Strain-dependent up-regulation of ephrin-B2 protein in periodontal ligament fibroblasts contributes to osteogenesis during tooth movement

Abstract

During orthodontic tooth movement, the application of adequate orthodontic forces allows teeth to be moved through the alveolar bone. These forces are transmitted through the periodontal ligaments (PDL) to the supporting alveolar bone and lead to deposition or resorption of bone, depending on whether the tissues are exposed to a tensile or compressive mechanical strain. Fibroblasts within the PDL (PDLF) are considered to be mechanoresponsive. The transduction mechanisms from mechanical loading of the PDLF to the initiation of bone remodeling are not clearly understood. Recently, members of the ephrin/Eph family have been shown to be involved in the regulation of bone homeostasis. For the first time, we demonstrate that PDLF exposed to tensile strain induce the expression of ephrin-B2 via a FAK-, Ras-, ERK1/2-, and SP1-dependent pathway. Osteoblasts of the alveolar bone stimulated with ephrin-B2 increased their osteoblastogenic gene expression and showed functional signs of osteoblastic differentiation. In a physiological setting, ephrin-B2-EphB4 signaling between PDLF and osteoblasts of the alveolar bone might contribute to osteogenesis at tension sites during orthodontic tooth movement.

FIGURE 1.

Ephrin and Eph receptor expression analysis. A, standard RT-PCR expression analysis of ephrin ligands and Eph receptors in primary human PDLF (upper panel) and primary human osteoblasts (lower panel). B and C, qRT-PCR analysis (TaqMan chemistry) for ephrin ligands and Eph receptors in primary human PDLF (B) and primary human osteoblasts (C). Data represent the summarized results of the analysis of the three PDLF and three osteoblast cell lines used in this study. Data are presented as mean ± S.D., n = 3. All experiments were performed in triplicate.

FIGURE 2.

Mechanical strain leads to spatial redistribution of FAK and α-actinin and to sustained FAK activation at focal adhesions in PDLF. PDLF were subjected to longitudinal cellular strain (2.5%) for 15 min and 1, 4, 24, and 72 h. The cellular distribution of FAK and α-actinin was assessed by means of immunofluorescent staining, and tyrosine-phosphorylated proteins were stained using an anti-phosphotyrosine-specific antibody. The application of mechanical stress induced lamellipodial protrusions at the cell periphery within 15 min. By 1 h, FAK was recruited to focal adhesions (arrowheads in FAK, C–F, 1–72 h) under the lamellipodial protrusion. In static control cells, α-actinin was primarily localized to regions near the end of actin bundles (α-actinin, A). After exposure to cellular strain, stress fibers formed, and α-actinin was more evenly distributed throughout the cell and along the stress fibers (α-actinin, A–F). p-FAK staining increased in the cellular peripheries (e.g. lamellipodia) with the prolonged application of cellular strain. Unstrained PDLF after 72 h in culture are presented as an inset in the p-FAK 72-h strained microphotograph for comparison. Strain directions are indicated (arrows). Scale bar, 10 μm.

FIGURE 3.

Ephrin-B2 transcription in PDLF is activated via a Ras- and ERK1/2-dependent pathway. Immunostaining for p-FAK suggested an increased phosphorylation status of FAK at sites of FA. Therefore, FAK was further analyzed using immunoblotting against t-FAK and for phosphorylated (Tyr⁵⁷⁶) FAK (A). t-FAK and p-FAK were normalization for the expression of β-actin (data not shown). Densitometric analysis revealed the p-FAK elevation beginning after 15 min of strain and increasing to 18.3% after 30 min. Phosphorylation was sustained between 24 and 72 h, peaking at 44.7% (24 h) elevation, as compared with static control cells. B, Ras activation assay. PDLF were subjected to mechanical strain. Ras was activated 15 min after the onset of mechanical strain, and a statistically significant activation was found after 24 h. C, Western blotting for total ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2). Induction of ERK1/2 phosphorylation was first detected 15 min after the onset of mechanical stress, being in line with the temporal onset of Ras activation. D, ChIP assays were performed to study the interactions between Sp1 and the ephrin-B2 promoter. Sp1 binding to the ephrin-B2 promoter was detected 4 h after the onset of mechanical strain and sustained until the end of the observation period (72 h). Values are represented as mean ± S.D.

FIGURE 4.

