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Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

Tai IC, Wang YH, Chen CH, Chuang SC, Chang JK, Ho ML - Int J Nanomedicine (2015)

Bottom Line: We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα.Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation.The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ; Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ; Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration.

No MeSH data available.


Related in: MedlinePlus

Simvastatin treatment increases active RhoA in D1 cells. Simvastatin constitutively activated RhoA, 0.5–24 hours after treatment.Notes: Peak activity was reached at 3 hours (A), with RhoA activity increasing in a dose-dependent manner (B), as determined using Western blots of immunoprecipitates. Simvastatin dose-dependently increased RhoA protein levels in the cytosol (aqueous phase) and reduced RhoA content in the membrane (detergent phase) at 3 hours (C). The amount of RhoA that coimmunoprecipitated with RhoGDIα was proportional to the amount of RhoGDIα in the lysate, because simvastatin treatment reduced RhoGDIα-associated RhoA at 3 hours (D). Simvastatin enhanced the ROCK substrate of phospho-MYPT (E) and pMLC (F) at 3 hours. The results are the mean values of three independent measurements, error bars: SEM. *P<0.05; **P<0.01 vs control; #P<0.05, ##P<0.01 vs SIM (1 µm).Abbreviations: SIM, simvastatin; ROCK, Rho-associated protein kinase; SEM, standard error of the mean; pMLC, phosphomyosin light chain; IP, immunoprecipitation.
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f1-ijn-10-5881: Simvastatin treatment increases active RhoA in D1 cells. Simvastatin constitutively activated RhoA, 0.5–24 hours after treatment.Notes: Peak activity was reached at 3 hours (A), with RhoA activity increasing in a dose-dependent manner (B), as determined using Western blots of immunoprecipitates. Simvastatin dose-dependently increased RhoA protein levels in the cytosol (aqueous phase) and reduced RhoA content in the membrane (detergent phase) at 3 hours (C). The amount of RhoA that coimmunoprecipitated with RhoGDIα was proportional to the amount of RhoGDIα in the lysate, because simvastatin treatment reduced RhoGDIα-associated RhoA at 3 hours (D). Simvastatin enhanced the ROCK substrate of phospho-MYPT (E) and pMLC (F) at 3 hours. The results are the mean values of three independent measurements, error bars: SEM. *P<0.05; **P<0.01 vs control; #P<0.05, ##P<0.01 vs SIM (1 µm).Abbreviations: SIM, simvastatin; ROCK, Rho-associated protein kinase; SEM, standard error of the mean; pMLC, phosphomyosin light chain; IP, immunoprecipitation.

Mentions: Active RhoA was detected using pull-down assays with or without the treatment of simvastatin. Simvastatin increased the levels of active RhoA within 30 minutes of treatment, with peak levels reaching within 3 hours of treatment followed by a slow decrease for 24 hours (Figure 1A). The effect of simvastatin was dose-dependent (Figure 1B). We used a membrane protein extraction assay to determine whether simvastatin alters the membrane anchoring of RhoA. Simvastatin dose-dependently increased the amount of cytosolic RhoA and reduced membrane-bound RhoA (Figure 1C). To further investigate the mechanism of RhoA activation by simvastatin, we assessed the association of RhoA with RhoGDIα by using immunoprecipitation. The amount of RhoA bound to RhoGDIα was lower in simvastatin-treated cells than in untreated cells (Figure 1D). The treatment with simvastatin increased the level of phospho-MYPT, a ROCK substrate molecule, suggesting increased ROCK activity (Figure 1E). Simvastatin increased the phosphorylation of MLC (Figure 1F).


Simvastatin enhances Rho/actin/cell rigidity pathway contributing to mesenchymal stem cells' osteogenic differentiation.

