Inflammatory Cytokines Associated with Degenerative Disc Disease Control Aggrecanase-1 (ADAMTS-4) Expression in Nucleus Pulposus Cells through MAPK and NF-κB
2013; Elsevier BV; Volume: 182; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2013.02.037
ISSN1525-2191
AutoresYe Tian, Wen Yuan, Nobuyuki Fujita, Jianru Wang, Hua Wang, Irving M. Shapiro, Makarand V. Risbud,
Tópico(s)Anesthesia and Pain Management
ResumoWe investigated TNF-α and IL-1β regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-κB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-α and IL-1β treatment. Treatment of cells with MAPK and NF-κB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38α, p38β2, and p38γ were involved in induction, ERK2 and p38δ played no role in TNF-α–dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-α, and shIKK-β significantly decreased TNF-α–dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-α–dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-κB signaling, TNF-α and IL-1β modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro. We investigated TNF-α and IL-1β regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-κB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-α and IL-1β treatment. Treatment of cells with MAPK and NF-κB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38α, p38β2, and p38γ were involved in induction, ERK2 and p38δ played no role in TNF-α–dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-α, and shIKK-β significantly decreased TNF-α–dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-α–dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-κB signaling, TNF-α and IL-1β modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro. The intervertebral disk is a unique tissue that that permits rotation, as well as flexion and extension of the spine. It consists of a gel-like nucleus pulposus (NP) surrounded circumferentially by a fibrocartilagenous annulus fibrosus. Cells of the NP are derived from the notochord,1Stemple D.L. Structure and function of the notochord: an essential organ for chordate development.Development. 2005; 132: 2503-2512Crossref PubMed Scopus (334) Google Scholar an embryonic tissue with limited blood supply. In common with chondrocytes, NP cells secrete a complex extracellular matrix that contains fibrillar collagens and the proteoglycan aggrecan. Assembly of these macromolecules provides a robust hydrodynamic system that accommodates applied biomechanical forces to the spine.2Feng H. Danfelter M. Strömqvist B. Heinegård D. Extracellular matrix in disc degeneration.J Bone Joint Surg Am. 2006; 88: 25-29Crossref PubMed Scopus (145) Google Scholar, 3Setton L.A. Chen J. Mechanobiology of the intervertebral disc and relevance to disc degeneration.J Bone Joint Surg Am. 2006; 88: 52-57Crossref PubMed Scopus (139) Google Scholar, 4Ng L. Grodzinsky A.J. Patwari P. Sandy J. Plaas A. Ortiz C. Individual cartilage aggrecan macromolecules and their constituent glycosaminoglycans visualized via atomic force microscopy.J Struct Biol. 2003; 143: 242-257Crossref PubMed Scopus (179) Google Scholar Intervertebral disk degeneration is characterized by increased expression of catabolic enzymes, decreased proteoglycan synthesis, and an overall shift toward synthesis of a fibrotic matrix. When this occurs, the water-binding capacity of the tissue is compromised, resulting in a failure to resist compressive forces and a reduction in disk height.5Urban J.P. Roberts S. Degeneration of the intervertebral disc.Arthritis Res Ther. 2003; 5: 120-130Crossref PubMed Google Scholar, 6Roberts S. Evans H. Trivedi J. Menage J. Histology and pathology of the human intervertebral disc.J Bone Joint Surg Am. 2006; 88: 10-14Crossref PubMed Scopus (336) Google Scholar Although a great deal is known about importance of proteoglycan secretion and function, the molecular mechanisms controlling aggrecan turnover in cells of the normal and the degenerated disk are not well understood. It has been reported that during disk degeneration and herniation, in addition to infiltrating immune cells, resident NP and annulus fibrosus cells produce high levels of the cytokines TNF-α and IL-1β.7Le Maitre C.L. Freemont A.J. Hoyland J.A. