parental cells transfected with an empty vector became comparable (Fig. 7C). We also examined the Akt signaling pathway after these cell lines were exposed to TNF-{alpha}. A significant activation of Akt was observed in HepG2 cells that had been stably transfected with caveolin-1{alpha}/{beta}, whereas TNF-{alpha} caused slight Akt activation in parental cells transfected caveolin-1{beta} and parental cells transfected with an empty vector (Fig. 7D).



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Fig. 7. HepG2 cells stably transfected with caveolin-1{alpha} respond differently to TNF-{alpha} than parental cells transfected with either caveolin-1{beta} or an empty vector. A: absence of caveolins in wild-type HepG2 as shown by RT-PCR. Total RNA obtained from L929 cells and mouse heart muscle cells (heart) were used as positive controls. B: differences in sensitivity to TNF-{alpha} and cyclohexamide among HepG2 cells stably transfected with caveolin-1{alpha}, parental cells transfected caveolin-1{beta}, and parental cells transfected with an empty vector. *P < 0.05 vs. HepG2 transfected with pcDNA3.1 and HepG2 transfected with caveolin-1{beta} (n = 6). C: LY-294002 abolished the differences in TNF-{alpha} sensitivity among the three types of cells. *P < 0.05 vs. HepG2 transfected with pcDNA3.1 and HepG2 transfected with caveolin-1{beta} (n = 4). D: HepG2 cells stably transfected with caveolin-1{alpha}, parental cells transfected with caveolin-1{beta}, and parental cells transfected with an empty vector were harvested 2 h after TNF-{alpha} and cycloheximide (CHX) treatment. Western blot analysis was carried out to assess the levels of p-Akt and Akt.

 
siRNA inhibited the expression of caveolin-1 and the phosphorylation of Akt, which was accompanied by an increased survival rate for L929 after treatment with TNF-{alpha}. We constructed two different plasmids to synthesize siRNAs for caveolin-1, pSUPER caveolin-1.1 and -1.2. As shown in Fig. 8A, pSUPER caveolin-1.2 gave a greater reduction of caveolin-1{alpha} protein expression than 1.1. pSUPER caveolin-1.2 also reduced the expression of caveolin-1{beta} (data not shown). This reduction was observed from 24 to 48 h after pSUPER caveolin-1.2 transfection (Fig. 8B). When L929 cells were transfected with these siRNAs and treated with TNF-{alpha}, the L929 cells transiently transfected with pSUPER caveolin-1.2 had a greater survival rate than those transfected with pSUPER control. However, when the cells were treated with both LY-294002 and TNF-{alpha}, the survival rates of L929 cells transfected with pSUPER caveolin-1.2 and those transfected with pSUPER control became comparable (Fig. 8C). We examined the Akt signaling pathway after the cells were exposed to TNF-{alpha}. A significant activation of Akt was observed in L929 cells transfected with pSUPER control, whereas TNF-{alpha} did not cause Akt activation in L929 cells transfected with pSUPER caveolin-1.2 (Fig. 8D).



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Fig. 8. Effect of small interfering RNA (siRNA) for caveolin-1 on the sensitivity of L929 cells to TNF-{alpha}. A: effect of pSUPER caveolin-1.1 and -1.2 on caveolin-1 protein expression. B: time course of the effect of pSUPER caveolin-1.2 on caveolin-1 protein expression. C: LY-294002 can abolish the differences in TNF-{alpha} sensitivity between pSUPER vector-transfected and pSUPER caveolin-1.2-transfected L929 cells. *P < 0.05 vs. pSUPER vector-transfected L929. (n = 4). D: L929 cells transfected with pSUPER vector and pSUPER caveolin-1.2 were harvested 1 h after treatment with TNF-{alpha}. Western blot analysis was carried out to assess the levels of p-Akt and Akt.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} is a proinflammatory cytokine that was originally identified and purified as a factor that leads to rapid hemorrhage necrosis of an established tumor (1). TNF-{alpha} can induce cell death in many different tumor cell lines (8) and is widely used to study the mechanisms of cell death (24). In vitro studies have shown that TNF-{alpha} can induce apoptosis in one cell type but necrosis in others (2, 8, 30), and the morphological characteristics of cells differ markedly among various TNF-treated cell lines. However, TNF-{alpha}-induced necrosis and apoptosis share some common signaling events downstream of the TNF-{alpha} receptor, such as the recruitment of TNF-receptor 1-associated death domain protein, Fas-associated death domain protein, and other cytosolic effector proteins to the cytosolic domains of TNF-{alpha} receptors (8, 19, 32).

