Inhibitory Effects of Insulin-like Growth Factor-1 and Osteogenic Protein-1 on Fibronectin Fragment- and Interleukin-1β-stimulated Matrix Metalloproteinase-13 Expression in Human Chondrocytes

Abstract

Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1β or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1β in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment- or IL-1β-stimulated transcription of the MMP-13 gene in a dose-dependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragment-stimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to down-regulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-κB and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis. Matrix metalloproteinase-13 (collagenase-3), a member of the family of matrix metalloproteinases (MMPs), plays a major pathological role in the cartilage destruction of arthritis. A dramatic up-regulation of MMP-13 by inflammatory cytokines such as interleukin (IL)-1β or by fibronectin fragments has been observed in chondrocytes. In this study, we investigated the inhibitory effects of insulin-like growth factor-1 (IGF-1) and osteogenic protein-1 (OP-1) on the expression of MMP-13, which was induced by fibronectin fragment or IL-1β in human immortalized or human primary chondrocytes. IGF-1 and OP-1 each significantly reduced the basal level as well as fibronectin fragment- or IL-1β-stimulated transcription of the MMP-13 gene in a dose-dependent fashion with the corresponding decreases in the protein level of MMP-13. The most prominent suppressive effect was observed by the combination of IGF-1 and OP-1, which decreased the basal promoter activity by 60% and almost completely abrogated the fibronectin fragment-stimulated MMP-13 promoter activity. OP-1 was found to enhance mRNA levels of IGF-1 and the IGF-1 receptor, the latter of which appeared to be responsible for the combined effect of IGF-1 and OP-1. The suppressive effect of IGF-1 and OP-1 on MMP-13 expression was due in part to down-regulation of the expression of pro-inflammatory cytokines and the activity of their intermediate molecules, including NF-κB and AP-1 factors. We propose that IGF-1 and OP-1 could be key physiological regulators of MMP-13 gene expression and that the combination of IGF-1 and OP-1 may be useful in controlling the excess catabolic activity in arthritis. Cartilage homeostasis is a well synchronized balance between anabolic and catabolic processes. During the development of osteoarthritis, this balance is disrupted resulting in progressive degradation of the articular cartilage (1Goldring M.B. Arthritis Rheum. 2000; 43: 1916-1926Crossref PubMed Scopus (632) Google Scholar). Studies have demonstrated that members of the matrix metalloproteinase (MMP) 1The abbreviations used are: MMP, matrix metalloproteinase; IL, interleukin; Fn-f, fibronectin fragment; IGF-1, insulin-like growth factor-1; OP-1, osteogenic protein-1; BMP, bone morphogenetic protein; IL-1Ra, IL-1 receptor antagonist; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RT, reverse transcription. family are the major pathophysiological mediators of the cartilage destruction process in osteoarthritis (2Billinghurst R.C. Dahlberg L. Ionescu M. Reiner A. Bourne R. Rorabeck C. Mitchell P. Hambor J. Diekmann O. Tschesche H. J. Clin. Invest. 1997; 99: 1534-1545Crossref PubMed Scopus (875) Google Scholar). Recently, in vitro, clinical, and transgenic studies have provided evidence that chondrocyte MMP-13 (collagenase-3) is a leading candidate enzyme mediating the degradation of type II collagen in osteoarthritis (2Billinghurst R.C. Dahlberg L. Ionescu M. Reiner A. Bourne R. Rorabeck C. Mitchell P. Hambor J. Diekmann O. Tschesche H. J. Clin. Invest. 1997; 99: 1534-1545Crossref PubMed Scopus (875) Google Scholar, 3Mitchell P.G. Magna H.A. Reeves L.M. Lopresti-Morrow L.L. Yocum S.A. Rosner P.J. Geoghegan K.F. Hambor J.E. J. Clin. Invest. 1996; 97: 761-768Crossref PubMed Scopus (833) Google Scholar, 4Dahlberg L. Billinghurst R.C. Manner P. Nelson F. Webb G. Ionescu M. Reiner A. Tanzer M. Zukor D. Chen J. Arthritis Rheum. 2000; 43: 673-682Crossref PubMed Scopus (211) Google Scholar). A dramatic up-regulation of MMP-13 gene expression in response to inflammatory cytokines, such as interleukin-1β (IL-1β) (5Shlopov B.V. Gumanovskaya M.L. Hasty K.A. 