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 JOINT3133
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
articular chondrocytes. HMOX1 was measured as an
example of specific SFN activity in human articular
chondrocytes not influenced by IL-1 treatment (Figure
4A). HMOX1 was highly expressed with SFN treatment,
beginning at 2 hours (P ? 0.001). Expression of I?B?
was significantly inhibited from 60 minutes to 4 hours,
but I?B? mRNA levels, with and without SFN treat-
ment, were equal by 8 hours (Figure 4A). SFN treatment
significantly inhibited the transcription of inflammatory
NF-?B–responsive genes induced by IL-1 (IL6, IL8,
iNOS, and MMP13) at 4 and 8 hours (Figure 4B). COX2
mRNA expression was not significantly affected. Expres-
sion of the cytoprotective gene A20 was completely
abolished with SFN treatment at 1–8 hours (Figure 4C).
Expression of SOD2 was more similar to that of I?B?,
with inhibition between 2 and 4 hours and mRNA levels
equal by 8 hours (Figure 4C).
Accumulation of SFN metabolites in chondro-
cytes. The 10-?M SFN treatment applied to chondro-
cytes in culture was not sufficient to inhibit the direct
NF-?B/DNA binding reaction on EMSA (Figure 3D).
We determined the intracellular concentration of SFN
and its metabolites in primary chondrocytes and SW-
1353 cells. The accumulation of SFN and its metabolites
was characterized within chondrocytes following the
addition of 10 ?M SFN to culture medium. Parallel
incubations of SFN were conducted in cell-free medium
(DMEM) to control for degradation of media matrix.
SFN–Cys, SFN–Cys-Gly, SFN–GSH, SFN–NAC, and
SFN were detected in primary chondrocytes and/or
culture medium (Figure 5A) and SW-1353 cells (results
not shown). Primary chondrocytes treated with 10 ?M
SFN for 0–2 hours showed a peak accumulation within
10–15 minutes, at 150–275 ?M (Figure 5B). SW-1353
cell accumulation occurred within 1 hour, at 1–1.6 mM
(results not shown). In both cell types, the predominant
intracellular form of SFN was SFN–GSH. Free SFN was
not detected intracellularly. Exogenous SFN–GSH or
exogenous SFN was titrated into nuclear extract/DNA–
binding reactions. Both SFN–GSH and SFN (Figure 5C)
diminished NF-?B–DNA binding. Incubation of SFN–
GSH or SFN with DTT prior to being added to the
DNA-binding reaction rescued NF-?B/DNA binding
Chondroprotective effect of SFN in vitro and
in vivo. We used a short-term in vitro bovine nasal
cartilage model of cartilage destruction to investigate
the chondroprotective activity of SFN in tissue (24). On
Figure 3. SFN regulates MAPK activation and NF-?B 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-?B in human articular chondrocytes treated with IL-1 (5
ng/ml) for 0–60 minutes, with or without 10 ?M 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 ?M SFN. Bars ? 100 ?m. 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-?B 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-?B transcriptional activation in chondrocytes by 10 ?M SFN, as determined by luciferase-linked ?B-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).
SULFORAPHANE IS PROTECTIVE IN THE ARTICULAR JOINT3135
day 2, IL-1/OSM–induced GAG release was significantly
repressed by 10 ?M SFN and was further repressed, in a
dose-dependent manner, by 15 and 20 ?M SFN (Figure
6A). IL-1/OSM–induced hydroxyproline release was also
significantly inhibited by 10 ?M SFN on day 11 (Figure
We sought to confirm the data in an in vivo
murine model of cartilage destruction. Among mice that
underwent DMM, those fed an SFN-rich diet (3 ?moles/
day) had significantly reduced cartilage destruction at 12
weeks compared with those fed a control diet (P ?
0.004) (Figure 6C). There was no significant alteration in
the body weight of mice across the experiment (data not
shown). Whole-genome microarray analysis of mouse
whole-joint tissue at 24 hours and the day 7 postopera-
tive time points identified a number of regulated
mRNAs. Candidate genes with expression changes of
?1.5-fold were validated using qRT-PCR. Procollagen,
Col2a1, and Col10a1 mRNA levels were significantly
increased in the destabilized joints compared with con-
trols (P ? 0.028 and P ? 0.035, respectively) on day 7.
Increased expression of Col2a1 and Col10a1 mRNA was
not observed in mice fed an SFN-rich diet (P ? 0.026
and P ? 0.015, respectively). The SFN diet had no effect
on basal Col2a1 and Col10a1 expression (Figure 6D).
