Intermittent Compressive Stress Enhanced Insulin-Like Growth Factor-1 Expression in Human Periodontal Ligament Cells

Abstract

Mechanical force was shown to promote IGF-1 expression in periodontal ligament both in vitro and in vivo. Though the mechanism of this effect has not yet been proved, here we investigated the molecular mechanism of intermittent mechanical stress on IGF-1 expression. In addition, the role of hypoxia on the intermittent compressive stress on IGF-1 expression was also examined. In this study, human periodontal ligament cells (HPDLs) were stimulated with intermittent mechanical stress for 24 hours. IGF-1 expression was examined by real-time polymerase chain reaction. Chemical inhibitors were used to determine molecular mechanisms of these effects. For hypoxic mimic condition, the CoCl2 supplementation was employed. The results showed that intermittent mechanical stress dramatically increased IGF-1 expression at 24 h. The pretreatment with TGF-β receptor I or TGF-β1 antibody could inhibit the intermittent mechanical stress-induced IGF-1 expression. Moreover, the upregulation of TGF-β1 proteins was detected in intermittent mechanical stress treated group. Correspondingly, the IGF-1 expression was upregulated upon being treated with recombinant human TGF-β1. Further, the hypoxic mimic condition attenuated the intermittent mechanical stress and rhTGF-β1-induced IGF-1 expression. In summary, this study suggests intermittent mechanical stress-induced IGF-1 expression in HPDLs through TGF-β1 and this phenomenon could be inhibited in hypoxic mimic condition.

Figure 1

Intermittent mechanical stress-induced IGF-1 expression. HPDLs were treated with intermittent mechanical stress for 2 h, 4 h, 8 h, and 24 h. The IGF-1 mRNA expression was determined using real-time PCR. The dot line represented the expression levels of the control. Asterisks indicated statistically significant difference.

Figure 2

Intermittent mechanical stress required the intermediate protein to induce IGF-1 expression. (a) Cycloheximide (CHX; 10 μM), (b) genistein (92.5 μM), and (c) monensin (100 μM) were pretreated 30 min prior to applying the intermittent mechanical stress for 24 h. IGF-1 mRNA expression was determined by real-time PCR. Asterisks indicated statistically significant difference. C: the control condition; S: the intermittent mechanical stress treatment condition.

Figure 3

TGF-β1 related to intermittent mechanical stress-induced IGF-1 expression. (a) SB431542 (10 μM) or (b) TGF-β1 neutralizing antibody (Ab) (5 μg/mL) was used to pretreat HPDLs 30 min before applying the intermittent mechanical stress for 24 h. The IGF-1 expression was measured by real-time PCR. (c) IGF-1 mRNA levels were examined after HPDLs were treated with rhTGF-β1 (2 ng/mL) for 24 h. (d) HPDLs were treated with cell culture medium from intermittent mechanical stress-treated group (CMS) or untreated group (CMC) 24 h. The IGF-1 mRNA levels were determined by real-time PCR. (e) The TGF-β1 protein in whole cell lysate was measured by ELISA assay. Asterisks indicated statistically significant difference. C: the control condition; S: the intermittent mechanical stress treatment condition.

Figure 4

CoCl₂ inhibited the effect of intermittent mechanical stress on IGF-1 expression. (a) HPDLs were treated with CoCl₂ (150 and 300 μM) in the presence or absence of intermittent mechanical stress stimulation for 24 h. The IGF-1 expression was evaluated by real-time PCR. (b) IGF-1 expression was examined after HPDLs were treated with rhTGF-β1 (2 ng/mL) with and without the CoCl₂ (150 μM) for 24 h. (c) The illustration represented the proposed signaling mechanism of the intermittent mechanical stress-induced IGF-1 expression by HPDLs. Asterisks indicated statistically significant difference. C: the control condition; S: the intermittent mechanical stress treatment condition.