Ephrin-B2 expression increases in PDL fibroblasts and decreases in osteoblasts exposed to mechanical strain. qRT-PCR analyses for ephrin-B2 (A) and EphB4 (B) expression after the application of mechanical strain in PDLF. Mechanical strain was applied for 1–72 h at 2.5% elongation. Significant overexpression for ephrin-B2, as compared with static control cells, was demonstrated after 24 h and sustained until 72 h (A). EphB4 was not significantly altered during the course of the experiment. Ephrin-B2 and EphB4 interactions might be evident within the osteoblast lineage. Therefore, osteoblasts were subjected to mechanical strain and tested for the transcription of ephrin-B2 and EphB4 by means of qRT-PCR (C and D). Ephrin-B2 and EphB4 expression was increased. It is noteworthy that temporal expression pattern of ephrin-B2 in osteoblasts was reciprocal to the temporal ephrin-B2 expression pattern observed in PDLF. Values are represented as mean ± S.D. *, p < 0.05 versus control, n = 3. qRT-PCR experiments were performed in triplicate. FAK might be a prominent mediator of mechano-induced alterations of ephrin-B2 expression in PDLF. siRNA against FAK was used to knock down FAK expression in PDLF. siRNA against FAK and a scrambled control siRNA were transfected to PDLF, and FAK expression was monitored on the mRNA level using qRT-PCR. E, siRNA against FAK attenuated FAK expression in PDLF after 12 h and attenuation remained significant up to 72 h. FAK attenuation mediated by siRNA was also monitored on the protein level using Western blotting against FAK (F), and probing for tubulin served as a loading control. Western blotting confirmed the siRNA-mediated knockdown of FAK in PDLF. PDLF attenuated for FAK using siRNA were subjected to mechanical strain for 1–72 h (G), and ephrin-B2 expression was assessed at discrete time points using qRT-PCR. Unlike WT PDLF, siRNA-transfected PDLF did not show an up-regulation of ephrin-B2 upon the application of mechanical strain.

FIGURE 5.

Ephrin-B2 activates Runx2 and ALPL transcription in osteoblasts of the alveolar bone via a Ras- and ERK1/2-dependent pathway. To prove that ephrin-B2-Fc causes EphB4 receptor phosphorylation in osteoblasts of the alveolar bone, osteoblasts were stimulated with 1 μg/ml ephrin-B2-Fc for 5–60 min. Tyrosine phosphorylation of the EphB4 receptor was detected after 5 min and sustained through the course of the experiment (A). To elucidate a putative signaling pathway linking ephrin-B2-Fc stimulation and downstream events, osteoblasts of the alveolar bone were stimulated with 1 μg/ml ephrin-B2-Fc for 2–10 min, and Ras activation was monitored. Ras was transiently activated after ephrin-B2-Fc stimulation (2 min) (B). Ras might transmit downstream signals via ERK1/2. To reveal a possible ERK1/2 dependence of ephrin-B2 downstream signaling, protein lysates of osteoblasts of the alveolar bone stimulated with ephrin-B2-Fc (1 μg/ml) for 5–60 min were probed with antibodies against ERK1/2 and pERK1/2. An increase in phosphorylated ERK1/2 was detected 5 min after stimulation, gradually decreasing to nearly base-line phosphorylation after 60 min (C). To detect further downstream effects of ephrin-B2-dependent signaling in osteoblasts of the alveolar bone, osteoblasts were stimulated with 2 and 4 μg/ml, respectively, of ephrin-B2-Fc for 6 days. qRT-PCR revealed the significant induction of Runx2, as well as ALPL in a dose-dependent manner (D). Although EphB4 is the only EphB receptor exhibiting a binding specificity for a single ligand, ephrin-B2 has binding affinity for other receptors of the EphB-class. To rule out the involvement of other EphB receptors for the ephrin-B2-dependent activation of Runx2 and ALP mRNA expression, we have used siRNA to knock down EphB4 in osteoblasts. siRNA against EphB4 effectively suppressed EphB4 expression in osteoblasts for up to 96 h (E). Stimulation of osteoblasts with 2 and 4 μg/ml ephrin-B2 in the presence of siRNA against EphB4 did not result in an up-regulation of Runx2 (F) or ALP (G) expression as observed in WT osteoblasts (D). The activation of ERK1/2 in osteoblasts upon ephrin-B2 stimulation (C) suggested the involvement of an ERK1/2-dependent pathway in the induction of osteoblastogenic gene expression after ephrin-B2 stimulation. We have used a specific MEK1/2 inhibitor, UO126, to block ERK1/2 activation in osteoblasts. Osteoblasts were stimulated with 2 or 4 μg/ml ephrin-B2 in the absence or presence of different concentrations of UO126. Runx2 (H and I) and ALP (J and K) were monitored by means of qRT-PCR. Already at a concentration of 10 μm, UO126 attenuated the ephrin-B2-dependent activation of Runx2 and its downstream target ALP in osteoblasts. These results suggested an involvement of an ERK1/2-dependent pathway in the ephrin-B2-dependent regulation of Runx2 in osteoblasts. Increased ALP transcription was accompanied by an increase in ALP activity (L). Alizarin Red staining for calcified nodules in osteoblasts stimulated for 6 days with ephrin-B2-Fc (2 and 4 μg/ml) further confirmed the osteoblastogenic effects of ephrin-B2-Fc stimulation. An untreated control is shown as an inset. Scale bar, 20 μm (M). Subpopulations of PDLF might contribute to osteogenesis at tension sites in orthodontic tooth movement. Therefore, we have tested for the expression of Runx2 and ALP in PDLF after stimulation with ephrin-B2-Fc by means of qRT-PCR. Both ALP and Runx2 transcription were up-regulated in a dose-dependent manner in PDLF (N). Up-regulation of ALPL and Runx2 transcription reached significance after stimulation with 4 μg/ml of ephrin-B2-Fc. Values are represented as mean ± S.D. *, p < 0.05 versus control, n = 3.

