Tumor Necrosis Factor-&alpha - American Journal of Pathology, The

The American Journal of Pathology, Vol. 184, No. 9, September 2014

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See related Commentary on page 2371 MATRIX PATHOBIOLOGY

Tumor Necrosis Factor-ae and Interleukin-1beDependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein KinaseeNF-kB Axis Implications in Inflammatory Disc Disease Xin Wang,*y Hua Wang,*z Hao Yang,*z Jun Li,*x Qiqing Cai,y Irving M. Shapiro,* and Makarand V. Risbud* From the Department of Orthopedic Surgery and the Graduate Program in Cell and Developmental Biology,* Thomas Jefferson University, Philadelphia, Pennsylvania; the Department of Bone and Soft Tissue,y The Affiliated Cancer Hospital of Zhengzhou University, Zhengzhou, China; the Department of Orthopedics,z First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China; and the Department of Orthopedic Surgery,x Changzheng Hospital, Second Military Medical University, Shanghai, China Accepted for publication June 4, 2014. Address correspondence to Makarand V. Risbud, Ph.D., Department of Orthopedic Surgery, Thomas Jefferson University, 1025 Walnut St, Suite 511 College Bldg, Philadelphia, PA 19107. E-mail: makarand.risbud@ jefferson.edu.

Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-kB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-kB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-kB, p38a/b2/g/d, and extracellular signaleregulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-IkB kinase b significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-b can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-a by counteracting the NF-kB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1e MAPKeNF-kB axis is required for TNF-aedependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism. (Am J Pathol 2014, 184: 2560e2572; http://dx.doi.org/10.1016/j.ajpath.2014.06.006)

Low back pain is one of the most prevalent and costly health problems facing the world population, with occurrence in 84% of the population; the total costs exceed $100 billion per year in the United States alone.1,2 Intervertebral disc degeneration (IVDD) is one of the major contributors of low back and neck pain and associated disability.3,4 Nucleus pulposus (NP) cells, which primarily secrete proteoglycan aggrecan and fibrillar collagens to Copyright ª 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajpath.2014.06.006

form the complex extracellular matrix (ECM), are key in maintaining a healthy disc.5 Loss of NP cells and their dysfunction, resulting in decreased proteoglycan synthesis, increased expression of catabolic enzymes, and a shift toward synthesis of fibrotic Supported by NIH grants AR050087, AR055655, and AR064733. X.W. and H.W. contributed equally to this work. Disclosures: None declared.

SDC4 Regulates MMP-3 Expression in NP matrix, are hallmarks of disc degeneration. All these pathological changes diminish the water-binding capacity of the disc, leading to failure to resist compressive loads in the spine.6 Despite the ubiquitous nature of the spinal pathologies, the molecular mechanisms of low back paineassociated IVDD have not been well investigated. Many studies have demonstrated that there was an increase in expression and activity of a range of matrix-degrading enzymes in IVDD, including the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) families.7e9 The MMP is a family of metal-dependent proteases capable of degrading all components of the ECM of connective tissues.10 It was demonstrated by many studies that elevated MMPs 1, 2, 3, and 13 have been found in degenerated IVD.8e11 Active MMP-3 has the ability to degrade core proteins of disc and cartilage connective matrix components, such as proteoglycans, fibronectin, laminin, elastin,12e14 and collagens II, IX, and X.15 Noteworthy, MMP-3 can indirectly affect the degradation of cartilagenous matrix by proteolysis of latent MMPs, including proeMMP1, proeMMP-7, and proeMMP-9 into the active forms,16e18 suggesting that MMP-3 may be important in disc pathologies. Elevated levels of the proinflammatory cytokines, including tumor necrosis factor (TNF)-a and IL-1b, have been reported in IVDD.19e21 Recent studies have shown that, in disc cells, MMP-3 expression is induced by these cytokines.22e27 However, only a handful of these studies have examined the mechanism of regulation of MMP-3 by cytokines.14,28 Likewise, little is known about the intricacies of the signaling pathways controlling cytokine-mediated MMP-3 expression during IVDD.29 TNF-aedependent elevated expression of syndecan 4 (SDC4), a cell surface heparan sulfate proteoglycan, plays a major role in matrix catabolism through activation of ADAMTS-5.19,30,31 Although synergistic actions of SDC4 on activity of several chemokines and cytokines have been demonstrated,32e34 in the context of inflammatory disc disease, it is not yet known if SDC4 contributes to the cellular actions of TNF-a and if a regulatory relationship exists between MMP-3 expression and SDC4. In the present study, using genetic approaches, we investigate the mechanisms by which cytokines TNF-a and IL-1b control expression of MMP-3 in human and rat NP cells. Our results indicate that, in addition to mitogenactivated protein kinase (MAPK)eNF-kB axis downstream of cytokine receptor, cell surface SDC4 is required for TNF-ae and IL-1bedependent MMP-3 expression in NP cells.

