Sulforaphane represses matrix-degrading proteases and protects cartilage from destruction in vitro and in vivo.
ABSTRACT Objective: Sulforaphane (SFN) has been reported to regulate signalling pathways relevant to chronic diseases. Our study investigated the impact of sulforaphane treatment on signalling pathways in chondrocytes and whether sulforaphane could block cartilage destruction in osteoarthritis. Methods: Gene expression, histone acetylation, transcription factors nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor kappaB (NF-κB) signalling were examined in vitro The bovine nasal cartilage explant model (BNC) and destabilisation of medial meniscus (DMM) murine model of osteoarthritis were used to assess chondroprotection at the tissue and whole animal level. Results: SFN inhibited cytokine-induced metalloproteinase expression in primary human articular chondrocytes (HACs) and in fibroblast-like synovial cells (FLS). SFN acts independently of the Nrf2 transcription factor and histone deacetylase activity in HACs to regulate metalloproteinase expression, but does mediate prolonged activation of Jun kinase (JNK) and p38 MAP kinase. SFN attenuates NF-κB signalling through at least inhibition of DNA binding in HACs with attenuation of expression of several NF-κB dependent genes. SFN abrogates cytokine-induced destruction of bovine nasal cartilage at the level of both proteoglycan and collagen breakdown (10µM compared to cytokines alone). A SFN-rich diet (3µmol daily dose SFN versus control chow) decreases arthritis score in the DMM murine model of osteoarthritis 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. © 2013 American College of Rheumatology.
- SourceAvailable from: Rosa Maria Borzi[Show abstract] [Hide abstract]
ABSTRACT: Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene α (GROα), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GROα. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GROα. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features.PLoS ONE 01/2014; 9(10):e109724. · 3.53 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Osteoarthritis (OA) is a degenerative joint disease for which there are no disease-modifying drugs. It is a leading cause of disability in the UK. Increasing age and obesity are both major risk factors for OA and the health and economic burden of this disease will increase in the future. Focusing on compounds from the habitual diet that may prevent the onset or slow the progression of OA is a strategy that has been under-investigated to date. An approach that relies on dietary modification is clearly attractive in terms of risk/benefit and more likely to be implementable at the population level. However, before undertaking a full clinical trial to examine potential efficacy, detailed molecular studies are required in order to optimise the design. This review focuses on potential dietary factors that may reduce the risk or progression of OA, including micronutrients, fatty acids, flavonoids and other phytochemicals. It therefore ignores data coming from classical inflammatory arthritides and nutraceuticals such as glucosamine and chondroitin. In conclusion, diet offers a route by which the health of the joint can be protected and OA incidence or progression decreased. In a chronic disease, with risk factors increasing in the population and with no pharmaceutical cure, an understanding of this will be crucial.Proceedings of The Nutrition Society 02/2014; · 3.67 Impact Factor
- Joint Bone Spine 07/2014; · 2.75 Impact Factor
ARTHRITIS & RHEUMATISM
Vol. 65, No. 12, December 2013, pp 3130–3140
© 2013 The Authors. Arthritis & Rheumatism is published by Wiley Periodicals, Inc. on
behalf of the American College of Rheumatology. This is an open access article under the
terms of the Creative Commons Attribution License, which permits use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Sulforaphane Represses Matrix-Degrading Proteases and
Protects Cartilage From Destruction In Vitro and In Vivo
Rose K. Davidson,1Orla Jupp,1Rachel de Ferrars,1Colin D. Kay,1Kirsty L. Culley,1
Rosemary Norton,1Clare Driscoll,2Tonia L. Vincent,2Simon T. Donell,3
Yongping Bao,1and Ian M. Clark1
Objective. Sulforaphane (SFN) has been reported
to regulate signaling pathways relevant to chronic dis-
eases. 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-?B 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 chon-
droprotection at the tissue and whole-animal levels.
Results. SFN inhibited cytokine-induced metallo-
proteinase 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 hu-
man articular chondrocytes but did mediate prolonged
activation of JNK and p38 MAPK. SFN attenuated
NF-?B signaling at least through inhibition of DNA
binding in human articular chondrocytes, with de-
creased expression of several NF-?B–dependent genes.
Compared with cytokines alone, SFN (10 ?M) abro-
gated cytokine-induced destruction of bovine nasal car-
tilage at both the proteoglycan and collagen breakdown
levels. An SFN-rich diet (3 ?moles/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, inde-
pendently of Nrf2, and blocks inflammation at the level
of NF-?B to protect against cartilage destruction in
vitro and in vivo.
