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Gtf2ird1-Dependent Mohawk Expression Regulates Mechanosensing Properties of the Tendon.

Kayama T, Mori M, Ito Y, Matsushima T, Nakamichi R, Suzuki H, Ichinose S, Saito M, Marumo K, Asahara H - Mol. Cell. Biol. (2016)

Bottom Line: In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover.Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation.Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Tokyo, Japan Department of Orthopaedic Surgery, The Jikei University School of Medicine, Tokyo, Japan.

No MeSH data available.


Cellular stretching induces nuclear translocation of Gtf2ird1. (A) Immunocytochemistry of a control rat tenocyte without stretching. Gtf2ird1 was seen predominantly within the cytoplasm in comparison with localization in the nucleus (green, Gtf2ird1; blue, Hoechst). (B) Immunocytochemistry of a rat tenocyte after cellular stretching. Gtf2ird1 expression was most concentrated in the nucleus and also moderately concentrated in the cytoplasm surrounding the nucleus (green, Gtf2ird1; blue, Hoechst). (C) Proportion of cells with nuclear translocation induced by stretching. Error bars represent standard errors of the means (***, P < 0.001, two-tailed Student's t test). (D) Western blot of fractionated protein extracted from stretched tenocytes confirms that Gtf2ird1 translocates into the nucleus. α/β-Tubulin and histone H3 were used as cytoplasmic and nuclear controls, respectively. (E) Gtf2ird1 knockdown using siRNA (siGtf2ird1) inhibits Mkx expression despite stretching, indicating the importance of Gtf2ird1 in mechanosensitive Mkx regulation. Error bars represent standard errors of the means (*, P < 0.05, ***P < 0.001, two-tailed Student's t test).
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Figure 5: Cellular stretching induces nuclear translocation of Gtf2ird1. (A) Immunocytochemistry of a control rat tenocyte without stretching. Gtf2ird1 was seen predominantly within the cytoplasm in comparison with localization in the nucleus (green, Gtf2ird1; blue, Hoechst). (B) Immunocytochemistry of a rat tenocyte after cellular stretching. Gtf2ird1 expression was most concentrated in the nucleus and also moderately concentrated in the cytoplasm surrounding the nucleus (green, Gtf2ird1; blue, Hoechst). (C) Proportion of cells with nuclear translocation induced by stretching. Error bars represent standard errors of the means (***, P < 0.001, two-tailed Student's t test). (D) Western blot of fractionated protein extracted from stretched tenocytes confirms that Gtf2ird1 translocates into the nucleus. α/β-Tubulin and histone H3 were used as cytoplasmic and nuclear controls, respectively. (E) Gtf2ird1 knockdown using siRNA (siGtf2ird1) inhibits Mkx expression despite stretching, indicating the importance of Gtf2ird1 in mechanosensitive Mkx regulation. Error bars represent standard errors of the means (*, P < 0.05, ***P < 0.001, two-tailed Student's t test).

Mentions: In order to assess whether the candidate genes play a critical role in the mechanical response of tendons, subcellular localization was studied with and without stretching in primary rat tenocytes. Mechanical loading in cells has been shown to induce cell shape changes (27, 33). Here, cyclic stretching resulted in elongated tenocytes that extended perpendicularly to the direction of stretch, as reported previously in mouse embryonic fibroblasts and muscle cells, rat vascular smooth muscle cells, xenopus kidney cells, and bovine aortic endothelial cells (34–37). Immunocytochemistry of Gtf2ird1, in particular, showed significant expression pattern changes as a result of cellular stretching. In cells without stretching, Gtf2ird1 showed cytoplasmic distribution (Fig. 5A). However, tenocytes exposed to mechanical stress revealed nuclear translocation of Gtf2ird1 (Fig. 5B). Almost none of the control tenocytes showed greater Gtf2ird1 expression in the nucleus, whereas more than 80% of stretched tenocytes demonstrated greater expression in the nucleus than in the cytoplasm (Fig. 5C). The proportion of nuclear translocation was also confirmed by Western blotting, where nuclear protein levels increased and cytoplasmic protein levels decreased (Fig. 5D). The result demonstrates that mechanical loading encourages nuclear translocation of Gtf2ird1, indicating that Gtf2ird1 is a mechanosensor in tenocytes.


Gtf2ird1-Dependent Mohawk Expression Regulates Mechanosensing Properties of the Tendon.

