Medial vascular calcification is a highly regulated process involving osteo-/chondrogenic trans differentiation of vascular smooth muscle cells. Both, protein kinase B (PKB) and serum- and glucocorticoid-inducible kinase I (SGK1) are involved in the intracellular signaling of vascular calcification and both phosphorylate and inactivate glycogen synthase kinase 3 (GSK-3). The present study explored whether PKB/SGK-dependent phosphorylation of GSK-3 alpha/beta is involved in vascular calcification. Experiments were performed in Gsk-3 alpha/beta double knockin mice lacking functional PKB/SGK phosphorylation sites (gsk-3(KI)) and corresponding wild-type mice (gsk-3(WT)) following high-dosed cholecalciferol treatment as well as ex vivo in aortic ring explants from gsk-3(KI) and gsk-3(WT) mice treated without and with phosphate. In gsk-3(WT) mice, high-dosed cholecalciferol induced vascular calcification and aortic osteo-/chondrogenic signaling, shown by increased expression of osteogenic markers Msx2, Cbfa1 and tissue-nonspecific alkaline phosphatase (Alpl). All these effects were suppressed in aortic tissue from gsk3(KI) mice. Cholecalciferol decreased aortic Gsk-3 alpha/beta phosphorylation (Ser(21/9)) in gsk-3(WT) mice, while no phosphorylation was observed in gsk-3(KI) mice. Moreover, the mRNA expression of type III sodium dependent phosphate transporter (Pit1) and plasminogen activator inhibitor 1 (Pai1) was increased following cholecalciferol treatment in aortic tissue of gsk-3(WT) mice, effects again blunted in gsk-3(KI) mice. In addition, phosphate treatment induced mineral deposition and osteogenic markers expression in aortic ring explants from gsk-3(WT) mice, effects reduced in aortic ring explants from gsk-3(KI) mice. In conclusion, vascular PKB/SGK-dependent phosphorylation of GSK-3 alpha/beta contributes to the osteoinductive signaling leading to vascular calcification. (C) 2018 Elsevier Inc. All rights reserved.