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 ?-catenin and stimulating osteogenesis by generating a stiffer cytoskeleton.
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 ?CT (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.
Mechanical factors play a crucial role in the development of articular cartilage in vivo. In this regard, tissue engineers have sought to leverage native mechanotransduction pathways to enhance in vitro stem cell-based cartilage repair strategies. However, a thorough understanding of how individual mechanical factors influence stem cell fate is needed to predictably and effectively utilize this strategy of mechanically-induced chondrogenesis. This article summarizes some of the latest findings on mechanically stimulated chondrogenesis, highlighting several new areas of interest, such as the effects of mechanical stimulation on matrix maintenance and terminal differentiation, as well as the use of multifactorial bioreactors. Additionally, the roles of individual biophysical factors, such as hydrostatic or osmotic pressure, are examined in light of their potential to induce mesenchymal stem cell chondrogenesis. An improved understanding of biomechanically-driven tissue development and maturation of stem cell-based cartilage replacements will hopefully lead to the development of cell-based therapies for cartilage degeneration and disease.
Mechanical strain provides an anti-adipogenic, pro-osteogenic stimulus to mesenchymal stem cells (MSC) through generating intracellular signals and via cytoskeletal restructuring. Recently, mTORC2 has been shown to be a novel mechanical target critical for the anti-adipogenic signal leading to preservation of ?-catenin. As mechanical activation of mTORC2 requires focal adhesions (FAs), we asked whether proximal signaling involved Src and FAK, which are early responders to integrin-FA engagement. Application of mechanical strain to marrow-derived MSCs was unable to activate mTORC2 when Src family kinases were inhibited. Fyn, but not Src, was specifically required for mechanical activation of mTORC2 and was recruited to FAs after strain. Activation of mTORC2 was further diminished following FAK inhibition, and as FAK phosphorylation (Tyr-397) required Fyn activity, provided evidence of Fyn/FAK cooperativity. Inhibition of Fyn also prevented mechanical activation of RhoA as well as mechanically induced actin stress fiber formation. We thus asked whether RhoA activation by strain was dependent on mTORC2 downstream of Fyn. Inhibition of mTORC2 or its downstream substrate, Akt, both prevented mechanical RhoA activation, indicating that Fyn/FAK affects cytoskeletal structure via mTORC2. We then sought to ascertain whether this Fyn-initiated signal pathway modulated MSC lineage decisions. siRNA knockdown of Fyn, but not Src, led to rapid attainment of adipogenic phenotype with significant increases in adipocyte protein 2, peroxisome proliferator-activated receptor gamma, adiponectin, and perilipin. As such, Fyn expression in mdMSCs contributes to basal cytoskeletal architecture and, when associated with FAs, functions as a proximal mechanical effector for environmental signals that influence MSC lineage allocation.
Mechanical signals can inactivate glycogen synthase kinase 3? (GSK3?), resulting in stabilization of ?-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 GSK3? in MSC. Mechanical strain (2% magnitude, 0.17 Hz) induced phosphorylation of Akt at Ser-473 and Thr-308 in parallel with phosphorylation of GSK3? at Ser-9. Inhibiting Akt (Akt1/2 kinase inhibitor treatment or Akt knockdown) prevented strain-induced phosphorylation of GSK3? at Ser-9. Inhibition of PI3K prevented Thr-308 phosphorylation, but strain-induced Ser-473 phosphorylation was measurable and induced phosphorylation of GSK3?, 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 GSK3? inactivation was prevented, whereas insulin inhibition of GSK3? 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 GSK3?. Thus, mechanical regulation of GSK3? 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.
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 3? (GSK3?) with ?-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 GSK3?, 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.
Failures of fracture repair (nonunions) occur in 10% of all fractures. The use of mesenchymal stem cells (MSC) in tissue regeneration appears to be rationale, safe, and feasible. The contributions of MSC to the reparative process can occur through autocrine and paracrine effects. The primary objective of this study is to find a novel mean, by transplanting primary cultures of bone marrow-derived MSCs expressing insulin-like growth factor-I (MSC(IGF)), to promote these seed-and-soil actions of MSC to fully implement their regenerative abilities in fracture repair and nonunions. MSC(IGF) or traceable MSC(IGF)-Lac-Z were transplanted into wild-type or insulin-receptor-substrate knockout (Irs1(-/-)) mice with a stabilized tibia fracture. Healing was assessed using biomechanical testing, microcomputed tomography (?CT), and histological analyses. We found that systemically transplanted MSC(IGF) through autocrine and paracrine actions improved the fracture mechanical strength and increased new bone content while accelerating mineralization. We determined that IGF-I adapted the response of transplanted MSC(IGF) to promote their differentiation into osteoblasts. In vitro and in vivo studies showed that IGF-I-induced osteoglastogenesis in MSCs was dependent of an intact IRS1-PI3K signaling. Furthermore, using Irs1(-/-) mice as a nonunion fracture model through altered IGF signaling, we demonstrated that the autocrine effect of IGF-I on MSC restored the fracture new bone formation and promoted the occurrence of a well-organized callus that bridged the gap. A callus that was basically absent in Irs1(-/-) left untransplanted or transplanted with MSCs. We provided evidence of effects and mechanisms for transplanted MSC(IGF) in fracture repair and potentially to treat nonunions.