Kayama T, Mori M, Ito Y, Matsushima T, Nakamichi R, Suzuki H, Ichinose S, Saito M, Marumo K, Asahara H - Mol. Cell. Biol. (2016)

Cellular stretching induces nuclear translocation of Gtf2ird1. (A) Immunocytochemistry of a control rat tenocyte without stretching. Gtf2ird1 was seen predominantly within the cytoplasm in comparison with localization in the nucleus (green, Gtf2ird1; blue, Hoechst). (B) Immunocytochemistry of a rat tenocyte after cellular stretching. Gtf2ird1 expression was most concentrated in the nucleus and also moderately concentrated in the cytoplasm surrounding the nucleus (green, Gtf2ird1; blue, Hoechst). (C) Proportion of cells with nuclear translocation induced by stretching. Error bars represent standard errors of the means (***, P < 0.001, two-tailed Student's t test). (D) Western blot of fractionated protein extracted from stretched tenocytes confirms that Gtf2ird1 translocates into the nucleus. α/β-Tubulin and histone H3 were used as cytoplasmic and nuclear controls, respectively. (E) Gtf2ird1 knockdown using siRNA (siGtf2ird1) inhibits Mkx expression despite stretching, indicating the importance of Gtf2ird1 in mechanosensitive Mkx regulation. Error bars represent standard errors of the means (*, P < 0.05, ***P < 0.001, two-tailed Student's t test).
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Figure 5: Cellular stretching induces nuclear translocation of Gtf2ird1. (A) Immunocytochemistry of a control rat tenocyte without stretching. Gtf2ird1 was seen predominantly within the cytoplasm in comparison with localization in the nucleus (green, Gtf2ird1; blue, Hoechst). (B) Immunocytochemistry of a rat tenocyte after cellular stretching. Gtf2ird1 expression was most concentrated in the nucleus and also moderately concentrated in the cytoplasm surrounding the nucleus (green, Gtf2ird1; blue, Hoechst). (C) Proportion of cells with nuclear translocation induced by stretching. Error bars represent standard errors of the means (***, P < 0.001, two-tailed Student's t test). (D) Western blot of fractionated protein extracted from stretched tenocytes confirms that Gtf2ird1 translocates into the nucleus. α/β-Tubulin and histone H3 were used as cytoplasmic and nuclear controls, respectively. (E) Gtf2ird1 knockdown using siRNA (siGtf2ird1) inhibits Mkx expression despite stretching, indicating the importance of Gtf2ird1 in mechanosensitive Mkx regulation. Error bars represent standard errors of the means (*, P < 0.05, ***P < 0.001, two-tailed Student's t test).
Mentions: In order to assess whether the candidate genes play a critical role in the mechanical response of tendons, subcellular localization was studied with and without stretching in primary rat tenocytes. Mechanical loading in cells has been shown to induce cell shape changes (27, 33). Here, cyclic stretching resulted in elongated tenocytes that extended perpendicularly to the direction of stretch, as reported previously in mouse embryonic fibroblasts and muscle cells, rat vascular smooth muscle cells, xenopus kidney cells, and bovine aortic endothelial cells (34–37). Immunocytochemistry of Gtf2ird1, in particular, showed significant expression pattern changes as a result of cellular stretching. In cells without stretching, Gtf2ird1 showed cytoplasmic distribution (Fig. 5A). However, tenocytes exposed to mechanical stress revealed nuclear translocation of Gtf2ird1 (Fig. 5B). Almost none of the control tenocytes showed greater Gtf2ird1 expression in the nucleus, whereas more than 80% of stretched tenocytes demonstrated greater expression in the nucleus than in the cytoplasm (Fig. 5C). The proportion of nuclear translocation was also confirmed by Western blotting, where nuclear protein levels increased and cytoplasmic protein levels decreased (Fig. 5D). The result demonstrates that mechanical loading encourages nuclear translocation of Gtf2ird1, indicating that Gtf2ird1 is a mechanosensor in tenocytes.

Bottom Line: In mammals, the tendon connective tissue experiences and resists physical forces, with tendon-specific mesenchymal cells called tenocytes orchestrating extracellular matrix (ECM) turnover.Furthermore, functional screening of the Mkx promoter region identified several upstream transcription factors that regulate Mkx In particular, general transcription factor II-I repeat domain-containing protein 1 (Gtf2ird1) that is expressed in the cytoplasm of unstressed tenocytes translocated into the nucleus upon mechanical stretching to activate the Mkx promoter through chromatin regulation.Here, we demonstrate that Gtf2ird1 is essential for Mkx transcription, while also linking mechanical forces to Mkx-mediated tendon homeostasis and regeneration.

View Article: PubMed Central - PubMed

Affiliation: Department of Systems BioMedicine, Tokyo Medical and Dental University, Tokyo, Japan Department of Orthopaedic Surgery, The Jikei University School of Medicine, Tokyo, Japan.

No MeSH data available.