miR-146a, an IL-1β responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4
Li et al. Arthritis Research & Therapy
miR-146a, an IL-1b responsive miRNA, induces vascular endothelial growth factor and chondrocyte apoptosis by targeting Smad4
Jing Li 0
Jingang Huang 0
Liming Dai 0
Xiaoling Zhang 0
Kerong Dai 0
0 The Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine , China, 200025
Introduction: miR-146a is one of the first identified miRNAs expressed differentially in osteoarthritis (OA) cartilage. However, the role it plays in OA pathogenesis is not clear. The aim of this study is to identify a molecular target of miR-146a, thereby elucidating its function in chondrocytes during OA pathogenesis. Methods: Primary chondrocytes from Sprague-Dawley rats were treated with IL-1b before the expression levels of miR-146a, Smad4 and vascular endothelial growth factor (VEGF) were quantified by real-time PCR and/or western blotting. The effect of miR-146a on cellular response to transforming growth factor (TGF)-b1 was quantified by a luciferase reporter harboring TGF-b1 responsive elements and by extracellular signal-regulated kinase assay. The effect of miR-146a on apoptosis was quantified by the TUNEL assay. OA pathogenesis was surgically induced with joint instability in rats, evaluated by histopathological analysis with safranin O staining, and the expression levels of miR-146a, Smad4, and VEGF were quantified using real-time PCR and/or immunohistochemistry. Results: IL-1b treatment of chondrocytes increased the expression levels of miR-146a and VEGF and decreased the levels of Smad4 in a time-dependent manner. miR-146a upregulated VEGF expression and downregulated Smad4 expression in chondrocytes, while a miR-146a inhibitor acted in a converse manner. Smad4, a common mediator of the TGF-b pathway, is identified as a direct target of miR-146a by harboring a miR-146a binding sequence in the 3'-UTR region of its mRNA. Mutation of the binding sequence significantly relieved the inhibition of the Smad4 reporter activity by miR-146a. Furthermore, miR-146a upregulation of VEGF is mediated by Smad4. Expression of miR-146a led to a reduction of cellular responsiveness to TGF-b and an increase of apoptosis rate in chondrocytes. In vivo, cartilage from surgically induced OA rats displayed higher levels of miR-146a and VEGF compared with the sham group. In contrast, Smad4 expression level was lower in the OA group than the sham group. Conclusion: IL-1b responsive miR-146a is overexpressed in an experimentally induced OA model, accompanied by upregulation of VEGF and downregulation of Smad4 in vivo. miR-146a may contribute to OA pathogenesis by increasing VEGF levels and by impairing the TGF-b signaling pathway through targeted inhibition of Smad4 in cartilage.
miRNAs have emerged as a novel class of gene
regulators in both animals and plants that regulate the
expression of more than one-third of human genes
posttranscriptionally . There is accumulating evidence
that miRNAs are multifunctional mediators in regulating
physiological processes, including development,
proliferation, differentiation, and apoptosis [2,3]. Although
most of them are widely distributed, the expression of
some miRNAs exhibits cell-type-specific, tissue-specific,
and developmental-stage-specific patterns . miRNAs
have also been reported to influence pathological
processes, such as cancer, diabetes, and cardiovascular
diseases . miRNAs act as key regulators in various types
of diseases because dysregulation of specific miRNAs
occurs prevalently under disease conditions [4,5]. Several
miRNAs have been identified, showing differential
expression patterns between osteoarthritis (OA) and
normal cartilage, and their postulated functions are
related to inflammatory and catabolic changes in OA
. miR-146a is one of the first identified miRNAs
associated with OA cartilage . miR-146a is expressed in
all layers of human articular cartilage, especially in the
superficial zone, and its expression is upregulated in OA
. However, the exact etiological mechanism of
miR146a in OA pathogenesis is not clear.
The imbalance of cartilage homeostasis between
catabolic and anabolic activities contributes to the etiology
of OA . A number of cytokines take part in this
process. Proinflammatory cytokines such as IL-1b and
TNFa are catabolic factors that lead to the breakdown
of articular cartilage , while anabolic factors such as
transforming growth factor (TGF)-b superfamily
members have been shown to exert a protective effect in OA
. Smad4, a common mediator of the TGF-b pathway
(co-Smad), plays an important role in transducing
TGFb signals by forming intracellular signaling complexes
with phosphorylated receptor-regulated Smads
(RSmads). The complexes then translocate into the
nucleus where they participate in the initiation or
repression of gene expression, thereby regulating the
transcription of target genes . In contrast, IL-1b
functions as a main catabolic factor in the OA process
and the elevation of IL-1b causes degradation of the
cartilage extracellular matrix .
In this study we present evidence that miR-146a is
upregulated in articular chondrocytes in response to
IL1b treatment in vitro and by destabilization of the knee
joints in vivo, and that Smad4 is a direct target of
miR146a. We find that the miR-146a inhibition of Smad4
results in upregulation of vascular endothelial growth
factor (VEGF) and apoptosis of chondrocytes.