Genetic mutation has been proven to be an efficient way to functionally identify genes in prokaryotes and lower eukaryotes. However, similar approaches in mammalian cells are not very successful, mostly because of the large and complex genome. In addition, the mutagens used in lower eukaryotes, which are usually chemicals or radiation, are not effective for genetic studies in mammalian cell cultures because of the difficulty of both controlling the mutation frequency and identifying the mutated gene (23). It has long been known that retroviral insertion can be used to generate genomic mutations. Because such insertion does not appear to prefer any particular site, the random disruption of genes can be achieved by retroviral infection (11).

Using random mutagenesis, we demonstrated here that disruption of the caveolin-1{alpha} gene produces resistance to TNF-{alpha}-induced cell death in L929 cells. HepG2 cells that overexpressed caveolin-1{alpha}/{beta} were more sensitive to the effects of TNF-{alpha} than cells that overexpressed caveolin-1{beta}. We further demonstrated that a reduced activation of the PI3K/Akt pathway underlies the loss of sensitivity to TNF-{alpha}-induced cell death in Caveolin-1{alpha}mut cells. The PI3K/Akt pathway was also more significantly activated in HepG2 cells that overexpressed caveolin-1{alpha}/{beta} than in cells that overexpressed caveolin-1{beta}.

Caveolin-1 is known to play an important role in regulating cell growth through its ability to modulate the activities of molecules involved in growth factor signaling. A large body of evidence suggests that caveolin-1, a putative tumor suppressor, interacts with and inactivates several signaling molecules along survival/proliferation pathways, such as epidermal growth factor receptor (5), platelet-derived growth factor receptor (34), Src (16), Raf (5), mitogen/extracellular signal-regulated kinase 1 (5, 10), and ERK (5, 10). The decision to survive and proliferate or to commit to apoptosis is determined by the net balance between anti- and proapoptotic signals that are linked in a tightly regulated manner. Therefore, these results imply that caveolin-1 expression may sensitize cells toward apoptosis.

The PI3K/Akt pathway is another important signaling pathway activated by growth factors that is critically involved in growth control and cell survival (3, 12, 33). The influence of caveolin on the PI3K/Akt pathway is not well understood. However, PI3K has been localized in caveolae in fibroblasts, endothelial cells, and myeloid-derived cells (17, 18, 35). This finding suggests that PI3K activity might be controlled by caveolin under certain conditions.

Multiple mechanisms can mediate the antiapoptotic effect of Akt, which has been studied intensively. Recently, Akt has been shown to phosphorylate the proapoptotic protein Bad, thereby inhibiting its proapoptotic function, which may account for the antiapoptotic effect of Akt (20). Therefore, we hypothesized that caveolin disruption would lead to the enhanced activation of Akt in response to TNF-{alpha} treatment. However, Akt activity was found to be suppressed in Caveolin-1{alpha}mut cells. Moreover, PI3K inhibitors increased the survival rate after treatment with TNF-{alpha} in L929 cells. The prodeath effects of Akt activation are also seen in other cells. Shack et al. (28) investigated the ability of caveolin-1 to modulate the cellular response to sodium arsenite and thereby alter survival of the human cell lines 293 and HeLa (34). Their experiments indicated that caveolin-induced upregulation of the PI3K/Akt signaling pathway sensitized cells to arsenite and H2O2.

The downstream targets responsible for Akt's death-promoting effects remain to be identified. Recently, Nemoto and Finkel (22) demonstrated that forkhead transcription factor plays an important role in scavenging reactive oxygen species and increases resistance to oxidative stress. Elevated Akt activity would be expected to result in increased forkhead phosphorylation and reduced forkhead activity. Therefore, forkhead transcription factors are plausible candidates for these targets.

