Mechanical Overloading Induced-Activation of mTOR Signaling in Tendon Stem/Progenitor Cells Contributes to Tendinopathy Development

Abstract

Despite the importance of mechanical loading in tendon homeostasis and pathophysiology, the molecular responses involved in the mechanotransduction in tendon cells remain unclear. In this study, we found that in vitro mechanical loading activated the mammalian target of rapamycin (mTOR) in rat patellar tendon stem/progenitor cells (TSCs) in a stretching magnitude-dependent manner. Application of rapamycin, a specific inhibitor of mTOR, attenuated the phosphorylation of S6 and 4E-BP1 and as such, largely inhibited the mechanical activation of mTOR. Moreover, rapamycin significantly decreased the proliferation and non-tenocyte differentiation of PTSCs as indicated by the reduced expression levels of LPL, PPARγ, SOX-9, collagen II, Runx-2, and osteocalcin genes. In the animal studies, mice subjected to intensive treadmill running (ITR) developed tendon degeneration, as evidenced by the formation of round-shaped cells, accumulation of proteoglycans, and expression of SOX-9 and collagen II proteins. However, daily injections of rapamycin in ITR mice reduced all these tendon degenerative changes. Collectively, these findings suggest that mechanical loading activates the mTOR signaling in TSCs, and rapamycin may be used to prevent tendinopathy development by blocking non-tenocyte differentiation due to mechanical over-activation of mTOR in TSCs.

Keywords: mTOR; mechanical loading; rapamycin; tendon stem cells; treadmill running.

FIGURE 1

Mechanical loading activates mTOR in a stretching magnitude-dependent manner and rapamycin abolishes the loading-induced activation. Western blot analysis shows higher levels of p-S6 in 4 and 8% stretched PTSCs. The addition of rapamycin to the culture medium largely blocks this effect (A). Quantification of the Western blot results shows that 4 and 8% stretching significantly increase p-S6 levels, but rapamycin negates this effect (B). Western blot analysis of p-S6 levels in PTSCs subjected to 4 and 8% mechanical stretching and cultured in medium without FBS shows that p-S6 levels are higher in stretched cells than the control cells (C). Quantification of the Western blot results confirms these findings (D). Control (Cont): PTSCs cultured under the same culture conditions as other groups but without stretching. Note that * denotes 4 and 8% compared to control, # denotes 8% + Rapa compared to 8% stretch in (B), * denotes 4 and 8% compared to control, # denotes 8% compared to 4% stretch in (D) (n = 3, and values are mean ± SD. *p < 0.05, ^#p < 0.05).

FIGURE 2

Rapamycin inhibits PTSC proliferation and differentiation in vitro. Rapamycin at the dose 500 nM significantly reduces PTSC proliferation (I; see also decreased cell density in B compared to A). Similarly, rapamycin inhibits PTSC differentiation in the adipogenic (C,D; see large amounts of lipid droplets in the inset of C but not in D), chondrogenic (E,F), and osteogenic (G,H) induction media. Moreover, the rapamycin treatment inhibits the expression of marker genes: PPARγ (J) and LPL (K) for adipocytes, SOX-9 (L) and collagen II (M) for chondrocytes, and Runx-2 (N) and osteocalcin (O) for osteocytes. Note that * denotes treatments compared to control, # denotes comparison between each differentiation medium treatment + Rapa with each medium treatment alone (n = 3, and values are mean ± SD. *p < 0.05, ^#p < 0.05). Black bars: 50 μm.

FIGURE 3

Rapamycin inhibits non-tenocyte differentiation of PTSCs induced by mechanical stretching. Rapamycin does not significantly decrease the collagen I expression induced by 4% stretching (4% + Rapa vs. 4%), but it does decrease the collagen I expression at 8% stretching (8% + Rapa vs. 8%) (A). Moreover, rapamycin treatment significantly reduces the expression of non-tenocyte related genes induced by 8% stretching, including LPL for adipocytes (B), collagen II for chondrocytes (C), and Runx-2 for osteocytes (D). Under 4% stretching, however, rapamycin does not cause changes in the expression of these non-tenocyte genes (B–D). Note that * denotes 4 and 8% compared to control, $ denotes 4% + Rapa compared to 4%, and # denotes 8% + Rapa compared to 8% (n = 3, and values are mean ± SD. *p < 0.05, ^#p < 0.05).

