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Am J Physiol Heart Circ Physiol 290: H2459-H2465, 2006. First published January 20, 2006; doi:10.1152/ajpheart.00750.2005
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Rapamycin antagonizes NF-{kappa}B nuclear translocation activated by TNF-{alpha} in primary vascular smooth muscle cells and enhances apoptosis

Arturo Giordano,1 Raffaella Avellino,2 Paolo Ferraro,1 Simona Romano,2 Nicola Corcione,1 and Maria Fiammetta Romano2

1Invasive Cardiology Unit, Clinica Pineta Grande, Castelvolturno; and 2Department of Biochemistry and Medical Biotechnology, University of Naples "Federico II," Naples, Italy

Submitted 17 July 2005 ; accepted in final form 14 January 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Several lines of evidence support the view that rapamycin inhibits NF-{kappa}B. TNF-{alpha}, a potent inducer of NF-{kappa}B, is released after artery injury (e.g., balloon angioplasty) and plays an important role in inflammation and restenosis. We investigated the effect of rapamycin on NF-{kappa}B activation and apoptosis in vascular smooth muscle cells (VSMCs) stimulated with TNF-{alpha}. Using EMSA, we found that TNF-{alpha} caused NF-{kappa}B nuclear translocation in VSMCs after 1 h of incubation. Rapamycin inhibited I{kappa}B{alpha} degradation, thereby preventing nuclear translocation. Activation of NF-{kappa}B was accompanied by an increase of Bcl-xL and Bfl-1/A1 proteins, detected by Western blot assay, whereas rapamycin prevented the TNF-{alpha}-induced enhancement of these antiapoptotic proteins. The extent of apoptosis of VSMCs exposed to TNF-{alpha} was significantly enhanced by rapamycin. The effect of rapamycin appeared to be independent of the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented I{kappa}B{alpha} degradation nor increased apoptosis of cells incubated with TNF-{alpha}. Finally, we demonstrate that the large immunophilin FK-506 binding protein FKBP51 is essential for TNF-{alpha}-induced NF-{kappa}B activation in VSMCs. Our findings show that rapamycin inhibits NF-{kappa}B activation and acts in concert with TNF-{alpha} in induction of VSMC apoptosis.

inflammation; restenosis; vascular injury

BALLOON ANGIOPLASTY IS A POTENT stimulus for smooth muscle cell proliferation (43). Rapamycin is a potent inhibitor of neointimal formation after artery injury. Indeed, the implantation of stents coated with a rapamycin-containing biopolymer has dramatically decreased the incidence of in-stent restenosis (39, 43). Rapamycin acts by binding to the ubiquitous cytosolic FK-506 binding protein FKBP12 to form a complex that inhibits the serine/threonine kinase function of the mammalian target of rapamycin (mTOR) (9, 15, 35, 38). mTOR is activated through the phosphatidylinositol 3-kinase/Akt-protein kinase B survival pathway (13) and controls the synthesis of proteins essential for cell cycle progression (14, 31) by phosphorylating two key translational regulators: the initiation factor 4E binding protein (4E-BPI) (14) and the 70-kDa S6 ribosomal protein kinase (p70S6k) (31). Rapamycin also increases levels of the cyclin-dependent kinase inhibitor p27Kip1 (21) and, reportedly, blocks the cell cycle at the G1-to-S transition in vascular smooth muscle cells (VSMCs) (19). However, the findings that cell cycle progression and proliferation can occur despite 4E-BPI dephosphorylation (22) and that rapamycin inhibited intimal hyperplasia in p27-knockout mice by decreasing proliferation and enhancing apoptosis (34) suggested that other mechanisms are involved in the capacity of rapamycin to prevent poststent restenosis.

Inhibition of NF-{kappa}B prevented neointimal formation after balloon injury in a rat carotid artery model (44). NF-{kappa}B is an important element in the activation of the inflammatory cytokines and adhesion molecule genes involved in lesion development after vascular injury (25). Moreover, NF-{kappa}B factors modulate expression of a number of genes that sustain cell survival (3, 10, 42, 46); among these, Bcl-xL and Bfl-1/A1 play an important role in the pathogenesis of vascular lesion formation (17, 28, 29, 39). Bcl-xL is expressed at high levels after artery injury and is considered a key regulator of VSMC apoptosis (17). Inhibition of Bcl-xL dramatically induces VSMC apoptosis (29), and differences in Bcl-xL expression may account for differences in sensitivity to apoptosis after VSMC injury (28). Also Bfl-1/A1 is involved in resistance to VSMC apoptosis and contributes to development of atherosclerotic lesions in diabetes (37). TNF-{alpha}, which is released at high levels after artery injury (45), stimulates expression of Bcl-xl and Bfl-1/A1 by activating NF-{kappa}B transcription factors (1, 36), and TNF-{alpha}-induced apoptosis, reportedly, is suppressed by these proteins (10, 46).

