p21 deficiency is susceptible to osteoarthritis through STAT3 phosphorylation
Preparation of human cartilage
Cartilage tissues were obtained during total hip joint replacement surgery from 11
patients with OA. Diagnosis of OA was based on clinical, laboratory, and radiographic
evaluations. Normal cartilage tissues were obtained during surgery for femoral neck
fractures from eight patients with no history of joint disease and with macroscopically
normal cartilage. All the samples were obtained in accordance with the World Medical
Association Declaration of Helsinki Ethical Principles for Medical Research Involving
Human Subjects. The study protocol was approved by Kobe University Graduate school
of Medicine Ethics Committee, and all patients gave their informed consent.
Cell culture
Primary chondrocytes were isolated and cultured from the cartilage tissues. Tissues
were minced and incubated with trypsin (0.5 mg/ml; Sigma-Aldrich, St. Louis, MO, USA)
for 15 minutes at 37 °C, after which the cartilage was treated with Dulbecco’s modified
Eagle’s medium (DMEM; Gibco/Life Technologies, Grand Island, NY, USA) containing 0.2
% collagenase (Sigma-Aldrich) at 37 °C for 15 h. Dissociated cells were cultured in
DMEM supplemented with 10 % fetal bovine serum (BioWhittaker FBS; Lonza, Walkersville,
MD, USA) and 100 U/ml penicillin-streptomycin. After overnight culture, nonadherent
cells were removed and adherent cells were further incubated on a 6-well plate in
fresh medium (3?×?10
5
cells/well). All experiments were conducted using first-passage cells. To characterize
the chondrocytes, we confirmed that the type II collagen showed higher expression
in the normal human hip chondrocytes than in the OA knee chondrocytes and that type
X collagen showed higher expression in the OA chondrocytes than in the normal chondrocytes.
Cell culture and exposure to cyclic tensile strain
Normal human knee chondrocytes (NHAC-kn; Cambrex, Charles City, IA, USA) were cultured
in a humidified atmosphere of 5 % CO
2
and 95 % air at 37 °C in a BulletKit (Cambrex). NHAC-kn were derived from a single-donor
knee articular cartilage and used as an established normal chondrocyte cell line 27], 28]. To characterize the chondrocytes, we confirmed that NHAC-kn expressed type II collagen
and sulfated proteoglycans, but not type X collagen, before use.
Before we performed the experiments, cells were grown to a subconfluent state (3?×?10
5
cells/well) in a 6-well plate and were then plated onto a silicon chamber coated with
fibronectin (Sigma-Aldrich) at a density of 3?×?10
5
cells/well in DMEM/F-12 supplemented with 10 % FBS and 100 U/ml penicillin-streptomycin.
Cyclic tensile strain experiments were performed using an ST-140 cyclic tensile strain
system (STREX, Osaka, Japan). Cyclic tensile strain was enforced at 3 %, 5 %, 8 %,
and 10 % elongation for 3 h (0.5 Hz) according to previous studies 23], 29], 30]. We used 5 % tensile strain as normal joint loading and 10 % tensile strain as excessive
loading to investigate the relationship between mechanical stress and cartilage metabolism.
Small interfering RNA transfection
Lipofectamine 2000 reagent was used to transfect p21 small interfering RNA (siRNA)
and nonspecific siRNA control into normal human knee chondrocyte monolayers according
to the recommendations of the manufacturer (Life Technologies, Carlsbad, CA, USA).
Briefly, 1 day before transfection, cells were plated in a 6-well plate in growth
medium without antibiotics to attain 30–50 % confluence at the time of transfection.
Subsequently, 100 pmol of siRNA and Lipofectamine 2000 complexes were prepared and
added to each well. After 12 h of transfection, the complexes were removed, and fresh
medium containing 10 % FBS was added. After an additional 12 h, the cells were replaced
onto a silicone chamber coated with fibronectin at a density of 3?×?10
5
cells/well, and cyclic tensile strain was applied.
