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.2013 Jul;33(14):2760-72.
doi: 10.1128/MCB.01519-12. Epub 2013 May 20.

Phospholipase D1 has a pivotal role in interleukin-1β-driven chronic autoimmune arthritis through regulation of NF-κB, hypoxia-inducible factor 1α, and FoxO3a

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Phospholipase D1 has a pivotal role in interleukin-1β-driven chronic autoimmune arthritis through regulation of NF-κB, hypoxia-inducible factor 1α, and FoxO3a

Dong Woo Kang et al. Mol Cell Biol.2013 Jul.

Abstract

Interleukin-1β (IL-1β) is a potent proinflammatory and immunoregulatory cytokine playing an important role in the progression of rheumatoid arthritis (RA). However, the signaling network of IL-1β in synoviocytes from RA patients is still poorly understood. Here, we show for the first time that phospholipase D1 (PLD1), but not PLD2, is selectively upregulated in IL-1β-stimulated synoviocytes, as well as synovium, from RA patients. IL-1β enhanced the binding of NF-κB and ATF-2 to the PLD1 promoter, thereby enhancing PLD1 expression. PLD1 inhibition abolished the IL-1β-induced expression of proinflammatory mediators and angiogenic factors by suppressing the binding of NF-κB or hypoxia-inducible factor 1α to the promoter of its target genes, as well as IL-1β-induced proliferation or migration. However, suppression of PLD1 activity promoted cell cycle arrest via transactivation of FoxO3a. Furthermore, PLD1 inhibitor significantly suppressed joint inflammation and destruction in IL-1 receptor antagonist-deficient (IL-1Ra(-/-)) mice, a model of spontaneous arthritis. Taken together, these results suggest that the abnormal upregulation of PLD1 may contribute to the pathogenesis of IL-1β-induced chronic arthritis and that a selective PLD1 inhibitor might provide a potential therapeutic molecule for the treatment of chronic inflammatory autoimmune disorders.

