FTY720 Attenuates LPS-Induced Inflammatory Bone Loss by Inhibiting Osteoclastogenesis via the NF- κ B and HDAC4/ATF Pathways

Abstract

Osteoclast (OC) abnormalities lead to many osteolytic diseases, such as osteoporosis, inflammatory bone erosion, and tumor-induced osteolysis. Exploring effective strategies to remediate OCs dysregulation is essential. FTY720, also known as fingolimod, has been approved for the treatment of multiple sclerosis and has anti-inflammatory and immunosuppressive effects. Here, we found that FTY720 inhibited osteoclastogenesis and OC function by inhibiting nuclear factor kappa-B (NF-κB) signaling. Interestingly, we also found that FTY720 inhibited osteoclastogenesis by upregulating histone deacetylase 4 (HDAC4) expression levels and downregulating activating transcription factor 4 (ATF4) expression levels. In vivo, FTY720 treatment prevented lipopolysaccharide- (LPS-) induced calvarial osteolysis and significantly reduced the number of tartrate-resistant acid phosphatase- (TRAP-) positive OCs. Taken together, these results demonstrate that FTY720 can inhibit osteoclastogenesis and ameliorate inflammation-induced bone loss. Which may provide evidence of a new therapeutic target for skeletal diseases caused by OC abnormalities.

Figure 1

FTY720 inhibits receptor activator of NF-κB ligand- (RANKL-) induced osteoclastogenesis without cytotoxicity. (a) The cell counting kit-8 (CCK-8) assay was used to detect the OD (optical density) value reflecting the level of cell proliferation under treatment with FTY720 for different times (48 h, 72 h, and 96 h) and at different concentrations (8-512 nM) (n = 3). (b) Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts (OCs) in the presence or absence of FTY720. Scale bar: 200 μm. (c) Quantitative analysis of TRAP-positive OCs with three or more nuclei (n = 3). (d) The molecular structure of FTY720. Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by one-way ANOVA with Tukey's post hoc test (a, c).

Figure 2

FTY720 downregulates the expression of osteoclast- (OC-) related genes. (a) RT–qPCR detection of the mRNA expression of OC-related genes, including tartrate-resistant acid phosphatase 5 (Acp5), recombinant human cathepsin K (CTSK), vacuolar ATPase d2 (V-ATPase d2), dendritic cell-specific transmembrane protein (DC-STAMP), c-Fos, nuclear factor of activated T cells c1 (NFATc1), and tumor necrosis factor receptor superfamily member 11a (TNFRSF11a). 18 s was selected as the control gene for the experiments (n = 3). (b) Western blotting of RANKL- (50 ng/mL) induced expression of c-Fos, NFATc1, and Integrin B3 on days 0, 1, 3, and 5 in the presence or absence of FTY720 (16 nM). B-Actin was selected as the control protein. (c) Quantitative statistics of the grayscale values of the c-Fos, NFATc1, and Integrin B3 protein bands (n = 3). Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by one-way ANOVA with Tukey's post hoc test (a) or two-way ANOVA with Bonserroni's post hoc test (c).

Figure 3

FTY720 suppresses the nuclear factor kappa-B (NF-κB) signaling pathway and upregulates histone deacetylase 4 (HDAC4) expression. (a) Western blotting of IκBα, NF-κB p65, and p-NF-κB p65 expression at 0, 5, 10,20, 30, and 60 min under RANKL (50 ng/mL) stimulation with or without FTY720 treatment. B-Actin was selected as the control protein. (b) Quantitative statistics of IκBa, NF-κB p65, and p-NF-κB p65 expression (n = 3). (c) Western blotting analysis of RANKL- (50 ng/mL) induced expression of HDAC4, activating transcription factor 4 (ATF4), and myocyte enhancer factor 2C (MEF2C) on days 0, 1, 3, and 5 in the presence or absence of FTY720. B-Actin was selected as the control protein. (d) Quantitative statistics of HDAC4, ATF4, and MEF2C expression (n = 3). Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by two-way ANOVA with Bonferroni's post hoc test (b, d).

Figure 4

FTY720 restored lipopolysaccharide- (LPS-) induced cranial osteolysis. (a) Reconstruction of cranial bone models by micro-CT upon treatment with PBS, LPS, or LPS and FTY720 (128 nM or 512 nM) together under different conditions. (a–d) 3D reconstructed images of the whole skull with a scale of 2 mm. ((a), A1–D1, A2–D2) The selected region of interest in the median part of the skull and the cranial suture images, respectively, corresponding to scales of 1 mm and 500 μm. (b) Quantitative analysis of the data from the PBS (n = 6), LPS (n = 6), and LPS with low (n = 5) or high (n = 5) FTY720 groups collected by micro-CT in the median cranial region for bone volume fraction (BV/TV), trabecular number (Tb.N), subchondral trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). (c) Quantitative analysis of the data from the PBS (n = 6), LPS (n = 6), and LPS with low (n = 5) or high (n = 5) FTY720 groups collected by micro-CT analysis of the cranial suture for BV/TV, Tb.N, Tb.Th, and Tb.Sp. Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by one-way ANOVA with Tukey's post hoc test (b, c).

Figure 5

FTY720 treatment partially restored cranial bone morphology and inhibited osteoclastogenesis in vivo. (a) Hematoxylin-eosin (H&E) staining of the region of interest. Median skull, scale bar, 200 μm (top panels), cranial suture, 200 μm (middle panels), and 50 μm (bottom panels). (b) TRAP staining of the region of interest. Median skull, scale bar, 200 μm (top panels), cranial suture, 200 μm (middle panels), and 50 μm (bottom panels). (c–e) Quantitative analysis of the skull middle bone marrow cavity area (c) and the number of TRAP-positive stained osteoclasts in the skull median (d) and on the skull cranial sutures (e) in the PBS (n = 6), LPS (n = 6), and LPS with low (n = 5) or high (n = 5) FTY720 groups. Data are expressed as the mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001 by one-way ANOVA with Tukey's post hoc test (b–d).

Figure 6

Schematic diagram of the mechanism by which FTY720 inhibits osteoclastogenesis.