Implication of HYBID (Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization) in Hyaluronan Degradation by Synovial Fibroblasts in Patients with Knee Osteoarthritis
2020; Elsevier BV; Volume: 190; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2020.01.003
ISSN1525-2191
AutoresJun Shiozawa, Susana de Vega, Mehmet Zeynel Cilek, Chiho Yoshinaga, Tomomi Nakamura, Shinya Kasamatsu, Hiroyuki Yoshida, Haruka Kaneko, Muneaki Ishijima, Kazuo Kaneko, Yasunori Okada,
Tópico(s)Fibroblast Growth Factor Research
ResumoCell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), plays a role in HA degradation. CEMIP2, also known as transmembrane protein 2 (TMEM2), possessing a sequence similarity with HYBID, is reported as a hyaluronidase in mice. However, the expression of these molecules in osteoarthritic synovium and their involvement in HA degradation in synovial fluid (SF) from patients with knee osteoarthritis remain elusive. This study examined their expression in synovial tissue and the relationship with molecular weight of HA in SF in knee osteoarthritis patients. Quantification of mRNA demonstrated that HYBID expression is significantly (5.5-fold) higher in osteoarthritic synovium than in normal control synovium, whereas TMEM2 expression level is similar between the two groups. By immunohistochemistry, HYBID was localized mainly to CD68-negative and fibroblast-specific protein 1–positive synovial lining cells and sublining fibroblasts in osteoarthritic synovium. The mRNA expression levels of HYBID, but not TMEM2, in osteoarthritic synovium positively correlated with distribution of lower-molecular-weight HA with below 1000 kDa in SF. HA-degrading activity in osteoarthritic synovial fibroblasts was abrogated by siRNA-mediated knockdown of HYBID. Among the 12 factors examined, IL-6 significantly up-regulated the HYBID expression and HA-degrading activity in osteoarthritic synovial fibroblasts. These data suggest that HYBID overexpressed by IL-6-stimulated synovial fibroblasts is implicated in HA degradation in osteoarthritic synovium. Cell migration-inducing hyaluronidase 1 (CEMIP), also known as hyaluronan (HA)-binding protein involved in HA depolymerization (HYBID), plays a role in HA degradation. CEMIP2, also known as transmembrane protein 2 (TMEM2), possessing a sequence similarity with HYBID, is reported as a hyaluronidase in mice. However, the expression of these molecules in osteoarthritic synovium and their involvement in HA degradation in synovial fluid (SF) from patients with knee osteoarthritis remain elusive. This study examined their expression in synovial tissue and the relationship with molecular weight of HA in SF in knee osteoarthritis patients. Quantification of mRNA demonstrated that HYBID expression is significantly (5.5-fold) higher in osteoarthritic synovium than in normal control synovium, whereas TMEM2 expression level is similar between the two groups. By immunohistochemistry, HYBID was localized mainly to CD68-negative and fibroblast-specific protein 1–positive synovial lining cells and sublining fibroblasts in osteoarthritic synovium. The mRNA expression levels of HYBID, but not TMEM2, in osteoarthritic synovium positively correlated with distribution of lower-molecular-weight HA with below 1000 kDa in SF. HA-degrading activity in osteoarthritic synovial fibroblasts was abrogated by siRNA-mediated knockdown of HYBID. Among the 12 factors examined, IL-6 significantly up-regulated the HYBID expression and HA-degrading activity in osteoarthritic synovial fibroblasts. These data suggest that HYBID overexpressed by IL-6-stimulated synovial fibroblasts is implicated in HA degradation in osteoarthritic synovium. The joint cavity is surrounded by articular cartilage and synovium and filled with synovial fluid, which is a mixture of hyaluronan (HA) and a protein-rich ultrafiltrate of blood plasma. In the normal joint, HA, which is an unbranched nonsulfated glycosaminoglycan composed of repeating disaccharide units of d-glucuronic acid and N-acetyl-d-glucosamine and synthesized by HA synthases (HAS1, HAS2, and HAS3), is present in the range of 1000 to 10,000 kDa, that is, high-molecular-weight HA (HMW-HA). HMW-HA plays an essential role in maintaining the lubricating property and shock absorption function of synovial fluid.1Uebelhart D. Williams J.M. Effects of hyaluronic acid on cartilage degradation.Curr Opin Rheumatol. 