The American Journal of Pathology, Vol. 182, No. 6, June 2013

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MUSCULOSKELETAL PATHOLOGY

Inflammatory Cytokines Associated with DegenerativeDisc Disease Control Aggrecanase-1 (ADAMTS-4)Expression in Nucleus Pulposus Cells through MAPK andNF-kBYe Tian,*y Wen Yuan,y Nobuyuki Fujita,*z Jianru Wang,*x Hua Wang,*x Irving M. Shapiro,* and Makarand V. Risbud*

From the Department of Orthopaedic Surgery and the Graduate Program in Cell and Developmental Biology,* Thomas Jefferson University, Philadelphia,Pennsylvania; the Department of Orthopaedic Surgery,y Changzheng Hospital, Second Military Medical University, Shanghai, China; the Department ofOrthopaedic Surgery,z Keio University School of Medicine, Tokyo, Japan; and the Department of Orthopaedics,x First Affiliated Hospital of Sun Yat-SenUniversity, Guangzhou, China

Accepted for publication

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February 19, 2013.

Address correspondence toMakarand V. Risbud, Ph.D.,Department of OrthopaedicSurgery, College Bldg.,Thomas Jefferson University,1025 Walnut St., Suite 511,Philadelphia, PA 19107.E-mail: makarand.risbud@jefferson.edu.

opyright ª 2013 American Society for Inve

ublished by Elsevier Inc. All rights reserved

ttp://dx.doi.org/10.1016/j.ajpath.2013.02.037

We investigated TNF-a and IL-1b regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells andits role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transienttransfections with rat NP cells and lentiviral silencing with human NP cells were performed to determinethe roles of MAPK and NF-kB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4expression and promoter activity increased in NP cells after TNF-a and IL-1b treatment. Treatment ofcells with MAPK and NF-kB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNAand protein expression. Although ERK1, p38a, p38b2, and p38g were involved in induction, ERK2 andp38d played no role in TNF-aedependent promoter activity. The inductive effect of p65 on ADAMTS4promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completelyblocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-a,and shIKK-b significantly decreased TNF-aedependent increase in ADAMTS-4 and -5 levels andaggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-aedependentaggrecan degradation in NP cells. By controlling activation of MAPK and NF-kB signaling, TNF-a andIL-1b modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to shownonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NPcells in vitro. (Am J Pathol 2013, 182: 2310e2321; http://dx.doi.org/10.1016/j.ajpath.2013.02.037)

Supported by NIH grants AR050087 (to I.M.S. and M.V.R.) andAR055655 (to M.V.R.).Y.T. and W.Y. contributed equally to this work.

The intervertebral disk is a unique tissue that that permitsrotation, as well as flexion and extension of the spine. It consistsof a gel-like nucleus pulposus (NP) surrounded circumferen-tially by a fibrocartilagenous annulus fibrosus. Cells of the NPare derived from the notochord,1 an embryonic tissue withlimited blood supply. In common with chondrocytes, NP cellssecrete a complex extracellular matrix that contains fibrillarcollagens and the proteoglycan aggrecan. Assembly of thesemacromolecules provides a robust hydrodynamic system thataccommodates applied biomechanical forces to the spine.2e4

Intervertebral disk degeneration is characterized by increasedexpression of catabolic enzymes, decreased proteoglycansynthesis, and an overall shift toward synthesis of a fibrotic

stigative Pathology.

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matrix. When this occurs, the water-binding capacity of thetissue is compromised, resulting in a failure to resistcompressive forces and a reduction in disk height.5,6 Althougha great deal is known about importance of proteoglycansecretion and function, the molecular mechanisms controllingaggrecan turnover in cells of the normal and the degenerateddisk are not well understood. It has been reported that duringdisk degeneration and herniation, in addition to infiltratingimmune cells, resident NP and annulus fibrosus cells produce

Control of ADAMTS-4 in Nucleus Pulposus

high levels of the cytokines TNF-a and IL-1b.7,8 These cyto-kines stimulate production of NGF, BDNF, and VEGF, mole-cules associated with nerve ingrowth and angiogenesis by NPcells.9 Moreover, both cytokines up-regulate expression by NPcells of catabolic matrix metalloproteinases (MMPs)3 and twomajor aggrecanases, A disintegrin and metalloproteinase withthrombospondin motifs 4 (ADAMTS-4) and 5 (ADAMTS-5).7,10e12 Among several members of the ADAMTS familythat cleave aggrecan in vitro, ADAMTS-4 (aggrecanase-1) andADAMTS-5 (aggrecanase-2) are the most likely to play a rolein aggrecan degradation and subsequent disk degeneration as inthe pathogenesis of osteoarthritis.13,14 ADAMTS-4 and -5produce fragments of aggrecan usually found in synovial fluidand cartilage by cleaving the protein following Glu373,Glu1545, Glu1714, Glu1819, and Glu1919.13,15,16

