Adiponectin Interacts In-Vitro With Cementoblasts Influencing Cell Migration, Proliferation and Cementogenesis Partly Through the MAPK Signaling Pathway

Abstract

Current clinical evidences suggest that circulating Adipokines such as Adiponectin can influence the ratio of orthodontic tooth movement. We aimed to investigate the effect that Adiponectin has on cementoblasts (OCCM-30) and on the intracellular signaling molecules of Mitogen-activated protein kinase (MAPK). We demonstrated that OCCM-30 cells express AdipoR1 and AdipoR2. Alizarin Red S staining revealed that Adiponectin increases mineralized nodule formation and quantitative AP activity in a dose-dependent manner. Adiponectin up-regulates the mRNA levels of AP, BSP, OCN, OPG, Runx-2 as well as F-Spondin. Adiponectin also increases the migration and proliferation of OCCM-30 cells. Moreover, Adiponectin induces a transient activation of JNK, P38, ERK1/2 and promotes the phosphorylation of STAT1 and STAT3. The activation of Adiponectin-mediated migration and proliferation was attenuated after pharmacological inhibition of P38, ERK1/2 and JNK in different degrees, whereas mineralization was facilitated by MAPK inhibition in varying degrees. Based on our results, Adiponectin favorably affect OCCM-30 cell migration, proliferation as well as cementogenesis. One of the underlying mechanisms is the activation of MAPK signaling pathway.

Keywords: MAPK; adiponectin; cementoblasts; cementogenesis; migration; proliferation.

FIGURE 1

Cementoblasts express Adiponectin receptors. (A) The expression of Adiponectin receptors (AdipoR1 and AdipoR2) in OCCM-30 mouse cementoblasts was examined by WB. β-actin is shown as internal control protein. (B) RT-PCR analysis show the mRNA expression of AdipoR1 and AdipoR2. Values are expressed as means ± SD. (C) Adiponectin receptors in OCCM-30 cells were visualized by immunofluorescence staining (Green color). DAPI staining was used for nuclei detection. Arrows show cellular receptor localization. AdipoR1 are located in the cytoplasm, cytomembrane and nucleus, while AdipoR2 are distributed mostly around the nucleus.

FIGURE 2

Adiponectin promotes cementogenesis in vitro. (A) Microscopic view of OCCM-30 cells after 14 days of stimulation with different concentrations of Adiponectin (Staining: Alizarin Red S). (B,C) Cells were cultivated with 0 (Control), 20, 40, and 80 ng/ml Adiponectin during 14 days. Mineralization grade was visualized and quantified using Alizarin Red S staining and by further dilution with Cetylpiridiumchlorid. Results are expressed as mg/ml Alizarin Red S (Mean ± SD of three independent results). (D) Adiponectin added to OCCM-30 cells increases the Alkaline Phosphatase (AP) enzymatic activity dose-dependently, reaching statistical significance after 24 h in comparison to the untreated group (Data are normalized to 1, *p < 0.05; **p < 0.01). (E) Kinetic analysis of relative mRNA expression of Alkaline Phosphatase (AP), Bone Sialoprotein (BSP), Osteoprotegerin (OPG), Osteocalcin (OCN), Runx-2 and F-Spondin on cementoblasts after Adiponectin (100 ng/ml) stimulation. Data are normalized to 1. GAPDH was used as housekeeping Gene. Values are expressed as means ± SD: Ns (not significant); *p < 0.05; **p < 0.01; ***p < 0.001 and ****p < 0.0001.

FIGURE 3

Increased migration and proliferation rates of OCCM-30 cells treated with Adiponectin. (A,B) Images show the migration effect that different concentrations of Adiponectin exert on OCCM-30 cells wounded monolayers at 0 and 24 h after standard scratching using a 100 µL pipet tip. The red lines indicate the wound edge at the beginning and at the end of the experiment. The migration rates were measured over a period of 24 h by ImageJ software. Data are presented as percentage of wound recovery. (C) The MTS assay showed that Adiponectin-treated cells during 24 h have an increased proliferation rate in comparison to untreated cells. This effect occurred dose-dependently, reaching statistical significance at a concentration of 0.4, 0.8, and 1.6 μg/ml Adiponectin. Values are expressed as means ± SD: ns (not significant); *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 4

Adiponectin promotes JNK, ERK1/2 and P38 phosphorylation on OCCM-30 cells. (A,B) The expression of JNK (46 and 54 kDa), ERK1/2 (42 and 44 kDa) and P38 (42 kDa) expression as well as their phosphorylated forms after Adiponectin (20 ng/ml) stimulation were analyzed by Western Blots. β-actin served as a loading control. Graphics show the relative expression of p-JNK, p-ERK1/2 and p-P38 compared to cells at time point 0 min. (C,D) Adiponectin (20 ng/ml) promotes STAT1 (98 KDa) and STAT3 (85 KDa) phosphorylation during a period of 30 min. Graphics show the relative expression of p-STAT1 and p-STAT3 compared to cells at time point 0 min. Values are expressed as means ± SD: Ns (not significant), *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 5

The MAP kinase pathway is involved in Adiponectin-induced migration and proliferation of OCCM-30 cementoblasts. (A,B) Cells were treated with the MAP kinase inhibitors: SB203580 (InvivoGen), FR180204 (Calbiochem) and SP600125 (InvivoGen) for 1 h and afterwards stimulated with Adiponectin during 24 h. As result, MAP kinase inhibition impairs cell proliferation (MTS assay). This effect was rescued after Adiponectin (100 ng/ml) stimulation. (C) Images of cell migration were captured prior to stimulation (0 h) and after 24 h. The recovered areas were calculated by ImageJ software comparing the scratched areas. Blockade of ERK1/2 (FR180204) and JNK (SP600125) over a period of 24 h significantly decreases cell migration whereas P38 inhibition did not significantly influence the migration rate. Values are expressed as means ± SD of three independent results: Ns (not significant), *p < 0.05; **p < 0.01; ***p < 0.001.

FIGURE 6

Inhibition of MAP kinases alter Adiponectin-induced cementogenesis. (A) Cells were treated with MAP kinase inhibitors in the presence or absence of Adiponectin (100 ng/ml) for 7 and 14 days, respectively. (B) Graphic shows the absorbance (OD₅₆₂) of Alizarin Red S dilution after induction of mineralization for 7 and 14 days. Mineralization grade does not differ significantly among the groups after 7 days induction. However, after 14 days induction, the sustained blockade of P38 (SB203580) and ERK1/2 (FR180204) facilitate mineralized nodule formation, effect that was partially counteracted by Adiponectin. This effect was not observed in the groups cultivated with the JNK inhibitor (SP600125) in the presence or absence of Adiponectin. Values are expressed as means ± SD: Ns (not significant), *p < 0.05; **p < 0.01; ***p < 0.001.