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Role of NF-╨Ю╤ФB transcription factors in antiinflammatory and proinflammatory actions of mechanical signals.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 11, November 2004, pp 3541–3548
DOI 10.1002/art.20601
© 2004, American College of Rheumatology
Role of NF-␬B Transcription Factors in Antiinflammatory and
Proinflammatory Actions of Mechanical Signals
Sudha Agarwal,1 James Deschner,1 Ping Long,2 Anupam Verma,2 Cynthia Hofman,2
Christopher H. Evans,3 and Nicholas Piesco2
Objective. The mechanisms by which chondrocytes convert biomechanical signals into intracellular
biochemical events are not well understood. In this
study, we sought to determine the intracellular mechanisms of the magnitude-dependent actions of mechanical signals.
Methods. Chondrocytes isolated from rabbit articular cartilage were grown on flexible membranes.
Cells were subjected to cyclic tensile strain (CTS) of
various magnitudes in the presence or absence of
interleukin-1␤ (IL-1␤), which was used as a proinflammatory signal for designated time intervals. The regulation of NF-␬B was measured by reverse transcriptase–
polymerase chain reaction, electrophoretic mobility
shift assay, and immunofluorescence.
Results. CTS of low magnitudes (4–8% equibiaxial strain) was a potent inhibitor of IL-1␤–dependent
NF-␬B nuclear translocation. Cytoplasmic retention of
NF-␬B and reduction of its synthesis led to sustained
suppression of proinflammatory gene induction. In contrast, proinflammatory signals generated by CTS of
high magnitudes (15–18% equibiaxial strain) mimicked
the actions of IL-1␤ and induced rapid nuclear translocation of NF-␬B subunits p65 and p50.
Conclusion. Magnitude-dependent signals of mechanical strain utilize the NF-␬B transcription factors
as common elements to abrogate or aggravate proin-
flammatory responses. Furthermore, the intracellular
events induced by mechanical overload are similar to
those that are initiated by proinflammatory cytokines in
arthritis.
The pathology of osteoarthritis (OA) is associated with excessive mechanical load and trauma experienced by the joint tissue, and the inability of this tissue
to tolerate that stress (1,2). In response to mechanical
loading, articular chondrocytes are exposed to compressive, tensile, and shear forces (3,4). These cells have the
necessary signaling and effector mechanisms to sense
and react to applied mechanical forces by mounting a
stream of cellular responses such as proliferation, matrix
catabolism, and matrix synthesis (5–9). An accumulating
body of evidence suggests that mechanical signaling
plays a key role in regulating cartilage damage and
repair. Exposure of cartilage to mechanical strain of high
magnitudes leads to inflammation and synthesis of mediators of tissue destruction, such as interleukin-1␤
(IL-1␤), tumor necrosis factor ␣ (TNF␣), inducible
nitric oxide synthase (iNOS), and matrix metalloproteinases (2,9–12). These mediators augment matrix degradation and inhibit the synthesis of matrix-associated
proteins (10,11,13). In contrast, lower levels of tensile
forces induce antiinflammatory and anabolic responses
(12,14,15). The intriguing question is how cartilage cells
adapt to mechanical loading, i.e., how intracellular signals generated by tensile strain of high magnitudes
manifest themselves as proinflammatory responses.
The signals induced by proinflammatory cytokines such as IL-1␤ and TNF␣ are transmitted to the
nucleus through activation of kinase cascades that lead
to phosphorylation, ubiquitination, and ultimate degradation of the inhibitor of NF-␬B (I␬B), a protein that
sequesters NF-␬B in the cytoplasm (16–20). Upon release from I␬B, NF-␬B, a multifunctional transcription
factor, translocates to the nucleus, where it binds to
Supported by NIH grants AR-48781, AT-00646, and HD40939.
1
Sudha Agarwal, PhD, James Deschner, DMD, PhD: Ohio
State University, Columbus; 2Ping Long, MD, Anupam Verma, MD,
Cynthia Hofman, PharmD, Nicholas Piesco, PhD: University of Pittsburgh, Pittsburgh, Pennsylvania; 3Christopher H. Evans, PhD: Harvard
Medical School, Boston, Massachusetts.
