ARTHRITIS & RHEUMATISMVol. 50, No. 11, November 2004, pp 3541–3548DOI 10.1002/art.20601© 2004, American College of Rheumatology

Role of NF-�B Transcription Factors in Antiinflammatory andProinflammatory 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 chondro-cytes convert biomechanical signals into intracellularbiochemical events are not well understood. In thisstudy, we sought to determine the intracellular mecha-nisms of the magnitude-dependent actions of mechani-cal signals.

Methods. Chondrocytes isolated from rabbit ar-ticular cartilage were grown on flexible membranes.Cells were subjected to cyclic tensile strain (CTS) ofvarious magnitudes in the presence or absence ofinterleukin-1� (IL-1�), which was used as a proinflam-matory signal for designated time intervals. The regu-lation of NF-�B was measured by reverse transcriptase–polymerase chain reaction, electrophoretic mobilityshift assay, and immunofluorescence.

Results. CTS of low magnitudes (4–8% equibi-axial strain) was a potent inhibitor of IL-1�–dependentNF-�B nuclear translocation. Cytoplasmic retention ofNF-�B and reduction of its synthesis led to sustainedsuppression of proinflammatory gene induction. In con-trast, proinflammatory signals generated by CTS ofhigh magnitudes (15–18% equibiaxial strain) mimickedthe actions of IL-1� and induced rapid nuclear trans-location of NF-�B subunits p65 and p50.

Conclusion. Magnitude-dependent signals of me-chanical strain utilize the NF-�B transcription factorsas common elements to abrogate or aggravate proin-

flammatory responses. Furthermore, the intracellularevents induced by mechanical overload are similar tothose that are initiated by proinflammatory cytokines inarthritis.

The pathology of osteoarthritis (OA) is associ-ated with excessive mechanical load and trauma experi-enced by the joint tissue, and the inability of this tissueto tolerate that stress (1,2). In response to mechanicalloading, articular chondrocytes are exposed to compres-sive, tensile, and shear forces (3,4). These cells have thenecessary signaling and effector mechanisms to senseand react to applied mechanical forces by mounting astream of cellular responses such as proliferation, matrixcatabolism, and matrix synthesis (5–9). An accumulatingbody of evidence suggests that mechanical signalingplays a key role in regulating cartilage damage andrepair. Exposure of cartilage to mechanical strain of highmagnitudes leads to inflammation and synthesis of me-diators of tissue destruction, such as interleukin-1�(IL-1�), tumor necrosis factor � (TNF�), induciblenitric oxide synthase (iNOS), and matrix metalloprotein-ases (2,9–12). These mediators augment matrix degra-dation and inhibit the synthesis of matrix-associatedproteins (10,11,13). In contrast, lower levels of tensileforces induce antiinflammatory and anabolic responses(12,14,15). The intriguing question is how cartilage cellsadapt to mechanical loading, i.e., how intracellular sig-nals generated by tensile strain of high magnitudesmanifest themselves as proinflammatory responses.

The signals induced by proinflammatory cyto-kines such as IL-1� and TNF� are transmitted to thenucleus through activation of kinase cascades that leadto phosphorylation, ubiquitination, and ultimate degra-dation of the inhibitor of NF-�B (I�B), a protein thatsequesters NF-�B in the cytoplasm (16–20). Upon re-lease from I�B, NF-�B, a multifunctional transcriptionfactor, translocates to the nucleus, where it binds to

Supported by NIH grants AR-48781, AT-00646, and HD-40939.

1Sudha Agarwal, PhD, James Deschner, DMD, PhD: OhioState University, Columbus; 2Ping Long, MD, Anupam Verma, MD,Cynthia Hofman, PharmD, Nicholas Piesco, PhD: University of Pitts-burgh, Pittsburgh, Pennsylvania; 3Christopher H. Evans, PhD: HarvardMedical School, Boston, Massachusetts.

Address correspondence and reprint requests to Sudha Agar-wal, PhD, Biomechanics and Tissue Engineering Laboratory, Sectionof Oral Biology, 4010 Postle Hall, The Ohio State University, 305 West12th Avenue, Columbus, OH 43210. E-mail: agarwal.61@osu.edu.

Submitted for publication March 2, 2004; accepted in revisedform July 28, 2004.

