Divergent responses of chondrocytes and endothelial
cells to shear stress: Cross-talk among COX-2,
the phase 2 response, and apoptosis
Zachary R. Healy†, Norman H. Lee‡, Xiangqun Gao§, Mary B. Goldring¶, Paul Talalay§, Thomas W. Kensler?,
and Konstantinos Konstantopoulos†,††
†Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, Baltimore, MD 21218;‡The Institute for Genomic Research,
Rockville, MD 20850;§Department of Pharmacology and Molecular Sciences, The Johns Hopkins University, School of Medicine, Baltimore, MD 21205;
¶Beth Israel Deaconess Medical Center, Rheumatology Division, and New England Baptist Bone and Joint Institute, Harvard Institutes of Medicine,
Boston, MA 02115; and?Department of Environmental Health Sciences, Bloomberg School of Public Health, The Johns Hopkins University,
Baltimore, MD 21205
Contributed by Paul Talalay, August 3, 2005
Fluid shear exerts anti-inflammatory and anti-apoptotic effects on
endothelial cells by inducing the coordinated expression of phase
2 detoxifying and antioxidant genes. In contrast, high shear is
pro-apoptotic in chondrocytes and promotes matrix degradation
and cartilage destruction. We have analyzed the mechanisms
regulating shear-mediated chondrocyte apoptosis by cDNA mi-
croarray technology and bioinformatics. We demonstrate that
shear-induced cyclooxygenase (COX)-2 suppresses phosphatidyl-
inositol 3-kinase (PI3-K) activity, which represses antioxidant re-
sponse element (ARE)?NF-E2 related factor 2 (Nrf2)-mediated tran-
scriptional response in human chondrocytes. The resultant
decrease in antioxidant capacity of sheared chondrocytes contrib-
utes to their apoptosis. Phase 2 inducers, and to a lesser extent
COX-2-selective inhibitors, negate the shear-mediated suppression
of ARE-driven phase 2 activity and apoptosis. The abrogation of
shear-induced COX-2 expression by PI3-K activity and?or stimula-
tion of the Nrf2?ARE pathway suggests the existence of PI3-K?
Nrf2?ARE negative feedback loops that potentially interfere with
c-Jun N-terminal kinase 2 activity upstream of COX-2. Reconstruct-
ing the signaling network regulating shear-induced chondrocyte
artificial cartilage in bioreactors and for developing therapeutic
strategies for arthritic disorders.
inflammation ? NAD(P)H:quinone oxidoreductase 1 ? NF-E2 related
factor 2 ? phosphatidylinositol 3-kinase ? sulforaphane
dependent manner. In the vasculature, high levels of laminar shear
atherogenic. Exposure of human aortic endothelial cells to high
laminar shear flow at 20 dynes (dyn)?cm2(1 dyn ? 10 ?N) induces
the expression of genes encoding for phase 2 detoxifying and
antioxidant genes including NAD(P)H:quinone oxidoreductase 1
(NQO1), heme oxygenase (HO)-1, and ?-glutamylcysteine ligase
(GCLR), which may be responsible for the resultant anti-
inflammatory responses (1, 2). Moreover, laminar shear flow
potently inhibits apoptosis in growth factor-starved human umbil-
ical vein endothelial cells (HUVECs) (3). Furthermore, low intra-
depolarization and apoptosis in multiple cell lines (4, 5), indicating
that the intracellular redox state is a key determinant of pro-
grammed cell death.
