Inflammatory Cytokines Induce Production of CHI3L1 by Articular Chondrocytes
2005; Elsevier BV; Volume: 280; Issue: 50 Linguagem: Inglês
10.1074/jbc.m510146200
ISSN1083-351X
AutoresAnneliese D. Recklies, Hua Ling, Chantal White, Suzanne M. Bernier,
Tópico(s)Immune Response and Inflammation
ResumoElevated levels of CHI3L1 (chitinase-3-like protein 1) are associated with disorders exhibiting increased connective tissue turnover, such as rheumatoid arthritis, osteoarthritis, scleroderma, and cirrhosis of the liver. This secreted protein is not synthesized in young healthy cartilage, but is produced in cartilage from old donors or patients with osteoarthritis. The molecular processes governing the induction of CHI3L1 are currently unknown. To elucidate the molecular events involved in CHI3L1 synthesis, we investigated two models of articular chondrocytes: neonatal rat chondrocytes, which do not express CHI3L1, and human chondrocytes, which express CHI3L1 constitutively. In neonatal rat chondrocytes, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-α for as little as 1 h was sufficient for sustained induction up to 72 h afterward. Using inhibitors selective for the major signaling pathways implicated in mediating the effects of TNF-α and interleukin-1, only inhibition of NF-κB activation was effective in curtailing cytokine-induced expression, including after removal of the cytokine, indicating that induction and continued production of CHI3L1 are controlled mainly by this transcription factor. Inhibition of NF-κB signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-κB. Selective induction of CHI3L1 by cytokines acting through NF-κB coupled with the known restriction of the catabolic responses by CHI3L1 in response to these inflammatory cytokines represents a key regulatory feedback process in controlling connective tissue turnover. Elevated levels of CHI3L1 (chitinase-3-like protein 1) are associated with disorders exhibiting increased connective tissue turnover, such as rheumatoid arthritis, osteoarthritis, scleroderma, and cirrhosis of the liver. This secreted protein is not synthesized in young healthy cartilage, but is produced in cartilage from old donors or patients with osteoarthritis. The molecular processes governing the induction of CHI3L1 are currently unknown. To elucidate the molecular events involved in CHI3L1 synthesis, we investigated two models of articular chondrocytes: neonatal rat chondrocytes, which do not express CHI3L1, and human chondrocytes, which express CHI3L1 constitutively. In neonatal rat chondrocytes, the inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1 potently induced steady-state levels of CHI3L1 mRNA and protein secretion. Treatment of chondrocytes with TNF-α for as little as 1 h was sufficient for sustained induction up to 72 h afterward. Using inhibitors selective for the major signaling pathways implicated in mediating the effects of TNF-α and interleukin-1, only inhibition of NF-κB activation was effective in curtailing cytokine-induced expression, including after removal of the cytokine, indicating that induction and continued production of CHI3L1 are controlled mainly by this transcription factor. Inhibition of NF-κB signaling also abolished constitutive expression by human chondrocytes. Thus, induction and continued secretion of CHI3L1 in chondrocytes require sustained activation of NF-κB. Selective induction of CHI3L1 by cytokines acting through NF-κB coupled with the known restriction of the catabolic responses by CHI3L1 in response to these inflammatory cytokines represents a key regulatory feedback process in controlling connective tissue turnover. CHI3L1 (chitinase-3-like protein 1; also known as HC-gp39 and YKL40) has been linked to both rheumatoid arthritis and osteoarthritis (1Johansen J.S. Stoltenberg M. Hansen M. Florescu A. Horslev-Petersen K. Lorenzen I. Price P.A. Rheumatology (Oxf.). 1999; 38: 618-626Crossref PubMed Scopus (167) Google Scholar) Elevated levels of this protein are present in the sera and synovial fluids of patients with these diseases, and some association with disease progression has been observed (1Johansen J.S. Stoltenberg M. Hansen M. Florescu A. Horslev-Petersen K. Lorenzen I. Price P.A. Rheumatology (Oxf.). 