Address correspondence and reprint requests to Sudha Agarwal, PhD, Biomechanics and Tissue Engineering Laboratory, Section
of Oral Biology, 4010 Postle Hall, The Ohio State University, 305 West
12th Avenue, Columbus, OH 43210. E-mail: [email protected]
Submitted for publication March 2, 2004; accepted in revised
form July 28, 2004.
3541
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AGARWAL ET AL
consensus sequences of several proinflammatory genes
to initiate their expression (16–18). Mechanosensing in
chondrocytes is closely related to inflammatory gene
induction (11). Furthermore, NF-␬B inhibition blocks
bone erosion associated with inflammatory arthritis (21).
Therefore, we speculated that pro- and antiinflammatory actions of cyclic tensile strain (CTS) are mediated
by NF-␬B transcription factors that are utilized by most
inflammation mediators in arthritis. In this study, we
found that chondrocytes perceive signals generated by
tensile strain and respond to them in a magnitudedependent manner. Furthermore, the antiinflammatory
effects of CTS of low magnitudes and the proinflammatory effects of CTS of high magnitudes are both regulated by the NF-␬B signal transduction pathway.
MATERIALS AND METHODS
Cell culture and materials. Chondrocytes were isolated from the 150–200-␮m-thick superficial layer of articular
cartilage from 14–16-week-old female NZW rabbits, as previously described (14,15,22). Briefly, cartilage pieces were
minced in Hank’s balanced salt solution (HBSS; Invitrogen,
Carlsbad, CA) and treated with 2.5% trypsin for 10 minutes.
Subsequently, the tissue was transferred to 1% collagenase
(Worthington, Lakewood, NJ) in HBSS and incubated at 37°C
for 2 hours. The cells were then centrifuged at 800g for 10
minutes. The pellet containing chondrocytes was washed twice
with HBSS, counted, and the cells were cultured in Ham’s F-12
medium (Mediatech, Herndon, VA) supplemented with
10% defined fetal calf serum (Hyclone, Logan, UT), 2 mM
L-glutamine (Invitrogen), 100 units/ml penicillin (Mediatech),
and 100 ␮g/ml streptomycin (Mediatech). All protocols were
approved by the Institutional Animal Care and Use Committee of the University of Pittsburgh. Chondrocytes that retained
their phenotype as shown by the expression of aggrecan, type
II collagen, and biglycan synthesis were used during the first 3
passages (22).
Application of cyclic equibiaxial strain. To study the
effects of tensile forces in vitro, chondrocytes (5 ⫻ 105/well)
were grown on pronectin-coated Bioflex II 6-well culture
plates (Flexcell International, Hillsborough, NC) to 80% confluence (7–8 days). Various magnitudes of cyclic equibiaxial
radial strain were applied to the cells at a rate of 0.05 Hz by a
Flexercell strain unit (Flexcell International). After loading of
the plates on a station (located in an incubator at 5% CO2 with
95% humidity), a vacuum deformed the membrane across the
postface to create uniform biaxial strain. The strain was
calculated as circumferential strain ⫽ 2␲(change in radius)/
2␲(original radius) ⫽ (change in radius)/(original radius) ⫽
radial strain. The relationship between vacuum level and strain
was linear. Cells grown on Bioflex II plates were assigned to 4
different treatment regimens: 1) untreated controls, 2) cells
treated with recombinant human IL-1␤ (rHuIL-1␤; EMD
Biosciences, San Diego, CA), 3) cells treated with CTS, or 4)
cells treated with rHuIL-1␤ and CTS.
Production of NO. NO production was measured
based upon the Griess reaction, as previously described
(14,23).