In marked contrast to the endothelium, extensive mechanical
loading of cartilage producing both hydrostatic pressure and fluid
shear (6) results in irreversible matrix erosion, chondrocyte apo-
ptosis (7), and osteoarthritis. Numerous in vitro and computational
studies support the view that hydrostatic pressure and low shear
(?5 dyn?cm2) is chondroprotective, whereas high shear promotes
luid shear is a critical physiological stimulus that modulates
intracellular signaling in a time-, magnitude-, and phenotype-
matrix degradation (6, 8, 9). Indeed, high fluid shear (16 dyn?cm2)
induces chondrocyte apoptosis (7), although the underlying mech-
anisms are unknown. We have recently shown that high shear (20
dyn?cm2) induces cyclooxygenase-2 (COX-2) expression in human
chondrocytic cells through a c-Jun N-terminal kinase 2 (JNK2)-
dependent pathway (10). Overexpression of COX-2 protein in
articular cartilage, an earmark of arthritis (11), has been associated
with inflammation and increased chondrocyte apoptosis by un-
The divergent effects of fluid shear on chondrocytes and endo-
to investigate whether shear stress regulates phase 2 gene expres-
sion in chondrocytes and to elucidate the signaling pathway of
shear-mediated chondrocyte apoptosis. Phase 2 enzymes protect
cells against oxidative and electrophilic stress. Regulation of basal
and inducible expression of cytoprotective phase 2 genes is medi-
ated by the antioxidant response element (ARE), a cis-acting
promoter element of these genes, and the cognate transcription
factor, NF-E2 related factor 2 (Nrf2) (1, 12). Nrf2 is normally
sequestered in the cytoplasm by binding to a repressor protein,
specific inducers disrupt the Nrf2?Keap1 complex, Nrf2 migrates
to the nucleus where it heterodimerizes with members of the small
Maf family, binds to the ARE, and enhances phase 2 gene
transcription (14). Interestingly, COX-2 mediates accumulation of
15d-PGJ2in sheared endothelial cells. This prostaglandin binds to
cysteine residues of Keap1, thereby releasing Nrf2 and activating
ARE-mediated transcription (2).
We show here that in human chondrocytes, shear-induced
COX-2 activity suppresses phosphoinositol 3-kinase (PI3-K) activ-
ity, which leads to decreased Nrf2 expression and ARE-mediated
transcription. The resultant decrease in antioxidant capacity may
then modulate apoptosis. Phase 2 inducers and to a lesser extent
specific inhibitors of COX-2 negate the shear-mediated repression
of ARE-regulated enzyme activity and apoptosis. We provide
evidence for the unexpected existence of negative feedback loops
by which PI3-K activity and?or phase 2 enzyme activity repress
COX-2 expression and inflammatory signaling. Elucidation of the
signaling networks regulating chondrocyte apoptosis may offer
novel approaches to therapy of arthritic pathology and provide
insight into the design of bioreactors for culturing cartilage.
Abbreviations: ARE, antioxidant response element; COX, cyclooxygenase; D3T, 1,2-
dithiole-3-thione; dyn, dyne; GCLR, ?-glutamylcysteine ligase regulatory unit; GSH, gluta-
thione; HO, heme oxygenase; HUVECs, human umbilical vein endothelial cells; JNK2, c-jun
N-terminal kinase 2; Keap1, Kelch-like ECH-associated protein 1; NQO1, NAD(P)H:quinone
oxidoreductase 1; Nrf2, NF-E2 related factor 2; PG, prostaglandin; PI3-K, phosphoinositol
3-kinase; SFN, sulforaphane.
††To whom correspondence should be addressed. E-mail: firstname.lastname@example.org.
© 2005 by The National Academy of Sciences of the USA
September 27, 2005 ?
vol. 102 ?
Chemicals. Sulforaphane (SFN) and 1,2-dithiole-3-thione (D3T)
were from LKT Laboratories (St. Paul, MN). NS398 and
CAY10404 were from Cayman Chemical (Ann Arbor, MI).
Cell Culture and Shear Stress Exposure. Human T?C28a2 chondro-
exposure, T?C28a2 cells were incubated for 24 h in serum-free
medium containing 1% Nutridoma-SP (Roche) (10, 15), a low-
Primary HUVECs were cultured as described in ref. 16.
T?C28a2 cells were exposed to shear stress in media containing
1% Nutridoma by use of a parallel-plate flow chamber with a
recirculating flow loop (37°C in 5% CO2) (10). HUVECs were
Cell Viability, NQO1 Activity, Glutathione Levels, and Prostaglandin
(PG)E2 Production. Cell viability was monitored with the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay (17).
NQO1 activity and total GSH (oxidized and reduced) levels of cell
lysates were determined in 96-well microtiter plates (17). PGE2
levels were determined in media by the Prostaglandin E2mono-
clonal EIA kit (Cayman Chemical).