Two key molecules that endow cartilage extracel-
lular matrix with its structural properties are type II
collagen and the proteoglycan aggrecan. The former
molecule is principally turned over by the action of
collagenolytic matrix metalloproteinases (MMPs; e.g.,
MMP-1 and MMP-13), while enzymes from the
ADAMTS family are responsible for metabolism of the
latter molecule (1). An imbalance between the activity
of key enzymes from these families and their inhibitors
is thought to underlie cartilage destruction in osteoar-
Epidemiology data suggest that high intake of
fruit and vegetables may protect against the onset and/or
progression of OA (2–4). Sulforaphane (1-isothiocya-
nato-4-methylsulphinylbutane; SFN) is a plant-derived
isothiocyanate obtained in the diet through consumption
of cruciferous vegetables, particularly broccoli (5). SFN
is a potent inducer of phase II (detoxification) metabo-
lism via activation of the transcription factor NF-E2–
Supported by the Biotechnology and Biological Sciences
Research Council (Diet and Health Research Industry Club grant
BB/I006060/1), the Dunhill Medical Trust (grant R73/0208), and
Arthritis Research UK (grant 19371).
1Rose K. Davidson, PhD, Orla Jupp, PhD, Rachel de Ferrars,
BSc, Colin D. Kay, PhD, Kirsty L. Culley, PhD, Rosemary Norton,
PhD, Yongping Bao, PhD, Ian M. Clark, PhD: University of East
Anglia, Norwich, UK;2Clare Driscoll, BSc, Tonia L. Vincent, MD,
PhD: Kennedy Institute of Rheumatology, London, UK, and Univer-
sity of Oxford, Oxford, UK;
Norwich University Hospital, Norfolk, UK.
Drs. Davidson and Jupp contributed equally to this work.
Address correspondence to Ian M. Clark, PhD, School of
Biological Sciences, University of East Anglia, Norwich Research
Park, Norwich NR4 7TJ, UK. E-mail: firstname.lastname@example.org.
Submitted for publication October 18, 2012; accepted in
revised form August 8, 2013.
3Simon T. Donell, MD: Norfolk and
related factor 2 (Nrf2), which binds to an antioxidant
response element in cognate genes (5,6). SFN can
impact on several signaling pathways in a cell type–
dependent manner. The antiinflammatory properties of
SFN have been reported previously (7–9), and these
effects were suggested to function through NF-?B, acti-
vator protein 1, and MAPK signaling. Modulation of
MMP expression in chondrocytes by SFN has been
previously described (9–11). The efficacy of SFN (at
high doses) in protecting mice with experimentally in-
duced inflammatory arthritis has been demonstrated,
and in vitro experiments using T cells from patients with
rheumatoid arthritis showed a reduction in the activa-
tion and production of interleukin-17 (IL-17) and tumor
necrosis factor ? (TNF?) (12). Epigenetic regulation by
SFN has also been reported in vitro and in vivo (13). We
previously showed that broad-spectrum histone deacety-
lase (HDAC) inhibitors are chondroprotective agents
(14), in part via repression of MMP expression, and this
finding was supported in animal models of arthritis
(15,16). In the current study, we sought to determine the
efficacy of SFN, which has been reported as a weak
HDAC inhibitor (17), as a chondroprotective agent.
MATERIALS AND METHODS
Materials. SFN and its metabolites were obtained from
Toronto Research Chemicals, except SFN–Cys-Gly, which was
synthesized by Dr. Sunil Sharma, University of East Anglia.
IL-1 and oncostatin M (OSM) were obtained from R&D
Systems. NF-?B p65 (catalog no. sc-109 X and no. sc-372), p50,
and c-Rel primary antibodies were obtained from Santa Cruz
Biotechnology. All other primary antibodies (phospho-p65;
catalog no. 3033), I?B? (catalog no. 4814S), acetylated histone
H3 (catalog no. 4353S), histone H3 (catalog no. 9715S),
acetylated Lys (catalog no. 9441S), GAPDH (catalog no.
