Download Regulation of plasminogen activator inhibitor 1 expression in human

ARTHRITIS & RHEUMATISM
Vol. 60, No. 8, August 2009, pp 2350–2361
DOI 10.1002/art.24680
© 2009, American College of Rheumatology
Regulation of Plasminogen Activator Inhibitor 1 Expression in
Human Osteoarthritic Chondrocytes by Fluid Shear Stress
Role of Protein Kinase C␣
Chih-Chang Yeh,1 Hsin-I Chang,2 Jui-Kun Chiang,3 Wang-Ting Tsai,2 Li-Ming Chen,2
Chean-Ping Wu,2 Shu Chien,4 and Cheng-Nan Chen2
Objective. To test a fluid flow system for the
investigation of the influence of shear stress on expression of plasminogen activator inhibitor 1 (PAI-1) in
human osteoarthritic (OA) articular chondrocytes
(from lesional and nonlesional sites) and human SW1353 chondrocytes.
Methods. Human SW-1353 chondrocytes and OA
and normal human articular chondrocytes were cultured on type II collagen–coated glass plates under
static conditions or placed in a flow chamber to form a
closed fluid-circulation system for exposure to different
levels of shear stress (2–20 dyn/cm2). Real-time polymerase chain reaction was used to analyze PAI-1 gene
expression, and protein kinase C (PKC) inhibitors and
small interfering RNA were used to investigate the
mechanism of shear stress–induced signal transduction
in SW-1353 and OA (lesional and nonlesional) articular
chondrocytes.
Results. There was a significant reduction in
PAI-1 expression in OA chondrocytes obtained from
lesional sites compared with those obtained from nonlesional sites. In SW-1353 chondrocytes subjected to 2
hours of shear flow, moderate shear stresses (5 and 10
dyn/cm2) generated significant PAI-1 expression, which
was regulated through PKC␣ phosphorylation and Sp-1
activation. These levels of shear stress also increased
PAI-1 expression in articular chondrocytes from nonlesional sites and from normal healthy cartilage through
the activation of PKC␣ and Sp-1 signal transduction,
but no effect of these levels of fluid shear stress was
observed on OA chondrocytes from lesional sites.
Conclusion. OA chondrocytes from lesional sites
and those from nonlesional sites of human cartilage
have differential responses to shear stress with regard
to PAI-1 gene expression, and therefore diverse functional consequences can be observed.
Supported by the Veterans Affairs Commission, Executive
Yuan, Taiwan (Geriatric Medicine Research Project grant
RVHCY96001), and the National Science Council, Taiwan (grants
NSC-96-2320-B-415-003 and NSC-97-2320-B-415-007-MY3).
1
Chih-Chang Yeh, MD: Chiayi Veterans Hospital, Chiayi,
Taiwan; 2Hsin-I Chang, PhD, Wang-Ting Tsai, MS, Li-Ming Chen,
MS, Chean-Ping Wu, PhD, Cheng-Nan Chen, PhD: National Chiayi
University, Chiayi, Taiwan; 3Jui-Kun Chiang, MD, MS: Buddhist Dalin
Tzu Chi General Hospital, Dalin, Chiayi, Taiwan; 4Shu Chien, MD,
PhD: University of California, San Diego, La Jolla.
Drs. Yeh and Chang contributed equally to this work.
Dr. Chien has received consulting fees, speaking fees, and/or
honoraria from the Institute of International Education, Tulane
University, Emory University, the American Heart Association, Baylor
College of Medicine, University of California, Berkley, Georgia Institute of Technology, and University of California, Los Angeles, and
holds a patent on the use of RasN17 for the inhibition of vascular
hypertrophy.
Address correspondence and reprint requests to Cheng-Nan
Chen, PhD, Department of Biochemical Science and Technology,
National Chiayi University, Chiayi 600, Taiwan. E-mail:
[email protected].
Submitted for publication October 28, 2008; accepted in
revised form April 15, 2009.
Osteoarthritis (OA) is the most common joint
disease among older persons. The most common sites of
clinical manifestations of degenerative cartilage in OA
are the hips, knees, and spine. The symptoms of OA
include pain, stiffness, and deformation of the knee
joints (1). OA occurs more frequently in women and
elderly individuals, but being overweight and engaging in
a regimen of intense exercise are also factors that convey
a high risk of OA. Excessive and repetitive mechanical
stresses on the articular joint generate cartilage wear
and may induce OA (2). Despite the widespread incidence of OA in the human population, its etiology is still
largely unknown.
Cartilage is a supporting connective tissue com2350
PKC␣ AND SHEAR-INDUCED PAI-1 EXPRESSION IN OA CHONDROCYTES
posed of small numbers of chondrocytes and large
amounts of extracellular matrix (ECM). The ECM synthesized by chondrocytes has a high water content and
contains collagen, proteoglycan (PG), metalloproteinases, and other small molecules; it plays an essential role
in cartilage structure and function (3). Many biochemical and genetic factors, as well as mechanical stress, can
modify the connection between chondrocytes and the
ECM and alter chondrocyte metabolism (4).
Plasmin is involved in various physiologic mechanisms, including thrombolysis, cell migration, metastasis, and arthritis formation. In addition, plasmin can
activate metalloproteinases and plays an important role
in modulating cartilage function (5). Urokinase plasminogen activator (uPA), tissue-type plasminogen activator
(tPA), and their inhibitor, plasminogen activator inhibitor 1 (PAI-1), are present in the cartilage to modulate
plasmin activation and the degradation of ECM. OA
cartilage has been shown to display increased plasmin
activity and elevated levels of uPA and tPA, as well as a
decrease in PAI-1 expression (6). It has been demonstrated that the activity of uPA is associated with the
degradation of cartilage, while the activity of PAI-1 is
associated with the synthesis of cartilage during pathophysiologic processes (7); hence, it is important to
understand the molecular mechanisms involved in the
regulation of PAI-1 expression in human cartilage.