1999; 11: 427-435Crossref PubMed Scopus (44) Google Scholar,2Tamer T.M. Hyaluronan and synovial joint: function, distribution and healing.Interdiscip Toxicol. 2013; 6: 111-125Crossref PubMed Scopus (145) Google Scholar These physicochemical properties of HA are lost when it is degraded to lower-molecular-weight HA (LMW-HA).3Vuorio E. Einola S. Hakkarainen S. Penttinen R. Synthesis of underpolymerized hyaluronic acid by fibroblasts cultured from rheumatoid and non-rheumatoid synovitis.Rheumatol Int. 1982; 2: 97-102Crossref PubMed Scopus (51) Google Scholar Other biological activities of HA are also dependent on its molecular weight4Noble P.W. Hyaluronan and its catabolic products in tissue injury and repair.Matrix Biol. 2002; 21: 25-29Crossref PubMed Scopus (464) Google Scholar,5Stern R. Asari A.A. Sugahara K.N. Hyaluronan fragments: an information-rich system.Eur J Cell Biol. 2006; 85: 699-715Crossref PubMed Scopus (863) Google Scholar: HMW-HA is anti-inflammatory and antiangiogenic, whereas LMW-HA promotes inflammatory and angiogenic reactions.6West D.C. Hampson I.N. Arnold F. Kumar S. Angiogenesis induced by degradation products of hyaluronic acid.Science. 1985; 228: 1324-1326Crossref PubMed Scopus (973) Google Scholar,7Rooney P. Kumar S. Ponting J. Wang M. The role of hyaluronan in tumour neovascularization (review).Int J Cancer. 1995; 60: 632-636Crossref PubMed Scopus (258) Google Scholar In osteoarthritis (OA), HA degradation is enhanced and so LMW-HA is accumulated in synovial fluid.3Vuorio E. Einola S. Hakkarainen S. Penttinen R. Synthesis of underpolymerized hyaluronic acid by fibroblasts cultured from rheumatoid and non-rheumatoid synovitis.Rheumatol Int. 1982; 2: 97-102Crossref PubMed Scopus (51) Google Scholar Therefore, HA in OA synovial fluid may not only enhance mechanical stress on articular cartilage due to decreased shock absorption function, but also promote synovitis, leading to the possible progression of cartilage destruction in the OA joint. Recently, the risk of knee OA progression has been associated with the preponderance of HA below 1000 kDa in synovial fluid, but not average HA molecular weight.8Band P.A. Heeter J. Wisniewski H.-G. Liublinska V. Pattanayak C.W. Karia R.J. Stabler T. Balazs E.A. Kraus V.B. Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression.Osteoarthritis Cartilage. 2015; 23: 70-76Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar Arthroscopic and histologic studies have shown that synovitis commonly occurs in knee OA,9Lindblad S. Hedfors E. Arthroscopic and immunohistologic characterization of knee joint synovitis in osteoarthritis.Arthritis Rheum. 1987; 30: 1081-1088Crossref PubMed Scopus (154) Google Scholar,10Ayral X. Pickering E.H. Woodworth T.G. Mackillop N. Dougados M. Synovitis: a potential predictive factor of structural progression of medial tibiofemoral knee osteoarthritis – results of a 1 year longitudinal arthroscopic study in 422 patients.Osteoarthritis Cartilage. 2005; 13: 361-367Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar and an immunohistochemical study reported that degree of synovial inflammation is more severe in early-stage knee OA than in late-stage OA.11Benito M.J. Veale D.J. FitzGerald O. van den Berg W.B. Bresnihan B. Synovial tissue inflammation in early and late osteoarthritis.Ann Rheum Dis. 2005; 64: 1263-1267Crossref PubMed Scopus (699) Google Scholar However, a growing amount of evidence using noninvasive imaging techniques such as magnetic resonance imaging demonstrated that synovial inflammation is present in many patients at all stages of OA12Sellam J. Berenbaum F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis.Nat Rev Rheumatol. 