The Role of Human HtrA1 in Arthritic Disease

Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms. Human HtrA1 belongs to a widely conserved family of serine proteases involved in various aspects of protein quality control and cell fate. Although HtrA1 has been implicated in the pathology of several diseases, its precise biological functions remain to be established. Through identification of potential HtrA1 targets, studies presented herein propose that within the context of arthritis pathology HtrA1 contributes to cartilage degradation. Elevated synovial HtrA1 levels were detected in fluids obtained from rheumatoid and osteoarthritis patients, with synovial fibroblasts identified as a major source of secreted HtrA1. Mass spectrometry analysis of potential HtrA1 substrates within synovial fluids identified fibronectin as a candidate target, and treatment of fibronectin with recombinant HtrA1 led to the generation of fibronectin-degradation products that may be involved in cartilage catabolism. Consistently, treatment of synovial fibroblasts with HtrA1 or HtrA1-generated fibronectin fragments resulted in the specific induction of matrix metalloprotease 1 and matrix metalloprotease 3 expression, suggesting that HtrA1 contributes to the destruction of extracellular matrix through both direct and indirect mechanisms. Human HtrA1 (L56) is a member of the HtrA 3The abbreviations used are: HtrA, high temperature requirement; MMP, matrix metalloprotease; OA, osteoarthritis; RA, rheumatoid arthritis; HSF, human synovial fibroblast; TIMP, tissue inhibitor of matrix metalloproteinase; ELISA, enzyme-linked immunosorbent assay.3The abbreviations used are: HtrA, high temperature requirement; MMP, matrix metalloprotease; OA, osteoarthritis; RA, rheumatoid arthritis; HSF, human synovial fibroblast; TIMP, tissue inhibitor of matrix metalloproteinase; ELISA, enzyme-linked immunosorbent assay. (High temperature requirement) family of serine proteases, a well defined group of proteases sharing many of the characteristics associated with bacterial HtrAs (1Clausen T. Southan C. Ehrmann M. Mol. Cell. 2002; 10: 443-455Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar). Such features include a highly conserved trypsin-like serine protease domain and at least one PDZ domain at the C terminus. In addition, HtrA1 contains an insulin-like growth factor-binding protein domain and a Kazal-type serine protease inhibitor motif at its N terminus (2Zumbrunn J. Trueb B. FEBS Lett. 1996; 398: 187-192Crossref PubMed Scopus (185) Google Scholar). Originally identified as a gene down-regulated in SV40-transformed fibroblasts (2Zumbrunn J. Trueb B. FEBS Lett. 1996; 398: 187-192Crossref PubMed Scopus (185) Google Scholar), HtrA1 has since been implicated in the modulation of various disease pathologies. Recent reports suggest that HtrA1 plays a protective role in various malignancies because of its tumorsuppressive properties (3Shridhar V. Sen A. Chien J. Staub J. Avula R. Kovats S. Lee J. Lillie J. Smith D.I. Cancer Res. 2002; 62: 262-270PubMed Google Scholar, 4Chien J. Staub J. Hu S.I. Erickson-Johnson M.R. Couch F.J. Smith D.I. Crowl R.M. Kaufmann S.H. Shridhar V. Oncogene. 2004; 23: 1636-1644Crossref PubMed Scopus (143) Google Scholar, 5Baldi A. De Luca A. Morini M. Battista T. Felsani A. Baldi F. Catricalà C. Amantea A. Noonan D.M. Albini A. Natali P.G. Lombardi D. Paggi M.G. Oncogene. 2002; 21: 6684-6688Crossref PubMed Scopus (167) Google Scholar, 6Nie G.