Quantitative reverse transcription polymerase chain reaction analysis
Chondrocytes were cultured in 6-well plates with various stimulations, and RNA was
extracted using a QIAshredder and RNeasy Mini Kit (Qiagen, Hilden, Germany) according
to the manufacturer’s protocol. Briefly, 1 ?g of total RNA was reverse-transcribed
to first-strand cDNA with 1.25 ?M oligo(dT) primer in 40 ?l of polymerase chain reaction
(PCR) buffer II containing 2.5 mM MgC1
2
, 0.5 mM deoxyribonucleotide triphosphate mix, 0.5 U of RNase inhibitor, and 1.25
U of murine leukemia virus reverse transcriptase (PerkinElmer/Applied Biosystems,
Foster City, CA, USA) at 42 °C for 60 minutes.
The relative expression levels of mRNA encoding human p21, collagen, type II, alpha 1 (COL2A1), ACAN, MMP3, MMP13, ADAMTS4, and ADAMTS5 were analyzed by SYBR Green real-time PCR using an ABI Prism 7700 sequence detection
system (Applied Biosystems). The relative expression of the genes of interest was
normalized against the GAPDH housekeeping gene by using the comparative cycle threshold (C
t
) method. The difference between the mean C
t
values of the gene of interest and the housekeeping gene is denoted as ?C
t
, and the difference between ?C
t
and the C
t
value of the calibrator sample is denoted as ??C
t
. The log
2
(??C
t
) value gives the relative level of gene expression. The primer sequence for detection
of human p21, COL2A1, ACAN, MMP3, MMP13, ADAMTS4, and ADAMTS5 are described in the Additional file 1.
Western blot analysis
Chondrocytes were washed three times with phosphate-buffered saline and lysed in a
hypotonic lysis buffer (25 mM Tris, 1 % Nonidet P-40, 150 mM NaCl, 1.5 mM ethylene
glycol tetraacetic acid) supplemented with a protease and phosphatase inhibitor mix
(Roche Diagnostics, Basel, Switzerland) on ice for 20 minutes 31]. The lysates were centrifuged at 15,000 rpm for 20 minutes to remove cellular debris,
and the supernatants were collected. Cytoplasmic proteins were quantified by using
the Bradford method with a protein assay reagent (Bio-Rad Laboratories, Hercules,
CA, USA) and diluted to an equal concentration with the hypotonic buffer. The expression
of p21 protein was detected using rabbit anti-p21 polyclonal antibody (Ab) (Cell Signaling
Technology, Danvers, MA, USA). The expression levels of phosphorylated STAT3 (phospho-STAT3
or p-STAT3) and STAT3 were detected using rabbit anti-phospho-STAT3 and anti-STAT3
Ab (Cell Signaling Technology).
Horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) Ab
(GE Healthcare Bio-Sciences, Piscataway, NJ, USA) or HRP-conjugated rabbit anti-mouse
IgG Ab (GE Healthcare Bio-Sciences) was used as a secondary antibody, and the signals
were visualized using Amersham ECL Plus reagent (GE Healthcare Life Sciences, Little
Chalfont, UK) with the LAS-3000mini chemiluminescent image analyzer (FUJIFILM Life
Science, Tokyo, Japan). Actin was used as a control to estimate protein loading on
the gel.
Generation of homozygous mice
Homozygous B6.129S6(Cg)-Cdkn1a tm1Led
/J mice were obtained from The Jackson Laboratory (Bar Harbor, ME, USA). We backcrossed
the mice against a C57BL/6 background and studied male mice at 10 weeks of age. All
the mice used in this study were backcrossed over 20 generations. p21 +/+
littermates were used as wild-type controls. Genotyping was performed by PCR amplification
of mouse-tail DNA by using allele-specific probes. Each experimental group contained
at least six mice.
In vivo mouse OA model
This study was carried out in strict accordance with the recommendations contained
in the Guide for the Care and Use of Laboratory Animals of the National Institutes
of Health. All procedures were approved by the Animal Studies Committee of Kobe University,
Japan (permit number P131104). Ten-week-old male p21
?/?
and p21
+/+
mice were used in these experiments. Mice were anesthetized using an intraperitoneal
injection of ketamine (100 mg/kg). Destabilization of the medial meniscus (DMM) was
induced in the right knee joint by transecting the anterior attachment of the medial
meniscotibial ligament as described previously 32]. For the control, surgery was performed on the right knee joints where the ligaments
were intact and termed sham. All mice were subjected to weight-bearing following recovery from anesthesia. The
mice were sacrificed 8 weeks after DMM or sham surgery, and their tissue was subjected
to histological evaluation. Six mice were analyzed for each group: p21
+/+
sham, p21
+/+
DMM, p21
?/?
sham, and p21
?/?