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consensus sequences of several proinflammatory genesto initiate their expression (16–18). Mechanosensing inchondrocytes is closely related to inflammatory geneinduction (11). Furthermore, NF-�B inhibition blocksbone erosion associated with inflammatory arthritis (21).Therefore, we speculated that pro- and antiinflamma-tory actions of cyclic tensile strain (CTS) are mediatedby NF-�B transcription factors that are utilized by mostinflammation mediators in arthritis. In this study, wefound that chondrocytes perceive signals generated bytensile strain and respond to them in a magnitude-dependent manner. Furthermore, the antiinflammatoryeffects of CTS of low magnitudes and the proinflamma-tory effects of CTS of high magnitudes are both regu-lated by the NF-�B signal transduction pathway.

MATERIALS AND METHODS

Cell culture and materials. Chondrocytes were iso-lated from the 150–200-�m-thick superficial layer of articularcartilage from 14–16-week-old female NZW rabbits, as previ-ously described (14,15,22). Briefly, cartilage pieces wereminced 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°Cfor 2 hours. The cells were then centrifuged at 800g for 10minutes. The pellet containing chondrocytes was washed twicewith HBSS, counted, and the cells were cultured in Ham’s F-12medium (Mediatech, Herndon, VA) supplemented with10% defined fetal calf serum (Hyclone, Logan, UT), 2 mML-glutamine (Invitrogen), 100 units/ml penicillin (Mediatech),and 100 �g/ml streptomycin (Mediatech). All protocols wereapproved by the Institutional Animal Care and Use Commit-tee of the University of Pittsburgh. Chondrocytes that retainedtheir phenotype as shown by the expression of aggrecan, typeII collagen, and biglycan synthesis were used during the first 3passages (22).

Application of cyclic equibiaxial strain. To study theeffects of tensile forces in vitro, chondrocytes (5 � 105/well)were grown on pronectin-coated Bioflex II 6-well cultureplates (Flexcell International, Hillsborough, NC) to 80% con-fluence (7–8 days). Various magnitudes of cyclic equibiaxialradial strain were applied to the cells at a rate of 0.05 Hz by aFlexercell strain unit (Flexcell International). After loading ofthe plates on a station (located in an incubator at 5% CO2 with95% humidity), a vacuum deformed the membrane across thepostface to create uniform biaxial strain. The strain wascalculated as circumferential strain � 2�(change in radius)/2�(original radius) � (change in radius)/(original radius) �radial strain. The relationship between vacuum level and strainwas linear. Cells grown on Bioflex II plates were assigned to 4different treatment regimens: 1) untreated controls, 2) cellstreated with recombinant human IL-1� (rHuIL-1�; EMDBiosciences, San Diego, CA), 3) cells treated with CTS, or 4)cells treated with rHuIL-1� and CTS.

Production of NO. NO production was measuredbased upon the Griess reaction, as previously described(14,23).

Reverse transcriptase–polymerase chain reaction (RT-PCR). Extraction of RNA was performed with an RNAextraction kit according to the manufacturer’s recommendedprotocols (Qiagen, Santa Clara, CA). A total of 1.0 �g of RNAwas reverse transcribed with 200 units of Moloney murineleukemia virus reverse transcriptase (Invitrogen) at 42°C for 25minutes followed by 65°C for 5 minutes. Complementary DNAwas amplified with 0.1 �g of specific primers in a reactionmixture (PCR supermix; Invitrogen) containing Taq DNApolymerase, Tris HCl, potassium chloride, magnesium chlo-ride, and deoxynucleoside triphosphates. Amplification wascarried out for 30 cycles of 45 seconds at 94°C, 45 seconds at59°C, and 60 seconds at 72°C with an Eppendorf DNA thermalcycler (Brinkmann, Westbury, NY). The sequence of sense andantisense rabbit primers was as follows: for GAPDH (293 bp),sense 5�-TCACCATCTTCCAGGAGCGA-3� and antisense5�-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�. Thebands of ethidium bromide–stained DNA products on agarosegels were photographed and digitized with a Kodak Imager1000 (Perkin Elmer, Emeryville, CA). The images were sub-jected to densitometric analysis and standardized with PCRproducts of GAPDH as an internal control. In some experi-ments, cells were incubated with various concentrations ofcaffeic acid phenethyl ester (CAPE; EMD Biosciences), acell-permeable inhibitor of NF-�B, for 10 minutes to inhibitthe nuclear translocation of NF-�B, and then subjected to CTSas described above.

