MMP-2 mediates local degradation and remodeling of collagen by annulus fibrosus cells of the intervertebral disc

Arthritis Research & Therapy

Anshu Rastogi²  Hyunchul Kim²  Julianne D Twomey²  Adam H Hsieh¹ 

[1] Department of Orthopaedics, School of Medicine; University of Maryland, Baltimore, 22 South Greene Street; Baltimore, MD 21201, USA

[2] Fischell Department of Bioengineering; University of Maryland, College Park, Jeong H. Kim Engineering Building, Rm 2330, College Park, MD 20742, USA

关键词: RNA interference;    Remodeling;    Collagen;    Matrix metalloproteinase;    Intervertebral disc;   

Others  :  799276
DOI  :  10.1186/ar4224

 received in 2012-06-19, accepted in 2013-04-27,  发布年份 2013

【 摘 要 】

Introduction

Degeneration of the intervertebral disc (IVD) is characterized by marked degradation and restructuring of the annulus fibrosus (AF). Although several matrix metalloproteinases (MMPs) have been found to be more prevalent in degenerate discs, their coordination and function within the context of the disease process are still not well understood. In this study, we sought to determine whether MMP-2 is associated with degenerative changes in the AF and to identify the manner by which AF cells use MMP-2.

Methods

Two established animal models of disc degeneration, static compression and transannular needle puncture of rodent caudal discs, were examined for MMP-2 immunopositivity. With lentiviral transduction of an shRNA expression cassette, we screened and identified an effective shRNA sequence for generating stable RNA interference to silence MMP-2 expression in primary rat AF cells. Gelatin films were used to compare gelatinase activity and spatial patterns of degradation between transduced cells, and both noninfected and nonsense shRNA controls. The functional significance of MMP-2 was determined by assessing the ability for cells to remodel collagen gels.

Results

Both static compression and 18-g annular puncture of rodent caudal discs stimulated an increase in MMP-2 activity with concurrent lamellar disorganization in the AF, whereas 22-g and 26-g needle injuries did not. To investigate the functional role of MMP-2, we established lentivirus-mediated RNAi to induce stable knockdown of transcript levels by as much as 88%, and protein levels by as much as 95% over a 10-day period. Culturing transduced cells on gelatin films confirmed that MMP-2 is the primary functional gelatinase in AF cells, and that MMP-2 is used locally in regions immediately around AF cells. In collagen gels, transduced cells demonstrated an inability to remodel collagen matrices.

Conclusions

Our study indicates that increases in MMP-2 observed in human degenerate discs are mirrored in experimentally induced degenerative changes in rodent animal models. AF cells appear to use MMP-2 in a very directed fashion for local matrix degradation and collagen remodeling. This suggests that MMP-2 may have a functionally significant role in the etiology of degenerative disc disease and could be a potential therapeutic target.

