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• W2019956407 abstract "Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels. Members of chitinase-like proteins (CLPs) have attracted much attention because of their ability to promote cell proliferation in insects (imaginal disc growth factors) and mammals (YKL-40). To gain insights into the molecular processes underlying the physiological control of growth and development in Lophotrochozoa, we report here the cloning and biochemical characterization of the first Lophotrochozoan CLP from the oyster Crassostrea gigas (Cg-Clp1). Gene expression profiles monitored by real time quantitative reverse transcription-PCR in different adult tissues and during development support the involvement of this protein in the control of growth and development in C. gigas. Recombinant Cg-Clp1 demonstrates a strong affinity for chitin but no chitinolytic activity, as was described for the HC-gp39 mammalian homolog. Furthermore, transient expression of Cg-Clp1 in primary cultures of rabbit articular chondrocytes as well as the use of both purified recombinant protein and conditioned medium from Cg-Clp1-expressing rabbit articular chondrocytes established that Cg-Clp1 stimulates cell proliferation and regulates extracellular matrix component synthesis, showing for the first time a possible involvement of a CLP on type II collagen synthesis regulation. These observations together with the fact that Cg-Clp1 gene organization strongly resembles that of its mammalian homologues argue for an early evolutionary origin and a high conservation of this class of proteins at both the structural and functional levels. Growth factors orchestrate growth and development in metazoan organisms. Despite the huge variety of growth factors characterized in vertebrates, only a few have been identified in Protostome lineages and are mainly restricted to the Ecdysozoan model organisms Caenorhabditis elegans and Drosophila melanogaster. Thus, the third branch of bilaterian, named Lophotrochozoa, is an obviously understudied group of animals, since none of the major model organisms presently belong to this clade (1Adoutte A. Balavoine G. Lartillot N. Lespinet O. Prud'homme B. de Rosa R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4453-4456Crossref PubMed Scopus (427) Google Scholar). Recent reports show that Lophotrochozoan animals exhibit biological characteristics that are considered ancestral, or at least less derived from the ancestral state than in other established systems that are known (2Tessmar-Raible K. Arendt D. Curr. Opin. Genet. Dev. 2003; 13: 331-340Crossref PubMed Scopus (84) Google Scholar). Since bivalve mollusks belong to the Lophotrochozoa, they are good candidates to characterize growth factors and then contribute to our understanding of the evolution of growth and developmental regulations in bilaterian animals. Furthermore, a better knowledge of the molecular control of mollusk physiology may help improve the hatchery production of these economically important animals. Unfortunately, the number of growth factor genes identified so far at the molecular level in bivalve mollusks is extremely limited. In the pacific oyster, Crassostrea gigas, only four members of the transforming growth factor-β superfamily and their corresponding receptors have already been described (3Lelong C. Mathieu M. Favrel P. Eur. J. Biochem. 2000; 267: 3986-3993Crossref PubMed Scopus (51) Google Scholar, 4Herpin A. Badariotti F. Rodet F. Favrel P. Biochim. Biophys. Acta. 2004; 1680: 137-144Crossref PubMed Scopus (18) Google Scholar, 5Herpin A. Lelong C. Becker T. Rosa F.M. Favrel P. Cunningham C. Mech. Dev. 2005; 122: 695-705Crossref PubMed Scopus (28) Google Scholar, 6Herpin A. Lelong C. Favrel P. Dev. Comp. Immunol. 