Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo

Abstract

Objective: Sulforaphane (SFN) has been reported to regulate signaling pathways relevant to chronic diseases. The aim of this study was to investigate the impact of SFN treatment on signaling pathways in chondrocytes and to determine whether sulforaphane could block cartilage destruction in osteoarthritis.

Methods: Gene expression, histone acetylation, and signaling of the transcription factors NF-E2-related factor 2 (Nrf2) and NF-κB were examined in vitro. The bovine nasal cartilage explant model and the destabilization of the medial meniscus (DMM) model of osteoarthritis in the mouse were used to assess chondroprotection at the tissue and whole-animal levels.

Results: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes and in fibroblast-like synovial cells. SFN acted independently of Nrf2 and histone deacetylase activity to regulate metalloproteinase expression in human articular chondrocytes but did mediate prolonged activation of JNK and p38 MAPK. SFN attenuated NF-κB signaling at least through inhibition of DNA binding in human articular chondrocytes, with decreased expression of several NF-κB-dependent genes. Compared with cytokines alone, SFN (10 μM) abrogated cytokine-induced destruction of bovine nasal cartilage at both the proteoglycan and collagen breakdown levels. An SFN-rich diet (3 μmoles/day SFN versus control chow) decreased the arthritis score in the DMM model of osteoarthritis in the mouse, with a concurrent block of early DMM-induced gene expression changes.

Conclusion: SFN inhibits the expression of key metalloproteinases implicated in osteoarthritis, independently of Nrf2, and blocks inflammation at the level of NF-κB to protect against cartilage destruction in vitro and in vivo.

Figure 1

Sulforaphane (SFN) inhibits cytokine-induced metalloproteinase expression in articular joint cells. Human articular chondrocytes, fibroblast-like synoviocytes, and the SW-1353 cell line were pretreated with 0–10 μM SFN and stimulated with or without interleukin-1 (IL-1; 5 ng/ml) and oncostatin M (OSM; 10 ng/ml) for 6 hours. A–C, SFN-induced inhibition of cytokine-induced ADAMTS4, ADAMTS5, MMP1, and MMP13 mRNA expression in human articular chondrocytes (A), MMP1 and MMP13 mRNA expression in fibroblast-like synoviocytes (B), and MMP1 and MMP13 mRNA expression in SW-1353 cells (C). D, Top, Human articular chondrocyte whole cell lysates immunoblotted for acetylated histone H3 (acH3), total histone H3, and acetylated lysine (acLys). Bottom, MMP28 mRNA expression in SW-1353 cells, as measured using quantitative reverse transcription–polymerase chain reaction. Values are the mean ± SEM (n = ≥3). RQ = relative quantification (expressed as ); N = sodium butyrate; C = negative control; I = IL-1; T = trichostatin A. ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.0001, SFN alone versus no treatment or SFN plus cytokines versus cytokines alone, by one-way analysis of variance with Dunnett’s post-test.

Figure 2

SFN does not require the NF-E2–related factor 2 (Nrf2) pathway to inhibit cytokine-induced metalloproteinase expression. Nrf2 targeting small interfering RNA (siRNA) was used to knock down Nrf2 expression in human articular chondrocytes, 24 hours prior to treatments. Human articular chondrocytes were treated with SFN (10 μM) for 30 minutes prior to the addition of IL-1 (5 ng/ml) and OSM (10 ng/ml), to induce gene expression for 6 hours. Relative mRNA gene expression was measured using quantitative reverse transcription–polymerase chain reaction and normalized to 18S ribosomal RNA expression. A, SFN-induced expression of HMOX1 mRNA. B, Silencing of Nrf2 expression using 25 nM targeting siRNA in human articular chondrocytes. C, Decreased HMOX1 expression following Nrf2 siRNA treatment. D, No impact of Nrf2 siRNA treatment on SFN-induced inhibition of cytokine-induced MMP1 expression. Values are the mean ± SEM (n = 3). ∗ = P < 0.05; ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001 versus 0 μM SFN (A) or as indicated. NT = nontargeting siRNA control (see Figure 1 for other definitions).