【 授权许可】

   
2013 Rastogi et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

【 参考文献 】

• [1]Iatridis JC, MacLean JJ, Roughley PJ, Alini M: Effects of mechanical loading on intervertebral disc metabolism in vivo. J Bone Joint Surg Am 2006, (88 Suppl 2):41-46.
• [2]Rutges JP, Kummer JA, Oner FC, Verbout AJ, Castelein RJ, Roestenburg HJ, Dhert WJ, Creemers LB: Increased MMP-2 activity during intervertebral disc degeneration is correlated to MMP-14 levels. J Pathol 2008, 214:523-530.
• [3]Salo J, Mackiewicz Z, Indahl A, Konttinen YT, Holm AK, Sukura A, Holm S: Plasmin-matrix metalloproteinase cascades in spinal response to an experimental disc lesion in pig. Spine 2008, 33:839-844.
• [4]Seguin CA, Pilliar RM, Madri JA, Kandel RA: TNF-alpha induces MMP2 gelatinase activity and MT1-MMP expression in an in vitro model of nucleus pulposus tissue degeneration. Spine 2008, 33:356-365.
• [5]Nagase H, Woessner JF Jr: Matrix metalloproteinases. J Biol Chem 1999, 274:21491-21494.
• [6]Akeda K, An HS, Pichika R, Patel K, Muehleman C, Nakagawa K, Uchida A, Masuda K: The expression of NG2 proteoglycan in the human intervertebral disc. Spine 2007, 32:306-314.
• [7]Levicoff EA, Kim JS, Sobajima S, Wallach CJ, Larson JW, Robbins PD, Xiao X, Juan L, Vadala G, Gilbertson LG, Kang JD: Safety assessment of intradiscal gene therapy II: effect of dosing and vector choice. Spine 2008, 33:1509-1516. discussion 1517
• [8]Shen B, Melrose J, Ghosh P, Taylor F: Induction of matrix metalloproteinase-2 and -3 activity in ovine nucleus pulposus cells grown in three-dimensional agarose gel culture by interleukin-1beta: a potential pathway of disc degeneration. Eur Spine J 2003, 12:66-75.
• [9]Kozaci LD, Guner A, Oktay G, Guner G: Alterations in biochemical components of extracellular matrix in intervertebral disc herniation: role of MMP-2 and TIMP-2 in type II collagen loss. Cell Biochem Funct 2006, 24:431-436.
• [10]Hsieh AH, Lotz JC: Prolonged spinal loading induces matrix metalloproteinase-2 activation in intervertebral discs. Spine 2003, 28:1781-1788.
• [11]MacLean JJ, Roughley PJ, Monsey RD, Alini M, Iatridis JC: In vivo intervertebral disc remodeling: kinetics of mRNA expression in response to a single loading event. J Orthop Res 2008, 26:579-588.
• [12]Aigner A: Delivery systems for the direct application of sirnas to induce rna interference (RNAi) in vivo. J Biomed Biotechnol 2006, 2006:71659.
• [13]Wall NR, Shi Y: Small RNA: can RNA interference be exploited for therapy? Lancet 2003, 362:1401-1403.
• [14]Dykxhoorn DM, Lieberman J: The silent revolution: RNA interference as basic biology, research tool, and therapeutic. Annu Rev Med 2005, 56:401-423.
• [15]Hsieh AH, Lotz JC: Time-course regulation of MMP-2 activity in loaded intervertebral discs. Presented at the International Society for the Study of the Lumbar Spine 30th Annual Meeting, 2003 2003.
• [16]Hsieh AH, Hwang D, Ryan DA, Freeman AK, Kim H: Degenerative anular changes induced by puncture are associated with insufficiency of disc biomechanical function. Spine 2009, 34:998-1005.
• [17]Primer3 [http://frodo.wi.mit.edu/] webcite
• [18]Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25:402-408.
• [19]Lotz JC, Colliou OK, Chin JR, Duncan NA, Liebenberg E: Compression-induced degeneration of the intervertebral disc: an in vivo mouse model and finite-element study. Spine 1998, 23:2493-2506.
• [20]Deryugina EI, Bourdon MA, Reisfeld RA, Strongin A: Remodeling of collagen matrix by human tumor cells requires activation and cell surface association of matrix metalloproteinase-2. Cancer Res 1998, 58:3743-3750.
• [21]Deryugina EI, Ratnikov B, Monosov E, Postnova TI, DiScipio R, Smith JW, Strongin AY: MT1-MMP initiates activation of pro-MMP-2 and integrin alphavbeta3 promotes maturation of MMP-2 in breast carcinoma cells. Exp Cell Res 2001, 263:209-223.
• [22]Silletti S, Kessler T, Goldberg J, Boger DL, Cheresh DA: Disruption of matrix metalloproteinase 2 binding to integrin alpha vbeta 3 by an organic molecule inhibits angiogenesis and tumor growth in vivo. Proc Natl Acad Sci USA 2001, 98:119-124.