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration.Arthritis Res Ther. 2005; 7: R732-R745Crossref PubMed Google Scholar, 8Lee S. Moon C.S. Sul D. Lee J. Bae M. Hong Y. Lee M. Choi S. Derby R. Kim B.J. Kim J. Yoon J.S. Wolfer L. Kim J. Wang J. Hwang S.W. Lee S.H. Comparison of growth factor and cytokine expression in patients with degenerated disc disease and herniated nucleus pulposus.Clin Biochem. 2009; 42: 1504-1511Crossref PubMed Scopus (114) Google Scholar These cytokines stimulate production of NGF, BDNF, and VEGF, molecules associated with nerve ingrowth and angiogenesis by NP cells.9Lee J.M. Song J.Y. Baek M. Jung H.Y. Kang H. Han I.B. Kwon Y.D. Shin D.E. Interleukin-1β induces angiogenesis and innervation in human intervertebral disc degeneration.J Orthop Res. 2011; 29: 265-269Crossref PubMed Scopus (167) Google Scholar Moreover, both cytokines up-regulate expression by NP cells of catabolic matrix metalloproteinases (MMPs)3Setton L.A. Chen J. Mechanobiology of the intervertebral disc and relevance to disc degeneration.J Bone Joint Surg Am. 2006; 88: 52-57Crossref PubMed Scopus (139) Google Scholar and two major aggrecanases, A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS-4) and 5 (ADAMTS-5).7Le Maitre C.L. Freemont A.J. Hoyland J.A. The role of interleukin-1 in the pathogenesis of human intervertebral disc degeneration.Arthritis Res Ther. 2005; 7: R732-R745Crossref PubMed Google Scholar, 10Séguin C.A. Pilliar R.M. Roughley P.J. Kandel R.A. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue.Spine (Phila Pa 1976). 2005; 30: 1940-1948Crossref PubMed Scopus (242) Google Scholar, 11Séguin C.A. Bojarski M. Pilliar R.M. Roughley P.J. Kandel R.A. Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration.Matrix Biol. 2006; 25: 409-418Crossref PubMed Scopus (112) Google Scholar, 12Millward-Sadler S.J. Costello P.W. Freemont A.J. Hoyland J.A. Regulation of catabolic gene expression in normal and degenerate human intervertebral disc cells: implications for the pathogenesis of intervertebral disc degeneration.Arthritis Res Ther. 2009; 11: R65Crossref PubMed Scopus (147) Google Scholar Among several members of the ADAMTS family that cleave aggrecan in vitro, ADAMTS-4 (aggrecanase-1) and ADAMTS-5 (aggrecanase-2) are the most likely to play a role in aggrecan degradation and subsequent disk degeneration as in the pathogenesis of osteoarthritis.13Tortorella M.D. Liu R.Q. Burn T. Newton R.C. Arner E. Characterization of human aggrecanase 2 (ADAM-TS5) substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4).Matrix Biol. 2002; 21: 499-511Crossref PubMed Scopus (122) Google Scholar, 14Malfait A.M. Liu R.Q. Ijiri K. Komiya S. Tortorella M.D. Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage.J Biol Chem. 2002; 277: 22201-22208Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar ADAMTS-4 and -5 produce fragments of aggrecan usually found in synovial fluid and cartilage by cleaving the protein following Glu373, Glu1545, Glu1714, Glu1819, and Glu1919.13Tortorella M.D. Liu R.Q. Burn T. Newton R.C. Arner E. Characterization of human aggrecanase 2 (ADAM-TS5) substrate specificity studies and comparison with aggrecanase 1 (ADAM-TS4).Matrix Biol. 2002; 21: 499-511Crossref PubMed Scopus (122) Google Scholar, 15Tortorella M.D. Pratta M. Liu R.Q. Austin J. Ross O.H. Abbaszade I. Burn T. Arner E. Sites of aggrecan cleavage by recombinant human aggrecanase1 (ADAMTS-4).J Biol Chem. 2000; 275: 18566-18573Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 16Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman Jr., M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Purification and cloning of aggrecanase-1: a member of the ADAMTS family of proteins.Science. 