Materials and Methods Reagents and Plasmids Plasmids were kindly provided by Wen-Ling Shih (Department of Life Science, Tzu Chi University, Hualien City, Taiwan) (MMP3-LUC, 2.3-kb human MMP3 promoter in pGL3),35 Jiahui Han (Scripps Institute, La Jolla, CA) (p38aAF, p38bAF, p38dAF, and p38gAF), Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX) (ERK-1K71R

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and ERK-2K52R), and Dr. Silvio Gutkind (NIH, Bethesda, MD) [activator protein (AP)-1 reporter]. Plasmids for short hairpin (sh)-p65 and sh-IkB kinase (IKK) b in lentiviral FSVsi vector that co-expresses yellow fluorescent protein (YFP) were gifts from Dr. Andree Yeremian (University of Lleida, Lleida, Spain). Lentiviral shRNA plasmids targeting rat Sdc4 were purchased from Genecoepia. Plasmids psPAX2 (number 12260) and pMD2.G (number 12259), developed by Dr. Didier Trono (École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland), and 3 nuclear factor for activated T cell (NFAT)eLuc (number 17870), developed by Dr. Gerald Crabtree (Stanford University, Stanford, CA), were obtained from Addgene (Cambridge, MA). pRL-TK (Promega, Madison, WI) containing the Renilla reniformis luciferase gene was used as an internal transfection control. IKKb and p65 antibodies were from Cell Signaling (Danvers, MA), MMP-3 and SDC4 antibodies were from Abcam (Cambridge, MA), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Novus Biologicals (Littleton, CO). Recombinant human TNF-a and IL-1b were purchased from PeproTech, Inc. (Rocky Hill, NJ).

Isolation of NP Cells and Treatments Rat (n Z 16 animals) and human (two samples: aged 33 years, female, autopsy, grade 1, and aged 39 years, male, surgical waste, grade 2) NP cells were isolated using a method previously described by Risbud et al.36 Collection of animal and human tissues for cell isolation was performed as per approved protocols by Thomas Jefferson University’s (Philadelphia, PA) Institutional Animal Care and Use Committee and Institutional Review Board, respectively. NP cells were passaged and maintained in monolayer in Dulbecco’s modified Eagle’s medium (DMEM), together with 10% fetal bovine serum (FBS), supplemented with antibiotics. To investigate the effect of cytokines, cells in some groups were treated with 10 ng/mL IL-1b or 25 to 50 ng/mL TNF-a for 24 hours, with or without 10 mmol/L signaling pathway inhibitors SM7368, SB203580, PD98059, and SP60025, and 100 nmol/L Go6983 (Calbiochem, Billerica, MA). Cytokine doses were based on our previous studies.19

Real-Time RT-PCR Analysis Total RNA was extracted from rat or human NP cells cultured in 6-cm plates (5 105 cells per plate) using RNAeasy mini columns (Qiagen, Valencia, CA) and treated with RNase-free DNase I before elution. Purified RNA (1 to 2 mg) was used to synthesize cDNA using EcoDry Premix (Clontech, Mountain View, CA). Reactions were set up in triplicate in 96-well plates using 1 mL cDNA together with Fast SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA) and gene-specific primers. Gene expression was quantified using the StepOnePlus real-time PCR system (Applied Biosystems), according to the manufacturer’s instructions. Hypoxanthine phosphoribosyltransferase 1 was used to normalize the expression of

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Protein Extraction and Western Blot Analysis After treatment of rat or human NP cells cultured in 10-cm plates (1 106 cells per plate), conditioned medium was collected and cells were immediately placed on ice and washed with ice-cold Hanks’ balanced salt solution. All of the wash buffers and the final cell lysis/resuspension buffers included 1 protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN), 5 mmol/L NaF, and 200 mmol/L Na3VO4. Conditioned medium was concentrated using 10-kDa cutoff protein concentrator columns, following the manufacturer’s instructions (Millipore, Billerica, MA), and stored at 80 C with protease inhibitors. Total cell or secreted proteins were resolved by electrophoresis on 8% to 12% SDSepolyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Berkeley, CA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with Tween 20 and incubated overnight at 4 C in 3% nonfat dry milk in Tris-buffered saline with Tween 20. The antibodies were cross-reactive against both human and rat MMP-3 (1:1000), SDC4 (1:1000), p65 (1:1000), IKKb (1:1000), or GAPDH (1:3000). Immunolabeling was detected using the electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ).