Protein kinase C (PKC) is an enzyme activated by
inositol phospholipid hydrolysis and acts as a key enzyme for signal transduction in various physiologic processes (8–10). The PKC family is composed of several
isoforms that are divided into 3 basic classes (conventional, novel, and atypical), according to the structure of
the regulatory domains and the methods of activation
(10). The role of PKC in articular chondrocytes is still
unclear, especially when these cells are exposed to
mechanical stress. Kimura et al (11) reported that PKC␣
can be detected in cultured bovine articular chondrocytes, that PG synthesis is stimulated in a concentrationdependent manner by a PKC activator and inhibited by
a PKC inhibitor, and that PKC␣-transfected chondrocytes produce elevated amounts of PG. In addition,
PKC␣ appears in chondrocytes in the early stages of OA
(12). It has been shown that PKC plays an essential role
in the metabolism of PG by chondrocytes, and that
mechanical stress exerted on chondrocytes or the cartilage matrix may modulate signal transduction of PKC
(13).
There is evidence suggesting that abnormal mechanical loading may be detrimental to cartilage tissue
(4,14). Previous studies have shown that chondrocytes of
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the superficial and transitional zones are exposed to high
and low fluid flow, respectively (15,16), suggesting that
mechanical shear stress may be a pathophysiologic process relevant in cartilage biology. Moreover, in cartilage
tissue engineering, the development of chondrocytes/
cartilage constructs is affected by different levels of
shear stress, ranging from ⬃1 dyn/cm2 to 23.2 dyn/cm2,
revealing that hydrodynamic shear stress may alter intercellular signaling pathways in chondrocytes (17,18). Despite the extensive number of studies on the effects of
mechanical forces on chondrocytes, the detailed mechanisms that transduce the mechanical stimuli of shear
stress to intracellular signaling, which ultimately leads to
regulation of downstream gene expression, remain unclear. Thus, studying the mechanotransduction in chondrocytes in response to shear stress may help to elucidate the mechanisms underlying the focal nature of OA.
In the present study, we investigated the effects
of fluid shear stress on the expression levels of PAI-1
messenger RNA (mRNA) by quantification of gene
expression with real-time polymerase chain reaction
(PCR). In order to elucidate the signaling pathways
involved in the shear-induced regulation of PAI-1 expression in human chondrocytes, we determined the
activation of PKC␣ and that of Sp-1 in response to shear
stress and examined the effects of specific inhibitors or
small interfering RNA (siRNA) specifically targeting
these signaling proteins on shear-induced PAI-1 expression. Our findings provide a molecular basis for the
mechanism by which fluid shear stress regulates signal
transduction and PAI-1 expression in human articular
chondrocytes, both from a chondrocytic cell line and
from patients with knee OA.
PATIENTS AND METHODS
Primary culture of normal and OA human chondrocytes. Human OA articular chondrocytes were isolated from
resected osteochondral specimens obtained during primary
total knee arthroplasty from patients with advanced knee OA
(n ⫽ 10; ages 65–80 years). The diagnosis in all patients
fulfilled the American College of Rheumatology criteria for
knee OA and corresponded to grade IV severity of knee OA
according to the Kellgren/Lawrence classification system
(19,20). OA chondrocytes from sites of lesions were derived
from residual pieces of degenerating cartilage (Outerbridge
grades III–IV) (21) over the medial condyle of the femur, and
OA chondrocytes from nonlesional sites were derived from
cartilage pieces over the lateral condyle of the femur in the
same patient. Normal human chondrocytes were isolated from
healthy cartilage pieces of the distal femoral condyles obtained
from patients undergoing above-the-knee amputation (n ⫽ 4;
ages 67–79 years) (Figure 1A). This study was performed with
the approval of the ethics committee of National Chiayi
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Figure 1. A, Schematic representation of the medial and lateral
condyle of the femur, depicting the sites of extraction of normal human
chondrocytes and human osteoarthritic (OA) chondrocytes from lesional and nonlesional cartilage. B and C, Plasminogen activator
inhibitor 1 (PAI-1) mRNA expression in samples of normal human
cartilage and lesional and nonlesional human OA cartilage, as detected
by real-time polymerase chain reaction. Bars show the mean and SEM
fold change in fluorescence intensity relative to OA chondrocytes from
nonlesional sites, with results normalized to the levels of GAPDH (B)
and 18S ribosomal RNA (C). ⴱ ⫽ P ⬍ 0.01.
University and Chiayi Veterans Hospital, and all patients
provided their informed consent.
Primary chondrocyte cultures were generated from
cartilage sections obtained from both the lesional and nonlesional areas of OA cartilage. The cartilage tissue samples were
minced and washed in Dulbecco’s modified Eagle’s medium
(DMEM) and subjected to 0.25% trypsin–EDTA digestion,
followed by overnight digestion in 0.2% type II collagenase.
The resulting cell suspension was filtered through 100-␮m
nylon meshes, washed repeatedly with phosphate buffered
YEH ET AL
saline (PBS), and centrifuged at 250g for 5 minutes. After the
cells had reached ⬃70% confluence, they were seeded in
monolayer, in DMEM supplemented with 10% fetal bovine
serum (FBS), onto glass slides (Corning, Corning, NY) coated
with type II collagen. To ensure that the cell phenotype was
maintained, only first-passage cultured chondrocytes were
used.
Reagents. All culture materials were purchased from
Gibco (Grand Island, NY). Bovine type II collagen was
purchased from BD Biosciences (San Diego, CA). Bisindolylmaleimide I (a pan-PKC inhibitor), calphostin C (an inhibitor
that specifically targets the conventional and novel, but not
atypical, PKC isoforms), and Gö 6976 (an inhibitor of conventional PKC isoforms) were purchased from Calbiochem (La
Jolla, CA). Mithramycin A (an inhibitor of Sp-1 binding) was
purchased from Biomol Research Laboratories (Plymouth
Meeting, PA). Rabbit polyclonal antibodies against phosphoPKC␣, PKC␣, and Sp-1 were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). The specific siRNA for Sp-1
and those for the PKC isoforms, as well as the control siRNA
(scrambled negative control containing random DNA sequences), were purchased from Invitrogen (Carlsbad, CA). All
other chemicals of reagent grade were obtained from Sigma
(St. Louis, MO).