1999; 284: 1664-1666Crossref PubMed Scopus (631) Google Scholar Unlike cartilage, in the NP both ADAMTS-4 and ADAMTS-5 expression is elevated in human degenerative disk disease.17Pockert A.J. Richardson S.M. Le Maitre C.L. Lyon M. Deakin J.A. Buttle D.J. Freemont A.J. Hoyland J.A. Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration.Arthritis Rheum. 2009; 60: 482-491Crossref PubMed Scopus (222) Google Scholar, 18Mwale F. Masuda K. Pichika R. Epure L.M. Yoshikawa T. Hemmad A. Roughley P.J. Antoniou J. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration.Arthritis Res Ther. 2011; 13: R120Crossref PubMed Scopus (71) Google Scholar, 19Wang J. Markova D. Anderson D.G. Zheng Z. Shapiro I.M. Risbud M.V. TNF-α and IL-1β promote a disintegrin-like and metalloprotease with thrombospondin type I motif-5-mediated aggrecan degradation through syndecan-4 in intervertebral disc.J Biol Chem. 2011; 286: 39738-39749Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar Surprisingly, despite the importance of these aggrecanases in the pathogenesis of osteoarthritis and disk disease, only a few studies have investigated regulation of ADAMTS transcription in NP cells,10Séguin C.A. Pilliar R.M. Roughley P.J. Kandel R.A. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue.Spine (Phila Pa 1976). 2005; 30: 1940-1948Crossref PubMed Scopus (242) Google Scholar, 11Séguin C.A. Bojarski M. Pilliar R.M. Roughley P.J. Kandel R.A. Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration.Matrix Biol. 2006; 25: 409-418Crossref PubMed Scopus (112) Google Scholar, 17Pockert A.J. Richardson S.M. Le Maitre C.L. Lyon M. Deakin J.A. Buttle D.J. Freemont A.J. Hoyland J.A. Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration.Arthritis Rheum. 2009; 60: 482-491Crossref PubMed Scopus (222) Google Scholar, 18Mwale F. Masuda K. Pichika R. Epure L.M. Yoshikawa T. Hemmad A. Roughley P.J. Antoniou J. The efficacy of Link N as a mediator of repair in a rabbit model of intervertebral disc degeneration.Arthritis Res Ther. 2011; 13: R120Crossref PubMed Scopus (71) Google Scholar and none have used promoter analysis. A clue to the mechanism lies in the findings that, in NP cells, NF-κB may contribute to TNF-α regulation of ADAMTS-4 and ADAMTS-5 expression,11Séguin C.A. Bojarski M. Pilliar R.M. Roughley P.J. Kandel R.A. Differential regulation of matrix degrading enzymes in a TNFalpha-induced model of nucleus pulposus tissue degeneration.Matrix Biol. 2006; 25: 409-418Crossref PubMed Scopus (112) Google Scholar and that TNF-α and IL-1β also modulate ADAMTS-5 enzymatic activity through syndecan-4.19Wang J. Markova D. Anderson D.G. Zheng Z. Shapiro I.M. Risbud M.V. TNF-α and IL-1β promote a disintegrin-like and metalloprotease with thrombospondin type I motif-5-mediated aggrecan degradation through syndecan-4 in intervertebral disc.J Biol Chem. 2011; 286: 39738-39749Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar These observations beg the question of how TNF-α and IL-1β control the expression of ADAMTS-4 and-5 and what is their relative contribution in NP cells in terms of aggrecan degradation. Although the present study addressed both ADAMTS-4 and -5, the mechanistic aspects of transcriptional control were studied using a 3.5-kb human ADAMTS4 promoter fragment. Here, we show for the first time that TNF-α and IL-1β control ADAMTS4 transcription in MAPK- and NF-κB–dependent fashion. Importantly, our results show that ADAMTS-4 and ADAMTS-5 are nonredundant and that both play a role in the cytokine-dependent degradation of aggrecan in human NP cells. A therapeutic strategy could conceivably target these enzymes for the structural preservation of the intervertebral disk. The 3.5-kb (−3109 to +406 bp) human ADAMTS4 promoter in pβ-gal-Basic vector was a kind gift from Dr. K. Thirunavukkarasu (Lilly Research Labs, Indianapolis, IN).20Thirunavukkarasu K. Pei Y. Moore T.L. Wang H. Yu X.P. Geiser A.G. Chandrasekhar S. Regulation of the human ADAMTS-4 promoter by transcription factors and cytokines.Biochem Biophys Res Commun. 