Unlike cartilage, in the NP both ADAMTS-4 andADAMTS-5 expression is elevated in human degenerativedisk disease.17e19 Surprisingly, despite the importance of theseaggrecanases in the pathogenesis of osteoarthritis and diskdisease, only a few studies have investigated regulation ofADAMTS transcription in NP cells,10,11,17,18 and none haveused promoter analysis. A clue to the mechanism lies in thefindings that, in NP cells, NF-kB may contribute to TNF-a regulation of ADAMTS-4 and ADAMTS-5 expression,11

and that TNF-a and IL-1b also modulate ADAMTS-5 enzy-matic activity through syndecan-4.19 These observations begthe question of howTNF-a and IL-1b control the expression ofADAMTS-4 and-5 andwhat is their relative contribution inNPcells in terms of aggrecan degradation. Although the presentstudy addressed both ADAMTS-4 and -5, the mechanisticaspects of transcriptional control were studied using a 3.5-kbhuman ADAMTS4 promoter fragment. Here, we show for thefirst time that TNF-a and IL-1b control ADAMTS4 transcrip-tion in MAPK- and NF-kBedependent fashion. Importantly,our results show that ADAMTS-4 and ADAMTS-5 arenonredundant and that both play a role in the cytokine-dependent degradation of aggrecan in human NP cells. Atherapeutic strategy could conceivably target these enzymes forthe structural preservation of the intervertebral disk.

Materials and Methods

Reagents and Plasmids

The 3.5-kb (�3109 to þ406 bp) human ADAMTS4 promoterin pb-gal-Basic vector was a kind gift from Dr. K.Thirunavukkarasu (Lilly Research Labs, Indianapolis, IN).20

The insert was recloned in basic pGL3 using XhoI and Hin-dIII digestion. pCMX-IkBM (catalog no. 12330), and RelA/p65 (catalog no. 20012), p50 (catalog no. 20018) developed byDr. Inder Verma and psPAX2 (catalog no. 12260) andpMD2G (catalog no. 12259) developed by Dr. Didier Tronowere obtained from the Addgene repository (Cambridge, MA).Plasmids DN-p38a, DN-p38b2, DN-p38g, and DN-p38dwere kindly provided by Jiahui Han (Scripps Research Insti-tute, La Jolla, CA); plasmids ERK-1K71R and ERK-2K52R,

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by Melanie Cobb (University of Texas Southwestern MedicalCenter, Dallas, TX); plasmids pLKO.1shADAMTS-4 andpLKO.1shADAMTS-5, by Dr. Mike Baker (University ofSheffield, Sheffield, UK); and plasmids shp65, shp52,shIKK-a, and shIKK-b in lentiviral FSVsi vector that coex-presses yellow fluorescent protein (YFP), by Dr. AndreeYeremian (University of Lleida, Lleida, Spain). The vectorpRL-TK (Promega, Madison, WI) containing the Renillaluciferase gene was used as an internal transfection control.

The amount of transfected plasmid, the pretransfectionperiod after seeding, and the post-transfection periodbefore harvesting were optimized for NP cells with pSVb-galactosidase plasmid (Promega).21 Wild-type and p65 nullcells were a kind gift from Dr. Denis Guttridge (Ohio StateUniversity, Columbus, OH). Antibody that recognizesADAMTS-dependent aggrecan degradation in interglobulardomain (anti-NITEGE) was a gift from Dr. Peter Roughley(Shriners Hospital for Children, Montreal, QC, Canada).Antibodies against ADAMTS-4, -5, and ADAMTS-generatedaggrecan neoepitope ARGSVIL were obtained from Abcam(Cambridge, MA). P-p38, p38, p52, P-p65, p65, IKK-a,IKK-b, P-ERK, ERK, P-JNK, and JNK antibodies were ob-tained from Cell Signaling Technology (Danvers, MA).b-Tubulin was obtained from the Developmental StudiesHybridoma Bank (University of Iowa, Iowa City, IA) andGAPDH from Novus Biologicals (Littleton, CO). TNF-a andIL-1b were purchased from PeproTech (Rocky Hill, NJ).

Isolation of NP Cells and Cytokine Treatments

Rat and humanNP cells were isolated using amethod reportedby Risbud et al.21 NP tissue from lumbar disks of three or fourrats was pooled for each isolation. NP cells weremaintained inDulbecco’smodified Eagle’smedium (DMEM) and 10% fetalbovine serum (FBS) supplemented with antibiotics and usedwithin the first three passages. To investigate the effect ofcytokines, cells were treated with 5 to 20 ng/mL IL-1b and 25to 100 ng/mL TNF-a for 24 hours in serum-free medium.

Human Tissue Collection and Grading

Both lumbar and cervical disk tissues were collected assurgical waste from individuals undergoing elective spinalsurgical procedures. Consistent with Thomas Jefferson Uni-versity’s Institutional Review Board guidelines, informedconsent for sample collection was obtained from each patient.Assessment of the disease state was performed using Pfirr-mann grading.22 This scheme uses T2-weighted magneticresonance imaging with image analysis by three independentobservers. Patient age, spinal level, and grade of NP tissuesused for cell isolation are listed in Supplemental Table S1.

