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A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension.

Wang AP, Li XH, Yang YM, Li WQ, Zhang W, Hu CP, Zhang Z, Li YJ - PLoS ONE (2015)

Bottom Line: The expression and activation of eIF2α, mTOR and c-myc were analyzed.In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression.These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, 410078, China; Department of Anatomy, School of Medicine, University of South China, Hengyang, 421001, China.

ABSTRACT
Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.

No MeSH data available.


Related in: MedlinePlus

mTOR siRNA inhibited mTOR, eIF2α and c-myc expression and PASMCs proliferation induced by hypoxia.The protein expression of mTOR (A), p-eIF2α and eIF2α (B), c-myc (C) was determined by western blot, statistical result of total gray value was estimated by the image analysis software. (D) Proliferation of PASMCs was measured by MTS assay. con: cells were treated with 21% O2 for 48 h; hypo: cells were treated with 3% O2 for 48 h; +Nc: cells were transfected with negative control 50nM for 4 h before stimulation with hypoxia for 48 h; +Veh: cells were transfected with transfection reagent (30uL/6 well) for 4 h before stimulation with hypoxia for 48 h; +mTOR siRNA 50 nM: cells were transfected with mTOR siRNA 50 nM for 4 h before stimulation with hypoxia for 48 h. Data represent the means ±S.E.M. n = 3. *P<0.05 vs. con; **P<0.01 vs. con; #P<0.05 vs. hypo; ##P<0.01 vs. hypo.
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pone.0130806.g006: mTOR siRNA inhibited mTOR, eIF2α and c-myc expression and PASMCs proliferation induced by hypoxia.The protein expression of mTOR (A), p-eIF2α and eIF2α (B), c-myc (C) was determined by western blot, statistical result of total gray value was estimated by the image analysis software. (D) Proliferation of PASMCs was measured by MTS assay. con: cells were treated with 21% O2 for 48 h; hypo: cells were treated with 3% O2 for 48 h; +Nc: cells were transfected with negative control 50nM for 4 h before stimulation with hypoxia for 48 h; +Veh: cells were transfected with transfection reagent (30uL/6 well) for 4 h before stimulation with hypoxia for 48 h; +mTOR siRNA 50 nM: cells were transfected with mTOR siRNA 50 nM for 4 h before stimulation with hypoxia for 48 h. Data represent the means ±S.E.M. n = 3. *P<0.05 vs. con; **P<0.01 vs. con; #P<0.05 vs. hypo; ##P<0.01 vs. hypo.

Mentions: As observed in our study, mTOR and eIF2α were up-regulated both in the media of pulmonary arteries and in hypoxia-induced cultured PASMCs. We next determined whether mTOR contributes to PASMCs proliferation and pulmonary vascular remolding via eIF2α. We performed immunofluorescence staining and immunohistochemical analysis of lung tissues from rats with chronic hypoxia-induced pulmonary vascular remodeling. Rapamycin markedly decreased mTOR, p-eIF2α and eIF2α levels (Fig 4A, 4D, 4F and 4G). At the same time, rapamycin decreased p-mTOR, p-eIF2α and eIF2α levels, and reduced cell proliferation in PASMCs under hypoxia (Fig 5A and 5B, 5D and 5E, 5G). In addition, p-eIF2α and total eIF2x expression was decreased and PASMCs proliferation by hypoxia-induced was inhibited after mTOR knockdown by siRNA (Fig 6A and 6B, 6D and 6E). These results were consistent with those obtained from the use of the mTOR pharmacologic blocker rapamycin, which strongly suggested that mTOR may act as an upstream positive regulator of eIF2α.


A Critical Role of the mTOR/eIF2α Pathway in Hypoxia-Induced Pulmonary Hypertension.