1999; 38: 618-626Crossref PubMed Scopus (167) Google Scholar, 2Johansen J.S. Cintin C. Jorgensen M. Kamby C. Price P.A. Eur. J. Cancer. 1995; 31: 1437-1442Abstract Full Text PDF Scopus (132) Google Scholar, 3Johansen J.S. Jensen H.S. Price P.A. Br. J. Rheumatol. 1993; 32: 949-955Crossref PubMed Scopus (214) Google Scholar). In addition, increased serum levels of CHI3L1 have been linked to other disease states associated with increased tissue fibrosis, such as cirrhosis of the liver (4Johansen J.S. Moller S. Price P.A. Bendtsen F. Junge J. Garbarsch C. Henriksen J.H. Scand. J. Gastroenterol. 1997; 32: 582-590Crossref PubMed Scopus (109) Google Scholar) and scleroderma (5La M.G. D'Angelo S. Valentini G. J. Rheumatol. 2003; 30: 2147-2151PubMed Google Scholar). Although CHI3L1 is often found in inflammatory environments, the factors triggering its production in these pathological conditions are currently unknown. CHI3L1 is a 39-kDa glycoprotein secreted by articular chondrocytes (6Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar), synoviocytes (7Nyirkos P. Golds E.E. Biochem. J. 1990; 269: 265-268Crossref PubMed Scopus (112) Google Scholar), and differentiated macrophages (8Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). It is a member of a family of mammalian proteins belonging structurally to glycohydrolase family 18 (9Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar), which includes bacterial and vertebrate as well as invertebrate chitinases. The mammalian group of this family consists of catalytically active members (chitinase-1 or chitotriosidase and acidic mammalian chitinase) and several inactive ones (CHI3L1 and CHI3L2 in humans and YM1 and YM2 in mice). The structures of the human CHI3L1 (10Houston D.R. Recklies A.D. Krupa J.C. van Aalten D.M.F. J. Biol. Chem. 2003; 278: 30206-30212Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 11Fusetti F. Pijning T. Kalk K.H. Bos E. Dijkstra B.W. J. Biol. Chem. 2003; 278: 37753-37760Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar) and murine YM1 (12Sun Y.J. Chang N.C. Hung S.I. Chang A.C. Chou C.C. Hsiao C.D. J. Biol. Chem. 2001; 276: 17507-17514Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 13Tsai M.L. Liaw S.H. Chang N.C. J. Struct. Biol. 2004; 148: 290-296Crossref PubMed Scopus (35) Google Scholar) proteins have been solved, demonstrating the conservation of the chitinase structural framework in the inactive members of this protein family. However, although a lectin-like function has been predicted, no physiological ligands have as yet been identified. CHI3L1 is synthesized constitutively by isolated human articular chondrocytes or in cartilage explants (6Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar), whereas its secretion from macrophages in vitro is associated with differentiation preceding induction and secretion of chitotriosidase (8Krause S.W. Rehli M. Kreutz M. Schwarzfischer L. Paulauskis J.D. Andreesen R. J. Leukocyte Biol. 1996; 60: 540-545Crossref PubMed Scopus (168) Google Scholar). CHI3L1 is secreted from human articular cartilage explants or isolated human chondrocytes in culture, although its expression in vivo is restricted to older and osteoarthritic cartilage (14Volck B. Ostergaard K. Johansen J.S. Garbarsch C. Price P.A. Scand. J. Rheumatol. 1999; 28: 171-179Crossref PubMed Scopus (75) Google Scholar). Secretion of CHI3L1 from cartilage explants diminishes with time in culture, but renewed cutting of the explants appears to restore secretion levels (15Johansen J.S. Olee T. Price P.A. Hashimoto S. Ochs R.L. Lotz M. Arthritis Rheum. 2001; 44: 826-837Crossref PubMed Scopus (77) Google Scholar), suggesting that CHI3L1 production is an injury response of the tissue. However, the factors that induce this response or how production is maintained for a relatively long time period is not known. Synthesis of CHI3L1 was reported to be enhanced by insulin-like growth factor 1 in isolated guinea pig chondrocytes (16De Ceuninck F. Pastoureau P. Bouet F. Bonnet J. Vanhoutte P.M. J. Cell. Biochem. 