Culture of SW-1353 chondrocytes. Human chondrosarcoma SW-1353 cells were obtained from American Type
Culture Collection (Rockville, MD) and cultured in DMEM
supplemented with 10% FBS. After the cells had reached
confluence (1–2 ⫻ 105 cells/cm2), they were trypsinized and
seeded onto glass slides (75 ⫻ 38 mm; Corning) precoated with
type II collagen (50 ␮g/ml in 0.1N acetic acid). The medium
was then changed to DMEM containing 2% FBS, and the cells
were incubated for a further 24 hours prior to being used in the
fluid flow experiment.
Fluid flow experiment. The glass slides were precoated
with type II collagen at 37°C for 1 hour, and chondrocytes were
then seeded onto glass slides for 24 hours. The glass slides with
cultured chondrocytes were mounted in a parallel-plate flow
chamber, as described in detail previously (22). The chamber
was connected to a perfusion loop system and kept in a
constant-temperature–controlled enclosure. The perfusate was
maintained at pH 7.4 by continuous gassing with a humidified
mixture of 5% CO2 in air. The fluid shear stress (␶) generated
on the cells by flow was estimated to be 2–20 dyn/cm2, unless
otherwise noted, using the formula ␶ ⫽ 6␮Q/wh2, where ␮ is
the dynamic viscosity of the perfusate, Q is the flow rate, and
w and h are the width and channel height, respectively.
Real-time quantitative PCR. Total RNA preparation
and the reverse transcription reaction were carried out as
described previously (23). PCRs were performed using an ABI
Prism 7900HT (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Amplification of specific PCR products was detected using SYBR Green PCR
Master Mix (Applied Biosystems). The designed primers in
this study were as follows: for PAI-1, forward 5⬘-CAT-CCCCCA-TCC-TAC-GTG-G-3⬘, reverse 5⬘-CCC-CAT-AGGGTG-AGA-AAA-CCA-3⬘; for PKC␣, forward 5⬘-ATT-CTATGC-GGC-AGA-GAT-TTC-C-3⬘, reverse 5⬘-TCC-TTCTGA-ATC-CAA-CAT-GAC-G-3⬘; for Sp-1, forward 5⬘-GGTGCC-TTT-TCA-CAG-GCT-C-3⬘, reverse 5⬘-CAT-TGGGTG-ACT-CAA-TTC-TGC-T-3⬘; for GAPDH, forward 5⬘-
PKC␣ AND SHEAR-INDUCED PAI-1 EXPRESSION IN OA CHONDROCYTES
GGG-GTC-ATT-GAT-GGC-AAC-AAT-A-3⬘, reverse 5⬘ATG-GGG-AAG-GTG-AAG-GTC-G-3⬘; and for 18S
ribosomal RNA (rRNA), forward 5⬘-CGG-CGA-CGA-CCCATT-CGA-AC-3⬘, reverse 5⬘-GAA-TCG-AAC-CCT-GATTCC-CCG-TC-3⬘. RNA samples were normalized to the levels
of GAPDH and 18S rRNA. All primer pairs had at least 1
primer crossing an exon–exon boundary.
The real-time PCR was performed in triplicate in a
total reaction volume of 25 ␮l containing 12.5 ␮l of SYBR
Green PCR Master Mix, 300 nM forward and reverse primers,
11 ␮l of distilled H2O, and 1 ␮l of complementary DNA from
each sample. Samples were heated for 10 minutes at 95°C and
amplified for 40 cycles of 95°C for 15 seconds and 60°C for 60
seconds. Quantification was performed using the 2⫺⌬⌬Ct
method (24), where the Ct value was defined as the threshold
cycle of the PCR at which amplified product was detected. The
⌬Ct value was obtained by subtracting the Ct value of the
housekeeping gene (GAPDH or 18S rRNA) from the Ct value
of the gene of interest (PAI-1). The present study used the ⌬Ct
value of controls as the calibrator. The fold change was
calculated according to the formula 2⫺⌬⌬Ct, where ⌬⌬Ct was
the difference between the ⌬Ct value and the ⌬Ct calibrator
value (which was assigned a value of 1 arbitrary unit).
Functional PAI-1 activity assay. The PAI-1 activity in
the conditioned medium of the flow system was determined
using a commercial enzyme-linked immunosorbent assay
(ELISA) kit (Molecular Innovations, Southfield, MI), following the manufacturer’s instructions (25). One unit of PAI-1
activity is defined as the amount of PAI-1 that inhibits 1 IU of
human single-chain tPA, as calibrated against the international
standard for tPA.
Western blot analysis. Chondrocytes were lysed with a
buffer containing 1% Nonidet P40, 0.5% sodium deoxycholate,
0.1% sodium dodecyl sulfate (SDS), and a protease inhibitor
mixture (phenylmethylsulfonyl fluoride, aprotinin, and sodium
orthovanadate). The total cell lysate (50 ␮g of protein) was
separated by SDS–polyacrylamide gel electrophoresis (12%
running, 4% stacking) and analyzed using the designated
antibodies and the Western-Light chemiluminescent detection
system (Bio-Rad, Hercules, CA), as described previously (26).
Reporter gene construct, siRNA, transfection, and
luciferase assays. The PAI-1 promoter construct (PAI-1-Luc)
contains 800 bp of PAI-1 5⬘-flanking DNA linked to the firefly
luciferase reporter gene of plasmid pGL4 (Promega, Madison,
WI). DNA plasmids at a concentration of 1 mg/ml were
transfected, using lipofectamine (Gibco), into SW-1353 cells
when the cells had reached 70% confluence. The pSV-␤galactosidase plasmid was cotransfected to normalize the
transfection efficiency. The cells were kept unstimulated as
static controls or were subjected to shear stress experiments 48
hours after transfection. For siRNA transfection, SW-1353
cells, which had reached 70% confluence, were transfected
with the designated siRNA, using the RNAiMAX transfection
kit (Invitrogen).
