Activation of the IGF1 pathway mediates changes in cellular contractility and motility in single-suture craniosynostosis

Abstract

Insulin growth factor 1 (IGF1) is a major anabolic signal that is essential during skeletal development, cellular adhesion and migration. Recent transcriptomic studies have shown that there is an upregulation in IGF1 expression in calvarial osteoblasts derived from patients with single-suture craniosynostosis (SSC). Upregulation of the IGF1 signaling pathway is known to induce increased expression of a set of osteogenic markers that previously have been shown to be correlated with contractility and migration. Although the IGF1 signaling pathway has been implicated in SSC, a correlation between IGF1, contractility and migration has not yet been investigated. Here, we examined the effect of IGF1 activation in inducing cellular contractility and migration in SSC osteoblasts using micropost arrays and time-lapse microscopy. We observed that the contractile forces and migration speeds of SSC osteoblasts correlated with IGF1 expression. Moreover, both contractility and migration of SSC osteoblasts were directly affected by the interaction of IGF1 with IGF1 receptor (IGF1R). Our results suggest that IGF1 activity can provide valuable insight for phenotype-genotype correlation in SSC osteoblasts and might provide a target for therapeutic intervention.

Keywords: Calvarial osteoblast; Cell migration; IGF1; IGF1R; Single-suture craniosynostosis; Traction force.

Fig. 1.

IGF1 expression in SSC osteoblasts. The selected 15 SSC cases and nine controls were assessed for ALP expression (marker of differentiation) and BrdU incorporation (marker of proliferation). (A) SSC osteoblasts exhibited an increase in ALP activity compared to the controls; however, it was not statistically significant (P>0.05). (B) Plot of ALP expression as a function of IGF1 expression for both SSC osteoblasts and controls reveal overlapping 95% confidence ellipses. However, the ellipse surrounding ALP expressions of SSC osteoblasts suggested a weak positive correlation with increasing IGF1 expression (R-value=0.397). (C) BrdU incorporation by SSC osteoblasts was found to be significantly reduced as compared to the controls (P=0.015). (D) This result was also corroborated by the 95% ellipse surrounding the BrdU incorporation in SSC osteoblast, where a marginally weak negative correlation was found between proliferation and IGF1 expression (R-value=−0.029). Data shown as mean±s.e.m. from three experimental replicates. *P<0.05 (Student's t-test). P-values for the 95% confidence ellipses were determined by Spearman–Rank correlations.

Fig. 2.

Increased contractility observed in SSC osteoblasts. Representative fluorescent images and traction forces of a fixed and stained (A) control and (B) SSC osteoblast cells. Red, microposts; green, actin; blue, RUNX2 nuclear signal. Traction forces were measured by analyzing the deflections of the microposts and are reported as force vectors (arrows). (C) Cell spread area remained unchanged for SSC osteoblasts and controls (P=0.439). (D) A plot of the cell area as a function of IGF1 expression for both SSC osteoblasts and controls revealed overlapping 95% confidence ellipses; however, a weak positive correlation of cell area with IGF1 expression was observed for SSC (R-value=0.257). (E) The traction forces of SSC osteoblasts appeared significantly more contractile than the controls (P=1.17×10⁻⁴). (F) The 95% confidence ellipse surrounding the traction forces of SSC osteoblasts demonstrated a positive correlation with IGF1 expression compared to the controls (R-value=0.532), suggesting that IGF1 is an important factor related to the disease. Data shown as mean±s.e.m. from three experimental replicates (Table S2). *P<0.05 (Student's t-test). P-values for the 95% confidence ellipses were determined by Spearman–Rank correlations.

Fig. 3.

Reduced migration observed in SSC osteoblasts. Time-lapse phase-contrast movies were obtained to observe the migration of (A) control and (B) SSC osteoblasts. Representative phase-contrast images were overlaid with the migration tracking data, where each color trace represents the path of a cell. Collecting the migration data for (C) controls and (D) SSC osteoblasts revealed that the controls explore a wider territory than SSC osteoblasts. (E) In addition, the migration speed was considerably reduced amongst SSC osteoblasts as compared to controls (P=1.15×10⁻³). (F) When comparing the migration speeds of the select SSC and control osteoblasts to IGF1 expression, the 95% confidence ellipse surrounding the SSC osteoblasts showed a negative correlation to IGF1 expression in SSC osteoblasts (R-value=−0.486). Data shown as mean±s.e.m. from three experimental replicates (Table S2). *P<0.05 (Student's t-test). P-values for the 95% confidence ellipses were determined by Spearman–Rank correlations.

