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Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

Popov C, Burggraf M, Kreja L, Ignatius A, Schieker M, Docheva D - BMC Mol. Biol. (2015)

Bottom Line: Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC.Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC.Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Experimental Surgery and Regenerative Medicine, Ludwig-Maximilians-University (LMU), Nussbaumstr. 20, D-80336, Munich, Germany. tzvetan.popov@med.uni-muenchen.de.

ABSTRACT

Background: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies.

Results: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC.

Conclusions: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

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Western blotting analysis for collagen-binding integrins, integrin-downstream kinases and matrix metalloproteinases upon 8% mechanical stimulation of TSPC. (A) Collagen I-binding integrins α1, α2 and α11 (ratio to GAPDH protein expression); (B) Phosphorylated and total levels of FAK, ERK, Akt, p38 and Jnk (ratio phospho-/total protein); (C) MMP9, 13 and 14 (ratio to GAPDH). (D) Schematic summary of the changes occurring in TSPC upon 8% biaxial mechanical loading. Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.
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Fig5: Western blotting analysis for collagen-binding integrins, integrin-downstream kinases and matrix metalloproteinases upon 8% mechanical stimulation of TSPC. (A) Collagen I-binding integrins α1, α2 and α11 (ratio to GAPDH protein expression); (B) Phosphorylated and total levels of FAK, ERK, Akt, p38 and Jnk (ratio phospho-/total protein); (C) MMP9, 13 and 14 (ratio to GAPDH). (D) Schematic summary of the changes occurring in TSPC upon 8% biaxial mechanical loading. Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.

Mentions: Based on the obtained mRNA data, we performed western blotting analyses for the major candidate genes as we investigated: 1) the collagen binding integrins α1, α2 and α11; 2) the activation of five key integrin downstream kinases and 3) the expression of MMP9, 13 and 14 responsible for the collagen degradation. For this analysis, TSPC were stimulated with 8% biaxial loading for 3 days, since this was the best condition according to our mRNA screening. Similar to the mRNA results (Figure 3B), stimulation with 8% mechanical strain resulted in significantly higher protein production for all three integrins. However, the increase in the protein expression was more pronounced for integrin α2 (1.5 folds) and α11 (1.9 folds) (Figure 4A). Next, we analyzed the changes in the activity of five integrin downstream kinases FAK, ERK, Akt, p38 and Jnk (Figure 5B). We found that upon mechanical loading, the activity of ERK (1.5 folds) and p38 (1.4 folds) kinases significantly increased, whereas the activity of Jnk (1.6 folds) was significantly reduced in the mechanically loaded TSPC. The other two kinases – FAK and Akt were not influenced by the mechanical loading. The analysis of MMP protein levels (Figure 5C) demonstrated that upon mechanical stimulation the expression of MMP9, 13 and 14 were significantly increased as the most pronounced changes were observed in the expression of MMP13 (1.4 folds) and 14 (1.8 folds) in comparison to the non-stimulated TSPC.Figure 5


Mechanical stimulation of human tendon stem/progenitor cells results in upregulation of matrix proteins, integrins and MMPs, and activation of p38 and ERK1/2 kinases.

Popov C, Burggraf M, Kreja L, Ignatius A, Schieker M, Docheva D - BMC Mol. Biol. (2015)

Western blotting analysis for collagen-binding integrins, integrin-downstream kinases and matrix metalloproteinases upon 8% mechanical stimulation of TSPC. (A) Collagen I-binding integrins α1, α2 and α11 (ratio to GAPDH protein expression); (B) Phosphorylated and total levels of FAK, ERK, Akt, p38 and Jnk (ratio phospho-/total protein); (C) MMP9, 13 and 14 (ratio to GAPDH). (D) Schematic summary of the changes occurring in TSPC upon 8% biaxial mechanical loading. Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4373449&req=5