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More recently, it has been demonstrated that OP-1 is expressed in human articular chondrocytes and in osteoarthritis cartilage (19Chubinskaya S. Merrihew C. Cs-Szabo G. Mollenhauer J. McCartney J. Rueger D.C. Kuettner K.E. J. Histochem. Cytochem. 2000; 48: 239-250Crossref PubMed Scopus (104) Google Scholar). Taken together, these findings imply potential involvement of endogenous OP-1 in cartilage repair mechanisms, either by enhancing matrix synthesis (18Koepp H.E. Sampath K.T. Kuettner K.E. Homandberg G.A. Inflamm. Res. 1999; 48: 199-204Crossref PubMed Scopus (31) Google Scholar) or by repressing the pro-inflammatory expression of cytokines, such as IL-6 and IL-1β, as well as the chemokines IL-8 and monocyte chemotatic protein-1 (20Gould S.E. Day M. Jones S.S. Dorai H. Kidney Int. 2002; 61: 51-60Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). It has been proposed that the effects of OP-1 in chondrocytes are mediated through type I serine/threonine kinase receptors (activin receptor-like kinase), ALK/BMPR-IB (21Haaijman A. Burger E.H. Goei S.W. Nelles L. Ten Dijke P. Huylebroeck D. Bronckers A.L. Growth Factors. 2000; 17: 177-192Crossref PubMed Scopus (20) Google Scholar). Synergistic stimulation of cell differentiation and proliferation by insulin-like growth factor (IGF-1) and OP-1 has been demonstrated in rat osteoblastic cells (22Yeh L.-C.C. Adamo M.L. Olson M.S. Lee J.C. Endocrinology. 1997; 138: 4181-4190Crossref PubMed Scopus (86) Google Scholar) and in human primary chondrocytes. 2Loeser, R. F., Pacione, C. A., and Chubinskaya, S. (2003) Arthritis Rheum., in press. Yeh and colleagues (17Yeh L.-C.C. Adamo M.L. Kitten A.M. Olson M.S. Lee J.C. Endocrinology. 1996; 137: 1921-1931Crossref PubMed Scopus (39) Google Scholar, 23Yeh L.-C.C. Adamo M.L. Duan C. Lee J.C. 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Arthritis Rheum. 2002; 46: 2368-2376Crossref PubMed Scopus (158) Google Scholar). The immortalized human chondrocyte cells provided by Bone and The primary and immortalized chondrocytes in fetal and and of human MMP-13 promoter and into a the of and The human promoter was into the the of and the cells to a of in and with of or of expression the The was used as a and a was as for the cells in and to for followed by with the Fn-f IL-1β IGF-1 or OP-1 by of IL-1β was a A to human IGF-1 or IGF-1 receptor and for to the The cells and activity was a of the in IGF-1 receptor and provided by R. The human IGF-1 promoter was provided by S. The AP-1 and and provided by NF-κB and of and and cellular and the and to the of the and transcription was with of cellular the for synthesis in of The for the and the for their are in the to be in the for the as M. G. J.M. L. D. J. Orthop. Res. PubMed Scopus Google Scholar). of the to the and of the of by the to potential to in was for The levels of mRNA with the of Fn-f, or growth was for the to that there was in the The was by of for for and for in the of of and and used is in on and by with and for the genes in to the to a and in a new MMP-13 by and chondrocytes and The protein was with and the of protein by followed by to for The of MMP-13 was by to MMP-13 and MMP-13 was provided by of of the of the MMP-13 in MMP-13 promoter for of transcription factors. the basal promoter activity of the MMP-13 we a of of MMP-13 and a the of The promoter of MMP-13 and into immortalized human chondrocytes. The of MMP-13 promoter the basal promoter activity. The promoter the promoter such as and Runx2 demonstrated of the basal promoter activity in The promoter activity to that of that the such as AP-1 or also the for transcription in the are to the basal promoter activity of the MMP-13 gene in chondrocytes. IGF-1 and OP-1 MMP-13 down-regulation of MMP-13 expression by IGF-1 has been in rat bone cell A.M. Rydziel S. Canalis E. Endocrinology. 1996; 137: PubMed Scopus Google Scholar) and in cartilage Rheum. 