Interleukin-1 Induces the Release of Lubricating Phospholipids from Human Osteoarthritic Fibroblast-like Synoviocytes

Abstract

(1) Background: Synovial fluid (SF) from knee joints with osteoarthritis (OA) has increased levels of phospholipids (PL). We have reported earlier that TGF-ß and IGF-1 stimulate fibroblast-like synoviocytes (FLS) to synthesize increased amounts of PLs. The current study examined whether IL-1ß induces the release of PLs in FLS and the underlying mechanism. (2) Methods: Cultured human OA FLS were treated with IL-1ß alone and with pathway inhibitors or with synthetic liver X receptor (LXR) agonists. Cholesterol hydroxylases, ABC transporters, apolipoproteins (APO), LXR, sterol regulatory binding proteins (SREBPs), and 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) were analyzed by RT-PCR, Western blot, and ELISA. The release of radiolabeled PLs from FLS was determined, and statistical analysis was performed using R (N = 5-9). (3) Results: Like synthetic LXR agonists, IL-1ß induced a 1.4-fold greater release of PLs from FLS. Simultaneously, IL-1ß upregulated the level of the PL transporter ABCA1 and of cholesterol hydroxylases CH25H and CYP7B1. IL-1ß and T0901317 stimulated the expression of SREBP1c, whereas only T0901317 enhanced SREBP2, HMGCR, APOE, LXRα, and ABCG1 additionally. (4) Conclusions: IL-1ß partially controls PL levels in OA-SF by affecting the release of PLs from FLS. Our data show that IL-1ß upregulates cholesterol hydroxylases and thus the formation of oxysterols, which, as natural agonists of LXR, increase the level of active ABCA1, in turn enhancing the release of PLs.

Keywords: ABCA1; CH25H; CYP7B1; FLS; LXR; cholesterol hydroxylase; interleukin; osteoarthritis; phospholipids; synovial fibroblasts.

Figure 1

IL-1ß and a synthetic LXR agonist induce the release of radiolabeled PLs from FLS. Release of [³H]-labeled PLs into the media is expressed as percentage of PLs in media from total PLs (media + cell lysate) after normalization to total cellular protein content. FLS were treated for 48 h with 1.0 ng/mL IL-1ß or 0.1 µM T0901317 ± 1.0 ng/mL IL-1ß. Data for biological replicates are shown as dot plots, with lines inside showing the mean ± SD (n = 5–9). Black circles represent data from IL-1ß-treated FLS versus control, whereas blue squares represent data from T0901317 ± IL-1ß versus vehicle control. ** p < 0.01, *** p < 0.001.

Figure 2

IL-1ß induces the expression of cholesterol hydroxylase CH25H and CYP7B1 in FLS. CH25H (A) and CYP7B1 (C) were measured by Western blot or enzyme-linked immunosorbent assay (B). The mRNA expression of CH25H and CYP7B1 (D) was determined by reverse transcription-polymerase chain reaction (RT-PCR) using the 2^−ΔΔCt method. Representative western blots show the protein expression of cultured FLS as the corresponding control (−) and of FLS treated (+) with IL-1ß or T0901317. All western blots were quantified using ImageJ (A,C). Treated FLS and corresponding controls derived from the same experiment, and patient and gels/blots were processed in parallel. FLS were treated with 1 ng/mL IL-1ß or 0.1 µM T0901317 for 24, 48, and 72 h. Data for biological replicates represent fold-change versus corresponding controls (= 1, indicated by broken horizontal lines) and are shown as dot plots, with lines inside showing the mean ± SD (n = 8). The y-axis represents the log of variable fold changes. Black circles show data from IL-1ß-treated FLS relative to untreated controls, whereas blue squares represent data from T0901317 relative to vehicle controls. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

T0901317 but not IL-1ß induces the expression of liver X receptor (LXR) and apolipoprotein E (APOE) in FLS. Western blots of LXRα (A) and APOE (C). The relative mRNA expression of LXRα and LXRß (B) and APOA1 and APOE (D) was determined by RT-PCR using the 2^−ΔΔCt method. For further details, see the legend for Figure 2, which is fully analogous, but here for LXRα and ß, APOA1, and APOE. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

IL-1ß and T0901317 induce the expression of ABCA1 but not ABCG1 in FLS. Western blots of ABCA1 (A) and ABCG1 (C). The mRNA expression of ABCA1 (B) and ABCG1 (D) was determined by RT-PCR using the 2^−ΔΔCt method. For further details, see the legend for Figure 2, which is fully analogous, but here for ABCA1 and ABCG1. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 5

Effect of IL-1ß and T0901317 on the expression of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding protein (SREBP) 1c and 2 in FLS. Western blots of SREBP1c (A) and HMGCR (C). The mRNA expression of SREBF1 and 2 (B) and HMGCR (D) was determined by RT-PCR using the 2^−ΔΔCt method. For further details, see the legend for Figure 2, which is fully analogous, but here for both SREBPs/SREBFs and HMGCR. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 6

Schematic of proposed mechanism of IL-1ß on phospholipid (PL) release from FLS during OA. IL-1ß induces the upregulation of cholesterol 25-hydroxylase (CH25H) and monooxygenase 25-hydroxycholesterol 7-alpha-hydroxylase, also called cytochrome P450 7B1 (CYP7B1), causing accumulation of oxysterols as endogenous ligands of nuclear liver X receptor (LXR). On activation of LXR by oxysterols or synthetic LXR agonists, such as T0901317, the protein level of active ATP-binding cassette transporter A1 (ABCA1) increases. The increased efflux of PLs to extracellular apolipoprotein (APO) A1 is mediated by ABCA1, which results in the formation of nascent HDL-c particles (nHDL). Oxysterols and T0901317 upregulate sterol regulatory element-binding protein (SREBP) 1c, suggesting that the biosynthesis of fatty acids that are needed for PLs is stimulated. Further, T0901317 enhances the expression of SREBP2, which binds as an important transcription factor to the promoter region of 3-hydroxy-3-methylglutaryl-coA reductase (HMGCR), the rate-limiting enzyme in cholesterol biosynthesis, to upregulate it. Dotted line: IL-1; solid line: T0901317; arrow: stimulation; dash: inhibition.