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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-induced osteogenic differentiation in an animal model of ectopic bone formation was reduced by cytoskeletal alteration agents.Notes: Immunofluorescence staining revealed that the F-actin, staining with phalloidin, was increased by simvastatin treatment and reduced by cotreatment with biochemical agents, scale bar represents 50 µm (A). Inhibiting actin filament organization or decreasing cell rigidity significantly inhibited simvastatin-induced osteogenic gene expression (B). Representative masses of ectopic bone formation were stained with Alizarin Red S, the 100× figures are represented as the boxed area in the 40× line, and the mineralization of masses was quantified using Image-Pro Plus software (C). The results are the mean values of three independent measurements, error bars: SEM. **P<0.01 vs control; #P<0.05; ##P<0.01 vs SIM (1 µM).Abbreviations: SIM, simvastatin; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
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f5-ijn-10-5881: Simvastatin-induced osteogenic differentiation in an animal model of ectopic bone formation was reduced by cytoskeletal alteration agents.Notes: Immunofluorescence staining revealed that the F-actin, staining with phalloidin, was increased by simvastatin treatment and reduced by cotreatment with biochemical agents, scale bar represents 50 µm (A). Inhibiting actin filament organization or decreasing cell rigidity significantly inhibited simvastatin-induced osteogenic gene expression (B). Representative masses of ectopic bone formation were stained with Alizarin Red S, the 100× figures are represented as the boxed area in the 40× line, and the mineralization of masses was quantified using Image-Pro Plus software (C). The results are the mean values of three independent measurements, error bars: SEM. **P<0.01 vs control; #P<0.05; ##P<0.01 vs SIM (1 µM).Abbreviations: SIM, simvastatin; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.

Mentions: We used an ectopic bone formation model in imprinting control region mice to confirm that Rho/actin/cell rigidity signaling is involved in simvastatin-induced osteogenesis. Confocal images revealed clear increases in density of actin fibril after treatment with simvastatin, and actin fibril was attenuated by Y-27632, blebbistatin, or cytochalasin D, 1 week after injection (Figure 5A). In addition, simvastatin-induced increases in expression of Runx2, BMP-2, and OC mRNA were also significantly reduced by the inhibitors, 1 week after injection (Figure 5B). Alizarin Red S images and quantification results demonstrated that simvastatin induced the mineralization of ectopic bone mass; this effect was decreased by treatment with Y-27632, blebbistatin, or cytochalasin D, 2 weeks after injection (Figure 5C).


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-induced osteogenic differentiation in an animal model of ectopic bone formation was reduced by cytoskeletal alteration agents.Notes: Immunofluorescence staining revealed that the F-actin, staining with phalloidin, was increased by simvastatin treatment and reduced by cotreatment with biochemical agents, scale bar represents 50 µm (A). Inhibiting actin filament organization or decreasing cell rigidity significantly inhibited simvastatin-induced osteogenic gene expression (B). Representative masses of ectopic bone formation were stained with Alizarin Red S, the 100× figures are represented as the boxed area in the 40× line, and the mineralization of masses was quantified using Image-Pro Plus software (C). The results are the mean values of three independent measurements, error bars: SEM. **P<0.01 vs control; #P<0.05; ##P<0.01 vs SIM (1 µM).Abbreviations: SIM, simvastatin; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
© Copyright Policy
Related In: Results  -  Collection

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

f5-ijn-10-5881: Simvastatin-induced osteogenic differentiation in an animal model of ectopic bone formation was reduced by cytoskeletal alteration agents.Notes: Immunofluorescence staining revealed that the F-actin, staining with phalloidin, was increased by simvastatin treatment and reduced by cotreatment with biochemical agents, scale bar represents 50 µm (A). Inhibiting actin filament organization or decreasing cell rigidity significantly inhibited simvastatin-induced osteogenic gene expression (B). Representative masses of ectopic bone formation were stained with Alizarin Red S, the 100× figures are represented as the boxed area in the 40× line, and the mineralization of masses was quantified using Image-Pro Plus software (C). The results are the mean values of three independent measurements, error bars: SEM. **P<0.01 vs control; #P<0.05; ##P<0.01 vs SIM (1 µM).Abbreviations: SIM, simvastatin; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
Mentions: We used an ectopic bone formation model in imprinting control region mice to confirm that Rho/actin/cell rigidity signaling is involved in simvastatin-induced osteogenesis. Confocal images revealed clear increases in density of actin fibril after treatment with simvastatin, and actin fibril was attenuated by Y-27632, blebbistatin, or cytochalasin D, 1 week after injection (Figure 5A). In addition, simvastatin-induced increases in expression of Runx2, BMP-2, and OC mRNA were also significantly reduced by the inhibitors, 1 week after injection (Figure 5B). Alizarin Red S images and quantification results demonstrated that simvastatin induced the mineralization of ectopic bone mass; this effect was decreased by treatment with Y-27632, blebbistatin, or cytochalasin D, 2 weeks after injection (Figure 5C).

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