Reverse transcriptase–polymerase chain reaction (RTPCR). Extraction of RNA was performed with an RNA
extraction kit according to the manufacturer’s recommended
protocols (Qiagen, Santa Clara, CA). A total of 1.0 ␮g of RNA
was reverse transcribed with 200 units of Moloney murine
leukemia virus reverse transcriptase (Invitrogen) at 42°C for 25
minutes followed by 65°C for 5 minutes. Complementary DNA
was amplified with 0.1 ␮g of specific primers in a reaction
mixture (PCR supermix; Invitrogen) containing Taq DNA
polymerase, Tris HCl, potassium chloride, magnesium chloride, and deoxynucleoside triphosphates. Amplification was
carried out for 30 cycles of 45 seconds at 94°C, 45 seconds at
59°C, and 60 seconds at 72°C with an Eppendorf DNA thermal
cycler (Brinkmann, Westbury, NY). The sequence of sense and
antisense rabbit primers was as follows: for GAPDH (293 bp),
sense 5⬘-TCACCATCTTCCAGGAGCGA-3⬘ and antisense
5⬘-CACAATGCCGAAGTGGTCGT-3⬘; for iNOS (243 bp),
sense 5⬘-CGCCCTTCCGCAGTTTCT-3⬘ and antisense 5⬘TCCAGGAGGACATGCAGCAC-3⬘; and for NF-␬B p65
(186 bp), sense 5⬘-CACTGCCGAGCTCAAGATCTGCC-3⬘
and antisense 5⬘-GTCGGCGTACGGAGGAGTCCG-3⬘. The
bands of ethidium bromide–stained DNA products on agarose
gels were photographed and digitized with a Kodak Imager
1000 (Perkin Elmer, Emeryville, CA). The images were subjected to densitometric analysis and standardized with PCR
products of GAPDH as an internal control. In some experiments, cells were incubated with various concentrations of
caffeic acid phenethyl ester (CAPE; EMD Biosciences), a
cell-permeable inhibitor of NF-␬B, for 10 minutes to inhibit
the nuclear translocation of NF-␬B, and then subjected to CTS
as described above.
Electrophoretic mobility shift assay (EMSA). To determine the nuclear translocation of NF-␬B, an EMSA was
performed, as previously described (24). Briefly, nuclear extracts (4 ␮g) were incubated at 37°C for 15 minutes with 8
fmoles of 32P end-labeled, 45-mer, double-stranded NF-␬B
oligonucleotide containing the NF-␬B binding site (5⬘TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAGGGAGGCGTGG-3⬘) (Stratagene, La Jolla, CA) from the
human immunodeficiency virus long terminal repeat. The
DNA protein complex was separated from free oligonucleotide
on 6% native polyacrylamide gels, and the specificity of
binding was analyzed by competition with unlabeled oligonucleotide. Prior to analyzing the complexes by EMSA, the
nuclear extracts were incubated with preimmune serum or antibodies against the NF-␬B components p50, p52, p65, RelB, or
c-Rel at room temperature for 30 minutes. The binding of NF-␬B
to its consensus sequences was visualized in dried gels, and the
bands were quantitatively analyzed by scintillation counting.
Western blot analysis. Determination of cytoplasmic
NF-␬B proteins was performed by Western blot analysis.
NF-␬B was analyzed from cytoplasmic extracts of cells (2 ⫻
106) subjected to the regimens described above and resolved
on sodium dodecyl sulfate–10% polyacrylamide gel electrophoresis gels under reducing conditions. After electrophoresis,
the proteins were electrotransferred to Immunolon membranes (New England Nuclear, Boston, MA), blocked with 5%
nonfat dry milk, probed with rabbit anti–NF-␬B p65 antibody
ROLE OF NF-␬B IN MECHANICAL SIGNALS
Figure 1. Magnitude-dependent response of chondrocytes to mechanical signals. A, Regulation of nitric oxide (NO) production by
various magnitudes of cyclic tensile strain (CTS) in the presence and
absence of interleukin-1␤ (IL-1␤) (1.0 ng/ml). Accumulation of NO in
the culture supernatants was assessed after 24 hours. Values are the
mean and SEM of triplicate determinations. ⴱ ⫽ P ⱕ 0.05 between
unstretched cells and cells subjected to CTS in the absence and
presence of IL-1␤, by Student’s t-test. B and C, The regulation of
inducible NO synthase (iNOS) mRNA expression by various magnitudes of CTS was determined in B, the absence and C, the presence of
IL-1␤ (1.0 ng/ml). Expression of iNOS mRNA was measured by
reverse transcriptase–polymerase chain reaction after 4 hours. Representative results from 1 of 3 separate experiments are shown.
(Santa Cruz Biotechnology, Santa Cruz, CA), and detected
with horseradish peroxidase (HRP)–conjugated goat antirabbit IgG (Santa Cruz Biotechnology). To visualize the
NF-␬B bands, Western Lightening chemiluminescence reagent
(Perkin Elmer, Boston, MA) was used as a substrate for HRP.
The bands were semiquantitatively assessed by densitometric
analysis using the Fluor-S Max imaging system (Bio-Rad,
Hercules, CA).
