EGF Positively Regulates the Proliferation and Migration, and Negatively Regulates the Myofibroblast Differentiation of Periodontal Ligament-Derived Endothelial Progenitor Cells through MEK/ERK- and JNK-Dependent Signals
Cellular Physiology and Biochemistry
This is an Open Access article licensed under the terms of the Creative Commons Attribution-NonCommercial 3.0 Unported license (CC BY-NC) (www.karger.com/OA-license), applicable to the online version of the article only. Distribution permitted for non-commercial purposes only. Abstract Background/Aims: Remodeling of fibrous and vascular tissues in the periodontal ligament (PDL) around the tooth root was observed during tooth movement by orthodontic force application. We previously demonstrated
... ously demonstrated that a single cell-derived culture (SCDC) of primarily cultured PDL fibroblasts, called SCDC2, has an endothelial progenitor cell (EPC)-like character and can form endothelial cell (EC) marker-positive blood vessel-like structures. However, the types of molecular mechanisms that control the in vivo kinetic properties and the differentiation of the PDL-derived EPC-like cells into myofibroblasts (MFs), which are known to expand fibrous tissues, require clarification. Methods: Using specific mitogen activated protein kinase (MAPK) inhibitors, we examined how epidermal growth factor (EGF)-mediated MAPK signals affected the proliferation, migration, and MF differentiation of these cells. Results: EGF induced SCDC2 cell proliferation in MAPK/extracellular signal-regulated kinase (ERK) kinase (MEK)-and c-Jun N-terminal kinase (JNK)-dependent manners. In addition, EGF suppressed the expression of MF differentiation markers in these cells in a MEK/ERK-dependent manner, and, moreover, stimulated the cell migration in a MEK/ERK-dependent manner. Conclusion: EGF regulates fibrous tissue remodeling in PDLs through MEK/ERK-and JNK-mediated signals by affecting the proliferation, migration, and MF differentiation of the PDL-derived EPC-like cells. Cellular Physiology and Biochemistry Cellular Physiology and Biochemistry into the low chamber, which contained Ham's F-12 media; this process was allowed to occur for 14 h at 37°C in the presence or absence of EGF, the MEK inhibitor U0126, or the JNK inhibitor SP600125 at the indicated concentrations. EGF, U0126, or SP600125 were added to the culture media in both the upper and lower chambers. The membrane was fixed in 4% paraformaldehyde in PBS and stained using the Diff-Quick stain kit (Sysmex Co., Kobe, Japan) for 2 h. Cells that had migrated onto the underside of the membrane were counted in 9 fields on the membrane, and the value was shown as the average of the 9 fields. The migration of the control was set to 100%, and all other datasets were measured against this value. Statistical analysis Data were presented as mean ± SD. Differences between treatments were evaluated using Student's t-test for single comparisons. The results shown in all experiments were representative of at least 3 separate experiments. A p value of <0.05 was considered statistically significant. EGF possibly affects fibrous tissue remodeling in mechanical tensile force-loaded PDL by controlling the MF differentiation of the PDL-derived EPC-like fibroblastic cells Using the tensile force application method against cultured cells on plastic tissue culture plates as described in Materials and Methods, we examined how tensile force affected the Fig. 3. EGF-induced MEK/ERK-mediated signals negatively regulate MF differentiation of the PDL-derived EPC-like fibroblastic cells. (A) Cells were treated with EGF (10 ng/mL) with or without MEK inhibitor U0126 (1-5 µM) and cultured for 3 days. Then, the total RNA was isolated, and the expression patterns of MF markers (a, α-SMA; b, h1-calponin; c, colIα1) were evaluated by using qRT-PCR as described in Materials and Methods. The mRNA expression levels of these genes were normalized to those of GAPDH, and the relative expression levels were shown as fold increases or decreases relative to the level in the control. Data are represented as the means ± SD for 4 dishes for each time point (*P < 0.01; **P < 0.05). (B) SCDC2 cells were treated with EGF (10 ng/mL) with or without U0126 (5 µM) and cultured for 3 days. Next, the cells were immunostained with (a) anti-α-SMA (red) or (b) anti-h1 calponin (red), and then the cells were labeled with phalloidin (green) and DAPI (blue) as described in Materials and Methods. Scale bar, 50 µm. (C) Cells were treated with EGF (10 ng/mL) with or without U0126 (5 µM) and JNK inhibitor SP600125 (7.5-10 µM) and cultured for 3 days. Then, the expression patterns of MF markers were evaluated as described in (A).