2010; 6: 625-635Crossref PubMed Scopus (854) Google Scholar,13Mathiessen A. Conaghan P.G. Synovitis in osteoarthritis: current understanding with therapeutic implications.Arthritis Res Ther. 2017; 19: 18Crossref PubMed Scopus (441) Google Scholar and supports the notion that synovitis contributes to the progression of OA by accelerating cartilage breakdown.12Sellam J. Berenbaum F. The role of synovitis in pathophysiology and clinical symptoms of osteoarthritis.Nat Rev Rheumatol. 2010; 6: 625-635Crossref PubMed Scopus (854) Google Scholar,14MacFarlane L.A. Yang H. Collins J.E. Jarraya M. Guermazi A. Mandl L.A. Martin S.D. Wright J. Losina E. Katz J.N. Association of changes in effusion-synovitis with progression of cartilage damage over eighteen months in patients with osteoarthritis and meniscal tear.Arthritis Rheumatol. 2019; 71: 73-81Crossref PubMed Scopus (24) Google Scholar Moreover, recent studies on the risk factors of knee OA have suggested that synovitis may be an independent cause of OA15Felson D.T. Niu J. Neogi T. Goggins J. Nevitt M.C. Roemer F. Torner J. Lewis C.E. Guermazi A. Synovitis and the risk of knee osteoarthritis: the MOST study.Osteoarthritis Cartilage. 2016; 24: 458-464Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar and an independent driver of radiographic OA onset and progression.13Mathiessen A. Conaghan P.G. Synovitis in osteoarthritis: current understanding with therapeutic implications.Arthritis Res Ther. 2017; 19: 18Crossref PubMed Scopus (441) Google Scholar Hyaluronidases (HYALs) composed of six different molecules were long thought to be key enzymes for HA degradation,16Csoka A.B. Frost G.I. Stern R. The six hyaluronidase-like genes in the human and mouse genomes.Matrix Biol. 2001; 20: 499-508Crossref PubMed Scopus (481) Google Scholar and among them, HYAL-1 and HYAL-2, the latter of which acts together with cell surface HA receptor CD44, were reported to play a key role in HA degradation. However, knockdown of these genes failed to change HA-degrading activity in skin fibroblasts.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar Therefore, we searched for new molecules related to HA degradation by microarray analysis, and demonstrated that hyaluronan-binding protein involved in hyaluronan depolymerization (HYBID), which was renamed later as cell migration-inducing hyaluronidase 1 (CEMIP) by HUGO Gene Nomnclature Committee, plays a key role in HA degradation independently from HYAL1 and HYAL2/CD44 in human fibroblasts.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar,18Nagaoka A. Yoshida H. Nakamura S. Morikawa T. Kawabata K. Kobayashi M. Sakai S. Takahashi Y. Okada Y. Inoue S. Regulation of hyaluronan (HA) metabolism mediated by HYBID (Hyaluronan-binding protein involved in HA depolymerization, KIAA1199) and HA synthases in growth factor-stimulated fibroblasts.J Biol Chem. 2015; 290: 30910-30923Crossref PubMed Scopus (60) Google Scholar HYBID was reported as a deafness gene of unknown function (KIAA1199)19Abe S. Usami S. Nakamura Y. Mutations in the gene encoding KIAA1199 protein, an inner-ear protein expressed in Deiters' cells and the fibrocytes, as the cause of nonsyndromic hearing loss.J Hum Genet. 2003; 48: 564-570Crossref PubMed Scopus (96) Google Scholar and also named as CEMIP (cell migration-inducing protein) by another group.20Evensen N.A. Li Y. Kuscu C. Liu J. Cathcart J. Banach A. Zhang Q. Li E. Joshi S. Yang J. Denoya P.I. Pastorekova S. Zucker S. Shroyer K.R. Cao J. Hypoxia promotes colon cancer dissemination through up-regulation of cell migration-inducing protein (CEMIP).Oncotarget. 2015; 6: 20723-20739Crossref PubMed Scopus (51) Google Scholar A previous study on skin fibroblasts has shown that HYBID is localized in clasthrin-coated vesicles and contributes to degradation of HMW-HA into LMW-HA fragments, which are released to the extracellular milieu.