• [23]Fujiwara A, Shibata E, Terashima H, Shishido A, Nishiki J, Yoshida K, Miyauchi K, Madachi A, Matsuura N: Evaluation of matrix metalloproteinase-2 (MMP-2) activity with film in situ zymography for improved cytological diagnosis of breast tumors. Breast Cancer 2006, 13:272-278.
• [24]Thomopoulos S, Fomovsky GM, Chandran PL, Holmes JW: Collagen fiber alignment does not explain mechanical anisotropy in fibroblast populated collagen gels. J Biomech Eng 2007, 129:642-650.
• [25]Nemoto O, Yamagishi M, Yamada H, Kikuchi T, Takaishi H: Matrix metalloproteinase-3 production by human degenerated intervertebral disc. J Spinal Disord 1997, 10:493-498.
• [26]Pockert AJ, Richardson SM, Le Maitre CL, Lyon M, Deakin JA, Buttle DJ, Freemont AJ, Hoyland JA: Modified expression of the ADAMTS enzymes and tissue inhibitor of metalloproteinases 3 during human intervertebral disc degeneration. Arthritis Rheum 2009, 60:482-491.
• [27]Rutges JP, Nikkels PG, Oner FC, Ottink KD, Verbout AJ, Castelein RJ, Creemers LB, Dhert WJ: The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc. Eur Spine J 2010, 19:1340-1346.
• [28]Weiler C, Nerlich AG, Zipperer J, Bachmeier BE, Boos N: 2002 SSE Award Competition in Basic Science: Expression of major matrix metalloproteinases is associated with intervertebral disc degradation and resorption. Eur Spine J 2002, 11:308-320.
• [29]Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF, Evans CH: Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 1996, 21:271-277.
• [30]Martin MD, Matrisian LM: The other side of MMPs: protective roles in tumor progression. Cancer Metastasis Rev 2007, 26:717-724.
• [31]Page-McCaw A, Ewald AJ, Werb Z: Matrix metalloproteinases and the regulation of tissue remodelling. Nature Rev Mol Cell Biol 2007, 8:221-233.
• [32]Tournier JM, Polette M, Hinnrasky J, Beck J, Werb Z, Basbaum C: Expression of gelatinase A, a mediator of extracellular matrix remodeling, by tracheal gland serous cells in culture and in vivo. J Biol Chem 1994, 269:25454-25464.
• [33]Aimes RT, Quigley JP: Matrix metalloproteinase-2 is an interstitial collagenase: inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem 1995, 270:5872-5876.
• [34]Lelongt B, Trugnan G, Murphy G, Ronco PM: Matrix metalloproteinases MMP2 and MMP9 are produced in early stages of kidney morphogenesis but only MMP9 is required for renal organogenesis in vitro. J Cell Biol 1997, 136:1363-1373.
• [35]Thrailkill KM, Clay Bunn R, Fowlkes JL: Matrix metalloproteinases: their potential role in the pathogenesis of diabetic nephropathy. Endocrine 2009, 35:1-10.
• [36]Bjorklund M, Koivunen E: Gelatinase-mediated migration and invasion of cancer cells. Biochim Biophys Acta 2005, 1755:37-69.
• [37]Inoue K, Mikuni-Takagaki Y, Oikawa K, Itoh T, Inada M, Noguchi T, Park JS, Onodera T, Krane SM, Noda M, Itohara S: A crucial role for matrix metalloproteinase 2 in osteocytic canalicular formation and bone metabolism. J Biol Chem 2006, 281:33814-33824.
• [38]Lieu S, Hansen E, Dedini R, Behonick D, Werb Z, Miclau T, Marcucio R, Colnot C: Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2. Dis Models Mech 2011, 4:203-211.
• [39]Okada Y, Naka K, Kawamura K, Matsumoto T, Nakanishi I, Fujimoto N, Sato H, Seiki M: Localization of matrix metalloproteinase 9 (92-kilodalton gelatinase/type IV collagenase = gelatinase B) in osteoclasts: implications for bone resorption. Lab Invest 1995, 72:311-322.
• [40]Galis ZS, Muszynski M, Sukhova GK, Simon-Morrissey E, Libby P: Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions. Ann NY Acad Sci 1995, 748:501-507.
• [41]Johnson JL, Baker AH, Oka K, Chan L, Newby AC, Jackson CL, George SJ: Suppression of atherosclerotic plaque progression and instability by tissue inhibitor of metalloproteinase-2: involvement of macrophage migration and apoptosis. Circulation 2006, 113:2435-2444.
• [42]Bell E, Ivarsson B, Merrill C: Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro. Proc Natl Acad Sci USA 1979, 76:1274-1278.
