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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 gene and protein expression was affected by cytoskeletal alteration agents.Notes: Disrupting the actin filament organization or decreasing cell rigidity significantly reduced the simvastatin-induced upregulation of osteogenic genes, including Runx2, BMP-2, and OC, 12 (A) and 24 hours (B) after SIM treatment. Immunofluorescence staining revealed simvastatin-induced osteogenic transcription factor Runx2 in the nucleus at 12 (C) and 24 hours (D) and the osteogenic proteins BMP-2 (C) and OC (E) in the cytosol at 24 hours were significantly abrogated by the biochemical agents. Results are 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; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
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f3-ijn-10-5881: Simvastatin-induced osteogenic gene and protein expression was affected by cytoskeletal alteration agents.Notes: Disrupting the actin filament organization or decreasing cell rigidity significantly reduced the simvastatin-induced upregulation of osteogenic genes, including Runx2, BMP-2, and OC, 12 (A) and 24 hours (B) after SIM treatment. Immunofluorescence staining revealed simvastatin-induced osteogenic transcription factor Runx2 in the nucleus at 12 (C) and 24 hours (D) and the osteogenic proteins BMP-2 (C) and OC (E) in the cytosol at 24 hours were significantly abrogated by the biochemical agents. Results are 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; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.

Mentions: Biochemical agents, including Y-27632, blebbistatin, and cytochalasin D, were used to determine whether RhoA/actin/cell rigidity signaling was involved in simvastatin-induced osteogenesis. Cells treated with Y-27632 or blebbistatin became stellate-shaped and lacked actin fibril organization. Cells treated with cytochalasin D exhibited disrupted actin fibril organization and were clustered into irregular aggregates, even in the cultures treated with simvastatin (Figure 2A). Confocal images revealed clear increases in focal adhesion after treatment with simvastatin and decreases after adding Y-27632, blebbistatin, and cytochalasin D (Figure 2B). These biochemical agents not only disrupted fibril organization but also reduced simvastatin-enhanced cell rigidity (Figure 2C). Next, we measured the mRNA and protein levels of osteogenic genes (Runx2, BMP-2, and OC) in D1 cells treated with simvastatin alone or in combination with Y-27632, blebbistatin, or cytochalasin D. We found that incubation with Y-27632 or blebbistatin or disrupting actin polymerization with cytochalasin D significantly reduced the simvastatin-induced gene expression of Runx2, BMP-2, and OC at 12 and 24 hours (Figure 3A and B). Confocal images showed that simvastatin-induced increases in Runx2 protein in nuclei were lower at 12 and 24 hours (Figure 3C) and that simvastatin-induced increases in BMP-2 and OC protein levels were lower at 12 hours of culture in Y-27632, blebbistatin, or cytochalasin D (Figure 3D and E). These blockers also inhibited simvastatin-induced ALP activity on Day 3 and mineralization effects on Day 5 after treatment with simvastatin (Figure 4A and B). These results demonstrated that simvastatin-induced osteogenesis involves RhoA signaling, actin rearrangement, and increased cell rigidity.


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 gene and protein expression was affected by cytoskeletal alteration agents.Notes: Disrupting the actin filament organization or decreasing cell rigidity significantly reduced the simvastatin-induced upregulation of osteogenic genes, including Runx2, BMP-2, and OC, 12 (A) and 24 hours (B) after SIM treatment. Immunofluorescence staining revealed simvastatin-induced osteogenic transcription factor Runx2 in the nucleus at 12 (C) and 24 hours (D) and the osteogenic proteins BMP-2 (C) and OC (E) in the cytosol at 24 hours were significantly abrogated by the biochemical agents. Results are 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; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
© Copyright Policy
Related In: Results  -  Collection

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

f3-ijn-10-5881: Simvastatin-induced osteogenic gene and protein expression was affected by cytoskeletal alteration agents.Notes: Disrupting the actin filament organization or decreasing cell rigidity significantly reduced the simvastatin-induced upregulation of osteogenic genes, including Runx2, BMP-2, and OC, 12 (A) and 24 hours (B) after SIM treatment. Immunofluorescence staining revealed simvastatin-induced osteogenic transcription factor Runx2 in the nucleus at 12 (C) and 24 hours (D) and the osteogenic proteins BMP-2 (C) and OC (E) in the cytosol at 24 hours were significantly abrogated by the biochemical agents. Results are 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; Bleb, blebbistatin; CD, cytochalasin D; SEM, standard error of the mean; OC, osteocalcin.
Mentions: Biochemical agents, including Y-27632, blebbistatin, and cytochalasin D, were used to determine whether RhoA/actin/cell rigidity signaling was involved in simvastatin-induced osteogenesis. Cells treated with Y-27632 or blebbistatin became stellate-shaped and lacked actin fibril organization. Cells treated with cytochalasin D exhibited disrupted actin fibril organization and were clustered into irregular aggregates, even in the cultures treated with simvastatin (Figure 2A). Confocal images revealed clear increases in focal adhesion after treatment with simvastatin and decreases after adding Y-27632, blebbistatin, and cytochalasin D (Figure 2B). These biochemical agents not only disrupted fibril organization but also reduced simvastatin-enhanced cell rigidity (Figure 2C). Next, we measured the mRNA and protein levels of osteogenic genes (Runx2, BMP-2, and OC) in D1 cells treated with simvastatin alone or in combination with Y-27632, blebbistatin, or cytochalasin D. We found that incubation with Y-27632 or blebbistatin or disrupting actin polymerization with cytochalasin D significantly reduced the simvastatin-induced gene expression of Runx2, BMP-2, and OC at 12 and 24 hours (Figure 3A and B). Confocal images showed that simvastatin-induced increases in Runx2 protein in nuclei were lower at 12 and 24 hours (Figure 3C) and that simvastatin-induced increases in BMP-2 and OC protein levels were lower at 12 hours of culture in Y-27632, blebbistatin, or cytochalasin D (Figure 3D and E). These blockers also inhibited simvastatin-induced ALP activity on Day 3 and mineralization effects on Day 5 after treatment with simvastatin (Figure 4A and B). These results demonstrated that simvastatin-induced osteogenesis involves RhoA signaling, actin rearrangement, and increased cell rigidity.

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