Electrophoretic mobility shift assay (EMSA). To de-termine the nuclear translocation of NF-�B, an EMSA wasperformed, as previously described (24). Briefly, nuclear ex-tracts (4 �g) were incubated at 37°C for 15 minutes with 8fmoles of 32P end-labeled, 45-mer, double-stranded NF-�Boligonucleotide containing the NF-�B binding site (5�-TTGTTACAAGGGACTTTCCGCTGGGGACTTTCCAG-GGAGGCGTGG-3�) (Stratagene, La Jolla, CA) from thehuman immunodeficiency virus long terminal repeat. TheDNA protein complex was separated from free oligonucleotideon 6% native polyacrylamide gels, and the specificity ofbinding was analyzed by competition with unlabeled oligo-nucleotide. Prior to analyzing the complexes by EMSA, thenuclear extracts were incubated with preimmune serum or anti-bodies against the NF-�B components p50, p52, p65, RelB, orc-Rel at room temperature for 30 minutes. The binding of NF-�Bto its consensus sequences was visualized in dried gels, and thebands were quantitatively analyzed by scintillation counting.

Western blot analysis. Determination of cytoplasmicNF-�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 resolvedon sodium dodecyl sulfate–10% polyacrylamide gel electro-phoresis gels under reducing conditions. After electrophoresis,the proteins were electrotransferred to Immunolon mem-branes (New England Nuclear, Boston, MA), blocked with 5%nonfat dry milk, probed with rabbit anti–NF-�B p65 antibody

3542 AGARWAL ET AL

(Santa Cruz Biotechnology, Santa Cruz, CA), and detectedwith horseradish peroxidase (HRP)–conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology). To visualize theNF-�B bands, Western Lightening chemiluminescence reagent(Perkin Elmer, Boston, MA) was used as a substrate for HRP.The bands were semiquantitatively assessed by densitometricanalysis using the Fluor-S Max imaging system (Bio-Rad,Hercules, CA).

Immunofluorescence. Nuclear translocation of NF-�Bwas analyzed by immunofluorescence using rabbit anti–NF-�Bp65 IgG (Santa Cruz Biotechnology) and Cy3-conjugated goatanti-rabbit IgG (The Jackson Laboratory, Bar Harbor, ME).Phalloidin–fluorescein isothiocyanate was used as a counter-stain to visualize F-actin (Santa Cruz Biotechnology). The cellsadhered to Bioflex membranes were mounted in phosphatebuffered saline with 20% glycerol. The cells were observedunder 20� or 40� objectives with a BX50 epifluorescencemicroscope (Olympus, Lake Success, NY). The images werecaptured with an air-cooled camera and Magnafire image-capturing software (Olympus).

RESULTS

Magnitude of CTS determines its antiinflamma-tory or proinflammatory actions on chondrocytes. IL-1�is intricately involved in the pathogenesis of OA as wellas rheumatoid arthritis. Since IL-1� up-regulates multi-ple proinflammatory genes such as iNOS, we usedIL-1�–dependent NO production to probe the intracell-ular target sites of CTS in the IL-1� signal transductionpathway. As shown in Figure 1A, under normal cellconditions, accumulation of NO was not observed in theculture supernatants of control cells at 24 hours. The

effects of CTS on chondrocytes were magnitude depen-dent. Over a period of 24 hours, NO production was notinduced by lower magnitudes (4% and 8%) of CTS. Incontrast, exposure of chondrocytes to CTS of highermagnitudes (12%, 15%, and 18%) resulted in an in-creased production of NO. As expected, IL-1� treat-ment also induced a significant up-regulation of NOsynthesis (Figure 1A). Coexposure of chondrocytes toCTS of lower magnitudes and IL-1� resulted in amagnitude-dependent inhibition (P � 0.05) of the IL-1�–induced NO production. The inhibitory effect ofCTS on IL-1�–induced NO production was decreased at12% CTS but was still significant.