Rapamycin has been shown to inhibit NF-{kappa}B in various cell types (20, 31, 41). We thus investigated whether it antagonizes the induction of Bcl-xl and Bfl-1/A1 in VSMCs stimulated with TNF-{alpha}. We also analyzed the effect of rapamycin on apoptosis of cells incubated with TNF-{alpha}. It is conceivable that, besides blocking proliferation, this drug limits the accumulation of neointimal cells in injured vessels by enhancing their apoptosis.

We attempted to clarify the role of the large immunophilin FK-506 binding protein FKBP51 (5), which is specifically inhibited by rapamycin (5), in NF-{kappa}B/Rel activation in VSMCs. Indeed, mapping of the TNF-{alpha}-NF-{kappa}B signal transduction pathway showed that FKBP51 is an IKK{alpha} cofactor essential for the function of the IKK kinase complex (7).


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and reagents. VSMCs from Wistar rat thoracic aorta (kindly provided by Dr. G. Esposito, Dept. of Cardiovascular and Immunological Sciences, University of Naples Federico II) (18) were cultured in medium 199 (ICN Biomedicals, Aurora, OH) supplemented with 10% heat-inactivated FCS (ICN Biomedicals). The experiments were performed when the cells were at passage 4–6. Rapamycin (Rapamune) was obtained from Wyeth Ayerst Laboratories (Marietta, PA), wortmannin from Sigma Aldrich (St. Louis, MO), and TNF-{alpha} from Roche Diagnostics (Basel, Switzerland).

Western blot analysis. For I{kappa}B{alpha} detection, cytoplasmic extracts were obtained from 1 x 106 cells resuspended in 100 µl of lysing buffer [10 mM HEPES, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM DTT, 1 mM PMSF, 50 µg/ml antipain, 40 µg/ml bestatin, 20 µg/ml chymostatin, and 0.2% (vol/vol) Nonidet P-40] for 15 min in ice. For Bcl-xL, Bfl-1/A1, p65/RelA, and FKBP51 detection, whole cell lysates were prepared by homogenization in modified RIPA buffer (150 mM NaCl, 50 mM Tris·HCl, pH 7.4, 1 mM EDTA, 1 mM PMSF, 1% Triton X-100, 1% sodium deoxycholic acid, 0.1% SDS, 5 µg/ml aprotinin, and 5 µg/ml leupeptin). Cell debris was removed by centrifugation. Protein concentration was determined by a protein assay (Bio-Rad, Richmond, CA). The cell lysate was boiled for 5 min in 1x SDS sample buffer (50 mM Tris·HCl, pH 6.8, 12.5% glycerol, 1% SDS, and 0.01% bromphenol blue) containing 5% beta-mercaptoethanol, run on a 10% SDS-polyacrylamide gel, transferred to a membrane filter (Cellulosenitrate, Schleicher and Schuell, Keene, NH), and incubated with the primary antibody. The antibodies against I{kappa}B{alpha} (a rabbit polyclonal antibody; Santa Cruz Biotechnology, Santa Cruz, CA), Bcl-xL (a mouse monoclonal antibody; Santa Cruz Biotechnology), Bfl-1/A1 (a rabbit polyclonal antibody; Santa Cruz Biotechnology), FKBP51 (a rabbit polyclonal antibody; Abcam, Cambridge, UK), and p65/RelA [a rabbit polyclonal antibody against COOH-terminal peptide (529–551) kindly provided by Dr. Shao-Cong Sun, Pennsylvania State University College of Medicine] were added to the incubation mixture at a dilution of 1:500. After a second incubation with peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) or anti-mouse IgG (Santa Cruz Biotechnology), the blots were developed with the enhanced chemiluminescence system (ECL, Amersham Pharmacia Biotech, Piscataway, NJ).