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar HYBID is a secreted protein composed of one G8, two GG, and four PbH1 domains, lacking substantial homology to HYAL enzymes, HA-binding proteins, and HA-link modules. It selectively binds to HA, but does not interact with sulfated glycosaminoglycans.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar A recent study has shown that a type II transmembrane protein with a sequence homology to HYBID, named as transmembrane protein 2 (TMEM2) [alias cell migration-inducing hyaluronidase 2 (CEMIP2)] acts as cell-surface hyaluronidase in mice and is ubiquitously expressed in adult mouse tissues.21Shimizu H. Shimoda M. Mochizuki S. Miyamae Y. Abe H. Chijiiwa M. Yoshida H. Shiozawa J. Ishijima M. Kaneko K. Kanaji A. Nakamura M. Toyama Y. Okada Y. Hyaluronan-binding protein involved in hyaluronan depolymerization is up-regulated and involved in hyaluronan degradation in human osteoarthritic cartilage.Am J Pathol. 2018; 188: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar However, no studies have shown the expression or function of TMEM2 in human tissues such as synovium. Although it is reported that HYBID is expressed by rheumatoid synovial lining and sublining cells,17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar little information is available for tissue or cellular localization responsible for the expression in OA synovial tissue. In addition, the relationship between the expression levels of HYBID or TMEM2 and the size distribution of HA in OA synovial fluids remains unknown. Recent studies on the regulation of the HYBID gene expression have demonstrated that transforming growth factor-β1 (TGF-β1) down-regulates the expression in skin fibroblasts,18Nagaoka A. Yoshida H. Nakamura S. Morikawa T. Kawabata K. Kobayashi M. Sakai S. Takahashi Y. Okada Y. Inoue S. Regulation of hyaluronan (HA) metabolism mediated by HYBID (Hyaluronan-binding protein involved in HA depolymerization, KIAA1199) and HA synthases in growth factor-stimulated fibroblasts.J Biol Chem. 2015; 290: 30910-30923Crossref PubMed Scopus (60) Google Scholar and tumor necrosis factor-α (TNF-α) up-regulates the expression in OA chondrocytes.8Band P.A. Heeter J. Wisniewski H.-G. Liublinska V. Pattanayak C.W. Karia R.J. Stabler T. Balazs E.A. Kraus V.B. Hyaluronan molecular weight distribution is associated with the risk of knee osteoarthritis progression.Osteoarthritis Cartilage. 2015; 23: 70-76Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar However, little is known about regulators of HYBID expression in OA synovial fibroblasts. The present study found that HYBID, but not TMEM2, is up-regulated in OA synovial tissue. This study also shows that HYBID is implicated in degradation of HMW-HA in OA synovial fluids, and that among the factors examined, IL-6 efficiently up-regulates the HYBID expression in OA synovial fibroblasts. Synovial tissue samples were obtained at the time of arthroplasty from joints of patients with knee OA (n = 27; mean ± SD age, 75.7 ± 7.3 years), which was diagnosed according to the criteria of the American College of Rheumatology.22Altman R. Asch E. Bloch D. Bole G. Borenstein D. Brandt K. Christy W. Cooke T.D. Greenwald R. Hochberg M. Howell D. Kaplan D. Koopman W. Longley III, S. Mankin H. McShane D.J. Medsger Jr., T. Meenan R. Mikkelsen W. Moskowitz R. Murphy W. Rothschild B. Segal M. Sokoloff L. Wolfe F. Development of criteria for the classification and reporting of osteoarthritis. Classification of osteoarthritis of the knee. Diagnostic and Therapeutic Criteria Committee of the American Rheumatism Association.Arthritis Rheum. 