Chromatin immunoprecipitation (ChIP) assay. After
crosslinking cells with 1% formaldehyde, the cells were centrifuged and then resuspended in lysis buffer for 3 cycles of
sonication, each for 15 seconds. Supernatants were recovered
by centrifugation. Immunoprecipitation was performed overnight with specific antibodies against Sp-1 at 4°C with rotation.
PCR was performed with primers that amplify the part of the
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human PAI-1 promoters that contains the Sp-1 binding sites;
these primers were 5⬘-TCA-GCA-AGT-CCC-AGA-GAGGG-3⬘ and 5⬘-GAT-GAA-CTC-ATG-TTC-CAG-CC-3⬘.
Sp-1 transcription factor ELISA. Nuclear extracts of
cells were prepared as described previously (27). Equal
amounts of nuclear extracts were used for quantitative measurements of Sp-1 activation, using commercially available
ELISA kits (Panomics, Redwood City, CA) that measure Sp-1
DNA binding activity.
Cellular DNA content analysis. Cell viability was analyzed by flow cytometry. Briefly, cells were harvested in PBS
containing 2 mM EDTA, washed once with PBS, and fixed for
30 minutes in cold ethanol (70%). Fixed cells were washed
once in PBS and permeabilized with 0.2% Tween 20 and 1
mg/ml RNase A in PBS for 30 minutes. The cells were then
washed once in PBS and stained with 50 ␮g/ml of propidium
iodide (Roche, Basel, Switzerland). Stained cells were analyzed with a FACSCalibur system (BD Biosciences), and the
data were analyzed using CellQuest software (BD Biosciences). At least 3 independent experiments were performed.
Statistical analysis. Results are expressed as the
mean ⫾ SEM. Statistical analysis was performed using an
independent Student’s t-test for comparisons of 2 groups of
data, and using analysis of variance followed by Scheffe’s test
for multiple comparisons. P values less than 0.05 were considered significant.
RESULTS
Down-regulation of PAI-1 in human OA chondrocytes from lesional sites. Expression of PAI-1
mRNA in human articular chondrocytes from the lesional and nonlesional sites of OA cartilage (n ⫽ 10) and
from normal healthy cartilage (n ⫽ 4) (Figure 1A) were
analyzed using real-time PCR. The internal reference
genes GAPDH and 18S rRNA were used for normalization of the results of real-time PCR. Healthy cartilage
showed wide variations in the expression of PAI-1
mRNA, reflecting individual differences. There was no
significant difference between healthy cartilage and nonlesional OA cartilage. However, expression of the PAI-1
gene was significantly down-regulated in OA chondrocytes from sites of lesions in comparison with OA
chondrocytes derived from nonlesional sites (P ⬍ 0.01)
(Figures 1B and C), indicating that PAI-1 expression is
modulated in OA chondrocytes. (The results in Figure
1B were normalized to GAPDH, while those in Figure
1C were normalized to 18S rRNA.)
Up-regulation of PAI-1 gene expression by moderate levels of shear stress. To study PAI-1 mRNA
expression under different shear stress conditions, human SW-1353 chondrocytes (used as a cell model) were
exposed to different fluid shear stresses (2, 5, 10, and 20
dyn/cm2) for 0.5, 1, 2, 3, or 4 hours, and the changes in
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YEH ET AL
Figure 2. Time course of expression of plasminogen activator inhibitor 1 (PAI-1) mRNA in human SW-1353 chondrocytes under shear stresses of
2 dyn/cm2 (A), 5 dyn/cm2 (B), 10 dyn/cm2 (C), and 20 dyn/cm2 (D). The chondrocytes were kept under static control (CL) conditions or were exposed
to the different shear stress levels for up to 4 hours. RNA samples were isolated at the indicated time periods and subjected to real-time polymerase
chain reaction. Bars show the mean and SEM fold change in fluorescence intensity relative to OA chondrocytes from nonlesional sites, with results
normalized to the levels of GAPDH. ⴱ ⫽ P ⬍ 0.05 versus static control.
PAI-1 mRNA expression were analyzed by real-time
PCR and normalized to the levels of GAPDH and 18S
rRNA. At shear stresses of 5 or 10 dyn/cm2, the PAI-1
mRNA level began to increase after 1 hour of shearing
and reached its highest level at 2 hours; thereafter, it
gradually reduced to the level of the static control
(Figures 2B and C). In contrast, a lower shear stress (2
dyn/cm2) or higher shear stress (20 dyn/cm2) had no
significant effect on the PAI-1 mRNA levels (Figures 2A
and D). (Data on exposure of the SW-1353 cells to
different levels of shear stress for 2 hours with results
normalized to 18S rRNA as the internal reference gene
are available from the corresponding author upon request).
A luciferase assay was conducted to confirm the
effects of shear stress on PAI-1 gene transcription. The
luciferase activity of SW-1353 cells exposed to shear
stresses of 5 and 10 dyn/cm2, but not 2 and 20 dyn/cm2,
for 2 hours was significantly higher than that under static
control conditions (Figure 3A).
We further examined functional PAI-1 activity in
SW-1353 cells under different shear stress conditions
with the use of a functional PAI-1 activity assay (Figure
3B). PAI-1 activity in conditioned medium subjected to
5 and 10 dyn/cm2 of the flow system was higher than that
under the static control conditions and at 2 and 20
dyn/cm2 shear stress. These data confirmed that 5 and 10
dyn/cm2 shear stress efficiently up-regulate PAI-1 transcription and protein activity. Notably, the effect of 10
dyn/cm2 was significantly higher than that of 5 dyn/cm2.
Mediation of the shear stress–induced upregulation of PAI-1 expression by the PKC␣ pathway.