To determine if the effects of CTS of lowermagnitudes were perceived at the transcriptional level,we measured the IL-1�–induced iNOS messenger RNA(mRNA) expression in the presence of CTS of variousmagnitudes at 4 hours. Unstretched chondrocytes andcells exposed to CTS of lower magnitudes did not showiNOS mRNA expression (Figure 1B). Interestingly,iNOS mRNA expression was markedly up-regulatedwith CTS at 10–18%. IL-1� (1.0 ng/ml) induced signifi-cant levels of iNOS mRNA expression (Figure 1C).More important, CTS of low magnitudes (CTS-L;2–8%) strongly suppressed the IL-1�–dependent iNOSmRNA expression. CTS of 10% and 12% was lesseffective in inhibiting iNOS mRNA induction, but theeffect was still significant. CTS of high magnitudes(CTS-H; 15% and 18%) did not suppress iNOS mRNAexpression. Collectively, these observations suggestedthat mechanical signals act on chondrocytes in amagnitude-dependent manner. Additionally, the intra-cellular target sites of both CTS-L and CTS-H lieupstream of iNOS mRNA expression and may involveproinflammatory pathways similar to those regulated byIL-1�.

Inhibition of IL-1�–induced NF-�B gene expres-sion by CTS-L. We next investigated the nuclear trans-location of NF-�B, the key transcription factor involvedin signal transduction of proinflammatory cytokines, toexplore the intracellular mechanisms by which CTS-Lattenuates IL-1�–induced proinflammatory responses.Analysis by EMSA did not reveal nuclear translocationof NF-�B in controls and cells treated with CTS-L (6%)over a period of 15–90 minutes (Figure 2A). Rapidnuclear translocation of NF-�B was observed within 15minutes of IL-1� treatment, and a further 6.2-foldincrease in the nuclear NF-�B was apparent after 90minutes (Figure 2B). In contrast, cells treated simulta-neously with CTS-L and IL-1� exhibited a time-dependent suppression of the IL-1�–induced NF-�B

Figure 1. Magnitude-dependent response of chondrocytes to me-chanical signals. A, Regulation of nitric oxide (NO) production byvarious magnitudes of cyclic tensile strain (CTS) in the presence andabsence of interleukin-1� (IL-1�) (1.0 ng/ml). Accumulation of NO inthe culture supernatants was assessed after 24 hours. Values are themean and SEM of triplicate determinations. � � P � 0.05 betweenunstretched cells and cells subjected to CTS in the absence andpresence of IL-1�, by Student’s t-test. B and C, The regulation ofinducible NO synthase (iNOS) mRNA expression by various magni-tudes of CTS was determined in B, the absence and C, the presence ofIL-1� (1.0 ng/ml). Expression of iNOS mRNA was measured byreverse transcriptase–polymerase chain reaction after 4 hours. Repre-sentative results from 1 of 3 separate experiments are shown.

ROLE OF NF-�B IN MECHANICAL SIGNALS 3543

nuclear translocation (Figures 2A and B). Quantitativeassessment of EMSA gels revealed that �98% of theIL-1�–induced nuclear translocation of NF-�B was in-hibited by CTS-L at 60 minutes, and the inhibitory effectwas sustained over the next 30 minutes.

NF-�B consists of heterodimers or homodimersof various types of Rel proteins. The composition ofthese dimers is important for the regulation of proin-flammatory gene expression. Therefore, we next exam-

ined the subunit structure of NF-�B involved in theactions of CTS-L. IL-1� induced nuclear translocationof NF-�B heterodimers composed of p65 and p50subunits. CTS-L directly inhibited the IL-1�–inducednuclear translocation of NF-�B, as evidenced by a 66%reduction in nuclear p65 and p50 subunits by supershiftEMSA (Figure 2C). CTS-L did not induce nucleartranslocation of RelB, p52, or c-Rel (results not shown).

Using immunofluorescence, we further con-

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 analysisof 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 p50subunits 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 wereused 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–conjugatedphalloidin (green). Representative results from 1 of 3 separate experiments are shown.

3544 AGARWAL ET AL

firmed the CTS-L–induced inhibition of NF-�B nucleartranslocation. These experiments revealed that CTS-Linhibited NF-�B activation by its sequestration in thecytoplasm. IL-1�–activated chondrocytes exhibitedtranslocation of NF-�B to the nucleus within 30 minutes.During the ensuing 150 minutes, a further increase ofNF-�B was observed, mainly in the nuclear compart-ment, which was also accompanied by an increase in thecytoplasmic chamber (Figure 2D). However, cells ex-posed to CTS-L (6%) and IL-1� simultaneously exhib-ited cytoplasmic retention of NF-�B during the first 30minutes. Thereafter, the effect of CTS-L on the IL-1�–dependent nuclear translocation of NF-�B resulted inalmost total inhibition during the next 150 minutes.Treatment of cells with 6% CTS alone did not inducenuclear translocation of NF-�B, with results found to besimilar to those in untreated control cells.

