American journal of rhinology & allergy · 2016
Rapamycin inhibits transforming growth factor beta 1 induced myofibroblast differentiation via the phosphorylated-phosphatidylinositol 3-kinase mammalian target of rapamycin signal pathways in nasal polyp-derived fibroblasts.
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Ko Dong-Yn, Shin Jae-Min, Um Ji-Young, Kang Byungjin, Park Il-Ho, Lee Heung-Man
Аннотация
PURPOSE: Rapamycin has antiproliferative and antifibrogenic effects in vitro and in vivo. The purpose of this study was to evaluate the effects of rapamycin on transforming growth factor (TGF) beta 1 induced myofibroblast differentiation (alpha smooth-muscle actin [SMA]), extracellular matrix production, and collagen contraction in nasal polyp-derived fibroblasts (NPDF). The underlying molecular mechanisms of rapamycin were also determined in NPDFs.
METHODS: NPDFs were grown in culture and transformed into myofibroblasts by using TGF beta 1 (5 ng/mL). For cytotoxicity evaluation, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. Expression levels of alpha SMA, phosphorylated phosphatidylinositol 3-kinase (PI3K), and phosphorylated mammalian target of rapamycin (mTOR) were determined by using Western blot, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The total amount of collagen was analyzed by using the Sircol collagen assay, and contractile activity was measured with a collagen gel contraction assay. Silencing mTOR with mTOR-specific small interference RNA was determined by using reverse transcription-polymerase chain reaction.
RESULTS: Whereas rapamycin (range, 0-400 nM) had no significant cytotoxic effects on TGF beta 1 induced NPDFs, it significantly reduced the expression levels of alpha-SMA in TGF beta 1 induced NPDFs in a dose-dependent manner. TGF beta 1 induced collagen production and collagen contraction were significantly inhibited by rapamycin treatment. Rapamycin also attenuated the TGF beta 1 induced activation of PI3K and mTOR, and its inhibitory effects were similar to those of mTOR silencing and a specific PI3K inhibitor.
CONCLUSIONS: Rapamycin inhibited TGF beta 1 induced myofibroblast differentiation, extracellular matrix production, and collagen contraction through the PI3K/mTOR signal pathways in NPDFs.
METHODS: NPDFs were grown in culture and transformed into myofibroblasts by using TGF beta 1 (5 ng/mL). For cytotoxicity evaluation, a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide assay was used. Expression levels of alpha SMA, phosphorylated phosphatidylinositol 3-kinase (PI3K), and phosphorylated mammalian target of rapamycin (mTOR) were determined by using Western blot, reverse transcription-polymerase chain reaction, and immunofluorescence staining. The total amount of collagen was analyzed by using the Sircol collagen assay, and contractile activity was measured with a collagen gel contraction assay. Silencing mTOR with mTOR-specific small interference RNA was determined by using reverse transcription-polymerase chain reaction.
RESULTS: Whereas rapamycin (range, 0-400 nM) had no significant cytotoxic effects on TGF beta 1 induced NPDFs, it significantly reduced the expression levels of alpha-SMA in TGF beta 1 induced NPDFs in a dose-dependent manner. TGF beta 1 induced collagen production and collagen contraction were significantly inhibited by rapamycin treatment. Rapamycin also attenuated the TGF beta 1 induced activation of PI3K and mTOR, and its inhibitory effects were similar to those of mTOR silencing and a specific PI3K inhibitor.
CONCLUSIONS: Rapamycin inhibited TGF beta 1 induced myofibroblast differentiation, extracellular matrix production, and collagen contraction through the PI3K/mTOR signal pathways in NPDFs.