Immunofluorescence. Nuclear translocation of NF-␬B
was analyzed by immunofluorescence using rabbit anti–NF-␬B
p65 IgG (Santa Cruz Biotechnology) and Cy3-conjugated goat
anti-rabbit IgG (The Jackson Laboratory, Bar Harbor, ME).
Phalloidin–fluorescein isothiocyanate was used as a counterstain to visualize F-actin (Santa Cruz Biotechnology). The cells
adhered to Bioflex membranes were mounted in phosphate
buffered saline with 20% glycerol. The cells were observed
under 20⫻ or 40⫻ objectives with a BX50 epifluorescence
microscope (Olympus, Lake Success, NY). The images were
captured with an air-cooled camera and Magnafire imagecapturing software (Olympus).
RESULTS
Magnitude of CTS determines its antiinflammatory or proinflammatory actions on chondrocytes. IL-1␤
is intricately involved in the pathogenesis of OA as well
as rheumatoid arthritis. Since IL-1␤ up-regulates multiple proinflammatory genes such as iNOS, we used
IL-1␤–dependent NO production to probe the intracellular target sites of CTS in the IL-1␤ signal transduction
pathway. As shown in Figure 1A, under normal cell
conditions, accumulation of NO was not observed in the
culture supernatants of control cells at 24 hours. The
3543
effects of CTS on chondrocytes were magnitude dependent. Over a period of 24 hours, NO production was not
induced by lower magnitudes (4% and 8%) of CTS. In
contrast, exposure of chondrocytes to CTS of higher
magnitudes (12%, 15%, and 18%) resulted in an increased production of NO. As expected, IL-1␤ treatment also induced a significant up-regulation of NO
synthesis (Figure 1A). Coexposure of chondrocytes to
CTS of lower magnitudes and IL-1␤ resulted in a
magnitude-dependent inhibition (P ⱕ 0.05) of the IL1␤–induced NO production. The inhibitory effect of
CTS on IL-1␤–induced NO production was decreased at
12% CTS but was still significant.
To determine if the effects of CTS of lower
magnitudes were perceived at the transcriptional level,
we measured the IL-1␤–induced iNOS messenger RNA
(mRNA) expression in the presence of CTS of various
magnitudes at 4 hours. Unstretched chondrocytes and
cells exposed to CTS of lower magnitudes did not show
iNOS mRNA expression (Figure 1B). Interestingly,
iNOS mRNA expression was markedly up-regulated
with CTS at 10–18%. IL-1␤ (1.0 ng/ml) induced significant levels of iNOS mRNA expression (Figure 1C).
More important, CTS of low magnitudes (CTS-L;
2–8%) strongly suppressed the IL-1␤–dependent iNOS
mRNA expression. CTS of 10% and 12% was less
effective in inhibiting iNOS mRNA induction, but the
effect was still significant. CTS of high magnitudes
(CTS-H; 15% and 18%) did not suppress iNOS mRNA
expression. Collectively, these observations suggested
that mechanical signals act on chondrocytes in a
magnitude-dependent manner. Additionally, the intracellular target sites of both CTS-L and CTS-H lie
upstream of iNOS mRNA expression and may involve
proinflammatory pathways similar to those regulated by
IL-1␤.
Inhibition of IL-1␤–induced NF-␬B gene expression by CTS-L. We next investigated the nuclear translocation of NF-␬B, the key transcription factor involved
in signal transduction of proinflammatory cytokines, to
explore the intracellular mechanisms by which CTS-L
attenuates IL-1␤–induced proinflammatory responses.
Analysis by EMSA did not reveal nuclear translocation
of NF-␬B in controls and cells treated with CTS-L (6%)
over a period of 15–90 minutes (Figure 2A). Rapid
nuclear translocation of NF-␬B was observed within 15
minutes of IL-1␤ treatment, and a further 6.2-fold
increase in the nuclear NF-␬B was apparent after 90
minutes (Figure 2B). In contrast, cells treated simultaneously with CTS-L and IL-1␤ exhibited a timedependent suppression of the IL-1␤–induced NF-␬B
3544
AGARWAL ET AL
Figure 2. Abrogation of interleukin-1␤ (IL-1␤)–induced nuclear translocation of NF-␬B by cyclic tensile strain of low magnitudes (CTS-L). A,
Nuclear proteins of untreated cells (control) and cells treated for 15, 30, 60, or 90 minutes with IL-1␤ (1.0 ng/ml) and/or CTS-L (6%) were extracted.