Fig5: Western blotting analysis for collagen-binding integrins, integrin-downstream kinases and matrix metalloproteinases upon 8% mechanical stimulation of TSPC. (A) Collagen I-binding integrins α1, α2 and α11 (ratio to GAPDH protein expression); (B) Phosphorylated and total levels of FAK, ERK, Akt, p38 and Jnk (ratio phospho-/total protein); (C) MMP9, 13 and 14 (ratio to GAPDH). (D) Schematic summary of the changes occurring in TSPC upon 8% biaxial mechanical loading. Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.
Mentions: Based on the obtained mRNA data, we performed western blotting analyses for the major candidate genes as we investigated: 1) the collagen binding integrins α1, α2 and α11; 2) the activation of five key integrin downstream kinases and 3) the expression of MMP9, 13 and 14 responsible for the collagen degradation. For this analysis, TSPC were stimulated with 8% biaxial loading for 3 days, since this was the best condition according to our mRNA screening. Similar to the mRNA results (Figure 3B), stimulation with 8% mechanical strain resulted in significantly higher protein production for all three integrins. However, the increase in the protein expression was more pronounced for integrin α2 (1.5 folds) and α11 (1.9 folds) (Figure 4A). Next, we analyzed the changes in the activity of five integrin downstream kinases FAK, ERK, Akt, p38 and Jnk (Figure 5B). We found that upon mechanical loading, the activity of ERK (1.5 folds) and p38 (1.4 folds) kinases significantly increased, whereas the activity of Jnk (1.6 folds) was significantly reduced in the mechanically loaded TSPC. The other two kinases – FAK and Akt were not influenced by the mechanical loading. The analysis of MMP protein levels (Figure 5C) demonstrated that upon mechanical stimulation the expression of MMP9, 13 and 14 were significantly increased as the most pronounced changes were observed in the expression of MMP13 (1.4 folds) and 14 (1.8 folds) in comparison to the non-stimulated TSPC.Figure 5

Bottom Line: Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC.Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC.Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

View Article: PubMed Central - PubMed

Affiliation: Department of Surgery, Experimental Surgery and Regenerative Medicine, Ludwig-Maximilians-University (LMU), Nussbaumstr. 20, D-80336, Munich, Germany. tzvetan.popov@med.uni-muenchen.de.

ABSTRACT

Background: Tendons are dense connective tissues subjected periodically to mechanical stress upon which complex responsive mechanisms are activated. These mechanisms affect not only the development of these tissues but also their healing. Despite of the acknowledged importance of the mechanical stress for tendon function and repair, the mechanotransduction mechanisms in tendon cells are still unclear and the elucidation of these mechanisms is a key goal in tendon research. Tendon stem/progenitor cells (TSPC) possess common adult stem cell characteristics, and are suggested to actively participate in tendon development, tissue homeostasis as well as repair. This makes them an important cell population for tendon repair, and also an interesting research target for various open questions in tendon cell biology. Therefore, in our study we focused on TSPC, subjected them to five different mechanical protocols, and investigated the gene expression changes by using semi-quantitative, quantitative PCR and western blotting technologies.

Results: Among the 25 different genes analyzed, we can convincingly report that the tendon-related genes - fibromodulin, lumican and versican, the collagen I-binding integrins - α1, α2 and α11, the matrix metalloproteinases - MMP9, 13 and 14 were strongly upregulated in TSPC after 3 days of mechanical stimulation with 8% amplitude. Molecular signaling analyses of five key integrin downstream kinases suggested that mechanical stimuli are mediated through ERK1/2 and p38, which were significantly activated in 8% biaxial-loaded TSPC.

Conclusions: Our results demonstrate the positive effect of 8% mechanical loading on the gene expression of matrix proteins, integrins and matrix metalloproteinases, and activation of integrin downstream kinases p38 and ERK1/2 in TSPC. Taken together, our study contributes to better understanding of mechanotransduction mechanisms in TPSC, which in long term, after further translational research between tendon cell biology and orthopedics, can be beneficial to the management of tendon repair.

Show MeSH