RT-qPCR Analysis

After treatment, total RNA was extracted from NP cells(5 � 105 cells per plate) using RNeasy mini spin columns

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(Qiagen, Valencia, CA). Before elution from the column, RNAwas treated with RNase-free DNase I. Two micrograms of totalDNA-free RNA was used to synthesize cDNA, using a Super-Scipt III cDNA synthesis kit (Life TechnologieseInvitrogen,Carlsbad, CA). Reactions were set up in triplicate in 96-wellplates using 1 mL cDNA with Fast SYBR Green PCR MasterMix (Life TechnologieseAppliedBiosystems, Foster City, CA)to which gene-specific forward and reverse PCR primers wereadded. Each set of samples included a template-free control.PCR reactions were performed in a StepOnePlus real-time PCRsystem (Life TechnologieseApplied Biosystems) accordingto the manufacturer’s instructions. Expression of the gene ofinterest was first normalized to the housekeeping gene hypo-xanthine phosphoribosyltransferase 1 (Hprt1), with data ex-pressed as relative to the corresponding control group. All of theprimers were synthesized by Integrated DNA Technologies(Coralville, IA) (Table 1).

Protein Extraction and Western Blotting

NP cells (1 � 106 cells per plate) were placed on ice immedi-ately after treatment and washed with ice-cold Hanks’ balancedsalt solution. All of the wash buffers and the final resuspensionbuffer included 1� protease inhibitor cocktail (Roche AppliedScience, Indianapolis, IN), 5 mmol/L NaF, and 200 mmol/LNa3VO4. Conditioned medium was collected and concentratedusing centrifugal filter units (EMD Millipore, Billerica, MA).For detecting aggrecan neoepitopes, protein lysates were pre-treated with 0.1 U/mL chondroitinase ABC (Sigma-Aldrich, St.Louis, MO) for 1 to 6 hours at 37�C. Proteins were resolved on8% to 12% SDS-PAGE gels and were transferred by electro-blotting to polyvinylidene difluoride membranes (Bio-RadLaboratories, Hercules, CA). The membranes were blockedwith 5% nonfat dry milk in Tris-buffered salineeTween(50 mmol/L Tris, pH 7.6, 150 mmol/L NaCl, 0.1% tween 20)and incubated overnight at 4�C in 3% nonfat dry milk in Tris-buffered salineeTween with the specific antibodies all ata dilution of 1:1000. Immunolabeling was detected usingAmersham ECL reagent (GE Healthcare, Little Chalfont, UK).

Transfections and Dual-Luciferase Reporter Assay

NP cells were transferred to 48-well plates at a density of2 � 104 cells per well, at 1 day before transfection. To inves-tigate the effect of NF-kB on ADAMTS4 promoter activity,

Table 1 Sequences of Primers Used in RT-qPCR

Target Primer sequence

HPRT1Forward 50-AGTCCCAGCGTCGTGATTAGTGAT-30

Reverse 50-GAGCAAGTCTTTCAGTCCTGTCCA-30

ADAMTS4Forward 50-ACAATGGCTATGGACACTGCCTCT-30

Reverse 50-TGTGGACAATGGCTTGAGTCAGGA-30

ADAMTS5Forward 50-GTCCAAATGCACTTCAGCCACGAT-30

Reverse 50-AATGTCAAGTTGCACTGCTGGGTG-30

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cells were cotransfected with 50 to 200 ng of p65, p50, or bothp65 and p50 with or without appropriate backbone vector and175 ng ADAMTS4 reporter and 175 ng pRL-TK plasmid. Toinvestigate the effects of p38 and ERK signaling, cells weretransfected with 50 to 150 ng of dominant-negative p38 (DN-p38) or DN-ERK plasmids. In some wells, cells were treatedwith the inhibitors for NF-kB (10 mmol/L sm-7368), p38(10 mmol/L SB203580), ERK (10 mmol/L PD98059), or JNK(10 mmol/L SP600125) (all Calbiochem, from EMD Milli-pore). In some experiments, cells were transfected with 250 ngofADAMTS4 reporter plasmids with 250 ng pRL-TK plasmid.Lipofectamine 2000 (Life TechnologieseInvitrogen) wasused as a transfection reagent. For each transfection, plas-mids were premixed with the transfection reagent. At 48hours after transfection, the cells were harvested and a dual-luciferase reporter assay system (Promega) was used forsequential measurements of firefly and Renilla luciferaseactivities. Quantification of luciferase activities and calcu-lation of relative ratios were performed using a luminometer(TD-20/20; Turner Designs, Sunnyvale, CA). At least threeindependent transfections were performed, and all analyseswere performed in triplicate.