1986; 29: 1039-1049Crossref PubMed Scopus (5285) Google Scholar Normal control synovial tissue samples were obtained at reconstruction surgery for anterior cruciate ligament from knee joints of patients with anterior cruciate ligament injury (n = 8; mean ± SD age, 37.3 ± 11.0 years). The OA synovial tissues were fixed with 4% paraformaldehyde and embedded in paraffin. Paraffin sections were stained with hematoxylin and eosin. Written informed consent was obtained from the patients for the experimental use of the surgical samples according to the hospital ethics guidelines. The study protocols, which complied with the principles outlined in the Declaration of Helsinki, were approved by the Ethical Committee Review Board in Juntendo University (No 15-074). Total RNA was isolated from OA and normal control synovial tissues using RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized from 2 μg of total RNA using the ReverTra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). The cDNA was amplified by quantitative real-time PCR using the THUNDERBIRD SYBR qPCR Mix (Toyobo) on a QuantStudio3 (Applied Biosystems, Foster City, CA). Nucleotide sequences for the primers were as follows: for HYBID 5′-TCACAGAGGACTCCTACCCG-3′ (forward) and 5′-ATTGGCCATCCAGAAGGTGG-3′ (reverse); and for TMEM2 5′-TTACGGCTTTCAGGGTGGTC-3′ (forward) and 5′-TTGGGAACGTCCTGTTCCTG-3′ (reverse). To examine the expression levels of HYBID and TMEM2 in synovial tissues, absolute quantification of mRNA copy numbers was performed using the standard curve method according to the methods described by Whelan et al.23Whelan J.A. Russell N.B. Whelan M.A. A method for the absolute quantification of cDNA using real-time PCR.J Immunol Methods. 2003; 278: 261-269Crossref PubMed Scopus (599) Google Scholar Briefly, purified plasmids containing the coding sequences of human HYBID [pcDNA3.1(−)-HYBID]17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar and human TMEM2 (RC224793; Origene, Rockville, MD) were digested with restriction enzymes, that is, EcoRI and NdeI (TAKARA, Kusatsu, Japan) for the HYBID plasmid and FauI (New England Biolabs, Ipswich, MA) for the TMEM2 plasmid, to cut out the inserts from the plasmids, both of which were purified using the QIAquick Nucleotide Removal Kit (Qiagen). Concentrations of the plasmid and insert were determined using the QuantiFluor dsDNA System (Promega, Madison, WI), and copy numbers were calculated according to the following formula: copy number (g/molecule) = (base pairs size of double-stranded plasmid containing the insert) × (330 Daltons × 2 nucleotides/base pairs) ÷ (Avogadro number 6.023 × 1023 molecules/mole). Standard curves of plasmid DNA containing the coding regions of HYBID and TMEM2 were generated with threshold cycle (CT) values obtained from quantitative real-time PCR of various copy numbers of the plasmids. The copy numbers of HYBID and TMEM2 molecules present in the synovial tissue samples were calculated by relating the PCR signal (CT value) of each synovial tissue sample to the standard curves. Paraffin sections of OA synovial tissues were autoclaved at 121°C for 10 minutes in Target Retrieval Solution (DAKO Cytomation, Tokyo, Japan) for antigen retrieval, and nonspecific reactions were blocked with BLOCK ACE (DS Pharma Biomedical, Osaka, Japan). The sections were also treated with 3% H2O2 to block peroxidase reactions. They were then immunostained with rabbit anti-HYBID (KIAA1199) antibody (SAB2105467; Sigma-Aldrich, St. Louis, MO), rabbit anti–IL-6 antibody (ab6672; Abcam, Cambridge, UK) or nonimmune control IgG (X0936; DAKO Cytomation), followed by incubation with biotinylated antibody against rabbit IgG according to the ABC method (Vector Laboratories, Burlingame, CA). Color was developed with 3-amino-9-ethylcarbazole chromogen (Vector Laboratories), and sections were counterstained with hematoxylin. Specificity of anti-HYBID (KIAA1199) antibody (SAB2105467; Sigma-Aldrich) was confirmed by the absorption test by incubating the antibody with the peptide antigen (KIAA1199 Blocking Peptide, 33R-7080; Fitzgerald Industries International, Acton, MA) prior to immunohistochemistry (data not shown). Paraffin sections of OA synovial tissues were subjected to antigen retrieval with Target Retrieval Solution (DAKO Cytomation), and nonspecific reactions and autofluorescence were blocked with BLOCK ACE (DS Pharma Biomedical). They were double-immunostained with mouse anti-CD68 antibody (clone PG-M1, M0876; DAKO Cytomation) and rabbit anti-HYBID (KIAA1199) antibody (SAB2105467; Sigma-Aldrich) or rabbit anti–IL-6 antibody (ab6672; Abcam, Cambridge, UK), followed by incubation with anti-mouse IgG antibody conjugated to Alexa Fluor 488 or anti-rabbit IgG antibody conjugated to Alexa Fluor 546 (Life Technologies, Carlsbad, CA). Similarly, double-immunofluorescence staining was performed with rabbit anti-HYBID (KIAA1199) antibody (SAB2105467; Sigma-Aldrich) and mouse anti–fibroblast-specific protein 1 (S100A4) antibody (ab218511; Abcam), followed by incubation with anti-rabbit IgG antibody conjugated to Alexa Fluor 546 or anti-mouse IgG antibody conjugated to Alexa Fluor 488 (Life Technologies). Nuclei were stained with Hoechst 33342 (Thermo Fisher Scientific, Tokyo, Japan), and images were obtained with a confocal microscope TCS-SP5 (Leica, Wetzlar, Germany). HA in synovial fluid from OA patients was determined using QnE Hyaluronic Acid enzyme-linked immunosorbent assay (ELISA) (Biotech Trading Partners, Encinitas, CA) according to the manufacture's protocol. The assay detected HA molecular weight forms as small as 20 to 30 kDa. The molecular weight of HA in OA synovial fluid was determined by size-exclusion gel filtration using Shodex OHpak SB-807 columns (Showa Denko, Tokyo, Japan). Fractions (0.5 mL) were collected, and the HA content was determined as described previously.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar The column was calibrated with HA species: sodium HA H2 (2500 kDa), M2 (1000 kDa), and L2 (150 kDa), all of which were purchased from PG Research (Tokyo, Japan). The HYBID protein concentrations in OA synovial fluids were evaluated by densitometric analysis of immunoblotting data according to a standard curve determined using recombinant HYBID.17Yoshida H. Nagaoka A. Kusaka-Kikushima A. Tobiishi M. Kawabata K. Sayo T. Sakai S. Sugiyama Y. Enomoto H. Okada Y. Inoue S. KIAA1199, a deafness gene of unknown function, is a new hyaluronan binding protein involved in hyaluronan depolymerization.Proc Natl Acad Sci U S A. 2013; 110: 5612-5617Crossref PubMed Scopus (174) Google Scholar Synovial fibroblasts were isolated from human OA synovium by enzymatic dissociation and cultured in Dulbecco's modified Eagle's medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum according to previously published methods.24Takizawa M. Ohuchi E. Yamanaka H. Nakamura H. Ikeda E. Ghosh P. Okada Y. Production of tissue inhibitor of metalloproteinases 3 is selectively enhanced by calcium pentosan polysulfate in human rheumatoid synovial fibroblasts.Arthritis Rheum. 2000; 43: 812-820Crossref PubMed Scopus (44) Google Scholar,25Fujita Y. Shiomi T. Yanagimoto S. Matsumoto H. Toyama Y. Okada Y. Tetraspanin CD151 is expressed in osteoarthritic cartilage and is involved in pericellular activation of pro-matrix metalloproteinase 7 in osteoarthritic chondrocytes.Arthritis Rheum. 2006; 54: 3233-3243Crossref PubMed Scopus (32) Google Scholar They were used for experiments at passages 1 to 4 (P1 to P4). Serum-starved OA synovial fibroblasts at P3 were treated with histamine (Wako, Osaka, Japan), TGF-β1 (R&D Systems, Minneapolis, MN), IL-6 (R&D Systems) plus soluble IL-6 receptor (sIL-6R) (R&D Systems), IL-1α (R&D Systems), IL-8 (R&D Systems), TNF-α (R&D Systems), insulin-like growth factor-1 (IGF-1) (Sigma-Aldrich), vascular endothelial growth factor165 (VEGF165) (R&D Systems), basic fibroblast growth factor (bFGF) (Sigma-Aldrich), prostaglandin E2 (PGE2) (Sigma-Aldrich), tumor necrosis factor-β (TNF-β) (R&D Systems), and C-C motif chemokine ligand 5 (CCL5) (R&D Systems) or vehicle alone in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum for 24 hours. The expression levels of HYBID and TMEM2 were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and determined by a SYBR Green real-time PCR assay (Invitrogen, Carlsbad, CA) according to the ΔΔCT method.26Chijiiwa M. Mochizuki S. Kimura T. Abe H. Tanaka Y. Fujii Y. Shimizu H. Enomoto H. Toyama Y. Okada Y. CCN1 (Cyr61) is overexpressed in human osteoarthritic cartilage and inhibits ADAMTS-4 (aggrecanase 1) activity.Arthritis Rheumatol. 