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. 2018 Sep;42(3):1484-1494.
doi: 10.3892/ijmm.2018.3714. Epub 2018 Jun 4.

IL‑1β and TNF‑α Suppress TGF‑β‑promoted NGF Expression in Periodontal Ligament‑derived Fibroblasts Through Inactivation of TGF‑β‑induced Smad2/3‑ and p38 MAPK‑mediated Signals

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Free PMC article

IL‑1β and TNF‑α Suppress TGF‑β‑promoted NGF Expression in Periodontal Ligament‑derived Fibroblasts Through Inactivation of TGF‑β‑induced Smad2/3‑ and p38 MAPK‑mediated Signals

Maiko Ohta et al. Int J Mol Med. .
Free PMC article

Abstract

Mechanosensitive (MS) neurons in the periodontal ligament (PDL) pass information to the trigeminal ganglion when excited by mechanical stimulation of the tooth. During occlusal tooth trauma of PDL tissues, MS neurons are injured, resulting in atrophic neurites and eventual degeneration of MS neurons. Nerve growth factor (NGF), a neurotrophic factor, serves important roles in the regeneration of injured sensory neurons. In the present study, the effect of pro‑inflammatory cytokines, including interleukin 1β (IL‑1β) and tumor necrosis factor α (TNF‑α), on transforming growth factor β1 (TGF‑β1)‑induced NGF expression was evaluated in rat PDL‑derived SCDC2 cells. It was observed that TGF‑β1 promoted NGF expression via Smad2/3 and p38 mitogen‑activated protein kinase (MAPK) activation. IL‑1β and TNF‑α suppressed the TGF‑β1‑induced activation of Smad2/3 and p38 MAPK, resulting in the abrogation of NGF expression. NGF secreted by TGF‑β1‑treated SCDC2 cells promoted neurite extension and the expression of tyrosine hydroxylase, a rate‑limiting enzyme in dopamine synthesis in rat pheochromocytoma PC12 cells. These results suggested that pro‑inflammatory cytokines suppressed the TGF‑β‑mediated expression of NGF in PDL‑derived fibroblasts through the inactivation of TGF‑β‑induced Smad2/3 and p38 MAPK signaling, possibly resulting in the disturbance of the regeneration of injured PDL neurons.