Subsequently, the presence of NF-␬B in the nuclear extract was determined by electrophoretic mobility shift assay (EMSA). B, Quantitative analysis
of net 32P associated with each band from the EMSA gel shown in A was performed by scintillation counting to assess NF-␬B nuclear translocation.
C, NF-␬B subunits involved in the actions of CTS-L were analyzed by supershift EMSA (SS-EMSA) using antibodies against p65 (RelA) and p50
subunits of NF-␬B. D, Chondrocytes were treated for 30, 60, 120, or 180 minutes with IL-1␤ (1.0 ng/ml) and/or CTS-L (6%). Untreated cells were
used as controls. Nuclear translocation of NF-␬B was assessed by immunofluorescence staining. NF-␬B was stained with rabbit anti–NF-␬B p65 IgG,
with Cy3-conjugated goat anti-rabbit IgG as secondary antibody (red). Cellular ␤-actin was stained with fluorescein isothiocyanate–conjugated
phalloidin (green). Representative results from 1 of 3 separate experiments are shown.
nuclear translocation (Figures 2A and B). Quantitative
assessment of EMSA gels revealed that ⬎98% of the
IL-1␤–induced nuclear translocation of NF-␬B was inhibited by CTS-L at 60 minutes, and the inhibitory effect
was sustained over the next 30 minutes.
NF-␬B consists of heterodimers or homodimers
of various types of Rel proteins. The composition of
these dimers is important for the regulation of proinflammatory gene expression. Therefore, we next exam-
ined the subunit structure of NF-␬B involved in the
actions of CTS-L. IL-1␤ induced nuclear translocation
of NF-␬B heterodimers composed of p65 and p50
subunits. CTS-L directly inhibited the IL-1␤–induced
nuclear translocation of NF-␬B, as evidenced by a 66%
reduction in nuclear p65 and p50 subunits by supershift
EMSA (Figure 2C). CTS-L did not induce nuclear
translocation of RelB, p52, or c-Rel (results not shown).
Using immunofluorescence, we further con-
ROLE OF NF-␬B IN MECHANICAL SIGNALS
firmed the CTS-L–induced inhibition of NF-␬B nuclear
translocation. These experiments revealed that CTS-L
inhibited NF-␬B activation by its sequestration in the
cytoplasm. IL-1␤–activated chondrocytes exhibited
translocation of NF-␬B to the nucleus within 30 minutes.
During the ensuing 150 minutes, a further increase of
NF-␬B was observed, mainly in the nuclear compartment, which was also accompanied by an increase in the
cytoplasmic chamber (Figure 2D). However, cells exposed to CTS-L (6%) and IL-1␤ simultaneously exhibited cytoplasmic retention of NF-␬B during the first 30
minutes. Thereafter, the effect of CTS-L on the IL-1␤–
dependent nuclear translocation of NF-␬B resulted in
almost total inhibition during the next 150 minutes.
Treatment of cells with 6% CTS alone did not induce
nuclear translocation of NF-␬B, with results found to be
similar to those in untreated control cells.
Examination by RT-PCR of NF-␬B mRNA expression revealed that the IL-1␤–induced nuclear translocation of NF-␬B was followed by a time-dependent
increase in NF-␬B mRNA synthesis (Figure 3A). Semiquantitative assessment of PCR products revealed that
CTS-L markedly inhibited 92% of the IL-1␤–induced
NF-␬B p65 mRNA expression within 30 minutes, and
this inhibition increased to 98% over the next 150
minutes. Densitometric analysis of Western blots of
cytoplasmic NF-␬B revealed that IL-1␤ treatment resulted in a near total depletion of NF-␬B p65 from
cytoplasm within the first 30 minutes (Figure 3B). Nevertheless, cytoplasmic NF-␬B was rapidly replenished
during the next 150 minutes, with a ⬎6-fold increase
over that present in untreated control cells. The densitometric analysis also showed that IL-1␤ failed to induce
up-regulation of NF-␬B p65 in the presence of CTS-L
(6%); instead, a nearly 30% reduction in NF-␬B was
observed over the period of 180 minutes when compared
with untreated control cells. Control cells and cells
exposed to CTS-L alone did not exhibit significant
changes in NF-␬B p65 levels during the experiment.