Caveolin-1 is expressed in two isoforms, caveolin-1{alpha} and caveolin-1{beta}. The {alpha}- and {beta}-isoforms of caveolin-1 have been reported to show distinct, but overlapping, distributions (27); however, no detailed study concerning their morphological and functional diversity has been performed. Recently, the morphological potentials of the two isoforms in mammalian cells were examined (9). Freeze-fracture immunoelectron microscopy of human fibroblasts suggested that the {alpha}-to-{beta} ratio in deep caveolae was higher than that in shallow ones. Their results obtained with HepG2 cells expressing different isoforms agree with those in human fibroblasts in that the {alpha}-isoform was more efficient than the {beta}-isoform at forming caveolae. Therefore, it is possible that caveolar formation is necessary for activation of the PI3K/Akt pathway and the promotion of cell death. There may also be some functional difference between deep and shallow caveolae. It is also possible that deep caveolae may activate the PI3K/Akt pathway more efficiently than shallow caveolae because deep caveolae could store some molecules in the lumen or could form an efficient structure for signal transduction. However, the detailed molecular mechanism about caveolar formation is still unknown. Moreover, there may be some counterpart molecules that interact with caveolin-1{alpha} and/or caveolin-1{beta}. The existence of this kind of molecules, which may change caveolar formation or interact with the PI3K/Akt pathway, can affect the sensitivity to TNF-{alpha}-induced cell death. Our current hypothesis about this signaling pathway is indicated in Fig. 9.



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Fig. 9. Proposed model of the TNF-mediated cell death signaling pathway. In the model, elevated Akt activity results in increased forkhead phophorylation and reduced forkhead activity. Caveolin-1{alpha} deficiency may affect caveolar formation and inhibit this signaling pathway. PI3K, phosphoinositide 3-kinase; FKHR, forkhead transcription factor.

 
To study more directly whether caveolin-1 is required for TNF-{alpha}-induced cell death, we suppressed its expression by RNA interference. We constructed two different plasmids for the synthesis of siRNAs for caveolin-1: pSUPER caveolin-1.1 and -1.2. Our pSUPER caveolin-1.2 significantly reduced the expression of caveolin-1, inhibited the phosphorylation of Akt, and prevented TNF-{alpha}-induced cell death in wild-type L929. Because we could not select a specific site for the reduction of caveolin-1 isoforms, the functional diversity of each isoform could not be detected by this method.

Interestingly, Caveolin-1{alpha}mut is selectively resistant to different death triggers. The selective resistance of Caveolin-1{alpha}mut cells to certain death stimuli, but not to others, further supports the idea that caveolin-1{alpha} mutation-mediated TNF-{alpha} resistance does not result from a generalized deleterious effect of cellular physiology but rather from a specific defect in a cell death pathway used by TNF-{alpha}.

Caveolin-1 mutant mice showed profound dysfunction of the vascular system and pronounced thickening of lung alveolar septa caused by an uncontrolled proliferation of cells (4). The abnormal septa showed immunostaining for Flk1, a marker of nondifferentiated endothelial and hematopoietic progenitors (4). The uncontrolled hyperpromotion of angioblastic cells implies that caveolin-1 may play a role in development and may be caused, at least in part, by some impairment of the cell death process.


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This work was supported by grants-in-aid from the Ministry of Science, Education, Culture, and Technology of Japan.


    ACKNOWLEDGMENTS
 
The valuable comments of Dr. Toru Kita, Dr. Toyoshi Fujimoto, and Dr. Naoharu Iwai are appreciated.


    FOOTNOTES
 

Address for reprint requests and other correspondence: K. Ono, Division of Translational Research, Kyoto Medical Center, National Hospital Organization, 1-1 Mukaihata-cho, Fukakusa, Fushimi-ku, Kyoto 612-8555, Japan (E-mail: kohono{at}kuhp.kyoto-u.ac.jp).

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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