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FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

Yu H, Herbert BA, Valerio M, Yarborough L, Hsu LC, Argraves KM - Lipids Health Dis (2015)

Bottom Line: This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining.Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. yuho@musc.edu.

ABSTRACT

Background: Periodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.

Methods: Murine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR).

Results: FTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts.

Conclusion: FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

No MeSH data available.


Related in: MedlinePlus

FTY720 significantly decreased Nfatc1, Ctsk, Acp5, and Oscar expressions in bone marrow-derived pre-osteoclasts with or without A. actinomycetemcomitans (Aa) stimulation. Bone marrow-derived pre-osteoclasts were treated as described in Methods. Cells were treated with vehicle (ethanol) or FTY720 (2 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (0.5 CFU/cell) in the presence of vehicle or FTY720 for 4 h (a-d) or for 24 h (e-h). a, e Nfatc1 mRNA, (b, f) Ctsk mRNA, (c, g) Acp5 mRNA, and (d, h) Oscar mRNA levels were normalized by an endogenous control GAPDH expression and expressed as fold change compared with control group. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)
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Fig4: FTY720 significantly decreased Nfatc1, Ctsk, Acp5, and Oscar expressions in bone marrow-derived pre-osteoclasts with or without A. actinomycetemcomitans (Aa) stimulation. Bone marrow-derived pre-osteoclasts were treated as described in Methods. Cells were treated with vehicle (ethanol) or FTY720 (2 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (0.5 CFU/cell) in the presence of vehicle or FTY720 for 4 h (a-d) or for 24 h (e-h). a, e Nfatc1 mRNA, (b, f) Ctsk mRNA, (c, g) Acp5 mRNA, and (d, h) Oscar mRNA levels were normalized by an endogenous control GAPDH expression and expressed as fold change compared with control group. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)

Mentions: To elucidate the mechanisms associated with the role of FTY720 in osteoclastogenesis, we analyzed the mRNA expressions of various kinds of osteoclastogenic factors, including Nfatc1, Ctsk, Acp5, Oscar, and RANKL in bone marrow-derived pre-osteoclasts treated for 4 or for 24 h with FTY720 (2 μM) or vehicle (ethanol), with or without A. actinomycetemcomitans stimulation. As shown in Fig. 4a-d, cells treated with both M-CSF and RANKL significantly increased the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar compared with those levels in cells treated with M-CSF alone. In cells treated with FTY720 for 4 h without bacterial stimulation (Fig. 4a), there was a 22.0 % significant reduction of Nfatc1 mRNA expression as compared with vehicle control. However, there were no significant differences of the mRNA levels of Ctsk, Acp5, and Oscar between FTY720-treated cells and vehicle-treated cells (Fig. 4b-d). In cells treated with FTY720 for 24 h without bacterial stimulation (Fig. 4. e-h), FTY720 significantly decreased the mRNA levels of Ctsk by 36.1 %, Acp5 by 51.5 %, and Oscar by 47.3 % compared with those levels in vehicle-treated cells. However, there was no significant difference in Nfatc1 mRNA expression between these two groups.Fig. 4


FTY720 inhibited proinflammatory cytokine release and osteoclastogenesis induced by Aggregatibacter actinomycetemcomitans.

Yu H, Herbert BA, Valerio M, Yarborough L, Hsu LC, Argraves KM - Lipids Health Dis (2015)

FTY720 significantly decreased Nfatc1, Ctsk, Acp5, and Oscar expressions in bone marrow-derived pre-osteoclasts with or without A. actinomycetemcomitans (Aa) stimulation. Bone marrow-derived pre-osteoclasts were treated as described in Methods. Cells were treated with vehicle (ethanol) or FTY720 (2 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (0.5 CFU/cell) in the presence of vehicle or FTY720 for 4 h (a-d) or for 24 h (e-h). a, e Nfatc1 mRNA, (b, f) Ctsk mRNA, (c, g) Acp5 mRNA, and (d, h) Oscar mRNA levels were normalized by an endogenous control GAPDH expression and expressed as fold change compared with control group. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)
© Copyright Policy - open-access
Related In: Results  -  Collection