Tai IC, Wang YH, Chen CH, Chuang SC, Chang JK, Ho ML - Int J Nanomedicine (2015)

Simvastatin treatment increases active RhoA in D1 cells. Simvastatin constitutively activated RhoA, 0.5–24 hours after treatment.Notes: Peak activity was reached at 3 hours (A), with RhoA activity increasing in a dose-dependent manner (B), as determined using Western blots of immunoprecipitates. Simvastatin dose-dependently increased RhoA protein levels in the cytosol (aqueous phase) and reduced RhoA content in the membrane (detergent phase) at 3 hours (C). The amount of RhoA that coimmunoprecipitated with RhoGDIα was proportional to the amount of RhoGDIα in the lysate, because simvastatin treatment reduced RhoGDIα-associated RhoA at 3 hours (D). Simvastatin enhanced the ROCK substrate of phospho-MYPT (E) and pMLC (F) at 3 hours. The results are the mean values of three independent measurements, error bars: SEM. *P<0.05; **P<0.01 vs control; #P<0.05, ##P<0.01 vs SIM (1 µm).Abbreviations: SIM, simvastatin; ROCK, Rho-associated protein kinase; SEM, standard error of the mean; pMLC, phosphomyosin light chain; IP, immunoprecipitation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4590348&req=5

f1-ijn-10-5881: Simvastatin treatment increases active RhoA in D1 cells. Simvastatin constitutively activated RhoA, 0.5–24 hours after treatment.Notes: Peak activity was reached at 3 hours (A), with RhoA activity increasing in a dose-dependent manner (B), as determined using Western blots of immunoprecipitates. Simvastatin dose-dependently increased RhoA protein levels in the cytosol (aqueous phase) and reduced RhoA content in the membrane (detergent phase) at 3 hours (C). The amount of RhoA that coimmunoprecipitated with RhoGDIα was proportional to the amount of RhoGDIα in the lysate, because simvastatin treatment reduced RhoGDIα-associated RhoA at 3 hours (D). Simvastatin enhanced the ROCK substrate of phospho-MYPT (E) and pMLC (F) at 3 hours. The results are the mean values of three independent measurements, error bars: SEM. *P<0.05; **P<0.01 vs control; #P<0.05, ##P<0.01 vs SIM (1 µm).Abbreviations: SIM, simvastatin; ROCK, Rho-associated protein kinase; SEM, standard error of the mean; pMLC, phosphomyosin light chain; IP, immunoprecipitation.
Mentions: Active RhoA was detected using pull-down assays with or without the treatment of simvastatin. Simvastatin increased the levels of active RhoA within 30 minutes of treatment, with peak levels reaching within 3 hours of treatment followed by a slow decrease for 24 hours (Figure 1A). The effect of simvastatin was dose-dependent (Figure 1B). We used a membrane protein extraction assay to determine whether simvastatin alters the membrane anchoring of RhoA. Simvastatin dose-dependently increased the amount of cytosolic RhoA and reduced membrane-bound RhoA (Figure 1C). To further investigate the mechanism of RhoA activation by simvastatin, we assessed the association of RhoA with RhoGDIα by using immunoprecipitation. The amount of RhoA bound to RhoGDIα was lower in simvastatin-treated cells than in untreated cells (Figure 1D). The treatment with simvastatin increased the level of phospho-MYPT, a ROCK substrate molecule, suggesting increased ROCK activity (Figure 1E). Simvastatin increased the phosphorylation of MLC (Figure 1F).

Bottom Line: We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα.Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation.The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration.

View Article: PubMed Central - PubMed

Affiliation: Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ; Department of Physiology, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan ; Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Recent studies have indicated that statins induce osteogenic differentiation both in vitro and in vivo. The molecular mechanism of statin-stimulated osteogenesis is unknown. Activation of RhoA signaling increases cytoskeletal tension, which plays a crucial role in the osteogenic differentiation of mesenchymal stem cells. We thus hypothesized that RhoA signaling is involved in simvastatin-induced osteogenesis in bone marrow mesenchymal stem cells. We found that although treatment with simvastatin shifts localization of RhoA protein from the membrane to the cytosol, the treatment still activates RhoA dose-dependently because it reduces the association with RhoGDIα. Simvastatin also increased the expression of osteogenic proteins, density of actin filament, the number of focal adhesions, and cellular tension. Furthermore, disrupting actin cytoskeleton or decreasing cell rigidity by using chemical agents reduced simvastatin-induced osteogenic differentiation. In vivo study also confirms that density of actin filament is increased in simvastatin-induced ectopic bone formation. Our study is the first to demonstrate that maintaining intact actin cytoskeletons and enhancing cell rigidity are crucial in simvastatin-induced osteogenesis. The results suggested that simvastatin, which is an osteoinductive factor and acts by increasing actin filament organization and cell rigidity combined with osteoconductive biomaterials, may benefit stem-cell-based bone regeneration.

No MeSH data available.


Related in: MedlinePlus