-Y. Hampton A. Li Y. Findlay J. Salamonsen L. Biochem. J. 2003; 371: 39-48Crossref PubMed Scopus (115) Google Scholar). Studies have shown that HtrA1 is down-regulated in cancerous tissue as compared with normal tissue and that overexpression results in the inhibition of tumor cell growth and proliferation both in vitro and in vivo (5Baldi A. De Luca A. Morini M. Battista T. Felsani A. Baldi F. Catricalà C. Amantea A. Noonan D.M. Albini A. Natali P.G. Lombardi D. Paggi M.G. Oncogene. 2002; 21: 6684-6688Crossref PubMed Scopus (167) Google Scholar). In contrast to tumor tissue, HtrA1 expression is up-regulated in skeletal muscle of Duchenne muscular dystrophy (7Bakay M. Zhao P. Chen J. Hoffman E. Neuromuscul. Disord. 2002; 12: S125-S141Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) and in cartilage of osteoarthritic joints (8Hu S.I. Carozza M. Klein M. Nantermet P. Luk D. Crowl R.M. J. Biol. Chem. 1998; 273: 34406-34412Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). Therefore, up-regulation of HtrA1 in osteoarthritic joints may contribute to the development of this debilitating disease. Progressive degradation of components of the extracellular matrix plays an important role in the pathogenesis of arthritic diseases (9Sandell L.J. Aigner T. Arthritis Res. 2001; 3: 107-113Crossref PubMed Scopus (662) Google Scholar, 10Roughley P.J. Arthritis Res. 2001; 3: 342-347Crossref PubMed Scopus (122) Google Scholar). The destruction of the major cartilage components is driven by members of all classes of proteases, including serine proteases, although the matrix metalloproteases (MMPs) are considered to be the primary instigators (11Vincenti M.P. Brinckerhoff C.E. Arthritis Res. 2002; 4: 157-164Crossref PubMed Scopus (590) Google Scholar, 12Mort J.S. Billington C.J. Arthritis Res. 2001; 3: 337-341Crossref PubMed Scopus (130) Google Scholar, 13Muller-Ladner U. Gay S. Ann. Rheum. Dis. 2002; 61: 957-959Crossref PubMed Scopus (48) Google Scholar). Elevated levels of various MMPs have been identified in the diseased joints of both osteoarthritis (OA) (14Lohmander L.S. Hoerrner L.A. Lark M.W. Arthritis Rheum. 1993; 36: 181-189Crossref PubMed Scopus (296) Google Scholar, 15Yoshihara Y. Nakamura H. Obata K. Yamada H. Hayakawa T. Fujikawa K. Okada Y. Ann. Rheum. Dis. 2000; 59: 455-461Crossref PubMed Scopus (485) Google Scholar, 16Kageyama Y. Miyamoto S. Ozeki T. Hiyohsi M. Suzuki M. Nagano A. Clin. Rheumatol. 2000; 19: 14-20Crossref PubMed Scopus (26) Google Scholar) and rheumatoid arthritis (RA) (17Walakovits L.A. Moore V.L. Bhardwaj N. Gallick G.S. Lark M.W. Arthritis Rheum. 1992; 35: 35-42Crossref PubMed Scopus (259) Google Scholar) patients, originating primarily from synovial fibroblasts and chondrocytes (9Sandell L.J. Aigner T. Arthritis Res. 2001; 3: 107-113Crossref PubMed Scopus (662) Google Scholar, 18Smith R.L. Front. Biosci. 1999; 4: D704-D712Crossref PubMed Google Scholar, 19Murphy G. Knauper V. Atkinson S. Butler G. English W. Hutton M. Stracke J. Clark I. Arthritis Res. 2002; 4: S39-S49Crossref PubMed Scopus (270) Google Scholar). Within the cartilage matrix, interstitial collagens are the main targets of degradative collagenases such as MMP-1 (collagenase-1) (11Vincenti M.P. Brinckerhoff C.E. Arthritis Res. 2002; 4: 157-164Crossref PubMed Scopus (590) Google Scholar, 12Mort J.S. Billington C.J. Arthritis Res. 2001; 3: 337-341Crossref PubMed Scopus (130) Google Scholar). The primary function of these MMPs is in the degradation of native fibrillar collagen, resulting in the generation of collagen fragments that are then further cleaved by gelatinases, MMP-2 and MMP-9, and stromelysin (MMP-3) (12Mort J.S. Billington C.J. Arthritis Res. 2001; 3: 337-341Crossref PubMed Scopus (130) Google Scholar). However, for collagenases to gain access to these substrates, small proteoglycans and interfibrillar cross-links must first be removed (12Mort J.S. Billington C.J. Arthritis Res. 2001; 3: 337-341Crossref PubMed Scopus (130) Google Scholar). Recently, it was suggested that several proteoglycans and glycoproteins in the extracellular matrix may serve as potential substrates for HtrA1 (20Tocharus J. Tsuchiya A. Kajikawa M. Ueta Y. Oka C. Kawaichi M. Dev. Growth Differ. 