Transforming Growth Factor β-activated Kinase-1 Promotes The Proliferation And Migration Of Vascular Smooth Muscle Cells Via Autophagy Regulation

Objective: Vascular Smooth Muscle Cells(VSMCs)are major components of blood vessel wall that keep vascular tension and mediate vasoconstriction and vasodilation.Hyperproliferation and migration of VSMCs are significant cytological elements in the development of several vascular proliferative diseases,such as restenosis,transplantation arteriosclerosis and atherosclerosis.Transforming growth factor β-activated kinase-1(TAK1),a serine/ threonine kinase,participates in the regulation of multiple cell functions,such as differentiation,proliferation,and migration,after activated by growth factors and cytokines.This study aims to investigate the role and mechanism of TAK1 in platelet derived growth factor-BB(PDGF-BB)-induced proliferation and migration of VSMCs.Methods: 1.Primary VSMCs were isolated form thoracic aorta of Sprague Dawley(SD)rats.Cultured VSMCs were stimulated with recommended PDGF-BB.Expression and phosphorylation level of TAK1 were detected by western Blot assay.q RT-PCR was used to investigate TAK1 m RNA change.2.Cultured VSMCs were transfected with TAK1-si RNA and pretreated with TAK1 inhibitor 5Z-7-Oxozeaenol,respectively,followed by PDGF-BB stimulation.1)CCK-8 assay was performed to detect cell viability.DNA synthesis was assessed by Ed U incorporation.Western Blot assay was performed to detect PCNA expression level.Cell migration was estimated by Transwell and Wound-healing assay.2)Flow cytometry was performed to determine the distribution of cell cycle stages.Expression level of cell cycle related proteins,including Cyclin D1,Cyclin E1,CDK2,and CDK4,as well as autophagy related proteins,including Beclin 1,LC3 B,and p62,were detected by Western Blot assay.Immunofluorescence staining was performed to explore intracellular expression and distribution of LC3 B.3.Autophagy inhibitor 3-Methyladenine(3-MA)and Spautin-1 were used on VSMCs,respectively,before PDGF-BB treatment.CCK-8 assay was performed to detect the cell viability.Wound-healing assay was performed to detect cell migration.Expression level of cell cycle related proteins and PCNA were detected by Western Blot assay.4.Cultured VSMCs were pretreated with both 5Z-7-Oxozeaenol and Spautin-1,or 5Z-7-Oxozeaenol alone,followed by PDGF-BB stimulation.Western Blot assay was performed to detect the expression level of PCNA and Beclin 1.Results: In VSMCs,PDGF-BB upregulated TAK1 m RNA and protein expression,induced TAK1 phosphorylation,and significantly promoted cell proliferation and migration.Gene knockdown through si RNA and inhibition by 5Z-7-Oxozeaenol,respectively,suppressed PDGF-BB-induced DNA synthesis and PCNA expression increase,as well effectively decreased VSMCs proliferation and migration.Further analysis shows that PDGF-BB upregulated expression level of cell cycle related proteins,including Cyclin D1,Cyclin E1,CDK2,and CDK4,promoting VSMCs S phase entrance from G1 phase,through activating TAK1.In addition,TAK1 regulated PDGF-BB-induced autophagy that expression of Beclin 1 and LC3 B were upregulated while p62 accumulation was downregulated.The use of autophagy inhibitor 3-MA and Spautin-1,respectively,attenuated the PDGFBB-induced upregulation of cell cycle related protein and PCNA,as well as VSMCs proliferation and migration.In VSMCs with PDGF-BB stimulation,autophagy inhibitor Spautin-1 and TAK1 inhibitor 5Z-7-Oxozeaenol presented a similar inhibition on cell proliferation,respectively,while Spautin-1 didn’t further enhance the proliferate inhibition of 5Z-7-Oxozeaenol.This result released that PDGF-BB promote VSMCs proliferation and migration through TAK1/autophagy pathway.Conclusions: TAK1 regulates PDGF-BB-induced VSMCs proliferation through mediating autophagy.The result demonstrated that TAK1 is an effective prevention and therapy target in vascular proliferate diseases.