Conversely, inhibiting miR-146a or overexpressing Smad4
reduces VEGF expression in chondrocytes. Furthermore,
we demonstrate that miR-146a upregulation in vivo is
accompanied by downregulation of Smad4 and
upregulation of VEGF in a surgically induced OA model of
Sprague-Dawley rats. Together, these findings suggest
that dysregulation of miR-146a may contribute to OA
pathogenesis by inhibiting Smad4, a key component in
the anabolic TGF-b pathway, by stimulating VEGF in
the angiogenesis, chondrocyte hypertrophy, and
extracellular matrix degradation pathways, and by inducing
Materials and methods
Primary cell culture
Primary chondrocytes were isolated from the femoral
condyles and tibial plateau of male Sprague-Dawley rats
(160 to 180 g). Rat articular cartilage was cut into small
fragments, followed by digestion first with 0.25% trypsin
(Gibco Invitrogen, Carlsbad, CA, USA) for 30 minutes
at 37°C and then with 0.2% collagenase (Sigma-Aldrich,
St Louis, MO, USA) for 5 hours at 37°C. After
dissociation, the cell suspension was filtered through a 40 μm
cell strainer (BD Falcon, Bedford, MA, USA), and cells
were collected by centrifugation at 800 × g for 10
minutes. Chondrocytes were then resuspended in
DMEM/F12 medium (Gibco Invitrogen) supplemented with 10%
fetal bovine serum (Gibco Invitrogen). Primary
chondrocytes were cultured according to a previous method
. Briefly, chondrocytes were placed in monolayer
culture in six-well plates (for RNA) or 12-well plates
(for protein) in DMEM/F-12 medium containing 10%
fetal bovine serum. Transfection experiments were
performed 1 day after seeding. Primary chondrocytes used
in the experiments were either freshly isolated or were
at passage 1. Either freshly isolated or at passage 1,
these chondrocytes do not express Col I - a marker of
dedifferentiation - as determined by real-time RT-PCR.
The observed effects of miR-146a are identical in
chondrocytes at the freshly isolated and passage 1 stage.
The miRNA expression profiles of the rat chondrocytes
treated with IL-1b (10 ng/ml; R&D Systems,
Minneapolis, MN, USA) at various time points were determined
by miRNA microarray analysis using the μParaflo™
microfluidic chips (LC Sciences, Houston, TX, USA),
which were based on Sanger miRBase Release 17.0 .
Total RNA was size-fractionated and the small RNAs (<
300 nucleotides) isolated were 3’-extended with a poly
(A) tail. Hybridization was performed overnight. Data
were analyzed by first subtracting the background and
then normalizing the signals using a LOWESS filter
(locally-weighted regression) . Normalized data were
further analyzed by one-way analysis of variance
followed by a Student-Newman-Keuls multiple comparison
test. miRNAs with P < 0.01 were considered
Construction of plasmids and site-directed mutagenesis
For plasmid DNA and miRNA co-transfection, primary
chondrocytes were transfected using the Human
Chondrocyte Nucleofector kit (Amaxa, Cologne, Germany)
following the manufacturer’s instructions . The
miR146a expression plasmid was created as previously
described . Briefly, the precursor sequence for
miR146a was amplified through PCR using genomic DNA
as the template, and the PCR products were cloned into
the pSuper vector (Oligoengine, Seattle, WA, USA).
Fragments harboring the 3’ UTR of Smad4 were cloned
into the XbaI site of the pGL3-control vector (Promega,
Madison, WI, USA) using the following primers: sense,
5’-CCGCTCGAGTGAAGGAATCATTCCAGTGCTAG3’; and antisense,
5’-TGCTCTAGACTTGGTAAAATTAACTCACCCACA-3’. The mutated 3’ UTR luciferase
reporter plasmid was generated by site-directed
mutagenesis using the QuikChange site-directed mutagenesis
kit (Stratagene, La Jolla, CA, USA). The following
primers were used: sense,
5’-TTAAAGGCAGAGAACAAGAGAAAGTTAATTCACC-3’; and antisense,
5’-GGTGAATTAACTTTCTCTTGTTCTCTGCCTTTAA-3’. All sequences of the amplified products were
confirmed by DNA sequencing.
Luciferase reporter assay
All plasmids for transfection were prepared using the
QIAGEN plasmid purification kit (QIAGEN, Hilden,
Germany). HEK293T cells were transiently transfected
using Lipofectamine 2000 (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s instructions, and
pRL-SV40 vector (Promega) was used as a control for
transfection efficiency. Twenty-four hours after
transfection, cells were lysed, and Firefly and Renilla luciferase
activities were measured using the Dual-Luciferase
Reporter Assay System (Promega) according to the
manufacturer’s protocol. C5.18 cells were co-transfected with
miR-146a mimics (GenePharma, Shanghai, China) and
p3TP-lux using DharmaFECT Duo transfection reagent
(Dharmacon Inc., Lafayette, CO, USA). The p3TP-lux
plasmid was a kind gift from Dr Regis J. O’Keefe (Center
for Musculoskeletal Research, University of Rochester,
Rochester, New York, USA). Twelve hours after
transfection, the cells were serum starved for 12 hours
followed by 4 hours treatment with or without TGF-b1 (10
ng/ml, R&D Systems). Cell lysates were extracted and
luciferase activities were measured using the
Dual-Luciferase Reporter Assay System (Promega). Each
experiment was repeated at least three times.
