Compression induces Ephrin-A2 in PDL fibroblasts via c-fos

Abstract

Ephrin-A2-EphA2 and ephrin-B2-EphB4 interactions have been implicated in the regulation of bone remodeling. We previously demonstrated a potential role for members of the Eph-ephrin family of receptor tyrosine kinases for bone remodeling during orthodontic tooth movement: compression-dependent upregulation of ephrin-A2 in fibroblasts of the periodontal ligament (PDL) attenuated osteogenesis in osteoblasts of the alveolar bone. However, factors affecting the regulation of ephrin-A2 expression upon the application of compressive forces remained unclear. Here, we report a mechano-dependent pathway of ephrin-A2 induction in PDL fibroblasts (PDLFs) involving extracellular signal-regulated kinases (ERK) 1/2 and c-fos. PDLF subjected to compressive forces (30.3 g/cm(2)) upregulated c-fos and ephrin-A2 mRNA and protein expression and displayed increased ERK1/2 phosphorylation. Inhibition of the MAP kinase kinase (MEK)/ERK1/2 pathway using the specific MEK inhibitor U0126 significantly reduced ephrin-A2 messenger RNA upregulation upon compression. Silencing of c-fos using a small interfering RNA approach led to a significant inhibition of ephrin-A2 induction upon the application of compressive forces. Interestingly, ephrin-A2 stimulation of PDLF induced c-fos expression and led also to the induction of ephrin-A2 expression. Using a reporter gene construct in murine 3T3 cells, we found that ephrin-A2 was able to stimulate serum response element (SRE)-dependent luciferase activity. As the regulation of c-fos is SRE dependent, ephrin-A2 might induce c-fos via SRE activation. Taken together, we provide evidence for an ERK1/2- and c-fos-dependent regulation of ephrin-A2 in compressed PDLF and suggest a novel pathway for ephrin-A2 induction emanating from ephrin-A2 itself. We showed previously that ephrin-A2 at compression sites might contribute to tooth movement by inhibiting osteogenic differentiation. The regulatory pathway of ephrin-A2 induction during tooth movement identified in this study might be accessible for pharmacological interventions.

Keywords: Eph family receptors; cellular mechanotransduction; mitogen-activated protein kinase 1; periodontal ligament; serum response element; tooth movement.

Figure 1.

The compression-dependent upregulation of ephrin-A2 in PDL is accompanied by ERK1/2 activation and the transcriptional induction of c-fos. We confirmed our previously reported data and showed compression-dependent upregulation of ephrin-A2 and EphA2 in a primary periodontal ligament (PDL) fibroblast population (PDLF III) (A, B). Primary human PDLFs (PDLF I, II, III) were subjected to compression (30.3 g/cm²) for 1, 4, 6 h. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis revealed a significant upregulation of c-fos in compressed PDLF I, II, and III (D, F, H). Significant upregulation of c-fos preceded the upregulation of ephrin-A2. The regulation of ephrin-A2 and c-fos, respectively, upon the application of compressive forces was confirmed on the protein level for PDLF I, II, and III (C, E, G, lower panels). For comparability, the Western blots were stripped and reprobed. Tubulin was used as a loading control. Compression experiments were performed in triplicates (n = 3). Static cells, time matched, served as controls. The qRT-PCR assays were performed in triplicates. Data are presented as mean ± SD. *P < 0.05 vs. control (one-way analysis of variance, Dunnett’s post hoc test).

Figure 2.

