Mechanical regulation of glycogen synthase kinase 3β (GSK3β) in mesenchymal stem cells is dependent on Akt protein serine 473 phosphorylation via mTORC2 protein

Abstract

Mechanical signals can inactivate glycogen synthase kinase 3β (GSK3β), resulting in stabilization of β-catenin. This signaling cascade is necessary for the inhibition of adipogenesis in mesenchymal stem cells (MSC) that is produced by a daily strain regimen. We investigated whether Akt is the mechanically activated kinase responsible for phosphorylation and inactivation of GSK3β in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3β at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3β at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3β, suggesting that Ser-473 phosphorylation is sufficient for the downstream mechanoresponse. As Rictor/mTORC2 (mammalian target of rapamycin complex 2) is known to transduce phosphorylation of Akt at Ser-473 by insulin, we investigated whether it contributes to strain-induced Ser-473 phosphorylation. Phosphorylation of Ser-473 by both mechanical and insulin treatment in MSC was prevented by the mTOR inhibitor KU0063794. When mTORC2 was blocked, mechanical GSK3β inactivation was prevented, whereas insulin inhibition of GSK3β was still measured in the absence of Ser-473 phosphorylation, presumably through phosphorylation of Akt at Thr-308. In sum, mechanical input initiates a signaling cascade that is uniquely dependent on mTORC2 activation and phosphorylation of Akt at Ser-473, an effect sufficient to cause inactivation of GSK3β. Thus, mechanical regulation of GSK3β downstream of Akt is dependent on phosphorylation of Akt at Ser-473 in a manner distinct from that of growth factors. As such, Akt reveals itself to be a pleiotropic signaling molecule whose downstream targets are differentially regulated depending upon the nature of the activating input.

FIGURE 1.

Mechanical strain induces rapid phosphorylation of Akt. A, mdMSC were subjected to strain for 0.5–2 h, and cellular proteins were immunoblotted for phosphorylated Akt and GSK3β. B, densitometric analysis of phosphorylated Akt (Ser-473) normalized to total Akt and phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = six experiments) for mdMSC subjected to strain for 45 min. *, significant difference from the unstrained control (p < 0.001). C, immunoblots of cultures treated with insulin (100 nm) for 0.5–2 h.

FIGURE 2.

Mechanical inhibition of GSK3β is dependent on Akt activation. A, immunoblots of mdMSC subjected to strain for 45 min following treatment with the Akt inhibitor Akti-1/2 (40 μm). B, densitometric analysis of phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = four experiments). *, significant difference from the unstrained control (p < 0.05). C, immunoblots of total cellular proteins from mdMSC transfected with nonsense siRNA (siSCR) or siRNA targeting Akt (siAkt) and then cultured for 3 days before application of strain for 45 min. D, densitometric analysis of phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = three experiments). *, significant difference from the unstrained control (p < 0.01).

FIGURE 3.

Strain-dependent phosphorylation of GSK3β does not require PI3K. A, immunoblots of mdMSC subjected to strain for 60 min following treatment with the PI3K inhibitor LY294002 (50 μm). B, immunoblots of cultures treated with LY294002 and then stimulated with insulin (50 nm) for 30 or 60 min.

FIGURE 4.

ILK does not contribute to mechanical activation of Akt. A, immunoblots of cellular proteins from mdMSC transfected with nonsense siRNA (siSCR) or siRNA targeting ILK (siILK) and then cultured for 3 days before application of strain for 30 or 60 min. B, densitometric analysis of phosphorylated Akt (Ser-473) normalized to total Akt and phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = three experiments) for mdMSC subjected to strain for 60 min. *, significant difference from the unstrained control (p < 0.05).

FIGURE 5.

PKC mediates Akt phosphorylation at Ser-473. A, immunoblots of mdMSC subjected to strain for 45 min following treatment with the PKC inhibitor calphostin C (1 μm). B, densitometric analysis of phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = three experiments). *, significant difference from the unstrained control (p < 0.01). C, immunoblots of cultures subjected to strain for 30 min following treatment with the conventional PKC inhibitor Gö6976.

FIGURE 6.

mTORC2 is required for mechanical activation of Akt. A, immunoblots of mdMSC subjected to strain for 45 min following treatment with the mTOR inhibitor KU0063794 (2 μm). B, densitometric analysis of phosphorylated GSK3β (Ser-9) normalized to total GSK3β (n = three experiments). *, significant difference from the unstrained control (p < 0.05). C, immunoblots of cultures subjected to strain for 45 min following treatment with rapamycin (30 nm) to inhibit mTORC1. D, immunoblots of mdMSC treated with KU0063794 and then stimulated with insulin (50 nm) for 60 min. E, immunoblots of cultures treated with rapamycin and then stimulated with insulin for 60 min.