Transfections and Dual-Luciferase Assay Cells were seeded to 48-well plates (2 104 cells per well) 1 day before transfection. To measure the effect of IL-1b and TNF-a on MMP-3, cells were transfected with 250 ng of MMP-3eLuc with 250 ng pRL-TK plasmid. To investigate the effect of NF-kB and MAPK on MMP-3 plasmid activity, cells were cotransfected with 50 to 200 ng of dominant-negative (DN)eNF-kB, DN-p38a/b/g/d, or DN-ERK1/ERK2, with or without appropriate backbone vector, 150 ng MMP-3 reporters, and 150 ng pRL-TK plasmids. In some experiments, cells were transfected with 250 ng of MMP3 reporter with 250 ng pRL-TK and treated with 10 mmol/L inhibitors for NF-kB (SM7368), p38 (SB203580), ERK (PD98059), or c-Jun N-terminal kinase (JNK; SP60025) (Calbiochem) in the presence of TNF-a or IL1b. Lipofectamine 2000 (Invitrogen, Grand Island, NY) was used as a transfection reagent. For each transfection, plasmids were premixed with the transfection reagent, and 24 hours after the transfection, some NP cells were treated with IL-1b and TNF-a. Forty-eight hours after the transfection (72 hours when cotransfected with sh-SDC4), NP cells were harvested, and a Dual-Luciferase reporter assay system (Promega) was used for sequential measurements of firefly and Renilla luciferase activities. Quantification of luciferase activities and calculation of relative ratios were performed using a luminometer (TD-20/20; Turner Designs, Sunnyvale, CA). At least three independent transfections were performed, and all analyses were performed in triplicate.

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Lentiviral Particle Production and Viral Transduction Human embryonic kidney (HEK) 293T cells were seeded in 10-cm plates (1.3 106 cells per plate) in DMEM with 10% heat-inactivated FBS 2 days before transfection. Cells were transfected with 2.5 mg of sh-control (Ctr), sh-p65, or sh-IKKb plasmids or sh-SDC4 plasmids along with 1.875 mg psPAX2 and 0.625 mg pMD2.G. After 16 hours, transfection medium from the HEK 293T cell plates was removed and replaced with DMEM with 5% heat-inactivated FBS and penicillinstreptomycin. Medium containing lentiviral particles was collected from the HEK 293T cell plates at 48 to 60 hours after transfection. Human or rat NP cells were plated in DMEM with 5% heat-inactivated FBS 1 day before transduction. NP cells in 10-cm plates were transduced with 5 mL of conditioned medium containing viral particles along with 6 mg/mL polybrene. Twenty-four hours later, the medium was removed and replaced with DMEM with 5% heat-inactivated FBS. Cells were harvested for mRNA and protein extraction 6 days after transduction. A transduction efficiency of 60% to 80% was achieved as determined from the number of YFP-positive cells.

Statistical Analysis All experiments were performed at least three independent times and contained three replicates. Data are presented as the means SEM. Differences between groups were assessed by Student’s t test and analysis of variance. P < 0.05 was considered statistically significant.

Results TNF-a and IL-1b Increase Mmp3 Gene Expression and Levels of Active MMP-3 Protein in NP Cells To test the premise that TNF-a and IL-1b regulate MMP-3 expression and activity in IVDD context, rat NP cells were treated with TNF-a and IL-1b, and Mmp3 expression was analyzed by real-time RT-PCR. Treatment with both TNF-a and IL-1b resulted in dose-dependent (Figure 1, A and B) and time-dependent (Figure 1, C and D) increases in Mmp3 mRNA levels. Western blot analysis showed that treatment of rat NP cells with TNF-a and IL-1b increased levels of a 50kDa product that corresponds to active MMP-3 protein in both the cellular fraction (Figure 1E) and the secreted fraction from the conditioned medium (Figure 1F). Corresponding densitometric analysis confirmed that an increase in active MMP-3 in both cellular (Figure 1G) and secreted (Figure 1H) fractions was significant. To investigate whether the regulation of MMP-3 expression is at the transcriptional level, we measured the activity of a 2.3-kb MMP3 promoter after cytokine treatment. This promoter fragment has been shown to contain several transcription factor binding sites, including NFAT, AP-1, cAMP response element binding protein, E26 transformation-specific, STAT, NF-kB, and specificity protein 1.33 Both TNF-a and IL-1b significantly increased the

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MMP3 promoter activity in a dose-dependent manner (Figure 1, I and J), although IL-1bedependent modulation of MMP3 promoter activity is only seen at a concentration of 10 ng/mL. Compared with IL-1b, MMP3 was much more responsive to TNF-a treatment, and TNF-a was, therefore, selected as a representative cytokine for further studies.

