Mechanical Loading Regulates NFATc1 and β-Catenin Signaling through a GSK3β Control Node

Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of β-catenin levels. We asked whether mechanical up-regulation of β-catenin was critical to reduction in adipogenesis as well as other mechanical events inducing alternate MSC lineage selection. In MSC cultured under strong adipogenic conditions, mechanical load (3600 cycles/day, 2% strain) inactivated GSK3β in a Wnt-independent fashion. Small interfering RNA targeting GSK3β prevented both strain-induced induction of β-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of β-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor γ and adiponectin, implicating β-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3β on the putative β-catenin target, COX2, was unaffected by β-catenin knockdown. GSK3β inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of β-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3β caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3β induces activation of both β-catenin and NFATc1 signaling, limiting adipogenesis via the former and promoting osteoblastic differentiation via NFATc1/COX2. Our novel findings suggest that mechanical loading regulates mesenchymal stem cell differentiation through inhibition of GSK3β, which in turn regulates multiple downstream effectors