Mechanical signals of both low and high intensity are inhibitory to fat and anabolic to bone in vivo, and have been shown to directly affect mesenchymal stem cell pools from which fat and bone precursors emerge. To identify an idealized mechanical regimen which can regulate MSC fate, low intensity vibration (LIV; <10 microstrain, 90 Hz) and high magnitude strain (HMS; 20,000 microstrain, 0.17 Hz) were examined in MSC undergoing adipogenesis. Two x twenty minute bouts of either LIV or HMS suppressed adipogenesis when there was at least a 1h refractory period between bouts; this effect was enhanced when the rest period was extended to 3h. Mechanical efficacy to inhibit adipogenesis increased with additional loading bouts if a refractory period was incorporated. Mechanical suppression of adipogenesis with LIV involved inhibition of GSK3? with subsequent activation of ?-catenin as has been shown for HMS. These data indicate that mechanical biasing of MSC lineage selection is more dependent on event scheduling than on load magnitude or duration. As such, a full day of rest should not be required to "reset" the mechanical responsiveness of MSCs, and suggests that incorporating several brief mechanical challenges within a 24h period may improve salutary endpoints in vivo. That two diverse mechanical inputs are enhanced by repetition after a refractory period suggests that rapid cellular adaptation can be targeted.
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 PPAR? and adiponectin. Strain increased ?-catenin and inhibition of adipogenesis required this effect. Under osteogenic conditions, strain activated ?-catenin signaling and increased expression of WISP1 and COX2. mdMSC were also generated from mice lacking caveolin-1, a protein known to sequester ?-catenin: caveolin-1((-/-)) mdMSC exhibited retarded differentiation along both adipogenic and osteogenic lineages but retained mechanical responses that involved ?-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 ?-catenin influencing phenotype. As such, these cells offer a useful model for studies of direct mechanical regulation of MSC differentiation and function.
Regulation of mesenchymal stem cell (MSC) lineage selection is important for the generation of bone mass. Inhibition of cyclooxygenase-2 (COX2) may increase adipogenesis at the cost of decreasing osteoprogenitor output. Here we investigated the role of COX2 and its products during MSC differentiation. Indomethacin stimulated adipogenesis (increased aP2, adiponectin and lipid droplets) of CH310T1/2 stem cells as well as marrow-derived MSCs to a degree similar to the PPAR?2 ligand, rosiglitazone. Unlike rosiglitazone, indomethacin significantly upregulated PPAR?2 expression. Indomethacin and the COX2 specific inhibitor celecoxib suppressed PGE2 production, but celecoxib did not induce adipogenesis. As well, addition of PGE2 failed to reverse indomethacin induced adipogenesis, indicating that indomethacins effects were prostaglandin independent. In MSCs over-expressing PPAR?2 and RXR?, indomethacin did not increase PPAR-induced transcription, while rosiglitazone and 15d-PGJ2 did (1.7- and 1.3-fold, respectively, P?0.001). We considered whether indomethacin might directly affect C/EBP? proximally to PPAR?2 induction. Indomethacin significantly increased C/EBP? expression and protein within 24?h of addition. These results indicate that indomethacin promotes adipogenesis by increasing C/EBP? and PPAR?2 expression in a prostaglandin-independent fashion. This effect of indomethacin is pertinent to potential deleterious effects of this commonly used anti-inflammatory drug on bone remodeling and tissue healing.
In the last 5 years a role for beta-catenin in the skeleton has been cemented. Beginning with mutations in the Lrp5 receptor that control beta-catenin canonical downstream signals, and progressing to transgenic models with bone-specific alteration of beta-catenin, research has shown that beta-catenin is required for normal bone development. A cell critical to bone in which beta-catenin activity determines function is the marrow-derived mesenchymal stem cell (MSC), where sustained beta-catenin prevents its distribution into adipogenic lineage. beta-Catenin actions are less well understood in mature osteoblasts: while beta-catenin contributes to control of osteoclastic bone resorption via alteration of the osteoprotegerin/RANKL ratio, a specific regulatory role during osteoblast bone synthesis has not yet been determined. The proven ability of mechanical factors to prevent beta-catenin degradation and induce nuclear translocation through Lrp-independent mechanisms suggests processes by which exercise might modulate bone mass via control of lineage allocation, in particular, by preventing precursor distribution into the adipocyte pool. Effects resulting from mechanical activation of beta-catenin in mature osteoblasts and osteocytes likely modulate bone resorption, but whether beta-catenin is involved in osteoblast synthetic function remains to be proven for both mechanical and soluble mediators. As beta-catenin appears to support the downstream effects of multiple osteogenic factors, studies clarifying when and where beta-catenin effects occur will be relevant for translational approaches aimed at preventing bone loss and terminal adipogenic conversion.
