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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 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (8 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (1.5 CFU/cell) for 30 to 120 min. a P-PI3K, PI3K, p-Akt, Akt, p-ERK, and ERK expressions were evaluated by Western blot. b P-PI3K protein density, (c) P-Akt protein density, and (d) P-ERK protein density were analyzed by Quantity One Software and normalized by total protein expression, respectively. Data are expressed as mean ± SEM (n = 3, *p < 0.05, *** p < 0.001)
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Fig2: FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (8 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (1.5 CFU/cell) for 30 to 120 min. a P-PI3K, PI3K, p-Akt, Akt, p-ERK, and ERK expressions were evaluated by Western blot. b P-PI3K protein density, (c) P-Akt protein density, and (d) P-ERK protein density were analyzed by Quantity One Software and normalized by total protein expression, respectively. Data are expressed as mean ± SEM (n = 3, *p < 0.05, *** p < 0.001)

Mentions: To further elucidate which signaling pathways were affected by FTY720 in regulating the immune response induced by A. actinomycetemcomitans, we performed Western blot assays in BMMs treated with vehicle (ethanol) or FTY720 (8 μM), with or without A. actinomycetemcomitans stimulation. As shown in Fig. 2a-d, FTY720 treatment decreased p-PI3K by 92.5 %, p-Akt by 65.9 %, and p-ERK by 54.0 % 30 min after bacterial stimulation compared with the control treatment. FTY720 reduced p-PI3K by 75.2 %, p-Akt by 76.9 %, and p-ERK by 59.1 % 60 min after bacterial stimulation compared with the control treatment. Additionally, FTY720 attenuated p-PI3K by 43.6 %, p-Akt by 59.2 %, and p-ERK by 50.0 % in cells without bacterial stimulation compared with the control treatment. The protein levels of p-NF-κB p65, p-JNK, p-p38 MAPK, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were similar between FTY720-treated cells and vehicle-treated cells before or after bacterial stimulation (data not shown). These results supported that FTY720 specifically attenuated the PI3K, Akt, and ERK signaling pathways, which could contribute to the down-regulation of the proinflammatory cytokine response stimulated by A. actinomycetemcomitans.Fig. 2


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 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (8 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (1.5 CFU/cell) for 30 to 120 min. a P-PI3K, PI3K, p-Akt, Akt, p-ERK, and ERK expressions were evaluated by Western blot. b P-PI3K protein density, (c) P-Akt protein density, and (d) P-ERK protein density were analyzed by Quantity One Software and normalized by total protein expression, respectively. Data are expressed as mean ± SEM (n = 3, *p < 0.05, *** p < 0.001)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4492085&req=5

Fig2: FTY720 attenuated p-PI3K, p-Akt, and p-ERK expressions induced by A. actinomycetemcomitans in BMMs. Murine BMMs were treated with vehicle (ethanol) or FTY720 (8 μM) for 30 min. Then the cells were either unstimulated or stimulated with A. actinomycetemcomitans (Aa) (1.5 CFU/cell) for 30 to 120 min. a P-PI3K, PI3K, p-Akt, Akt, p-ERK, and ERK expressions were evaluated by Western blot. b P-PI3K protein density, (c) P-Akt protein density, and (d) P-ERK protein density were analyzed by Quantity One Software and normalized by total protein expression, respectively. Data are expressed as mean ± SEM (n = 3, *p < 0.05, *** p < 0.001)
Mentions: To further elucidate which signaling pathways were affected by FTY720 in regulating the immune response induced by A. actinomycetemcomitans, we performed Western blot assays in BMMs treated with vehicle (ethanol) or FTY720 (8 μM), with or without A. actinomycetemcomitans stimulation. As shown in Fig. 2a-d, FTY720 treatment decreased p-PI3K by 92.5 %, p-Akt by 65.9 %, and p-ERK by 54.0 % 30 min after bacterial stimulation compared with the control treatment. FTY720 reduced p-PI3K by 75.2 %, p-Akt by 76.9 %, and p-ERK by 59.1 % 60 min after bacterial stimulation compared with the control treatment. Additionally, FTY720 attenuated p-PI3K by 43.6 %, p-Akt by 59.2 %, and p-ERK by 50.0 % in cells without bacterial stimulation compared with the control treatment. The protein levels of p-NF-κB p65, p-JNK, p-p38 MAPK, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were similar between FTY720-treated cells and vehicle-treated cells before or after bacterial stimulation (data not shown). These results supported that FTY720 specifically attenuated the PI3K, Akt, and ERK signaling pathways, which could contribute to the down-regulation of the proinflammatory cytokine response stimulated by A. actinomycetemcomitans.Fig. 2

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