Key points are not available for this paper at this time.
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. 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. 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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. 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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. 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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. K. R. T. A. Roughley P.J. J. The of as a of in a model of intervertebral disc degeneration.Arthritis Res Ther. 2011; PubMed Scopus Google Scholar, J. D. TNF-α and IL-1β a and with thrombospondin aggrecan degradation through in intervertebral disc.J Biol Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar the importance of these in the pathogenesis of and disk a studies investigated regulation of ADAMTS in NP 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, 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. K. R. T. A. Roughley P.J. J. The of as a of in a model of intervertebral disc degeneration.Arthritis Res Ther. 2011; PubMed Scopus Google Scholar and promoter A to the in the in NP cells, NF-κB to TNF-α regulation of ADAMTS-4 and ADAMTS-5 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 modulate ADAMTS-5 activity through J. D. TNF-α and IL-1β a and with thrombospondin aggrecan degradation through in intervertebral disc.J Biol Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar These the of TNF-α and IL-1β the expression of ADAMTS-4 and is their contribution in NP cells in of aggrecan degradation. Although the study both ADAMTS-4 and the of were a human ADAMTS4 promoter show for the first that TNF-α and IL-1β ADAMTS4 in and our show that ADAMTS-4 and ADAMTS-5 are nonredundant and that both play a role in the degradation of aggrecan in human NP cells. A these enzymes for the of the intervertebral The to human ADAMTS4 promoter in was a from K. K. Y. Wang H. S. Regulation of the human ADAMTS-4 promoter by and Res 2006; PubMed Scopus Google Scholar The was in and and p50 by and and by were from the and were by Han plasmids and by of plasmids and by of and plasmids shp65, shp52, shIKK-α, and shIKK-β in lentiviral that protein by of The the gene was as an The of the after and the were for NP cells with A. D. pulposus cells a to the intervertebral disc Biochem. 2006; PubMed Scopus Google Scholar and p65 cells were a from that aggrecan degradation in was a from Roughley for and aggrecan were from and were from was from the of and from TNF-α and IL-1β were from and human NP cells were a reported by A. D. pulposus cells a to the intervertebral disc Biochem. 2006; PubMed Scopus Google Scholar NP tissue from of or was for NP cells were in and with and the first To the effect of cells were with 5 to IL-1β and to TNF-α for in and disk were as from with for was from of the disease was performed A. M. J. of intervertebral disc (Phila Pa 1976). PubMed Scopus Google Scholar with by and of NP for are in was from NP cells cells from the was with I. of was to a synthesis were in in 1 with to and were of a were performed in a system to the of the gene of was first to the gene 1 with as to the of the were by of in in a NP cells cells were on after and with of the and the inhibitor 5 and was and aggrecan protein were with for 1 to were on to and were by to The were blocked with in and in in with the a of was NP cells were to a of 2 cells 1 To the effect of NF-κB on ADAMTS4 promoter cells were with to of or both p65 and p50 with or and ADAMTS4 and To the of and signaling, cells were with to of or In cells were with the inhibitors for NF-κB or from In cells were with of ADAMTS4 plasmids with was as a plasmids were with the after the cells were and a system was for of and of and of were performed a transfections were and were performed in human embryonic cells cells were in in with 2 Cells were with of shRNA or shRNA with and the was and with with and Lentiviral were and after NP cells cells were in with 1 Cells in were with 5 of with the was and with with Cells were for protein 5 after were are as were by and of was of ADAMTS-4 in rat was and Western with the expression of ADAMTS4 mRNA in NP and in annulus fibrosus tissue is A and NP tissue ADAMTS-4 and To the that cytokines with disk degeneration ADAMTS-4 rat NP cells were with TNF-α and and expression of ADAMTS-4 was Treatment with both TNF-α and IL-1β resulted in increase in ADAMTS4 mRNA levels and In the of ADAMTS-4 protein in of NP cells by Western significantly increased ADAMTS-4 protein expression in both rat NP cells and and human NP cells To the regulation of expression is the the activity of a ADAMTS4 promoter after cytokine treatment. cytokines significantly increased the promoter activity To determine MAPK NF-κB is for the of ADAMTS-4 in rat NP cells, first activation of these after with TNF-α and with TNF-α or IL-1β was a increase in protein was 5 to and was no in the