Lentiviral Particle Production and Viral Transduction

HEK 293T human embryonic kidney cells (1.3 � 106 cellsper plate) were seeded in 10-cm plates in DMEM with 10%heat-inactivated FBS, at 2 days before transfection. Cellswere transfected with 2.5 mg of shRNA control sequence orgene-specific shRNA plasmids, along with 1.875 mg psPAX2(a packaging vector) and 0.625 mg pMD2.G (an envelopevector). After 16 hours, the transfection medium wasremoved and replaced with DMEMwith 5% heat-inactivatedFBS and penicillinestreptomycin. Lentiviral particles wereharvested at 48 and 60 hours after transfection. Human NPcells (1� 106 cells per plate) were plated in DMEMwith 5%heat-inactivated FBS, at 1 day before transduction. Cells in10-cm plates were transduced with 5 mL of medium con-taining viral particles, along with 6 mg/mL polybrene. After24 hours, the medium was removed and replaced withDMEM with 5% heat-inactivated FBS. Cells were harvestedfor protein extraction at 5 days after viral transduction.

Statistical Analysis

All experiments were repeated independently three times.Data are presented as means � SEM. Differences betweengroups were analyzed by Student’s t-test and analysis ofvariance. P < 0.05 was considered significant.

Results

Expression of ADAMTS-4 Is Regulated by TNF-a andIL-1b in NP Cells

Expression of ADAMTS-4 in mature rat tissues was studiedusing real-time PCR and Western blot analysis. Compared

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Control of ADAMTS-4 in Nucleus Pulposus

with Hprt1, the basal expression of ADAMTS4 mRNA inhealthy NP and in annulus fibrosus tissue is very low(Figure 1, A and B). NP tissue shows weak ADAMTS-4bands at approximately 58 and 73 kDa (Figure 1B). Toexplore the premise that cytokines concerned with diskdegeneration regulate ADAMTS-4 expression, rat NP cellswere treated with TNF-a and IL-1b, and expression ofADAMTS-4 was analyzed. Treatment with both TNF-a andIL-1b resulted in dose-dependent increase in ADAMTS4mRNA levels (Figure 1, C and D). In addition, we measuredthe level of ADAMTS-4 protein in conditioned medium oftreated NP cells by Western blot analysis. Cytokine treat-ment significantly increased ADAMTS-4 protein expression

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in both rat NP cells (Figure 1, E and F) and human NP cells(Supplemental Figure S1A). To investigate whether theregulation of expression is at the transcriptional level, wemeasured the activity of a 3.5-kb ADAMTS4 promoter(Figure 1G) after cytokine treatment. Both cytokinessignificantly increased the promoter activity (Figure 1H).

TNF-a and IL-1b Promote ADAMTS-4 and -5 Expressionthrough Activation of MAPK and NF-kB Signaling

To determine whether MAPK and/or NF-kB signalingis required for the cytokine-dependent induction ofADAMTS-4 in rat NP cells, we first evaluated activation of

Figure 1 Expression and cytokine dependencyof ADAMTS-4 in rat NP cells. A: RT-qPCR analysisshows ADAMTS-4 was expressed at a very low levelin adult rat NP and annulus fibrosus (AF) tissues.B: Western blot analysis of ADAMTS4 expression inadult disk tissues reveals bands at 58 and 73 kDa,representing mature/processed protein. C and D:RT-qPCR analysis of ADAMTS4 expression by rat NPcells treated with the cytokines TNF-a (C) and IL-1b (D) for 24 hours. There was a dose-dependentincrease in ADAMTS4 mRNA expression by thecytokine treatment. E and F: Western blot analysisof NP cells indicates increased expression ofADAMTS-4 after TNF-a and IL-1b treatment. G:Schematic of ADAMTS4 promoter constructs,showing important transcription factor and regu-latory elements. H: Treatment of NP cells with TNF-a and IL-1b resulted in significant induction ofADAMTS4 promoter activity. Data are expressed asmeans � SEM from three independent experi-ments. *P < 0.05. Ctrl, control.

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these signaling pathways after treatment with TNF-a andIL-1b. After treatment with TNF-a (Figure 2A) or IL-1b(Figure 2B), there was a rapid increase in P-p65 proteinlevels. Activation was maximal at 5 to 30 minutes and thendeclined rapidly. As expected, there was no appreciablechange in the level of total p65 during the treatment period.We also examined levels of the phosphorylated MAPK iso-forms P-p38, P-ERK1/2, and P-JNK. Again, there wasa rapid increase in all three isoforms, among which ERKexhibited more sustained levels of phosphorylation. Toascertain whether the cytokine-induced expression ofADAMTS-4 and -5 requires NF-kB and/or MAPK signaling,rat NP cells were pretreated with pathway-specific inhibitors.Pretreatment caused a significant suppression in TNF-a andIL-1b induction of both ADAMTS4 and ADAMTS5 mRNA

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levels (Figure 2, CeF). Similarly, a pronounced decrease incytokine-mediated increase in levels of ADAMTS-4 protein(58 and 73 kDa) was seen in the presence of MAPK and NF-kB pathway inhibitors (Figure 2, G and H).