Examination by RT-PCR of NF-�B mRNA ex-pression revealed that the IL-1�–induced nuclear trans-location of NF-�B was followed by a time-dependentincrease in NF-�B mRNA synthesis (Figure 3A). Semi-quantitative assessment of PCR products revealed thatCTS-L markedly inhibited 92% of the IL-1�–inducedNF-�B p65 mRNA expression within 30 minutes, andthis inhibition increased to 98% over the next 150minutes. Densitometric analysis of Western blots ofcytoplasmic NF-�B revealed that IL-1� treatment re-sulted in a near total depletion of NF-�B p65 fromcytoplasm within the first 30 minutes (Figure 3B). Nev-ertheless, cytoplasmic NF-�B was rapidly replenishedduring the next 150 minutes, with a �6-fold increaseover that present in untreated control cells. The densi-tometric analysis also showed that IL-1� failed to induceup-regulation of NF-�B p65 in the presence of CTS-L(6%); instead, a nearly 30% reduction in NF-�B wasobserved over the period of 180 minutes when comparedwith untreated control cells. Control cells and cellsexposed to CTS-L alone did not exhibit significantchanges in NF-�B p65 levels during the experiment.These observations are consistent with the results of theimmunofluoresence analysis shown in Figure 2D, inwhich IL-1� induced a dramatic time-dependent in-crease in cytoplasmic NF-�B p65.

Involvement of NF-�B nuclear translocation andsynthesis in proinflammatory gene induction by CTS-H.We next examined whether CTS-H also utilizes theNF-�B signaling pathway for its proinflammatory ac-tions. The time course of NF-�B nuclear translocation,as determined by EMSA, revealed that CTS-H (15%) ortreatment with IL-1� resulted in a progressive increasein nuclear NF-�B accumulation over a period of 30–180

minutes (Figure 4A). RT-PCR analysis of cells exposedto CTS-H (15%) showed that nuclear translocation ofNF-�B was paralleled by a time-dependent up-regulation of NF-�B mRNA expression (Figure 4B).CTS-H induced NF-�B mRNA expression within 30minutes, and a further 2.8-, 5.8-, and 6.4-fold increase inthe NF-�B mRNA expression was found at 60, 120, and180 minutes, respectively, by densitometric analysis.

Similar to IL-1�, the heterodimers of NF-�B thattranslocated to the nucleus in response to CTS-H werecomposed of p65 and p50 subunits. CTS-H did notinduce a nuclear translocation of RelB, p52, or c-Rel, asrevealed by supershift EMSA (Figure 4C). Immunoflu-orescence analysis confirmed that CTS-H induced arapid and sustained nuclear translocation of NF-�Bbetween 30 and 120 minutes, which was also paralleledby an increase of NF-�B in the cytoplasm (Figure 4D).

Figure 3. Inhibition of IL-1�–induced NF-�B mRNA expression andNF-�B synthesis by CTS-L. A, IL-1�–induced NF-�B p65 mRNAexpression over a period of 30–180 minutes in the absence andpresence of CTS-L (6%) was analyzed by reverse transcriptase–polymerase chain reaction. B, Cytoplasmic NF-�B p65 in untreatedcells and cells treated with IL-1� (1.0 ng/ml) and/or CTS-L (6%) overa period of 30–180 minutes was determined by densitometric analysisof Western blots. Representative results from 1 of at least 3 separateexperiments are shown. See Figure 2 for definitions.

ROLE OF NF-�B IN MECHANICAL SIGNALS 3545

To further confirm that CTS-H actions weremediated by NF-�B, chondrocytes were either untreatedor treated with CAPE for 10 minutes prior to beingsubjected to CTS-H (15%). Quantitative assessment ofEMSA gels demonstrated that CAPE (100 �M) inhib-

ited 53% of CTS-H–induced nuclear translocation ofNF-�B within 30 minutes and led to near-completeinhibition 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 adose dependent manner (5, 25, 50, and 100 �M) (Fig-ures 5B and C). In parallel experiments, iNOS expres-sion induced by IL-1� and/or CTS-H was also inhibitedby CAPE.