2118S), JNK (catalog no. 9258S), ERK (catalog no. 9102),
p38 (catalog no. 9212), phospho-JNK (catalog no. 4668S),
phospho-ERK (catalog no. 9101S), and phospho-p38 (catalog
no. 4511S) were from Cell Signaling Technology. Small inter-
fering RNA (siRNA) against Nrf2 (Ambion) and AllStars
nontargeting siRNA were from Qiagen; staurosporine was
obtained from Sigma-Aldrich; and trichostatin A and sodium
butyrate were from Calbiochem. NF-?B consensus sequence
IRDye 700–labeled oligos were from Li-Cor. The I?B? pro-
moter reporter plasmid was a gift from Prof. Derek Mann,
Newcastle University, UK (originally from Prof. Ronald Hay,
University of Dundee, UK).
Cell culture and treatments. The SW-1353 human
chondrosarcoma cell line was purchased from ATCC. Primary
human articular chondrocytes were isolated from the cartilage
of patients with OA who underwent knee replacement surgery,
as previously described (16). All human articular chondrocytes
were used at passages 1–2. Fibroblast-like synovial cells (FLS)
were cultured from the synovial tissue of patients with OA,
with tissue dissected into ?1-cm3pieces and placed in culture
to allow cell outgrowth. These cells were seeded for the
experiments, as described. This study was performed with
ethics approval (Norfolk Ethics Committee), and all patients
provided informed consent.
Cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM; GlutaMAX) supplemented with 10% fetal
calf serum volume/volume, 1,000 IU/ml penicillin, and 100
?g/ml streptomycin at 37°C in an atmosphere of 5% CO2. Cells
were plated at 1.2 ? 104cells/cm2, left to adhere overnight, and
serum-starved overnight prior to treatment. Cells or cartilage
tissue specimens were preincubated with SFN for 30 minutes
prior to cytokine stimulation.
Complementary DNA (cDNA) synthesis and quantita-
tive reverse transcription–polymerase chain reaction (qRT-
PCR). Whole cell lysates were harvested into 30 ?l of Cells-
to-cDNA II Cell Lysis Buffer (Ambion). Lysates (8 ?l) treated
with DNase I (Ambion) were reverse transcribed in a total
volume of 20 ?l, using 200 ng random primers and 100 units
Moloney murine leukemia virus reverse transcriptase (Invitro-
gen), according to the manufacturer’s instructions, in the
presence of 40 units RNasin (Promega).
Relative quantification of genes was performed using
an ABI Prism 7500 Sequence Detection System (Applied
Biosystems). PCRs used 5 ?l of reverse-transcribed RNA (a
10-fold dilution of cDNA was used for 18S analyses). The
MMP and ADAMTS primers and probes were previously
described (18,19). The primers and probes for IL8, IL6, INOS,
Nrf2, A20, I?B? , COX2, SOD2, and HMOX1 were designed
using the Universal Probe Library (Roche). Relative quantifi-
cation is expressed as 2??Ct, and all data were normalized to
18S ribosomal RNA expression.
Total RNA was extracted and purified from whole
mouse joints, using TRIzol reagent (Invitrogen) according to
the manufacturer’s instructions. RNA quality was analyzed
using an Agilent 2100 Bioanalyzer. Sample replicates were
pooled and hybridized to an Illumina Mouse WG-6 whole-
genome array (Source BioScience). Probe signal underwent
quantile normalization, and messenger RNA (mRNA) levels
were validated by qRT-PCR in replicates.
Gene silencing. Human articular chondrocytes were
transfected using DharmaFECT 1 (Thermo Scientific) with 25
nM siRNA against Nrf2 or nontargeting AllStars siRNA
(Qiagen) for 24 hours prior to SFN and cytokine treatments.
All treatments were carried out in quadruplicate. Gene expres-
sion was measured using qRT-PCR.
Western blotting. Whole cell lysates were harvested
into ice-cold radioimmunoprecipitation assay buffer (50 mM
Tris HCl, pH 7.6, 150 mM NaCl, 1% v/v Triton X-100, 1%
weight/volume sodium deoxycholate, 0.1% w/v sodium dodecyl
sulfate, 10 mM NaF, 2 mM Na3VO4, 1? protease inhibitor
cocktail [Fisher Scientific]). Cytosolic and nuclear cell fractions
were obtained by adding 500 ?l hypotonic buffer (20 mM Tris
HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2) to cell pellets and
incubated for 15 minutes on ice. Nonidet P40 (NP40; 25 ?l
10% v/v) was added and vortexed for 10 seconds. Samples were
centrifuged for 10 minutes at 300g. Supernatant was collected
(cytosolic fraction) and stored at ?20°C. Fifty microliters of
nuclear extraction buffer (100 mM Tris HCl, pH 7.4, 100 mM
NaCl, 1% v/v Triton X-100, 1 mM EDTA, 1 mM EGTA, 10%
v/v glycerol, 0.1% w/v sodium dodecyl sulfate [SDS], 0.5% w/v
deoxycholate, 1? protease inhibitor cocktail, and phosphatase
SULFORAPHANE IS PROTECTIVE IN THE ARTICULAR JOINT3131
inhibitors) was added to the pellets and incubated for 30
minutes on ice, with vortexing every 10 minutes. Samples were
centrifuged at 14,000g for 3 minutes at 4°C. Supernatants were
stored at ?80°C. Samples were separated on reducing SDS–
polyacrylamide gel electrophoresis gels, transferred to PVDF
membranes, and probed overnight at 4°C. Proteins were
detected using horseradish peroxidase–conjugated secondary
antibodies (Dako). Bands were visualized using LumiGLO
reagent (New England Biolabs) and exposure to Kodak Bio-
Max MS film (Sigma-Aldrich).
