Role Of PUMA In Pericyte Migration Induced By Methamphetamine

Objective: Mounting evidence indicates that methamphetamine causes blood-brain barrier damage,with emphasis on endothelial cells.The role of pericytes in methamphetamine-induced BBB damage remains unknown.Our study demonstrated that methamphetamine increased the migration of pericytes from the endothelial basement membrane and resulted in BBB dysfunction.However,the detailed mechanisms underlying this process remain poorly understood.Thus,we examined the molecular mechanisms involved in methamphetamine-induced pericyte migration.Methods: The expression of p53 up-regulated modulator of apoptosis(PUMA)was examined by western blotting and immunofluorescence staining.The expression of integrin ?1 and integrin ?3 was examined by western blotting.In addition,the phosphorylation of proteins involved in cell signaling pathways was detected by western blotting,and cell migration was examined by wound-healing assay and transwell migration assay for a pericyte-like cell line,C3H/10T1/2,transduced with a lentivirus expressing green fluorescent protein(LV-GFP),as well as for primary human brain vascular pericytes(HBVPs).The role of PUMA in pericyte migration was validated using a si RNA approach.PUMA KO mice were used to further validate the role of PUMA in methamphetamine-mediated pericyte migration using immunostaining and electron microscopy.Results: 1)Exposure of C3H/10T1/2 cells and HBVPs to methamphetamine increased the expression of PUMA;2)PUMA participated in pericyte migration induced by methamphetamine.Knockdown of PUMA in pericytes transduced with PUMA si RNA attenuated the methamphetamine-induced increase in cell migration;3)Activation of MAPKs and Akt/PI3 K pathways was involved in methamphetamine-mediated up-regulation of PUMA;4)Exposure of C3H/10T1/2 cells and HBVPs to methamphetamine resulted increased the expression of integrin ?1 and ?3 in a time-dependent manner.Pretreatment of primary HBVPs with either neutralizing anti-?1 integrin or anti-?3 integrin resulted in the significant suppression of methamphetamine-mediated pericyte migration;5)Tyrosine kinases Src and Pyk2 were involved in methamphetamine-mediated pericyte migration;6)In vivo relevance of this study was further confirmed in PUMA KO genetic mice,methamphetamine administration induced the migration of pericyte in WT mice,but not in PUMA KO mice.Conclusions: PUMA was involved in methamphetamine-mediated pericyte migration via activation of MAPKs and Akt/PI3 K pathways,followed by the up-regulation of integrin ?1 and ?3 and the phosphorylation of tyrosine kinases Src and Pyk2,leading to the increased expression of PUMA.