The regulation of catabolic factors in more than
one joint tissue is important in OA (25). Our data for
primary chondrocytes demonstrate that SFN dose-
dependently inhibited IL-1/OSM–induced expression of
collagenases (MMP1 and MMP13) and aggrecanases
(ADAMTS4 and ADAMTS5). These findings support an
earlier report that SFN inhibited IL-1/TNF?–induced
collagenase expression (10). We also observed SFN-
induced inhibition of collagenase expression in OA FLS.
In rheumatoid synovial cells, SFN was reported
to inhibit hyperplasia and induce apoptosis (12), al-
though SFN was administered at high doses in vivo via
intraperitoneal injection. Our measurements in pri-
mary human articular chondrocytes, SW-1353 cells, and
FLS (data not shown) are consistent with previous
reports (10,26) that SFN is not cytotoxic at concentra-
tions attained in the plasma from dietary intake (27).
We initially hypothesized that SFN was chondro-
protective via the inhibition of HDAC activity, because
SFN has been reported to be a weak HDAC inhibitor in
other cell types (17,28). We did not observe any evi-
dence to support SFN as an inhibitor of HDAC in
chondrocytes, either directly or as an inducer of MMP28.
SFN is a potent inducer of the transcription
factor Nrf2, and Nrf2 is an important mediator of SFN
activity in several cell types (6). Interestingly, Guille ´n et
al showed up-regulation of HMOX1 (an Nrf2-regulated
gene) by cobalt protoporphyrin IX in chondrocytes
coincided with a reduction in MMP-1 and MMP-13
Figure 4. SFN treatment inhibits expression of known NF-?B–
responsive genes. Human articular chondrocytes were treated with 5
ng/ml IL-1 for 0–8 hours, with (?) or without (▫) 10 ?M SFN. Gene
expression was measured using real-time quantitative reverse
transcription–polymerase chain reaction and normalized to 18S ribo-
somal RNA expression. A, Expression of HMOX1 and I?B?. 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.
3136 DAVIDSON ET AL
expression and an increase in type II collagen and
aggrecan expression (29). Our knockdown of Nrf2
showed that SFN remained able to inhibit cytokine-
induced MMP1 expression, independently of Nrf2; how-
ever, activation of Nrf2 by SFN will likely contribute
chondroprotective effects through the induction of cyto-
protective genes, including HMOX1 (30–33).
Regulation of NF-?B signaling by SFN has been
reported. Inhibition of I?B phosphorylation and/or de-
gradation, IKK phosphorylation, and NF-?B nuclear
translocation by SFN are described in various cell types,
including chondrocytes (11,34–36). We did not observe
any evidence demonstrating that SFN regulated these
mechanisms in primary human articular chondrocytes.
Moreover, our studies showed that SFN treatment af-
fected the reaccumulation kinetics of I?B?, likely due to
postactivation inhibition of NF-?B (37). This was sup-
ported by the complete ablation of I?B? and A20
expression required for signaling through the negative
feedback loop of NF-?B.
In accordance with our data, Heiss et al (37)
reported that SFN inhibited NF-?B/DNA binding and
did not affect I?B degradation or NF-?B nuclear trans-
location in response to lipopolysaccharide. Although the
trans-activating inhibitory activity of NF-?B by SFN
cannot be ruled out (38), our evidence suggests that
SFN-induced inhibition of NF-?B signaling is generic,
given that the expression of both proinflammatory and
cytoprotective genes regulated by NF-?B was decreased,
albeit with distinct gene-dependent kinetics.
NF-?B is a known redox-sensitive transcription
factor requiring a reducing environment for DNA bind-
ing. Heiss et al proposed that SFN could inhibit NF-?B
directly by forming dithiocarbamates with NF-?B Cys
residues or indirectly via inhibiting, e.g., thioredoxin/
thioredoxin reductase, thereby modulating redox poten-
Figure 5. Sulforaphane (SFN) accumulates primarily as SFN–glutathione (SFN–GSH) in chondrocytes and can directly inhibit NF-?B binding to
the consensus DNA sequence. Cell-free medium (control) and primary chondrocytes were treated with 10 ?M 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-?B 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 ?M
exogenous SFN–GSH or exogenous SFN and 17.9 mM DTT). m/z ? mass/charge (see Figure 3 for other definitions).
