Cyclic stretch promotes osteogenesis-related gene expression in osteoblast-like cells through a cofilin-associated mechanism

Abstract

Osteoblasts have the capacity to perceive and transduce mechanical signals, and thus, regulate the mRNA and protein expression of a variety of genes associated with osteogenesis. Cytoskeletal reconstruction, as one of the earliest perception events for external mechanical stimulation, has previously been demonstrated to be essential for mechanotransduction in bone cells. However, the mechanism by which mechanical signals induce cytoskeletal deformation remains poorly understood. The actin‑binding protein, cofilin, promotes the depolymerization of actin and is understood to be important in the regulation of activities in various cell types, including endothelial, neuronal and muscle cells. However, to the best of our knowledge, the importance of cofilin in osteoblastic mechanotransduction has not been previously investigated. In the present study, osteoblast‑like MG‑63 cells were subjected to physiological cyclic stretch stimulation (12% elongation) for 1, 4, 8, 12 and 24 h, and the expression levels of cofilin and osteogenesis-associated genes were quantified with reverse transcription‑quantitative polymerase chain reaction, immunofluorescence staining and western blotting analyses. Additionally, knockdown of cofilin using RNA interference was conducted, and the mRNA levels of osteogenesis‑associated genes were compared between osteoblast‑like cells in the presence and absence of cofilin gene knockdown. The results of the present study demonstrated that cyclic stretch stimulates the expression of genes associated with osteoblastic activities in MG‑63 cells, including alkaline phosphatase (ALP), osteocalcin (OCN), runt‑related transcription factor 2 (Runx2) and collagen‑1 (COL‑1). Cyclic stretch also regulates the mRNA and protein expression of cofilin in MG‑63 cells. Furthermore, stretch‑induced increases in the levels of osteogenesis-associated genes, including ALP, OCN, Runx2 and COL‑1, were reduced following cofilin gene knockdown. Together, these results demonstrate that cofilin is involved in the regulation of mechanical load‑induced osteogenesis and, to the best of our knowledge, provides the first evidence demonstrating the importance of cofilin in osteoblastic mechanotransduction.

Figure 1

Evaluation for the mRNA expression levels of osteogenesis-associated genes ALP, OCN, Runx2 and COL-1 in MG-63 cells under cyclic stretch (12% elongation, 0.1 Hz) via reverse transcription-quantitative polymerase chain reaction analysis. The effect of cyclic mechanical stretch on (A) ALP, (B) OCN, (C) Runx2 and (D) COL-1 mRNA levels were measured at 1, 4, 8, 12 and 24 h. Data are presented as the mean ± standard deviation, n=6. ^*P<0.05, ^**P<0.01 vs. control. ALP, alkaline phosphatase; OCN, osteocalcin; Runx2, runt related transcription factor 2; COL-1, collagen 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control.

Figure 2

Cyclic mechanical stretch activates cofilin in osteoblast-like MG-63 cells. (A) Western blot analyses and quantification of stretch-induced protein expression of (B) cofilin and (C) p-cofilin in MG-63 cells under cyclic stretch (n=6). (D) Evaluation of the mRNA expression levels of cofilin under cyclic stretch via reverse transcription-quantitative polymerase chain reaction analysis (n=6). Data are presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01 vs. control; ^##P<0.05 vs. 4 h. (E) Immunofluorescence analyses for stretch-induced protein expression of cofilin (scale bar = 50 µm). p-cofilin, phosphorylated cofilin; Ctrl, control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Cofilin shRNA fragments inhibit stretch-induced cofilin activation in osteoblast-like MG-63 cells. (A) Immunofluorescence analyses for different shRNA fragments (fragments 1, 2, 3, 4 and, 5; left to right) transfected in osteoblast-like MG-63 cells (scale bar = 100 µm). (B) The effects of different shRNA were evaluated by measuring the mRNA expression of cofilin via reverse transcription-quantitative polymerase chain reaction (n=5). (C) Western blot analyses and (D) densitometry to determine the effects of four transfected shRNAs on cofilin protein expression levels (n=5). Data are presented as the mean ± standard deviation. ^**P<0.01 vs. control. shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control.

Figure 4

Inhibition of cofilin suppresses stretch-induced decreased expression of osteogenesis-associated genes ALP, OCN, Runx2 and COL-1 in osteoblast-like MG-63 cells. (A) shRNA knockdown of cofilin suppressed the cyclic mechanical stretch-induced mRNA expression of (A) ALP, (B) OCN, (C) Runx2 and (D) COL-1 under cyclic stretch for 1, 4, 8, 12 and 24 h, measured by reverse transcription-quantitative polymerase chain reaction (n=6). Data are presented as the mean ± standard deviation. ^*P<0.05, ^**P<0.01, comparisons indicated by brackets. ALP, alkaline phosphatase; OCN, osteocalcin; Runx2, runt-related transcription factor 2; COL-1, collagen 1; shRNA, short hairpin RNA; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; Ctrl, control.