EMSA. Cell nuclear extracts were prepared from 1 x 106 cells by cell pellet homogenization in two volumes of 10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 10% glycerol. Nuclei were centrifuged at 1,000 g for 5 min, washed, and resuspended in two volumes of the above-specified solution. KCl (3 M) was added to reach 0.39 M KCl. Nuclei were lysed at 4°C for 1 h and centrifuged at 10,000 g for 30 min. The supernatants clarified by centrifugation were collected and stored at –80°C. Protein concentration was determined with a protein assay (Bio-Rad). The NF-{kappa}B consensus 5'-CAACGGCAGGGGAATCTCCCTCTCCTT-3' (32) oligonucleotide was end-labeled with [{gamma}-32P]ATP (Amersham Pharmacia Biotech) using a polynucleotide kinase (Roche). End-labeled DNA fragments were incubated at room temperature for 20 min with 5 µg of nuclear protein in the presence of 1 µg of poly(dI-dC) in 20 µl of a buffer consisting of 10 mM Tris·HCl, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM DTT, and 5% glycerol. In competition assays, a 50x molar excess of NF-{kappa}B or nuclear factor of activated T cells (NF-AT) cold oligonucleotide was added to the incubation mixture. The rabbit antibody against p65 (see above) or a preimmune antibody was used in the supershift assay. Protein-DNA complexes were separated from free probe on a 6% polyacrylamide gel run in 0.25x Tris-borate buffer at 200 mV for 3 h at room temperature. The gels were dried and exposed to X-ray film (Kodak AR).

Cell transfection and small interfering RNA. A small interfering (si) RNA was designed for the rat FKBP51 gene [5'-AGGCGAGAUCUGCCACUUA-3' (sense); Dharmacon Research, Boulder, CO]. The siRNA for the human p65 gene [5-GCCCUAUCCCUUUACGU-3 (sense)] (40), which shares 77% homology with the rat gene, was kindly provided by Prof. M. C. Turco (DIFARMA, Fisciano, Italy). Scrambled duplexes were used as controls (Dharmacon Research). At 24 h before transfection of the oligonucleotide, the cells were incubated in six-well plates in medium without antibiotics at 5 x 105 cells/ml. The siRNA or the scrambled oligonucleotide was transfected at the final concentration of 50 nM using Metafectene (Biontex, Munich, Germany) according to the manufacturer's instructions. After 2 days, 20 ng/ml of TNF-{alpha} were added to the culture medium, and 1 or 2 h later, the cells were harvested and processed by Western blot assay or EMSA.

Analysis of apoptosis. Phosphatidylserine externalization was investigated by annexin V staining. Briefly, 1 x 105 cells were resuspended in 100 µl of binding buffer (10 mM HEPES-NaOH, pH 7.5, 140 mM NaCl, and 2.5 mM CaCl2) containing 5 µl of annexin V-FITC (Pharmigen/Becton Dickinson, San Diego, CA) for 15 min at room temperature in the dark. Then 400 µl of the same buffer were added to each sample, and the cells were analyzed with a flow cytometer (FACScan, Becton Dickinson). A TdT-mediated dUTP nick end-labeling assay was performed with the In Situ Cell Death Detection Kit (Roche) using TMR red according to the manufacturer's instructions. Briefly, 1 x 106 cells were fixed with 2% (vol/vol) paraformaldehyde in PBS for 15 min at room temperature, washed, and permeabilized with 0.1% Triton X-100 in PBS for 2 min in ice. After a second wash, the cells were incubated with TMR red labeling solution for 1 h at 37°C and analyzed by flow cytometry.

Statistical analysis. Values are means and SD of independent experiments. The statistical significance of differences between means was estimated using the paired Student's t-test, because the two series of samples compared were the same and differed only in the treatment applied. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Rapamycin inhibits TNF-{alpha}-induced NF-{kappa}B/Rel activity in VSMCs. High levels of TNF-{alpha} have been detected after balloon angioplasty (45). To investigate whether neointimal cells were sensitive to signals induced by TNF-{alpha} receptor triggering, we evaluated the induction of NF-{kappa}B/Rel nuclear activity by EMSA in VSMCs incubated with TNF-{alpha} for 30, 60, and 90 min. NF-{kappa}B proteins appeared in VSMC nuclei after 60 min of incubation with TNF-{alpha} (Fig. 1A). The band indicated by the arrow in Fig. 1 corresponds to specific NF-{kappa}B complexes, because it was supershifted by anti-p65 (RelA) antibodies. This finding suggests that the band consists of RelA dimers. On the contrary, incubation with the preimmune antibody did not change the migration pattern of the band (Fig. 1B). To investigate whether rapamycin inhibited NF-{kappa}B/Rel nuclear activity induced by TNF-{alpha}, we incubated VSMCs with and without TNF-{alpha} and with and without rapamycin for 1 h and then analyzed the nuclear extracts by EMSA. Addition of rapamycin to TNF-{alpha}-stimulated VSMCs caused a decrease in NF-{kappa}B/Rel nuclear levels compared with cells stimulated with TNF-{alpha} alone (Fig. 1C).


