title: PI3Kα isoform-dependent activation of RhoA regulates Wnt5a-induced osteosarcoma cell migration.
abstract: BACKGROUND
  We have reported that the phosphatidylinositol-3 kinase (PI3K)/Akt signaling pathway mediated Wnt5a-induced osteosarcoma cell migration. However, the signaling pathways regulating Wnt5a/PI3K/Akt-mediated cell migration remains poorly defined in osteosarcoma cells.
  
  
  METHODS
  We evaluated the activations of RhoA, Rac1 and Cdc42 in osteosarcoma MG-63 and U2OS cells with small G-protein activation assay. Boyden chamber assays were used to confirm the migration of cells transfected indicated constructs or siRNA specific against RhoA. A panel of inhibitors of PI3K and Akt treated osteosarcoma cells and blocked kinase activity. Western blotting and RhoA activation assay were employed to measure the effect of kinase inhibitors and activations of RhoA and Akt.
  
  
  RESULTS
  We found that Wnt5a had a potent stimulatory effect on RhoA activation, but not on Rac1 and Cdc42 activations. Wnt5a-induced cell migration was largely abolished by siRNA specific against RhoA. DN-RhoA (GFP-RhoA-N19) was also capable of retarding Wnt5a-induced cell migration, but the overexpression of CA-RhoA (GFP-RhoA-V14) was not able to accelerate cell migration. The Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of LY294002 (PI3K inhibitor) and MK-2206 (Akt inhibitor). Furthermore, we found that the Wnt5a-induced activation of RhoA was mostly blocked by pretreatment of HS-173 (PI3Kα inhibitor). Lastly, the phosphorylation of Akt (p-Ser473) was not altered by transfection with siRNA specific against RhoA or DN-RhoA (GFP-RhoA-N19).
  
  
  CONCLUSIONS
  Taken together, we demonstrate that RhoA acts as the downstream of PI3K/Akt signaling (specific PI3Kα, Akt1 and Akt2 isoforms) and mediated Wnt5a-induced the migration of osteosarcoma cells.
authors.affiliation: Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, 213003 Jiangsu China.
authors.forename: Ailiang
authors.initials: A
authors.lastname: Zhang
authors.affiliation: Safety Assessment and Research Center for Drug, Pesticide and Veterinary Drug of Jiangsu Province, School of Public Health, Nanjing Medical University, Nanjing, 211166 Jiangsu China.
authors.forename: Ting
authors.initials: T
authors.lastname: Yan
authors.affiliation: Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, 213003 Jiangsu China.
authors.forename: Kun
authors.initials: K
authors.lastname: Wang
authors.affiliation: Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, 213003 Jiangsu China.
authors.forename: Zhihui
authors.initials: Z
authors.lastname: Huang
authors.affiliation: Spine Surgery, Third Affiliated Hospital of Soochow University, Changzhou, 213003 Jiangsu China.
authors.forename: Jinbo
authors.initials: J
authors.lastname: Liu
keywords: Akt; Migration; Osteosarcoma; PI3K; RhoA; Wnt5a