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p38 MAPK signaling in postnatal tendon growth and remodeling.

Schwartz AJ, Sarver DC, Sugg KB, Dzierzawski JT, Gumucio JP, Mendias CL - PLoS ONE (2015)

Bottom Line: By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon.Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth.The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, Michigan, United States of America; Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

No MeSH data available.


p38 MAPK blot.Representative immunoblot of overloaded plantaris tendons demonstrating the ability of SB203580 to prevent the phosphorylation of p38 MAPK. Total p38 MAPK protein is shown as a loading control.
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pone.0120044.g001: p38 MAPK blot.Representative immunoblot of overloaded plantaris tendons demonstrating the ability of SB203580 to prevent the phosphorylation of p38 MAPK. Total p38 MAPK protein is shown as a loading control.

Mentions: While SB203580 has previously been used to block p38 MAPK activation in other tissues in vivo, to verify that SB203580 is effectively able to block p38 MAPK in tendon, rats received a single 3mg/kg dose of SB203580 or vehicle as described above. Plantaris overload surgeries were performed, and tissue was collected for analysis 6 hours later. Overloaded plantaris tendons were snap frozen in liquid nitrogen and made into a fine powder with a mortar and pestle. The tendon was further homogenized in 400μL of T-PER (Thermo Scientific, Waltham, MA) with a 1:100 protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was determined using a Bradford protein assay (Thermo Scientific), and 7.5 μg of protein was diluted in Laemmli sample buffer with 1:20 beta-mercaptoethanol, placed in boiling water for 2 minutes, and then loaded into AnyKD mini-gels (BioRad, Berkeley, CA). Proteins were separated, then transferred from gels onto 0.45 μm nitrocellulose membranes (BioRad) using electrophoresis, blocked with 1% goat serum, and incubated with primary antibodies against either p38 MAPK or phospho-p38 MAPK (Cell Signaling Technology, Beverly, MA). After primary antibody incubation, membranes were rinsed and incubated with HRPO-conjugated goat anti-rabbit secondary antibodies (Abcam, Cambridge, MA). Proteins were detected using enhanced chemiluminescent reagents (BioRad) and visualized using a digital chemiluminescent documentation system (BioRad). Catalog numbers and dilutions of antibodies are listed in Table 1. Representative results are shown in Fig. 1.


p38 MAPK signaling in postnatal tendon growth and remodeling.

Schwartz AJ, Sarver DC, Sugg KB, Dzierzawski JT, Gumucio JP, Mendias CL - PLoS ONE (2015)

p38 MAPK blot.Representative immunoblot of overloaded plantaris tendons demonstrating the ability of SB203580 to prevent the phosphorylation of p38 MAPK. Total p38 MAPK protein is shown as a loading control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359143&req=5

pone.0120044.g001: p38 MAPK blot.Representative immunoblot of overloaded plantaris tendons demonstrating the ability of SB203580 to prevent the phosphorylation of p38 MAPK. Total p38 MAPK protein is shown as a loading control.
Mentions: While SB203580 has previously been used to block p38 MAPK activation in other tissues in vivo, to verify that SB203580 is effectively able to block p38 MAPK in tendon, rats received a single 3mg/kg dose of SB203580 or vehicle as described above. Plantaris overload surgeries were performed, and tissue was collected for analysis 6 hours later. Overloaded plantaris tendons were snap frozen in liquid nitrogen and made into a fine powder with a mortar and pestle. The tendon was further homogenized in 400μL of T-PER (Thermo Scientific, Waltham, MA) with a 1:100 protease and phosphatase inhibitor cocktail (Thermo Scientific). Protein concentration was determined using a Bradford protein assay (Thermo Scientific), and 7.5 μg of protein was diluted in Laemmli sample buffer with 1:20 beta-mercaptoethanol, placed in boiling water for 2 minutes, and then loaded into AnyKD mini-gels (BioRad, Berkeley, CA). Proteins were separated, then transferred from gels onto 0.45 μm nitrocellulose membranes (BioRad) using electrophoresis, blocked with 1% goat serum, and incubated with primary antibodies against either p38 MAPK or phospho-p38 MAPK (Cell Signaling Technology, Beverly, MA). After primary antibody incubation, membranes were rinsed and incubated with HRPO-conjugated goat anti-rabbit secondary antibodies (Abcam, Cambridge, MA). Proteins were detected using enhanced chemiluminescent reagents (BioRad) and visualized using a digital chemiluminescent documentation system (BioRad). Catalog numbers and dilutions of antibodies are listed in Table 1. Representative results are shown in Fig. 1.

Bottom Line: By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon.Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth.The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, Michigan, United States of America; Department of Molecular & Integrative Physiology, University of Michigan Medical School, Ann Arbor, Michigan, United States of America.

ABSTRACT
Tendon is a dynamic tissue whose structure and function is influenced by mechanical loading, but little is known about the fundamental mechanisms that regulate tendon growth and remodeling in vivo. Data from cultured tendon fibroblasts indicated that the p38 MAPK pathway plays an important role in tendon fibroblast proliferation and collagen synthesis in vitro. To gain greater insight into the mechanisms of tendon growth, and explore the role of p38 MAPK signaling in this process, we tested the hypotheses that inducing plantaris tendon growth through the ablation of the synergist Achilles tendon would result in rapid expansion of a neotendon matrix surrounding the original tendon, and that treatment with the p38 MAPK inhibitor SB203580 would prevent this growth. Rats were treated with vehicle or SB203580, and subjected to synergist ablation by bilateral tenectomy of the Achilles tendon. Changes in histological and biochemical properties of plantaris tendons were analyzed 3, 7, or 28 days after overload, and comparisons were made to non-overloaded animals. By 28 days after overload, tendon mass had increased by 30% compared to non-overloaded samples, and cross-sectional area (CSA) increased by around 50%, with most of the change occurring in the neotendon. The expansion in CSA initially occurred through the synthesis of a hyaluronic acid rich matrix that was progressively replaced with mature collagen. Pericytes were present in areas of active tendon growth, but never in the original tendon ECM. Inhibition of p38 MAPK resulted in a profound decrease in IL6 expression, and had a modest effect on the expression of other ECM and cell proliferation genes, but had a negligible impact on overall tendon growth. The combined results from this study provided novel insights into tendon mechanobiology, and suggest that p38 MAPK signaling does not appear to be necessary for tendon growth in vivo.

No MeSH data available.