RNA and quantitative real-time PCR
Total RNA, including miRNA, was extracted using the
miRNeasy Mini Kit (QIAGEN) according to the
manufacturer’s instructions. Then 1 μg total RNA was
reverse-transcribed with a specific stem-loop primer for
miRNA and with a random primer for mRNA,
respectively. After RT reaction, real-time PCR was performed
by an ABI 7900HT system using SYBR Premix Ex
Taq™ (Takara, Madison,. WI, USA). b-actin and small
nuclear RNA U6 were used as internal controls for
cDNA and miRNA, respectively. Primer sequences used
for real-time PCR are presented in Table 1.
Whole-cell lysates were prepared with ice-cold lysis
buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1%
NP40, and 0.1% sodium dodecyl sulfate) supplemented with
protease inhibitors (Complete Tablet; Roche, Mannheim,
Germany). Proteins were size-fractionated by SDS-PAGE
and transferred to a PVDF membrane (Hybond-P;
Amersham Biosciences, Amersham, UK). Membranes
were hybridized with antibodies against Smad4 (1:1,000;
Santa Cruz, Santa Cruz, CA, USA), VEGF (1:1,000;
Santa Cruz), extracellular signal-regulated kinase (ERK)
1/2 (1:1,000; Cell Signaling Technology, Danvers, MA,
USA), phospho-ERK1/2 (1:1,000; Cell Signaling
Technology) and GAPDH (1:5,000; Kangcheng, Shanghai,
China). Densitometric analysis of immunoblots was
performed using the ImageJ software provided by the
National Institutes of Health (Developed by National
Institutes of Health, Bethesda, Maryland, USA).
Smad4 knockdown by siRNA
RNA interference was performed using siGENOME
SMARTpool siRNA (Dharmacon Inc.) targeting rat
Smad4. Transfection for primary chondrocytes was
carried out using Lipofectamine RNAiMAX reagent
(Invitrogen) according to the manufacturer’s protocol.
Chondrocytes were fixed for 20 minutes at room
temperature with 4% paraformaldehyde in PBS 48 hours post
transfection, and apoptosis was assessed using the In Situ
Cell Death Detection Kit - Fluorescein (Roche) according
to the manufacturer’s instructions. The number of
TUNEL-positive cells (green) and the number of Hoechst
33342-positive cells (blue nuclear stain) were visually
counted. All samples were analyzed with at least three
independent replicates, and five fields from each replicate
were randomly selected for counting the TUNEL-positive
cells and the Hoechst 33342-positive cells. The observer
who performed the cell counts and immunofluorescence
quantitation was blinded to the types of the samples.
Surgical induction of osteoarthritis
Animal handling and experimental procedures were
performed following approval from the Institute of Health
Sciences Institutional Animal Care and Use Committee.
Eight-week-old male Sprague-Dawley rats (200 g) were
randomized into two groups of 20 rats each. OA was
induced by medial collateral ligament transection and
medial meniscal tear of the knee joints, as previously
described . Briefly, animals were anesthetized and
surgery was performed to transect the medial collateral
ligament and to cut the medial meniscus through the
full thickness to induce joint destabilization of the right
knee. Sham animals underwent the same surgical
procedure without any ligament transection or meniscal tear.
After surgery, each rat was given penicillin once per day
for the first 3 days. Animals were sacrificed at 8 weeks
Table 1 Primer sequences used for real-time PCR
Rat b-actin [GenBank:NM_031144.2]
Rat U6 [GenBank:K00784]
Rat Smad4 [GenBank:NM_019275.2]
Rat VEGF [GenBank:NM_031836.2]
VEGF, vascular endothelial growth factor.
post surgery, and samples of the knee joints were
collected for further molecular and histological analyses.