DMM (total of 24 analyzed mice).
Histological evaluation for cartilage degeneration
Mouse knee joints were fixed with 4 % paraformaldehyde for 24 h, decalcified with
14 % ethylenediaminetetraacetic acid for 7 days, and embedded in paraffin. Coronal
histological sections were taken through the joint at 80-?m intervals and stained
with Safranin O and Fast Green. OA histopathology was evaluated by using the Osteoarthritis
Research Society International (OARSI) cartilage OA histopathology scoring system
33]. Histological scores were measured in four quadrants (medial femoral condyle, medial
tibial plateau, lateral femoral condyle, and lateral tibial plateau) of the knee joints
at all sectioned levels (eight sections per knee). A summed OA score was calculated
from all four quadrants for all sections, which represented the changes across the
whole joint.
Immunohistochemistry
Deparaffinized sections were digested with proteinase (Dako, Glostrup, Denmark) for
10 minutes and treated with 3 % hydrogen peroxide (Wako Pure Chemical Industries,
Osaka, Japan) to block endogenous peroxidase activity. The sections were treated with
a 1:100 dilution of antiphosphorylated STAT3 (Cell Signaling Technology), anti-MMP-13
(Abcam, Cambridge, UK), and anti-F4/80 (AbD Serotec, Kidlington, UK) antibodies at
4 °C overnight and were subsequently treated with peroxidase-labeled antirabbit Ig
(Histofine Simple Stain MAX PO; Nichirei Bioscience, Tokyo, Japan) at room temperature
for 30 minutes. The signal was developed as a brown reaction product by using the
peroxidase substrate 3,3?-diaminobenzidine (Histofine Simple Stain DAB Solution; Nichirei
Bioscience), and the sections were examined microscopically. Methyl green stain was
used as a counterstain. One sagittal section from the center of the most severe OA
lesion in each tibial plateau was scored. The number of stained cells was counted
in three areas of high-magnification fields at both superficial and deep zones of
the cartilage tissue individually by three blinded observers. The average percentage
of p-STAT3– and MMP-13–positive cells/total cells were calculated. One sagittal section
each from six mice was evaluated in each group. These positive cells were included
superior to the tidemark and were included for both femur and tibia.
Statistical analysis
Statistical analysis was performed using one-way (Fig. 1a, c) or two-way (Figs. 2, 3e, 4e and 5e) analysis of variance with Tukey’s post hoc test for multiple comparisons of paired
samples. The Mann–Whitney U test was used for comparisons between the two groups (Fig. 6a, b). P values less than 0.05 were considered significant. The results are presented as mean
values with 95 % confidence intervals (95 % CIs) and were considered statistically
significant at P??0.05.
Fig. 1. Expression of p21, ACAN, and MMP-13 without and with STAT3 inhibitor. a Expression levels of p21 mRNA were quantified by real-time PCR. Expression ratios of p21 are shown. Columns represent mean ratios with 95 % CI at 3, 5, 8, and 10 % strain/nonstrain
control. b p21 and phospho-STAT3 were analyzed by Western blotting. The results shown represent
three independent experiments. c The effect of a STAT3-specific inhibitor on p21-regulated ACAN and MMP-13 expression.
Chondrocytes were transfected with p21 siRNA or nonspecific control siRNA for 12 h,
and 5 % cyclic stretch stress was introduced for 3 h in the presence of 50 ?M DMSO
or STAT3-specific inhibitor. Expression levels of p21, ACAN, and MMP13 mRNAs were quantified by real-time PCR. Expression ratios of ACAN and MMP13 are shown. Columns represent mean ratios with 95 % CI against untreated control.
