Abstract
Aim:
Failure of transplanted cartilage or allogenic chondrocytes is attributed mainly to immunological rejection and cartilage degradation. A major feature is the loss of aggrecan from the cartilage matrix, primarily due to the action of the specific proteinases aggrecanase-1 and aggrecanase-2. The aim of this in vitro study was to determine whether the specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi would mitigate aggrecan loss from cultured chondrocytes.
Methods:
Expression plasmid vectors of shRNA targeting aggrecanase-1 and aggrecanase-2 were constructed and transfected into cultured rattus costochondral chondrocytes. The transfected cells were induced with interleukin-1β (IL-1β). Gene mRNA levels were analyzed by RT—PCR. Aggrecan and collagen II content were measured by immunohistochemistry and Western blotting.
Results:
As the chondrocytes underwent dedifferentiation, agggrecanase-1 increased significantly. The specific inhibition of aggrecanase-1 and aggrecanase-2 by RNAi had no negative effect on the morphology and growth velocity of the chondrocytes. The mRNA of aggrecanase-1 and aggrecanase-2 decreased significantly. The α-2-macroglobulin expression level was increased by the shRNA specific for aggrecanase-1. Other genes of the chondrocytic extracellular matrix were not affected. RNAi significantly increased the aggrecan and collagen II content of chondrocytes treated with IL-1β.
Conclusion:
The results suggest that inhibition of aggrecanase-1 and aggrecanase-2 by RNAi can mitigate aggrecan degradation, without interfering with chondrocytic gene phenotype recovery. RNAi technology can be a useful tool for studying degenerative processes in cartilage.
Similar content being viewed by others
Article PDF
References
Hardingham TE, Fosang AJ . Proteoglycans: many forms and many functions. FASEB J 1992; 6: 861–70.
Vertel BM . The ins and outs of aggrecan. Trends Cell Biol 1995; 5: 458–64.
Kouji K, Yasunori O, Hiroto K, Hiroyuki N, Masayuki M, Hiroshi O, et al. ADAMTS-1 cleaves a cartilage proteoglycan, aggrecan. FEBS Lett 2000; 478: 241–5.
Zheng XM, Yang ZQ, Hou CQ, Wang Y, Chang XF, Xu YC, et al. The effects of cartilage matrix on cartilage implantation mechanisms. Chin J Plast Surg 1999; 15: 74–5 ( in Chinese).
Yang ZQ, Hou CQ, Chang XF, Wang Y, Xu YC, Zheng XM . Experimental observation on the dynamic changes of cartilage matrix in homografts. Chin J Aesthetic Med 1997; 6: 14–6 ( in Chinese).
Hou CQ, Yang ZQ, Chang XF, Wang Y, Xu YC, Yang XP, et al. An experimental study on changes in collagen content after homologous cartilage implantation. Chin J Plast Surg 1995; 11: 274–6 ( in Chinese).
Zheng XM, Yang ZQ, Hou CQ, Feng B, Wang Y, Xu YC . Experimental observations of the quantitative change of proteoglycan in homogenous cartilage graft in the guinea pig. Chin J Plast Surg 1995; 11: 129–31 ( in Chinese).
Romaniuk A, Malejczyk J, Kubicka U, Hyc A, Olszewski W, Moskalewski S . Rejection of cartilage formed by transplanted allogeneic chondrocytes: evaluation with monoclonal antibodies. Transplant Immunol 1995; 3: 251–7.
Mankin HJ, Lippiello L . Biochemical and metabolic abnormalities in articular cartilage from osteo-arthritic human hips. J Bone Joint Surg Am 1970; 52: 424–34.
Lohmander LS, Neame PJ, Sandy JD . The structure of aggrecan fragments in human synovial fluid evidence that aggrecanase mediates cartilage degradation in inflammatory joint disease, joint injury, and osteoarthritis. Arthritis Rheum 1993; 36: 1214–22.
Doege K, Sasaki M, Horigan E, Hassell JR, Yamada Y . Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones. J Biol Chem 1987; 262: 17 757–67.