2001; PubMed Scopus Google Scholar). We investigated the of IGF-1, OP-1, or the combination of IGF-1 and OP-1 is of the basal level of the MMP-13 expression in human articular chondrocytes. in the of MMP-13 promoter demonstrated the response to IGF-1 or OP-1 with in the promoter activity. and in the of the inhibitory effect of IGF-1 and OP-1 by the promoter to that a the MMP-13 promoter are responsible for the inhibitory effect of IGF-1 OP-1. The combination of IGF-1 and OP-1 reduced the basal promoter activity of MMP-13 with The inhibitory effects of IGF-1, OP-1, and the combination of the growth on MMP-13 transcription by primary human chondrocytes and The inhibitory effect of IGF-1 and OP-1 on MMP-13 promoter activity was The inhibitory activity of IGF-1 or OP-1 was observed the of the with of either IGF-1 or OP-1 dose-dependent inhibitory effect on MMP-13 expression of IGF-1 or OP-1 the of effect on the MMP-13 promoter activity. However, it was that a of IGF-1 or OP-1 was the of of the growth factor was to down-regulate MMP-13 promoter activity. that growth factor the cellular for the growth IGF-1 and OP-1 and MMP-13 of MMP-13 expression in response to Fn-f or IL-1β has been observed in chondrocytes. we IGF-1 and OP-1 suppressive effects on or IL-1β-stimulated MMP-13 expression by IGF-1 and OP-1 and in combination a inhibitory effect on MMP-13 promoter activity the activity induced by IL-1β The combination of IGF-1 and OP-1 almost completely activity and IL-1β-stimulated activity of MMP-13 promoter in cells with the promoter Taken together, we that IGF-1 and OP-1 a effect in the of basal as well as or IL-1β-stimulated MMP-13 gene expression in human chondrocytes. The of IGF-1 and OP-1 on MMP-13 to of the IGF-1 by with the of either IGF-1 or OP-1 inhibitory effect on the promoter activity of MMP-13 In the of IGF-1 or OP-1 the cells to as to a as of IGF-1 or OP-1 by the cellular studies to the mechanisms by which the combination of growth IGF-1 and OP-1, the most prominent effect on the expression of MMP-13 gene in chondrocytes. The mRNA levels of rat IGF-1 and proteins IGF-1 receptors by human OP-1 have been in rat cells L.-C.C. Adamo M.L. Duan C. Lee J.C. J. 1998; PubMed Scopus Google Scholar). we that IGF-1 and OP-1 may each the cellular for either IGF-1 or OP-1. the of OP-1 on the expression of the IGF-1 a human IGF-1 promoter a gene was of the IGF-1 followed by the with OP-1 the IGF-1 promoter activity in immortalized chondrocytes and 60% in primary human chondrocytes The of IGF-1 expression by OP-1 was by The of OP-1 the mRNA levels of IGF-1 also the IGF-1 receptor in immortalized chondrocytes with IGF-1 for on the effect on the mRNA or protein levels of OP-1 or OP-1 receptors including and Taken together, these that OP-1 may the IGF-1 the level of transcription in human chondrocytes. the of IGF-1 caused the of the MMP-13 we the effect of a to the IGF-1 receptor and IGF-1 on the inhibitory activity of with was as a the with either the to IGF-1 receptor or the IGF-1 completely the IGF-1 effect on MMP-13 promoter activity The with the MMP-13 promoter activity. we the effect of the on the inhibitory activity of OP-1. we found that the also the inhibitory activity of OP-1 on MMP-13 gene by studies by human IGF-1 receptor which blocks cellular The expression of the almost completely cellular to IGF-1 also OP-1 The that was used for effect on the promoter activity. that OP-1 was through stimulation of the IGF-1 to down-regulate MMP-13 gene However, it was that the to IGF-1, IGF-1 receptor, or the of the effects of IGF-1 and OP-1 on MMP-13 expression The that the of IGF-1 inhibitory effect on MMP-13 with the that OP-1 the IGF-1 receptor that the IGF-1 receptor could be a factor in this the cellular level of IGF-1 receptor is the factor in the response to IGF-1, we may dose-dependent down-regulation of MMP-13 gene expression by expression of the IGF-1 to human IGF-1 receptor followed by the of IGF-1 to in chondrocytes. We observed cellular response to IGF-1 by the expression of IGF-1 receptor of IGF-1 with the expression of IGF-1 receptor a cellular of the IGF-1 effect on MMP-13 promoter activity was observed the OP-1 receptor was expressed followed by the of the the expression of either IGF-1 receptor or OP-1 receptor the inhibitory effect of OP-1 on MMP-13 promoter activity Taken together, we that up-regulation of IGF-1 and the IGF-1 receptor, the IGF-1 was responsible in part for the effect of IGF-1 and OP-1 on MMP-13 expression in human chondrocytes. IGF-1 and OP-1 of in has been that IGF-1 and OP-1 inflammatory expression in primary human cells or in (20Gould S.E. Day M. Jones S.S. Dorai H. Kidney Int. 2002; 61: 51-60Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, M. O. 