SDC4 Serves as TNF-a Coreceptor and Is Required for Cytokine-Dependent Induction of MMP-3 To ascertain if SDC4 is required for cytokine-induced MMP-3 expression in NP cells, we stably suppressed


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AeD: Cytokine-dependent expression of MMP-3 in rat NP cells. Real-time RT-PCR analysis shows that TNF-a and IL-1b up-regulate the expression of MMP-3 in a dose- and time-dependent manner. For time-dependent studies, 50 ng/mL TNFa and 10 ng/mL IL-1b were used. Western blot (E and F) and corresponding densitometric (G and H) analyses show that treatment of NP cells with TNF-a or IL-1b significantly increases the level of MMP-3 in cellular protein (E and G) and secreted protein (F and H) in condition medium. The activity of MMP3 promoter construct (MMP-3eLUC) is significantly induced after treatment of NP cells with TNF-a (I) and IL-1b (J) in a dose-dependent manner. Values shown are means SEM of three independent experiments. *P < 0.05.

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SDC4 expression in human NP cells by transduction with lentivirus-expressing SDC4-shRNA. There was a robust green fluorescent protein (GFP) expression by the virally transduced cells, indicating high-level transduction efficiency and transgene expression (Figure 2A). As expected, there was a significant decrease in SDC4 mRNA (Figure 2B) and protein (Figure 2, C and D) expression in NP cells transduced with SDC4-shRNA. Furthermore, we observed that SDC4 was required for TNF-aedependent MMP-3 induction. Suppression of SDC4 significantly decreases the inductive effect of TNF-a on the expression level of MMP-3 in cellular (Figure 2, E and G) and secreted

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Wang et al (Figure 2, F and H) protein fractions, as seen by Western blot and corresponding densitometric analyses.

SDC4 Regulation of MMP-3 Expression Is Independent of NF-kB and PKC Signaling We then determined if SDC4 mediates its effect on MMP-3 expression through NF-kB. For this purpose, we first treated rat NP cells with surfen, a potent heparan sulfate inhibitor, and in parallel silenced Sdc4 expression by Sdc4-shRNA. Then, we measured activities of a prototypic NF-kBeresponsive reporter and MMP3 promoter (Figure 3A) in the presence or absence of TNF-a. Surprisingly, heparan sulfate inhibition (Figure 3, B and C) or silencing of Sdc4 (Figure 3, D and E) did not affect TNF-aedependent induction in activities of both the prototypic NF-kB and MMP3 reporter. Likewise, activities of prototypic AP-1 and NFAT reporters were unaffected by Sdc4 suppression (data not shown), suggesting lack of their involvement in MMP-3

regulation. To further determine whether protein kinase C (PKC) was responsible for mediating SDC4 action, we measured cytokine-dependent Mmp3 expression in the presence of PKC inhibitor, Go6983. Noteworthy, PKC inhibition did not result in significant changes in cytokinedependent Mmp3 mRNA expression (data not shown).

Cytokines Promote MMP-3 Expression through MAPK and NF-kB We next investigated the involvement of MAPK and NF-kB signaling pathways in TNF-ae and IL-1bemediated induction of MMP-3. For these studies, rat NP cells were treated with pathway-specific inhibitors before the exposure of TNF-a and IL-1b. RT-PCR analysis showed that pretreatment with inhibitors results in a significant suppression in cytokine-dependent Mmp3 mRNA expression (Figure 4, A and B). Although the p38/JNK/ERK inhibitor partially counteracted the inductive effects of TNF-a and IL-1b on

Figure 2

SDC4 controls the cytokine-mediated increase in levels of active MMP-3 protein. A: Immunofluorescence detects GFP in human NP cells transduced with lentivirus co-expressing GFP and either Sdc4-shRNA (LV-shSDC4) or Ctr-shRNA (LV-shC), showing a high transduction efficiency. RT-PCR (B), Western blot (C), and corresponding densitometric (D) analyses of cells transduced with LV-shC and LV-shSdc4. Expression of SDC4 is significantly blocked by the Sdc4-shRNA compared with the control. Western blot (E and F) and densitometric (G and H) analyses of MMP-3 expression in cells transfected with LV-shC and LV-shSdc4 after TNF-a treatment. The TNF-ae dependent induction in MMP-3 cellular protein (E and G) and secreted protein (F and H) is significantly blocked by suppression of SDC4 levels. Values shown are means SEM of three independent experiments. *P < 0.05. Original magnification, 20 (A). CM, conditioned medium.

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Figure 3 Cytokine-mediated up-regulation of MMP-3 by SDC4 is independent of NF-kB activity. A: Schematic showing the organization and binding sites for kB and other transcription factors in synthetic prototypic NF-kB (NF-kBeLUC) and MMP3 promoter constructs (MMP-LUC). Treatment of rat NP cells with heparan sulfate inhibitor surfen (B and C) or cotransfection with Sdc4-shRNA (D and E) does not block the inductive effect of TNF-a on activities of the prototypic NF-kB reporter (NF-kBeLUC) (B and D) or MMP3 promoter construct (MMP3-LUC) (C and E). Values shown are means SEM of three independent experiments. *P < 0.05.