Activation of PKC is known to be an important participant in mechanically induced signaling cascades in chondrocytes (9). To determine the role of PKC isoforms in
the shear-induced PAI-1 expression in SW-1353 chondrocytes, the cells were incubated with the specific
inhibitor for pan-PKC (bisindolylmaleimide I, 20 ␮M),
that for the conventional and novel PKC isoforms
(calphostin C, 100 nM), and that for the conventional
PKC isoforms (Gö 6976, 5 ␮M) (19) for 1 hour, and then
exposed to shear stress of 10 dyn/cm2 for 2 hours in the
presence of each inhibitor. Induction of PAI-1 by 10
dyn/cm2 of shear stress was significantly inhibited by
bisindolylmaleimide I, calphostin C, and Gö 6976, as
shown in Figure 4A with results normalized to GAPDH
(results normalized to 18S rRNA are available from the
corresponding author upon request), suggesting that the
PKC␣ AND SHEAR-INDUCED PAI-1 EXPRESSION IN OA CHONDROCYTES
2355
Figure 3. Analysis of PAI-1 promoter activity by luciferase assay (A) and PAI-1 protein activity by enzyme-linked immunosorbent assay (B) under
different levels of shear stress. SW-1353 chondrocytes were transfected with the PAI-1-Luc plasmid and exposed to 2–20 dyn/cm2 shear stress for
2 hours; results are expressed as the fold change relative to the static control, normalized to ␤-galactosidase activity (A). PAI-1 activity was assessed
in conditioned medium from the flow system under the different levels of shear stress for 2 hours (B). Bars show the mean and SEM. ⴱ ⫽ P ⬍ 0.05
versus static control; # ⫽ P ⬍ 0.05 versus 5 dyn/cm2 shear stress. See Figure 2 for definitions.
conventional PKC pathway is involved in shear-induced
PAI-1 gene expression.
The involvement of the conventional PKC pathway in shear-induced PAI-1 expression was further
confirmed by our experiments showing that PAI-1 ex-
pression was inhibited by transfecting the cells with PKC
isoform–specific siRNA (100 ␮g/ml) prior to the application of shear stress at 10 dyn/cm2 for 2 hours. Transfection of SW-1353 chondrocytes with PKC␣ siRNA
resulted in a significant inhibition of the shear-induced
Figure 4. Changes in PAI-1 expression and protein kinase C␣ (PKC␣) phosphorylation in SW-1353 chondrocytes. A, Effects of 1 hour of
pretreatment of chondrocytes with DMSO or the PKC inhibitors bisindolylmaleimide I (Bis), calphostin C (Cal), or Gö 6976 (Go) on PAI-1 mRNA
levels in SW-1353 chondrocytes before and after 2 hours of shear stress at 10 dyn/cm2. Controls were left untreated under static conditions (CL).
B, Effects of 48 hours of transfection of siRNA targeting PKC␣ (si-␣), PKC␤1 (si-␤1), or PKC␥ (si-␥) on the levels of PAI-1 mRNA in chondrocytes
before and after 2 hours of shear stress at 10 dyn/cm2. Controls were cells without transfection under static conditions (CL) or cells transfected with
control siRNA (si-CL). Bars in A and B show the mean and SEM fold change in fluorescence intensity relative to the static control, with results
normalized to the levels of GAPDH. ⴱ ⫽ P ⬍ 0.05 versus static control; # ⫽ P ⬍ 0.05 versus control under shear stress (DMSO in A, si-CL in B).
C and D, Effects of duration of shear stress (2–60 minutes at 10 dyn/cm2) (C) and shear stress level (2–20 dyn/cm2 for 10 minutes) (D) on PKC␣
phosphorylation in SW-1353 cells, as assessed by Western blotting (top) and as the fold change in phosphorylation levels (bottom). Bars in C and
D are the mean and SEM fold change in band density relative to static control, normalized to total protein levels. See Figure 2 for other definitions.
2356
PAI-1 gene expression. Transfection of cells with
PKC␤1 or PKC␥ siRNA had a minor suppressive effect
on the shear-induced PAI-1 expression (Figure 4B,
showing results normalized to GAPDH; results normalized to 18S rRNA are available from the corresponding
author upon request). Minor suppressive effects were
also observed in cells transfected with PKC␦, PKC␧, and
PKC␫ siRNA (results not shown). (Data indicating the
inhibitory effect of all of these siRNA on PKC␣ phosphorylation and the excellent gene-silencing efficiency
and high cell viability after 48 hours of transfection are
available from the corresponding author upon request.)
Induction of PKC␣ phosphorylation by shear
stress. To investigate the effect of shear stress on the
mechanotransduction of PKC␣, SW-1353 cells were
exposed to 10 dyn/cm2 shear stress, or kept in static
conditions as a control, for 0, 2, 5, 10, 30, and 60 minutes.
The activation of PKC␣ was determined by Western
blotting with specific anti–phospho-PKC␣ antibodies,
with results expressed as the extent of phosphorylation.
The phosphorylation of PKC␣ in SW-1353 chondrocytes
increased rapidly (within 2 minutes) after exposure to 10
dyn/cm2 shear stress and reached a maximal level at 10
minutes; the levels of phosphorylation decreased thereafter but still remained higher than that in the static
control at 60 minutes (Figure 4C). SW-1353 chondrocytes exposed to shear stress at 5 dyn/cm2 for 10 minutes
also significantly increased the phosphorylation of
PKC␣, although to a lesser degree than that after
exposure to 10 dyn/cm2 (Figure 4D). These findings
show that PKC␣ phosphorylation is involved in the
regulation of PAI-1 gene expression.
Mediation of Sp-1 activation by shear stress–
induced PAI-1 expression. Because the promoter region
of the PAI-1 gene contains the Sp-1 binding domain,
which is responsible for the modulation of gene expression (28), we tested whether Sp-1 activation is involved
in the signal transduction pathway leading to shearinduced PAI-1 gene expression. SW-1353 chondrocytes
were transfected with Sp-1 siRNA or incubated with the
specific inhibitor for Sp-1 (mithramycin A, 100 nM) (29)
for 1 hour, followed by application of shear stress of 10
dyn/cm2 for 2 hours. The shear stress–induced PAI-1
mRNA expression was significantly reduced by Sp-1
inhibition with mithramycin A and Sp-1 siRNA (Figure
5A, showing results normalized to GAPDH; results
normalized to 18S rRNA are available from the corresponding author upon request), indicating that Sp-1 is
involved in the regulation of PAI-1 gene expression.