Figures

Figure 1
Figure 1
TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells through its type I receptor in a dose-dependent manner. After 24-h culture in growth medium, SCDC2 cells were starved for 24 h. The starved cells were then treated with (A) TGF-β1 at various concentrations for 24 h, or (B) pretreated with or without TGF-β type I receptor inhibitor SB-431542 (10 µM) for 30 min and then with or without TGF-β1 (10 ng/ml) for 24 h. (C) Starved cells were treated with or without TGF-β1 (10 ng/ml) for the indicated times. The relative expression level of NGF was evaluated using reverse transcription-quantitative polymerase chain reaction. Data represent the mean ± standard deviation (n=6). *P<0.05. TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture.
Figure 2
Figure 2
TGF-β1 promoted the mRNA expression of NGF in SCDC2 cells in Smad2/3-dependent and p38 MAPK-dependent manners. Effects of (A) SIS3 (10 µM), and (B) SB203580 (10 µM) on expression of NGF mRNA were evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). *P<0.05. (C) Phosphorylation status of Smad2/3 and p38 MAPK in cells stimulated with TGF-β1 (10 ng/ml) for the indicated times, evaluated using western blot analysis. (D) After 24-h starvation, cells were pretreated with Smad3 inhibitor SIS3 (10 µM) for 30 min and then treated with or without TGF-β1 (10 ng/ml) for 30 min, and the status of nuclear translocation of Smad2/3 following TGF-β1 stimulation was examined using immunofluorescence analysis (×200 magnification; scale bar, 50 µm). (E) Phosphorylation status of MAPKAPK-2 evaluated using western blot analysis in cells stimulated with TGF-β1 (10 ng/ml) and/or with the inhibitor SB203580. (F) Effect of SP600125 (10 µM) on expression of NGF mRNA was evaluated as described in Materials and methods. Data represent the mean ± standard deviation (n=6). *P<0.05. TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture; RT-qPCR, reverse transcription-quantitative polymerase chain reaction; MAPK, mitogen-activated protein kinase; MAPKAPK-2, MAPK-activated protein kinase 2.
Figure 3
Figure 3
IL-1β and TNF-α suppressed the TGF-β1-induced mRNA expression of NGF in SCDC2 cells by abrogating Smad2/3 and p38 MAPK activities. The effects of IL-1β and TNF-α on TGF-β1-induced mRNA expression of NGF in SCDC2 cells were evaluated using RT-qPCR. The cells were treated with or without (A) IL-1β alone or (B) TNF-α alone at indicated concentrations, (C) TGF-β1 (10 ng/ml) and/or IL-1β (10 ng/ml), and (D) TGF-β1 (10 ng/ml) and/or TNF-α (10 ng/ml). Data represent the mean ± standard deviation (n=6). *P<0.05. Phosphorylation status of (E) Smad2/3 and (F) p38 MAPK was evaluated using western blot analysis in cells treated with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for the indicated times. (G) NGF protein concentration secreted into the culture medium was determined using ELISA in cells cultured with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for 5 days. (H) Nuclear translocation status of NF-κB p65 (red) was evaluated using immunofluorescence analysis (blue, nuclei; green, filamentous actin) in SCDC2 cells treated with or without IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 24 h (×200 magnification; scale bar, 50 µm). IL, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture; MAPK, mitogen-activated protein kinase.
Figure 3
Figure 3
IL-1β and TNF-α suppressed the TGF-β1-induced mRNA expression of NGF in SCDC2 cells by abrogating Smad2/3 and p38 MAPK activities. The effects of IL-1β and TNF-α on TGF-β1-induced mRNA expression of NGF in SCDC2 cells were evaluated using RT-qPCR. The cells were treated with or without (A) IL-1β alone or (B) TNF-α alone at indicated concentrations, (C) TGF-β1 (10 ng/ml) and/or IL-1β (10 ng/ml), and (D) TGF-β1 (10 ng/ml) and/or TNF-α (10 ng/ml). Data represent the mean ± standard deviation (n=6). *P<0.05. Phosphorylation status of (E) Smad2/3 and (F) p38 MAPK was evaluated using western blot analysis in cells treated with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for the indicated times. (G) NGF protein concentration secreted into the culture medium was determined using ELISA in cells cultured with or without TGF-β1 (10 ng/ml) alone, TGF-β1 (10 ng/ml) + IL-1β (10 ng/ml), or TGF-β1 (10 ng/ml) + TNF-α (10 ng/ml) for 5 days. (H) Nuclear translocation status of NF-κB p65 (red) was evaluated using immunofluorescence analysis (blue, nuclei; green, filamentous actin) in SCDC2 cells treated with or without IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 24 h (×200 magnification; scale bar, 50 µm). IL, interleukin; TNF, tumor necrosis factor; TGF, transforming growth factor; NGF, nerve growth factor; SCDC, single cell-derived culture; MAPK, mitogen-activated protein kinase.
Figure 4
Figure 4
Nerve growth factor secreted by SCDC2 cells following TGF-β1 stimulation promoted neurite extension from the surface of ATPγS-treated PC12 cells. (A) Neurite extension of PC12 cells was visualized by immunostaining (×200 magnification; scale bar, 50 µm) with anti-neurofilament H antibody (red) and nuclei were stained with DAPI (blue). SCDC2 cells (2×104 cells) and rat pheochromocytoma cells PC12 (1×104 cells) were co-cultured and treated with or without TGF-β1 (10 ng/ml) for 4 days. Cells were also treated with TGF-β type I receptor inhibitor SB-431542 (10 µM), TrkA inhibitor GW441756 (2 nM), IL-1β (10 ng/ml), or TNF-α (10 ng/ml) from the beginning of the co-culture. In addition, ATPγS (100 µM) was added to all cultures during cell seeding. Dimethyl sulfoxide was added to cell cultures as a vehicle control for SB-431542 and GW441756, respectively. (B) Statistical assessment of neurite extension in PC12 cells co-cultured with SCDC2 cells. Data represent the mean ± standard deviation (n=8). *P<0.05. TGF, transforming growth factor; SCDC, single cell-derived culture; IL, interleukin; TNF, tumor necrosis factor; ATPγS, adenosine 5′-O-(3-thio)triphosphate; TrkA, tropomyosin receptor kinase A.
Figure 5
Figure 5
Nerve growth factor secreted by SCDC2 cells subsequent to TGF-β1 stimulation promoted the expression of TH mRNA in PC12 cells. SCDC2 cells (7×104 cells) and rat pheochromocytoma cells PC12 cells (3.5×104 cells) were co-cultured and stimulated with or without TGF-β1 (10 ng/ml) for 24 h. The relative expression level of TH was evaluated using reverse transcription-quantitative polymerase chain reaction in cells also treated with (A) ATPγS (100 µM), and with (B) TGF-β type I receptor inhibitor SB-431542 (10 µM), TrkA inhibitor GW441756 (2 nM), IL-1β (10 ng/ml) or TNF-α (10 ng/ml) during the co-culture. Dimethyl sulfoxide was added to cell cultures as a vehicle control for SB-431542 and GW441756, respectively. Data represent the mean ± standard deviation (n=6). *P<0.05. TGF, transforming growth factor; SCDC, single cell-derived culture; IL, interleukin; TNF, tumor necrosis factor; ATPγS, adenosine 5′-O-(3-thio)triphosphate; TrkA, tropomyosin receptor kinase A; TH, tyrosine hydroxylase.

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