Figure 3

SFN regulates MAPK activation and NF-κB signaling. A, Effect of SFN on phosphorylation of JNK, ERK, and p38 MAPK in human articular chondrocytes treated with 5 ng/ml IL-1 for 0–60 minutes. B, Activation of NF-κB in human articular chondrocytes treated with IL-1 (5 ng/ml) for 0–60 minutes, with or without 10 μM SFN. C, Top, Intracellular localization of p65 in human articular chondrocytes treated with IL-1 (5 ng/ml) for 45 minutes, with or without 10 μM SFN. Bars = 100 μm. Bottom, Immunoblotting of cytoplasmic and nuclear fractions from human articular chondrocytes probed for p65. Translocation of p65 to the nucleus was unaffected by SFN treatment. D, Left, NF-κB binding to consensus DNA-binding sequence, as determined by electrophoretic mobility shift assay. Nuclear extracts from human articular chondrocytes were treated as described in C. Right, Inhibition of NF-κB transcriptional activation in chondrocytes by 10 μM SFN, as determined by luciferase-linked κB-reporter assay. Bars show the mean ± SEM (n = 3). ∗∗∗ = P < 0.0001. LD = loading dye; WT = wild-type; x = exogenous; cc = competitor oligos; acLys = acetylated lysine; RLU = relative light units (see Figure 1 for other definitions).

Figure 4

SFN treatment inhibits expression of known NF-κB–responsive genes. Human articular chondrocytes were treated with 5 ng/ml IL-1 for 0–8 hours, with (▪) or without (□) 10 μM SFN. Gene expression was measured using real-time quantitative reverse transcription–polymerase chain reaction and normalized to 18S ribosomal RNA expression. A, Expression of HMOX1 and IκBα. B, Expression of the inflammatory genes IL6, IL8, iNOS, MMP13, and COX2. C, Expression of the cytoprotective genes A20 and SOD2. Values are the mean ± SEM (n = 3). ∗ = P < 0.05; ∗∗ = P < 0.01; ∗∗∗ = P < 0.001, by two-way analysis of variance with Bonferroni post-test. See Figure 1 for other definitions.

Figure 5

Sulforaphane (SFN) accumulates primarily as SFN–glutathione (SFN–GSH) in chondrocytes and can directly inhibit NF-κB binding to the consensus DNA sequence. Cell-free medium (control) and primary chondrocytes were treated with 10 μM SFN for 0–2 hours, with iberin used as an internal control. A, Expression of SFN–Cys, SFN–Cys-Gly, SFN–GSH, iberin, SFN–N-acetylcysteine (SFN–NAC), and SFN, as determined by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). B, Intracellular accumulation of SFN–GSH in primary chondrocytes, as quantified by HPLC-MS/MS. Values are the mean ± SEM. C, Titration of exogenous SFN–GSH (top) or exogenous SFN (bottom) into DNA-binding reaction mixtures. The binding reaction mixtures contained nuclear extracts from human articular chondrocytes stimulated with interleukin-1 (IL-1; 5 ng/ml) for 45 minutes and the NF-κB consensus DNA sequence. D, Preincubation of exogenous SFN–GSH or exogenous SFN with the reducing agent dithiothreitol (DTT) for 15 minutes rescued inhibition of nuclear extract/DNA binding (final concentrations 250 μM exogenous SFN–GSH or exogenous SFN and 17.9 mM DTT). m/z = mass/charge (see Figure 3 for other definitions).

Figure 6

SFN inhibits cartilage destruction in a bovine explant model and protects against surgically induced osteoarthritis in mice in vivo. A and B, Release of glycosaminoglycan (GAG) (A) and hydroxyproline (HP) (B) into culture medium. Bovine nasal cartilage explants were treated with 0–20 μM SFN, with or without IL-1 (0.5 ng/ml) and OSM (5 ng/ml), for 14 days. Values are the mean ± SEM (n = 4). ∗∗ = P < 0.001; ∗∗∗ = P < 0.0001 versus no treatment. C, Left, Summed scores of each histologic section obtained through the joints of C57BL/6 mice that were fed a control diet or a diet containing 0.18 gm/kg SFN ad libitum, 2 weeks prior to and following destabilization of the medial meniscus (DMM). Data are shown as box plots. Each box represents the 25th to 75th percentiles. Lines inside the boxes represent the median. Lines outside the boxes represent the 10th and the 90th percentiles. Right, Representative Safranin O–stained histologic sections (8 μm) obtained from the medial joint compartment of mice fed a control diet and those fed an SFN-rich diet. Original magnification × 40. D, Expression of Col2a1 mRNA (top) and Col10a1 mRNA (bottom) in the joints of unoperated control mice (n = 13) and mice that underwent DMM (n = 17), that were fed a control diet or an SFN-rich diet (0.6 gm/kg), as determined by quantitative reverse transcription–polymerase chain reaction. Values are the mean ± SEM (n = 3). See Figure 1 for other definitions. Color figure can be viewed in the online issue, which is available at http://onlinelibrary.wiley.com/doi/10.1002/art.38133/abstract.