Histology and immunohistochemistry
Knee joints from the model animals were fixed
overnight with 4% paraformaldhyde in PBS and then
embedded in paraffin. Tissue sections (5 μm) were
deparaffinized in xylene, serially rehydrated in ethanol,
and washed with PBS. Sections were stained with
safranin O/fast green to identify proteoglycan loss . For
immunohistochemistry, sections in 10 mM sodium
citrate buffer (pH 6.0) were heated in a microwave oven
and kept at 95°C for 10 minutes. Slides were cooled for
30 minutes at room temperature after antigen
unmasking. Endogenous peroxidase activity was blocked with
3% hydrogen peroxide, followed by rinsing several times
in PBS. After blocking nonspecific protein binding with
5% BSA in PBS for 30 minutes at room temperature,
sections were incubated overnight at 4°C with primary
antibodies against Smad4 (Santa Cruz) and VEGF (Santa
Cruz). The slides were rinsed in PBS and then incubated
with secondary antibody (EnVision detection kit,
Peroxidase/DAB, Rabbit/mouse; Dako Cytomation,
Carpinteria, CA, USA) according to the manufacturer’s
protocol. Sections were counterstained with Mayer’s
hematoxylin (Sigma, St. Louis, MO, USA). After
washing, the slides were stained with 3,3’-diaminobenzidine
tetrahydrochloride (EnVision detection kit, Peroxidase/
DAB, Rabbit/mouse; Dako Cytomation). Staining with
normal IgG and staining without primary antibodies
were also performed as negative controls. For
immunohistochemistry, sections were quantified using ImagePro
Plus version 5.0 (Media Cybernetics, Bethesda, MD,
USA). Three fields of view per section were analyzed
from each animal. Mean values and variances of
Smad4positive and VEGF-positive cells in each group were
calculated from 20 animals per group.
Results are expressed as mean ± standard deviation.
Statistical analysis was carried out using Student’s t test
between two groups or one-way analysis of variance
followed by Student-Newman-Kuels test for multiple
comparisons. P < 0.05 were considered statistically
IL-1b treatment increases expression of miR-146a and
VEGF and decreases Smad4 expression in chondrocytes
To identify the miRNAs involved in pathogenesis of OA,
we screened for miRNAs responsive to treatment of the
proinflammatory cytokine IL-1b (10 ng/ml) in primary
rat chondrocytes. This is an established cell culture
model to mimic inflammation and other molecular
events related to the progression of OA in chondrocytes
. Expression of miRNAs in IL-1b-stimulated
chondrocytes was investigated by microarray analysis [GEO:
GSE33310] . A series of miRNAs changed their
expression levels in response to IL-1b treatment (Figure
S1 in Additional file 1). Of particular interest, miR-146a
was chosen for further investigation because previous
studies have revealed that miR-146a mediates
inflammation response , and its expression is higher in OA
cartilage than in normal cartilage .
Treatment of IL-1b rapidly induced miR-146a within 6
hours in primary rat chondrocytes, and its expression
gradually increased over a 24-hour time course (Figure
1A), which is consistent with the microarray results. In
parallel with the increase of miR-146a level, IL-1b
treatment stimulated VEGF mRNA (Figure 1C) and protein
levels (Figure 1D) in a time-dependent manner. In
contrast, IL-1b treatment inhibited Smad4 mRNA (Figure
1B) and protein levels (Figure 1D) in a time-dependent
miR-146a directly inhibits Smad4 expression through a
seed site in the 3’-UTR of Smad4 mRNA
To determine whether miR-146a regulates the
expression of Smad4 and VEGF, we transfected miR-146a into
primary chondrocytes. Overexpression of miR-146a
inhibited Smad4 protein levels and stimulated VEGF
protein levels (Figure 2A). Conversely, transfection of a
miR-146a inhibitor stimulated Smad4 protein levels and
inhibited VEGF protein levels in chondrocytes (Figure
2D). miR-146a thus regulates the expression of Smad4
and VEGF in an opposite manner.
Using miRNA target prediction software , we
identified a potential miR-146a binding sequence in the 3’
UTR of Smad4 (Figure 2G). To determine whether
miR146a inhibits Smad4 expression through this seed
sequence, we constructed luciferase reporter plasmids
Figure 1 IL-1b stimulates miR-146a and vascular endothelial growth factor along with downregulation of Smad4. Primary rat
chondrocytes were treated with IL-1b (10 ng/ml) for the indicated time periods. Expression levels of miR-146a, Smad4, and vascular endothelial
growth factor (VEGF) were monitored by quantitative real-time PCR and western blotting. (A) miR-146a expression was significantly increased
over a 24-hour time course. (B) mRNA levels of Smad4 were reduced after treatment with IL-1b. (C) VEGF was upregulated by IL-1b. Values are
the mean ± standard deviation (SD) of three independent experiments. (D) Protein levels of Smad4 and VEGF were respectively decreased and
increased in chondrocytes stimulated with IL-1b. The relative expression levels of protein are shown at the bottom of the bands as normalized
by the GAPDH level. (E) Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three
independent experiments. (F) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of
three independent experiments. Control samples (Con) were not treated with IL-1b.
Figure 2 Smad4 is a direct target of miR-146a. (A) Overexpression of miR-146a inhibits Smad4 expression and increases vascular endothelial
growth factor (VEGF) expression in primary chondrocytes, as assayed by immunoblotting. NC, negative control. (B) Densitometric analysis of
immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± standard deviation (SD) of three independent experiments. (C)
Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments.
