Effects of Melatonin and Its Underlying Mechanism on Ethanol-Stimulated Senescence and Osteoclastic Differentiation in Human Periodontal Ligament Cells and Cementoblasts

Abstract

The present study evaluated the protective effects of melatonin in ethanol (EtOH)-induced senescence and osteoclastic differentiation in human periodontal ligament cells (HPDLCs) and cementoblasts and the underlying mechanism. EtOH increased senescence activity, levels of reactive oxygen species (ROS) and the expression of cell cycle regulators (p53, p21 and p16) and senescence-associated secretory phenotype (SASP) genes (interleukin [IL]-1β, IL-6, IL-8 and tumor necrosis factor-α) in HPDLCs and cementoblasts. Melatonin inhibited EtOH-induced senescence and the production of ROS as well as the increased expression of cell cycle regulators and SASP genes. However, it recovered EtOH-suppressed osteoblastic/cementoblastic differentiation, as evidenced by alkaline phosphatase activity, alizarin staining and mRNA expression levels of Runt-related transcription factor 2 (Runx2) and osteoblastic and cementoblastic markers (glucose transporter 1 and cementum-derived protein-32) in HPDLCs and cementoblasts. Moreover, it inhibited EtOH-induced osteoclastic differentiation in mouse bone marrow⁻derived macrophages (BMMs). Inhibition of protein never in mitosis gene A interacting-1 (PIN1) by juglone or small interfering RNA reversed the effects of melatonin on EtOH-mediated senescence as well as osteoblastic and osteoclastic differentiation. Melatonin blocked EtOH-induced activation of mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), mitogen-activated protein kinase (MAPK) and Nuclear factor of activated T-cells (NFAT) c-1 pathways, which was reversed by inhibition of PIN1. This is the first study to show the protective effects of melatonin on senescence-like phenotypes and osteoclastic differentiation induced by oxidative stress in HPDLCs and cementoblasts through the PIN1 pathway.

Keywords: PIN1; cementoblasts; human periodontal ligament cells; melatonin; osteoclast differentiation; senescence.

Figure 1

Effect of ethyl alcohol (EtOH) on cell viability (A) and cell death in human periodontal ligament cells (HPDLCs) and cementoblast. Cells are incubated with indicated concentration of EtOH for indicated times (A) and 3 days (B); Cell viability and death were examined by MTT assay and flow cytometry, respectively. These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05).

Figure 2

Effect of ethyl alcohol (EtOH) on characterization of cellular senescence by senescence-associated β-galactosidase (β-gal) staining (A), β-gal activity (B), cell cycle analysis (C,D) and expression of senescence-associated proteins (E) in periodontal ligament cells (PDLCs) and cementoblasts. Cells are incubated with indicated concentration of EtOH for 3 days (A–E); (A,B) SA-β-Gal activity was evaluated using a staining kit. Cell cycle and protein analysis were assessed by flow cytometry (C,D) and Western blot (E), respectively. Flow-cytometric frequency histograms of progenitors stained with propidium iodide (PI) for DNA content. These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). Arrows in Figure 2A represent β-gal (+) cells.

Figure 3

Effect of ethyl alcohol (EtOH) on characterization of cellular senescence by reactive oxygen species (ROS) production (A,B) and mRNA expression of senescence-associated secretory phenotype (SASP) factors (C) in PDLCs and cementoblasts. Cells are incubated with indicated concentration of EtOH for 3 days (A–C). ROS production and mRNA analysis were assessed by flow cytometry (A,B) and RT-PCR (C), respectively. These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05).

Figure 4

Effect of melatonin on EtOH-induced cellular senescence in PDLCs and cementoblasts. Cells are incubated with indicated concentration of melatonin (μM) and EtOH (25 mM) for 3 days (A–C). Senescence was examined by β-gal activity (A), ROS production (B,C) and expression of senescence-associated proteins (D) and mRNAs (E). These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 5

Involvement of PIN1 pathway on effects of melatonin in EtOH-induced cellular senescence of PDLCs and cementoblasts. Cells are pretreated with juglone or PIN1 siRNA and then incubated with melatonin (100 μM) and EtOH (25 mM) for 3 days (A–F). mRNA and protein expression were accessed by Western blot and RT-PCR (A,B,E,F), respectively. Senescence was examined by β-gal activity (C), ROS production (D,E) and expression of senescence-associated proteins (E) and mRNAs (F). These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 5