We further evaluated shear stress–induced Sp-1
activation using a ChIP assay. SW-1353 chondrocytes
YEH ET AL
exposed to 10 dyn/cm2 shear stress showed a timedependent increase in Sp-1 binding activity on the PAI-1
promoter from 10 minutes to 30 minutes after exposure,
and the effect lasted for at least 2 hours (Figure 5B,
panel I). To further confirm these results, we performed
quantitative analysis for Sp-1 binding activity using the
Sp-1 transcription factor ELISA. Results of the ELISA
also showed that exposure of SW-1353 chondrocytes to
10 dyn/cm2 shear stress increased Sp-1 DNA binding
activity beginning at 10 minutes after exposure, and the
activity remained elevated for at least 2 hours (Figure
5B, panel II).
Pretreatment of cells with PKC inhibitors or
transfection of cells with PKC␣ siRNA significantly
inhibited the increase in Sp-1 DNA binding activity, but
there was no suppression of Sp-1 binding activity in cells
transfected with PKC␤1 siRNA or PKC␥ siRNA (Figure
5C). Application of 10 dyn/cm2 shear stress for 2 hours
caused an increase in promoter activity in SW-1353
chondrocytes transfected with the PAI-1-Luc plasmid.
Pretreatment of cells with bisindolylmaleimide I, Gö
6976, or mithramycin A or transfection of cells with
PKC␣ siRNA and Sp-1 siRNA resulted in a marked
inhibition of shear stress–induced PAI-1 promoter activity. However, transfection with PKC␤1, PKC␥, or control siRNA had little effect on the induction of PAI-1
promoter activity by shear stress (Figure 5D). These
results provide additional evidence that the PKC␣ and
Sp-1 pathways play an important role in the regulation of
shear stress–induced PAI-1 expression in chondrocytes.
Differential regulation of PAI-1 expression by
shear stress in human OA chondrocytes. Because PAI-1
mRNA expression in human OA chondrocytes from the
sites of lesions was significantly down-regulated in comparison with that in OA chondrocytes from nonlesional
sites (Figure 1), we examined the effects of shear stress
on the regulation of PAI-1 gene expression in human
articular chondrocytes, using real-time PCR; the internal reference genes GAPDH and 18S rRNA were used
to normalize the results. As shown in Figures 6A and B,
exposure to 10 dyn/cm2 shear stress for 2 hours significantly increased the expression ratio of shear stress–
induced PAI-1 mRNA to static control PAI-1 mRNA in
normal chondrocytes and in OA chondrocytes derived
from nonlesional sites, but the same level of shear stress
had a minor effect on the PAI-1 mRNA expression ratio
in OA chondrocytes from lesional sites. (The results in
Figure 6A were normalized to GAPDH, while those in
Figure 6B were normalized to 18S rRNA.) These data
suggest that 10 dyn/cm2 shear stress can further increase
the PAI-1 expression in normal and OA lesional chon-
PKC␣ AND SHEAR-INDUCED PAI-1 EXPRESSION IN OA CHONDROCYTES
2357
Figure 5. Roles of Sp-1 and protein kinase C␣ (PKC␣) in shear stress–induced PAI-1 mRNA expression and Sp-1 activation in SW-1353
chondrocytes. A, PAI-1 mRNA levels were determined in SW-1353 chondrocytes pretreated for 1 hour with vehicle control (DMSO) or the Sp-1
inhibitor mithramycin A (MMA) or transfected for 48 hours with control siRNA (si-CL) or siRNA for Sp-1 (si-Sp-1), and then exposed to 10 dyn/cm2
shear stress for 2 hours. Cells left unexposed under static conditions served as controls (CL). B, SW-1353 chondrocytes were left under static
conditions (CL) or exposed to 10 dyn/cm2 shear stress for the amounts of time indicated. Sp-1 activation was then determined by chromatin
immunoprecipitation (ChIP) assay (I) or by transcription factor enzyme-linked immunosorbent assay (II). Chromatin was immunoprecipitated with
anti–Sp-1 antibody. One percent of the precipitated chromatin was assayed to verify equal loading (INPUT). C, SW-1353 chondrocytes were
pretreated for 1 hour with DMSO or the PKC inhibitors bisindolylmaleimide I (Bis) or Gö 6976 (Go) or transfected for 48 hours with si-CL or
specific siRNA for PKC␣ (si-␣), PKC␤1 (si-␤1), or PKC␥ (si-␥), and then exposed to 10 dyn/cm2 shear stress for 30 minutes. Sp-1 activation was
then determined by ChIP assay. D, SW-1353 chondrocytes were pretreated with DMSO, Bis, Go, or MMA for 1 hour or transfected with si-CL, si-␣,
si-␤1, si-␥, or si-Sp-1, and then exposed to 10 dyn/cm2 shear stress for 2 hours. Cells were cotransfected with the PAI-1-Luc plasmid, and PAI-1
promoter activity was measured by luciferase assay. Bars show the mean and SEM fold change relative to static control. ⴱ ⫽ P ⬍ 0.05 versus static
control; # ⫽ P ⬍ 0.05 versus DMSO-treated cells under shear stress; & ⫽ P ⬍ 0.05 versus control siRNA transfection. See Figure 2 for other
definitions.
drocytes. In contrast, chondrocytes from lesional cartilage, which already exhibits a reduction in PAI-1 expression, will lose the ability to respond to the same shear
stress.