Transient Transfection and Plasmid Constructs. T?C28a2 cells were
transfected with 10 ?g of plasmid and 2 ?g of control by using
Lipofectamine with Plus Reagent (Invitrogen). Cells were allowed
to recover for 3 h, were incubated overnight in medium containing
1% Nutridoma, and were exposed to the indicated treatments.
Efficiency was assessed by flow cytometry with pEGFP-N2 (BD
Biosciences). pCMV-mNrf2 and pNQO1?ARE-luc constructs
constructs were provided by A. Klippel (Atugen, Berlin) (20).
Promoter Activity Assay. T?C28a2 cells were transfected with 10 ?g
of pNQO1?ARE-luc and 1 ?g each of pEGFP-N2 and pSV40-
hRL2 (Promega) to normalize transfection efficiency. Firefly and
Renilla luciferase activities were measured by using the Dual-
Luciferase Reporter Assay kit (Promega).
Target Generation and Purification for Microarray Analysis. Total
RNA (10 ?g) isolated by using TRIzol (Invitrogen) were reverse
transcribed in the presence of aminoallyl(aa)-dUTP with Super-
Script II RT (Invitrogen) (10). The RNA template was hydrolyzed
(0.2 M NaOH?0.1 M EDTA) at 70°C for 15 min, and unincorpo-
rated nucleotides were removed with a Microcon YM30 column
(Millipore). aa-dUTP-labeled targets were dried, resuspended in
0.1 M Na2CO3 buffer (pH 9.0) (10, 21), and coupled to either
NHS-Cy3 or NHS-Cy5 (Amersham Pharmacia) for 1 h at 25°C.
Uncoupled label was removed with a QIAquick column (Qiagen).
Microarray Hybridization and Analysis. Cy-3- and Cy-5-labeled tar-
gets were mixed, dried, resuspended in hybridization buffer (50%
formamide?10? SSC?0.2% SDS?COT-1 DNA?Poly(A)-DNA),
with a set of 32,448 or 39,936 ESTs, allowed to hybridize at 42°C
ratios were derived by using TIGR Spotfinder (10, 21). Differen-
tially expressed genes were identified by Significance Analysis of
Microarrays, and analyzed with the software TMEV (10).
Quantitative Real-Time PCR (qRT-PCR). qRT-PCR was used to verify
DNA microarray data. Incorporation of SYBR Green into PCR
products was monitored with the 7900HT detection system. Addi-
tional information is provided in Table 2, which is published as
supporting information on the PNAS web site.
Intracellular Protein Staining and Western Blots. T?C28a2 cells were
fixed in 1.0% formaldehyde for 10 min at 37°C, permeabilized in
90% methanol for 20 min on ice, and incubated at 25°C for 10 min
in blocking buffer (0.5% BSA in PBS). Specimens were then
incubated with fluorophore-conjugated monoclonal antibodies
specific for COX-1 (COX-1?FITC) and COX-2 (COX-2?PE)
(Cayman Chemical) or isotype controls (BD Biosciences) for 30
min, washed twice in blocking buffer, and analyzed by flow cytom-
etry. For Western blots, total cell lysates were subjected to SDS?
PAGE, transferred to a membrane, and probed with caspase-9 and
?-actin antibodies (Upstate Biotechnology).
DNA Fragmentation and Mitochondrial Depolarization. For DNA
fragmentation, cells were fixed in 4% paraformaldehyde for 1 h at
25°C, washed twice in PBS, and permeabilized briefly in 0.1%
Triton-X100?0.1% sodium citrate on ice. Subsequently, cells were
washed twice in PBS, labeled by using the In Situ Cell Death Kit
(Roche), and analyzed by flow cytometry. To quantify mitochon-
drial membrane potential, cells were labeled by using the Mito-
Probe JC-1 Kit (Molecular Probes).
High Fluid Shear Protects Endothelial Cells but Is Proinflammatory in
(20 dyn?cm2) induces mRNA expression of a battery of ARE-
mediated genes including those encoding for NQO1, HO-1, and
the antioxidant capacity of T?C28a2 chondrocytic cells. Inspection
of microarray data revealed that exposure of T?C28a2 cells to 20
dyn?cm2for 1.5 or 24 h did not result in significant changes in the
mRNA levels of ARE-mediated genes (Table 2), nor did it appre-
ciably affect NQO1 activity or total GSH levels, common markers
of antioxidant capacity, relative to static controls (Fig. 1 A and B).