Involvement of PIN1 pathway on effects of melatonin in EtOH-induced cellular senescence of PDLCs and cementoblasts. Cells are pretreated with juglone or PIN1 siRNA and then incubated with melatonin (100 μM) and EtOH (25 mM) for 3 days (A–F). mRNA and protein expression were accessed by Western blot and RT-PCR (A,B,E,F), respectively. Senescence was examined by β-gal activity (C), ROS production (D,E) and expression of senescence-associated proteins (E) and mRNAs (F). These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 6

Involvement of PIN1 pathway on effects of melatonin in EtOH-suppressed osteoblastic/cementoblastic differentiation in PDLCs and cementoblasts. Cells are pretreated with juglone (50 nM) or PIN1 siRNA (30 nM) and then incubated with melatonin (100 μM) and EtOH (25 mM) for 14 days (A–C). Differentiation was accessed by ALP activity (A), RT-PCR (B) and Alizarin red staining (C). These data are representative of three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 7

Indirect effects of melatonin on EtOH-induced osteoclastic differentiation in PDLCs and cementoblasts. Cells are pretreated with juglone (50 nM) or PIN1 siRNA (30 nM) and then incubated with melatonin (100 μM) and EtOH (25 mM) for 3 days (A) in PDLCs and cementoblasts and conditioned medium (CM) were prepared. The bone-marrow derived macrophage (BMM) cells were incubated with M-CSF (10 ng/mL) and RANKL (50 ng/mL) or 20% CM collected from PDLCs and cementoblasts. After 48 h of culture, the cells were fixed and osteoclast-like cells were identified by TRAP staining; (B,C) Representative pictures of TRAP staining (B) and actin ring (C); The numbers of osteoclasts per well were counted (D); mRNA expression of osteoclast-specific marker genes was assessed by RT-PCR (A,E). Representative immunofluorescence of NFATc1 and F-actin expression (F) for CM from PDLCs. Similar data were obtained from three independent experiments. Red color is for F-actin. Green color is for NFATc1. Merged color is for F-actin & NFATc1. NFATc1 expression was examined by RT-PCR (E) and immunofluorescence (F). * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 7

Indirect effects of melatonin on EtOH-induced osteoclastic differentiation in PDLCs and cementoblasts. Cells are pretreated with juglone (50 nM) or PIN1 siRNA (30 nM) and then incubated with melatonin (100 μM) and EtOH (25 mM) for 3 days (A) in PDLCs and cementoblasts and conditioned medium (CM) were prepared. The bone-marrow derived macrophage (BMM) cells were incubated with M-CSF (10 ng/mL) and RANKL (50 ng/mL) or 20% CM collected from PDLCs and cementoblasts. After 48 h of culture, the cells were fixed and osteoclast-like cells were identified by TRAP staining; (B,C) Representative pictures of TRAP staining (B) and actin ring (C); The numbers of osteoclasts per well were counted (D); mRNA expression of osteoclast-specific marker genes was assessed by RT-PCR (A,E). Representative immunofluorescence of NFATc1 and F-actin expression (F) for CM from PDLCs. Similar data were obtained from three independent experiments. Red color is for F-actin. Green color is for NFATc1. Merged color is for F-actin & NFATc1. NFATc1 expression was examined by RT-PCR (E) and immunofluorescence (F). * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 8

Direct effects of melatonin on EtOH-induced osteoclastic differentiation in BMMs. BMMs were stimulated with RANKL in the presence of juglone (50 nM) or PIN1 siRNA (30 nM), EtOH (25 mM) and melatonin (100 μM) for 5 days. In vitro osteoclatogenesis was accessed by TRAP staining (A) and actin ring staining (B), counting of osteoclast (C), mRNA expression of osteoclast-specific marker genes (D). Similar data were obtained from three independent experiments. * statistically significant difference compared to the control groups (p < 0.05). ^# statistically significant difference in each group.

Figure 9

Involvement of AMPK, mTOR and MAPK pathway on effects of melatonin in EtOH-induced senescence or differentiation in PDLCs and cementoblasts. Cells are pretreated with juglone (50 nM) or PIN1siRNA (30 nM) and then incubated with melatonin (100 μM) and EtOH (25 mM) for 60 min (A,B) and 45 min (C). Signal pathways was accessed by Western blot analysis. These data are representative of three independent experiments.