Mmp3 mRNA expression, NF-kB inhibitors blocked Mmp3 mRNA expression almost to the basal level. To ascertain the mechanism of action, cytokine-dependent MMP3 promoter activity was measured in the presence of pathway inhibitors. Results showed a strong suppression of promoter activity by all of the inhibitors in the presence of TNF-a (Figure 4C) and IL-1b (Figure 4D), indicating that the regulation of MMP-3 expression by MAPK and NF-kB pathways is at the transcriptional level.

Isoform Specificity of MAPK and Contribution of NF-kB Pathway in Regulation of MMP3 Promoter Activity We next used a genetic approach to confirm these findings and determined if MAPK regulates MMP-3 expression in an isoform-specific manner. Rat NP cells were transfected with DN-ERK1 or DN-ERK2, or DN-p38a/b2/g/d expression plasmids and MMP3 promoter activity was measured in the presence of TNF-a or IL-1b. Although DN-ERK1 (Figure 5A) had little or no effect on the induction of promoter activity by TNF-a and IL-1b, cotransfection with DNERK2 (Figure 5B) inhibited cytokine-dependent induction of MMP3 promoter. In contrast to isoform-specific differences of


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ERK, cotransfection with DN-P38a (Figure 5C), DN-p38b2 (Figure 5D), DN-p38d (Figure 5E), or DN-p38g (Figure 5F) significantly inhibited the cytokine-dependent induction of MMP3 promoter. Because NF-kB pathway inhibitor robustly inhibits the cytokine-dependent induction of MMP3 expression, we first evaluated responsiveness of MMP3-Luc to increasing doses of NF-kB inhibitor. Results showed that MMP3 promoter activity, induced by TNF-a, is completely blocked by NF-kB pathway inhibitors, even at the concentration of 10 mmol/L (Figure 5G). Moreover, when rat NP cells were cotransfected with IkBaM/(DN)eNF-kB, TNFaemediated induction in MMP3 promoter activity was repressed to the basal level (Figure 5H). Both these experiments suggested a role of canonical NF-kB pathway in this regulation.

MAPK and NF-kB Pathway Inhibitors Suppress Cytokine-Dependent Induction of MMP-3 Protein Levels We determined if treatment with MAPK and NF-kB pathway-specific inhibitors results in suppression in levels of active MMP-3 protein. Western blot analysis of cellular (Figure 6A) and conditioned (Figure 6C) media and

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Figure 4

MAPK and NF-kB regulation of MMP3 expression in rat NP cells. Real-time RT-PCR analysis of Mmp3 expression in NP cells after TNF-a (A) and IL-1b (B) treatment for 24 hours, with or without 10 mmol/L of SB203580, PD98059, SP60025, or SM7368 (p38, ERK, JNK, and NF-kB inhibitors, respectively). A significant blocking of cytokine-dependent induction in Mmp3 mRNA expression is seen by all inhibitors. Increased activation of MMP3 reporter by TNF-a (C) or IL-1b (D) is suppressed by both MAPK and NF-kB inhibitors. Values shown are means SEM of three independent experiments. *P < 0.05.

corresponding densitometric analysis (Figure 6, B and D), respectively, showed that TNF-aedependent induction in both cellular and secreted MMP-3 protein levels in rat NP cells was suppressed by MAPK and NF-kB pathway inhibitors. Similarly, a pronounced decrease in the levels of IL-1bemediated MMP-3 induction in both cellular and secreted fractions was seen in the presence of MAPK and NF-kB pathway inhibitors (Figure 6, EeH). Although the p38/JNK/ERK inhibitor partially counteracted the inductive effects of cytokine on MMP-3 cell protein expression, NFkB inhibitors totally blocked MMP-3 cell protein expression to the basal level.

Canonical IKKb-p65/RelA Signaling Controls CytokineMediated MMP-3 Expression in Human NP Cells To further elucidate if the canonical NF-kB signaling pathway was involved in regulation of MMP-3 expression, we silenced expression of p65 and its upstream controller IKKb. Human NP cells were transduced with lentivirus coexpressing YFP and IKKb-shRNA, p65-shRNA, or CtrshRNA. MMP-3 expression was then measured after TNF-a treatment. There was a robust YFP expression in the virally transduced cells, indicating high-level transduction efficiency and transgene expression (Figure 7A). As expected, in cells transduced with sh-IKKb and sh-p65, there was a significant decrease in the expression of IKKb and p65 mRNA (Figure 7B) and protein (Figure 7, C and D), respectively, when compared with cells transduced with control shRNA. More important, suppression of individual NF-kB signaling components IKKb and p65 significantly decreased the inductive effect of TNF-a on the expression

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level of MMP-3 cellular (Figure 7, E and G) and secreted (Figure 7, F and H) protein fractions.