In contrast, exposure of HUVECs to 20 dyn?cm2increased NQO1
protein activity (Fig. 1A) and GSH levels (Fig. 1B) in a time-
dependent manner, in agreement with previous findings (1). We
next examined the effects of longer shear duration on phase 2
response. In contrast to HUVECs, prolonged exposure (48 h) of
T?C28a2 cells to 20 dyn?cm2significantly decreased both NQO1
activity (Fig. 1A) and GSH levels (Fig. 1B), which correlates well
with NQO1 and GCLR mRNA levels (Table 1).
To examine whether the reduced phase 2 response of shear-
stimulated T?C28a2 cells was an artifact of prolonged shear
exposure (48 h), cells were subjected to a low shear level (5
increase in NQO1 activity (Fig. 1A) and GSH levels (Fig. 1B).
Moreover, shear stress exposure of 40 dyn?cm2for 24 h resulted
in a profound reduction in NQO1 activity (Fig. 1A) and GSH
levels (Fig. 1B). These findings suggest that shear stress alters the
antioxidant capacity of chondrocytes in a time- and intensity-
Human Chondrocytic Cells. Priorworkhasestablishedthekeyroleof
the ARE, a cis-acting DNA regulatory element of phase 2 genes,
Microarray analysis revealed that prolonged exposure (48 h) of
T?C28a2 cells to a shear level of 20 dyn?cm2resulted in a marked
reduction in Nrf2 and phase 2 transcript expression, including
NQO1, HO-1, GST ?1, and GCLR (Table 1). The accuracy of the
microarray transcriptional profiling was confirmed by qRT-PCR
(Fig. 9, which is published as supporting information on the PNAS
web site). Based on these findings, we propose that the decreased
Healy et al.
September 27, 2005 ?
vol. 102 ?
no. 39 ?
Nrf2 mRNA expression is responsible for shear-mediated suppres-
sion of phase 2 genes.
To establish involvement of the ARE in shear-mediated regu-
lation of phase 2 genes, T?C28a2 cells were transfected with a
of T?C28a2 cells to 20 dyn?cm2for 48 h resulted in a substantial
reduction of the ARE-driven promoter activity (Fig. 1C). In
contrast, application of low shear (5 dyn?cm2) for 48 h moderately
increased promoter activity (Fig. 1C Left), in agreement with
enzyme activities (Fig. 1A). Having established (Fig. 10, which is
published as supporting information on the PNAS web site) that
T?C28a2 chondrocytic cells, like other cells, respond to chemical
inducers of phase 2 activity, the functionality of the ARE element
was confirmed in positive control experiments with SFN and D3T
(12, 22) (Fig. 1C Right). Taken together, these data suggest that
shear-mediated decrease of Nrf2 leads to repression of ARE-
transcriptional activity in chondrocytes.
Phase 2 Inducers Restore ARE-Regulated Enzyme Activity and Reduce
COX-2 Expression and PGE2 Production in Shear-Treated Chondro-
cytes. We next wished to reverse the reduction in antioxidant
capacity and corresponding pro-inflammatory state generated in
shear-treated chondrocytes. Recently, activation of the Nrf2?ARE
pathway has been shown to correlate with suppression of inflam-
mation (1, 14, 23), thus prompting the use of phase 2 inducers.
Treatment of T?C28a2 cells with SFN (1.25 ?M), a potent phase 2
inducer found in edible plants (22), abolished the shear-induced
suppression of NQO1 activity and intracellular GSH levels (Fig. 2).
Furthermore, D3T (5 ?M), a specific inducer of the Nrf2?ARE
pathway (12), was likewise effective in suppressing the shear-
mediated reduction of phase 2 enzyme activity (Fig. 2).