2004; 28: 461-485Crossref PubMed Scopus (169) Google Scholar). This lack of knowledge is above all the consequence of both the paucity of genomic sequence information and the difficulty in developing functional bioassays in these anatomically complex animals. Many growth factors are considered to be phylogenetically conserved between Deuterostomes and Protostomes. Examples include the superfamilies of insulin-related peptides (7Smit A.B. Vreugdenhil E. Ebberink R.H.M. Geraerts W.P.M. Klootwijk J. Joose J. Nature. 1988; 331: 535-538Crossref PubMed Scopus (231) Google Scholar), epidermal growth factor-related proteins (8Hermann P.M. van Kesteren R.E. Wildering W.C. Painter S.D. Reno J.M. Smith J.S. Kumar S.B. Geraerts W.P. Ericsson L.H. Smit A.B. Bulloch A.G. Nagle G.T. J. Neurosci. 2000; 20: 6355-6364Crossref PubMed Google Scholar), and transforming growth factor-β-related proteins (6Herpin A. Lelong C. Favrel P. Dev. Comp. Immunol. 2004; 28: 461-485Crossref PubMed Scopus (169) Google Scholar). This opens the possibility of identifying growth factor-like molecules in oysters by homology searching. Among growth factors, some of them are more attractive, since they are able to promote cell proliferation. Except for one growth factor family isolated in D. melanogaster (9Kawamura K. Shibata T. Saget O. Peel D. Bryant P.J. Development. 1999; 126: 211-219Crossref PubMed Google Scholar), no proliferating polypeptides have been shown to have direct mitogenic activity in invertebrates. These new soluble mitogenic growth factors named imaginal disc growth factors (IDGFs) 2The abbreviations used are: IDGF, imaginal disc growth factor; CLP, chitinase-like protein; RACE, rapid amplification of cDNA ends; MF1, microfilaria-1; CHO-K1, Chinese hamster ovary, line K1; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; PG, proteoglycan; GAG, glycosaminoglycan; GH, glycosyl hydrolase; RAC, rabbit articular chondrocyte(s); pNPG, p-nitrophenyl β-d-N-acetylglucosamine; PMSF, phenylmethylsulfonyl fluoride; RT, reverse transcription; FCS, fetal calf serum. cooperate with insulin to stimulate proliferation, polarization, and mobility of imaginal disc cells (9Kawamura K. Shibata T. Saget O. Peel D. Bryant P.J. Development. 1999; 126: 211-219Crossref PubMed Google Scholar). These proteins are structurally related to chitinases but show an amino acid substitution that is known to abrogate catalytic activity. This suggests that they may have evolved from chitinases and have subsequently acquired a new growth-promoting function that does not require chitinase catalytic activity. Interestingly, homologues of chitinase-like proteins (CLPs) have also been described in mammals. Among these factors, human HC-gp39 protein (human cartilage glycoprotein-39) and its mammalian orthologues (YKL-40) also display mitogenic activity. Recklies et al. (10Recklies A.D. White C. Ling H. Biochem. J. 2002; 365: 119-126Crossref PubMed Scopus (317) Google Scholar) have recently reported that purified HC-gp39 stimulates growth of connective tissue cells in concentration ranges similar to those effective for insulin-like growth factor-1 (10Recklies A.D. White C. Ling H. Biochem. J. 2002; 365: 119-126Crossref PubMed Scopus (317) Google Scholar). This mitogenic activity is mediated by signaling through the mitogen-activated protein kinase and the phosphatydylinositol 3-kinase pathways. Prominent sites of HC-gp39 production are observed in the degenerated articular cartilage and inflamed or hyperplastic synovium (11Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar), fibrotic liver tissue (12Tran A. Benzaken S. Saint-Paul M.C. Guzman-Granier E. Hastier P. Pradier C. Barjoan E.M. Demuth N. Longo F. Rampal P. Eur. J. Gastroenterol. Hepatol. 