DISCUSSION

The mechanisms by which chondrocytes convertbiomechanical signals into intracellular events have be-come an area of intense interest in orthopedic research.The results presented here describe intracellular mech-anisms by which biomechanical signals are convertedinto biochemical events. Our foremost findings are thatchondrocytes perceive mechanical signals and respondto them in a magnitude-dependent manner. Mechanicalsignals of low magnitude (2–8% CTS) were not per-ceived as inflammatory signals and did not affect iNOSor NO synthesis. However, the actions of CTS-L were

Figure 4. Induction of NF-�B nuclear translocation and synthesis byCTS 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� (1.0ng/ml) and/or CTS-H (15%) were extracted. Subsequently, the presenceof NF-�B in the nuclear extract was determined by EMSA. Quantitativeanalysis of net 32P associated with each band determined by EMSA wasperformed to assess NF-�B nuclear translocation. Each point is the meanof 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 analyzedby reverse transcriptase–polymerase chain reaction. C, NF-�B subunitsinvolved in CTS-H actions (15%, 30 minutes) were analyzed by supershiftEMSA using antibodies against p65, p50, p52, RelB, and c-Rel subunits ofNF-�B. D, Immunofluorescence staining of chondrocytes subjected toCTS-H (15%) for 30, 60, or 120 minutes was performed in the presenceor 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-conjugatedgoat anti-rabbit IgG as secondary antibody (red). Cellular �-actin wasstained by fluorescein isothiocyanate–conjugated phalloidin (green). Rep-resentative results from 1 of 3 separate experiments are shown. See Figure2 for other definitions.

Figure 5. Abrogation of CTS of high magnitudes (CTS-H)–inducednuclear translocation of NF-�B by caffeic acid phenethyl ester(CAPE). A, Induction of CTS-H–dependent nuclear translocation ofNF-�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 thepresence or absence of IL-1� (1.0 ng/ml). Subsequently, the presenceof NF-�B in the nuclear extract was determined by EMSA. Theradioactivity associated with bands determined by EMSA was mea-sured with a scintillation counter. Values are the mean and SEM oftriplicate determinations. B, Effect of various concentrations of CAPEon inducible nitric oxide synthase (iNOS) mRNA expression inducedby IL-1� and/or CTS-H. Chondrocytes were exposed to variousconcentrations of CAPE (0, 5, 25, 50, or 100 �M) for 10 minutes priorto being treated with IL-1� (1.0 ng/ml) and/or CTS-H (15%) for 4hours. The iNOS mRNA expression was analyzed by reversetranscriptase–polymerase chain reaction (RT-PCR). C, Densitometricanalysis of the RT-PCR gels shown in B. Representative results from1 of 3 separate experiments are shown. See Figure 2 for otherdefinitions.

3546 AGARWAL ET AL

evident in the presence of IL-1�, i.e., CTS-L directlyabolished the actions of inflammatory insults by inhibit-ing IL-1�–induced iNOS mRNA expression and NOproduction. CTS-L also inhibits chondrocytic transcrip-tion of a number of other proinflammatory genes, suchas cyclooxygenase 2 and matrix metalloproteinases(15,22). Exposure of chondrocytes to 15% dynamiccompression also inhibits IL-1�–induced prostaglandinE2 production in chondrocytes in vitro (25). In vivo,during normal movement, articular chondrocytes expe-rience compression loads of 15%, which leads to 5%elongation of chondrocytes. Collectively, these observa-tions suggest that signals generated by CTS-L may beequivalent to those experienced by compressive loadingof chondrocytes in vivo.

In contrast, CTS-H (15–18%) is associated withproinflammatory gene expression, as evidenced by en-hanced iNOS mRNA expression and NO production.Mechanical signals of high magnitudes also markedlyup-regulate matrix degradation and decrease matrixsynthesis (1,2,6,7). Since these actions of mechanicalstrain can also be induced by IL-1� or TNF�(10,11,13,19,20), CTS-H and proinflammatory media-tors appear to exert similar effects to up-regulate inflam-matory responses. These findings further suggest thatthe magnitude of mechanical strain is a critical determi-nant of chondrocytic responses.