(D) On the protein level, knockdown of miR-146a results in the upregulation of Smad4 and the downregulation of VEGF. Relative expression
levels of protein shown at the bottom of the bands as normalized by GAPDH level. (E) Densitometric analysis of immunoblot band intensities
for Smad4 normalized by GAPDH. Data are mean ± SD of three independent experiments. (F) Densitometric analysis of immunoblot band
intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments. (G) Schematic representation of the Smad4
3’ UTR indicating the binding site of miR-146a. WT, wildtype; MT, mutant. (H) miR-146a inhibits Smad4 3’ UTR luciferase activity. HEK293T cells
were transfected with either pGL3 luciferase vector containing a fragment of Smad4 3’ UTR harbouring binding sites for miR-146a, or the
corresponding mutant constructs. Ectopic expression of miR-146a led to a remarkable reduction of the luciferase activity of reporter with the
wildtype 3’ UTR but not that of the mutant reporter. Values are mean ± SD of three independent experiments. *P < 0.05 versus vector. **P <
0.01 versus vector.
harboring the wildtype 3’ UTR and the mutant 3’ UTR
in which the putative miR-146a binding site is mutated
(Figure 2G). While the reporter activity of the wildtype
3’ UTR is significantly inhibited by miR-146a, this
inhibition is greatly reduced in the mutant 3’ UTR (Figure
2H). Smad4 is thus a direct target of miR-146a.
IL-1b regulates Smad4 and VEGF expression
To elucidate the role of miR-146a in mediating IL-1b
signaling, we used a specific miR-146a hairpin inhibitor
to block its expression. Chondrocytes were treated with
IL-1b for 24 hours in the presence or absence of the
miR-146a inhibitor. Knockdown of endogenous
miR146a with the inhibitor significantly suppressed the
IL1b upregulation of miR-146a expression (Figure 3A).
While IL-1b treatment inhibited Smad4 mRNA levels,
transfection of the miR-146a inhibitor markedly
increased Smad4 mRNA despite the presence of IL-1b
(Figure 3B). While IL-1b treatment greatly increased the
VEGF mRNA levels, the miR-146a inhibitor significantly
reduced this increase (Figure 3C). Knockdown of
miR146a caused similar effects on the IL-1b regulation of
Smad4 and VEGF protein levels as on their mRNA
levels (Figure 3D). miR-146a is thus involved in IL-1b
regulation of Smad4 and VEGF expression.
Upregulation of VEGF by miR-146a is mediated by Smad4
To determine whether Smad4 mediates the upregulation
of VEGF by miR-146a, RNA interference with Smad4
siRNA was performed in rat chondrocytes.
Chondrocytes were transfected with siRNA against Smad4. This
Smad4 siRNA transfection reduced the levels of both
Smad4 mRNA (Figure 4A) and protein (Figure 4B).
Knockdown of Smad4 increased VEGF protein levels
(Figure 4B), while overexpression of Smad4 significantly
reduced miR-146a stimulation of VEGF protein levels
(Figure 4E). Smad4 thus mediates upregulation of VEGF
miR-146a attenuates TGF-b signaling pathway
Because Smad4 is a common mediator of the TGF-b
signaling pathway, we next addressed the question of
whether miR-146a affects the cellular responses to
TGFb. C5.18 cells were co-transfected with miR-146a and
p3TP-luciferase reporter plasmid (p3TP-lux, possessing
TGF-b response elements) followed by treatment with
TGF-b1 (10 ng/ml). As shown in Figure 5A,
overexpression of miR-146a led to a decrease in both basal and
TGF-b1-stimulated activity of the p3TP-luciferase
reporter, suggesting that miR-146a significantly inhibits
TGFb signaling transduction. To further investigate the role
of miR-146a in TGF-b signaling, we conducted a
timecourse study of ERK activation by TGF-b1 in
chondrocytes transfected with miR-146a. Western blot
analysis revealed time-dependent activation of ERK with
maximal activation occurring at 30 minutes post
treatment (Figure 5B). Overexpression of miR-146a reduced
the levels of phospho-ERK 1/2 at all time points (Figure
5B), whereas the total ERK levels remained relatively
constant (Figure 5B).
miR-146a increases apoptosis in chondrocytes
Since IL-1b stimulates apoptosis in chondrocytes 
and the loss of cellularity is a hallmark of OA cartilage
, we examined whether the expression of miR-146a
affects chondrocyte apoptosis. Overexpression of
miR146a in chondrocytes caused a significant increase of
the percentage of TUNEL-positive cells (Figure 6),
indicating that miR-146a takes part in mediating
IL-1binduced apoptosis in chondrocytes.
Co-regulation of miR-146a with Smad4 and VEGF in OA
cartilage in vivo
To determine whether expression of miR-146a, Smad4
and VEGF is co-regulated in OA cartilage in vivo, we
surgically induced OA through joint instability in
Sprague-Dawley rats (Figure 7B, C). The expression of
miR146a was significantly upregulated in OA cartilage
compared with normal cartilage (Figure 7A).
