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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.


Expression of genes related to cell specification and proliferation.Target gene expression was normalized to the stable housekeeping gene beta 2 microglobulin (B2M), and further normalized to plantaris tendons that were not subjected to synergist ablation. Values are mean±SD, N = 8 tendons for each group. Differences between groups were tested using a two-way ANOVA (α = 0.05) followed by Newman-Keuls post hoc sorting: a, different (P<0.05) from 3D vehicle; b, different (P<0.05) from 3D p38 MAPK inhibitor; c, different (P<0.05) from 7D vehicle; d, different (P<0.05) from 7D p38 MAPK inhibitor; e, different (P<0.05) from 28D vehicle.
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pone.0120044.g009: Expression of genes related to cell specification and proliferation.Target gene expression was normalized to the stable housekeeping gene beta 2 microglobulin (B2M), and further normalized to plantaris tendons that were not subjected to synergist ablation. Values are mean±SD, N = 8 tendons for each group. Differences between groups were tested using a two-way ANOVA (α = 0.05) followed by Newman-Keuls post hoc sorting: a, different (P<0.05) from 3D vehicle; b, different (P<0.05) from 3D p38 MAPK inhibitor; c, different (P<0.05) from 7D vehicle; d, different (P<0.05) from 7D p38 MAPK inhibitor; e, different (P<0.05) from 28D vehicle.

Mentions: We next sought to identify different types of cell populations present in overloaded tendons. We used immunohistochemistry to determine the presence of these cells (Fig. 9), and gene expression analysis to determine quantitative changes in the markers for these cells and in the factors that regulate their activity (Figs. 9–11). Representative immunohistochemistry from 7 day vehicle treated samples are shown in Fig. 9, although these cells and markers were found in all groups throughout the course of overload. Ki67 is a marker of proliferating cells, CD146 is a marker of pericytes, and F4/80 is a marker of macrophages, and cells expressing all three markers were observed in the neotendon but not in the original tendon (Fig. 9). Type I procollagen is a marker of fibroblasts, and was observed in both the original tendon and neotendon (Fig. 9). Hyaluronic acid, identified with the use of biotinylated hyaluronic acid binding protein (HABP), was found in areas immediately surrounding fibroblasts in the original tendon, but was found throughout the matrix of the neotendon (Fig. 9). For quantitative measures of genes involved in cell proliferation (Fig. 9), Ki67 expression was greatest in both vehicle and treatment groups at 3 days, and steadily decreased over time. CD146 expression was greatest at 7 days, and decreased with p38 MAPK inhibition at the 7 and 28 day time points. Transcripts for the basic helix-loop-helix (bHLH) transcription factor scleraxis (Scx) and the type II transmembrane glycoprotein tenomodulin (Tnmd), which play important roles in fibroblast proliferation and differentiation into the tendon lineage, were both expressed at their greatest at the 7 day time point. Inhibition of p38 MAPK increased scleraxis expression at 7 days and decreased expression at 28 days, but there was no observed influence of treatment on the abundance of tenomodulin transcripts. Hypoxia inducible factor-1 alpha (HIF1a), which can promote fibroblast proliferation, also had generally similar changes in expression with time and treatment. The expression of alpha-smooth muscle actin (SMA), which is a marker of myofibroblasts, was slightly elevated in vehicle and p38 MAPK inhibited groups at 7 days, and returned to baseline levels by 28 days. Transcripts for two other fibroblast markers, fibroblast specific protein-1 (FSP1) and mohawk (Mkx), displayed little to no effect of treatment nor time. Egr1 and Egr2 are transcriptional regulatory proteins involved in tendon development, and while Egr1 was downregulated in all groups compared to non-overloaded samples and displayed no effect of treatment nor time, Egr2 expression peaked at 7 days and was significantly decreased with p38 MAPK inhibition at this time point. The expression of the canonical epithelial-to-mesenchymal transition (EMT) genes Snail1 (Snai1), Slug, Goosecoid (Gsc), and Twist1 were generally greatest at 7 days and remained elevated at all observed time points following overload. The inhibition of p38 MAPK significantly decreased the expression of Snail1 and Twist1 at 3 days, and Snail1, Twist1, and Goosecoid at 7 days.


p38 MAPK signaling in postnatal tendon growth and remodeling.