Figure 1
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  		Fig. 1. Rapamycin inhibits TNF-{alpha}-induced NF-{kappa}B/Rel activity in vascular smooth muscle cells (VSMCs). A: effect of TNF-{alpha} on NF-{kappa}B/Rel nuclear translocation in VSMCs. Nuclear extracts prepared from VSMCs cultured for 30–90 min with 20 ng/ml TNF-{alpha} were incubated with [{gamma}-32P]ATP-end-labeled NF-{kappa}B consensus and subjected to EMSA. B: supershift analysis of nuclear complexes induced by TNF-{alpha} in rat VSMCs. Nuclear extracts prepared from VSMCs cultured for 1 h with 20 ng/ml TNF-{alpha} were incubated with [{gamma}-32P]ATP-end-labeled NF-{kappa}B consensus with and without anti-p65 or preimmune antibody and subjected to EMSA. C: effect of rapamycin on TNF-{alpha}-induced NF-{kappa}B/Rel nuclear translocation in VSMCs. Nuclear extracts prepared from VSMCs cultured for 1 h with and without 20 ng/ml TNF-{alpha} and with and without 100 ng/ml rapamycin were incubated with [{gamma}-32P]ATP-end-labeled NF-{kappa}B consensus and subjected to EMSA. Results from 3 different experiments show that intensity of the NF-{kappa}B band, expressed as integrated optical density and determined using NIH Image 1.61 for Macintosh, was significantly increased in TNF-{alpha} samples compared with control (P = 0.006) or rapamycin + TNF-{alpha} specimens (P = 0.02).

 
NF-{kappa}B nuclear translocation is preceded by I{kappa}B{alpha} degradation (3). We therefore investigated whether rapamycin prevented this process by measuring I{kappa}B{alpha} cytoplasmic levels in Western blot. TNF-{alpha} induced I{kappa}B{alpha} degradation after 45 min, and I{kappa}B{alpha} levels were restored after 120 min (Fig. 2A). Rapamycin did not cause the disappearance of I{kappa}B{alpha}, which indicates that the NF-{kappa}B inhibitor was not degraded. In contrast to rapamycin, wortmannin, a specific phosphatidylinositol 3-kinase inhibitor, did not prevent TNF-{alpha}-induced I{kappa}B{alpha} degradation (Fig. 2B). Thus our findings suggest that rapamycin inhibits NF-{kappa}B independently of blockage of the phosphatidylinositol 3-kinase/Akt pathway.


Figure 2
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  		Fig. 2. Effect of rapamycin on TNF-{alpha}-induced I{kappa}B{alpha} degradation in VSMCs. A: Western blot analysis of I{kappa}B{alpha} levels in cytoplasmic lysates prepared from cells cultured for 0–120 min with 20 ng/ml TNF-{alpha} and with and without 100 ng/ml rapamycin. B: Western blot analysis of I{kappa}B{alpha} levels in cytoplasmic lysates prepared from cells cultured for 1 h with and without 20 ng/ml TNF-{alpha} and with and without 100 ng/ml rapamycin or 1 µM wortmannin. Densitometry analysis of I{kappa}B{alpha} levels in 3 different experiments, determined by NIH Image 1.61 for Macintosh, show decreased levels in TNF-{alpha} specimens compared with control (P = 0.05) or rapamycin + TNF-{alpha} samples (P = 0.02). No difference was detected between samples treated with TNF-{alpha} alone and those treated with TNF-{alpha} + wortmannin.