FIGURE 4

Rapamycin blocks the ITR-induced cellular morphological changes in mouse patellar tendons. H&E staining shows the normal elongated shape of the tendon cells in cage control (A–C) and rapamycin injected mouse tendons (D–F, white arrows in C,F). Many round shape cells are present in ITR tendons (G-I; yellow arrows in I). With rapamycin injection prior to ITR, markedly fewer round shaped cells are shown in the tendon tissues (J–L). Semi-quantification analysis indicates that more than 55% of the cells in ITR tendon, but less than 10% of the cells in ITR + Rapa treated tendons are round (M). Note that * denotes ITR compared to control, # denotes ITR + Rapa compared to ITR (*p < 0.05, ^#p < 0.05). Green bar: 500 μm; Yellow bars: 100 μm; Black bars: 25 μm.

FIGURE 5

Rapamycin inhibits ITR-induced degenerative changes in mouse tendons. Histochemical analysis by Safranin O and Fast Green staining shows tendon cells in elongated shape in collagen tissues from tendons of the cage control (A–C, white arrows in C) and tendons with rapamycin injections (D–F, white arrows in F). However, many round-shaped cells are positively stained with Safranin O in tendons of the ITR group (red in G,H,I; yellow arrows in I). Rapamycin injection blocks the ITR-induced degenerative changes in the tendons (J–L). Semi-quantification analysis indicates that more than 30% of the cells in ITR tendon are positively stained with Safranin O, but less than 7% of the cells in ITR + Rapa treated tendons are positively stained with Safranin O (M). Note that * denotes ITR compared to control, # denotes ITR + Rapa compared to ITR (*p < 0.05, ^#p < 0.05). Black bars: 500 μm; Yellow bars: 100 μm; Red bars: 25 μm.

FIGURE 6

Rapamycin reduces ITR-induced tendon tissue degeneration. Masson trichrome staining results indicate that the collagen fascicles in the patellar tendons of cage control mice are dense connective tissues, which are well organized, and positively stained with Biebrich scarlet-acid fuchsin (red in A). Similar results are found in the tendon tissues of the mice treated with rapamycin injection daily for 12 weeks (B). However, in ITR mouse tendons, loose and disorganized connective tissues are present and positively stained with aniline blue (C). Finally, rapamycin injection reduces the degenerative changes in mouse tendons induced by ITR, as shown by decreased blue area and increased the red area (D). Semi-quantification analysis confirms these findings (E). Note that * denotes ITR compared to control, # denotes ITR + Rapa compared to ITR (*p < 0.05, ^#p < 0.05). Black bars: 100 μm.

FIGURE 7

Rapamycin blocks ITR-induced SOX-9 and collagen II expression in mouse tendons. The cells of cage control tendons (A,B,I,J) and tendons from the rapamycin injection group (C,D,K,L) exhibit an elongated shape with negative SOX-9 staining (white arrows in B,D) and minimal levels of collagen II staining (K,L). However, many cells are positively stained with SOX-9 (red cells in E,F) and collagen II (red cells in M,N) in tendons of the ITR group. Rapamycin injection blocks the ITR-induced degenerative changes in the tendon, as evidenced by markedly fewer cells positively stained with SOX-9 (G,H) or collagen II (O,P). Semi-quantification analysis indicates that more than 55% of the cells in ITR tendons are positively stained either with SOX-9 (Q) or with collagen II (R). Note that * denotes ITR compared to cage control, # denotes ITR + Rapa compared to ITR (*p < 0.05, ^#p < 0.05). White bars: 200 μm; Yellow bars: 25 μm.