The next most striking finding is that the NF-�Bsignal transduction pathway is central to the proinflam-matory and antiinflammatory actions of mechanicalstrain. NF-�B transcription factors have an establishedrole in cytosolic signaling of proinflammatory cytokinesthrough their nuclear translocation and subsequenttransactivation of a plethora of genes (16,18). Theantiinflammatory signals generated by CTS-L act di-rectly on the NF-�B pathway and abrogate the IL-1�–induced nuclear translocation of NF-�B in a sustainedmanner. The time course of the CTS-L–mediated inhi-bition of IL-1�–induced NF-�B nuclear translocationwas 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 demonstratethat CTS-L–mediated suppression of NF-�B nucleartranslocation also resulted in the inhibition of its mRNAexpression and synthesis. Hence, the antiinflammatoryactions of CTS-L were mediated both by suppression ofIL-1�–induced NF-�B nuclear translocation and by in-hibition of IL-1�–induced NF-�B synthesis.

NF-�B/Rel proteins exist as homo- or het-erodimers of 5 different subunits of NF-�B, includingp65 (RelA), c-Rel, RelB, p50, and p52, which constitute

different NF-�B DNA binding complexes (16). Targeteddisruption studies provide evidence that combinatorialinteractions between different NF-�B subunits exhibitdistinguishable DNA binding specificity and transcrip-tional activity. For example, p50 homodimers lack thetransactivation domain and act as repressors of geneexpression (16,18,27). In our experiments, we investi-gated the subunit composition of the inducible NF-�Bcomplexes using specific antibodies against differentNF-�B proteins. Exposure of chondrocytes to CTS-Lresulted in the suppression of the IL-1�–induced nucleartranslocation of NF-�B consisting of p65 and p50. Sincea clear inhibition of IL-1�–induced p65 and p50 subunitsof NF-�B was observed, CTS-L appears to directlyintercept IL-1�–induced nuclear translocation of theseNF-�B subunits to attenuate IL-1�–induced proinflam-matory gene induction. This is also supported by immu-nofluorescence analysis, which demonstrates cyto-plasmic retention of NF-�B p65 in cells exposedsimultaneously to IL-1� and CTS-L. Nevertheless, a rolefor p50 homodimers in inhibiting proinflammatory re-sponses cannot yet be completely excluded.

CTS-H is a potent proinflammatory signal, andthus it is not surprising that its actions are mediated byNF-�B nuclear translocation and synthesis. In this re-spect, the actions of CTS-H are similar to classic proin-flammatory cytokines that involve NF-�B transcriptionfactors to initiate inflammation. Consistent with ourobservation that CTS-H actions were mediated byNF-�B nuclear translocation was the finding that CAPEcompletely abrogated the CTS-H–induced NF-�B nu-clear translocation and ultimately iNOS mRNA expres-sion. Furthermore, the finding that CTS-H induced thenuclear translocation of p65 and p50 heterodimers ofNF-�B also suggests that CTS-H utilizes proinflamma-tory pathways for its signal transduction. Thus, despitebeing a physical signal, CTS-H acts in a manner similarto molecular activators that stimulate the transcriptionalactivity of NF-�B.

Taken together, these findings show that chon-drocytes can perceive biomechanical signals and convertthem into biochemical events that regulate proinflam-matory gene induction. In this process, the NF-�Bpathway is critical in regulating the antiinflammatoryand proinflammatory actions of mechanical signals. Al-though the effects of low and high magnitudes ofmechanical strain are diametrically opposed, their sig-nals interact with the same Rel proteins to elicit verydifferent physiologic responses. Low levels of CTS gen-erate signals that inhibit NF-�B nuclear translocation tolimit IL-1�–inducible expression of proinflammatorygenes. In contrast, CTS of high magnitudes generates

ROLE OF NF-�B IN MECHANICAL SIGNALS 3547

signals that are similar to IL-1� in that it employs NF-�Btranscription factors to initiate expression of proinflam-matory genes involved in soft and hard tissue destruc-tion. How different magnitudes of cyclic strain on chon-drocytes could lead to opposite effects is perplexing. It isconceivable that low and high magnitudes of mechanicalstrain act on different upstream kinases of the NF-�Bsignal transduction cascade. Alternatively, these signalsmay act on pathways that are known to regulate NF-�Bnuclear translocation.

While these studies clearly show a role of theNF-�B signal transduction pathway in mechanotrans-duction, a role for other signaling pathways involved inproinflammatory and antiinflammatory signaling cannotbe ruled out. Continued studies will allow us to addresswhether signals generated by CTS of low and highmagnitudes have similar target sites upstream of theNF-�B translocation that regulate the magnitude-dependent responses to mechanical signals.

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