TGF-b Suppresses the Induction of MMP-3 by TNF-a in NP Cells by Counteracting NF-kB Signaling Next, we investigated whether the induction of MMP-3 by TNF-a can be modulated by transforming growth factor (TGF)-b. We transfected rat NP cells with MMP3-LUC (Figure 8A) and prototypic NF-kB (Figure 8B) reporters and measured their activities after TGF-b and/or TNF-a treatment. As expected, TNF-a increased the activity of both reporters. In contrast, TGF-b not only decreased the basal activities, but also completely blocked the inductive effect of TNF-a (Figure 8, A and B), suggesting that TGF-b inhibits NF-kB signaling. In addition to its effect on NF-kB, TGF-b blocked elevated expression of Sdc4 in NP cells treated with TNF-a (Figure 8C). More important, mRNA expression of Mmp3, induced by TNF-a, was blocked by TGF-b to nearly a basal level (Figure 8C). Likewise, an increase in levels of active MMP-3 protein by TNF-a in both the cellular (Figure 8, D and E) and secreted (Figure 8, F and G) protein fractions was blocked in the presence of TGF-b.

Discussion This study clearly showed that the expression of MMP-3, a key enzyme involved in degradation of many prominent ECM components of the intervertebral disc,11,29 is regulated by the inflammatory cytokines TNF-a and IL-1b. More important, our experiments, for the first time to our knowledge,

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Figure 5

MAPK and NF-kB regulation of cytokine-mediated induction of MMP3 promoter activity in rat NP cells. NP cells were cotransfected with MMP-3eLUC, along with DN-ERK1 (A), DNERK2 (B), DN-p38a (C), DN-p38b2 (D), DN-p38d (E), DN-p38g (F), or the empty backbone vector. Cells were treated with TNF-a or IL-1b, and reporter activity was measured 24 hours after the treatment. Cotransfection of DN-ERK1 does not block cytokine-mediated induction of the reporter activity, whereas cotransfection of DN-ERK2, DNp38a, DN-p38b2, DN-p38d, and DN-p38g results in robust suppression of cytokine-dependent induction of the MMP3 promoter activity. G: Induction of MMP3 by TNF-a can be blocked completely by NF-kB inhibitor SM7368, even at a lower dose. H: Cotransfection of DNeNF-kB completely blocks TNF-aedependent induction in MMP3 promoter activity by NP cells. Values shown are means SEM of three independent experiments. *P < 0.05.

demonstrate that, in NP cells, SDC4 is necessary to mediate cytokine-dependent induction of MMP-3 expression. Although our results confirm that signaling through MAPK and NF-kB is necessary, it is evident that a separate signaling event downstream of SDC4 is also required to mediate cytokine-dependent MMP-3 expression. Our studies address the intricacies of the signaling network used by inflammatory cytokines to mediate their catabolic effects on NP cells and may provide novel targets for therapies to mitigate or delay the onset of intervertebral disc degeneration. Because MMP-3 has a diverse substrate specificity that includes aggrecan, fibronectin, and various collagens,12e15 and because it indirectly activates several latent MMPs,16e18 many authorities consider it as the key catabolic enzyme in promoting matrix degradation during disc degeneration.37,38


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Kanemoto et al37 demonstrated that the number of MMP-3e positive cells was significantly correlated with the magnetic resonance imagingebased grade of disc degeneration, and that the ratio of MMP-3epositive cells in prolapsed lumbar IVDs was significantly higher than in non-prolapsed discs. Studies by Takahashi et al38 suggested that the 5A allele of the MMP3 promoter linked to regulation of gene expression is a likely risk factor for accelerated degenerative changes in the lumbar disc of elderly persons, whereas Aulisa et al39 suggest that the 5A/5A promoter genotype is associated with a higher risk of adolescent idiopathic scoliosis, a deformative condition affecting the spine. Despite a strong connection of MMP-3 to these disc pathologies, little is understood of the complexities of the pathways that control MMP-3 expression and activity in the NP cells. We and others

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Figure 6 Cytokines promote MMP-3 expression through MAPK and NF-kB pathways in rat NP cells. Western blot (A and C) and corresponding densitometric (B and D, respectively) analyses show that TNF-aedependent induction of MMP-3 in cellular (A and B) and secreted (C and D) protein levels in NP cells is blocked by MAPK and NF-kB pathway inhibitors. Measurement of MMP-3 levels on IL-1b treatment in both cellular (E and F) and secreted (G and H) protein fraction shows that MAPK and NF-kB pathway inhibitors exert a strong suppressive effect. Values shown are means SEM of three independent experiments. *P < 0.05. CM, conditioned medium; PD, PD98059 (ERK inhibitor); SB, SB203580 (p38 inhibitor); SM, SM7368 (NF-kB inhibitor); SP, SP60025 (JNK inhibitor).