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Figures

Fig 1
Fig 1
PLD1 is upregulated in the RA synovium and is selectively induced by inflammatory responses in RAFLS. (A and B) The expression of PLD was analyzed by qPCR (A) and immunoblotting with an Ab specific to PLD (B) in RAFLS and OAFLS. (C) Cells were labeled with [3H]myristate for 12 h, after which PLD activity was measured. (D) RAFLS and OAFLS were transfected with or without the PLD1 promoter and stimulated with TNF-α (1 ng/ml), IL-1β (10 ng/ml), or IL-6 (10 ng/ml) for 36 h, after which the expression of PLD isozymes was analyzed by qPCR (left) and promoter assay (right). (E) Cells were treated with cytokines for 36 h, after which the lysates were immunoblotted with an anti-PLD Ab. (B and E) Relative PLD1 protein levels were quantitated by densitometer analysis. The data shown are representative of at least three independent experiments. (F) Cells were treated with the cytokines for 30 min, and the enzymatic activity of PLD was examined. (G) IHC staining of PLD and IL-1β (×400, left) and quantification of PLD1- or IL-1β-positive cells in the synovium from mild or severe RA and OA patients (right). *,P < 0.01; **,P < 0.05. The data presented are the means ± SDs of four independent experiments.
Fig 2
Fig 2
IL-1β-induced PLD1 expression is mediated by the TRAF6/ERK/NF-κB and TRAF6/p38/ATF-2 signaling pathways. (A) RAFLS were cotransfected with pGL4-PLD1 and dn-TRAF6 and treated with IL-1β (10 ng/ml) for 36 h. A luciferase activity assay was performed (left). PLD1 expression was analyzed by Western blotting (right). (B) RAFLS were pretreated with FTase I, U0126, AG126, PDTC, SP600125 (SP), SB203580 (SB), and wortmannin (Wort) for 30 min and treated with IL-1β for 36 h, after which the lysates were analyzed by Western blotting with an anti-PLD Ab. (C) RAFLS were transfected with dn-TRAF6 and treated with IL-1β. HA, hemagglutinin. (D) RAFLS were pretreated with the indicated inhibitors and treated with IL-1β. (E) RAFLS were transfected with the indicated dominant negative constructs and then stimulated with IL-1β for 36 h. (C to E) Lysates were immunoblotted with the indicated Abs. (A to E) Relative PLD1 protein levels were quantitated by densitometer analysis. The data shown are representative of three independent experiments. (F) RAFLS were pretreated with PDTC for 30 min and then treated with or without IL-1β for 12 h. A ChIP assay was performed with preimmune IgG, anti-NF-κB, or anti-ATF-2 Ab, and the product was analyzed by qPCR. Arrows indicate the positions of the primers used in the ChIP experiment. (G) RAFLS were cotransfected with WT pGL4-PLD1, one or two NF-κB or ATF-2 binding site mutant forms (mut) of pGL4-PLD1 and then treated with or without IL-1β. A luciferase activity assay was then performed. (H) IHC staining of the indicated proteins in synovium from mild or severe RA and OA patients (×400). *,P < 0.01; **,P < 0.05. The data presented are the means ± SDs of four independent experiments.
Fig 3
Fig 3
PLD1 activity is required for IL-1β-induced expression of proinflammatory mediators, matrix-degrading enzymes, and adhesion molecules via inhibition of NF-κB. (A) RAFLS were pretreated with the PLD1 inhibitor VU0155069 or transfected with two kinds of siRNAs specific to PLD1 and then stimulated with IL-1β for 24 h. Secretion of IL-6, IL-15, and MCP-1 was quantified by ELISA. (B, C) RAFLS were transfected with siRNA specific to PLD1 and then treated with IL-1β for 36 h. Lysates were immunoblotted by the indicated Abs, and the activity of MMP-2 was analyzed by gelatin zymography of the conditioned medium. (D) RAFLS were pretreated with VU0155069 and stimulated with IL-1β. Lysates were immunoblotted with the indicated Abs. (E) Luciferase assay for NF-κB in RAFLS. (F) RAFLS were pretreated with VU0155069 for 30 min and then stimulated with IL-1β for 36 h. The lysate was fractionated into the cytosol and nucleus and then analyzed by immunoblotting with the indicated Abs (left). NF-κB-positive cells identified in different fields by IHC analysis were quantitated as described in Materials and Methods (right). Mean scores ± SDs (error bars) are shown. (B to D, F) The relative levels of the indicated proteins were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (G) ChIP assay of the binding of NF-κB to the promoters of its target genes. RAFLS were pretreated with a PLD1 inhibitor and stimulated with IL-1β for 12 h. A ChIP assay was then performed with preimmune IgG or an anti-NF-κB Ab, followed by qPCR. *,P < 0.05; **,P < 0.01. The data presented are the means ± SDs of four independent experiments.
Fig 4
Fig 4
PLD1 activity is required for IL-1β-induced expression of angiogenic factors via binding of HIF-1α to the promoters of its target genes. (A) RAFLS were pretreated with VU0155069 or transfected with two kinds of siRNAs specific to PLD1, followed by stimulation with IL-1β for 24 h. Secretion of VEGF and IL-8 was quantified by ELISA. (B and C) RAFLS were transfected with siRNA specific to PLD1, pretreated with or without VU0155069, and stimulated with IL-1β for 36 h. Cell lysates were immunoblotted with the indicated Abs. The relative levels of the indicated proteins were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (D) RAFLS were transfected with HRE-Luc, pretreated with VU0155069, and stimulated with IL-1β for 36 h, and then luciferase activity was determined. (E) RAFLS were pretreated with VU0155069 or cycloheximide (CHX, 5 μM) for 30 min, treated with IL-1β for 36 h, and then treated with MG132 (20 μM) for 6 h. The HIF-1α protein level was determined by Western blotting. Relative HIF-1α protein levels were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (F and G) RAFLS were pretreated with VU0155069 for 30 min and treated with IL-1β for 12 h, and a ChIP assay was performed with preimmune IgG, anti-HIF-1α, and anti-NF-κB Abs, followed by qPCR. *,P < 0.01; **,P < 0.05. The data presented are the means ± SDs of four independent experiments.