MAPK and C/EBP-b Control ADAMTS4 Promoter Activityin NP Cells

To investigate the mechanism of MAPK regulation ofADAMTS-4 expression, we transfected rat NP cells withdominant-negative (DN) DN-p38a, DN-p38b2, DN-p38g,DN-p38d, or DN-ERK1 or DN-ERK2 expression plasmidsand measured ADAMTS4 promoter activity. DN-p38d(Supplemental Figure S1B) and DN-ERK2 (SupplementalFigure S1, C and D) did not suppress promoter activity. In

Figure 2 Modulation of cytokine-dependentexpression of ADAMTS-4 and -5 expression by NF-kB and MAPK signaling in rat NP cells. A and B:Western blot analysis of NF-kB and MAPK signalingproteins after treatment of NP cells with TNF-a (A)and IL-1b (B). Cytokine treatment induced phos-phorylation of p65, p38, ERK, JNK within the first15 minutes. No appreciable change was observedin expression of p65, p38, ERK, and JNK. CeF: RT-qPCR analysis of ADAMTS4 and ADAMTS5 expressionby NP cells after treatment with TNF-a (C and E) orIL-1b (D and F) for 24 hours with or withoutinhibitors for NF-kB (SM7368, 10 mmol/L), p38(SB203580, 10 mmol/L), ERK (PD98059, 10 mmol/L), and JNK (SP600125, 10 mmol/L). Inhibition ofNF-kB signaling and MAPK signaling resulted ina significant blocking of cytokine-dependentinduction in ADAMTS4 and ADAMTS5 mRNAexpression. G and H: Western blot analysis indi-cates that treatment with NF-kB and MAPK inhib-itors completely abolished ADAMTS-4 proteininduction (the bands are indicated by asterisk) byTNF-a (G) and IL-1b (H). Data are expressed asmeans � SEM from three independent experi-ments. *P < 0.05.

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Control of ADAMTS-4 in Nucleus Pulposus

contrast, cytokine-dependent induction in ADAMTS4 promoteractivitywas significantly suppressedbycotransfectionwithDN-P38a (Figure 3,AandB), p38b2 (Figure3C), p38g (Figure3D),andDN-ERK1 (Figure 3, E and F). Because CCAAT enhancer-binding protein b (C/EBP-b; alias LAP2) has been shown tocontrol IL-1be and TNF-aedependent transcription in chon-drocytes,23 we investigated whether a similar regulatory systemexists in cells of the NP. We transfected rat NP cells with liver-enriched inhibitory protein (LIP), a functional LAP antagonist,and measured cytokine-dependent ADAMTS4 promoter ac-tivity. Surprisingly, suppression ofC/EBP-b function resulted infurther inductionof thepromoter activity byTNF-a (Figure3G).On the other hand, cotransfection with LAP2 resulted insuppression of the basal promoter activity (Figure 3H).

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NF-kB Signaling Controls ADAMTS4 Promoter Activityin the NP Cells

To investigate the role of NF-kB in the transcriptionalregulation of ADAMTS4, we first used JASPAR databaseanalysis (performed January 2012)24 (http://jaspar.genereg.net) for evidence of putative NF-kB binding motifs in thepromoter. Sequence analysis revealed four putative bindingsites. JASPAR analysis provides a quantitative score for eachpotential binding site, based on the probability of observingeach nucleotide at each position of the binding motifcompared to the consensus sequences of known bindingsites. The raw score is normalized to a range of 0-1 to providea relative score.24 The sequence, the location in the promoter

Figure 3 MAPK signaling controls ADAMTS4promoter activity in rat NP cells. A and B:Cotransfection of rat NP cells with DN-p38aabolished TNF-aedependent (A) and IL-1bedependent (B) induction in ADAMTS4 promoteractivity. C and D: IL-1bedependent increase inADAMTS4 promoter activity was blocked by DN-p38b2 (C) and DN-p38g (D). E and F: DN-ERK1suppressed induction in ADAMTS4 promoteractivity by TNF-a (C) and IL-1b (D) treatment. ForIL-1b treatment, inhibition was seen only at thehighest dose (150 ng). G: NP cells were cotrans-fected with LIP and ADAMTS4 promoter, andluciferase activity was measured after TNF-a treat-ment. Addition of LIP further increased TNF-aedependent ADAMTS-4 reporter activity. H: ADAMTS4promoter activity was measured after cotransfectionwith LAP2. LAP2 suppressed ADAMTS4 promoteractivity. Data are expressed as means � SEM fromthree independent experiments. *P < 0.05.