have reported increased expression of both IL-1b and TNF-a in human degenerate NP.20,21 More important, studies have shown that both these cytokines promote matrix degradation through increased production of degradative enzymes, including MMPs and ADAMTS-4/5.22,24,40,41 We, therefore, first confirmed that MMP-3 expression by healthy NP cells was modulated under the inflammatory conditions. A robust stimulatory effect of cytokine exposure on MMP-3 expression suggested that the elevation of MMP-3 during degeneration might be the result of the NP cell stimulation by these agents. Results of these studies are in agreement with previous reports that showed MMP-3 production is increased by IL-1b and TNF-a in NP cells.22e27 More important, our promoter studies showed that the regulation was at the transcript level. Our earlier study had hinted that SDC4, a cell surface heparan sulfate proteoglycan, may be required for cytokine-

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dependent MMP-3 expression and activation.19 More important, SDC4 expression itself is influenced by TNF-a and IL-1b through NF-kB, and it is shown to contribute to aggrecan catabolism by promoting ADAMTS-5 activation.19,31 To confirm if a regulatory relationship between MMP-3 and SDC4 exists and to delineate the mode of this interaction, lentiviral SDC4-shRNA silencing studies were performed in NP cells. Suppression of SDC4 completely blocked the cytokine-dependent induction of the MMP-3 protein level. These results are in agreement with known function of SDC4 in controlling activities of several growth factors and chemokines in a heparan sulfateedependent manner.32e34,42,43 Noteworthy, binding of chemokines, such as CXCL12 and chemokine ligand 5, to SDC4 is shown to enhance their interaction with cognate receptors and formation of heteromeric complexes.33,34

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Figure 7

NF-kB regulation of MMP-3 expression. A: Immunofluorescence detection of YFP in human NP cells transduced with lentivirus (LV) coexpressing YFP and either shp65 or shIkkb shows high transduction efficiency. RT-PCR (B) and Western blot (C) and corresponding densitometric (D) analyses of cells transduced with LV-shC, LVshp65, and LV-shIkkb. Expression of p65 and Ikkb is significantly blocked by the respective shRNAs compared with the control. Western blot (E and F) and densitometric (G and H) analyses of MMP-3 expression in cells transfected with LV-shC, LVshp65, or LV-shIkkb after TNF-a treatment. The TNF-aedependent induction in MMP-3 cellular (E and G) and secreted (F and H) protein is significantly blocked by suppression of components of the NF-kB pathway. Values shown are means SEM of three independent experiments. *P < 0.05. Original magnification, 20 (A). CM, conditioned medium.

We then tested if cross talk between SDC4 and NF-kB and MAPK pathways downstream to TNF-a was responsible for its action. Although NF-kB regulation of SDC4 expression has been previously demonstrated,19 to our surprise, when NP cells were transduced by SDC4-shRNA or treated with surfen, the inductive effect of TNF-a on MMP3 promoter and prototypic NF-kB reporter activity remained unaltered, indicating that SDC4 did not modulate NF-kB and MAPK pathways. Similarly, failure of PKC inhibitor Go6983 to affect TNF-aemediated MMP-3 expression suggested that PKC was not involved. Likewise, unresponsiveness of AP-1 and NFAT reporters to TNF-a and SDC4 suppression and presence of known AP-1, NFAT, cAMP response element binding protein, STAT, and SP1 binding sites within 2.3 kb of MMP3 promoter suggested


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that these transcription factors did not mediate SDC4 action on MMP-3 expression.35 Taken together, these results suggest that SDC4 contributes in mediating TNF-a action on MMP-3 expression and that, in addition to canonical NFkB and MAPK, a yet unknown, independent downstream signaling event to SDC4 is necessary for controlling MMP3 transcription. Future experiments are directed at delineating complexities of signaling through SDC4 in NP cells and at delineating the identity and nature of the SDC4 regulatory motif that may possibly lie outside the 2.3-kb promoter used in this analysis. There is some evidence implicating that cytokinedependent MMP-3 expression in NP cells may be regulated by MAPK and NF-kB signaling, although the details of the transduction pathway are not known.14,28,44