 
Rapamycin counteracts the TNF-{alpha}-induced increase of antiapoptotic proteins. NF-{kappa}B/Rel transcription factors regulate the expression of genes that protect cells against death. Among these genes, Bcl-xL (10) and Bfl-1/A1 (46) are induced by TNF-{alpha} and are responsible for resistance to TNF-{alpha}-triggered cell death. Furthermore, both genes are involved in the control of smooth muscle cell apoptosis (17, 29, 30, 37). We therefore evaluated the effect of TNF-{alpha} and rapamycin on Bcl-xL and Bfl-1/A1 expression in VSMCs by Western blot assay. Representative results of two different experiments are shown in Fig. 3. TNF-{alpha} enhanced the basal expression of Bcl-xL by twofold, whereas it induced the expression of Bfl-1/A1, which was negligible in control cells. Coincubation of rapamycin and TNF-{alpha} caused a ≥50% decrease in Bcl-xL and Bfl-1/A1 protein levels compared with samples incubated with TNF-{alpha} alone. This finding is in agreement with inhibition of NF-{kappa}B.


Figure 3
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  		Fig. 3. Effect of TNF-{alpha} and rapamycin on induction of Bcl-xL and Bfl-1/A1 in VSMCs. Bcl-xL and Bfl-1/A1 expression levels in whole cell lysates prepared from cells cultured for 2 h with and without 20 ng/ml TNF-{alpha} and with and without 100 ng/ml rapamycin were analyzed by Western blot. Expression levels were quantified by densitometry and expressed vs. baseline (Bcl-XL) or TNF-{alpha}-induced level (Bfl-1/A1).

 
Rapamycin enhances apoptosis of VSMCs stimulated with TNF-{alpha}. NF-{kappa}B inhibition sensitizes many cell types to TNF-{alpha}-induced apoptosis (10, 42). Consequently, it is feasible that rapamycin, which inhibited NF-{kappa}B, modulates apoptosis of VSMCs stimulated with TNF-{alpha}. To address this issue, we cultured VSMCs with TNF-{alpha} in the presence and absence of rapamycin and analyzed cell death. Furthermore, because rapamycin also inhibits phosphatidylinositol 3-kinase/Akt through blockage of mTOR kinase activity, we examined the effect of the specific phosphatidylinositol 3-kinase inhibitor wortmannin on apoptosis of VSMCs incubated with TNF-{alpha}. Annexin V stained 31.5% (SD 6.2) of cells cultured in 10% FCS-medium 199 and 40.0% (SD 6.6) of cells cultured in the presence of TNF-{alpha} (P = 0.1; Fig. 4A). Addition of rapamycin (100 ng/ml) to TNF-{alpha}-stimulated cells significantly increased cell death [60.1% (SD 1.1), P < 0.03; Fig. 4A]. Rapamycin alone at ≤200 ng/ml caused a slight increase of basal apoptosis (P > 0.09). Wortmannin also slightly increased basal apoptosis (P = 0.1) but did not act in concert with TNF-{alpha}. Indeed, the extent of apoptosis of VSMCs cultured with wortmannin + TNF-{alpha} was similar to that of cells cultured with the cytokine alone (Fig. 4A). These findings suggest that the cooperative effect of rapamycin and TNF-{alpha} on VSMC apoptosis involved mechanisms unrelated to phosphatidylinositol 3-kinase/Akt inhibition. This cooperative effect was confirmed by TdT-mediated dUTP nick end-labeling assay. The percentage of cells that incorporated dUTP in their DNA increased after addition of rapamycin to TNF-{alpha}-stimulated cells (Fig. 4B).


Figure 4
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  		Fig. 4. Rapamycin enhances apoptosis of VSMCs stimulated with TNF-{alpha}. A: effect of rapamycin and wortmannin on apoptosis of VSMCs cultured in the absence and presence of TNF-{alpha}. Cell death values (means ± SD) were measured by annexin V staining and flow cytometry. VSMCs were cultured for 24 h in 10% FCS-medium 199 with and without 20 ng/ml TNF-{alpha} and with and without rapamycin or wortmannin. Data are from 4 different experiments, each done in triplicate. *Significantly different (P < 0.03): rapamycin (≥100 ng/ml) + TNF-{alpha} vs. TNF-{alpha} alone. B: effect of rapamycin on apoptosis of VSMCs cultured with TNF-{alpha}. Flow cytometry diagrams show tetramethylrhodamine (TMR)-dUTP incorporation in VSMCs cultured for 24 h with 20 ng/ml TNF-{alpha} and/or 100 ng/ml rapamycin.