In confirmation of earlier work that application of ?20 dyn?cm2
to chondrocytes increases COX-2 transcript levels (Table 1) (10),
we observed that high, but not low, shear induced COX-2 protein
expression in T?C28a2 cells (Fig. 3). Moreover, high shear sub-
determined. Data are relative to static controls. Bars are mean ? SEM (n ? 4–7,*, P ? 0.01, and §, P ? 0.05, with respect to static controls). (C) ARE-driven NQO1
promoter activity in response to shear stress stimulation and phase 2 inducers. (Left) T?C28a2 cells were transfected with pNQO1?ARE-luc vector and exposed
to either static conditions or laminar shear flow (5 or 20 dyn?cm2) for 24 or 48 h . (Right) To determine the efficacy of phase 2 inducers, transfected cultures were
and GFP expression. Data are relative to static controls (n ? 4,*, P ? 0.01, and §, P ? 0.05, with respect to the static control)
Phenotype-specific effects of shear stress duration and intensity on phase 2 response. HUVECs and T?C28a2 chondrocytic cells were subjected to either
Table 1. Effects of phase 2 inducer D3T or transfection with PI3-K expression plasmid on
mRNA transcript ratios in chondrocytes
Signaling molecules of interest
Transcript ratio (shear?static)
Protein kinases and transcription factors
JNK2 (C-jun N-terminal kinase 2)
PI3-K (phosphoinositol 3-kinase)
Transcription Factor AP-1 (c-jun)
Nrf2 (NF-E2 related factor 2)
Catalytic and effector proteins
Phase 2 and antioxidative proteins
NQO1 (NAD(P)H:quinone reductase-1)
HO-1 (heme oygenase-1)
HO-2 (heme oxygenase-2)
GST ?1 (glutathione S-transferase)
GCLR (?-glutamylcysteine ligase)
7.2 ? 0.5
0.3 ? 0.1
4.0 ? 0.4
0.7 ? 0.1
1.6 ? 0.1
0.8 ? 0.2
1.4 ? 0.1
1.3 ? 0.1
1.3 ? 0.1
3.9 ? 0.7
1.0 ? 0.2
0.9 ? 0.1
3.5 ? 0.2
1.1 ? 0.1
1.5 ? 0.1
1.3 ? 0.2
1.3 ? 0.1
0.8 ? 0.1
1.7 ? 0.2
0.8 ? 0.2
0.8 ? 0.1
0.7 ? 0.1
0.6 ? 0.1
1.1 ? 0.1
0.5 ? 0.1
0.7 ? 0.1
0.5 ? 0.1
0.4 ? 0.1
1.3 ? 0.1
1.2 ? 0.1
0.9 ? 0.1
1.6 ? 0.1
1.0 ? 0.1
1.6 ? 0.1
2.8 ? 0.1
1.4 ? 0.1
1.9 ? 0.2
1.1 ? 0.1
1.3 ? 0.1
1.8 ? 0.2
2.0 ? 0.1
2.4 ? 0.4
All values represent transcript ratios for sheared (20 dyn?cm2, 48 h) to paired static controls (0 dyn?cm2, 48 h)
of T?C28a2 cells. Paired treatments consisted of no treatment (†); shear treated with 5 ?M D3T and static treated
with 0.1% DMSO (‡); and shear transfected with pBJ M?p110* (shear) and static transfected with vector only
(static) (§). Data represent mean ? SD (n ? 5–8).
www.pnas.org?cgi?doi?10.1073?pnas.0506620102 Healy et al.
stantially increases COX-2-dependent PGE2 production in
T?C28a2 cells in a time-dependent fashion, whereas only basal
levels were detected in specimens subjected to 5 dyn?cm2(Fig. 4).
Interestingly, D3T nearly abrogated both COX-2 protein expres-
sion (Fig. 3) and PGE2production (Fig. 4) in chondrocytes sub-
jected to high shear. To establish involvement of Nrf2 in this
process, chondrocytes were transfected with the pCMV plasmid
containing murine Nrf2. This intervention substantially reduced
PGE2production (53%) in sheared T?C28a2 cells (Fig. 4), consis-
tent with the 33% transfection efficiency of pCMV-mNrf2.