SULFORAPHANE IS PROTECTIVE IN THE ARTICULAR JOINT3137
tial (37,38). This proposition was supported by the
observation that a reducing agent reversed the SFN-
induced inhibition of transcription factor binding (39),
although it is in opposition with thioredoxin being an
Nrf2-responsive gene (40). SFN has been reported to
accumulate intracellularly via conjugation with glutathi-
one in a cell-dependent manner (41,42). We show that
SFN at high concentrations (between 150–275 ?M)
accumulates primarily as SFN–GSH in primary human
chondrocytes, and this concentration of SFN–GSH can
directly inhibit NF-?B/DNA binding to the NF-?B con-
Kim et al previously reported that SFN inhibited
cytokine-induced NF-?B DNA binding and JNK activa-
tion (10). Our results corroborate these data in part,
although we did not observe consistent inhibition of
JNK activation in primary human articular chondrocytes
across donors. We did observe that compared with IL-1
alone, the addition of SFN consistently prolonged JNK
and p38 MAPK phosphorylation. Extended MAPK ac-
tivation has been attributed to a lack of NF-?B feed-
back and TNF-induced accumulation of reactive oxygen
species (43). In other cell types, this leads to cell death,
which was not observed in our models. It has been
reported that direct phosphorylation of Nrf2 by the
p38? MAPK isoform promoted the association between
Nrf2 and Kelch-like ECH-associated protein 1 proteins
and subsequent inhibition of Nrf2 activity (44). Our
data suggest that SFN can inhibit an isoform of p38
MAPK; if this is confirmed, it would be consistent with
the prosurvival/antiapoptotic properties of SFN reported
in chondrocytes as p38 dependent (26). It remains un-
clear whether the effects of SFN on MAPK signaling are
additive, synergistic, or a result of altered NF-?B signaling.
The protective effect of SFN at the tissue level in
bovine nasal cartilage explants supports recent findings
by Kim et al (11). We now show that SFN obtained from
the diet is protective against OA in vivo. Because mice
feed by grazing rather than by consuming a daily meal,
there is no easy means with which to compare mice with
humans. However, the dosage chosen (3 ?moles/day)
gives an overall delivery that is deemed to be the high
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 ?M 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 ?m) 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/
3138DAVIDSON ET AL
end of physiologic (?2.3–7.4 ?moles/liter in plasma in
humans) (27,45). A whole-genome array analysis of
mouse whole knee joint tissue uncovered a number of
regulated genes. Full analysis of these data is beyond the
scope of this study; however, regulation of Col2a1 and
Col10a1 was of particular interest. The increased expres-
sion of Col2a1 and Col10a1 on day 7 after DMM was
performed may represent facets of both protective and
injury responses, respectively.
COL2A1 expression is regulated cooperatively by
SOX9, L-SOX5, and SOX6 (46–48), and it is reported
that NF-?B p65 activity may precede that of SOX9 to
initiate chondrogenic differentiation (49). More re-
cently, p65 was shown to specifically bind the COL2A1
intronic enhancer to regulate COL2A1 as well as SOX9
expression (50). Hypoxia-inducible factor 2?1, encoded
by EPAS1, has been reported as the most potent trans-
activator of COL10A1 expression in cultured chondro-
cytes, and EPAS1 itself is strongly induced by NF-?B p65
activity (51). Mice fed the SFN-rich diet did not show
increased expression of Col2a1 or Col10a1 mRNA in
response to DMM surgery, potentially because of a
dampened NF-?B response in vivo. Our data therefore
show that a high-glucosinolate diet may be a useful
measure either to prevent or to slow the progression of
We thank Dr. Stuart Rushworth (University of East
Anglia) for his advice on NF-?B studies and Dr. Maria
O’Connell (University of East Anglia) for sharing her expertise
regarding Nrf2. We thank Linh Le for purifying mRNA from
mouse knee joints. We also thank Adele Cooper (Norfolk and
Norwich University Hospitals) for her work enabling us to
obtain human tissue samples and Olga Boruc (KIR, UK) for
her expertise in performing DMM in mice. Thanks to H. G.
Blake (Costessey, Norfolk, UK) for the generous gift of bovine
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Clark had full access to all of the
data in the study and takes responsibility for the integrity of the data
and the accuracy of the data analysis.
Study conception and design. Davidson, Kay, Culley, Bao, Clark.
Acquisition of data. Davidson, Jupp, de Ferrars, Norton, Driscoll,
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