 
FKBP51 controls TNF-{alpha}-induced NF-{kappa}B activation in VSMCs. Components of the FKBP family are the first target of rapamycin (5, 11, 12). These abundant cytosolic proteins are endowed with peptidyl-prolyl-isomerase activity, which is required for many cellular processes and is specifically inhibited by rapamycin (5). Among these, FKBP51 catalyzes the isomerization of peptidyl-prolyl-imide bonds in the {alpha}-subunit of the IKK kinase complex and is essential for IKK function (7). To assess the role of FKBP51 in the NF-{kappa}B activation pathway in VSMCs, using the siRNA approach, we downmodulated immunophilin levels in primary rat VSMCs. FKBP51 protein levels were clearly lower in VSMCs transfected with FKBP51 siRNA than in cells incubated with control medium or transfected with a scrambled oligonucleotide (Fig. 5A). We then investigated the ability of TNF-{alpha} to induce I{kappa}B degradation and NF-{kappa}B nuclear translocation when FKBP51 was downmodulated. I{kappa}B levels decreased in control cells and in scrambled-oligonucleotide-transfected cells cultured with TNF-{alpha} (Fig. 5B), which indicated I{kappa}B degradation. On the contrary, the levels of the inhibitor did not decrease when FKBP51 was downmodulated. Accordingly, EMSA showed the absence of NF-{kappa}B complexes in nuclear extracts from FKBP51 siRNA-transfected cells stimulated with TNF-{alpha} (Fig. 5C). These findings suggest that FKBP51 controls NF-{kappa}B activation in VSMCs. A competition assay showed the specificity of the NF-{kappa}B band. This band disappeared when the extract was incubated with the 32P-labeled NF-{kappa}B oligonucleotide in the presence of a 50x molar excess of unlabeled NF-{kappa}B oligonucleotide, but not in the presence of unlabeled NF-AT.


Figure 5
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  		Fig. 5. FK-506 binding protein (FKBP51) controls TNF-{alpha}-induced NF-{kappa}B activation in VSMCs. A: effect of small interfering RNA (siRNA) transfection on FKBP51 levels in VSMCs. FKBP51 expression levels in cell lysates obtained from nontransfected VSMCs and VSMCs transfected with specific siRNA or scrambled oligonucleotide as control were assayed by Western blot. Expression levels were quantified by densitometry and expressed vs. baseline level. B: effect of FKBP51 depletion on TNF-{alpha}-induced I{kappa}B{alpha} degradation in VSMCs. I{kappa}B{alpha} levels in cytoplasmic lysates prepared from nontransfected VSMCs and VSMCs transfected with FKBP51 siRNA or scrambled oligonucleotide as control and cultured for 1 h with and without 20 ng/ml TNF-{alpha} were assayed by Western blot. Expression levels were quantified by densitometry and expressed vs. baseline level. C: effect of FKBP51 depletion on TNF-{alpha}-induced NF-{kappa}B/Rel nuclear translocation in VSMCs. Nuclear extracts prepared from VSMCs cultured for 1 h with 20 ng/ml TNF-{alpha} were incubated with [{gamma}-32P]ATP-end-labeled NF-{kappa}B consensus and subjected to EMSA. A competition assay, performed with the same NF-{kappa}B cold oligonucleotide or an unrelated oligonucleotide, demonstrated specificity of the NF-{kappa}B band (arrow). Results are representative of 2 different experiments. NF-AT, nuclear factor of activated T cells.

 
Lack of induction of Bcl-xL in p65- or FKBP51-depleted cells. To verify that the induction of antiapoptotic proteins by TNF-{alpha} depended on NF-{kappa}B activation, we used the siRNA technique to downmodulate the levels of p65 and used Western blot to analyze the expression levels of Bcl-xL in VSMCs stimulated with TNF-{alpha}. TNF-{alpha} caused a twofold increase in Bcl-xL basal levels in cells transfected with the control oligonucleotide, but not when p65 was downmodulated (Fig. 6A). This result suggests that the trans-activating property of p65/RelA is essential for the induction of Bcl-xL stimulated by TNF-{alpha}. Similar results were obtained in FKBP51-depleted cells, notwithstanding the stable levels of p65. These findings support the concept that FKBP51 is important in NF-{kappa}B signaling (7).