These observations are consistent with the results of the
immunofluoresence analysis shown in Figure 2D, in
which IL-1␤ induced a dramatic time-dependent increase in cytoplasmic NF-␬B p65.
Involvement of NF-␬B nuclear translocation and
synthesis in proinflammatory gene induction by CTS-H.
We next examined whether CTS-H also utilizes the
NF-␬B signaling pathway for its proinflammatory actions. The time course of NF-␬B nuclear translocation,
as determined by EMSA, revealed that CTS-H (15%) or
treatment with IL-1␤ resulted in a progressive increase
in nuclear NF-␬B accumulation over a period of 30–180
3545
Figure 3. Inhibition of IL-1␤–induced NF-␬B mRNA expression and
NF-␬B synthesis by CTS-L. A, IL-1␤–induced NF-␬B p65 mRNA
expression over a period of 30–180 minutes in the absence and
presence of CTS-L (6%) was analyzed by reverse transcriptase–
polymerase chain reaction. B, Cytoplasmic NF-␬B p65 in untreated
cells and cells treated with IL-1␤ (1.0 ng/ml) and/or CTS-L (6%) over
a period of 30–180 minutes was determined by densitometric analysis
of Western blots. Representative results from 1 of at least 3 separate
experiments are shown. See Figure 2 for definitions.
minutes (Figure 4A). RT-PCR analysis of cells exposed
to CTS-H (15%) showed that nuclear translocation of
NF- ␬ B was paralleled by a time-dependent upregulation of NF-␬B mRNA expression (Figure 4B).
CTS-H induced NF-␬B mRNA expression within 30
minutes, and a further 2.8-, 5.8-, and 6.4-fold increase in
the NF-␬B mRNA expression was found at 60, 120, and
180 minutes, respectively, by densitometric analysis.
Similar to IL-1␤, the heterodimers of NF-␬B that
translocated to the nucleus in response to CTS-H were
composed of p65 and p50 subunits. CTS-H did not
induce a nuclear translocation of RelB, p52, or c-Rel, as
revealed by supershift EMSA (Figure 4C). Immunofluorescence analysis confirmed that CTS-H induced a
rapid and sustained nuclear translocation of NF-␬B
between 30 and 120 minutes, which was also paralleled
by an increase of NF-␬B in the cytoplasm (Figure 4D).
3546
AGARWAL ET AL
To further confirm that CTS-H actions were
mediated by NF-␬B, chondrocytes were either untreated
or treated with CAPE for 10 minutes prior to being
subjected to CTS-H (15%). Quantitative assessment of
EMSA gels demonstrated that CAPE (100 ␮M) inhib-
Figure 5. Abrogation of CTS of high magnitudes (CTS-H)–induced
nuclear translocation of NF-␬B by caffeic acid phenethyl ester
(CAPE). A, Induction of CTS-H–dependent nuclear translocation of
NF-␬B was inhibited by CAPE over a period of 30–180 minutes.
Chondrocytes were either untreated or were treated with CAPE (100
␮M) for 10 minutes prior to being subjected to CTS-H (15%) in the
presence or absence of IL-1␤ (1.0 ng/ml). Subsequently, the presence
of NF-␬B in the nuclear extract was determined by EMSA. The
radioactivity associated with bands determined by EMSA was measured with a scintillation counter. Values are the mean and SEM of
triplicate determinations. B, Effect of various concentrations of CAPE
on inducible nitric oxide synthase (iNOS) mRNA expression induced
by IL-1␤ and/or CTS-H. Chondrocytes were exposed to various
concentrations of CAPE (0, 5, 25, 50, or 100 ␮M) for 10 minutes prior
to being treated with IL-1␤ (1.0 ng/ml) and/or CTS-H (15%) for 4
hours. The iNOS mRNA expression was analyzed by reverse
transcriptase–polymerase chain reaction (RT-PCR). C, Densitometric
analysis of the RT-PCR gels shown in B. Representative results from
1 of 3 separate experiments are shown. See Figure 2 for other
definitions.
