Buer Sen

Mechanical Strain Inhibits Adipogenesis in Mesenchymal Stem Cells by Stimulating a Durable β-Catenin Signal

The ability of exercise to decrease fat mass and increase bone mass may occur through mechanical biasing of mesenchymal stem cells (MSCs) away from adipogenesis and toward osteoblastogenesis. C3H10T1/2 MSCs cultured in highly adipogenic medium express peroxisome proliferator-activated receptor gamma and adiponectin mRNA and protein, and accumulate intracellular lipid. Mechanical strain applied for 6 h daily inhibited expression of peroxisome proliferator-activated receptor gamma and adiponectin mRNA by up to 35 and 50%, respectively, after 5 d. A decrease in active and total beta-catenin levels during adipogenic differentiation was entirely prevented by daily application of mechanical strain; furthermore, strain induced beta-catenin nuclear translocation. Inhibition of glycogen synthase kinase-3beta by lithium chloride or SB415286 also prevented adipogenesis, suggesting that preservation of beta-catenin levels was important to strain inhibition of adipogenesis. Indeed, mechanical strain inactivated glycogen synthase kinase-3beta, which was preceded by Akt activation, indicating that strain transmits antiadipogenic signals through this pathway. Cells grown under adipogenic conditions showed no increase in osteogenic markers runt-related transcription factor (Runx) 2 and osterix (Osx); subsequent addition of bone morphogenetic protein 2 for 2 d increased Runx2 but not Osx expression in unstrained cultures. When cultures were strained for 5 d before bone morphogenetic protein 2 addition, Runx2 mRNA increased more than in unstrained cultures, and Osx expression more than doubled. As such, mechanical strain enhanced MSC potential to enter the osteoblast lineage despite exposure to adipogenic conditions. Our results indicate that MSC commitment to adipogenesis can be suppressed by mechanical signals, allowing other signals to promote osteoblastogenesis. These data suggest that positive effects of exercise on both fat and bone may occur during mesenchymal lineage selection.

β-Catenin Levels Influence Rapid Mechanical Responses in Osteoblasts

Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/β-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce β-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on β-catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active β-catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/β-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of β-catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3β, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of β-catenin or up-regulation of Wisp1 and Cox2 expression. Conditions that increased basal β-catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3β to allow β-catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, β-catenin serves as both a modulator and effector of mechanical signals in bone cells.

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.

Bone marrow fat accumulation accelerated by high fat diet is suppressed by exercise

Marrow adipose tissue (MAT), associated with skeletal fragility and hematologic insufficiency, remains poorly understood and difficult to quantify. We tested the response of MAT to high fat diet (HFD) and exercise using a novel volumetric analysis, and compared it to measures of bone quantity. We hypothesized that HFD would increase MAT and diminish bone quantity, while exercise would slow MAT acquisition and promote bone formation. Eight week-old female C57BL/6 mice were fed a regular (RD) or HFD, and exercise groups were provided voluntary access to running wheels (RD-E, HFD-E). Femoral MAT was assessed by μCT (lipid binder osmium) using a semi-automated approach employing rigid co-alignment, regional bone masks and was normalized for total femoral volume (TV) of the bone compartment. MAT was 2.6-fold higher in HFD relative to RD mice. Exercise suppressed MAT in RD-E mice by more than half compared with RD. Running similarly inhibited MAT acquisition in HFD mice. Exercise significantly increased bone quantity in both diet groups. Thus, HFD caused significant accumulation of MAT; importantly running exercise limited MAT acquisition while promoting bone formation during both diets. That MAT is exquisitely responsive to diet and exercise, and its regulation by exercise appears to be inversely proportional to effects on exercise induced bone formation, is relevant for an aging and sedentary population.

Caveolin-1 knockout mice have increased bone size and stiffness

The skeletal phenotype of the cav‐1−/− mouse, which lacks caveolae, was examined. μCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties. Cell studies showed that cav‐1 deficiency leads to increased osteoblast differentiation. These results suggest that cav‐1 helps to maintain osteoblast progenitors in a less differentiated state.

Cell mechanosensitivity to extremely low-magnitude signals is enabled by a LINCed nucleus

A cells ability to recognize and adapt to the physical environment is central to its survival and function, but how mechanical cues are perceived and transduced into intracellular signals remains unclear. In mesenchymal stem cells (MSCs), high‐magnitude substrate strain (HMS, ≥2%) effectively suppresses adipogenesis via induction of focal adhesion (FA) kinase (FAK)/mTORC2/Akt signaling generated at FAs. Physiologic systems also rely on a persistent barrage of low‐level signals to regulate behavior. Exposing MSC to extremely low‐magnitude mechanical signals (LMS) suppresses adipocyte formation despite the virtual absence of substrate strain (<0.001%), suggesting that LMS‐induced dynamic accelerations can generate force within the cell. Here, we show that MSC response to LMS is enabled through mechanical coupling between the cytoskeleton and the nucleus, in turn activating FAK and Akt signaling followed by FAK‐dependent induction of RhoA. While LMS and HMS synergistically regulated FAK activity at the FAs, LMS‐induced actin remodeling was concentrated at the perinuclear domain. Preventing nuclear‐actin cytoskeleton mechanocoupling by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complexes inhibited these LMS‐induced signals as well as prevented LMS repression of adipogenic differentiation, highlighting that LINC connections are critical for sensing LMS. In contrast, FAK activation by HMS was unaffected by LINC decoupling, consistent with signal initiation at the FA mechanosome. These results indicate that the MSC responds to its dynamic physical environment not only with “outside‐in” signaling initiated by substrate strain, but vibratory signals enacted through the LINC complex enable matrix independent “inside–inside” signaling. Stem Cells 2013;33:2063–2076