Inhibition of ERK1/2 phosphorylation or small interfering (siRNA)–mediated silencing of c-fos reduced the compression-dependent upregulation of ephrin-A2 in periodontal ligament fibroblasts (PDLFs). To test whether the activation of ERK1/2 (p44/p42) MAPkinase and the induction of c-fos expression are causal for ephrin-A2 upregulation in PDLFs, we selectively blocked ERK1/2 activation or c-fos transcriptional induction in PDLFs subjected to compressive forces. For the inhibition experiments, only PDLF III were used. siRNA targeting c-fos was used to perturb c-fos expression at the transcriptional level. siRNA with a scrambled sequence served as a control. As revealed by quantitative reverse transcription polymerase chain reaction (qRT-PCR), the compression-dependent induction of c-fos (A) and ephrin-A2 (B) were significantly decreased by siRNA-mediated c-fos silencing. U0126, a selective small-molecule inhibitor of MEK, the upstream kinase of ERK1/2, was used to block ERK1/2 activation in PDLFs. (C) To prove the inhibitory effects of U0126 on ERK1/2 phosphorylation in compressed PDLFs, Western blotting was performed. Lysates of compressed PDLFs treated with U0126 (10 µM and 40 µM) were probed with antibodies against ERK1/2 and pERK1/2. U0126 at 10 µM led to a marked inhibition of ERK1/2 phosphorylation (C, middle panel). At 40 µM, ERK1/2 phosphorylation was undetectable (C, right panel). qRT-PCR for c-fos (D) and ephrin-A2 (E) in compressed PDLFs. U0126 at both concentrations significantly prevented the compression-dependent induction of c-fos and of ephrin-A2 in PDLFs. These data provide further evidence for a putative inductive pathway involving ERK1/2 and c-fos leading to compression-dependent ephrin-A2 regulation in PDLFs. Compression experiments were performed in triplicates (n = 3). Static cells, time matched, served as controls. The qRT-PCR assays were performed in triplicates. Data are presented as mean ± SD. *P < 0.05 vs. control (one-way analysis of variance, Dunnett’s post hoc test).

Figure 3.

Ephrin-A2 is involved in its own induction in compressed periodontal ligament fibroblasts (PDLFs). EphA2 stimulation by different ephrin-As was shown to promote the activation of ERK kinases, which, via ternary complex transcription factor (TCFs), could lead to the induction of serum response element (SRE), the pivotal cis-element involved in the regulation of the transcription of immediate early genes, including c-fos. Therefore, ephrin-A2 via ERK1/2 SRE activation might be involved in the regulation of c-fos, which could lead to its own transcriptional activation. To test this, PDLFs were stimulated with preclustered ephrin-A2–Fc and incubated for 1, 4, and 6 h. (A, D, G) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that ephrin-A2–Fc significantly induced the transcription of ephrin-A2 in the 3 analyzed PDLF populations (PDLF I, II, and III) 4 h after stimulation; transcription remained enhanced until 6 h after stimulation. (B, E, H) qRT-PCR demonstrated that the activation of ephrin-A2 transcription was preceded by a significant, transient induction of c-fos 1 h after stimulation with ephrin-A2–Fc. (C, F, I) Ephrin-A2–Fc stimulation might activate ephrin-A2 transcription via ERK1/2 phosphorylation. Western blotting for ERK1/2 and pERK1/2 showed that ERK1/2 phosphorylation was evident in PDLFs 5 min after stimulation, returning to background levels after 20 min. To test for EphA2 receptor phosphorylation after ephrin-A2–Fc stimulation, immunoprecipitation of EphA2 and subsequent probing with an anti–phosphotyrosine antibody (PY) was performed (A, D, G, lower panel). The regulation of ephrin-A2 and c-fos after ephrin-A2–Fc stimulation was confirmed on the protein level (B, E, H, lower panel). Tubulin served as a loading control. (J) To test whether ephrin-A2–Fc stimulation leading to ephrin-A2 transcriptional activation involves the activation of SRE, a reporter construct (pSRE-Luc) was transfected into murine 3T3 fibroblasts. 3T3 cells were stimulated with preclustered ephrin-A2–Fc for 10, 20, 30, and 60 min. Enhanced SRE-dependent luciferase activity was measured 10 min after stimulation, reached significance after 20 min, and remained significantly upregulated until the end of the observation period at 60 min. Stimulation experiments were performed in triplicates (n = 3). Cells treated with the anti–Fc antibody (0.1 µg/mL), time matched, served as controls. Luciferase reporter gene assays were performed twice in triplicates. The qRT-PCR assays were performed in triplicates. Data are presented as mean ± SD. *P < 0.05 vs. control (one-way analysis of variance, Dunnett’s post hoc test).

Figure 4.

Putative signaling pathways involved in the regulation of ephrin-A2 in periodontal ligament fibroblasts (PDLFs). Please see Discussion section for details. Solid lines: Involvement was shown in this study. Dashed lines: Involvement was shown or suggested elsewhere.