Figure 6
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  		Fig. 6. Effect of TNF-{alpha} on induction of Bcl-xL in VSMCs depleted of p65 or FKBP51. A: Western blot assay of Bcl-xL, p65, and FKBP51 expression levels in whole cell lysates from VSMCs transfected with specific p65 siRNA or scrambled oligonucleotide (oligo) as control and cultured for 2 h with and without 20 ng/ml TNF-{alpha}. B: Western blot assay of Bcl-xL, p65, and FKBP51 expression levels in whole cell lysates from VSMCs transfected with specific FKBP51 siRNA or scrambled oligonucleotide as control and cultured for 2 h with and without 20 ng/ml TNF-{alpha}.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Various signaling pathways, activated during vascular injury by a number of cytokines (6), concur in determining postangioplasty restenosis (43). NF-{kappa}B contributes to angioplasty-induced lumen loss by inducing an inflammatory response (23, 25, 44) and a decreased rate of apoptosis (23), whereas the phosphatidylinositol 3-OH-kinase cascade controls VSMC proliferation (23). Among the cytokines released at the site of artery injury, TNF-{alpha}, which is mainly produced by infiltrating monocytes/macrophages (24), is particularly intriguing. TNF-{alpha} is a pleiotropic cytokine (4), and its receptor binding leads to activation of apoptosis, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-OH-kinase, and IKK (16), thereby eliciting a broad spectrum of cellular responses. Suppression of apoptosis, which is NF-{kappa}B dependent (4, 23, 42, 46), augments the inflammatory response to TNF-{alpha} (23). We investigated the effects of TNF-{alpha} on NF-{kappa}B activation and apoptosis in VSMCs and their modulation by rapamycin. Our study suggests that rapamycin interferes with IKK function and, hence, prevents TNF-{alpha}-induced NF-{kappa}B activation. This notion is supported by the finding that rapamycin exerted an anti-inflammatory effect in a human model of angioplasty (27). Accordingly, we found that induction of the prosurvival Bcl-2 homologs Bcl-XL and Bfl-1/A1, which are transcriptional targets of NF-{kappa}B (10, 46), was inhibited by rapamycin, and, as expected, the extent of apoptosis of VSMCs cultured with rapamycin + TNF-{alpha} was greater than that of cells cultured with the cytokine alone. Taken together, these findings suggest that, besides blocking cell proliferation, rapamycin controls neointimal hyperplasia by cooperating with endogenous TNF-{alpha} to induce VSMC apoptosis. This concept is consistent with the finding of apoptosis in a porcine coronary angioplasty model treated with rapamycin (33).

Although TNF-{alpha} through TNF-{alpha} receptor type 1 (TNFR1) induces tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-OH-kinase (16), thereby activating the phosphatidylinositol 3-kinase/Akt pathway, the effect of rapamycin on NF-{kappa}B activation and apoptosis seemed to be independent of this signaling pathway, because the phosphatidylinositol 3-kinase inhibitor wortmannin neither prevented I{kappa}B{alpha} degradation nor increased apoptosis of VSMCs cultured with TNF-{alpha}. In contrast, we demonstrate that the large immunophilin FKBP51 (5) is required for TNF-{alpha}-induced NF-{kappa}B activation in VSMCs. Rapamycin very specifically binds to FKBP51 and inhibits its isomerase activity (5), which is required for function of the IKK kinase complex (7). Therefore, it is reasonable to assume that it counteracts NF-{kappa}B activation by affecting the IKK{alpha} cofactor (7). The finding that rapamycin reduces the phosphorylating activity of IKK on its I{kappa}B substrate (31) supports this hypothesis.

Data from our present study may have an impact on the synthesis of natural-product derivatives, in particular small molecules that are able to modulate the action of the target and improve the bioavailability of a drug (2). Indeed, a low tissue distribution of the drug has been cited as a cause of the unsuccessful outcome of oral rapamycin therapy for recalcitrant restenosis (8). Thus far, the rapamycin derivatives used in clinical trials have been tested for their ability to inhibit mTOR (26). Our finding that IKK is also a target of rapamycin raises the possibility that molecules specifically targeting IKK may improve clinical outcome after balloon dilatation.

In conclusion, our study shows that rapamycin inhibits NF-{kappa}B activity in VSMCs and downmodulates antiapoptotic proteins possibly responsible for reduced sensitivity to death signals that might counteract smooth muscle cell growth after artery injury.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by funds from the Cardiovascular Service.


   ACKNOWLEDGMENTS
 
We are grateful to Jean Gilder for editing the text.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Romano, Dept. of Biochemistry and Medical Biotechnology, Univ. of Naples "Federico II," via S. Pansini 5, 80131 Naples, Italy (e-mail: romano{at}dbbm.unina.it)

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.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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