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. 2014 Jan;29(1):78-89.
doi: 10.1002/jbmr.2031.

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

Buer Sen  1 , Zhihui Xie, Natasha Case, William R Thompson, Gunes Uzer, Maya Styner, Janet Rubin
Affiliations

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

Buer Sen et al. J Bone Miner Res. 2014 Jan.

Abstract

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.

Keywords: ADIPOCYTE; AKT; OSTEOBLAST; RICTOR; VINCULIN.

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Conflict of interest statement

Disclosures - All authors state that they have no conflicts of interest

Figures

Figure 1
Figure 1. mTORC2 is required for mechanical Akt activation and strain induced cytoskeletal reorganization
a. After 48 h of treatment with siRNA based on the rictor (siRictor) or mutated (siCTL) sequences. mdMSC were strained x 10 min. Immunoblot showed a loss of mechanical Akt activation when rictor is knocked down. Shown to the right, the mTOR inhibitor KU63794 (2mM) prevented mechanical Akt activation. b. Strain-induced focal adhesion assembly (vinculin = red fluorescence) and F-actin (green) and actin reorganization failed to occur when mTOR was inhibited by KU63794. Scale bars = 25 µm. c. Rictor knock down, as opposed to treatment with control siRNA, prevented strain-induced cytoskeletal reorganization. d. Mechanical strain x 10 min activated Akt in both murine embryonic fibroblasts (MEF) and embryonic MSC (C3H10T1/2); this was inhibited by pretreatment with KU63794.
Figure 2
Figure 2. mTORC2 is associated with strain-induced FAs
a. 3 h after 100 strain cycles, cell bodies were removed with hydrodynamic force and remaining adherent FA proteins scraped for Western blot. Strained cells generated more focal adhesion macromolecular complexes. Densitometry of vinculin and rictor bands from 3 separate experiments in series is shown at the right; * = p < 0.05 different from control. b, c. IF of flushed membranes shows increases in punctate vinculin and rictor (b) or mTOR (c) staining after strain. Scale bars = 25 µm.
Figure 3
Figure 3. Strain induces mTORC2 association with FA proteins, dependent on mTOR activity
a. MSC cell lysates were immunoprecipitated with antibody to vinculin 10 min post-strain and analyzed by immunoblot, normalized to vinculin. Strain increased the association of rictor, mTOR and total and phosphorylated Akt with vinculin. Densitometry of rictor and total Akt bands is shown at the right; * = p < 0.05 different from control, n=3. b. Lysates prior to vinculin immunoprecipitation showed equivalent amounts of proteins of interest. c. Paxillin immunoprecipitation of strained cells showed that association with vinculin, rictor, mTOR and Akt was increased by strain. Densitometry of rictor and Akt bands is shown to the right; * = p < 0.05 different from control, n=3. d. IP with vinculin shows that strain failed in increase the association of rictor and Akt with vinculin when mTOR was inhibited with KU63794 (2mM). Densitometry confirms mTOR activity is necessary for association of rictor and Akt bands with vinculin; * = p < 0.05 different from control, n=3.
Figure 4
Figure 4. Akt must be activated to effect cytoskeletal reorganization
a. Addition of the Akt inhibitor, Akti1/2 (40 µM) prevented cytoskeletal reorganization 3h post strain. Vinculin = red, F-actin = green; scale bars = 25 µm. b. Akt activity is required for strain induced association of rictor and Akt with IP’d vinculin. Densitometry of rictor (top) and total Akt (bottom) bands are shown below a representative Western blot; * = p < 0.05 different from control, n=3. c. IF of adherent proteins after removing cell bodies with hydrodynamic force. Akt is associated with increased punctate vinculin membrane staining in strained cells. Scale bars = 25 µm.
Figure 5
Figure 5. mTORC2 association with FA elements is necessary for strain activation of Akt
a. siRNA targeting vinculin, but not control siRNA (siCTL) blocked strain-induced activation of Akt, shown as an increase phospho-473 Akt. Densitometry confirms the failure to significantly increase pAkt when vinculin is knocked down; * = p < 0.05 different from control, n=3. b. Lysates from siCTL and siVin cells shows immunoblot for vinculin and tubulin (top figure) before pull down with antibody to Paxillin (lower figure). Rictor association with paxillin increases only when vinculin is present. c. Blebbistatin (50µM) delivered prior to strain prevented strain-induced rictor, Akt and actin association with vinculin pull down. d. Blebbistatin prevented strain induced mTORC2 activity measured by increase in pAkt. e. Strain was applied at the beginning (strain 1) and/or end (strain 2) of a 3 hour period, at which point all lysates were made. pAkt rises after strain (comparing no strain, 1st lane, to strain 2, 2nd lane). pAkt returns to basal levels 3 h after strain (strain 1, 3rd lane). A 2nd strain application (strain 1+2, 4th lane) shows increased pAkt. Densitometry confirms pAkt return to baseline, and amplification at the second strain application; * ¹ control, * ¹ #, (n=4).
Figure 6
Figure 6. Insulin activation of Akt induces neither cytoskeletal reorganization nor mTORC2 association with vinculin
a. Insulin (100 ng/ml x 1 h) does not induce cytoskeletal reorganization, as does 100 cycles of strain, shown by vinculin (red) and actin (green) staining. Scale bars = 25 µm. b. Vinculin pull down after strain (str) or insulin (Ins): insulin does not induce rictor, mTOR or Akt association with vinculin as does strain. Densitometry of rictor and total Akt bands is shown at the right; * = p < 0.05 different from control, n=3. c. siRNA targeting vinculin failed to prevented insulin activation of Akt, shown as an increase phosphor-473 Akt.
Figure 7
Figure 7. Rictor deficiency limits osteogenesis and promotes adipogenesis
a. Western blot shows Rictor protein 4 d after transfection with siRNA targeting rictor or mutated rictor-like RNA sequence. b. mdMSC cultured in osteogenic media x 4d prior to RT-PCR for osteogenic genes. Rictor knock-down cells had significantly less osteocalcin and osterix expression, * = p < 0.05 different from cultures treated with siCTL, n=3. c,d. After 7d in osteogenic medium, stain for alkaline phosphatase and alkaline phosphatase activity are reduced in rictor knock-down cultures. e,f. mdMSC ± rictor knock-down were cultured in adipogenic media and assayed for adipogenic genes (e) and proteins (f) at 6 days; both repeated x 2. Rictor deficiency enhanced acquisition of adipogenic phenotype. g. Oil red O positive cells were increased in cultures with knock-down of rictor, low magnification shows bright lipid droplets.
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