2006; 345: 197-204Crossref PubMed Scopus (53) Google Scholar The insert was recloned in basic pGL3 using XhoI and HindIII digestion. pCMX-IκBM (catalog no. 12330), and RelA/p65 (catalog no. 20012), p50 (catalog no. 20018) developed by Dr. Inder Verma and psPAX2 (catalog no. 12260) and pMD2G (catalog no. 12259) developed by Dr. Didier Trono were obtained from the Addgene repository (Cambridge, MA). Plasmids DN-p38α, DN-p38β2, DN-p38γ, and DN-p38δ were kindly provided by Jiahui Han (Scripps Research Institute, La Jolla, CA); plasmids ERK-1K71R and ERK-2K52R, by Melanie Cobb (University of Texas Southwestern Medical Center, Dallas, TX); plasmids pLKO.1shADAMTS-4 and pLKO.1shADAMTS-5, by Dr. Mike Baker (University of Sheffield, Sheffield, UK); and plasmids shp65, shp52, shIKK-α, and shIKK-β in lentiviral FSVsi vector that coexpresses yellow fluorescent protein (YFP), by Dr. Andree Yeremian (University of Lleida, Lleida, Spain). The vector pRL-TK (Promega, Madison, WI) containing the Renilla luciferase gene was used as an internal transfection control. The amount of transfected plasmid, the pretransfection period after seeding, and the post-transfection period before harvesting were optimized for NP cells with pSV β-galactosidase plasmid (Promega).21Risbud M.V. Guttapalli A. Stokes D.G. Hawkins D. Danielson K.G. Schaer T.P. Albert T.J. Shapiro I.M. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment.J Cell Biochem. 2006; 98: 152-159Crossref PubMed Scopus (218) Google Scholar Wild-type and p65 null cells were a kind gift from Dr. Denis Guttridge (Ohio State University, Columbus, OH). Antibody that recognizes ADAMTS-dependent aggrecan degradation in interglobular domain (anti-NITEGE) was a gift from Dr. Peter Roughley (Shriners Hospital for Children, Montreal, QC, Canada). Antibodies against ADAMTS-4, -5, and ADAMTS-generated aggrecan neoepitope ARGSVIL were obtained from Abcam (Cambridge, MA). P-p38, p38, p52, P-p65, p65, IKK-α, IKK-β, P-ERK, ERK, P-JNK, and JNK antibodies were obtained from Cell Signaling Technology (Danvers, MA). β-Tubulin was obtained from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IA) and GAPDH from Novus Biologicals (Littleton, CO). TNF-α and IL-1β were purchased from PeproTech (Rocky Hill, NJ). Rat and human NP cells were isolated using a method reported by Risbud et al.21Risbud M.V. Guttapalli A. Stokes D.G. Hawkins D. Danielson K.G. Schaer T.P. Albert T.J. Shapiro I.M. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment.J Cell Biochem. 2006; 98: 152-159Crossref PubMed Scopus (218) Google Scholar NP tissue from lumbar disks of three or four rats was pooled for each isolation. NP cells were maintained in Dulbecco's modified Eagle's medium (DMEM) and 10% fetal bovine serum (FBS) supplemented with antibiotics and used within the first three passages. To investigate the effect of cytokines, cells were treated with 5 to 20 ng/mL IL-1β and 25 to 100 ng/mL TNF-α for 24 hours in serum-free medium. Both lumbar and cervical disk tissues were collected as surgical waste from individuals undergoing elective spinal surgical procedures. Consistent with Thomas Jefferson University's Institutional Review Board guidelines, informed consent for sample collection was obtained from each patient. Assessment of the disease state was performed using Pfirrmann grading.22Pfirrmann C.W. Metzdorf A. Zanetti M. Hodler J. Boos N. Magnetic resonance classification of lumbar intervertebral disc degeneration.Spine (Phila Pa 1976). 