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Figure 4 NF-kB regulation of ADAMTS-4expression. A: Rat NP cells were transfected withRelA/p65, and ADAMTS4 promoter activity wasmeasured. There was a dose-dependent increase inpromoter activity up to 100 ng of p65. B: Cotrans-fection with RelB and c-Rel had no effect onADAMTS4 promoter activity in rat NP cells. C: Rat NPcells were cotransfected with RelA/p65 and/or p50,and promoter activity was measured. When p65 andp50 were added together, p50 significantly blockedp65-mediated induction in promoter activity. D:Rat NP cells were cotransfected with p65 alone andwith increasing doses of p50. Even at 50 ng, p50completely blocked p65-mediated induction ofpromoter activity. E and F: Rat NP cells cotrans-fected with p50, and ADAMTS4 promoter activitywas measured after TNF-a (E) and IL-1b (F) treat-ment. Cytokine-mediated induction in promoteractivity was completely blocked by p50. G: TNF-aeand IL-1bemediated induction in promoter activitywas completely blocked by the NF-kB inhibitorSM7368. H: Cotransfection of cells with DN-NF-kB/IkBaM resulted in a significant inhibition of IL-1bedependent ADAMTS4 promoter activity. Data areexpressed as means � SEM from three independentexperiments. *P < 0.05.

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relative to transcription start site, and relative score was asfollows: GGCAAGTCCC, �159 relative to �150 bp, score0.90; GGGGATTCTC, �1042 relative to �1033 bp, score0.88; GGGATTCTCC, �1043 relative to �1034 bp, score0.88; andGGGGATTTCC,�1394 relative to�1385 bp, score0.98.We then examined the effect of overexpression ofNF-kBsubunits on ADAMTS4 promoter activity in rat NP cells.Cotransfection with p65 resulted in a dose-dependentincrease in ADAMTS4 promoter activity (Figure 4A). Onthe other hand, neither the RelB nor the c-Rel subunitinfluenced ADAMTS4 promoter activity (Figure 4B).Although p50 alone had no effect on ADAMTS4 promoteractivity, it blocked the inductive effect of p65 even at a low

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dose (Figure 4, C and D). Notably, p50 completely sup-pressed the inductive effect of both cytokines on theADAMTS4 promoter (Figure 4, E and F).To confirm that ADAMTS4 promoter activity is respon-

sive to NF-kB signaling, we performed loss-of-functionstudies. When cells were treated with the NF-kB inhibitorSM7368 (Figure 4G) or cotransfected with DN-NF-kB/IkBaM (Figure 4H), cytokine-mediated induction inADAMTS4 promoter activity was completely abolished.Specificity of the NF-kB inhibitors SM7368 and IkBaMwas validated by measuring the activity of a well char-acterized NF-kB responsive reporter (SupplementalFigure S2A). To further validate the role of p65/RelA and

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Figure 5 Regulation of ADAMTS-4 and -5 expression by NF-kB. A: Immunofluorescence detection of YFP in human NP cells transduced with lentiviruscoexpressing YFP and NF-kB pathwayespecific shRNAs (LV-shp65, LV-shp52, LV-shIKKa, LV-shIKKb) show high transduction efficiency. BeG: Western blotanalysis of cells transduced with control lentivirus LV-shC and LV-shp65 (B), LV-shp52 (C), LV-shIKKa (E), and LV-shIKKb (F). Expression of p65, p52, IKK-a, andIKK-b was suppressed by corresponding shRNAs, compared with cells transduced with a lentivirus expressing control shRNA. Densitometric analysis of p65 andp52 in cells transduced with LV-shp65 and LV-shp52 (D) and of IKK-a and IKK-b in cells transduced with LV-shIKKa and LV-shIKKb (G). H:Western blot analysis ofADAMTS-4 (AD-4), ADAMTS-5 (AD-5), and aggrecan neoepitope (ARGSVIL) in human NP cells infected with LV-shC and LV-shp65, LV-shp52, LV-shIKKa, and LV-shIKKb after TNF-a treatment. Note that the TNF-aedependent increase in ADAMTS-4 (the band is indicated by an asterisk), ADAMTS-5, and aggrecan neo-epitope (ARGSVIL) levels is significantly blocked by suppression of components of the NF-kB signaling pathway. Data are expressed as means � SEM from threeindependent experiments. *P < 0.05. Original magnification, �20.

Control of ADAMTS-4 in Nucleus Pulposus

to determine whether there is cell type specificity, wemeasured ADAMTS4 promoter activity in RelA null andwild-type mouse embryonic fibroblasts. Only in wild-typecells was the promoter activity cytokine inducible(Supplemental Figure S2, B and C).

NF-kB Signaling Controls TNF-aeDependent ADAMTS-4and -5 Expression and Aggrecan Degradation in NP Cells

Given that IL-1b and TNF-a regulated ADAMTS-4 expres-sion using similar signaling pathways, we performedlentiviral-mediated gene silencing studies using TNF-a asa representative cytokine. We first silenced the expression ofindividual NF-kB signaling components and then measuredADAMTS-4 expression in human NP cells. There was robustYFP expression by the virally transduced cells (Figure 5A),indicating high levels of transduction efficiency and transgeneexpression. In cells transduced with plasmids shp65 andshp52, there was a significant decrease in the protein levels of

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p65 and p52, respectively, compared with cells transducedwith control shRNA (Figure 5, BeD). Similarly, transductionof human NP cells with plasmids shIKK-a and shIKK-b resulted in decreased levels of IKK-a and IKK-b protein,respectively (Figure 5, EeG). Importantly, suppression ofindividual NF-kB signaling components significantly blockedthe inductive effect of TNF-a on the expression of ADAMTS-4 and -5 protein levels, as well as aggrecan degradation asmeasured by neoepitope generation (Figure 5H).