Immunocytochemical analysis. Human articular chon-
drocytes were grown on chamber slides at a density of 3.75 ?
104/cm2and treated with SFN (10 ?M) for 30 minutes prior to
stimulation with IL-1 (5 ng/ml) for 45 minutes. Cells were
probed for NF-?B p65 rabbit polyclonal antibody (Santa Cruz
Biotechnology) at 1:100 dilution, followed by the secondary
antibody, Cy3-conjugated goat anti-rabbit IgG (Abcam) at
1:200 dilution. Nuclei were stained with DAPI and examined
using a Zeiss AxioPlan 2IE fluorescence microscope at 20?
magnification. Negative controls omitted the primary anti-
body. Images were acquired and analyzed with AxioVision
version 4.7 software.
Electrophoretic mobility shift assay (EMSA). For the
preparation of nuclear extracts, cells were lysed in 0.1% v/v
NP40 in phosphate buffered saline on ice for 1 minute, and
then centrifuged. Thereafter, the pellets were suspended in 3?
volume high-salt buffer (25 mM HEPES, pH 7.8, 500 mM KCl,
0.5 mM MgSO4, 1 mM dithiothreitol [DTT], protease, and
phosphatase inhibitors]), and incubated for 20 minutes on ice,
with occasional mixing. Samples were centrifuged at high
speed for 2 minutes, and supernatant was stored at ?80°C.
Protein was quantified using Bradford Reagent (Bio-Rad).
Nuclear extracts were analyzed for DNA binding using the
Li-Cor protocol for NF-?B IRDye 700–labeled oligos. Nuclear
extracts containing 5 ?g total protein were added to the binding
reactions at room temperature for 20 minutes in the dark. DNA
binding was visualized using an Odyssey infrared imaging
system (Li-Cor). The NF-?B consensus sequence (mutant
G/C) was 5?-AGTTGAGGG/CGACTTTCCCAGGC-3?.
Transfection and gene promoter reporter assay. SW-
1353 cells were plated at 2 ? 104/well in a 24-well plate and left
to adhere. Transfections were carried out using 200 ng plasmid
DNA and 0.5 ?l Lipofectamine 2000 (Fisher Scientific) for 24
hours. The culture medium was changed to serum-free over-
night, after which the cells were treated for 6 hours. Fifty
microliters of luciferin substrate (Promega) was added to 10 ?l
cell lysate, and luminescence was measured immediately using
an EnVision Multilabel Plate Reader (PerkinElmer).
High-performance liquid chromatography tandem
mass spectrometry (HPLC-MS/MS) analysis. Primary chon-
drocytes or SW-1353 cells were seeded at a density of 1.7 ?
104/cm2and grown to confluence. Medium was replaced with
phenol-free/serum-free DMEM containing 10 ?M SFN and
incubated for 0–2 hours. Samples were acidified with formic
acid, and the internal standard iberin (10 ?M) was added.
HPLC-MS/MS analysis was carried out using an Agi-
lent 1200 Series HPLC System linked to an AB Sciex Q-Trap
3200 MS/MS system. Separation was performed using a Kine-
tex pentafluorophenyl reverse-phase HPLC column (2.6 ?m,
100 ? 4.60 mm; Phenomenex) at 37°C. The flow rate was 1
ml/minute, using 0.1% v/v formic acid in water and 0.1% v/v
formic acid in acetonitrile; the initial gradient was 5% and
increased to 35% over 12 minutes.
