Yonsei Med J.  2008 Aug;49(4):592-600. 10.3349/ymj.2008.49.4.592.

The Inhibition of Insulin-stimulated Proliferation of Vascular Smooth Muscle Cells by Rosiglitazone Is Mediated by the Akt-mTOR-P70S6K Pathway

• Park S
• Lim S
• Chang W
• Song H
• Lee S
• Song BW
• Kim HJ
• Cha MJ
• Choi E
• Jang Y
• Chung N
• Cho SY
• Hwang KC

• KMID: 1793194
• DOI: http://doi.org/10.3349/ymj.2008.49.4.592

Abstract

PURPOSE
Thiazolidinediones (TZDs) are known to inhibit the proliferation of vascular smooth muscle cell (VSMC) by increasing the activity of p27(Kip1) and retinoblastoma protein (RB). However, the upstream signaling mechanisms associated with this pathway have not been elucidated. The Akt-mTOR-P70S6 kinase pathway is the central regulator of cell growth and proliferation, and increases cell proliferation by inhibiting the activities of p27(Kip1) and retinoblastoma protein (RB). Therefore, we hypothesized in this study that rosiglitazone inhibits VSMC proliferation through the inhibition of the Akt-TOR-P70S6K signaling pathway. MATERIALS and METHODS: Rat aortic smooth muscle cells (RAoSMCs) were treated with 10microM of rosiglitazone 24 hours before the addition of insulin as a mitogenic stimulus. Western blot analysis was performed to determine the inhibitory effect of rosiglitazone treatment on the Akt-mTOR-P70S6K signaling pathway. Carotid balloon injury was also performed in Otsuka Long-Evans Tokushima Fatty (OLETF) diabetic rats that were pretreated with 3 mg/kg of rosiglitazone. RESULTS: Western blot analysis demonstrated significant inhibition of activation of p-Akt, p-m-TOR, and p-p70S6K in cells treated with rosiglitazone. The inhibition of the activation of the p-mTOR-p-p70S6K pathway seemed to be mediated by both the upstream PI3K pathway and MEK-ERK complex. CONCLUSION: The inhibitory effect of rosiglitazone on RAoSMC proliferation in vitro and in vivo is mediated by the inhibition of the Akt-mTOR-P70S6K pathway.

Keyword

Rosiglitazone; smooth muscle cells; mammalian target of rapamycin; insulin

MeSH Terms

Animals
Cell Proliferation/drug effects
Cells, Cultured
Cytoprotection/drug effects
Enzyme Activation/drug effects
Insulin/*pharmacology
Male
Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors/metabolism
Muscle, Smooth, Vascular/drug effects/*metabolism
Myocytes, Smooth Muscle/drug effects/*metabolism
Phosphorylation
Protein Kinase Inhibitors/pharmacology
Protein Kinases/*metabolism
Proto-Oncogene Proteins c-akt/antagonists & inhibitors/*metabolism
Rats
Ribosomal Protein S6 Kinases, 70-kDa/*metabolism
Signal Transduction/drug effects
Thiazolidinediones/*pharmacology

Figure

• 

  Fig. 1 Effect of rosiglitazone on the proliferation of insulin-induced RAoSMCs (A). Quiescent RAoSMCs (0.5 × 104 cells per well) were stimulated with 1 µM of insulin. After 3 days in culture in the absence or presence of rosiglitazone, cell viability was determined by MTT assay. Effect of rosiglitazone on the expression of mTOR in RAoSMCs (B). Western blot analysis of p-mTOR in RAoSMCs exposed to 10 µM of rosiglitazone for 3 days in DMEM with 0.5% FBS. Each signal was quantified by scanning densitometry, and the figure shows the levels of each activity as the relative value of the maximal level of p-mTOR. Each Western blot was repeated 3 times. RAoSMCs, rat aortic smooth muscle cells; MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mTOR, mammalian target of rapamycin; DMEM, Dulbecco's modified Eagle medium; FBS, fetal bovine serum. *p < 0.05, †p < 0.01.

• 

  Fig. 2 Effect of rosiglitazone on activation of mTOR in balloon-injured OLETF rats. After exposure of the left carotid artery to a 2F Fogarty balloon catheter, the expresson of p-mTOR was detected through immunostaining of sections of tissue cut through the areas of balloon injury after treatment of 3 mg/kg of rosiglitazone. The immunostain was visualized with Texas Red-conjugated goat anti-rabbit IgG secondary antibody. mTOR, mammalian target of rapamycin; OLETF, Otsuka Long-Evans Tokushima Fatty. *p < 0.05, †p < 0.01.

• 

  Fig. 3 Effect of rosiglitazone on the phosphorylation of Akt, mTOR, and p70S6K in RAoSMCs with 10 µM of rosiglitazone. Equivalent amounts of protein lysate from RAoSMCs were subjected to SDS-PAGE, transferred to a PVDF membrane, and incubated with antibody. The immunoreactive protein was visualized by the use of an alkaline phosphatase detection system. mTOR, mammalian target of rapamycin; RAoSMCs, rat aortic smooth muscle cells; SDS-PAGE, sodium dodecyl sulfate-polyacryamide gel electrophoresis; PVDF, polyvinylidene fluoride. *p < 0.05, †p < 0.01.

• 

  Fig. 4 Effect of rosiglitazone on neointimal formation in balloon-injured OLETF rats. Injured and uninjured (intact) femoral arteries were excised at 21 days after injury. Three mg/kg of rosiglitazone was administered for 7 days before balloon injury and 21 days after balloon injury. The sections were stained with H & E, and neointima formation was evaluated. Representative photographs of H & E staining are shown in the upper, and bar graphs show neointima quantified by Imagescope 2.5. Data are mean ± SEM (each n = 5 to 8). OLETF, Otsuka Long-Evans Tokushima Fatty. *p < 0.05.

• 

  Fig. 5 Effect of rosiglitazone on the phosphorylation of P70S6K after treatment with U0126 (A) or wortmannin (B) in insulin-stimulated RAoSMCs. Equivalent amounts of protein lysate from RaoSMCs were subjected to SDS-PAGE, transferred to a PVDF membrane, and incubated with antibody. The immunoreactive protein was visualized by the use of an alkaline phosphatase detection system. RAoSMCs, rat aortic smooth muscle cells; SDS-PAGE, sodium dodecyl sulfate-polyacryamide gel electrophoresis; PVDF, polyvinylidene fluoride. *p < 0.05, †p < 0.01.

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