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.
Exercise prevents marrow mesenchymal stem cell (MSC) adipogenesis, reversing trends that accompany aging and osteoporosis. Mechanical input, the in-vitro analogue to exercise, limits PPAR? expression and adipogenesis in MSC. We considered whether C/EBP? might be mechanoresponsive as it is upstream to PPAR?, and also is known to upregulate endoplasmic reticulum (ER) stress. MSC (C3H10T1/2 pluripotent cells as well as mouse marrow-derived MSC) were cultured in adipogenic media and a daily mechanical strain regimen was applied. We demonstrate herein that mechanical strain represses C/EBP? mRNA (0.6-fold ±0.07, p<0.05) and protein (0.4-fold ±0.1, p<0.01) in MSC. SiRNA silencing of ?-catenin prevented mechanical repression of C/EBP?. C/EBP? overexpression did not override strains inhibition of adipogenesis, which suggests that mechanical control of C/EBP? is not the primary site at which adipogenesis is regulated. Mechanical inhibition of C/EBP?, however, might be critical for further processes that regulate MSC health. Indeed, overexpression of C/EBP? in MSC induced ER stress evidenced by a dose-dependent increase in the pro-apoptotic CHOP (protein 4-fold ±0.5, p<0.05) and a threshold reduction in the chaperone BiP (protein 0.6-fold ±0.1, p?=?0.2; mRNA 0.3-fold ±0.1, p<0.01). ChIP-seq demonstrated a significant association between C/EBP? and both CHOP and BiP genes. The strain regimen, in addition to decreasing C/EBP? mRNA (0.5-fold ±0.09, p<0.05), expanded ER capacity as measured by an increase in BiP mRNA (2-fold ±0.2, p<0.05) and protein. Finally, ER stress induced by tunicamycin was ameliorated by mechanical strain as demonstrated by decreased C/EBP?, increased BiP and decreased CHOP protein expression. Thus, C/EBP? is a mechanically responsive transcription factor and its repression should counter increases in marrow fat as well as improve skeletal resistance to ER stress.
Exercise-generated signals are pro-osteogenic and anti-adipogenic within the marrow. In vitro studies indicate that mechanical signals directly block adipogenic differentiation through activation of ?-catenin and by limiting PPAR?2 expression. Whether mechanically generated ?-catenin can inhibit adipogenesis during PPAR? transactivation is unknown. We evaluated the ability of mechanical signals to limit adipogenesis in marrow derived mesenchymal stem cells (mdMSC) distal to activation of PPAR?. First, we established that mdMSC attained an adipogenic phenotype within 2-4 days in the presence of rosiglitazone (1-25 ?M) and that ?-catenin activation via GSK3? inhibition interfered with this process. Similarly, mechanical strain (3600 cycles, 2% strain daily) inhibited adipogenesis at 3 days, preventing rosiglitazone-induced PPAR? upregulation as well as aP2 and adiponectin protein expression. To assess whether a reduction in PPAR? expression was necessary for anti-adipogenic action, PPAR?2 was overexpressed: both mechanical strain and GSK3? inhibition prevented expression of aP2 and adiponectin proteins despite abundant PPAR?2 and its ligand. To understand the fate of single cells experiencing mechanical strain we generated mdMSC from aP2-GFP reporter expressing mice. Rosiglitazone treatment for 3 days induced GFP expression in more than 80% of cells. Sorting by GFP expression revealed that the highest 20% of aP2-GFP expressing cells was responsible for the majority of adipogenic protein expression. This highly expressing GFP fraction had a reduced ability to respond to an osteogenic stimulus: BMP-2 treatment increased osterix by 12-fold in contrast to the 42-fold increase in osterix expression that resulted from BMP-2 treatment of the bottom 75% of GFP expressing cells. This suggested that highly expressing aP2-GFP cells represented more terminally differentiated adipocytes, with reduced multipotentiality. Application of mechanical strain to aP2-GFP mdMSC treated with rosiglitazone caused a two-fold decrease in the size of the upper cell fraction, suggesting that mechanical strain preserved MSC in a multipotent state. Our data show that mechanical strain restricts adipogenesis both by limiting PPAR?2 expression and by preventing PPAR? action, protecting the potential of MSC to enter other lineages.
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