Fig. 4.

IGF1R regulates osteoblast contractility. A control osteoblast line with the lowest expression of IGF1 and a SSC osteoblast line with the highest IGF1 expression were chosen for studying the role of IGF1 signaling on traction force. (A) Controls with low IGF1 expression appeared significantly more contractile upon stimulation with exogenous IGF1 (population size of experimental repetition, n₁=13, n₂=9, n₃=12; P=0.006) as compared to their untreated controls (n₁=14, n₂=3, n₃=10). Selectively inhibiting IGF1R (with NVP) (n₁=12, n₂=9, n₃=10) or myosin-II ATPase (with blebbistatin, Blebb) (n₁=12, n₂=9, n₃=11) did not affect traction forces. Stimulating NVP-treated (n₁=9, n₂=13, n₃=19), or blebbistatin-treated cells (n₁=12, n₂=10, n₃=9), with exogenous IGF1 resulted in no change in traction force. (B) Compared to the untreated SSC cells (population size of experimental repetition, n₁=10, n₂=12, n₃=11), SSC osteoblasts stimulated with IGF1 showed a significant increase in traction forces (n₁=9, n₂=14, n₃=9; P=0.012). When selectively inhibiting IGF1R, a loss in traction forces was observed (n₁=11, n₂=10, n₃=16; P=6.30×10⁻⁴). NVP-treated cells subsequently treated with exogenous IGF1 remained unchanged compared to the NVP-treated cells; however, their traction forces were significantly lower than those of untreated SSC osteoblasts (P=0.003). Blebbistatin also caused the traction forces of SSC osteoblasts to be significantly reduced (n₁=12, n₂=12, n₃=9; P=8.08×10⁻⁵). Stimulating the blebbistatin-treated SSC osteoblasts with exogenous IGF1 resulted in a small but significant increase in force (n₁=13, n₂=13, n₃=8; P=0.029); however, this increase still remained significantly lower than the forces of untreated SSC osteoblasts (P=2.06×10⁻⁴). Data shown as mean±s.e.m. from three experimental replicates. *P<0.05 (one-way ANOVA with a Bonferroni's and Fischer post-hoc adjustment).

Fig. 5.

IGF1R regulates osteoblast migration. A control osteoblast line with the lowest expression of IGF1 and a SSC osteoblast line with the highest IGF1 expression were chosen for studying the role of IGF1 signaling on migration. (A) Controls with low IGF1 expression appeared significantly less migratory upon IGF1 stimulation (population size of experimental repetition, n_1,2,3=3; P=0.017) compared to their untreated controls (n_1,2,3=3). Upon selectively inhibiting IGF1R (with NVP) (n_1,2,3=3) or myosin-II ATPase (with blebbistatin, Blebb) (n_1,2,3=3), the cells remained migratory. Furthermore, even after supplementing NVP-treated (n₁=2, n_2,3=3), or blebbistatin-treated cells (n_1,2,3=3) with exogenous IGF1, these cells still remained migratory. (B) Untreated SSC cells (n₁=4, n₂=3, n₃=2) and those stimulated with exogenous IGF1 (n₁=4, n₂=4, n₃=3) showed a decrease in migration speed as compared to untreated controls. When selectively inhibiting IGF1R, these cells were more migratory (n₁=3, n₂=2, n₃=4; P=0.009). Stimulating with IGF1 (n₁=4, n₂=4, n₃=3) did not lead to a further increase in migration; however, this increase still remained significantly higher to the migration speed of untreated SSC osteoblasts (P=0.037). Upon inhibiting myosin-II ATPase with blebbistatin (n_1,2,3=4), their migration speeds appeared significantly greater than the untreated SSC osteoblasts (P=0.001), even after IGF1 stimulation (n₁=4, n₂=3, n₃=4; P=7.62×10⁻⁴). Data shown as mean±s.e.m. from three experimental replicates. *P<0.05 (one-way ANOVA with a Bonferroni's and Fischer post-hoc adjustment).