2001; 8: PubMed Scopus Google Scholar). The pro-inflammatory cytokines are also for Res. 2001; PubMed Scopus Google Scholar, A. J. M. Matrix Biol. 2002; PubMed Scopus Google Scholar, J. F. M. J. 2000; Google Scholar). we that the inhibitory effects of IGF-1 and OP-1 on MMP-13 expression may in part the of inflammatory expression with the inflammatory intermediate gene in human chondrocytes. IL-1β or Fn-f stimulation significantly the mRNA level of MMP-13 as well as inflammatory cytokines in immortalized chondrocytes IL-1β, and Upon the of the combination of IGF-1 and OP-1, we observed decreased expression of endogenous inflammatory cytokines that induced by Fn-f or IL-1β and studies have demonstrated that the of inflammatory AP-1 and NF-κB transcription MMP-13 gene expression in cells or human chondrocytes A. J. M. Matrix Biol. 2002; PubMed Scopus Google Scholar, J. F. M. J. 2000; Google Scholar), the effects of these in MMP-13 we the of NF-κB or of AP-1 transcription for the of MMP-13 by IL-1 S. Biol. 2002; PubMed Scopus Google Scholar), the IGF-1 and OP-1 effect on MMP-13 of which NF-κB the basal level as well as the or MMP-13 promoter activity The of NF-κB could in chondrocytes K. M. Arthritis Rheum. 2001; PubMed Scopus Google Scholar). we to that the reduced promoter activity of MMP-13 was due to cell We observed of the cells with In the of AP-1 such as and abrogated the and of MMP-13 promoter activity In with the or AP-1 which of NF-κB or AP-1 was as a for the to IL-1β, or the AP-1 expression the basal expression of these was significantly in response to IGF-1, OP-1, or the combination of the growth factors. However, chondrocytes with Fn-f or IL-1β, the combination of IGF-1 and OP-1 significantly reduced or activity of and The of which blocks the NF-κB completely the IL-1β-stimulated to a promoter activity of the of AP-1 the promoter activity of In the of AP-1 abrogated the suppressive effect of IGF-1 and OP-1 on the promoter activity of Taken together, these that NF-κB and AP-1 are regulators in the stimulation of MMP-13 expression by Fn-f or IL-1β in human chondrocytes. the down-regulation of inflammatory expression their intermediate may be part of the by which IGF-1 and OP-1 their suppressive effects on MMP-13 expression in human chondrocytes. The of the on or IL-1β-stimulated MMP-13 in have been on the MMP-13 expression is studies have that Fn-f expression IL-1 Arthritis Rheum. 2002; 46: PubMed Scopus Google Scholar, G.A. F. C. C. K. H. K. A. J. 1997; PubMed Scopus Google Scholar). the expression of mediated by has been H. L. J. 2002; PubMed Scopus (104) Google Scholar). we that the IL-1 may in part MMP-13 expression (6Forsyth C.B. Pulai J. Loeser R.F. Arthritis Rheum. 2002; 46: 2368-2376Crossref PubMed Scopus (158) Google Scholar), the between Fn-f and IL-1 leading to the stimulation of MMP-13 we the effect of on MMP-13 gene expression with that of IL-1β in the or of IGF-1 and OP-1. almost completely MMP-13 promoter it decreased MMP-13 expression in immortalized human chondrocytes combined with IGF-1 and OP-1, reduced or MMP-13 promoter activity to the basal In in cells with the promoter by with the the of significantly reduced MMP-13 and to a MMP-13 mRNA combined with IGF-1 and OP-1 MMP-13 mRNA completely the of MMP-13 expression by Fn-f or Taken with findings in which significantly reduced MMP-13 protein level to a MMP-13 protein expression by human primary chondrocytes (6Forsyth C.B. Pulai J. Loeser R.F. Arthritis Rheum. 2002; 46: 2368-2376Crossref PubMed Scopus (158) Google Scholar), the that the of MMP-13 through mechanisms, and In the we investigated the inhibitory effects of IGF-1 and OP-1 on MMP-13 expression in human chondrocytes. We found that the suppressive effect of IGF-1 and OP-1 on the MMP-13 promoter activity was dose-dependent the level with a corresponding in the level of MMP-13 We that IGF-1 and OP-1 each reduced the basal expression of MMP-13 and also the induced expression of these genes stimulation by IL-1β or The most prominent of basal and induced MMP-13 expression was observed with IGF-1 and OP-1 together, the a of In we observed that the combined suppressive effect of IGF-1 and OP-1 was on MMP-13 promoter activity that induced by the MMP-13 promoter activity that induced by that the Fn-f that MMP-13 expression be by and (6Forsyth C.B. Pulai J. Loeser R.F. Arthritis Rheum. 