Fig 5
Fig 5
PLD1 activity is involved in IL-1β-induced angiogenesis and migration. (A, B) RAFLS were preincubated with 10 μM VU0155069 and treated with IL-1β for 36 h. Conditioned medium was collected and applied to HUVECs, and then tube formation (A) and migration (B) were measured. RAFLS were transfected with siRNAs specific to PLD1 (C) or pretreated with or without 10 μM VU0155069 (D), seeded into collagen type I (0.1% solution)-coated migration chambers, and then stimulated with IL-1β for 36 h. The extent of migration is expressed as the average number of cells per microscopic field. *,P < 0.01; **,P < 0.01; ***,P < 0.05. The data presented are the means ± SDs of four independent experiments.
Fig 6
Fig 6
Inhibition of PLD1 suppresses IL-1β-induced phosphorylation of FoxO3a and enhances cell cycle arrest via transactivation of FoxO3a. (A) RAFLS were pretreated with VU0155069 (10 μM) and stimulated with IL-1β for 30 min. (B) RAFLS were pretreated with a PI3K inhibitor (wortmannin [Wort], LY294002 [LY]) and stimulated with PA (50 μM) for 30 min. (A and B) Lysates were immunoblotted with the indicated Abs. The relative levels of the indicated proteins were quantitated by densitometer analysis. The data shown are representative of four independent experiments. (C and D) RAFLS were transfected with FoxO-Luc and treated with the indicated drugs, and a luciferase activity assay was performed. (E) RAFLS were pretreated with VU0155069 and treated with IL-1β for 30 min. Lysate was fractionated into the cytosol and nucleus and then immunoblotted with the indicated Abs (left). Relative FoxO3a protein levels were quantitated by densitometer analysis. The data shown are representative of four independent experiments. The FoxO3a-positive cells identified in different fields by IHC were quantitated (right). (F) RAFLS were pretreated with VU0155069 and treated with IL-1β for 12 h, and a ChIP assay was performed with the indicated Abs. (G) RAFLS were transfected with or without siRNA specific to FoxO3a and subjected to real-time qPCR (left) and immunoblotting (right). a.u., arbitrary units. The relative levels of the indicated proteins were quantitated by densitometer analysis. The data shown are representative of four independent experiments. RAFLS were treated with or without VU0155069 and IL-1β for 36 h and then analyzed by FACS (H) and BrdU incorporation assay (I). (J) IHC staining of p-FoxO3a and p27Kip1 in the synovium from mild/severe RA and OA patients. *,P < 0.01; **,P < 0.05. The data presented are the means ± SDs of four independent experiments.
Fig 7
Fig 7
PLD1 inhibitor suppresses the pathogenesis of spontaneous arthritis, decreases the expression of NF-κB and HIF-1α target genes, and promotes the expression of FoxO3a target genes in IL-1Ra-deficient mice. (A) IL-1Ra−/− mice were treated with intraperitoneal injections of PLD1 inhibitor (10 mg/kg;n = 8) or vehicle (n = 8). Tissue sections from joints of each mouse were stained with H&E, toluidine blue, or safranin O. Representative photographs from each group are shown. The histological scores of mice treated with PLD1 inhibitor or vehicle are shown at the bottom. The data represent individual values and the average value of five individual mice in each group. *,P < 0.05 compared with the vehicle-treated group. (B to D) Tissue sections from the joints of mice treated with PLD1 inhibitor or vehicle were stained with the indicated Abs. The cells stained with each Ab are shown in brown (×400). (E) Tissue sections from the spleens of IL-1Ra-deficient mice were stained with 4′,6-diamidino-2-phenylindole (DAPI; blue) or anti-p-FoxO3a Ab (green). Tissues were monitored with a Zeiss LSM 510 confocal microscope. Anti-p-FoxO3a- and DAPI-stained images were overlaid to visualize the nuclear and cytoplasmic localization of p-FoxO3a. The data shown are representative of three independent experiments. (F) Model illustrating the roles of PLD1 in RAFLS. IL-1β binds to its receptor and stimulates the TRAF6-ERK/NF-κB and TRAF6/p38/ATF-2 signaling pathways, leading to selective PLD1 expression. PLD1-derived PA is involved in the production of proinflammatory mediators and angiogenic factors. PLD1 inhibitor abolishes the expression of molecules involved in RA pathogenesis by suppressing the binding of NF-κB/HIF-1α to the promoters of IL-1β target genes and enhances the expression of cell cycle arrest genes (p27Kip1 and p21Cip1 genes) via transactivation of FoxO3a. The abnormal upregulation of PLD1 in RAFLS may contribute to the pathogenesis of chronic arthritis and thus provide a potential target for the control of inflammatory arthritis.
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