Figure 4. Induction of NF-␬B nuclear translocation and synthesis by
CTS of high magnitudes (CTS-H). A, Nuclear proteins of untreated cells
(control) and cells treated for 30, 60, 120, or 180 minutes with IL-1␤ (1.0
ng/ml) and/or CTS-H (15%) were extracted. Subsequently, the presence
of NF-␬B in the nuclear extract was determined by EMSA. Quantitative
analysis of net 32P associated with each band determined by EMSA was
performed to assess NF-␬B nuclear translocation. Each point is the mean
of triplicate values. B, NF-␬B p65 mRNA expression induced by CTS-H
(15%) and/or IL-1␤ (1.0 ng/ml) over a period of 180 minutes was analyzed
by reverse transcriptase–polymerase chain reaction. C, NF-␬B subunits
involved in CTS-H actions (15%, 30 minutes) were analyzed by supershift
EMSA using antibodies against p65, p50, p52, RelB, and c-Rel subunits of
NF-␬B. D, Immunofluorescence staining of chondrocytes subjected to
CTS-H (15%) for 30, 60, or 120 minutes was performed in the presence
or absence of IL-1␤ (1.0 ng/ml). Untreated cells were used as controls.
NF-␬B was stained with rabbit anti–NF-␬B p65 IgG, with Cy3-conjugated
goat anti-rabbit IgG as secondary antibody (red). Cellular ␤-actin was
stained by fluorescein isothiocyanate–conjugated phalloidin (green). Representative results from 1 of 3 separate experiments are shown. See Figure
2 for other definitions.
ited 53% of CTS-H–induced nuclear translocation of
NF-␬B within 30 minutes and led to near-complete
inhibition by 60 minutes (Figure 5A). Additionally,
CAPE inhibited the ability of NF-␬B to drive CTS-H–
induced transcription of iNOS mRNA at 4 hours, in a
dose dependent manner (5, 25, 50, and 100 ␮M) (Figures 5B and C). In parallel experiments, iNOS expression induced by IL-1␤ and/or CTS-H was also inhibited
by CAPE.
DISCUSSION
The mechanisms by which chondrocytes convert
biomechanical signals into intracellular events have become an area of intense interest in orthopedic research.
The results presented here describe intracellular mechanisms by which biomechanical signals are converted
into biochemical events. Our foremost findings are that
chondrocytes perceive mechanical signals and respond
to them in a magnitude-dependent manner. Mechanical
signals of low magnitude (2–8% CTS) were not perceived as inflammatory signals and did not affect iNOS
or NO synthesis. However, the actions of CTS-L were
ROLE OF NF-␬B IN MECHANICAL SIGNALS
evident in the presence of IL-1␤, i.e., CTS-L directly
abolished the actions of inflammatory insults by inhibiting IL-1␤–induced iNOS mRNA expression and NO
production. CTS-L also inhibits chondrocytic transcription of a number of other proinflammatory genes, such
as cyclooxygenase 2 and matrix metalloproteinases
(15,22). Exposure of chondrocytes to 15% dynamic
compression also inhibits IL-1␤–induced prostaglandin
E2 production in chondrocytes in vitro (25). In vivo,
during normal movement, articular chondrocytes experience compression loads of 15%, which leads to 5%
elongation of chondrocytes. Collectively, these observations suggest that signals generated by CTS-L may be
equivalent to those experienced by compressive loading
of chondrocytes in vivo.
In contrast, CTS-H (15–18%) is associated with
proinflammatory gene expression, as evidenced by enhanced iNOS mRNA expression and NO production.
Mechanical signals of high magnitudes also markedly
up-regulate matrix degradation and decrease matrix
synthesis (1,2,6,7). Since these actions of mechanical
strain can also be induced by IL-1 ␤ or TNF ␣
(10,11,13,19,20), CTS-H and proinflammatory mediators appear to exert similar effects to up-regulate inflammatory responses. These findings further suggest that
the magnitude of mechanical strain is a critical determinant of chondrocytic responses.
The next most striking finding is that the NF-␬B
signal transduction pathway is central to the proinflammatory and antiinflammatory actions of mechanical
strain. NF-␬B transcription factors have an established
role in cytosolic signaling of proinflammatory cytokines
through their nuclear translocation and subsequent
transactivation of a plethora of genes (16,18). The
antiinflammatory signals generated by CTS-L act directly on the NF-␬B pathway and abrogate the IL-1␤–
induced nuclear translocation of NF-␬B in a sustained
manner. The time course of the CTS-L–mediated inhibition of IL-1␤–induced NF-␬B nuclear translocation
was rapid and could be observed within 15 minutes.
