Abstract
Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of beta-catenin levels. We asked whether mechanical up-regulation of beta-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 GSK3beta in a Wnt-independent fashion. Small interfering RNA targeting GSK3beta prevented both strain-induced induction of beta-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of beta-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor gamma and adiponectin, implicating beta-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3beta on the putative beta-catenin target, COX2, was unaffected by beta-catenin knockdown. GSK3beta inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of beta-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3beta caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3beta induces activation of both beta-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 GSK3beta, which in turn regulates multiple downstream effectors.
Highlights
Mechanical Effect on -Catenin Is Critical for Inhibition of Adipogenesis—As mesenchymal stem cells (MSC) enter the adipogenic lineage and express PPAR␥ and adiponectin, the levels of both active and total -catenin drop rapidly as we have previously shown [6]
To further understand mechanical control of adipocyte differentiation, we first wished to ascertain whether the mechanical effect was separate from Wnt signaling, which has been implicated by others as essential to mechanical signaling [30]
Mechanical Stimulation of COX2 Does Not Require -Catenin Activation—We examined the effect of mechanical loading on COX2, a gene that responds to multiple types of mechanical stimulation in bone cells, as we and others [10, 12, 16] have reported
Summary
Reagents—Fetal bovine serum was from Atlanta Biologicals (Atlanta, GA). Culture media, trypsin-EDTA reagent, antibiotics, Lipofectamine 2000, reverse transcriptase, and Taq polymerase were from Invitrogen. Key experiments were replicated in a marrow-derived mesenchymal stem cell line generated from C56/BL6 wild-type mice using the procedure of Peister et al [25]. These cells readily undergo differentiation into osteogenic, adipogenic, or alternative lineages using standard modifiers. Chromatin pellets were sonicated to an average of 300 –500-bp fragments of DNA, centrifuged, and diluted into chromatin immunoprecipitation buffer (16.7 mM Tris-HCl, pH 8.1,150 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA). DNA fragments were purified using Qiagen QIAquick Spin Kits (Valencia, CA) and subjected to PCR techniques using primers Densitometry data, where given, were compiled from at least three separate experiments
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