2001; 26: 1873-1878Crossref PubMed Scopus (2864) Google Scholar This scheme uses T2-weighted magnetic resonance imaging with image analysis by three independent observers. Patient age, spinal level, and grade of NP tissues used for cell isolation are listed in Supplemental Table S1. After treatment, total RNA was extracted from NP cells (5 × 105 cells per plate) using RNeasy mini spin columns (Qiagen, Valencia, CA). Before elution from the column, RNA was treated with RNase-free DNase I. Two micrograms of total DNA-free RNA was used to synthesize cDNA, using a SuperScipt III cDNA synthesis kit (Life Technologies–Invitrogen, Carlsbad, CA). Reactions were set up in triplicate in 96-well plates using 1 μL cDNA with Fast SYBR Green PCR Master Mix (Life Technologies–Applied Biosystems, Foster City, CA) to which gene-specific forward and reverse PCR primers were added. Each set of samples included a template-free control. PCR reactions were performed in a StepOnePlus real-time PCR system (Life Technologies–Applied Biosystems) according to the manufacturer's instructions. Expression of the gene of interest was first normalized to the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (Hprt1), with data expressed as relative to the corresponding control group. All of the primers were synthesized by Integrated DNA Technologies (Coralville, IA) (Table 1).Table 1Sequences of Primers Used in RT-qPCRTargetPrimer sequenceHPRT1 Forward5′-AGTCCCAGCGTCGTGATTAGTGAT-3′ Reverse5′-GAGCAAGTCTTTCAGTCCTGTCCA-3′ADAMTS4 Forward5′-ACAATGGCTATGGACACTGCCTCT-3′ Reverse5′-TGTGGACAATGGCTTGAGTCAGGA-3′ADAMTS5 Forward5′-GTCCAAATGCACTTCAGCCACGAT-3′ Reverse5′-AATGTCAAGTTGCACTGCTGGGTG-3′ Open table in a new tab NP cells (1 × 106 cells per plate) were placed on ice immediately after treatment and washed with ice-cold Hanks' balanced salt solution. All of the wash buffers and the final resuspension buffer included 1× protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN), 5 mmol/L NaF, and 200 μmol/L Na3VO4. Conditioned medium was collected and concentrated using centrifugal filter units (EMD Millipore, Billerica, MA). For detecting aggrecan neoepitopes, protein lysates were pretreated with 0.1 U/mL chondroitinase ABC (Sigma-Aldrich, St. Louis, MO) for 1 to 6 hours at 37°C. Proteins were resolved on 8% to 12% SDS-PAGE gels and were transferred by electroblotting to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Hercules, CA). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline–Tween (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 saline–Tween with the specific antibodies all at a dilution of 1:1000. Immunolabeling was detected using Amersham ECL reagent (GE Healthcare, Little Chalfont, UK). NP cells were transferred to 48-well plates at a density of 2 × 104 cells per well, at 1 day before transfection. To investigate the effect of NF-κB on ADAMTS4 promoter activity, cells were cotransfected with 50 to 200 ng of p65, p50, or both p65 and p50 with or without appropriate backbone vector and 175 ng ADAMTS4 reporter and 175 ng pRL-TK plasmid. To investigate the effects of p38 and ERK signaling, cells were transfected with 50 to 150 ng of dominant-negative p38 (DN-p38) or DN-ERK plasmids. In some wells, cells were treated with the inhibitors for NF-κB (10 μmol/L sm-7368), p38 (10 μmol/L SB203580), ERK (10 μmol/L PD98059), or JNK (10 μmol/L SP600125) (all Calbiochem, from EMD Millipore). In some experiments, cells were transfected with 250 ng of ADAMTS4 reporter plasmids with 250 ng pRL-TK plasmid. Lipofectamine 2000 (Life Technologies–Invitrogen) was used as a transfection reagent. For each transfection, plasmids were premixed with the transfection reagent. At 48 hours after transfection, the 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. HEK 293T human embryonic kidney cells (1.3 × 106 cells per plate) were seeded in 