Wang AP, Li XH, Yang YM, Li WQ, Zhang W, Hu CP, Zhang Z, Li YJ - PLoS ONE (2015)

mTOR siRNA inhibited mTOR, eIF2α and c-myc expression and PASMCs proliferation induced by hypoxia.The protein expression of mTOR (A), p-eIF2α and eIF2α (B), c-myc (C) was determined by western blot, statistical result of total gray value was estimated by the image analysis software. (D) Proliferation of PASMCs was measured by MTS assay. con: cells were treated with 21% O2 for 48 h; hypo: cells were treated with 3% O2 for 48 h; +Nc: cells were transfected with negative control 50nM for 4 h before stimulation with hypoxia for 48 h; +Veh: cells were transfected with transfection reagent (30uL/6 well) for 4 h before stimulation with hypoxia for 48 h; +mTOR siRNA 50 nM: cells were transfected with mTOR siRNA 50 nM for 4 h before stimulation with hypoxia for 48 h. Data represent the means ±S.E.M. n = 3. *P<0.05 vs. con; **P<0.01 vs. con; #P<0.05 vs. hypo; ##P<0.01 vs. hypo.
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pone.0130806.g006: mTOR siRNA inhibited mTOR, eIF2α and c-myc expression and PASMCs proliferation induced by hypoxia.The protein expression of mTOR (A), p-eIF2α and eIF2α (B), c-myc (C) was determined by western blot, statistical result of total gray value was estimated by the image analysis software. (D) Proliferation of PASMCs was measured by MTS assay. con: cells were treated with 21% O2 for 48 h; hypo: cells were treated with 3% O2 for 48 h; +Nc: cells were transfected with negative control 50nM for 4 h before stimulation with hypoxia for 48 h; +Veh: cells were transfected with transfection reagent (30uL/6 well) for 4 h before stimulation with hypoxia for 48 h; +mTOR siRNA 50 nM: cells were transfected with mTOR siRNA 50 nM for 4 h before stimulation with hypoxia for 48 h. Data represent the means ±S.E.M. n = 3. *P<0.05 vs. con; **P<0.01 vs. con; #P<0.05 vs. hypo; ##P<0.01 vs. hypo.
Mentions: As observed in our study, mTOR and eIF2α were up-regulated both in the media of pulmonary arteries and in hypoxia-induced cultured PASMCs. We next determined whether mTOR contributes to PASMCs proliferation and pulmonary vascular remolding via eIF2α. We performed immunofluorescence staining and immunohistochemical analysis of lung tissues from rats with chronic hypoxia-induced pulmonary vascular remodeling. Rapamycin markedly decreased mTOR, p-eIF2α and eIF2α levels (Fig 4A, 4D, 4F and 4G). At the same time, rapamycin decreased p-mTOR, p-eIF2α and eIF2α levels, and reduced cell proliferation in PASMCs under hypoxia (Fig 5A and 5B, 5D and 5E, 5G). In addition, p-eIF2α and total eIF2x expression was decreased and PASMCs proliferation by hypoxia-induced was inhibited after mTOR knockdown by siRNA (Fig 6A and 6B, 6D and 6E). These results were consistent with those obtained from the use of the mTOR pharmacologic blocker rapamycin, which strongly suggested that mTOR may act as an upstream positive regulator of eIF2α.

Bottom Line: The expression and activation of eIF2α, mTOR and c-myc were analyzed.In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression.These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, School of Pharmaceutical Sciences, Central South University, Changsha, 410078, China; Department of Anatomy, School of Medicine, University of South China, Hengyang, 421001, China.

ABSTRACT
Enhanced proliferation of pulmonary arterial vascular smooth muscle cells (PASMCs) is a key pathological component of vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Mammalian targeting of rapamycin (mTOR) signaling has been shown to play a role in protein translation and participate in the progression of pulmonary hypertension. Eukaryotic translation initiation factor-2α (eIF2α) is a key factor in regulation of cell growth and cell cycle, but its role in mTOR signaling and PASMCs proliferation remains unknown. Pulmonary hypertension (PH) rat model was established by hypoxia. Rapamycin was used to treat rats as an mTOR inhibitor. Proliferation of primarily cultured rat PASMCs was induced by hypoxia, rapamycin and siRNA of mTOR and eIF2α were used in loss-of-function studies. The expression and activation of eIF2α, mTOR and c-myc were analyzed. Results showed that mTOR/eIF2α signaling was significantly activated in pulmonary arteries from hypoxia exposed rats and PASMCs cultured under hypoxia condition. Treatment with mTOR inhibitor for 21 days attenuated vascular remodeling, suppressed mTOR and eIF2α activation, inhibited c-myc expression in HPH rats. In hypoxia-induced PASMCs, rapamycin and knockdown of mTOR and eIF2α by siRNA significantly abolished proliferation and increased c-myc expression. These results suggest a critical role of the mTOR/eIF2αpathway in hypoxic vascular remodeling and PASMCs proliferation of HPH.

No MeSH data available.


Related in: MedlinePlus