The expression of PKC␣ mRNA was downregulated in OA chondrocytes from the sites of lesions
compared with OA chondrocytes derived from nonlesional sites (Figure 6C, panel I). Exposure of OA
chondrocytes from nonlesional cartilage to 10 dyn/cm2
shear stress for 10 minutes increased the phosphorylation of PKC␣, but this effect was not observed in OA
chondrocytes derived from lesional cartilage (Figure 6C,
panel II). Although the expression of Sp-1 mRNA
appeared higher in OA lesional chondrocytes, the difference compared with OA nonlesional chondrocytes
was not statistically significant (Figure 6D, panel I). OA
chondrocytes from lesional cartilage and OA chondrocytes from nonlesional cartilage showed different Sp-1
DNA binding activities induced by shear stress (Figure
6D, panel II), again indicating that the PKC␣/Sp-1
pathway plays an important role in the regulation of
shear stress–induced PAI-1 expression in human OA
chondrocytes.
DISCUSSION
Biochemical and genetic factors, as well as mechanical stress, contribute to the OA lesion in human
cartilage by disrupting chondrocyte–matrix associations
and altering metabolic responses in the chondrocyte
(30). The major biochemical factors involved in the
pathogenesis of OA include cytokines (e.g.,
interleukin-1␤ [IL-1␤] and tumor necrosis factor ␣),
chemokines (e.g., stromal cell–derived factor 1), metalloproteinases, tissue inhibitors of metalloproteinases,
and the uPA/PAI-1 system (30). PAI-1 is the main
physiologic inhibitor of uPA and tPA in vivo. Temporal
changes in the expression and relative activity levels of
the PA/PAI pair may influence cell function, either as a
direct consequence of ECM-barrier proteolysis or by
modulating cellular adhesive interactions with the ECM
(31).
2358
YEH ET AL
Figure 6. Effects of 10 dyn/cm2 shear stress on PAI-1, PKC␣, and Sp-1 mRNA expression, PKC␣ phosphorylation, and Sp-1 activation in samples
of normal human cartilage and lesional and nonlesional osteoarthritic (OA) cartilage. A and B, Cells were kept under static control conditions (CL)
or exposed to 10 dyn/cm2 shear stress (SS) for 2 hours. Results are expressed as the mean and SEM ratio of shear stress–induced mRNA expression
to static control mRNA expression for each type of sample, normalized to GAPDH (A) and 18S rRNA (B). ⴱ ⫽ P ⬍ 0.05. C, PKC␣ mRNA
expression was assessed in lesional and nonlesional OA cartilage. Results are expressed as the mean and SEM fold induction, normalized to GAPDH
(I). ⴱ ⫽ P ⬍ 0.05 versus OA nonlesional chondrocytes. Phosphorylation of PKC␣ in human OA chondrocytes (nonlesional and lesional) was assessed
by Western blotting of cells kept under static control conditions or exposed to 10 dyn/cm2 shear stress for 10 minutes (II). Results are the mean and
SEM fold change in band densities relative to nonlesional static controls, normalized to total protein levels. ⴱ ⫽ P ⬍ 0.05 versus nonlesional static
controls. D, Sp-1 mRNA expression was detected in samples of lesional and nonlesional OA human cartilage by real-time polymerase chain reaction
(I). Results are the mean and SEM fold induction, normalized to GAPDH. Sp-1 activation was assessed by chromatin immunoprecipitation (ChIP)
assay (II). Human OA chondrocytes were kept under static control conditions or exposed to 10 dyn/cm2 shear stress for 30 minutes, and ChIP assay
for Sp-1 was performed. Chromatin was immunoprecipitated with anti–Sp-1 antibody. One percent of the precipitated chromatin was assayed to
verify equal loading (INPUT). See Figure 2 for other definitions.
Cell type–specific synthesis and subcellular targeting of PAI-1 and uPA appear to play an important
role in modulating the progression of OA (6). Multiple
factors, e.g., IL-1␤ (32,33), have been shown to regulate
PAI-1 expression in chondrocytes, but the role of shear
stress in regulating PAI-1 gene expression in chondrocytes is unclear. The present study assessing the SW1353 human chondrocytic cell line demonstrated that
shear stress regulates PAI-1 expression at the transcriptional level. Moreover, analysis of human PAI-1 promoter activity revealed that Sp-1 functions as the cis
element for shear stress responsiveness via PKC␣ phosphorylation.
Chondrocytes show different responses to distinct levels of mechanical stress. Previous studies indicated that 5–10 dyn/cm2 shear stress has anticatabolic
effects on articular cartilage, and that shear stresses
higher than 16 dyn/cm2 can induce chondrocyte apoptosis, inflammation, and cartilage degradation (34–37). In
our study, we assessed SW-1353 chondrocytes in a fluid
flow chamber to investigate the effect of shear stress on
the signaling pathway leading to PAI-1 gene expression.
In this system, shear stresses of 5 dyn/cm2 and 10
dyn/cm2 were found to activate the PKC␣ signal transduction pathway, followed by an increase in PAI-1
promoter activity through regulation of Sp-1 DNA binding, and these events led to increased PAI-1 expression
on chondrocytes. Healy et al (37) also showed that
cyclooxygenase 2 (COX-2) expression in human chondrocytes is detected only at levels above the threshold
shear stress level of 5 dyn/cm2, and a further increase in
COX-2 levels occurs after exposure to 10 dyn/cm2.
According to these findings, shear stress applied at a
level of ⬃10 dyn/cm2 effectively decreases the catabolic
effects in chondrocytes. In contrast, abnormally high
mechanical loading, such as 20 dyn/cm2 shear stress, may
induce COX-2–mediated inflammation (37) and reduce
PAI-1–mediated inhibition of uPA, tPA, and cartilage
degradation.
The present study characterized a novel mechanism in which moderate levels of shear stress play an
important role in the regulation of PAI-1 expression in
normal human chondrocytes, but this regulatory response is deficient in human OA chondrocytes at sites of
PKC␣ AND SHEAR-INDUCED PAI-1 EXPRESSION IN OA CHONDROCYTES
lesions. Thus, in normal chondrocytes and in OA chondrocytes from nonlesional sites, shear stress at 10 dyn/
cm2 induces the up-regulation of PAI-1 expression via
activation of the PKC␣ and Sp-1 signaling pathways. In
contrast, human OA articular chondrocytes from lesional sites not only have significantly lower PAI-1
expression under static conditions (Figure 1) but also do
not possess the ability to respond to moderate shear
stresses to increase PAI-1 expression (Figure 6A).