Immunohistochemical analysis showed a decrease of Smad4-positive
cells (Figure 7D, E) and an increase of VEGF-positive
cells (Figure 7F, G) in OA cartilage than in normal
cartilage (sham). The percentage of chondrocytes positive
for Smad4 was substantially decreased in the OA group
(31.5 ± 5.1%) compared with the sham group (61.1 ±
4.6%), while the percentage of VEGF-positive cells in the
sham and OA groups (8.3 ± 2.1% and 63.7 ± 5.4%,
respectively) indicated a statistically significant increase
in OA cartilage (P < 0.01). The induction of miR-146a
expression in OA cartilage is thus correlated with the
upregulation of VEGF and the downregulation of Smad4
in rat joints with surgically induced OA.
miR-146a is one of the first identified miRNAs
upregulated in human OA cartilage. However, it was not clear
whether this is a coincidence or miR-146a plays a role
in OA pathogenesis. We provide several lines of
evidence here to demonstrate that miR-146a may be an
important regulator in OA.
First, we demonstrate for the first time that miR-146a
is upregulated by experimentally induced OA
pathogenesis in a well-established OA animal model of
SpragueDawley rats in vivo. The induction of miR-146a
expression in articular cartilage is thus caused by OA. In
addition to miR-146a, other miRNAs may also play
important roles in OA pathogenesis: miR-140, a
Figure 3 miR-146a suppression impairs IL-1b downregulation of Smad4 and upregulation of vascular endothelial growth factor.
Chondrocytes were transfected with miR-146a inhibitors and treated with or without IL-1b for 24 hours. (A) miR-146a inhibitors decreased
IL-1binduced upregulation of miR-146a. NC, negative control. (B) The inhibitory effect of IL-1b on Smad4 expression was counteracted by miR-146a
inhibitors. (C) Vascular endothelial growth factor (VEGF) expression was significantly reduced by miR-146a inhibitors with IL-1b treatment at the
RNA level. Values are the mean ± standard deviation (SD) of at least three independent experiments. **P < 0.01. (D) On the protein level,
miR146a inhibitors counteracted IL-1b-induced downregulation of Smad4 and upregulation of VEGF. The relative expression levels of protein are
shown at the bottom of the bands as normalized by GAPDH level. (E) Densitometric analysis of immunoblot band intensities for Smad4
normalized by GAPDH. Data are mean ± SD of three independent experiments. (F) Densitometric analysis of immunoblot band intensities for
VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments.
Figure 4 Vascular endothelial growth factor production is regulated by Smad4. Knockdown of Smad4 by RNAi increased vascular
endothelial growth factor (VEGF) expression at the protein level. Chondrocytes were transfected with siRNA specific for Smad4 or with control
(Ctrl) siRNA. (A) siRNA targeting Smad4 efficiently reduced endogenous Smad4 expression at the RNA level. Values are the mean ± standard
deviation (SD) of at least three independent experiments. **P < 0.01. (B) Inhibition of Smad4 increased the protein level of VEGF. (C)
Densitometric analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three independent
experiments. (D) Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three
independent experiments. (E) Co-transfection of miR-146a and Smad4 expression plasmid reversed miR-146a-mediated upregulation of VEGF.
Relative expression levels of protein shown at the bottom of the bands as normalized by GAPDH level. NC, negative control. (F) Densitometric
analysis of immunoblot band intensities for Smad4 normalized by GAPDH. Data are mean ± SD of three independent experiments. (G)
Densitometric analysis of immunoblot band intensities for VEGF normalized by GAPDH. Data are mean ± SD of three independent experiments.
cartilage-specific miRNA, regulates gene expression of
ADAMTS-5 in chondrocytes ; and miR-140-/- mice
display an OA-like phenotype . miR-140 may also be
involved in the formation and maintenance of cartilage
through targeting HDAC4 . In addition, miR-27a
affects the expression of matrix metalloproteinase
(MMP)-13 and IGFBP-5 , and miR-27b inhibits the
IL-1b-induced upregulation of MMP-13 in human
osteoarthritic chondrocytes .
Second, we demonstrate that miR-146a is induced by
IL-1b treatment of chondrocytes in a time-dependent
manner in vitro. We focused our study on miR-146a
after it came up in our screening for IL-1b upregulated
miRNAs in chondrocytes. Our observation and the
previous literature suggest that the responsiveness to IL-1b
and/or other inflammatory cytokines is a hallmark of
miR-146a. The expression of miR-146a/b was elevated
after treatment with lipopolysaccharide and
proinflammatory mediators . Stanczyk and colleagues reported
that the expression of miR-146 is increased in
rheumatoid arthritis synovial fibroblasts . Nakasa and
colleagues reported increased miR-146a/b expression in
synovial tissue from rheumatoid arthritis patients .
miR-146a operates as a negative regulator in innate
immunity by affecting IL-1R-associated kinase-1 and
TNF-receptor-associated factor 6. In human OA tissue
samples, miR-146a may be involved in both
proinflammatory cytokine response and modulation [7,32].