mTORC2 regulates mechanically induced cytoskeletal reorganization and lineage selection in marrow-derived mesenchymal stem cells.

The cell cytoskeleton interprets and responds to physical cues from the microenvironment. Applying mechanical force to mesenchymal stem cells induces formation of a stiffer cytoskeleton, which biases against adipogenic differentiation and toward osteoblastogenesis. mTORC2, the mTOR complex defined by its binding partner rictor, is implicated in resting cytoskeletal architecture and is activated by mechanical force. We asked if mTORC2 played a role in mechanical adaptation of the cytoskeleton. We found that during bi‐axial strain‐induced cytoskeletal restructuring, mTORC2 and Akt colocalize with newly assembled focal adhesions (FA). Disrupting the function of mTORC2, or that of its downstream substrate Akt, prevented mechanically induced F‐actin stress fiber development. mTORC2 becomes associated with vinculin during strain, and knockdown of vinculin prevents mTORC2 activation. In contrast, mTORC2 is not recruited to the FA complex during its activation by insulin, nor does insulin alter cytoskeletal structure. Further, when rictor was knocked down, the ability of mesenchymal stem cells (MSC) to enter the osteoblastic lineage was reduced, and when cultured in adipogenic medium, rictor‐deficient MSC showed accelerated adipogenesis. This indicated that cytoskeletal remodeling promotes osteogenesis over adipogenesis. In sum, our data show that mTORC2 is involved in stem cell responses to biophysical stimuli, regulating both signaling and cytoskeletal reorganization. As such, mechanical activation of mTORC2 signaling participates in mesenchymal stem cell lineage selection, preventing adipogenesis by preserving β‐catenin and stimulating osteogenesis by generating a stiffer cytoskeleton.

Mechanical Regulation of Glycogen Synthase Kinase 3β (GSK3β) in Mesenchymal Stem Cells Is Dependent on Akt Protein Serine 473 Phosphorylation via mTORC2 Protein

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.

Mechanical activation of β-catenin regulates phenotype in adult murine marrow-derived mesenchymal stem cells

Regulation of skeletal remodeling appears to influence the differentiation of multipotent mesenchymal stem cells (MSC) resident in the bone marrow. As murine marrow cultures are contaminated with hematopoietic cells, they are problematic for studying direct effects of mechanical input. Here we use a modified technique to isolate marrow‐derived MSC (mdMSC) from adult mice, yielding a population able to differentiate into adipogenic and osteogenic phenotypes that is devoid of hematopoietic cells. In pure mdMSC populations, a daily strain regimen inhibited adipogenic differentiation, suppressing expression of PPARγ and adiponectin. Strain increased β‐catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated β‐catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin‐1, a protein known to sequester β‐catenin: caveolin‐1(−/−) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved β‐catenin activation. Interestingly, caveolin‐1(−/−) mdMSC failed to express bone sialoprotein and did not form mineralized nodules. In summary, mdMSC from adult mice respond to both soluble factors and mechanical input, with mechanical activation of β‐catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function.

Mechanically Induced Focal Adhesion Assembly Amplifies Anti‐Adipogenic Pathways in Mesenchymal Stem Cells

The fate of pluripotent mesenchymal stem cells (MSC) is determined through integration of chemical, spatial, and physical signals. The suppression of MSC adipogenesis by mechanical stimuli, which requires Akt‐induced inhibition of glycogen synthase kinase 3β (GSK3β) with β‐catenin activation, can be enhanced by repetitive dosing within a single day. Here, we demonstrate that reapplication of cyclic strain within a 24‐hour period leads to amplification of both Akt activation and its subsequent inhibition of GSK3β, such that total cycle number can be reduced while still inhibiting adipogenesis. Amplification of Akt signaling is facilitated by a dynamic restructuring of the cell in response to mechanical signals, as evidenced by a transient increase in focal adhesion (FA) number and increased RhoA activity. Preventing FA assembly or development of tension blocks activation of Akt by mechanical signals, but not by insulin. This indicates that the FA infrastructure is essential to the physical, but not necessarily the chemical, sensitivity, and responsiveness of the cell. Exploiting the transient nature of cytoskeletal remodeling may represent a process to enhance cell responsiveness to mechanical input and ultimately define the fate of MSCs with a minimal input. STEM CELLS 2011;29:1829–1836