2002; 46: 2368-2376Crossref PubMed Scopus (158) Google Scholar). with IGF-1 and OP-1 a suppressive effect on the and MMP-13 as by a down-regulation of promoter activity and mRNA to the basal the that has of a suppressive effect on the MMP-13 activity on the MMP-13 we that the combination of IGF-1 and OP-1 with for controlling MMP-13 gene that the combined effect of IGF-1 and OP-1 on MMP-13 expression is in part by the stimulation of the IGF-1 cellular in human chondrocytes and the of IGF-1 and OP-1 to the expression of inflammatory cytokines and the activity of the inflammatory intermediate gene is on human OP-1 the transcription of IGF-1 and the IGF-1 receptor, of OP-1 or OP-1 receptor mRNA synthesis was observed by excess of to IGF-1 and the IGF-1 receptor or expression of the human IGF-1 receptor completely the inhibitory effect of IGF-1 as well as OP-1 on MMP-13 promoter activity. expression of the human IGF-1 receptor the inhibitory effect of IGF-1 and expression of OP-1 receptor of the IGF-1 effect on MMP-13 promoter activity. be for OP-1 receptor such as or The OP-1 effect IGF-1 receptor protein levels by expression may be due to the IGF-1 by OP-1, which cellular to the combination of IGF-1 and OP-1. expression of inflammatory cytokines or the reduced of the inflammatory gene by the combination of IGF-1 and OP-1 may the combined effect of IGF-1 and OP-1. also that a process through between the of IGF-1 and OP-1 in chondrocytes may be in the combined effects of IGF-1 and OP-1. excess of to IGF-1 and the IGF-1 receptor completely the inhibitory effect of IGF-1 and OP-1 blocks the combined effect of IGF-1 and OP-1. the of cells with IGF-1 followed by a in the of OP-1 or cells to OP-1 or chondrocytes by either IGF-1 or OP-1 to to of as as of IGF-1 or OP-1. that IGF-1 significantly gene with the OP-1 C. Pacione, S. Chubinskaya, and R. F. that IGF-1 may also in the OP-1 through mechanisms in human chondrocytes. the for the between the IGF-1 and OP-1 be as studies for of the inhibitory of IGF-1 and OP-1 on MMP-13 gene expression in chondrocytes. of a of promoter of MMP-13 into immortalized or human primary chondrocytes the activity by the MMP-13 The promoter for transcription including the for AP-1 and as well as of Runx2 and family of the we observed decreased MMP-13 promoter activity. that the are for basal promoter activity G. M. Hambor J.E. J. 1997; PubMed Scopus Google Scholar), the in the of MMP-13 gene may be to MMP-13 promoter activity in human chondrocytes. also that the of MMP-13 promoter was most to the of IGF-1 and OP-1. The response was decreased as the promoter The promoter the response to the growth factors. that the inhibitory effects of the growth may be mediated by a response are mediated through in the promoter of the MMP-13 the of AP-1 in the of MMP-13 gene is well known G. M. Hambor J.E. J. 1997; PubMed Scopus Google Scholar, G. S. M. Res. 19: PubMed Scopus Google Scholar, R.C. N. G. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), the of including the on MMP-13 promoter is gene the NF-κB in the MMP-13 promoter between and and we propose that this be a potential candidate to for the MMP-13 gene Res. 2001; PubMed Scopus Google Scholar) that MMP-13 In either primary or immortalized human promoter activity of MMP-13 was to with The IL-1β-stimulated MMP-13 promoter activity was the of IL-1Ra, which is known to activity of the IL-1 that the MMP-13 promoter is to with The in the may be due to either the cell used for the or the in which the We a which is the that was used by the In the demonstrates the inhibitory effects of IGF-1 and OP-1 on basal as well as on the and expression of the MMP-13 gene in immortalized human chondrocytes in a The combination of these growth prominent of MMP-13 expression the level with corresponding decreases in MMP-13 The mechanisms by which OP-1 and IGF-1 their combined effects on MMP-13 appeared to be in part through stimulation of the IGF-1 and the of pro-inflammatory cytokines by IGF-1 and OP-1 in human chondrocytes. findings that IGF-1 and OP-1 could be physiological regulators of MMP-13 and the combination of IGF-1 and OP-1 may be useful in controlling the catabolic activity in arthritis. We the of and and for human We for OP-1, for IL-1Ra, for IGF-1 and for We also S. and for