The abnormal response of lesion-derived OA
chondrocytes to excessive loading is a likely contributor
to the dysregulation of chondrocyte function, favoring
disequilibrium between the catabolic and anabolic activities of the chondrocyte in remodeling the cartilage
ECM by the production of metalloproteinases and aggrecanase (38). The progressive degradation of the
cartilage matrix that occurs in OA indicates that there is
a local imbalance in the proteinase–inhibitor content
(6). Shlopov et al (30) found that in the joint of the same
OA patient, the levels of matrix metalloproteinase 13
(MMP-13) and MMP-1 were higher in chondrocytes
from lesional sites than in chondrocytes from nonlesional sites, and their results suggested that aberrant
mechanical forces led to elevated levels of MMPs to
enhance degradation.
Although previous studies have shown that OA
chondrocytes from lesional articular cartilage and those
from nonlesional articular cartilage have different levels
of gene expression under static conditions (19,39), there
have been no reports on their differential gene expression in response to external stimuli (e.g., shear stress). It
is known that vascular endothelial cells contain mechanoreceptors on the cell surface, and several studies
have shown that shear stress can alter the gene expression in endothelial cells through the activation of intracellular signal transduction pathways (40,41). In primary
culture human chondrocytes, we observed that the influence of moderate shear stresses on healthy and OA
lesional chondrocytes was similar to the results in SW1353 chondrocytes. In contrast, in OA lesional chondrocytes, such moderate shear stresses failed to activate
PKC␣ and had no effect on the Sp-1 DNA binding
activity; thus, this signal transduction pathway is suppressed in OA lesional chondrocytes. These findings
serve to explain the differential regulation of PAI-1
expression on OA chondrocytes from lesional sites compared with those from nonlesional sites.
To respond to mechanical environments such as
shear stress, chondrocytes may have putative
mechanosensors on the cell surface, and different levels
of shear stress may activate different mechanosensors
2359
and/or different signaling pathways. According to our
findings, the process of mechanotransduction in response to moderate shear stresses may be malfunctional
in chondrocytes obtained from the OA lesion.
It has been reported that shear stress can activate
MAPK pathways to regulate early and late inflammatory
responses in OA chondrocytes (12), but there is no
report on the activation of PKC by shear stress in
chondrocytes. PKC␣ plays an essential role in chondrocyte dedifferentiation and redifferentiation (42), and
there are several reports on the possible linkage between
PKC isoforms and the pathogenesis of OA. Hamanishi
et al (43) found that the activation of PKC can inhibit
the pathogenesis of OA in animal studies. Thus, the
regulation of PKC in chondrocytes by shear stress could
have physiologic and pathophysiologic significance. Our
findings of the decrease in PKC␣ expression and the lack
of a shear stress–induced phosphorylation response of
PKC␣ in OA chondrocytes from lesional articular cartilage suggest that these molecular derangements may
lead to dysfunction of the chondrocytes at lesional sites.
The PAI-1 promoter has different binding sites for
transcription factors such as Sp-1, Ets-1, and activator
protein 1 (28). In the present study, we used a transcription factor ELISA and a ChIP assay to demonstrate that
the regulation of PAI-1 gene expression on chondrocytes
is mediated by the activation of Sp-1 and by an increase
in Sp-1 DNA binding activity following PKC␣ phosphorylation. Nonlesional chondrocytes from OA articular
cartilage can activate PKC␣ and increase Sp-1 DNA
binding activity in response to shear stress, but such
shear stress–induced effects are absent in OA chondrocytes from lesional cartilage.
Cartilage-specific matrix components (type II
collagen) play a major role in the regulation of chondrocyte differentiation and the expression of chondrocyte
phenotype (44). When grown on type II collagen, the
chondrocytes maintain their round phenotype, produce
type II collagen and fibronectin, and express ␤1 integrins
on their cell membrane. Results from that previous
study also indicated that the influence of type II collagen
on cellular behavior depends on the integrins participating in a chondrocyte–type II collagen interaction (44). In
addition, OA chondrocytes show a strong activation of
synthetic activity in the increased expression of type II
collagen (45).
Although the literature contains extensive reports on the effects of fluid shear stress on chondrocytes,
the detailed mechanisms that transduce mechanical
stimuli to intracellular signals to regulate the downstream gene expression remain unclear. Integrins, as the
2360
YEH ET AL
main receptors that connect the cytoskeleton and the
ECM, have been shown to play important roles in
transmitting mechanical stresses into chemical signals in
a wide variety of cells seeded on the ECM (46). Recent
studies indicate that shear stress–induced signal transduction and gene expression are dependent on the
specific integrin and its cognate ECM protein to which
the cells are adhered (47,48). Therefore, elucidation of
the role of integrins in these events requires further
studies on OA lesional, OA nonlesional, and normal
articular chondrocytes with the use of different ECM
proteins and by manipulation of the integrin activities.
In summary, our present study revealed that, in
comparison with nonlesional cartilage from OA patients
and normal healthy cartilage, lesional cartilage from OA
patients exhibits a decrease in PAI-1 mRNA expression
and a loss of induction of PAI expression by shear stress
through PKC␣ phosphorylation, Sp-1 activation, and
Sp-1 DNA binding. Our findings provide a molecular
basis for understanding the mechanisms contributing to
the function of chondrocytes in a healthy state and to the
dysfunction of chondrocytes in the progression of a
disease such as OA.
AUTHOR CONTRIBUTIONS
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. Cheng-Nan Chen 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. Chien, C-N Chen.
Acquisition of data. Yeh, Chang, Chiang, Tsai, L-M Chen, Wu.
Analysis and interpretation of data. Yeh, Chang, Chiang, Tsai, L-M
Chen, Wu.
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