2000; 12: 989-993Crossref PubMed Scopus (87) Google Scholar), and gliomas, where a correlation of HC-gp39 production with malignancy has been reported (13Tanwar M.K. Gilbert M.R. Holland E.C. Cancer Res. 2002; 62: 4364-4368PubMed Google Scholar). Based on the reported tissue distribution, a role of HC-gp39 in tissue remodeling has been proposed (11Hakala B.E. White C. Recklies A.D. J. Biol. Chem. 1993; 268: 25803-25810Abstract Full Text PDF PubMed Google Scholar, 14Sun 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). The guinea pig orthologue stimulates synthesis of the cartilage matrix component, aggrecan, in chondrocytes, in addition to cell growth (15De 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), suggesting that this protein acts generally to promote anabolic events in connective tissues. With mammalian and insect homologues, the CLP family seems to be phylogenetically conserved at both the structural and functional levels. Nevertheless, no member of this singular mitogenic growth factor family is identified at the moment in Lophotrochozoa. The characterization of homologous genes in this clade should help decipher the functionalities of this important but poorly understood family of growth factors. In this paper, we report the structural and functional characterization of the first Lophotrochozoan “chitinase-Like protein” from the oyster C. gigas. Cg-Clp1 (C. gigas chitinase-like protein 1) tissue distribution and temporal pattern of expression was established by real time PCR in order to assess its biological function in vivo. Phylogenetic analysis, gene structure comparison with homologues, and biochemical properties of Cg-Clp1 recombinant protein indicate that Cg-Clp1 is more closely related to mammalian YKL-40 than to insect IDGFs. Furthermore, we show that Cg-Clp1 activities on mammalian chondrocytes are identical to its mammalian closest homolog YKL-40. For the first time, the involvement of a CLP on type II collagen synthesis regulation was also demonstrated. Adult oysters C. gigas were purchased from a local oyster farm (Normandie, France). Embryo and larval stages were produced in the IFREMER shellfish laboratory of Argenton (France). Reverse transcription was carried out using oligo(dT)17 as primer, 1 μg of mRNA and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega). cDNAs were used as templates for PCR amplifications using two degenerated primers designed to anneal to conserved consensus regions of Drosophila imaginal disc growth factors. The sense primer corresponding to the LK(I/M)L(F/L)(S/T/R/C/W)VGG amino acid sequence was 5′-CTN AAR ATN CTN YTN WSN GTN GGN GG-3′, whereas the antisense primer corresponding to the FDGLDLA amino acid sequence was 5′-GGC NAG RTC NAG NCC RTC RAA-3′ (where Y represents C or T, R is A or G, S is C or G, W is A or T, and N is A, C, G, or T). PCR was performed in a total volume of 50 μl with 10 ng of mantle edge cDNA in 10 mm Tris/HCl, pH 9.0, containing 50 mm KCl, 0.1% Triton X-100, 0.2 mm each dNTP, 1 μm each primer, 2.5 mm MgCl2, and 1 unit of TaqDNA polymerase (Eurogentec). The reaction was cycled between 94, 50, and 72 °C (45, 60, and 90 s, respectively), followed by an extension step at 72 °C for 5 min. After 40 cycles, a resulting 147-bp fragment was isolated. Full-length cDNA was generated by 5′ and 3′ RACE using the Marathon cDNA amplification kit (Clontech). Double-stranded cDNA from oyster mantle edges was ligated to adaptors, and 25 ng of this template was used to PCR-amplify 5′- and 3′-RACE fragments using adaptor-specific primers and gene-specific primers deduced from the initial 147-bp fragment sequence. PCR products were subcloned into pGEM-T easy vector using a TA cloning kit (Promega) and sequenced using ABI cycle sequencing chemistry. Sequences used in the phylogenetic analyses were chosen to represent a range of chitinases and chitinase-like family proteins from both Ecdysozoans and Deuterostomes. The sequences were aligned using ClustalX version 1.81 and by manual inspection. From these alignments, distance-based phylogenetic trees were constructed using the minimum evolution method of the PAUP (Phylogenetic Analysis Using Parsimony) package version 4.0b4a. One thousand bootstrap trials were run using the neighbor-joining algorithm for each node. Quantitative RT-PCR analysis was performed using the iCycler apparatus (Bio-Rad). Total RNA was isolated from adult tissues using Tri-Reagent (Sigma) according to the manufacturer's instructions. After treatment for 20 min at 37 °C with 1 unit of DNase I (Sigma) to