1998; 69: 414-424Crossref PubMed Scopus (23) Google Scholar). However, like human chondrocytes, these cells produce CHI3L1 constitutively in culture; and thus, the effect may be pleiotropic. Some insight into possible physiological roles for CHI3L1 has been gained by the observation that this protein stimulates growth of connective tissue cells such as chondrocytes, synoviocytes, and skin fibroblasts (17Recklies A.D. White C. Ling H. Biochem. J. 2002; 365: 119-126Crossref PubMed Scopus (317) Google Scholar, 18De Ceuninck F. Gaufillier S. Bonnaud A. Sabatini M. Lesur C. Pastoureau P. Biochem. Biophys. Res. Commun. 2001; 285: 926-931Crossref PubMed Scopus (159) Google Scholar). CHI3L1 was reported to promote adhesion and migration of vascular endothelial cells (19Malinda K.M. Ponce L. Kleinman H.K. Shackelton L.M. Millis A.J. Exp. Cell Res. 1999; 250: 168-173Crossref PubMed Scopus (226) Google Scholar, 20Nishikawa K.C. Millis A.J. Exp. Cell Res. 2003; 287: 79-87Crossref PubMed Scopus (169) Google Scholar), suggesting a role in angiogenesis. De Ceuninck et al. (18De Ceuninck F. Gaufillier S. Bonnaud A. Sabatini M. Lesur C. Pastoureau P. Biochem. Biophys. Res. Commun. 2001; 285: 926-931Crossref PubMed Scopus (159) Google Scholar) also reported that CHI3L1 increases proteoglycan synthesis in guinea pig chondrocytes. In addition, CHI3L1 dampens the response of chondrocytes and synovial cells to the inflammatory cytokines tumor necrosis factor-α (TNF-α) 2The abbreviations used are: TNF-αtumor necrosis factor-αIL-1interleukin-1DMEMDulbecco's modified Eagle's mediumRTreverse transcriptionGAPDHglyceraldehyde 3-phosphate dehydrogenaseMAPKmitogen-activated protein kinaseERKextracellular signal-regulated kinasePI3Kphosphatidylinositol 3-kinaseJAKJanus kinase. and interleukin-1 (IL-1), decreasing the production of matrix metalloproteases and chemokines (21Ling H. Recklies A.D. Biochem. J. 2004; 380: 651-659Crossref PubMed Scopus (194) Google Scholar). These observations suggest that CHI3L1 may play a protective role in inflammatory environments, limiting degradation of the extracellular matrix and thus controlling tissue damage. However, increased levels of CHI3L1 may also contribute to the development of tissue fibrosis. tumor necrosis factor-α interleukin-1 Dulbecco's modified Eagle's medium reverse transcription glyceraldehyde 3-phosphate dehydrogenase mitogen-activated protein kinase extracellular signal-regulated kinase phosphatidylinositol 3-kinase Janus kinase. In contrast to human articular chondrocytes, neonatal rat articular chondrocytes do not express CHI3L1 in primary culture. These cells therefore provide an ideal system to study the regulation of CHI3L1 expression. We report here that synthesis of CHI3L1 is induced in rat articular chondrocytes by the inflammatory cytokines TNF-α and IL-1 and that this process is uniquely dependent on the activity of the transcription factor NF-κB. These results indicate that production of CHI3L1 is a component of the inflammatory response of articular chondrocytes. By feeding back to modulate the extent of the response of cells to such inflammatory cytokines, the regulated expression of CHI3L1 functions to limit the degradative response in connective tissue. Exogenous Factors, Plasmids, and Inhibitors—Cells and explants were treated with factors in serum-free RPMI 1640 medium (Invitrogen, Burlington, Ontario, Canada) containing 100 units/ml penicillin, 100 units/ml streptomycin, and 10 mm HEPES. Recombinant human TNF-α, recombinant human IL-6 and its soluble receptor, and recombinant mouse epidermal growth factor were obtained from Sigma (Mississauga, Ontario). Recombinant rat IL-1β, recombinant human IL-1β, and human parathyroid hormone-(1–34) were purchased from Pierce, R&D Systems (Minneapolis, MN), and Peptide Institute, Inc. (Louisville, KY), respectively. The vehicle for the cytokines and factors was phosphate-buffered saline and 1 mg/ml bovine serum albumin (Roche Diagnostics, Laval, Quebec, Canada). A constitutively expressed construct of wild-type IκB (pSVK3-IκB) and a dominant-negative form of IκB (pSVK3-IκB-2NΔ4) were a generous gift from Dr. J. Hiscott (Lady Davis Institute for Medical Research, McGill University) (22Algarte M. Nguyen H. Heylbroeck C. Lin R. Hiscott J. J. Virol. 