10-cm plates in DMEM with 10% heat-inactivated FBS, at 2 days before transfection. Cells were transfected with 2.5 μg of shRNA control sequence or gene-specific shRNA plasmids, along with 1.875 μg psPAX2 (a packaging vector) and 0.625 μg pMD2.G (an envelope vector). After 16 hours, the transfection medium was removed and replaced with DMEM with 5% heat-inactivated FBS and penicillin–streptomycin. Lentiviral particles were harvested at 48 and 60 hours after transfection. Human NP cells (1 × 106 cells per plate) were plated in DMEM with 5% heat-inactivated FBS, at 1 day before transduction. Cells in 10-cm plates were transduced with 5 mL of medium containing viral particles, along with 6 μg/mL polybrene. After 24 hours, the medium was removed and replaced with DMEM with 5% heat-inactivated FBS. Cells were harvested for protein extraction at 5 days after viral transduction. All experiments were repeated independently three times. Data are presented as means ± SEM. Differences between groups were analyzed by Student's t-test and analysis of variance. P < 0.05 was considered significant. Expression of ADAMTS-4 in mature rat tissues was studied using real-time PCR and Western blot analysis. Compared with Hprt1, the basal expression of ADAMTS4 mRNA in healthy NP and in annulus fibrosus tissue is very low (Figure 1, A and B). NP tissue shows weak ADAMTS-4 bands at approximately 58 and 73 kDa (Figure 1B). To explore the premise that cytokines concerned with disk degeneration regulate ADAMTS-4 expression, rat NP cells were treated with TNF-α and IL-1β, and expression of ADAMTS-4 was analyzed. Treatment with both TNF-α and IL-1β resulted in dose-dependent increase in ADAMTS4 mRNA levels (Figure 1, C and D). In addition, we measured the level of ADAMTS-4 protein in conditioned medium of treated NP cells by Western blot analysis. Cytokine treatment significantly increased ADAMTS-4 protein expression in both rat NP cells (Figure 1, E and F) and human NP cells (Supplemental Figure S1A). To investigate whether the regulation of expression is at the transcriptional level, we measured the activity of a 3.5-kb ADAMTS4 promoter (Figure 1G) after cytokine treatment. Both cytokines significantly increased the promoter activity (Figure 1H). To determine whether MAPK and/or NF-κB signaling is required for the cytokine-dependent induction of ADAMTS-4 in rat NP cells, we first evaluated activation of these signaling pathways after treatment with TNF-α and IL-1β. After treatment with TNF-α (Figure 2A) or IL-1β (Figure 2B), there was a rapid increase in P-p65 protein levels. Activation was maximal at 5 to 30 minutes and then declined rapidly. As expected, there was no appreciable change in the level of total p65 during the treatment period. We also examined levels of the phosphorylated MAPK isoforms P-p38, P-ERK1/2, and P-JNK. Again, there was a rapid increase in all three isoforms, among which ERK exhibited more sustained levels of phosphorylation. To ascertain whether the cytokine-induced expression of ADAMTS-4 and -5 requires NF-κB and/or MAPK signaling, rat NP cells were pretreated with pathway-specific inhibitors. Pretreatment caused a significant suppression in TNF-α and IL-1β induction of both ADAMTS4 and ADAMTS5 mRNA levels (Figure 2, C–F). Similarly, a pronounced decrease in cytokine-mediated increase in levels of ADAMTS-4 protein (58 and 73 kDa) was seen in the presence of MAPK and NF-κB pathway inhibitors (Figure 2, G and H). To investigate the mechanism of MAPK regulation of ADAMTS-4 expression, we transfected rat NP cells with dominant-negative (DN) DN-p38α, DN-p38β2, DN-p38γ, DN-p38δ, or DN-ERK1 or DN-ERK2 expression plasmids and measured ADAMTS4 promoter activity. DN-p38δ (Supplemental Figure S1B) and DN-ERK2 (Supplemental Figure S1, C and D) did not suppress promoter activity. In contrast, cytokine-dependent induction in