prevent genomic DNA contamination, 1 μg of total RNA was reverse transcribed using 1 μg of random hexanucleotidic primers (Promega), 0.5 mm dNTPs and 200 units of Moloney murine leukemia virus reverse transcriptase (Promega) at 37 °C for 1 h in the appropriate buffer. The reaction was stopped by incubation at 70 °C for 10 min. The iQ™ SYBR Green super-mix PCR kit (Bio-Rad) was used for real time monitoring of amplification (5 ng of cDNA template, 40 cycles: 95 °C/15s, 60 °C/15s) with the following primers: QsCgClp1 (5′-CTTCCTCCGCTTCCATGA-3′) and QaCgClp1 (5′-CCATGAAGTCCGCGAATC-3′) as sense and antisense primers, respectively. Accurate amplification of the target amplicon was checked by performing a melting curve. Using QsGAPDH (5′-TTCTCTTGCCCCTCTTGC-3′) and QaGAPDH (5′-CGCCCAATCCTTGTTGCTT-3′) or QsACT (5′-GCCCTGGACTTCGAACAA-3′) and QaACT (5′-CGTTGCCAATGGTGATGA-3′) primers, respectively, a parallel amplification of oyster glyceraldehyde-3-phosphate dehydrogenase transcript (EMBL CGI548886) or oyster actin transcript (AF026063) was carried out to normalize the expression data of the Cg-Clp1 transcript. The relative level of Cg-Clp1 expression was calculated for 100 copies of the reference housekeeping genes by using the following formula: n = 100 × 2(Ct reference – Ct Cg-Clp1). COL2A1 expression was performed using the following primers: COL2A1 sense, 5′-GGC AAT AGC AGG TTC ACG TAC A-3′; COL2A1 antisense, 5′-CGA TAA CAG TCT TGC CCC ACT T-3′. Comparison of COL2A1 expression is based on a comparative CT method (ΔΔCT) (16Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (124899) Google Scholar), and the relative COL2A1 expression can be quantified according to the formula of 2–ΔΔCT with 18 S RNA used as the internal control gene. A genomic library of C. gigas was constructed in λ-DASHII (Stratagene) according to the manufacturer's instructions. A total of 1.8 × 106 independent clones were recovered (17Herpin A. Favrel P. Cunningham C. Gene (Amst.). 2002; 301: 21-30Crossref PubMed Scopus (26) Google Scholar). After amplification, a total of 105 recombinant λ-DASH phages were plated, adsorbed to nylon membranes, and screened at high stringency with a random primed 32P-labeled full-length cDNA (specific activity 109 dpm/μg). Positive clones were purified as described by Sambrook (18Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY1989Google Scholar), subjected to restriction analysis and Southern blot hybridization using the original probe to confirm that the λ-DASH clones contained Cg-Clp1-specific genomic sequences. The genomic organization of the Cg-Clp1 gene was subsequently determined by sequencing subcloned positive genomic fragments. Exon/intron boundaries were determined by comparing the genomic sequence to the Cg-Clp1 cDNA sequence. A 1420-bp fragment corresponding to the most 3′ end of Cg-Clp1 was subcloned in pGEMT easy. This recombinant plasmid was used as a template for the synthesis of biotin-labeled sense and antisense cRNA probes according to the manufacturer's instructions (PerkinElmer Life Sciences). Dissected C. gigas mantle edge was fixed, dehydrated in increasing alcohol series and xylene, and embedded in paraplast. Seven-μm sections were cut and mounted on aminosilane-coated slides. Sections were rehydrated, and endogenous peroxidase activity was blocked by incubating sections in 0.3% hydrogen peroxide in methanol for 30 min at room temperature. Slides were then washed and incubated in a blocking solution according to the manufacturer's instructions. Hybridization was performed overnight at 55 °C. Biotin-labeled probes were detected using a streptavidin-horseradish peroxidase conjugate. Peroxidase activity was revealed by diaminobenzidine substrate (Sigma). Cg-Clp1 cDNA coding region (without signal sequence) was generated by PCR using a gene-specific sense primer containing a BamHI site and an antisense primer containing a HindIII site. Both the pQE30 expression vector (Qiagen) and PCR-amplified Cg-Clp1 cDNA fragment were digested by BamHI and