Mechanobiological stimuli,such as low-intensity pulsed ultrasound(LIPUS),have been shown to promote bone regeneration and fresh fracture repair,but the fundamental biophysical mechanisms involved remain elusive.Here,w...Mechanobiological stimuli,such as low-intensity pulsed ultrasound(LIPUS),have been shown to promote bone regeneration and fresh fracture repair,but the fundamental biophysical mechanisms involved remain elusive.Here,we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes.This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment.Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection.MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation,and knockdown of Piezo1 restricted the increase in cell migration and proliferation.After labeling with Fluo-8,MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation.No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells,which was similar to the results in the GsMTx4-treated group.The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased(P<0.01)after LIPUS stimulation.In addition,Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation,continued for 5 min,and then returned to their initial levels at 30 min.These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium.The influx of Ca2+serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization,which regulate the proliferation of MC3T3-E1 cells.展开更多
Background:Despite integrin being highlighted as a stiffness-sensor molecule in matrix stiffness-driven angiogenesis,other stiffness-sensor molecules and their mechanosensory pathways related to angiogenesis in hepato...Background:Despite integrin being highlighted as a stiffness-sensor molecule in matrix stiffness-driven angiogenesis,other stiffness-sensor molecules and their mechanosensory pathways related to angiogenesis in hepatocellular carcinoma(HCC)remain obscure.Here,we explored the interplay between Piezo1 and integrinβ1 in the mechanosensory pathway and their effects on HCC angiogenesis to better understand matrix stiffness-induced angiogenesis.Methods:The role of Piezo1 in matrix stiffness-induced angiogenesis was investigated using orthotopic liver cancer SD rat models with high liver stiffness background,and its clinical significance was evaluated in human HCC tissues.Matrix stiffness-mediated Piezo1 upregulation and activation were assayed using an in vitro fibronectin(FN)-coated cell culture system with different stiffness,Western blotting and Ca^(2+)probe.The effects of shPiezo1-conditioned medium(CM)on angiogenesis were examined by tube formation assay,wound healing assay and angiogenesis array.The underlying mechanism by which Piezo1 participated in matrix stiffness-induced angiogenesis was analyzed by microRNA quantitative real-time polymerase chain reaction(qRT-PCR),matrix stiffness measurement,dual-luciferase reporter assay,ubiquitination assay and co-immunoprecipitation.Results:Increased matrix stiffness significantly upregulated Piezo1 expression at both cellular and tissue levels,and high expression of Piezo1 indicated an unfavorable prognosis.High matrix stiffness also noticeably enhanced the activation level of Piezo1,similar to its expression level.Piezo1 knockdown significantly suppressed tumor growth,angiogenesis,and lung metastasis of HCC rat models with high liver stiffness background.shPiezo1-CM from HCC cells attenuated tube formation and migration abilities of vascular endothelial cells remarkably,and analysis of differentially expressed pro-angiogenic factors revealed that Piezo1 promoted the expression and secretion of vascular endothelial growth factor(VEGF),CXC chemokine ligand 16(CXCL16)an展开更多
基金supported by the National Institute of Health(R01AR052379 and R01AR61821,YXQ)GZ is partially supported by a fellowship from the Dental School of the Chinese Medical University during his studies at Stony Brook University.
文摘Mechanobiological stimuli,such as low-intensity pulsed ultrasound(LIPUS),have been shown to promote bone regeneration and fresh fracture repair,but the fundamental biophysical mechanisms involved remain elusive.Here,we propose that a mechanosensitive ion channel of Piezo1 plays a pivotal role in the noninvasive ultrasound-induced mechanical transduction pathway to trigger downstream cellular signal processes.This study aims to investigate the expression and role of Piezo1 in MC3T3-E1 cells after LIPUS treatment.Immunofluorescence analysis shows that Piezo1 was present on MC3T3-E1 cells and could be ablated by shRNA transfection.MC3T3-E1 cell migration and proliferation were significantly increased by LIPUS stimulation,and knockdown of Piezo1 restricted the increase in cell migration and proliferation.After labeling with Fluo-8,MC3T3-E1 cells exhibited fluorescence intensity traces with several high peaks compared with the baseline during LIPUS stimulation.No obvious change in the fluorescence intensity tendency was observed after LIPUS stimulation in shRNA-Piezo1 cells,which was similar to the results in the GsMTx4-treated group.The phosphorylation ratio of ERK1/2 in MC3T3-E1 cells was significantly increased(P<0.01)after LIPUS stimulation.In addition,Phalloidin-iFluor-labeled F-actin filaments immediately accumulated in the perinuclear region after LIPUS stimulation,continued for 5 min,and then returned to their initial levels at 30 min.These results suggest that Piezo1 can transduce LIPUS-induced mechanical signals into intracellular calcium.The influx of Ca2+serves as a second messenger to activate ERK1/2 phosphorylation and perinuclear F-actin filament polymerization,which regulate the proliferation of MC3T3-E1 cells.
基金ational Natural Science Foundation of China,Grant/Award Number:81972910。
文摘Background:Despite integrin being highlighted as a stiffness-sensor molecule in matrix stiffness-driven angiogenesis,other stiffness-sensor molecules and their mechanosensory pathways related to angiogenesis in hepatocellular carcinoma(HCC)remain obscure.Here,we explored the interplay between Piezo1 and integrinβ1 in the mechanosensory pathway and their effects on HCC angiogenesis to better understand matrix stiffness-induced angiogenesis.Methods:The role of Piezo1 in matrix stiffness-induced angiogenesis was investigated using orthotopic liver cancer SD rat models with high liver stiffness background,and its clinical significance was evaluated in human HCC tissues.Matrix stiffness-mediated Piezo1 upregulation and activation were assayed using an in vitro fibronectin(FN)-coated cell culture system with different stiffness,Western blotting and Ca^(2+)probe.The effects of shPiezo1-conditioned medium(CM)on angiogenesis were examined by tube formation assay,wound healing assay and angiogenesis array.The underlying mechanism by which Piezo1 participated in matrix stiffness-induced angiogenesis was analyzed by microRNA quantitative real-time polymerase chain reaction(qRT-PCR),matrix stiffness measurement,dual-luciferase reporter assay,ubiquitination assay and co-immunoprecipitation.Results:Increased matrix stiffness significantly upregulated Piezo1 expression at both cellular and tissue levels,and high expression of Piezo1 indicated an unfavorable prognosis.High matrix stiffness also noticeably enhanced the activation level of Piezo1,similar to its expression level.Piezo1 knockdown significantly suppressed tumor growth,angiogenesis,and lung metastasis of HCC rat models with high liver stiffness background.shPiezo1-CM from HCC cells attenuated tube formation and migration abilities of vascular endothelial cells remarkably,and analysis of differentially expressed pro-angiogenic factors revealed that Piezo1 promoted the expression and secretion of vascular endothelial growth factor(VEGF),CXC chemokine ligand 16(CXCL16)an