2015; 67: 1557-1567Crossref PubMed Scopus (26) Google Scholar Nucleotide sequences for the primers were as follows: for HYBID 5′-AGGGAAGCAGGTCAGAGTGA-3′ (forward) and 5′-TCTCGGCTACAGACCCAGAG-3′ (reverse); for TMEM2 5′-ACTTGGTGGCTGGCATGTTC-3′ (forward) and 5′-CATGAGCTGGGCCTGAGTTG-3′ (reverse); and for GAPDH 5′-GCACCGTCAAGGCTGAGAAC-3′ (forward) and 5′-TGGTGAAGACGCCAGTGGA-3′ (reverse). The expression of HYAL1, HYAL2, HAS1, HAS2, and HAS3 in IL-6–treated OA synovial fibroblasts was also examined according to the method described above using the primers as follows: for HYAL1 5′-CCCAAGGTTGCACAGCAAGA-3′ (forward) and 5′-ACTCAGTAGGAGTGCAAGGGCTGTA-3′ (reverse); for HYAL2 5′-ACCATGCACTCCCAGTCTACGTC-3′ (forward) and 5′-TCGCCAATGGTAGAGATGAGGTC-3′ (reverse); for HAS1 5′-ATCCTGCATCAGCGGTCCTC-3′ (forward) and 5′-CTGGTTGTACCAGGCCTCAAGAA-3′(reverse); for HAS2 5′-GTAGGCATCCAGCACTGGACAA-3′ (forward) and 5′-CCAGTAAATTCAGGCCACAGAACA-3′ (reverse); and for HAS3 5′-TTGCAATCCAGGCTGTTCTCA-3′ (forward) and 5′-GCTGTCCACCTTAGTGCTGGTTC-3′(reverse). In addition, OA synovial fibroblasts (P3) treated with IL-6 (0, 10, 50, or 100 ng/mL) in the presence of sIL-6R (100 ng/mL) for 48 hours were subjected to immunoblotting with anti-HYBID antibody (Proteintech, Rosemont, IL) and anti-GAPDH antibody (as a loading control) (ab125247; Abcam). Serum-starved OA synovial fibroblasts at P3 were treated with humanized anti–IL-6R antibody (tocilizumab; 25 μg/mL) (Actemra; Chugai Pharmaceutical Co., Tokyo, Japan) or nonimmune human IgG (25 μg/mL; R&D Systems) in Dulbecco's modified Eagle's medium containing 1% fetal bovine serum for 1 hour prior to stimulation with IL-6 (0 or 100 ng/mL) and sIL-6R (100 ng/mL) for 48 hours, and then subjected to immunoblotting with anti-HYBID antibody (Proteintech). The effect of tocilizumab on HYBID mRNA and protein expression in serum-starved OA synovial fibroblasts at P1 was also tested by SYBR Green real-time PCR and immunoblotting analyses of the cells treated with nonimmune human IgG (25 μg/mL) or tocilizumab (1, 5, or 25 μg/mL) for 24 hours and 48 hours, respectively. A knockdown experiment was performed using two different siRNAs designed to target HYBID and nonsilencing control RNAs, both of which were purchased from Thermo Fisher Scientific (Waltham, MA). OA synovial fibroblasts were transfected with these siRNAs using Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific), and used for the experiment at 48 hours after transfection. Knockdown of the HYBID expression was confirmed by immunoblotting with anti-HYBID antibody (Proteintech) and anti-GAPDH antibody (Abcam). Cellular HA-degrading activity was examined by culturing siRNA-transfected or IL-6–treated OA synovial fibroblasts in medium containing fluoresceinamine-labeled HA H1 (FA-HA H1; 10 μg/mL) for 48 hours and applying the harvested medium to a Sepharose CL-2B column (1 × 60 cm; GE Healthcare, Tokyo, Japan) equilibrated with 0.5 mol/L NaCl in distilled water.21Shimizu H. Shimoda M. Mochizuki S. Miyamae Y. Abe H. Chijiiwa M. Yoshida H. Shiozawa J. Ishijima M. Kaneko K. Kanaji A. Nakamura M. Toyama Y. Okada Y. Hyaluronan-binding protein involved in hyaluronan depolymerization is up-regulated and involved in hyaluronan degradation in human osteoarthritic cartilage.Am J Pathol. 2018; 188: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Calibration was performed using the FA-HA species including H1 (1800 kDa; average molecular size), M1 (1000 kDa), L1 (200 kDa), and S1 (50 kDa).21Shimizu H. Shimoda M. Mochizuki S. Miyamae Y. Abe H. Chijiiwa M. Yoshida H. Shiozawa J. Ishijima M. Kaneko K. Kanaji A. Nakamura M. Toyama Y. Okada Y. Hyaluronan-binding protein involved in hyaluronan depolymerization is up-regulated and involved in hyaluronan degradation in human osteoarthritic cartilage.Am J Pathol. 2018; 188: 2109-2119Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar Concentrations of HA in the medium fr
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