HindIII, purified, ligated, and subsequently transfected into a XL1 Blue MRF′ strain (Stratagene). Correct insertion of the coding fragment was checked by sequencing. Selected recombinant colonies were propagated in LB containing 50 μg/ml of ampicillin at 37 °C. When culture grew at logarithmic phase, an optimum concentration of 0.1 mm isopropyl-β-d-1-thiogalactopyranoside was added to induce the expression of the Cg-Clp1 gene. After 3 h of induction, the cultured cells were harvested by centrifugation, lysed overnight with a denaturation buffer containing 20 mm NaHPO4/Na2PO4, pH 8,500 mm NaCl, and 8 m urea. After centrifugation for 10 min at 10,000 × g, supernatant containing Cg-Clp1 was analyzed by SDS-PAGE. Flp-In™-CHO cells (Invitrogen) used in this study harbor in their genome an FRT site, which allows an efficient Flp recombinase-mediated DNA integration of an FRT-tagged construction plasmid. These cells were transfected according to the manufacturer's instructions in 10-cm2 culture dishes with a mixture of Lipofectamine 2000 (10 μl) in serum-free Ham's F-12 medium and 4 μg of plasmid DNA (3.6 μg of pOG44 Flp recombinase expression vector (Invitrogen) and 0.4 μg of the expression plasmid pSecTag-CgClp1). pSeTag-CgClp1 was obtained by cloning Cg-Clp1 coding sequence (without signal sequence) in frame with the Igκ signal sequence of the pSeqTag/FRT/V5 His plasmid (Invitrogen). After 7 h, fetal calf serum was added to a final concentration of 10%, and the incubation continued for 24 h, following which hygromycin (500 μg/ml) was added to select Cg-Clp1 recombinant cells. After 20 days of selection, hygromycin-resistant transfected cells were lysed in 20 mm Hepes, pH 8.0, 500 mm NaCl, 0.1 mm EDTA, and 1 mm PMSF by repeated freeze thaw cycles. This cell extract was centrifuged for 10 min at 20,000 × g and analyzed by SDS-PAGE. Recombinant Cg-Clp1 was detected by Western blot analyses using a V5 tag-specific antibody. Recombinant Cg-Clp1 protein was purified using the Pro-BondTM purification system under native conditions according to the manufacturer's protocol (Invitrogen). About 3 × 108 nontransfected Flp-In™-CHO cells (used as a negative control) and 3 × 108 cells expressing Cg-Clp1 protein were each suspended in 11 ml of binding buffer (1 mm PMSF, 20 mm sodium phosphate, 500 mm sodium chloride, pH 7.4) and lysed by three freeze-thaw cycles using a liquid nitrogen and a 42 °C water bath. For both lysates, DNA was sheared by passing the preparation through an 18-gauge needle several times, and cell debris was spin down at 20,000 × g for 10 min. Clear lysates were incubated with 2 ml of Ni2+-nitrilotriacetic acid-equilibrated resin for 20 min. The resin columns were washed three times with 10 ml of A buffer (30 mm imidazole, 1 mm PMSF, 20 mm sodium phosphate, 500 mm sodium chloride, pH 6.0). Finally, the columns were eluted with 12 ml of B buffer (30 mm imidazole, 1 mm PMSF, 20 mm sodium phosphate, 500 mm sodium chloride, pH 6.0), and elution fractions were then monitored by dot blot analyses using a V5 tag-specific antibody. Immunopositive fractions and corresponding fractions from nontransfected cells were each pooled and then desalted, buffer-exchanged to 100 mm acetic acid, and concentrated to one-tenth volume by several repeated ultrafiltration steps using centrifugal filter membranes (Centricon concentrator YM50; Amicon; Millipore Corp.), which removed molecules smaller than 50 kDa. Concentration of recombinant Cg-Clp1 protein was evaluated by comparing the intensity of the signal in Western blot with those obtained for given quantities of a control protein (Positope™; Invitrogen). RAC were prepared from the shoulders and the knees of 3-week-old rabbits, as previously described (19Galera P. Redini F. Vivien D. Bonaventure J. Penfornis H. Loyau G. Pujol J.P. Exp. Cell Res. 1992; 200: 379-392Crossref PubMed Scopus (84) Google Scholar, 20Benya P.D. Padilla S.R. Nimni M.E. Biochemistry. 1977; 16: 865-872Crossref PubMed Scopus (257) Google Scholar, 21Galera P. Vivien D. Pronost S. Bonaventure J. Redini F. Loyau G. Pujol J.P. J. Cell. Physiol. 