2004; 46: 257-274Crossref PubMed Scopus (117) Google Scholar, 21Murwantoko Yano M. Ueta Y. Murasaki A. Kanda H. Oka C. Kawaichi M. Biochem. J. 2004; 381: 895-904Crossref PubMed Scopus (74) Google Scholar, 22Tsuchiya A. Yano M. Tocharus J. Kojima H. Fukumoto M. Kawaichi M. Oka C. Bone. 2005; 37: 323-336Crossref PubMed Scopus (118) Google Scholar) and that this protease may therefore be pivotal in the onset of destructive joint pathology seen in arthritic disease. In the present study, we have demonstrated a potential direct and indirect involvement of HtrA1 in cartilage destruction in arthritic diseases. Materials—Dulbecco's modified eagle medium, Dulbecco's calcium- and magnesium-free phosphate-buffered saline, heat-inactivated bovine calf serum, l-glutamine, penicillin and streptomycin, collagenase Type I were obtained from Invitrogen. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, thiazolyl blue, 3,3′,5,5′tetramethylbenzidine, hydrocortisone, insulin, and transferrin were obtained from Sigma. Human recombinant interleukin 1β was obtained from R 258: 8848-8854Abstract Full Text PDF PubMed Google Scholar). Polyclonal HtrA1 antibody was produced by injecting purified recombinant HtrA1 (amino acids 141–480) from Escherichia coli into rabbits. Isolation of Human Synovial Fibroblasts (HSF)—HSF were isolated, harvested, and cultured using a method previously described (24Dayer J.M. Krane S.M. Russell R.G. Robinson D.R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 945-949Crossref PubMed Scopus (485) Google Scholar). Briefly, synovial tissue was obtained after synovectomy from patients with osteoarthritis or rheumatoid arthritis under approval of the local Ethics Committees. Samples were washed with Dulbecco's calcium- and magnesium-free phosphate-buffered saline prior to digestion with collagenase (750 units/ml in phosphate-buffered saline) for 1 h at 37°C. After digestion, the synovial fibroblasts were expanded in culture flasks containing Dulbecco's modified Eagle's medium and nutrient mix F12 (1:1) supplemented with 10% fetal calf serum, penicillin (50 international units/ml), streptomycin (50 μg/ml), l-glutamine (0.3 mg/ml), hydrocortisone (4 μg/ml), insulin (250 μg/ml), and transferrin (250 μg/ml). Cells were grown in a humidified incubator at 37 °C containing 5% CO2 in air. At least four separate cell lines were cultured and used between passages 3 and 5. 3-(4,5-Dimethylthiazol-2-yl)-2,5 diphenyltetrazolium Bromide Cell Proliferation Assay—Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assay as previously described (25van de Loosdrecht A.A. Nennie E. Ossenkoppele G.J. Beelen R.H. Langenhuijsen M.M. J. Immunol. Methods. 1991; 141: 15-22Crossref PubMed Scopus (191) Google Scholar). This assay was used throughout cell culture experiments as standard to confirm that cell numbers/viability were comparable. HtrA1 ELISA—HtrA1 protein levels within synovial fluid and cultured supernatant samples were determined using an HtrA1-specific ELISA developed in-house. Briefly, ELISA plates were coated overnight with monoclonal α-HtrA1 (1:100) and blocked with 5% bovine serum albumin/phosphate-buffered saline. Plates were washed with 0.05% Tween/phosphate-buffered saline and incubated with samples for 2 h