To examine the potential contribution of shear-induced COX-2
activity to the pro-inflammatory state of the T?C28a2 cells, these
cells were treated with a highly selective COX-2 inhibitor,
dyn?cm2for 48 h). This treatment reduced the shear-induced
down-regulation of NQO1 activity and intracellular GSH levels
(Fig. 5). Similar results were obtained with another COX-2 inhib-
itor, NS398 (30 ?M, data not shown). Collectively, these data
provide evidence that, in chondrocytes, shear-dependent up-
regulation of COX-2 activity is abrogated by induction of the
Nrf2-dependent phase 2 response and indicates important cross-
talk between these signaling pathways.
Shear-Induced Chondrocyte Apoptosis Is Suppressed by Phase 2
Inducers and COX-2 Specific Inhibitors. Microscopic inspection of
shear-stimulated (20 dyn?cm2for 48 h) T?C28a2 cells showed cell
shrinkage and membrane blebbing, providing morphological evi-
dence of apoptosis. Moreover, transcriptional profiling (Table 1)
effector molecule activated by mitochondrial depolarization, indi-
on apoptosis by measuring DNA fragmentation (TUNEL, Fig. 6A)
and mitochondrial membrane depolarization (MMP, Fig. 6B). The
presence of D3T (5 ?M) essentially abrogated shear-induced
apoptosis, whereas treatment with the COX-2 specific inhibitors
CAY10404 (6.75 ?M) and NS398 (30 ?M) resulted in a marked
reduction in apoptosis markers (Fig. 6). Additionally, the role of
caspase-9 in shear-mediated apoptosis was determined by immu-
noblot analysis, which revealed that high shear (20 dyn?cm2)
increased the expression of both the proform (46 kDa) and active
selective inhibitors substantially reduced expression (Fig. 6C).
Together, these results indicate that shear-mediated apoptosis
proceeds by disruption of mitochondrial integrity, which is regu-
lated by COX-2 and phase 2 enzyme activity.
High Shear Represses PI3-K Activity That Down-Regulates Phase 2
Enzymes and Increases Apoptosis in Chondrocytes. To identify po-
tential signaling partners involved in the down-regulation of phase
2 genes in shear-activated chondrocytes, differentially expressed
genes were clustered by using support trees. Analysis established
that transcriptional regulation of PI3-K (p85) paralleled that of
Nrf2 and phase 2 genes, indicating that PI3-K may be involved in
the shear-mediated repression of ARE-regulated transcriptional
prostaglandin E2(PGE2) production in chondrocytes. Cells were treated with
either solvent (0.1% DMSO) or D3T (5 ?M) or transfected with pCMV-null or
pCMV-mNrf2 (24 h), and then exposed to fluid shear (48 h) in the presence of
the agent. PGE2levels were determined in culture media at the indicated
times. Data are relative to paired static controls at t ? 0 (n ? 4, transfection
efficiency ? 32.8 ? 4.5%).
Effects of phase 2 induction on shear-induced COX-2-dependent
response in chondrocytes. Cells were treated with CAY10404 (6.75 ?M) or
control solvent (0.1% DMSO) for 2 h, exposed to static conditions or laminar
shear flow (20 dyn?cm2) for 48 h in the presence of agent, and NQO1 enzyme
activities (A) and total GSH levels (B) were determined. Data obtained with
NS398 (30 ?M) were indistinguishable from those with CAY10404. Data are
relative to static controls (n ? 3–9,*, P ? 0.01 with respect to static controls).
Effects of inhibition of COX-2 activity on shear-dependent phase 2
chondrocytes. Cells were treated with DMSO (0.1%), SFN (1.25 ?M), or D3T (5
?M) for 24 h, subjected to either static conditions or laminar shear flow (20 or
40 dyn?cm2) for 24 or 48 h in the presence of the agent, and NQO1 enzyme
activity (A) and GSH levels (B) were determined. Data are relative to static
controls (n ? 3–9,*, P ? 0.01 with respect to shear stress-paired solvent-
Effects of phase 2 induction on shear-dependent phase 2 response in
shear-activated chondrocytes. Cells, treated with either solvent (0.1% DMSO)
COX-2 expression. COX-1 expression remains unchanged (n ? 3).
Effects of the phase 2-inducer D3T on COX-2 protein levels in
Healy et al.
September 27, 2005 ?
vol. 102 ?
no. 39 ?
activity and the onset of apoptosis. To examine the role of PI3-K in
this signaling cascade, T?C28a2 cells were transfected with a
constitutively active PI3-K mutant, M?p110* and exposed to shear.