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Fig4: FTY720 significantly decreased Nfatc1, Ctsk, Acp5, and Oscar expressions in bone marrow-derived pre-osteoclasts with or without A. actinomycetemcomitans (Aa) stimulation. Bone marrow-derived pre-osteoclasts were treated as described in Methods. Cells were treated with vehicle (ethanol) or FTY720 (2 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (0.5 CFU/cell) in the presence of vehicle or FTY720 for 4 h (a-d) or for 24 h (e-h). a, e Nfatc1 mRNA, (b, f) Ctsk mRNA, (c, g) Acp5 mRNA, and (d, h) Oscar mRNA levels were normalized by an endogenous control GAPDH expression and expressed as fold change compared with control group. Data are expressed as mean ± SEM (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001)
Mentions: To elucidate the mechanisms associated with the role of FTY720 in osteoclastogenesis, we analyzed the mRNA expressions of various kinds of osteoclastogenic factors, including Nfatc1, Ctsk, Acp5, Oscar, and RANKL in bone marrow-derived pre-osteoclasts treated for 4 or for 24 h with FTY720 (2 μM) or vehicle (ethanol), with or without A. actinomycetemcomitans stimulation. As shown in Fig. 4a-d, cells treated with both M-CSF and RANKL significantly increased the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar compared with those levels in cells treated with M-CSF alone. In cells treated with FTY720 for 4 h without bacterial stimulation (Fig. 4a), there was a 22.0 % significant reduction of Nfatc1 mRNA expression as compared with vehicle control. However, there were no significant differences of the mRNA levels of Ctsk, Acp5, and Oscar between FTY720-treated cells and vehicle-treated cells (Fig. 4b-d). In cells treated with FTY720 for 24 h without bacterial stimulation (Fig. 4. e-h), FTY720 significantly decreased the mRNA levels of Ctsk by 36.1 %, Acp5 by 51.5 %, and Oscar by 47.3 % compared with those levels in vehicle-treated cells. However, there was no significant difference in Nfatc1 mRNA expression between these two groups.Fig. 4

Bottom Line: This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining.Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Health Sciences and the Center for Oral Health Research, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. yuho@musc.edu.

ABSTRACT

Background: Periodontitis is a bacteria-driven inflammatory bone loss disease. Previous studies showed that the oral pathogen Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) stimulated the generation of sphingosine 1 phosphate (S1P). In addition, S1P signaling regulated the migration of osteoclast precursors and affected osteoclastogenesis. Furthermore, treatment with FTY720 (also called fingolimod, a modulator of multiple S1P receptors) alleviated osteoporosis and suppressed arthritis in animals. This study determined the effect of FTY720 on proinflammatory cytokine production and osteoclastogenesis in murine bone marrow cells with or without A. actinomycetemcomitans stimulation.

Methods: Murine bone marrow-derived monocytes and macrophages (BMMs) were treated with vehicle ethanol or FTY720, and were either unstimulated or stimulated for 0.5 to 6 h with A. actinomycetemcomitans. The protein levels of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α in the media of BMMs were quantified by enzyme-linked immunosorbent assay (ELISA). Protein expressions, including phosphorylated phosphoinositide 3-kinase (p-PI3K), p-Akt, p-extracellular signal-regulated kinase (p-ERK), PI3K, Akt, and ERK were evaluated by Western blot. In addition, murine bone marrow-derived pre-osteoclasts were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa-B ligand (RANKL) for three days. Then the cells were treated with either vehicle or FTY720 and were either unstimulated or stimulated with A. actinomycetemcomitans for 4 to 24 h. Control cells were treated with M-CSF alone with or without bacterial stimulation. Osteoclasts were stained by tartrate-resistant acid phosphatase (TRAP) staining. The mRNA levels of osteoclastogenic factors, including nuclear factor of activated T-cells cytoplasmic calcineurin-dependent 1 (Nfatc1), cathepsin K (Ctsk), acid phosphatase 5 (Acp5), osteoclast-associated receptor (Oscar), and RANKL were quantified by quantitative real-time polymerase chain reaction (PCR).

Results: FTY720 dose-dependently inhibited IL-1β, IL-6, and TNF-α protein levels induced by A. actinomycetemcomitans in BMMs compared with controls. Additionally, FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans. Furthermore, FTY720 suppressed osteoclastogenesis in bone marrow-derived pre-osteoclasts with or without bacterial stimulation and reduced the mRNA levels of Nfatc1, Ctsk, Acp5, and Oscar, but not RANKL in bone marrow-derived pre-osteoclasts.

Conclusion: FTY720 inhibited proinflammatory cytokine production and suppressed osteoclastogenesis, supporting FTY720 as a potential therapy for inflammatory bone loss diseases.

No MeSH data available.


Related in: MedlinePlus