Role of PKB/SGK-dependent phosphorylation of GSK-3α/β in vascular calcification during cholecalciferol overload in mice.

Medial vascular calcification is a highly regulated process involving osteo-/chondrogenic transdifferentiation of vascular smooth muscle cells. Both, protein kinase B (PKB) and serum- and glucocorticoid-inducible kinase 1 (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-3α/β is involved in vascular calcification. Experiments were performed in Gsk-3α/β 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 gsk-3 ^KI mice. Cholecalciferol decreased aortic Gsk-3α/β 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-3α/β contributes to the osteoinductive signaling leading to vascular calcification.
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