This intervention prevented the shear-mediated suppression of
Nrf2, phase 2 genes, and the induction of procaspase-9 (Table 1).
Similarly, constitutively active PI3-K was sufficient to enhance
NQO1 activity and GSH levels in static cultures and ablate their
down-regulation in sheared chondrocytes (Fig. 7). Intriguingly,
shear-induced COX-2 mRNA expression was markedly suppressed
in chondrocytes transfected with the constitutively active form of
PI3-K (Table 1) but not the wild-type construct (Table 3, which is
published as supporting information on the PNAS web site).
Cumulatively, these data suggest that high shear suppresses PI3-K
activity, leading to the down-regulation of Nrf2 and phase 2 genes
and, ultimately, to increased chondrocyte apoptosis. More impor-
tantly, our data suggest a negative feedback loop by which PI3-K
activity, either directly or by activation of the Nrf2?ARE-regulated
pathway, represses COX-2 expression (Fig. 8) by modulating up-
stream JNK2 activity (Table 1).
repair of articular cartilage. Mechanical loading of cartilage pro-
duces hydrostatic pressure and fluid shear, both of which modulate
chondrocyte function (6). Although hydrostatic pressure and low
shear tend to be protective by increasing matrix production (6, 8),
high fluid shear exerts pro-inflammatory effects on chondrocytes,
potentially leading to matrix degradation and arthritic pathology.
Additionally, recent work has linked chondrocyte apoptosis (24),
aberrant COX-2 expression (11), and depleted glutathione levels
(25) to the pathogenesis and progression of arthritic disorders.
Therefore, from a molecular perspective, delineating the shear-
may offer novel avenues for combating arthritic pathology.
This report examines the phenotype-specific response of chon-
drocytes to fluid shear by characterizing the pathway regulating
shear-induced apoptosis and by identifying the roles of COX-2 and
phase 2 enzymes in this process. In chondrocytes, laminar shear
tional activity; the decreased antioxidant capacity directly contrib-
chondrocytes. Cells were transfected with pBJ M?p110* (constitutively active
conditions or laminar flow (20 dyn?cm2) for 48 h, and NQO1 enzyme activity
(A) and total GSH levels (B) were determined. Data are relative to null-
transfected static cultures (n ? 4,*, P ? 0.01 with respect to static controls; ‡,
P ? 0.05 with respect to null-transfected shear).
Effects of PI3-K activity on shear-dependent phase 2 response in
DNA fragmentation, mitochondrial membrane permeabilization, and
caspase-9 protein levels. T?C-28a2 cells were treated with the solvent (0.1%
using DNA fragmentation (TUNEL, A), mitochondrial membrane potential
(MMP, B), and caspase-9 expression (C).
Effects of phase 2 inducers and COX-2 inhibitors on shear-mediated
sion through a JNK2?c-jun-dependent pathway, leading to PGE2accumula-
tion and suppression of PI3-K activity and Nrf2?ARE-mediated phase 2 re-
sponse. The resultant decrease in antioxidant capacity permits disruption of
mitochondrial integrity, caspase-9 activation, and apoptosis. Phase 2 inducer,
PI3-K activity, and COX-2 inhibitors restore phase 2 activity. Moreover, acti-
vation of PI3-K and?or the Nrf2?ARE pathway abrogates COX-2 expression
and apoptosis through negative feedback, potentially by suppressing up-
stream JNK2?c-jun signaling.
In chondrocytes, high shear flow (20 dyn?cm2) induces COX-2 expres-
www.pnas.org?cgi?doi?10.1073?pnas.0506620102 Healy et al.