1992; 153: 596-606Crossref PubMed Scopus (77) Google Scholar). Cells were seeded at 2 × 104 cells/cm2 in either 6-well plates, 100-mm dishes, or 75-, 150-, and 175-cm2 flasks and cultured in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine (2 mm), penicillin (100 IU/ml), streptomycin (100 μg/ml), and Fungizone (0.25 μg/ml) in a 5% CO2 atmosphere. The medium was changed twice a week. Chondrocytes seeded at a density of 2 × 104 cells/cm2 in 6-well plates were transiently transfected at 80% confluence by the calcium phosphate precipitation method (22Ghayor C. Chadjichristos C. Herrouin J.F. Ala-Kokko L. Suske G. Pujol J.P. Galera P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 23Ghayor C. Herrouin J.F. Chadjichristos C. Ala-Kokko L. Takigawa M. Pujol J.P. Galera P. J. Biol. Chem. 2000; 275: 27421-27438Abstract Full Text Full Text PDF PubMed Google Scholar). Different amounts of pSegTag-Cg-Clp1 expression vector (Invitrogen) and the insertless corresponding vector used as a complement to 15 μg were cotransfected with a pSV40 β-galactosidase expression vector (2 μg) as an internal control of transfection efficiency and, in some experiments, with 15 μg of COL2A1-luciferase reporter plasmid. After overnight transfection, the medium was replaced by a fresh one, and cells were harvested 24 h later. Protein amount, β-galactosidase, and luciferase activities were determined. Luciferase activity was measured on total cell extracts (kit from Promega) in a luminometer (Berthold Lumat LB 9501). β-Galactosidase activity was assayed with a colorimetric assay (24Goldberg H. Helaakoski T. Garrett L.A. Karsenty G. Pellegrino A. Lozano G. Maity S. de Crombrugghe B. J. Biol. Chem. 1992; 267: 19622-19630Abstract Full Text PDF PubMed Google Scholar), whereas the protein amount was determined by the Bradford colorimetric method (Bio-Rad). Luciferase activities were normalized to protein amount and expressed in relative luciferase activity as the mean ± S.D. of three independent samples. The COL2A1-luciferase reporter vectors (pGL2–3.774kb, pGL2–0.387kb, and pGL2–0.110kb) have been previously described (25Chadjichristos C. Ghayor C. Herrouin J.F. Ala-Kokko L. Suske G. Pujol J.P. Galera P. J. Biol. Chem. 2002; 277: 43903-43917Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Nuclear extracts from transfected RAC were prepared by a minipreparation procedure (26Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar), and gel retardation assays were performed with the following nucleotides: +2392/+2415 α1(II), 5′-AGCCCCATTCATGAGAGACGAGGT-3′ (SOX9 binding site is indicated in italic type); +2817/+2845 α1(II), 5′-AGCGCAGCCTGGCCCCGCCCCTGCGCCGGCCGCGTCGGACCGGGGCGGGGACGCGGCCGC-3′ (known to contain Sp-responsive elements (22Ghayor C. Chadjichristos C. Herrouin J.F. Ala-Kokko L. Suske G. Pujol J.P. Galera P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 27Chadjichristos C. Ghayor C. Kypriotou M. Martin G. Renard E. Ala-Kokko L. Suske G. de Crombrugghe B. Pujol J.P. Galera P. J. Biol. Chem. 2003; 278: 39762-39772Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). They were end-labeled with [γ-32P]dATP (PerkinElmer Life Sciences) using T4 polynucleotide kinase (Invitrogen). RAC nuclear extracts (7 or 15 μg) were incubated for 30 min at room temperature with the probe (2 fmol) in 20 μl of a specific binding buffer (for +2392/+2415 probe, 20 mm HEPES, pH 7.9, 50 mm KCl, 10% (v/v) glycerol, 0.5 mm EDTA, 0.5 mm DTT, 1 mm PMSF, 0.05% (v/v) Nonidet P-40; for +2817/+2845 probe, 10% glycerol, 0.5 mm DTT, 0.05 Nonidet P-40, 0.05 mm EDTA, 25 mm HEPES, pH 7.9, 50 mm NaCl) and in the presence of 1 μg of poly(dG-dC)·poly(dG-dC) (Amersham Biosciennces), used as DNA nonspecific competitor. Samples were fractionated by electrophoresis for 1.5 h at 150 V on a 5% polyacrylamide gel (acrylamide/bis at 30:1) in 0.5 × TBE (45 mm Tris borate, 1 mm Na2EDTA) and visualized by autoradiography. The total amount of labeled GAG/PG was estimated after [35S]sulfate (Amersham Biosciences) labeling as previously described (28Beauchef G. Kypriotou M. Chadjichristos C. Widom R.L. Poree B. Renard E. Moslemi S. Wegrowski Y. Maquart F.X. Pujol J.P. Galera P. Biochem. Biophys. Res. Commun. 