1999; 73: 2694-2702Crossref PubMed Google Scholar). The specific antibody for IκB was purchased from New England Biolabs Inc. (Beverly, MA). Mouse anti-human NF-κB p65 and p50 monoclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Radiolabeled [32P]CTP was obtained from ICN Biomedicals (Aurora, OH), and the 3′-end labeling kits were from Amersham Biosciences (Montreal). In some experiments, pharmacologic inhibitors were used to selectively block individual signaling pathways. AG490, BAY 11-7085, BAY 11-7082, bisindolylmaleimide I, cyclosporin A, FK506, lactacystin, LY294002, PD98059, wortmannin, SN50, and inactive analogs of bisindolylmaleimide I (bisindolylmaleimide V) and U0126 (U0124) were purchased from EMD Biosciences (San Diego, CA). U0126 was obtained from Promega Corp. (Madison, WI). Cell Cultures—Chondrocytes were harvested from the distal femoral condyles of 1-day-old Sprague-Dawley rats as described previously (23Séguin C.A. Bernier S.M. J. Cell. Physiol. 2003; 197: 356-369Crossref PubMed Scopus (115) Google Scholar). Typically, 8–12 × 105 cells were obtained per condyle. Cells were cultured in RPMI 1640 medium supplemented with 100 units/ml penicillin, 100 units/ml streptomycin, 10 mm HEPES, and 5% fetal bovine serum (Invitrogen). Cells were plated at 450–550 cells/mm2 in 60-mm dishes (BD Biosciences, Mississauga). The medium was changed every 3 days and was replaced with serum-free medium 1 day prior to experiments except where indicated. All experiments were carried out on either primary or first passage cultures that retained expression of chondrocytic phenotypic markers. Fibroblast cultures were established by placing minced fragments of neonatal rat skin in culture dishes to allow outgrowth of fibroblasts, which occurs within a couple of days. Osteoblasts were prepared by timed sequential collagenase digestions of neonatal rat calvaria (24Bellows C.G. Aubin J.E. Heersche J.N. Antosz M.E. Calcif. Tissue Int. 1986; 38: 143-154Crossref PubMed Scopus (779) Google Scholar). Fractions 2/3 and 4/5 represent early and late stage osteoblasts, respectively. Skin fibroblasts and osteoblasts were seeded at 500 cells/mm2 and allowed to expand to near confluence before exposure to cytokines. Rat femoral heads were used for explant culture of intact articular cartilage. They were dissected clear of the ligamentum teres, washed three times with phosphate-buffered saline containing 100 units/ml penicillin and 100 units/ml streptomycin, and cultured in RPMI 1640 medium supplemented with 5% fetal bovine serum. Human juvenile chondrocytes had been prepared previously from knee cartilage obtained at autopsy (6Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar) and maintained as frozen stocks after one passage. The cells were revived and expanded in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 units/ml streptomycin. For expression studies, trypsinized cells were plated at 500 cells/mm2 and allowed to adhere for 48 h in serum-containing medium unless indicated otherwise. The human chondrocytes used in this study were between passages 3 and 5. Although type I collagen mRNA transcripts could be detected by reverse transcription (RT)-PCR, the cells still expressed mRNA for type II collagen and aggrecan, indicating that they retained a chondrocytic phenotype. RNA Extraction and RT-PCR—Confluent cultures were serum-deprived for 24 h prior to addition of exogenous factors in the presence or absence of pharmacologic inhibitors. Total RNA was collected from cells after 24 or 48 h using the acid/guanidium/phenol/chloroform extraction method (TRIzol, Invitrogen) according to the manufacturer's instructions. Levels of CHI3L1 transcripts were analyzed by RT-PCR using oligonucleotides CGCCCTCGACCATTCCCTGTGCACC (upstream primer) and TGTCCTGCTGGCCTCGGAAGAGG (downstream primer), giving rise to a 550-bp amplification product. Levels of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcripts were evaluated as housekeeping gene using upstream primer GCAGCCCAGAACATCATCCCTGCA and downstream primer CATTGTCATACCAGGAAATGAGCTT, resulting in a 350-bp amplification product. For analysis of rat MMP-13, oligonucleotides