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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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Gene expression of matrix metalloproteinases upon mechanical stimulation of TSPC. (A) RT-PCR analysis for MMP1 and MMP2. (B) Quantitative PCR analysis for MMP3, MMP9, MMP13 and MMP14 (ratio to HPRT housekeeping gene). 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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Fig4: Gene expression of matrix metalloproteinases upon mechanical stimulation of TSPC. (A) RT-PCR analysis for MMP1 and MMP2. (B) Quantitative PCR analysis for MMP3, MMP9, MMP13 and MMP14 (ratio to HPRT housekeeping gene). Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.

Mentions: Next, we examined the effect of mechanical loading on the gene expression changes of matrix metalloproteinases responsible for collagen degradation (MMP1, 2, 3, 9, 13 and 14). The expression of MMP1, 2 and 3 in TSPC did not respond to the mechanical stimulation as their gene levels remained similar to the non-stimulated cells at day 1 and 3 (Figure 4A and B). In contrast, the expression of MMP9, 13 and 14 increased when cells were stretched for 3 days (Figure 4B). The MMP9 expression was affected significantly by the 5% mechanical loading for 3 days as the gene levels increased with approximately 100 folds. When stimulated with 1, 5 or 8% loading strain, TSPC clearly upregulated MMP13 and 14, but only at day 3. In particular, MMP13 was increased with 2 folds at any of the applied loading strain. Similarly, MMP14 expression was elevated with at least 1.5 folds at day 3 in comparison to the non-stimulated TSPC. In conclusion, our data suggested that biaxial mechanical stimulation of TSPC for 3 days period of time upregulates the MMP9, 13 and 14 and is independently of the applied strain magnitude.Figure 4


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)

Gene expression of matrix metalloproteinases upon mechanical stimulation of TSPC. (A) RT-PCR analysis for MMP1 and MMP2. (B) Quantitative PCR analysis for MMP3, MMP9, MMP13 and MMP14 (ratio to HPRT housekeeping gene). 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

Fig4: Gene expression of matrix metalloproteinases upon mechanical stimulation of TSPC. (A) RT-PCR analysis for MMP1 and MMP2. (B) Quantitative PCR analysis for MMP3, MMP9, MMP13 and MMP14 (ratio to HPRT housekeeping gene). Data is representative of 3 donors, each used in 3 independent experiments; *p < 0.1, **p < 0.05, ***p < 0.01.
Mentions: Next, we examined the effect of mechanical loading on the gene expression changes of matrix metalloproteinases responsible for collagen degradation (MMP1, 2, 3, 9, 13 and 14). The expression of MMP1, 2 and 3 in TSPC did not respond to the mechanical stimulation as their gene levels remained similar to the non-stimulated cells at day 1 and 3 (Figure 4A and B). In contrast, the expression of MMP9, 13 and 14 increased when cells were stretched for 3 days (Figure 4B). The MMP9 expression was affected significantly by the 5% mechanical loading for 3 days as the gene levels increased with approximately 100 folds. When stimulated with 1, 5 or 8% loading strain, TSPC clearly upregulated MMP13 and 14, but only at day 3. In particular, MMP13 was increased with 2 folds at any of the applied loading strain. Similarly, MMP14 expression was elevated with at least 1.5 folds at day 3 in comparison to the non-stimulated TSPC. In conclusion, our data suggested that biaxial mechanical stimulation of TSPC for 3 days period of time upregulates the MMP9, 13 and 14 and is independently of the applied strain magnitude.Figure 4

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