of p65 during the We levels of the MAPK and was a increase in levels of To the expression of ADAMTS-4 and -5 NF-κB MAPK signaling, rat NP cells were with a in TNF-α and IL-1β of both ADAMTS4 and mRNA levels a in cytokine-mediated increase in levels of ADAMTS-4 protein and was in the of MAPK and NF-κB inhibitors and To the of MAPK regulation of ADAMTS-4 rat NP cells with or or expression plasmids and ADAMTS4 promoter activity. and and not promoter activity. In in ADAMTS4 promoter activity was significantly by with A and p38γ and and protein has been to and TNF-α–dependent in J.L. E. protein and high expression of and by human in to Biol Chem. Full Text Full Text PDF PubMed Scopus Google Scholar investigated a system in cells of the We rat NP cells with protein a and ADAMTS4 promoter activity. of function resulted in of the promoter activity by TNF-α the with resulted in of the promoter activity To the role of NF-κB in the regulation of first A. for the of 5: PubMed Scopus Google Scholar for of NF-κB motifs in the provides a quantitative for on the of of the to the of known The is to a of to a A. for the of 5: PubMed Scopus Google Scholar The the in the promoter to and was as to to to and to We the effect of of NF-κB on ADAMTS4 promoter activity in rat NP cells. Cotransfection with p65 resulted in a increase in ADAMTS4 promoter activity the the the ADAMTS4 promoter activity Although p50 no effect on ADAMTS4 promoter blocked the inductive effect of p65 a and p50 completely the inductive effect of both cytokines on the ADAMTS4 promoter and To that ADAMTS4 promoter activity is to NF-κB signaling, performed loss-of-function studies. When cells were with the NF-κB inhibitor or with cytokine-mediated in ADAMTS4 promoter activity was completely of the NF-κB inhibitors and was by the activity of a well characterized NF-κB To the role of and to determine is ADAMTS4 promoter activity in and embryonic in cells was the promoter activity cytokine and that IL-1β and TNF-α ADAMTS-4 expression performed gene silencing studies TNF-α as a We first the expression of NF-κB and ADAMTS-4 expression in human NP cells. was robust expression by the cells high levels of transduction and expression. In cells with plasmids and shp52, was a in the protein levels of p65 and with cells with shRNA transduction of human NP cells with plasmids and shIKK-β resulted in decreased levels of and of NF-κB significantly blocked the inductive effect of TNF-α on the expression of ADAMTS-4 and -5 protein as well as aggrecan degradation as by We the effect of silencing of ADAMTS-4 and ADAMTS-5 expression on aggrecan degradation in human NP cells. ADAMTS protein levels in the NP cells or the were significantly was no increase in either ADAMTS-4 or -5 the ADAMTS was We and to aggrecan degradation in ADAMTS cells. of ADAMTS-4 and -5 expression resulted in a in aggrecan degradation in NP cells and these and cells with shRNA an increase in aggrecan after TNF-α and The of matrix aggrecan degradation was In the protein with cells, silencing of ADAMTS-4 and ADAMTS-5 blocked aggrecan degradation after TNF-α treatment. The for the first that expression of an with aggrecan is by the cytokines TNF-α and IL-1β through the MAPK and NF-κB in NP cells. A major is by ADAMTS-4 and -5 expression and these cytokines aggrecan both ADAMTS-4 and -5 were for the aggrecan degradation in human NP cells, and their function to a role in intervertebral disk to our gene and protein expression ADAMTS-4 is in of the intervertebral The on Western that a of the is in the of expression in the rat the of matrix turnover in the Although ADAMTS-4 is in of aggrecan is involved with a of Moreover, the of regulation of expression is understood. been reported on the expression of ADAMTS-4 in and studies show that ADAMTS-4 is by TNF-α and M.D. A.M. C. Arner E. The role of ADAM-TS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage in Full Text PDF PubMed Scopus Google Scholar, E. in with necrosis factor induces a aggrecanase that is from ADAMTS or 2005; PubMed Scopus Google Scholar, T. S. M. M. A. T. Y. H. Y. Y. expression of ADAMTS4 (aggrecanase-1) in human osteoarthritic 2009; PubMed Scopus Google Scholar show that cytokine not ADAMTS-4 P.J. S. A. The of matrix metalloproteinase and gene expression in human by interleukin-1 and a study quantitative Rheum. 