Both ADAMTS-4 and ADAMTS-5 Contribute toTNF-aeInduced Aggrecan Degradation in NP Cells

We examined the effect of silencing of ADAMTS-4 andADAMTS-5 expression on aggrecan degradation inhuman NP cells. ADAMTS protein levels in the silencedNP cells or the conditioned medium were significantlyreduced (Figure 6, AeC). Furthermore, there was nocompensatory increase in either ADAMTS-4 or -5 when

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Figure 6 ADAMTS-4 and ADAMTS-5 promote aggrecan degradation in human NP cells. A and B: Western blot analysis of human NP cells infected withcontrol lentivirus (LV-shC) and lentivirus expressing shRNA ADAMTS-4 (LV-shADAMTS4) (A) and shRNA ADAMTS-5 (LV-shADAMTS5) (B) plasmids. Comparedwith control cells, expression of ADAMTS-4 and ADAMTS-5 was suppressed by shRNA shADAMTS-4 and shADAMTS-5 in both the conditioned medium and cellprotein. C: Densitometric analysis of multiple blots from the experiment presented in panels A and B. D and E: Western blot (D) and corresponding densi-tometric analysis (E) of aggrecan neoepitope (ARGSVIL) in conditioned medium of cells treated with TNF-a. The level of ARGSVIL was significantly reducedafter cytokine treatment in cells transduced with LV-shADAMTS-4 (LV-shA4) and LV-shADAMTS-4 (LV-shA5), compared with control (LV-shC). F and G: Westernblot (F) and corresponding densitometric analysis (G) of aggrecan neoepitope (NITEGE) in concentrated conditioned medium of cells treated with TNF-a. Thelevel of NITEGE was significantly reduced after TNF-a treatment in ADAMTS-4 and ADAMTS-5 silenced NP cells. H: Western blot analysis of NITEGE in cell-associated protein (the band is indicated by an asterisk) shows a significant reduction in levels after cytokine treatment in ADAMTS-4 and ADAMTS-5silenced cells, compared with controls. Data are expressed as means � SEM from three independent experiments. *P < 0.05. Cell Pro, cell protein; CM,conditioned medium.

Tian et al

the other ADAMTS was silenced. We then usedneoepitope-specific antibodies anti-ARGSVIL and anti-NITEGE to measure aggrecan degradation in ADAMTSsilenced cells. Suppression of ADAMTS-4 and -5 expressionresulted in a significant inhibition in TNF-aemediatedaggrecan degradation in NP cells (Figure 6, D and F).Densitometric analysis validated these findings (Figure 6, Eand G). As expected, cells transduced with control shRNAexhibited an increase in aggrecan neoepitope generation afterTNF-a treatment (Figure 6, D and F). The level of cell-associated versus pericellular matrix aggrecan degradationwas also evaluated. In the cell-associated protein fraction,compared with control cells, silencing of ADAMTS-4

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and ADAMTS-5 blocked aggrecan degradation after TNF-a treatment.

Discussion

The experiments described here demonstrated for the firsttime that expression of ADAMTS-4, an important enzymeconcerned with aggrecan degradation, is regulated by theinflammatory cytokines TNF-a and IL-1b through theMAPK and NF-kB signaling pathways in NP cells. Asecond major observation is that, by regulating ADAMTS-4and -5 expression and activity, these inflammatory cytokines

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Control of ADAMTS-4 in Nucleus Pulposus

controlled aggrecan turnover. Importantly, both ADAMTS-4 and -5 were required for the cytokine-dependent aggrecandegradation in human NP cells, and their function appears tobe nonredundant, suggesting a possible role in intervertebraldisk pathologies.

According to our gene and protein expression studies,ADAMTS-4 is expressed in tissues of the intervertebraldisk. The 73-kDa product on Western blot suggests thata mature/processed form of the protease is present in healthydiscal tissues; the level of zymogen expression in the ratprobably reflects the low level of matrix (aggrecan) turnoverin the healthy disk. Although ADAMTS-4 is consideredchiefly in terms of aggrecan catabolism, it is involved witha number of diverse physiological functions. Moreover, themechanism of regulation of expression is incompletelyunderstood. Conflicting data have been reported on theexpression of ADAMTS-4 in chondrocytes and fibroblasts;some studies show that ADAMTS-4 is up-regulated byTNF-a and IL-1b,25e27 whereas others show that cytokinetreatment does not affect ADAMTS-4 expression.28,29 In thepresent study, treatment of NP cells with TNF-a and IL-1bclearly induced ADAMTS-4 expression. Moreover, ourpromoter studies showed that regulation was at the transcriptlevel. These results are consistent with previous reports thatthe inflammatory cytokines induce ADAMTS4 mRNAexpression in the NP.10,11,19,30 Although the mechanism isunknown, there is some evidence to indicate that TNF-aedependent ADAMTS-4 expression and aggrecanaseactivity in the NP may be regulated by ERK and NF-kBsignaling.11 We confirmed that both cytokines promotedMAPK and p65/RelA activation and that these signalingpathways controlled ADAMTS-4 expression. Furthermore,mechanistic insights into regulation were forthcoming fromgain and loss-of-function transfection studies that measuredADAMTS4 promoter activity. Our results clearly showedisoform specificity for both MAPK signaling pathways.Although p38a, p38b2, p38g, and ERK1 positivelycontrolled cytokine-dependent ADAMTS4 promoter activity,p38d and ERK2 were not involved.