Heinegård D, Hascall VC . Aggregation of cartilage proteoglycans. 3. Characteristics of the proteins isolated from trypsin digests of aggregates. J Biol Chem 1974; 249: 4250–6.
Hardingham TE . The role of link-protein in the structure of cartilage proteoglycan aggregates. Biochem J 1979; 177: 237–47.
Malfait AM, Liu RQ, Ijiri K, Komiya S, Tortorella M . Inhibition of ADAM-TS4 and ADAM-TS5 prevents aggrecan degradation in osteoarthritic cartilage. J Biol Chem 2002; 277: 22 201–8.
Collins-Racie LA, Flannery CR, Zeng W, Corcoran C, Annis-Freeman B, Agostino MJ, et al. ADAMTS-8 exhibits aggrecanase activity and is expressed in human articular cartilage. Matrix Biol 2004; 23: 219–30.
Tortorella MD, Pratta M, Liu RQ, Austin J, Ross OH, Abbaszade I, et al. Sites of aggrecan cleavage by recombinant human aggrecanase-1 (ADAMTS-4). J Biol Chem 2000; 275: 18 566–73.
Rodriguez-Manzaneque J, Westling J, Thai S, Luque A, Knauper V, Murphy G, et al. ADAMTS-1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors. Biochem Biophys Res Commun 2002; 293: 501–8.
Tortorella MD, Liu RQ, Burn T, Newton RC, Arner E . Characterization of human aggrecanase 2 (ADAMTS5): substrate specificity studies in comparison with aggrecanase 1 (ADAMTS4). Matrix Biol 2002; 21: 499–511.
Somerville RP, Longpre J, Jungers K, Engle J, Ross M, Evanko S, et al. Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans gon-1. J Biol Chem 2003; 278: 9503–13.
Pratta MA, Tortorella MD, Arner EC . Age related changes in aggrecan glycosylation affect cleavage by aggrecanase. J Biol Chem 2000; 275: 39 096–102.
Arner EC, Pratta MA, Trzaskos JM, Decicco CP, Tortorella MD . Generation and characterization of aggrecanase. A soluble, cartilage-derived aggrecan degrading activity. J Biol Chem 1999; 274: 6594–601.
Hannon GJ . RNA interference. Nature 2002; 418: 244–51.
Caplen NJ . Gene therapy progress and prospects: down-regulating gene expression: the impact of RNA interference. Gene Ther 2004; 11: 1241–8.
Mello CC, Conte D Jr . Revealing the world of RNA interference. Nature 2004; 431: 338–42.
Elbashir SM, Harborth J, Lendeckel W, Yalcin A, Weber K, Tuschl T . Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 2001; 411: 428–9.
Caplen NJ, Parrish S, Imani F, Fire A, Morgan RA . Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci USA 2001; 98: 9742–7.
Gou D, Jin N, Liu L . Gene silencing in mammalian cells by PCR based short hairpin RNA. FEBS Lett 2003; 548: 113–8.
Wang ZH, Li GG, Yang ZQ, Wang L, Li LX, He XJ . Culture of rat costochondral chondrocytes and effect of aging on matrix catabolism of cultured chondrocytes. J Tissue Eng Reconstr Surg 2007; 5: 312–6 ( in Chinese).
Wang ZH, Yang ZQ, He XJ, Li GG, Wang L, Li LX . Construction and sequencing of recombinant lentiviral plasmid affecting rattus aggrecanase-1, 2 gene by RNA interfering. Chin J Aesth Med 2007; 16: 1679–82 ( in Chinese).
Sandy JD, Flannery CR, Neame PJ, Lohmander LS . The structure of aggrecan fragments in human synovial fluid. Evidence for the involvement in osteoarthritis of a novel proteinase which cleaves the Glu 373-Ala 374 bond of the interglobular domain. J Clin Invest 1992; 89: 1512–6.