2002; PubMed Scopus Google Scholar, Y. D.L. M. R.A. Tortorella M.D. Arner E.C. and regulation of aggrecanase in the role of 2002; PubMed Scopus Google Scholar In the of NP cells with TNF-α and IL-1β ADAMTS-4 expression. Moreover, our promoter studies that regulation was the These are with that the cytokines ADAMTS4 mRNA expression in the 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, J. D. TNF-α and IL-1β a and with thrombospondin aggrecan degradation through in intervertebral disc.J Biol Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, T. K. H. Y. Y. Y. in expression of tissue inhibitor of associated with from the nucleus pulposus to the nucleus pulposus in intervertebral (Phila Pa 1976). PubMed Scopus Google Scholar Although the is is to that TNF-α–dependent ADAMTS-4 expression and aggrecanase activity in the NP by and NF-κB 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 We confirmed that both cytokines MAPK and activation and that these ADAMTS-4 expression. regulation were from gain and loss-of-function studies that ADAMTS4 promoter activity. specificity for both MAPK Although p38α, p38β2, and ADAMTS4 promoter p38δ and ERK2 were not The of NF-κB motifs in the ADAMTS4 promoter of this factor in controlling Y. K. Y. K. I. Characterization of of human (ADAMTS-4) Biol 2000; PubMed Scopus Google Scholar that the and is for activity of the human ADAMTS4 the NF-κB with the is this In for the role of NF-κB in ADAMTS4 promoter our studies that the inductive effect is to p65 and and that and not play a p50 the inductive effect of p65 on ADAMTS4 The that the and of ADAMTS-4 expression is by p50 is with of function of p50 in controlling expression of a of and A.M. F. F. J. The complex of Full Text Full Text PDF PubMed Scopus Google Scholar, C. C. C. R.M. R.A. F. of a role for in 2009; PubMed Scopus Google Scholar, F. J. S. D.E. C. S. S.J. H. J.P. the and to 2005; Full Text Full Text PDF PubMed Scopus Google Scholar as well as the from our that p50 as a of J. Y. R.A. Le Maitre C.L. Tumor necrosis factor and of expression by nucleus pulposus cells through Rheum. PubMed Scopus Google Scholar the our has that p65 and p50 to the expression of the gene of the of TNF-α and IL-1β in NP J. D. TNF-α and IL-1β a and with thrombospondin aggrecan degradation through in intervertebral disc.J Biol Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar These the importance of both and specificity in the of NP cells. It is not to that of p50 and their to the motifs in of as to the ADAMTS4 A.M. F. F. J. The complex of Full Text Full Text PDF PubMed Scopus Google Scholar A to the and of this The that cells to ADAMTS4 promoter with provides of the importance of NF-κB in promoter Moreover, the silencing studies that of TNF-α–dependent ADAMTS-4 and -5 expression after of several NF-κB the importance of this in controlling ADAMTS-4 expression. the of these studies by controlling the activity of both MAPK and signaling, cytokines the expression of ADAMTS-4 in NP cells. to this of of ADAMTS4 K. Y. Wang H. S. Regulation of the human ADAMTS-4 promoter by and Res 2006; PubMed Scopus Google Scholar that and ADAMTS4 promoter through and in cells. Y. K. Y. K. I. Characterization of of human (ADAMTS-4) Biol 2000; PubMed Scopus Google Scholar that factor is involved in the regulation of the human ADAMTS4 promoter activity in that and to of the promoter for its activity. the ADAMTS4 promoter contains several and is known to in Y. induces expression of cytokines and in human via and Rheum. PubMed Scopus Google Scholar was to their In to its role in chondrocytes, our as a of ADAMTS4 in the the unique regulation of this gene in NP cells. We and increased expression of ADAMTS4 and mRNA and protein during disk 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, J. D. TNF-α and IL-1β a and with thrombospondin aggrecan degradation through in intervertebral disc.J Biol Chem. 2011; Full Text Full Text PDF PubMed Scopus Google Scholar, Sandy K. K. T. K. and fragments in the human intervertebral disc and of disc (Phila Pa 1976). PubMed Scopus Google Scholar Sandy K. K. T. K. and fragments in the human intervertebral disc and of disc (Phila Pa 1976). PubMed Scopus Google Scholar and S. Y. K. Y. K. C. A. T. of for ADAM-TS5 on intervertebral disc degeneration in the Res Ther. 2009; 11: PubMed Scopus Google Scholar in both human and ADAMTS-4 protein levels increase with of the ADAMTS-5 levels are and Moreover, S. Y. K. Y. K. C. A. T. of for ADAM-TS5 on intervertebral disc degeneration in the Res Ther. 2009; 11: PubMed Scopus Google Scholar that silencing of ADAMTS-5 is to aggrecan degradation in to these in the study expression of ADAMTS-4 was to TNF-α in NP cells from intervertebral of degenerate silencing of either ADAMTS-4 or ADAMTS-5 in 2 human NP cells resulted in of aggrecan on these is not to that ADAMTS-4 and -5 are nonredundant and that of the activity of of these to of the matrix and degenerative disk with Western of ADAMTS4 and 5 in human nucleus pulposus (NP) cells from degenerated and ADAMTS-4 with after TNF-α treatment. of MAPK in controlling ADAMTS-4 promoter activity in NP cells. NP cells were with and ADAMTS-4 promoter activity was after IL-1β treatment. Unlike p38δ no role in controlling ADAMTS-4 promoter activity. and Cotransfection of cells with no effect on TNF-α–dependent and in ADAMTS-4 promoter activity. are as from with in activity in rat NP cells was completely blocked by both and the NF-κB inhibitor and and were with ADAMTS-4 and with TNF-α and cells increase in ADAMTS-4 activity. are as from with
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