The presence of four putative NF-kB motifs in theADAMTS4 promoter indicated functional involvement ofthis factor in controlling transcription. Mizui et al31 sug-gested that the region between �383 and þ10 is required forfull activity of the human ADAMTS4 promoter. Notably, theNF-kB motif with the second-highest relative score is con-tained within this region. In further support for the roleof NF-kB in ADAMTS4 promoter regulation, our studiesclearly indicated that the inductive effect is restricted to p65and p52 and that subunits RelB and c-Rel do not playa regulatory role. Interestingly, p50 suppressed the inductiveeffect of p65 on ADAMTS4 promoter. The observation thatthe cytokine- and p65-dependent induction of ADAMTS-4expression is suppressed by p50 is consistent withprevious reports of repressive function of p50 homodimersin controlling expression of a number of genes, includingCCL2, CXCL10, GMCSF, and MMP13,32e34 as well as the

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recent report from our research group that clearly identifiedp50 as a negative regulator of cytokine-dependent CCL3transcription.35 On the other hand, our group has also shownthat p65 and p50 act synergistically to induce the expressionof the syndecan-4 gene (SDC4), one of the target genes ofTNF-a and IL-1b in NP cells.19 These results highlight theimportance of both context and target-gene specificity in theregulatory machinery of NP cells. It is not unreasonable toassume that formation of p50 homodimers and their bindingto the kB motifs results in recruitment of transcriptionalrepressor such as HDAC1 to the ADAMTS4 promoter,thereby suppressing RelA/p65 response.32 A detailedinvestigation would be required to further elucidate themechanism and significance of this interesting finding. Theobservation that RelA null cells failed to induce ADAMTS4promoter activity, even when treated with cytokines,provides further validation of the importance of NF-kBsignaling in promoter regulation. Moreover, the silencingstudies that demonstrated inhibition of TNF-aedependentADAMTS-4 and -5 expression after suppression of severalNF-kB signaling highlight the importance of this pathway incontrolling ADAMTS-4 expression. Taken together, theresults of these functional studies indicate that, by control-ling the activity of both MAPK and NF-kB, especiallyRelA/p65 signaling, cytokines control the expression ofADAMTS-4 in NP cells.

Relevant to this discussion of control of ADAMTS4transcription, Thirunavukkarasu et al20 showed that IL-1aand oncostatin induced ADAMTS4 promoter activity,possibly through NFATp and Runx2 in chondrocytic cells.Mizui et al31 showed that nuclear factor I (NFI) is involvedin the negative regulation of the human ADAMTS4 promoteractivity in chondrocytes; they speculated that Sp1 and AP2sites located within �382 to þ10 of the promoter may berequired for its full activity. Because the ADAMTS4promoter contains several CCAAT enhancer elements andbecause C/EBP-b is known to promote cytokine-dependenttranscription in chondrocytes,36 it was also important toconsider their possible regulatory roles. In contrast to its rolein chondrocytes, our present data clearly identify C/EBP-bas a negative regulator of cytokine-dependent ADAMTS4transcription in the NP, thus highlighting the uniquecell-type-specific regulation of this gene in NP cells.

We and others have demonstrated increased expressionof ADAMTS4 and ADAMTS5mRNA and protein during diskdegeneration.17,19,37 Patel et al37 and Seki et al38 showed that,in both human and rabbit, although ADAMTS-4 proteinlevels increase with severity of the disease, ADAMTS-5levels are similar at early and late stages. Moreover, Sekiet al38 showed that silencing ofADAMTS-5 alone is sufficientto block aggrecan degradation in rabbit disks. Lendingsupport to these earlier reports, in the present study expressionof ADAMTS-4 was more responsive to TNF-a in NP cellsfrom intervertebral disks of more degenerate grades; never-theless, silencing of either ADAMTS-4 or ADAMTS-5in grade 2 human NP cells resulted in inhibition of

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TNF-aeinduced aggrecan neoepitope generation. Based onthese findings, it is not unreasonable to conclude thatADAMTS-4 and -5 are nonredundant and therefore thattherapeutic blocking of the activity of even one of theseproteases could be expected to limit breakdown of theaggrecan-rich matrix and possibly mitigate degenerative diskdisease.

Supplemental Data

Supplemental material for this article can be found athttp://dx.doi.org/10.1016/j.ajpath.2013.02.037.

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