Tortorella MD, Malfait AM, Deccico C, Arner E . The role of ADAMTS4 (aggrecanase-1) and ADAM-TS5 (aggrecanase-2) in a model of cartilage degradation. Osteoarthritis Cartilage 2001; 9: 539–52.
Arner EC, Pratta MA, Decicco CP, Xue CB, Newton RC, Trzaskos JM, et al. Aggrecanase a target for the design of inhibitors of cartilage degradation. Ann NY Acad Sci 1999; 30: 92–107.
Stokes DG, Liu G, Coimbra IB, Piera-Velazquez S, Crowl RM, Jimenez SA . Assessment of the gene expression profile of differentiated and dedifferentiated human fetal chondrocytes by microarray analysis. Arthritis Rheum 2002; 46: 404–19.
Li Q, Lu JH, Liu W, Xia WR, Liu DL, Cui L, et al. The effects of aging on aggrecan catabolism in cultured chondrocytes. Chin J Clin Rehab 2003; 7: 216–8 ( in Chinese).
Schiffelers RM, Xu J, Storm G, Woodle MC, Scaria PV . Effects of treatment with small interfering RNA on joint inflammation in mice with collagen-induced arthritis. Arthritis Rheum 2005; 52: 1314–8.
Inoue A, Takahashi KA, Mazda O, Terauchi R, Arai Y, Kishida T, et al. Electro-transfer of small interfering RNA ameliorated arthritis in rats. Biochem Biophys Res Commun 2005; 336: 903–8.
Song RH, Tortorella MD, Malfait AM, Alston JT, Yang ZY, Elizabeth CA, et al. Aggrecan degradation in human articular cartilage explants is mediated by both ADAMTS-4 and ADAMTS-5. Arthritis Rheum 2007; 56: 575–85.
Arner EC . Aggrecanase-mediated cartilage degradation. Curr Opin Pharmacol 2002; 2: 322–9.
Tortorella MD, Arner EC, Hills R, Gormley J, Fok K, Pegg L, et al. Alpha2-macroglobulin is a novel substrate for ADAMTS-4 and ADAMTS-5 and represents an endogenous inhibitor of these enzymes. J Biol Chem 2004; 279: 17 554–61.
Micky DT, Elizabeth CA, Robert H, Jennifer G, Kam F, Lyle P, et al. ADAMTS-4 (aggrecanase-1): N-Terminal activation mechanisms. Arch Biochem Biophys 2005; 444: 34–44.
Gakuji H, Takanori A, Hiroyuki N, Kazuhiko T, Yasunori O . Inhibition of ADAMTS4 (aggrecanase-1) by tissue inhibitors of metalloproteinases (TIMP-1, 2, 3 and 4). FEBS Lett 2001; 494: 192–5.
Homandberg G . Potential regulation of cartilage metabolism in osteoarthritis by fibronectin fragments. Front Biosci 1999; 15: D713–30.
Zhou HW, Lou SQ, Zhang K . Recovery of function in osteoarthritic chondrocytes induced by p16INK48-specific siRNA in vitro. Rheumatology 2004; 43: 555–68.
Author information
Authors and Affiliations
Corresponding authors
Additional information
This study was supported by the National Natural Science Foundation of China (No 30572052) and the International Cooperation Project of Shaan'xi Province, China (No 2007KW-07).
Rights and permissions
About this article
Cite this article
Wang, Zh., Yang, Zq., He, Xj. et al. Effects of RNAi-mediated inhibition of aggrecanase-1 and aggrecanase-2 on rat costochondral chondrocytes in vitro. Acta Pharmacol Sin 29, 1215–1226 (2008). https://doi.org/10.1111/j.1745-7254.2008.00856.x
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1111/j.1745-7254.2008.00856.x
Keywords
This article is cited by
-
A composite scaffold of MSC affinity peptide-modified demineralized bone matrix particles and chitosan hydrogel for cartilage regeneration
Scientific Reports (2015)
-
Role of Sp1 transcription factor in Interleukin-1-induced ADAMTS-4 (aggrecanase-1) gene expression in human articular chondrocytes
Rheumatology International (2013)