Third, we demonstrate that miR-146a is induced by
joint instability resulting from medial collateral ligament
transection and medial meniscal tear of the knee joints
in vivo. The inductive factors for miR-146a may be
more complex in vivo. In addition to the
proinflammatory cytokines resulting from the medial collateral
ligament transection and medial meniscal tear, mechanical
instability is also a major cause of OA pathogenesis in
this animal model . Mechano-responsive miRNAs
are beginning to be identified in chondrocytes. miR-365
is the first identified mechanically responsive miRNA in
chondrocytes, which regulates chondrocyte
differentiation through inhibiting HDAC4 . In addition,
miR222 was postulated as a potential regulator of the
articular cartilage mechanotransduction pathway, since its
expression patterns in articular cartilage are higher in
the weight-bearing anterior medial condyle as compared
with the posterior nonweight-bearing medial condyle
Figure 5 miR-146a attenuates the transforming growth
factorb signaling pathway. (A) C5.18 cells were co-transfected with
miR146a and p3TP-lux reporter plasmid followed by treatment with or
without exogenous transforming growth factor (TGF)-b1 (10 ng/ml)
for 4 hours. The cell lysates were obtained to determine the
luciferase activity. Values are the mean ± standard deviation of at
least three independent experiments. **P < 0.01. NC, negative
control. (B) Overexpression of miR-146a blocked TGF-b1-stimulated
activation of extracellular signal-regulated kinase (ERK).
Chondrocytes were transfected with miR-146a mimics, and, after
serum starvation, cells were treated with TGF-b1 (10 ng/ml) for the
indicated periods. Cell lysates were analyzed for phosphorylated and
total ERK levels in chondrocytes.
. It remains to be tested whether miR-146a is
responsive to alteration of mechanical load in addition
to proinflammatory cytokine.
Fourth, we have for the first time identified a direct
molecular target of miR-146a in chondrocytes. We show
that the expression levels of Smad4, a key transcription
factor mediating the TGF-b family member signaling
pathway, are inversely related to miR-146a levels both in
vitro and in vivo. Similar results were obtained from
cultured human chondrocytes (data not shown). Mutation
of the miR-146a binding site in the 3’ UTR of Smad4
mRNA unequivocally identified Smad4 as a direct target
of miR-146a for post-transcriptional regulation.
Furthermore, miR-146a is critical for IL-1b downregulation of
Smad4 in chondrocytes.
Our data suggest that miR-146a regulates
chondrocytes and OA pathogenesis by inhibiting Smad4, a
pivotal mediator of the TGF-b signaling pathway.
Interestingly, the extent of miR-146a inhibition of
Smad4 protein levels is more than the extent of
miR146a inhibition of Smad4 mRNA levels. This indicates
that miR-146a targets Smad4 through both mRNA
degradation and translational repression. Smad4 plays
important roles in regulating chondrocyte differentiation
by inhibiting hypertrophy and cell apoptosis. In the
cartilage-specific Smad4 knockout mice, chondrocyte
proliferation is reduced, hypertrophic differentiation is
accelerated, and apoptosis is increased .
Furthermore, IL-1b inhibits Smad4 in a chondrocytic cell line
(SW1353), indicating that the antagonistic effect of
IL1b on TGF-b may be mediated by blocking the
expression of Smad4 . TGF-b may counteract some
IL-1binduced effects on cartilage deterioration by preserving
chondrocyte phenotypes, suppressing the expression of
MMPs, such as MMP-1 and MMP-3, and promoting the
synthesis of extracellular matrix of cartilage [37-39].
Loss of TGF-b and its downstream signaling molecules
often corresponds with skeletal abnormalities and
destruction of articular cartilage. For example,
overexpression of a functionless TGF-b type II receptor
accelerates terminal chondrocyte differentiation .
Moreover, Smad3 mutant mice display a phenotype
resembling human OA, which is accompanied by the
extensive progression of chondrocyte hypertrophy and
osteophyte formation .
We demonstrate that miR-146a inhibits chondrocyte
response to TGF-b by suppressing transcriptional
activity of a promoter harboring TGF-b responsive elements
and by suppressing TGF-b induction of ERK activity.
The activation of ERK mitogen-activated protein kinases
represents a downstream molecular event in response to
TGF-b in chondro-progenitor cells, which is required
for TGF-b-induced aggrecan expression . ERK not
only directly promotes phosphorylation of R-Smads, but
also affects co-activators or co-repressors that mediate
Smad DNA binding . It has been shown previously
that TGF-b stimulation of ERK activity is Smad4
dependent . Knockdown of Smad4 by miR-146a may
therefore inhibit ERK phosphorylation. Similar to
miR146a, other miRNAs have been implicated in regulating
TGF-b pathways by targeting Smads in chondrocytes.
For example, miR-199a* was reported to inhibit
early chondrogenic differentiation by targeting Smad1
We demonstrate that miR-146a results in an increase
of the apoptosis rate in articular chondrocytes. Reduced
cellularity in articular cartilage contributes to the onset
and development of OA. A higher proportion of
apoptotic cells was observed in the cartilage from OA patients
compared with that from normal people .
Expressions of apoptotic molecular markers, such as caspase-3
and caspase-8, were elevated in human osteoarthritic
cartilage . These are consistent with our hypothesis
that miR-164a contributes to OA pathogenesis by
inducing chondrocyte apoptosis.