CTGCACCCTCAGCAGGTTGA and CTCATAGACAGCATCTACTTTGTC were used for upstream and downstream priming, respectively, resulting in a 372-bp amplification product. Expression of GRO-1 was assessed using upstream primer GGCAGGGATTCACTTCAAGA and downstream primer GCCATCGGTGCAATCTATCT, giving rise to a 205-bp product. All primers were synthesized in the Biotechnology Core Facility of the Shriners Hospital for Children. All primers were designed for a melting temperature of ∼60 °C to allow parallel amplification of different primer sets. RNA prepared from involuting rat mammary gland was used as a positive control for RT-PCR amplification of CHI3L1 because it is expressed at high levels in corresponding mouse tissue (25Morrison B.W. Leder P. Oncogene. 1994; 9: 3417-3426PubMed Google Scholar). Mammary glands from lactating rats were harvested from dams with litters, used by Dr. Lee (Shriners Hospital for Children, Montreal, Canada) in a study requiring harvesting of neonatal rat bones. Mammary gland tissue was collected when no pups remained, and the dams were killed. RNA was prepared from the tissue as described above. Collection and Analysis of Conditioned Culture Media—Confluent cultures of chondrocytes, fibroblasts, and osteoblasts or explant cultures of three femoral heads/well were serum-deprived for 24 h prior to addition of exogenous factors in the presence or absence of pharmacologic inhibitors. Culture media were collected after 24 or 48 h, and proteins were precipitated overnight by addition of 2 volumes of cold acetone. The precipitate was recovered by centrifugation. Pellets were redissolved in 0.1 volume of SDS sample buffer unless indicated otherwise, separated by 12% SDS-PAGE, and analyzed by Western blotting using a polyclonal antibody recognizing rat CHI3L1 prepared as described below. For detection of human CHI3L1, a polyclonal antibody raised against the purified protein was used as described previously (6Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar). Antibody dilution was 1:1000 unless stated otherwise. For detection of rat CHI3L1, a polyclonal antibody was prepared using the C-terminal peptide sequence CGGKEALAVA. The N-terminal sequence CGG was added to provide a linker for coupling to ovalbumin for immunization. All peptides were synthesized and purified in the Biotechnology Core Facility at the Shriners Hospital for Children. The antiserum was shown by Western blotting to be specific for rat CHI3L1; it did not react with the human or murine protein. The reactivity could be absorbed with the unconjugated peptide used for immunization, but not with C-terminal peptides based on the human and murine sequences. This antiserum was used for all Western blot analysis at 1000-fold dilution. Bound immunoglobulin was visualized by enhanced chemiluminescence (ECL, Amersham Biosciences, Baie d'Urfé, Quebec). To identify the protein species, bands were excised from the SDS-polyacrylamide gels and subjected to peptide fragmentation and analysis by mass spectrometry, performed at Génome Québec (Montreal). Transfection of Human Chondrocytes with IκB Constructs—The plasmids pSVK3-IκB, pSVK3-IκB-2NΔ4, and pCMVS-EGFP were transfected into human articular chondrocytes using Magnetofection™ (OZ Biosciences, Marseilles, France) to enhance transfection efficiency. Chondrocytes (5 × 105 cells/well) were seeded into 6-well plates and allowed to attach for 48 h in the presence of DMEM supplemented with 10% fetal bovine serum. The cell layers were washed three times with serum-free DMEM to remove residual serum components. 