• [43]Azzam HS, Thompson EW: Collagen-induced activation of the M(r) 72,000 type IV collagenase in normal and malignant human fibroblastoid cells. Cancer Res 1992, 52:4540-4544.
• [44]Nguyen M, Arkell J, Jackson CJ: Three-dimensional collagen matrices induce delayed but sustained activation of gelatinase A in human endothelial cells via MT1-MMP. Int J Biochem Cell Biol 2000, 32:621-631.
• [45]Seltzer JL, Lee AY, Akers KT, Sudbeck B, Southon EA, Wayner EA, Eisen AZ: Activation of 72-kDa type IV collagenase/gelatinase by normal fibroblasts in collagen lattices is mediated by integrin receptors but is not related to lattice contraction. Exp Cell Res 1994, 213:365-374.
• [46]Tomasek JJ, Halliday NL, Updike DL, Ahern-Moore JS, Vu TK, Liu RW, Howard EW: Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton. J Biol Chem 1997, 272:7482-7487.
• [47]Wang DR, Sato M, Li LN, Miura M, Kojima N, Senoo H: Stimulation of pro-MMP-2 production and activation by native form of extracellular type I collagen in cultured hepatic stellate cells. Cell Structure Function 2003, 28:505-513.
• [48]Allenberg M, Weinstein T, Li I, Silverman M: Activation of procollagenase IV by cytochalasin D and concanavalin A in cultured rat mesangial cells: linkage to cytoskeletal reorganization. J Am Soc Nephrol 1994, 4:1760-1770.
• [49]Ellerbroek SM, Wu YI, Overall CM, Stack MS: Functional interplay between type I collagen and cell surface matrix metalloproteinase activity. J Biol Chem 2001, 276:24833-24842.
• [50]Giannelli G, Bergamini C, Fransvea E, Marinosci F, Quaranta V, Antonaci S: Human hepatocellular carcinoma (HCC) cells require both alpha3beta1 integrin and matrix metalloproteinases activity for migration and invasion. Lab Invest 2001, 81:613-627.
• [51]Yan L, Moses MA, Huang S, Ingber DE: Adhesion-dependent control of matrix metalloproteinase-2 activation in human capillary endothelial cells. J Cell Sci 2000, 113:3979-3987.
• [52]Zigrino P, Drescher C, Mauch C: Collagen-induced proMMP-2 activation by MT1-MMP in human dermal fibroblasts and the possible role of alpha2beta1 integrins. Eur J Cell Biol 2001, 80:68-77.
• [53]Bergknut N, Rutges JP, Kranenburg HJ, Smolders LA, Hagman R, Smidt HJ, Lagerstedt AS, Voorhout G, Hazewinkel HH, Grinwis GC, Creemers LB, Meij BP, Dhert WJ: The dog as an animal model for intervertebral disc degeneration? Spine 2012, 37(5):351-358. 011, as originally listed), and the complete reference info is now provided]
• [54]Pushparaj PN, Aarthi JJ, Manikandan J, Kumar SD: siRNA, miRNA, and shRNA: in vivo applications. J Dent Res 2008, 87:992-1003.
• [55]Kuhlmann I: The prophylactic use of antibiotics in cell culture. Cytotechnology 1996, 19:95-105.
• [56]Nagy V, Watzele M: FuGENE 6 Transfection Reagent: minimizing reagent-dependent side effects as analyzed by gene-expression profiling and cytotoxicity assays. Nature Methods 2006, 3(5):iii-v.
• [57]Sledz CA, Williams BR: RNA interference and double-stranded-RNA-activated pathways. Biochem Soc Trans 2004, 32:952-956.
• [58]Nishida K, Doita M, Takada T, Kakutani K, Miyamoto H, Shimomura T, Maeno K, Kurosaka M: Sustained transgene expression in intervertebral disc cells in vivo mediated by microbubble-enhanced ultrasound gene therapy. Spine 2006, 31:1415-1419.
• [59]Seki S, Asanuma-Abe Y, Masuda K, Kawaguchi Y, Asanuma K, Muehleman C, Iwai A, Kimura T: Effect of small interference RNA (siRNA) for ADAMTS5 on intervertebral disc degeneration in the rabbit anular needle-puncture model. Arthritis Res Ther 2009, 11:R166. BioMed Central Full Text
• [60]Tsung AJ, Kargiotis O, Chetty C, Lakka SS, Gujrati M, Spomar DG, Dinh DH, Rao JS: Downregulation of matrix metalloproteinase-2 (MMP-2) utilizing adenovirus-mediated transfer of small interfering RNA (siRNA) in a novel spinal metastatic melanoma model. Int J Oncol 2008, 32:557-564.
• [61]Sun Y, Liu M, Yang B, Li B, Lu J: Role of siRNA silencing of MMP-2 gene on invasion and growth of laryngeal squamous cell carcinoma. Eur Arch Otorhinolaryngol 2008, 265:1385-1391.