at 30 °C. After washing, polyclonal α-HtrA1 (1:500) was added for 1 h at 30 °C followed by a biotin-conjugated swine α-rabbit (1:5000) (Dako Cytomation) for 1 h at 30 °C. HtrA1 was detected using horseradish peroxidase-conjugated streptavidin (1:500). Plates were developed using 3,3′,5,5′tetramethylbenzidine in 100 mm citric acid, 0.1% H2O2, pH 3.95. The reaction was stopped with 7% H2SO4 and optical densities determined at 450 nm using a plate reader (Dynex). Purified recombinant HtrA1 (amino acids 141–480) was tested at concentrations ranging from 156 pg/ml to 10 ng/ml to generate a standard curve. Cloning, Expression, and Purification of HtrA1—The expression vector pCTH1 is a derivative of pQE60 (Qiagen) containing the gene M. S. S. N. W. Ehrmann M. Mol. Biol. 2004; 21: PubMed Scopus Google Scholar). of under control was by a into E. coli recombinant HtrA1 (amino acids 141–480) was by in I (50 mm pH mm 1 followed by at for 30 cell was a (Qiagen) at a of The was washed with (50 mm pH mm and (50 mm pH mm mm prior to in (50 mm pH mm mm of 1 were and by Purified protein was in in (50 mm pH mm 10% at °C. levels in purified HtrA1 were to be at levels as determined by the assay. was a containing as and developed the of HtrA1 in Synovial fluids identified as containing levels of HtrA1 were incubated with or recombinant HtrA1 for 3 h at and then by protein substrates were then from the and identified using of HtrA1 by HtrA1 inhibitor obtained from was determined by of HtrA1 with various concentrations of the inhibitor and prior to the Samples were incubated overnight at 37 °C in the The reaction was stopped by and the supernatant with of pH The was determined at of fibronectin was determined by of various of recombinant HtrA1 and with 10 of fibronectin in mm pH mm for 3 or h at 37 °C. and HtrA1 were incubated under the and as HtrA1 inhibitor was with HtrA1 for at temperature at a of or prior to the of fibronectin Samples were a of by from human synovial fibroblasts was using to the was from using as previously described P.J. N. 1999; PubMed Scopus (26) Google Scholar). for of mm 10 mm pH 0.1% and mm 2 mm 1 of of and of was a Human specific for the gene was and used as an were for and and were °C for an 2 followed by 30 for and or for MMP-1 and of °C for °C for 30 and °C for 1 The reaction was with a at °C. The for were for the and for the MMP-1 the and the MMP-2 the and the the and the for the and for the for the and for the bromide were using the of protein levels in culture were determined using an ELISA to the Systems of and fibronectin 10 of fibronectin was incubated with of HtrA1 for h at 37 °C in in mm pH mm these samples were with and HtrA1 inhibitor to synovial fibroblasts for h and levels by was used to between of was considered are as the Hu (8Hu S.I. Carozza M. Klein M. Nantermet P. Luk D. Crowl R.M. J. Biol. 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In expression of and by HtrA1 and of HtrA1 were of HtrA1 inhibitor and suggesting that the of HtrA1 is for up-regulation of In the HtrA1 inhibitor the of in to the interleukin 1 the of the HtrA1 the of HtrA1 been at the protein was by a specific was secreted by both and and levels were in ng/ml and in pg/ml with of HtrA1 for h and with these of HtrA1 were by the of HtrA1 of by confirm that the up-regulation of MMP-1 and by HtrA1 was through the generation of fibronectin were incubated with human of with fibronectin fragments for h resulted in a in the expression of MMP-1 and were to fibronectin as fibronectin or with HtrA1 inhibitor that these were because of the fibronectin fragments and HtrA1. fragments or expression that in the of HtrA1 