2.5 μg of freshly purified DNA (using a plasmid purification kit, Promega Corp.) was added to 0.2 ml of DMEM, followed by 2 μl of PolyMag II transfection reagent (OZ Biosciences). This mixture was added to 0.5 ml of DMEM covering the cell layer, and the plates were exposed to a magnetic field for 10 min at room temperature, followed by an additional 6-h incubation at 37 °C. The medium was replaced with fresh DMEM containing 10% fetal calf serum, and the cells were allowed to recover overnight. To determine levels of synthesis and secretion of CHI3L1, the cells were exposed to serum-free medium for 24 h. Responsiveness to TNF-α was determined by addition of 30 ng/ml TNF-α during the last 30 min of the culture period. Cell layers were harvested and analyzed for the presence of CHI3L1 and IκB by SDS-PAGE and Western blotting. Stimulation with TNF-α is expected to result in decreased cytoplasmic levels of IκB. The culture medium was collected at the end of the incubation period and prepared as described above for analysis of secreted CHI3L1. Analysis of NF-κB Binding to the Human CHI3L1 Promoter—Confluent human chondrocytes were treated with TNF-α (50 ng/ml) or IL-1β (10 ng/ml) in serum-free medium for 30 min, and nuclear extracts were prepared and analyzed for NF-κB binding by electrophoretic mobility shift assay as described by Sakai et al. (26Sakai T. Kambe F. Mitsuyama H. Ishiguro N. Kurokouchi K. Takigawa M. Iwata H. Seo H. J. Bone Miner. Res. 2001; 16: 1272-1280Crossref PubMed Scopus (60) Google Scholar). Cells were washed once with Ca2+- and Mg2+-free phosphate-buffered saline and then harvested with lysis buffer A (10 mm HEPES-KOH (pH 7.8), 10 mm KCl, 0.1 mm EDTA, 0.25% (v/v) Nonidet P-40, 1 mm dithiothreitol, and protease inhibitors (0.5 mm phenylmethylsulfonyl fluoride, 5 μm pepstatin, 10 μm leupeptin, and 1 mm sodium vanadate)). After centrifugation at 1200 rpm for 30 min at 4 °C, the nuclear pellets were washed once with 0.5 ml of Ca2+- and Mg2+-free phosphate-buffered saline and resuspended in an equal volume of lysis buffer B (50 mm HEPES-KOH (pH 7.8), 420 mm KCl, 0.1 mm EDTA, 5 mm MgCl2, and 2% (v/v) glycerol). Protein content was determined using the Bradford protein assay (Bio-Rad, Mississauga). Aliquots containing 10 μg of protein were incubated in the presence of 1 μm dithiothreitol and protease inhibitors as described above with 32P-labeled DNA fragments corresponding to positions -571 to -702 of the human CHI3L1 promoter (27Rehli M. Krause S.W. Andreesen R. Genomics. 1997; 43: 221-225Crossref PubMed Scopus (235) Google Scholar). The fragments were generated by PCR and 3′-end-labeled following the manufacturer's instructions. In control experiments, a 100-fold excess of unlabeled DNA fragment was added. Binding specificity was also investigated using a DNA fragment with a C-to-G substitution in the consensus sequence GGGAATTTCCC of the NF-κB/Rel DNA-binding motif at the underlined position. This fragment was generated by site-directed mutagenesis using the overlap extension method (28Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6833) Google Scholar). To identify NF-κB subunits, a supershift analysis was performed by adding 1 μg of anti-human NF-κB p65 or p50 antibody or an unrelated IgG to the incubation mixtures. All samples were incubated for 16 h at 4 °C and analyzed by electrophoresis on 6% (w/v) native polyacrylamide gels. The gels were dried and exposed overnight to Hyperfilm (Amersham Biosciences) at -80 °C. Induction and Secretion of CHI3L1 from Isolated Rat Articular Chondrocytes—Synthesis and secretion of CHI3L1 are detectable almost immediately following isolation of human articular chondrocytes (6Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar). However, upon screening a large number of cellular RNA preparations from primary neonatal rat chondrocytes, no evidence for its presence was detected. However, neonatal rat chondrocytes are responsive to various pro-inflammatory cytokines. As CHI3L1 is often found in inflammatory environments, neonatal rat chondrocytes therefore provide an excellent model system in which to characterize induction of CHI3L1 production by cytokines. Rat chondrocytes were treated with factors associated with inflammation (IL-1β, IL-6, TNF-α, and epidermal growth factor) or with a factor involved in cartilage development (parathyroid hormone). Of the cytokines tested, only TNF-α and IL-1β induced mRNA expression for CHI3L1 (Fig. 1A). IL-6 in the presence of its soluble receptor (parathyroid hormone) and epidermal growth factor at concentrations known to elicit cellular responses in these cells were not effective in increasing CHI3L1 transcript levels (data not shown). CHI3L1 mRNA was clearly detectable 24 h after addition of TNF-α, and levels remained elevated at 48 