Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation

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Abstract

Mechanical strain plays a critical role in the proliferation, differentiation and maturation of bone cells. As mechanical receptor cells, osteoblasts perceive and respond to stress force, such as those associated with compression, strain and shear stress. However, the underlying molecular mechanisms of this process remain unclear. Using a four-point bending device, mouse MC3T3-E1 cells was exposed to mechanical tensile strain. Cell proliferation was determined to be most efficient when stimulated once a day by mechanical strain at a frequency of 0.5 Hz and intensities of 2500 με with once a day, and a periodicity of 1 h/day for 3 days. The applied mechanical strain resulted in the altered expression of 1992 genes, 41 of which are involved in the mitogen-activated protein kinase (MAPK) signaling pathway. Activation of ERK by mechanical strain promoted cell proliferation and inactivation of ERK by PD98059 suppressed proliferation, confirming that ERK plays an important role in the response to mechanical strain. Furthermore, the membrane-associated receptors integrin β1 and integrin β5 were determined to regulate ERK activity and the proliferation of mechanical strain-treated MC3T3-E1 cells in opposite ways. The knockdown of integrin β1 led to the inhibition of ERK activity and cell proliferation, whereas the knockdown of integrin β5 led to the enhancement of both processes. This study proposes a novel mechanism by which mechanical strain regulates bone growth and remodeling. © 2012 Yan et al.

Figures

  • Figure 1. The best efficiently stimulation of mechanical strain was determined by MTT in MC3T3-E1 cells. (A) The proliferation was evaluated by MTT assay under mechanical strains of 2500 me at 0.5 Hz with different strain time (0.5 h–2.5 h) and periodicity (once or twice a day) and strain cycle (1 day–3 day). Data are represented as the mean 6 SD of at least three biological replicates, *P,0.05, **P,0.01 compared with treat time (0 h) group. (B) The proliferation was evaluated by MTT assay in different intensities (1000–5000 me) at 0.5 Hz, once a day for 1 hour over 3
  • Table 1. Cont.
  • Table 1. Microarry analyses the main signaling pathways in MC3T3-E1 cells with mechanical strain.
  • Figure 3. The KEGG MAPK signaling pathway showing genes differentially expressed in MC3T3-E1 cells exposed to mechanical strain. Mechanical strains of 2500 me, once a day at 0.5 Hz, and a periodicity of 1 h/day for 3 days. Orange frame indicates changed genes. KEGG, Kyoto Encyclopedia of Genes and Genomes. doi:10.1371/journal.pone.0035709.g003
  • Figure 4. Effect of mechanical strain on the MAPK signaling pathway. The expression values of the selected 5 genes from the MAPK signaling pathway was measured by cDNA microarray and quantitative real-time RT-PCR (qRT-PCR). MC3T3-E1 cells were stimulated under mechanical strains of 2500 me, once a day at 0.5 Hz, and a periodicity of 1 h/day for 3 days. Data are represented as the mean 6 SD of at least three biological replicates, * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0035709.g004
  • Table 2. The changed genes of the MAPK signaling pathway under mechanical strain of 2500 me, at 0.5 Hz, once a day for 1 h over 3 consecutive days in MC3T3-E1 cells.
  • Figure 5. Mechanical strain promotes MC3T3-E1 cells proliferation through the ERK signaling pathway. (A–C) The protein expression of ERK and ERK-phosphorylation in different treat groups (with or without 20 mM MEK1/2 inhibitors PD98059) was detected by Western blotting with anti-ERK1/2 and anti-p-ERK1/2. GAPDH was used as an internal control. Data are represented as mean 6 SD of at least three biological replicates, * P,0.05, ** P,0.01. (D) The proliferation of cells treated with or without PD98059 under mechanical strain was evaluated by MTT assay. Data are represented as the mean 6 SD, of at least three biological replicates, * P,0.05, ** P,0.01. doi:10.1371/journal.pone.0035709.g005
  • Figure 6. Integrin b1 and Integrin b5 have opposite effects on the phosphorylation of ERK and the proliferation in MC3T3-E1 cells. (A) and (B) Mechanical strain induces Integrin b1 and Integrin b5 expression. The mRNA and protein expressions of Integrin b1 (A) and Integrin b5 (B) were analysised by cDNA microarray, qPCR and immunofluorescence (IF) with anti-Itgb1 and anti-Itgb5. All data are represented as mean 6 SD of at least three biological replicates. * P,0.05, ** P,0.01 versus Control. (C–E) The protein expressions of ERK and ERK-phosphorylations in different treated groups (knockdown of Integrinb1, Integrinb5 or both simultaneously with siRNA transfection) under mechanical strain were detected by Western blotting with anti-ERK1/2 and anti-p-ERK1/2. GAPDH was used as an internal control. All data represent the mean 6 SD of at least three biological replicates, * P,0.05, ** P,0.01 between the indicated groups. (F) The proliferation of cells treated with Integrin b1-siRNA, Integrin b5siRNA or both under mechanical strain was evaluated by MTT assay. All data represent the mean 6 SD of at least three biological replicates, * P,0.05, ** P,0.01 between the indicated groups. doi:10.1371/journal.pone.0035709.g006

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Yan, Y. xian, Gong, Y. wei, Guo, Y., Lv, Q., Guo, C., Zhuang, Y., … Zhang, X. zheng. (2012). Mechanical strain regulates osteoblast proliferation through integrin-mediated ERK activation. PLoS ONE, 7(4). https://doi.org/10.1371/journal.pone.0035709

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