ADAMTS4 promoter activity was significantly suppressed by cotransfection with DN-P38α (Figure 3, A and B), p38β2 (Figure 3C), p38γ (Figure 3D), and DN-ERK1 (Figure 3, E and F). Because CCAAT enhancer-binding protein β (C/EBP-β; alias LAP2) has been shown to control IL-1β– and TNF-α–dependent transcription in chondrocytes,23Zhang Z. Bryan J.L. DeLassus E. Chang L.W. Liao W. Sandell L.J. CCAAT/Enhancer-binding protein beta and NF-kappaB mediate high level expression of chemokine genes CCL3 and CCL4 by human chondrocytes in response to IL-1beta.J Biol Chem. 2010; 285: 33092-33103Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar we investigated whether a similar regulatory system exists 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 activity. Surprisingly, suppression of C/EBP-β function resulted in further induction of the promoter activity by TNF-α (Figure 3G). On the other hand, cotransfection with LAP2 resulted in suppression of the basal promoter activity (Figure 3H). To investigate the role of NF-κB in the transcriptional regulation of ADAMTS4, we first used JASPAR database analysis (performed January 2012)24Wasserman W.W. Sandelin A. Applied bioinformatics for the identification of regulatory elements.Nat Rev Genet. 2004; 5: 276-287Crossref PubMed Scopus (878) Google Scholar (http://jaspar.genereg.net) for evidence of putative NF-κB binding motifs in the promoter. Sequence analysis revealed four putative binding sites. JASPAR analysis provides a quantitative score for each potential binding site, based on the probability of observing each nucleotide at each position of the binding motif compared to the consensus sequences of known binding sites. The raw score is normalized to a range of 0-1 to provide a relative score.24Wasserman W.W. Sandelin A. Applied bioinformatics for the identification of regulatory elements.Nat Rev Genet. 2004; 5: 276-287Crossref PubMed Scopus (878) Google Scholar The sequence, the location in the promoter relative to transcription start site, and relative score was as follows: GGCAAGTCCC, −159 relative to −150 bp, score 0.90; GGGGATTCTC, −1042 relative to −1033 bp, score 0.88; GGGATTCTCC, −1043 relative to −1034 bp, score 0.88; and GGGGATTTCC, −1394 relative to −1385 bp, score 0.98. We then examined the effect of overexpression of NF-κB subunits on ADAMTS4 promoter activity in rat NP cells. Cotransfection with p65 resulted in a dose-dependent increase in ADAMTS4 promoter activity (Figure 4A). On the other hand, neither the RelB nor the c-Rel subunit influenced ADAMTS4 promoter activity (Figure 4B). Although p50 alone had no effect on ADAMTS4 promoter activity, it blocked the inductive effect of p65 even at a low dose (Figure 4, C and D). Notably, p50 completely suppressed the inductive effect of both cytokines on the ADAMTS4 promoter (Figure 4, E and F). To confirm that ADAMTS4 promoter activity is responsive to NF-κB signaling, we performed loss-of-function studies. When cells were treated with the NF-κB inhibitor SM7368 (Figure 4G) or cotransfected with DN-NF-κB/IκBαM (Figure 4H), cytokine-mediated induction in ADAMTS4 promoter activity was completely abolished. Specificity of the NF-κB inhibitors SM7368 and IκBαM was validated by measuring the activity of a well characterized NF-κB responsive reporter (Supplemental Figure S2A). To further validate the role of p65/RelA and to determine whether there is cell type specificity, we measured ADAMTS4 promoter activity in RelA null and wild-type mouse embryonic fibroblasts. Only in wild-type cells was the promoter activity cytokine inducible (Supplemental Figure S2, B and C). Given that IL-1β and TNF-α regulated ADAMTS-4 expression using similar signaling pathways, we performed lentiviral-mediated gene silencing studies using TNF-α as a representative cytokine. We first silen
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