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

Abstract

Mechanical stimulation can prevent adipogenic and improve osteogenic lineage allocation of mesenchymal stem cells (MSC), an effect associated with the preservation of ␤-catenin levels. We asked whether mechanical up-regulation of ␤-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 GSK3␤ in a Wnt-independent fashion. Small interfering RNA targeting GSK3␤ prevented both strain-induced induction of ␤-catenin and an increase in COX2, a factor associated with increased osteoprogenitor phenotype. Small interfering RNA knockdown of ␤-catenin blocked mechanical reduction of peroxisome proliferator-activated receptor ␥ and adiponectin, implicating ␤-catenin in strain inhibition of adipogenesis. In contrast, the effect of both mechanical and pharmacologic inhibition of GSK3␤ on the putative ␤-catenin target, COX2, was unaffected by ␤-catenin knockdown. GSK3␤ inhibition caused accumulation of nuclear NFATc1; mechanical strain increased nuclear NFATc1, independent of ␤-catenin. NFATc1 knockdown prevented mechanical stimulation of COX2, implicating NFATc1 signaling. Finally, inhibition of GSK3␤ caused association of RNA polymerase II with the COX2 gene, suggesting transcription initiation. These results demonstrate that mechanical inhibition of GSK3␤ induces activation of both ␤-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 GSK3␤, which in turn regulates multiple downstream effectors.The output of differentiating cells from the marrow mesenchymal stem cell (MSC) 2 pool reflects a reciprocal relationship between the numbers of adipocytes and osteoblasts (1). This suggests that regulation of MSC differentiation should be an important point where control of osteoprogenitor and adipocyte output is delineated by microenvironmental factors. It is known that marrow is replaced by fat in sedentary and aged individuals (2, 3). Exercise, with subsequent physical loading of marrow MSC, increases bone mass, and generally represses fat (4, 5). Indeed, MSC are highly responsive to mechanical signals during differentiation, and mechanical load can apprehend adipogenesis even under strong adipogenic culture conditions (6). As such, the effects of mechanical strain or exercise to promote bone formation and limit adipogenesis are conveyed, at the very least, through direct effects on MSC differentiation.Our previous work suggests that ␤-catenin signaling is largely responsible for mechanical limitation of adipocyte differentiation (6). ␤-Catenin is known to regulate adipogenesis at multiple loci, including attenuation of PPAR␥ expression (7) and a negative effect on PPAR␥ activation of gene targets (8). The fall in ␤-catenin that accompanies the rising levels of...

Glycogen synthase kinase-3α/β inhibition promotes in vivo amplification of endogenous mesenchymal progenitors with osteogenic and adipogenic potential and their differentiation to the osteogenic lineage

“…This is in agreement with in vitro studies on the preadipocyte cell line ST2 (15) and is in line with studies on sustained expression of b-catenin by expression of Wnt10b in preadipocytes or following stimulation by mechanical loading, where adipogenesis is blocked in favor of osteogenesis. (13,24,25) Of interest is our data on osteoclast maturation and activation. In contrast to what was reported by Glass and colleagues, (20) where it was shown that Wnt signaling promoted the ability of differentiated osteoblasts to inhibit osteoclast differentiation, we have shown a transient increase in the number of TRACP þ osteoclasts, most likely owing to an increase in the number of hematopoietic progenitors, from which osteoclasts are derived.…”

Mechanosignaling in Bone Health, Trauma and Inflammation

“…Loading controls b-catenin levels by regulating phosphorylation of GSK3, thereby inhibiting b-catenin degradation and increasing its levels in the cytoplasm; thus, driving osteoblastic differentiation (14,103). The Wnt signals are reported to be transduced through the Lrp 5/ 6 receptor; thus, mutations in this receptor results in a constitutive ''ON'' signal, leading to a state of high bone mass (10,55,69), while loss of function mutations in the Lrp5 receptor lead to poor mechanical response to loading (70,101).…”

Vinculin promotes nuclear localization of TAZ to inhibit ECM stiffness-dependent differentiation into adipocytes

“…For instance, MSCs can be differentiated into adipocytes by stimulation with bone morphogenetic protein (BMP) 4 (Tang et al, 2004), and into osteoblasts or chondrocytes by treatment with BMP2 (Noël et al, 2004). In addition to soluble factors, MSC lineage specifications can be directed by physical changes induced by the extracellular environment, including cell area and strain, and by extracellular matrix (ECM) stiffness (Dupont et al, 2011;Engler et al, 2006;McBeath et al, 2004;Nobusue et al, 2014;Sen et al, 2009;Swift et al, 2013). MSCs cultured on soft substrates differentiate into adipocytes efficiently, whereas cells on rigid substrates preferentially differentiate into osteoblasts (Dupont et al, 2011;Engler et al, 2006).…”