Because NF-␬B regulates its own gene expression (26),
we investigated whether CTS-L also inhibits IL-1␤–
induced NF-␬B induction. Our findings demonstrate
that CTS-L–mediated suppression of NF-␬B nuclear
translocation also resulted in the inhibition of its mRNA
expression and synthesis. Hence, the antiinflammatory
actions of CTS-L were mediated both by suppression of
IL-1␤–induced NF-␬B nuclear translocation and by inhibition of IL-1␤–induced NF-␬B synthesis.
NF-␬B/Rel proteins exist as homo- or heterodimers of 5 different subunits of NF-␬B, including
p65 (RelA), c-Rel, RelB, p50, and p52, which constitute
3547
different NF-␬B DNA binding complexes (16). Targeted
disruption studies provide evidence that combinatorial
interactions between different NF-␬B subunits exhibit
distinguishable DNA binding specificity and transcriptional activity. For example, p50 homodimers lack the
transactivation domain and act as repressors of gene
expression (16,18,27). In our experiments, we investigated the subunit composition of the inducible NF-␬B
complexes using specific antibodies against different
NF-␬B proteins. Exposure of chondrocytes to CTS-L
resulted in the suppression of the IL-1␤–induced nuclear
translocation of NF-␬B consisting of p65 and p50. Since
a clear inhibition of IL-1␤–induced p65 and p50 subunits
of NF-␬B was observed, CTS-L appears to directly
intercept IL-1␤–induced nuclear translocation of these
NF-␬B subunits to attenuate IL-1␤–induced proinflammatory gene induction. This is also supported by immunofluorescence analysis, which demonstrates cytoplasmic retention of NF-␬B p65 in cells exposed
simultaneously to IL-1␤ and CTS-L. Nevertheless, a role
for p50 homodimers in inhibiting proinflammatory responses cannot yet be completely excluded.
CTS-H is a potent proinflammatory signal, and
thus it is not surprising that its actions are mediated by
NF-␬B nuclear translocation and synthesis. In this respect, the actions of CTS-H are similar to classic proinflammatory cytokines that involve NF-␬B transcription
factors to initiate inflammation. Consistent with our
observation that CTS-H actions were mediated by
NF-␬B nuclear translocation was the finding that CAPE
completely abrogated the CTS-H–induced NF-␬B nuclear translocation and ultimately iNOS mRNA expression. Furthermore, the finding that CTS-H induced the
nuclear translocation of p65 and p50 heterodimers of
NF-␬B also suggests that CTS-H utilizes proinflammatory pathways for its signal transduction. Thus, despite
being a physical signal, CTS-H acts in a manner similar
to molecular activators that stimulate the transcriptional
activity of NF-␬B.
Taken together, these findings show that chondrocytes can perceive biomechanical signals and convert
them into biochemical events that regulate proinflammatory gene induction. In this process, the NF-␬B
pathway is critical in regulating the antiinflammatory
and proinflammatory actions of mechanical signals. Although the effects of low and high magnitudes of
mechanical strain are diametrically opposed, their signals interact with the same Rel proteins to elicit very
different physiologic responses. Low levels of CTS generate signals that inhibit NF-␬B nuclear translocation to
limit IL-1␤–inducible expression of proinflammatory
genes. In contrast, CTS of high magnitudes generates
3548
AGARWAL ET AL
signals that are similar to IL-1␤ in that it employs NF-␬B
transcription factors to initiate expression of proinflammatory genes involved in soft and hard tissue destruction. How different magnitudes of cyclic strain on chondrocytes could lead to opposite effects is perplexing. It is
conceivable that low and high magnitudes of mechanical
strain act on different upstream kinases of the NF-␬B
signal transduction cascade. Alternatively, these signals
may act on pathways that are known to regulate NF-␬B
nuclear translocation.
While these studies clearly show a role of the
NF-␬B signal transduction pathway in mechanotransduction, a role for other signaling pathways involved in
proinflammatory and antiinflammatory signaling cannot
be ruled out. Continued studies will allow us to address
whether signals generated by CTS of low and high
magnitudes have similar target sites upstream of the
NF-␬B translocation that regulate the magnitudedependent responses to mechanical signals.
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