utes to their apoptosis, characterized by cell shrinkage, membrane Download full-text
blebbing, DNA fragmentation, mitochondrial depolarization, and
specific inhibitors, restore ARE-driven phase 2 response and
prevent apoptosis, highlighting the cross-talk between COX-2 and
phase 2 enzymes. The use of a constitutively active PI3-K mutant
also restored Nrf2?ARE-regulated activity, thus establishing the
role of PI3-K in shear-induced Nrf2 regulation. Moreover, the
abrogation of shear-induced COX-2 expression by the use of phase
2 inducers or constitutively active PI3-K suggests the existence of
PI3-K?Nrf2?ARE-mediated negative feedback loops, which po-
tentially interfere with JNK2?c-jun signaling activity upstream of
Negative feedback control is an essential regulatory mechanism
by which cells maintain physiological homeostasis. We have iden-
tified a negative feedback loop in chondrocytic cells regulating the
expression of pro- and anti-inflammatory molecules in response to
fluid shear stress. Time-course transcriptional profiling reveals that
high fluid shear activates the JNK2?c-jun?COX-2 pathway after
only 1.5 h (Table 2), whereas the suppression of PI3-K activity and
5), indicates that the JNK2?c-jun?COX-2 pathway functions up-
stream of PI3-K and Nrf2 (26) in the signaling cascade (Fig. 8);
by D3T, a selective inducer of the Nrf2?ARE pathway (12), points
to a negative feedback loop by which Nrf2 and?or Nrf2-regulated
enzymes repress JNK2?c-jun?COX-2 expression (Table 1), poten-
tially by interfering with JNK2?c-jun signaling or through modu-
lation of unknown elements in the COX-2 promoter.
Functional genomics revealed that the gene expression profile of
PI3-K (p85) paralleled that of Nrf2 and several phase 2 genes,
suppression of ARE-regulated genes. Transfection of cells with a
constitutively active PI3-K mutant, but not a wild-type construct,
markedly increased Nrf2?ARE-mediated phase 2 transcription
(Table 1) and enzyme activity (Fig. 7) in static and sheared
chondrocytes, supporting the view that PI3-K is a key regulator of
Nrf2-mediated transcription (27, 28). These findings, coupled with
time-course transcriptional profiling, suggest that PI3-K may me-
diate communication between COX-2 and the phase 2 response.
The existence of such cross-talk is supported by the findings that
COX-2 expression regulates PI3-K activity in UVB-irradiated
human lung adenocarcinoma cells (29). Interestingly, constitutively
active PI3-K also attenuated shear-induced JNK2?c-jun?COX-2
up-regulation (Table 1), potentially through interaction with JNK2
(30); however, because PI3-K activity also stimulates ARE-driven
gene expression, it is unclear whether PI3-K affects COX-2 expres-
sion directly, through expression of phase 2 enzymes (Fig. 8) or
through other mediators.
In addition to its role in detoxification and cellular defense, Nrf2
has been implicated in the regulation of inflammation in macro-
phages (14) and endothelial cells (1, 23). The ability of phase 2
inducers to abrogate shear-induced COX-2 expression and PGE2
production in chondrocytes presents a previously undescribed
approach for the prevention of cartilage inflammation. This view is
supported by similar work in IFN-?-stimulated macrophages show-
ing that phase 2 inducers can suppress COX-2 and iNOS transcrip-
tion, and that the potency of NQO1 induction and COX-2 sup-
pression is highly correlated (14). Moreover, recent evidence that
the shear-induced up-regulation of phase 2 genes in human aortic
endothelial cells is attenuated by COX-2-specific inhibitors (2) may
inhibitors reduce inflammation in cartilage, their presence in the
vasculature prevents the accumulation of COX-2-derived 15d-
PGJ2, which is associated with the atheroprotective nature of
laminar flow. Consequently, phase 2 inducers represent an attrac-
tive and safe alternative to COX-2 inhibitors based on their
anti-inflammatory potential and their highly beneficial antioxida-
These findings highlight the existing cross-talk among COX-2
expression, reduced antioxidant capacity, and increased apoptosis
chondrocyte apoptosis in response to high shear stress may identify
potential therapeutic targets for controlling arthritic pathogenesis
and?or progression and may be useful in the design of bioreactors
for cartilage culture.
We thank J. Quackenbush for insightful discussions, N. Wakabayashi for
the pNQO1?ARE-luc and pCMV-mNrf2 constructs, and A. Klippel for
Professor Award (to K.K.), a National Science Foundation Graduate
Fellowship (to Z.R.H.). Establishment of the cell line was supported by
National Institutes of Health Grant R01-AG22021 (to M.B.G.).
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