Figure 6 miR-146a leads to apoptosis in chondrocytes. (A) to (D) TUNEL assays were performed 2 days following the addition of negative
control (NC) or miR-146a to chondrocytes. miR-146a increases apoptosis in chondrocytes. The nuclei of apoptotic cells are labeled by TUNEL
with green fluorescence. All cell nuclei are counterstained by Hoechst 33342 (blue). Original magnification: (A), (B) ×200; (C), (D) ×600. (E)
Quantification of the percentage of TUNEL-positive cells shows a 40.3% increase in the percentage of apoptotic cells in chondrocytes transfected
with miR-146a. **P < 0.01.
Lastly, our data indicate that at least some of the
effects of miR-146a on OA pathogenesis may be exerted
by VEGF. We demonstrate that VEGF expression is
upregulated by induction of OA pathogenesis with joint
instability, treatment of IL-1b, overexpression of
miR146a, or knockdown of Smad4. Furthermore, induction
of VEGF by IL-1b at least partially depends on
upregulation of miR-146a; and its induction by miR-146a
depends on Smad4 downregulation. Smad4 has been
shown previously to inhibit VEGF expression and
suppress tumorigenicity through inhibition of angiogenic
activity in human pancreatic carcinoma cells .
Figure 7 miR-146a and Smad4 are expressed reciprocally in surgically induced osteoarthritis in rats. (A) The level of miR-146a increases
in cartilage from the osteoarthritis (OA) group compared with the sham group. Values are the mean ± standard deviation of 10 animals per
group. *P < 0.05. Femoral condyles from OA and Sham rats were stained with safranin O and subjected to immunohistochemistry for Smad4
and vascular endothelial growth factor (VEGF). (B), (C) Femoral condyles from OA rats exhibited reduced safranin O staining. (D), (E) Cartilage
from normal and osteoarthritic rats were immunostained for Smad4 protein. (F), (G) Cartilage from normal and osteoarthritic rats were
immunostained for VEGF protein. Original magnification: (B), (C) ×40; (D) to (G) ×400.
Interestingly, while the miR-146a inhibitor significantly
affects the IL-1b regulation of Smad4 and VEGF,
inhibition of miR-146a could not completely eliminate
IL-1bcaused stimulation of VEGF and suppression of Smad4.
This suggests that, in addition to miR-146a, other
factors are involved in mediating IL-1b regulation of VEGF
The induction of VEGF expression by miR-146a may
affect angiogenesis and inflammation during OA
pathogenesis. VEGF is increased in the osteoarthritic
synovium  and OA cartilage . Upregulation of VEGF
contributes to inflammation and pathological
angiogenesis in OA [51,52]. On the other hand, the upregulation
of VEGF may also lead to chondrocyte hypertrophy,
matrix degradation, and cell death - a series of critical
events during endochondral ossification that is
recapitulated during OA pathogenesis [53,54]. VEGF,
upregulated by hypertrophic chondrocytes, may in turn induce
the invasion of blood vessels to cartilage, secretion of
MMPs, extracellular matrix remodeling, and, ultimately,
cell death .
We demonstrate that miR-146a may be involved in a
novel signaling cascade critical for a series of
IL-1binduced pathologic features of OA including reduced
cellular response to TGF-b, elevated VEGF expression,
and increased chondrocyte apoptosis. Our results
demonstrate for the first time that Smad4 is a direct
target of miR-146a, and a critical mediator of miR-146a
regulation of VEGF expression. Our results provide
deeper insights into the roles of miRNA in OA
pathogenesis and raise the possibility that miR-146a may be a
therapeutic target for the treatment of OA.
Additional file 1: Figure S1 showing a heatmap of miRNA
expression profiles of chondrocytes stimulated with IL-1b.
Statistically significant miRNAs (P < 0.01, selected by analysis of variance
test) are presented. Green and red denotes downregulated and
upregulated expression, respectively.
BSA: bovine serum albumin; DMEM: Dulbecco’s modified Eagle’s medium;
ERK: extracellular signal-regulated kinase; IL: interleukin; miRNA: microRNA;
MMP: matrix metalloproteinase; OA: osteoarthritis; PBS: phosphate-buffered
saline; PCR: polymerase chain reaction; RNAi: RNA interference; RT: reverse
transcription; siRNA: small interfering RNA; TGF: transforming growth factor;
TNF: tumor necrosis factor; TUNEL: terminal deoxynucleotidyl transferase
dUTP nick end labeling; UTR: untranslated region; VEGF: vascular endothelial
The authors thank Prof. Regis J. O’Keefe (Center for Musculoskeletal Research,
University of Rochester) who provided the p3TP-lux plasmid. This work was
JL participated in the design of the study, carried out the experiments and
statistical analysis, and drafted the manuscript. JGH and LMD assisted in
performing immunohistochemistry. DGY assisted with in vivo experiments.
QC was involved in data interpretation and manuscript preparation. KRD and
XLZ conceived of the study, participated in its design and coordination, and
helped to draft the manuscript. All authors read and approved the final
manuscript for publication.
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