The results shown are the average of four individual experiments. Values are normalized
to GAPDH expression. ACAN aggrecan, CI confidence interval, DMSO dimethyl sulfoxide, MMP matrix metalloproteinase, PCR polymerase chain reaction, siRNA small interfering RNA, STAT3-I signal transducer and activator of transcription 3–specific inhibitor
Fig. 2. Knockdown efficiency of p21 siRNA transfection in human normal chondrocytes in response
to mechanical strain. The concentration of p21 siRNA was 100 nM. Expression levels
of p21, COL2A1, ACAN, MMP3, MMP13, ADAMTS4, and ADAMTS5 mRNAs were quantified by real-time PCR. Values are normalized to GAPDH expression. Columns represent mean ratios with 95 % confidence intervals against
untreated and nonloaded control. The results shown are the average of four individual
experiments. White columns: untreated samples, Black columns: nonspecific control
siRNA samples, and gray columns: p21 siRNA samples. ACAN aggrecan, ADAMTS a disintegrin and metalloproteinase with thrombospondin motifs, COL2A1 collagen, type II, alpha 1, MMP matrix metalloproteinase; PCR polymerase chain reaction, siRNA small interfering RNA
Fig. 3. p21 levels influence the summed OARSI scores in DMM mice. Cartilage tissue samples
were collected from the right knee (sham or DMM surgery) of (a) p21
+/+
(sham), (b) p21
?/?
(sham), (c) p21
+/+
(DMM), and (d) p21
?/?
(DMM) mice. The sections are stained with Safranin O and Fast Green. e Average summed OARSI scores with 95 % CIs from all four quadrants and eight sections.
Six mice were analyzed for each group: p21
+/+
sham, p21
+/+
DMM, p21
?/?
sham, and p21
?/?
DMM. CI confidence interval, DMM destabilization of the medial meniscus, OARSI Osteoarthritis Research Society International
Fig. 4. p21 levels influence the number of p-STAT3-positive cells in DMM mice. Cartilage tissue
samples were collected from the right knee (sham or DMM surgery) of (a) p21
+/+
(sham), (b) p21
?/?
(sham), (c) p21
+/+
(DMM), and (d) p21
?/?
(DMM) mice. e Percentages of pSTAT3-positive stained cells (number of positive cells/number of
total cells) with 95 % CI. The sections are stained for p-STAT3 antibody and counterstained
with methyl green. Six mice were analyzed for each of the following groups: p21
+/+
sham, p21
+/+
DMM, p21
?/?
sham, and p21
?/?
DMM. CI confidence interval, DMM destabilization of the medial meniscus, p-STAT3 phosphorylated signal transducer and activator of transcription 3
Fig. 5. p21 levels influence the number of MMP-13–positive cells in DMM mice. Cartilage tissue
samples were collected from the right knee (sham or DMM surgery) of (a) p21
+/+
(sham), (b) p21
?/?
(sham), (c) p21
+/+
(DMM), and (d) p21
?/?
(DMM) mice. e Percentages of MMP-13–positive stained cells (number of positive cells/number of
total cells) with 95 % CI. The sections are stained for MMP-13 antibody and counterstained
with methyl green. Six mice were analyzed for each group: p21
+/+
sham, p21
+/+
DMM, p21
?/?
sham, and p21
?/?
DMM. CI confidence interval, DMM destabilization of the medial meniscus, MMP matrix metalloproteinase
Fig. 6. Differential expression of p21, STAT3, and p-STAT3 in normal and OA chondrocytes.
a Expression of p-STAT3 and total STAT3 in normal (three samples) and OA (three samples)
primary chondrocytes analyzed by Western blotting. b Columns represent the ratios of protein expression levels (p-STAT3/actin, STAT3/actin)
determined by semiquantification of the digitally captured image. The results shown
are the averages of three individual samples. c Expression levels of p21 mRNA levels were quantified by real-time PCR. Expression
ratios of p21 are shown. Columns represent mean ratios with 95 % CIs of primary OA
chondrocytes/primary normal chondrocytes. The results shown are the averages of five
individual samples. Values are normalized to GAPDH expression. CI confidence interval, OA osteoarthritis, p-STAT3 phosphorylated signal transducer and activator of transcription 3, STAT3 signal transducer and activator of transcription 3