h, indicating that this is not a transient phenomenon. Low levels of CHI3L1 transcripts were detectable in cultures maintained for 48 h in serum-free medium. TNF-α and IL-1β also induced CHI3L1 expression in osteoblast cultures (prepared from neonatal rat calvaria) at both early and late stages of differentiation (Fig. 1B), but not in neonatal rat skin fibroblasts (Fig. 1C). Levels of transcripts for both MMP-13 and the chemokine GRO-1 (the rodent equivalent of IL-8), known targets of TNF-α and IL-1β, were increased following exposure to the cytokines, indicating that the lack of CHI3L1 induction is not a consequence of a lack of response of the fibroblasts to either TNF-α or IL-1β. To determine whether the stimulatory effect of TNF-α and IL-1 at the gene level translates to increased secretion of CHI3L1 protein, an antibody was raised against the C-terminal peptide of rat CHI3L1. This antibody detected a protein doublet migrating at ∼39 kDa in the culture medium from cells stimulated with TNF-α or IL-1β, but not in control medium (Fig. 2, A and B). The immune reactivity could be absorbed with the immunizing peptide, but not with similar peptides from the C terminus of either mouse or human CHI3L1 (data not shown). In addition, analysis by peptide fragmentation and mass spectrometry indicated that both bands were indeed rat CHI3L1. The lower molecular mass band may represent a glycosylation variant or an unglycosylated form of the protein. Based on these observations, the question arose as to whether or not CHI3L1 secretion could be induced in intact cartilage. Femoral heads from neonatal rats consisting mainly of epiphyseal and articular cartilage were harvested and cultured in the presence or absence of cytokines. Culture media were analyzed for secreted CHI3L1 by Western blotting (Fig. 2C). As was observed for the isolated cells, no protein was detectable at 24 or 48 h in control cultures. Both TNF-α and IL-1β induced production of the protein in femoral heads, and secretion was maintained over a 48-h culture period. Thus, the capacity of cytokines to initiate synthesis of CHI3L1 did not result from removal of the chondrocytes from their intact tissue environment, but is an inherent response of these cells in their native environment. Short Exposure to Cytokines Is Sufficient for Prolonged CHI3L1 Production—As we have previously found that short exposures of chondrocytes to TNF-α (1–4 h) result in persistent activation of NF-κB signaling pathways and reduction of type II collagen and link protein synthesis in rat chondrocytes (23Séguin C.A. Bernier S.M. J. Cell. Physiol. 2003; 197: 356-369Crossref PubMed Scopus (115) Google Scholar), the requirement for the continued presence of cytokines with respect to CHI3L1 production was investigated. Isolated rat chondrocytes were exposed to TNF-α for 4 h, followed by removal of the cytokine and continuation of culture in unsupplemented medium. Cells were harvested at 24, 48, or 72 h and analyzed for the presence of CHI3L1 mRNA by RT-PCR (Fig. 3A). Elevated transcript levels were evident up to 72 h after removal of the cytokine. Similarly, CHI3L1 protein levels increased between 48 and 72 h (Fig. 3B), suggesting that the cells continued to secrete the protein. CHI3L1 synthesis was induced after only 1 h of exposure to cytokines, as reflected by the presence of the protein in the culture medium harvested 24 h after cytokine removal, and the increased production was maintained for at least 48 h (Fig. 3C). To facilitate detection of CHI3L1 protein, media were concentrated 20-fold for this series of experiments. Similar results were obtained for IL-1β, although this cytokine appeared less efficient after a 1-h exposure compared with TNF-α, with which the amount of secreted protein appeared similar after 1- and 4-h exposures (given the quantitative limitations of the method of analysis). Low levels of CHI3L1 protein were detectable in the control culture medium from untreated cells at 48 h, but not at 24 h, consistent with induction of mRNA (Fig. 3A). These results demonstrate that a short pulse of cytokine exposure induces a long-term effect w
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