Analytes were detected with electrospray ionization
using multiple reaction monitoring in the positive mode, based
on the following precursor and product ions: SFN (mass/
charge [m/z] 178, 119, 114, 72, 55), SFN–glutathione (SFN–
GSH; m/z 485, 356, 308, 179, 114), SFN–Cys (m/z 299, 178, 136,
114), SFN–Cys-Gly (m/z 356, 179, 162, 136, 1,140), and SFN–
N-acetylcysteine (SFN–NAC; m/z 341, 212, 178, 130, 114).
Iberin (m/z 164, 105, 77, 72) was used as an internal standard.
Acquisition and quantification were performed using Analyst
software (Applied Biosystems).
In vitro cartilage degradation assays. Cartilage ex-
plants were pretreated with 0–30 ?M SFN. The cytokines IL-1
or IL-1/OSM (0.5 ng/ml and 5 ng/ml, respectively) were added
to induce cartilage breakdown. The treatments were refreshed
every 2 days over 14 days. All treatments were performed in
quadruplicate. The remaining cartilage was papain-digested
overnight at 65°C. Glycosaminoglycan (GAG) and hydroxypro-
line were measured in the medium as described previously
(20,21) and expressed as the percent release of the total.
Animals used in the experiments. C57BL/6 mice were
purchased from Harlan UK. The animal experiments were
performed following ethics and statutory approval, in accor-
dance with local policy. The mice were maintained at 21°C in
standard, individually ventilated cages holding 3–6 mice per
cage. The mice were fed a certified mouse diet (RM3; Special
Dietary Systems) and water ad libitum. The diets were changed
to AIN-93G or AIN-93G containing 0.18 or 0.6 gm/kg SFN
(Research Diets) for 2 weeks prior to and following surgery,
until the mice were killed.
Destabilization of the medial meniscus (DMM) model.
Ten-week-old male mice were anesthetized by inhalation of
isoflurane (3% for induction and 1.5–2% for maintenance) in
oxygen (1.5–2 liters/minute). All mice received a subcutaneous
injection of buprenorphine (Alstoe Animal Heath) postsur-
gery. The mice were fully mobile within 4–5 minutes after
withdrawal of isoflurane.
DMM was performed as previously described (22), and
sham surgery consisted of capsulotomy only (23). The con-
tralateral (left) knees for both procedures served as unoper-
ated controls. OA was scored by 2 individuals (TLV and one
other) in a blinded manner, using a validated histologic scoring
system, as described previously (22), and results were ex-
pressed as the summed score (sum of the 3 highest total section
scores for any given joint [minimum of 8 sections per joint, 80
microns apart]) (16,22,23).
Statistical analysis. Student’s t-test and one-way and
two-way analysis of variance (ANOVA) with Dunnett’s or
Bonferroni post-test, respectively, were performed using
GraphPad Prism version 5.00 for Windows. One-way ANOVA
was used when testing for differences between ?3 groups.
Two-way ANOVA was used when testing for an effect of 2
factors (e.g., treatment and time).
Inhibition of cytokine-induced MMP expression
in chondrocytes and synovial cells by SFN. OA affects
all of the tissue in the joint. We sought to determine
3132 DAVIDSON ET AL
quantitatively whether SFN could regulate key aggreca-
nases and collagenases in chondrocytes and synovial
cells. SFN inhibited cytokine-induced MMP expression
in human articular chondrocytes, FLS, and SW-1353
cells in a a dose-dependent manner (Figures 1A–C). In
human articular chondrocytes, 2.5 ?M SFN significantly
inhibited cytokine-induced ADAMTS4 and ADAMTS5
expression, 2.5 ?M SFN inhibited MMP1, and 5 ?M SFN
inhibited MMP13 (Figure 1A). Inhibition of gene ex-
pression in FLS or SW-1353 chondrosarcoma cells ap-
peared to be less sensitive. In FLS, 5 ?M SFN inhibited
MMP1, and 10 ?M inhibited MMP13 (Figure 1B). In
SW-1353 cells, 10 ?M SFN inhibited MMP1, and 5 ?M
inhibited MMP13 (Figure 1C). FLS and the SW-1353
cell line did not express robust levels of ADAMTS4 or
ADAMTS5, and therefore these were not measured.
SFN did not affect the expression of MMP2 in SW-1353
cells (data not shown).
Effect of SFN on histone deacetylase inhibition
in chondrocytes. We investigated the potential of SFN as
a HDAC inhibitor in human articular chondrocytes.