FIGURE 5.

Ephrin-B2 activates Runx2 and ALPL transcription in osteoblasts of the alveolar bone via a Ras- and ERK1/2-dependent pathway. To prove that ephrin-B2-Fc causes EphB4 receptor phosphorylation in osteoblasts of the alveolar bone, osteoblasts were stimulated with 1 μg/ml ephrin-B2-Fc for 5–60 min. Tyrosine phosphorylation of the EphB4 receptor was detected after 5 min and sustained through the course of the experiment (A). To elucidate a putative signaling pathway linking ephrin-B2-Fc stimulation and downstream events, osteoblasts of the alveolar bone were stimulated with 1 μg/ml ephrin-B2-Fc for 2–10 min, and Ras activation was monitored. Ras was transiently activated after ephrin-B2-Fc stimulation (2 min) (B). Ras might transmit downstream signals via ERK1/2. To reveal a possible ERK1/2 dependence of ephrin-B2 downstream signaling, protein lysates of osteoblasts of the alveolar bone stimulated with ephrin-B2-Fc (1 μg/ml) for 5–60 min were probed with antibodies against ERK1/2 and pERK1/2. An increase in phosphorylated ERK1/2 was detected 5 min after stimulation, gradually decreasing to nearly base-line phosphorylation after 60 min (C). To detect further downstream effects of ephrin-B2-dependent signaling in osteoblasts of the alveolar bone, osteoblasts were stimulated with 2 and 4 μg/ml, respectively, of ephrin-B2-Fc for 6 days. qRT-PCR revealed the significant induction of Runx2, as well as ALPL in a dose-dependent manner (D). Although EphB4 is the only EphB receptor exhibiting a binding specificity for a single ligand, ephrin-B2 has binding affinity for other receptors of the EphB-class. To rule out the involvement of other EphB receptors for the ephrin-B2-dependent activation of Runx2 and ALP mRNA expression, we have used siRNA to knock down EphB4 in osteoblasts. siRNA against EphB4 effectively suppressed EphB4 expression in osteoblasts for up to 96 h (E). Stimulation of osteoblasts with 2 and 4 μg/ml ephrin-B2 in the presence of siRNA against EphB4 did not result in an up-regulation of Runx2 (F) or ALP (G) expression as observed in WT osteoblasts (D). The activation of ERK1/2 in osteoblasts upon ephrin-B2 stimulation (C) suggested the involvement of an ERK1/2-dependent pathway in the induction of osteoblastogenic gene expression after ephrin-B2 stimulation. We have used a specific MEK1/2 inhibitor, UO126, to block ERK1/2 activation in osteoblasts. Osteoblasts were stimulated with 2 or 4 μg/ml ephrin-B2 in the absence or presence of different concentrations of UO126. Runx2 (H and I) and ALP (J and K) were monitored by means of qRT-PCR. Already at a concentration of 10 μm, UO126 attenuated the ephrin-B2-dependent activation of Runx2 and its downstream target ALP in osteoblasts. These results suggested an involvement of an ERK1/2-dependent pathway in the ephrin-B2-dependent regulation of Runx2 in osteoblasts. Increased ALP transcription was accompanied by an increase in ALP activity (L). Alizarin Red staining for calcified nodules in osteoblasts stimulated for 6 days with ephrin-B2-Fc (2 and 4 μg/ml) further confirmed the osteoblastogenic effects of ephrin-B2-Fc stimulation. An untreated control is shown as an inset. Scale bar, 20 μm (M). Subpopulations of PDLF might contribute to osteogenesis at tension sites in orthodontic tooth movement. Therefore, we have tested for the expression of Runx2 and ALP in PDLF after stimulation with ephrin-B2-Fc by means of qRT-PCR. Both ALP and Runx2 transcription were up-regulated in a dose-dependent manner in PDLF (N). Up-regulation of ALPL and Runx2 transcription reached significance after stimulation with 4 μg/ml of ephrin-B2-Fc. Values are represented as mean ± S.D. *, p < 0.05 versus control, n = 3.