2005; 333: 1123-1131Crossref PubMed Scopus (16) Google Scholar). Chondrocytes seeded at a density of 2 × 106 cells/55-cm2 culture plates were transiently transfected at 80% confluence by the calcium phosphate precipitation method with 10 μg of pSegTag-Cg-Clp1 expression vector (Invitrogen) or 10 μg of the insertless corresponding vector as a negative control. The resulting conditioned media (5 ml/55 cm2) were collected 24 h later and stored at –80 °C until used for their effect on the neosynthesis of collagen. A measurement of total collagen neosynthesis was performed as previously described (22Ghayor C. Chadjichristos C. Herrouin J.F. Ala-Kokko L. Suske G. Pujol J.P. Galera P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). In addition to transfection experiments, both nickel-purified recombinant Cg-Clp1 and conditioned medium were tested. Recombinant Cg-Clp1 was added to the [3H]proline-labeled culture medium at a final concentration of 1 nm (60 ng/ml). As a negative control, a corresponding protein extract purified from nontransfected cells was used. Alternatively, the labeled culture medium was prepared with conditioned medium (1.5 ml/9.6 cm2) instead of fresh culture medium. Tritiated thymidine (74.5 Ci/mmol; PerkinElmer Life Sciences) was added to the culture medium of proliferating chondrocytes at a final concentration of 1 μCi/ml. After 24 h, the medium was removed, and the monolayers were rinsed with PBS. This rinse medium was aspirated, and the culture dishes were placed on an ice block while 1 ml of ice-cold 5% trichloroacetic acid was added slowly. The culture dishes were placed in a cold room at 4 °C for 30 min, whereupon the trichloroacetic acid was removed, and the-fixed monolayer was carefully rinsed three additional times with ice-cold 5% trichloroacetic acid. Then 0.5 ml of 0.1 m NaOH was added to each well for 1 h at 50°C to solubilize the precipitated material. Aliquots of 0.4 ml of NaOH extracts were removed from each well for liquid scintillation counting. Western blot analysis of type II collagen was performed on RAC as previously described (22Ghayor C. Chadjichristos C. Herrouin J.F. Ala-Kokko L. Suske G. Pujol J.P. Galera P. J. Biol. Chem. 2001; 276: 36881-36895Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). 15 μg of cell layer-associated proteins/well was used instead of 50 μg. Anti-type II collagen antibody was from Novotec (Lyon, France). Zymogram Detection—Chitinolytic enzyme activities were examined after polyacrylamide gel electrophoresis. A chitinase assay was carried out as described by Filho et al. (29Filho B.P. Lemos F.J. Secundino N.F. Pascoa V. Pereira S.T. Pimenta P.F. Insect. Biochem. Mol. Biol. 2002; 32: 1723-1729Crossref PubMed Scopus (85) Google Scholar). To visualize the active enzyme, 0.01% glycol chitin was incorporated into 6% (w/v) polyacrylamide minigels. After electrophoresis, the gel was incubated in 1% Triton X-100, 0.1 m sodium acetate, pH 5.0, at 37 °C for 3 h at room temperature. The gel was transferred in a fresh solution of 0.01% calcofluor white M2R (Sigma), a specific fluorescent chitin stain, in 0.5 m Tris-HCl, pH 9.0. After 5 min, the gel was removed and washed several times in distilled water for 1 h. Areas of activity with digested chitin were visualized as dark bands on a fluorescent background under a UV transilluminator and photographed. Spectrophotometric Enzyme Activity Measurement—N-Acety" @default.
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• W2019956407 title "The Phylogenetically Conserved Molluscan Chitinase-like Protein 1 (Cg-Clp1), Homologue of Human HC-gp39, Stimulates Proliferation and Regulates Synthesis of Extracellular Matrix Components of Mammalian Chondrocytes" @default.
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