Whole cell lysates from chondrocytes were immuno-
blotted for histone H3 acetylation and general lysine
acetylation. Acetylation of histone H3 or general lysine
acetylation were unaltered by 0–30 ?M SFN (Figure
1D). Similar results were seen in SW-1353 cells (data not
shown). MMP28 is known to be regulated by HDAC
inhibition in SW-1353. MMP28 mRNA levels were not
affected by SFN (Figure 1D).
Cell viability. Cytotoxicity and activation of
caspases 3/7 were measured in primary chondrocytes,
FLS, and SW-1353 cells (n ? 3) treated with 0–50 ?M
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 ?M 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
2??Ct); 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.
SULFORAPHANE IS PROTECTIVE IN THE ARTICULAR JOINT 3133
SFN or 10 ?M staurosporine, in quadruplicate for 6
city or caspase activation by SFN in these cells (data not
Effect of Nrf2 knockdown on MMP expression in
chondrocytes. The Nrf2 signaling pathway is a major
mediator of SFN activity. We examined whether knock-
down of Nrf2 could affect SFN-induced inhibition of
MMP expression in chondrocytes. Treatment with SFN
significantly induced expression of HMOX1 (an Nrf2-
regulated gene) in human articular chondrocytes in a
dose-dependent manner (Figure 2A). Small interfering
RNA against Nrf2 reduced Nrf2 expression in human
articular chondrocytes (Figure 2B), and SFN-induced
HMOX1 expression was significantly reduced by Nrf2
siRNA compared with that induced by nontarget control
(Figure 2C). IL-1/OSM–induced MMP1 expression was
inhibited with SFN treatment, and Nrf2 knockdown did
not reverse the SFN inhibition of cytokine-induced
MMP1 expression (Figure 2D).
Prolongation of MAPK activation by SFN. We
examined the effects of SFN on MAPK activation in
primary human articular chondrocytes. SFN affected the
phosphorylation kinetics of both JNK and p38 MAPK.
Phosphorylation of JNK and p38 MAPK was sustained
for a longer period of time with SFN pretreatment
compared with IL-1 treatment alone. An unidentified
higher–molecular weight band for phosphorylated p38
MAPK was seen in IL-1–treated samples, which was
inhibited by pretreatment with SFN between 15 minutes
and 60 minutes. SFN treatment did not affect ERK
signaling in human articular chondrocytes (Figure 3A).
Direct modulation of NF-?B signaling in human
articular chondrocytes by SFN. We examined the effect
of SFN on NK-?B signaling in chondrocytes. SFN
treatment of human articular chondrocytes delayed the
reaccumulation of I?B? following NF-?B activation by
IL-1 (Figure 3B). However, phosphorylation of p65
(Ser536) (Figure 3B) and translocation of p65 to the
nucleus (Figure 3C) were unaffected by SFN treatment
in human articular chondrocytes. EMSAs for DNA
binding were performed in human articular chondro-
cytes (Figure 3D) and SW-1353 cells (data not shown).
Specific binding of the NF-?B consensus sequence was
detected by the appearance of 2 bands in human artic-
ular chondrocytes when incubated with nuclear extracts
from human articular chondrocytes treated with IL-1 for
45 minutes. These 2 bands were blocked with the
addition of anti-p65 or anti–p50 NF-?B antibodies,
respectively. These bands could also be competed with
unlabeled wild-type but not mutant oligonucleotide,
demonstrating specificity. Nuclear extracts from human
articular chondrocytes pretreated with SFN prior to
treatment with IL-1 showed substantially diminished
binding of the upper band and complete abrogation of
binding to the lower band. Acetylated proteins in the
upper of the 2 bands containing p65 and p50 complexes
were detected, whereas the lower band remained largely
unaffected by the addition of pan–acetylated lysine
antibody. The addition of 10 ?M exogenous SFN directly
into the DNA-binding reaction did not affect NF-?B
binding (Figure 3D). A luciferase-linked ?B-promoter
reporter assay showed that pretreatment with 10 ?M
SFN significantly inhibited IL-1–induced NF-?B signal-
ing (P ? 0.0001) (Figure 3D).
Effect of SFN on the expression kinetics of a
panel of known NF-?B–responsive genes in human
articular chondrocytes. The regulation of NF-?B signal-
ing by SFN was confirmed by investigating the expres-
sion of NF-?B–responsive genes in cultured human
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 ?M) 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 ribo-
somal 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 ?M SFN (A) or as indicated.
NT ? nontargeting siRNA control (see Figure 1 for other definitions).
3134 DAVIDSON ET AL