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

Expression of genes related to cell specification and proliferation.Target gene expression was normalized to the stable housekeeping gene beta 2 microglobulin (B2M), and further normalized to plantaris tendons that were not subjected to synergist ablation. Values are mean±SD, N = 8 tendons for each group. Differences between groups were tested using a two-way ANOVA (α = 0.05) followed by Newman-Keuls post hoc sorting: a, different (P<0.05) from 3D vehicle; b, different (P<0.05) from 3D p38 MAPK inhibitor; c, different (P<0.05) from 7D vehicle; d, different (P<0.05) from 7D p38 MAPK inhibitor; e, different (P<0.05) from 28D vehicle.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0120044.g009: Expression of genes related to cell specification and proliferation.Target gene expression was normalized to the stable housekeeping gene beta 2 microglobulin (B2M), and further normalized to plantaris tendons that were not subjected to synergist ablation. Values are mean±SD, N = 8 tendons for each group. Differences between groups were tested using a two-way ANOVA (α = 0.05) followed by Newman-Keuls post hoc sorting: a, different (P<0.05) from 3D vehicle; b, different (P<0.05) from 3D p38 MAPK inhibitor; c, different (P<0.05) from 7D vehicle; d, different (P<0.05) from 7D p38 MAPK inhibitor; e, different (P<0.05) from 28D vehicle.
Mentions: We next sought to identify different types of cell populations present in overloaded tendons. We used immunohistochemistry to determine the presence of these cells (Fig. 9), and gene expression analysis to determine quantitative changes in the markers for these cells and in the factors that regulate their activity (Figs. 9–11). Representative immunohistochemistry from 7 day vehicle treated samples are shown in Fig. 9, although these cells and markers were found in all groups throughout the course of overload. Ki67 is a marker of proliferating cells, CD146 is a marker of pericytes, and F4/80 is a marker of macrophages, and cells expressing all three markers were observed in the neotendon but not in the original tendon (Fig. 9). Type I procollagen is a marker of fibroblasts, and was observed in both the original tendon and neotendon (Fig. 9). Hyaluronic acid, identified with the use of biotinylated hyaluronic acid binding protein (HABP), was found in areas immediately surrounding fibroblasts in the original tendon, but was found throughout the matrix of the neotendon (Fig. 9). For quantitative measures of genes involved in cell proliferation (Fig. 9), Ki67 expression was greatest in both vehicle and treatment groups at 3 days, and steadily decreased over time. CD146 expression was greatest at 7 days, and decreased with p38 MAPK inhibition at the 7 and 28 day time points. Transcripts for the basic helix-loop-helix (bHLH) transcription factor scleraxis (Scx) and the type II transmembrane glycoprotein tenomodulin (Tnmd), which play important roles in fibroblast proliferation and differentiation into the tendon lineage, were both expressed at their greatest at the 7 day time point. Inhibition of p38 MAPK increased scleraxis expression at 7 days and decreased expression at 28 days, but there was no observed influence of treatment on the abundance of tenomodulin transcripts. Hypoxia inducible factor-1 alpha (HIF1a), which can promote fibroblast proliferation, also had generally similar changes in expression with time and treatment. The expression of alpha-smooth muscle actin (SMA), which is a marker of myofibroblasts, was slightly elevated in vehicle and p38 MAPK inhibited groups at 7 days, and returned to baseline levels by 28 days. Transcripts for two other fibroblast markers, fibroblast specific protein-1 (FSP1) and mohawk (Mkx), displayed little to no effect of treatment nor time. Egr1 and Egr2 are transcriptional regulatory proteins involved in tendon development, and while Egr1 was downregulated in all groups compared to non-overloaded samples and displayed no effect of treatment nor time, Egr2 expression peaked at 7 days and was significantly decreased with p38 MAPK inhibition at this time point. The expression of the canonical epithelial-to-mesenchymal transition (EMT) genes Snail1 (Snai1), Slug, Goosecoid (Gsc), and Twist1 were generally greatest at 7 days and remained elevated at all observed time points following overload. The inhibition of p38 MAPK significantly decreased the expression of Snail1 and Twist1 at 3 days, and Snail1, Twist1, and Goosecoid at 7 days.

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.