or fibronectin fragments the levels of detected in from patients be and The for the is although be by the of the for by in disease between E. R.G. Ann. Rheum. Dis. 2002; 61: PubMed Scopus Google Scholar). HtrA1 levels in cartilage from patients have previously been shown to be as compared with cartilage from (8Hu S.I. Carozza M. Klein M. Nantermet P. Luk D. Crowl R.M. J. Biol. Chem. 1998; 273: 34406-34412Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar). In addition, in vitro studies have demonstrated that cartilage a in HtrA1 by chondrocytes (20Tocharus J. Tsuchiya A. Kajikawa M. Ueta Y. Oka C. Kawaichi M. Dev. Growth Differ. 2004; 46: 257-274Crossref PubMed Scopus (117) Google Scholar). using an HtrA1-specific ELISA, we have demonstrated that HtrA1 levels are in the synovial fluids from both and patients, with a of HtrA1 detected in synovial HtrA1 levels serve as an for of disease. that from and joint tissue HtrA1. This up-regulation of HtrA1 to be disease as human fibroblasts secreted HtrA1 and The of HtrA1 by and that the synovial may be an important source of HtrA1 within the arthritic in to the Although both and it is the highly of chondrocytes that for the seen in the levels of HtrA1 in and synovial fluid (20Tocharus J. Tsuchiya A. Kajikawa M. Ueta Y. Oka C. Kawaichi M. Dev. Growth Differ. 2004; 46: 257-274Crossref PubMed Scopus (117) Google Scholar). of cartilage is a of and (9Sandell L.J. Aigner T. Arthritis Res. 2001; 3: 107-113Crossref PubMed Scopus (662) Google Scholar, 10Roughley P.J. Arthritis Res. 2001; 3: 342-347Crossref PubMed Scopus (122) Google Scholar). identified the extracellular matrix fibronectin as a of suggesting a direct role of HtrA1 in matrix degradation. HtrA1 purified human fragments of various including several fragments ranging from to and Elevated levels of fibronectin fragments ranging from to have been identified both in and synovial fluid in concentrations Front. Biosci. 1999; 4: PubMed Google Scholar, D. Clin. PubMed Scopus Google Scholar) and are involved in the of R. 1993; PubMed Google Scholar, W. U. G. M. J. Clin. PubMed Scopus Google Scholar). The involvement of fibronectin fragments in cartilage is by to D. F. Biochem. 1993; PubMed Scopus Google Scholar) and the of several MMPs Front. Biosci. 1999; 4: PubMed Google Scholar, H. L. Biochem. J. 2002; PubMed Scopus Google Scholar). In the present we have demonstrated that HtrA1 has the potential to expression and in arthritic joints through of of MMP-1 and expression in by HtrA1 was shown to be the of fibronectin Therefore, we suggest that HtrA1 fibronectin present within the cell culture and the resulting fibronectin fragments the expression and of was from the that HtrA1 fibronectin fragments and expression by as has previously been Front. Biosci. 1999; 4: PubMed Google Scholar). The present for a role of HtrA1 in both and RA, to a for its biological functions in this HtrA1 have the potential to cartilage through of extracellular matrix components such as cartilage matrix and collagen (20Tocharus J. Tsuchiya A. Kajikawa M. Ueta Y. Oka C. Kawaichi M. Dev. Growth Differ. 2004; 46: 257-274Crossref PubMed Scopus (117) Google Scholar, 21Murwantoko Yano M. Ueta Y. Murasaki A. Kanda H. Oka C. Kawaichi M. Biochem. J. 2004; 381: 895-904Crossref PubMed Scopus (74) Google Scholar, 22Tsuchiya A. Yano M. Tocharus J. Kojima H. Fukumoto M. Kawaichi M. Oka C. Bone. 2005; 37: 323-336Crossref PubMed Scopus (118) Google Scholar), it to through its to the of MMPs by synovial Therefore, specific inhibition of HtrA1 or in arthritic joints may serve as a for treatment of arthritic diseases. for HtrA1 inhibitor and and Crowl for