Fangwei Li1,2, Jian Wang1, Yanting Zhu1, Lu Liu1, Wei Feng1, Wenhua Shi1, Qingting Wang1, Qianqian Zhang1, Limin Chai1, Manxiang Li3. 1. Department of Respiratory Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, China. 2. Department of Respiratory Medicine, Lanzhou University Second Hospital, Lanzhou, China. 3. Department of Respiratory Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an, Chinamanxiangli@hotmail.com.
Abstract
BACKGROUND/AIMS: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue. METHODS: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay. RESULTS: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes. CONCLUSION: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).
BACKGROUND/AIMS: The underlying molecular mechanisms involved in sphingosine kinase 1 (SphK1)/sphingosine 1-phosphate (S1P) mediation of platelet-derived growth factor (PDGF)-induced pulmonary arterial smooth muscle cell (PASMC) proliferation are still unclear, and the present study aims to address this issue. METHODS: Small interfering RNA (siRNA) and microRNA inhibitor transfection was performed to block the expression of SphK1, bone morphogenetic protein receptor II (BMPRII) and microRNA-21 (miR-21). Gene expression levels of SphK1, BMPRII and inhibitor of DNA binding 1 (Id1) were detected by immunoblotting, miR-21 expression level was examined with qRT-PCR, and S1P production was measured by ELISA. Additionally, PASMC proliferation was determined by BrdU incorporation assay. RESULTS: Our results indicated that PDGF increased the expression of SphK1 protein and S1P production, up-regulated miR-21 expression, reduced BMPRII and Id1 expression, and promoted PASMCs proliferation. Pre-silencing of SphK1 with siRNA reversed PDGF-induced S1P production, miR-21 up-regulation, BMPRII and Id1 down-regulation, as well as PASMC proliferation. Pre-inhibition of miR-21 also blocked BMPRII and Id1 down-regulation as well as PASMC proliferation caused by PDGF. Knockdown of BMPRII down-regulated Id1 expression in PASMCs. We further found that inhibition of PI3K/Akt and ERK signaling pathways, particularly ERK cascade, suppressed PDGF-induced above changes. CONCLUSION: Our study indicates that SphK1/S1P pathway plays an important role in PDGF-induced PASMC proliferation via miR-21/BMPRII/Id1 axis and targeting against SphK1/S1P axis might be a novel strategy in the prevention and treatment of pulmonary arterial hypertension (PAH).