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Figure 8 TGF-b suppresses the induction of MMP-3 by TNF-a in rat NP cells by modulation of NF-kB. Transfection shows that TGF-b blocks the inductive effect of TNF-a on activities of MMP-3eLUC (A) and NF-kBeLUC (B). C: TGF-b blocks cytokine-mediated induction in Sdc4 and Mmp3 expression induced by TNF-a to a nearly basal level. Western blot analysis shows that an increase in MMP-3 level in cellular (D) and secreted fraction in condition medium (F) by TNF-a can be inhibited by TGF-b. Densitometric analysis of the experiment described in D and F above shows a significant suppression in TNF-aedependent increase in MMP-3 in cellular (E) and secreted (G) fraction by TGF-b. GAPDH was used as a loading control and to calculate relative expression. Values shown are means SEM of three independent experiments. H: A model of TNF-aemediated expression of MMP-3 through SDC4 and MAPK/NF-kB axis in NP cells. Binding of TNF-a to TNF receptor 1 activates downstream MAPK (p38a/b2/d/g, ERK2, and JNK) and NF-kB signaling pathway, resulting in expression of MMP-3 and SDC4. SDC4 is required for TNF-aemediated induction of MMP3 through both MAPK/NF-kB axis and an independent, yet unknown, signaling event. TGF-b inhibits NF-kB signaling, thereby suppressing both SDC4 and MMP-3 expression, which ultimately leads to prevention of matrix degradation. *P < 0.05. CM, conditioned medium.

Noteworthy, the activity of the MAPK pathway is responsive to TNF-a and IL-1b in NP cells, and these pathways have been shown to modulate NF-kB signaling.40,45 We, therefore, first explored the contribution of this pathway to the MMP-3 regulatory system by using both inhibitor and genetic loss-of-function experiments. A significant suppression of TNF-ae and IL-1bemediated MMP-3 expression by p38-, JNK-, and ERK-specific inhibitors suggested that the regulation of MMP3 transcription by cytokines is more complex than what was originally thought. This was further evident when MMP3 promoter activity showed differential sensitivity to MAPK isoforms; p38a/b2/d/g and ERK2 positively controlled cytokine-dependent induction of MMP-3, whereas ERK1 was not involved in the response. On the basis of these findings, it is not unreasonable to assume that, by

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controlling the activity of specific MAPK isoforms, cytokines regulate the expression of MMP-3 in NP cells. Inhibitor studies by Wuertz and colleagues28,44 have suggested that NF-kB activity may play a role in IL1bedependent MMP-3 up-regulation in NP cells. To delineate if canonical NF-kB signaling through the IKKbp56/RelA axis is involved in this regulation, we first treated NP cells with canonical pathway-specific inhibitor in the presence of cytokines. Results showed that pretreatment with NF-kB inhibitor results in a significant suppression in MMP3 mRNA and protein expression and promoter activity to almost the basal level. This notion was further supported by loss-of-function genetic experiments using shRNAs targeting p65 and IKKb. The observed inhibition of TNF-ae dependent MMP-3 expression after suppression of p65 and

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SDC4 Regulates MMP-3 Expression in NP IKKb signaling highlighted the importance of the canonical NF-kB pathway in controlling MMP3 gene expression. In addition to the transcriptional induction of MMP-3 by TNF-a and IL-1b, its expression has also been shown to respond to a variety of other stimuli, such as nitric oxide46,47 and TGF-b.48 TGF-b is known to control ECM synthesis and matrix degradation in NP cells.49 However, the role of TGF-b in cytokine-dependent MMP-3 expression in NP cells is unknown. Significant suppression of TNF-aemediated induction in MMP-3 expression by TGF-b was likely due to an inhibitory effect on NF-kB signaling. Likewise, this NF-kB modulation also explains blocking of cytokine-dependent induction in SDC4 levels in TGF-betreated cells.19 Both these results suggest that an increase in TGF-b levels seen during disc degeneration would be expected to preserve ECM levels by controlling expression and activity of MMP3. Taken together, our results clearly suggest that, in NP cells, TNF-a and IL-1b promote the expression of MMP-3 through MAPK and NF-kB pathways. Moreover, cytokine TNF-ae and IL-1bedependent MMP-3 expression requires SDC4. Modulation of these signaling pathways using small molecular inhibitors may provide a new approach to treat intervertebral disc degeneration and mitigate the catabolic action of MMP-3.

Acknowledgments We thank Wen-Ling Shih (Tzu Chi University, Hualien City, Taiwan) for providing MMP3-LUC; Jiahui Han (Scripps Institute, La Jolla, CA) for providing p38aAF, p38bAF, p38dAF, and p38gAF; Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX) for providing ERK-1K71R and ERK-2K52R; Dr. Silvio Gutkind (NIH, Bethesda, MD) for providing AP-1; and Dr. Andree Yeremian (University of Lleida, Lleida, Spain) for providing FSVsi vector. X.W., H.W., H.Y., and J.L. performed experiments and analyzed the data. M.V.R., I.M.S., and Q.C. designed experiments and analyzed the data. M.V.R. and I.M.S. secured the funding. All authors were involved in writing of the paper and approval of the submitted manuscript.

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