The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix

Abstract

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely α1β1, α2β1, α10β1, and α11β1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express α1β1, α2β1, or α10β1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant αI domains in Escherichia coli. For each of the four αI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both α1I- and α2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by α2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the α2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix. Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely α1β1, α2β1, α10β1, and α11β1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express α1β1, α2β1, or α10β1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant αI domains in Escherichia coli. For each of the four αI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both α1I- and α2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by α2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the α2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix. Collagen IX was the first member to be discovered of a subgroup of collagens, now known as the fibril-associated collagens with interrupted triple helix (FACITs) 1The abbreviations used are: FACITs, fibril-associated collagen with interruptions in triple helix; RT, reverse transcription; GAG, glycosaminoglycan; s, sense; as, antisense; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; BSA, bovine serum albumin.1The abbreviations used are: FACITs, fibril-associated collagen with interruptions in triple helix; RT, reverse transcription; GAG, glycosaminoglycan; s, sense; as, antisense; GST, glutathione S-transferase; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; BSA, bovine serum albumin. (1Olsen B.R. Int. J. Biochem. Cell Biol. 1997; 29: 555-558Crossref PubMed Scopus (83) Google Scholar). At the present, FACITs also include collagens XII, XIV, XVI, XIX, XX, XXI, and XXII. Collagen IX is composed of three different α chains, termed α1(IX), α2(IX), and α3(IX). Structurally the collagen IX molecule can be divided into three triple helical domains (COL1, COL2, and COL3) separated and flanked by non-triple helical (NC) domains. Collagen IX is expressed in cartilage and in a limited number of other locations, including developing eye. Collagen IX can be covalently cross-linked to collagen II, and typically it covers the large fibrils formed by collagens II and XI (2Eyre D.R. Apon S. Wu J.J. Ericsson L.H. Walsh K.A. FEBS Lett. 1987; 220: 337-341Crossref PubMed Scopus (179) Google Scholar, 3van der Rest M. Mayne R. J. Biol. Chem. 1988; 263: 1615-1618Abstract Full Text PDF PubMed Google Scholar, 4Wu J.J. Woods P.E. Eyre D.R. J. Biol. Chem. 1992; 267: 23007-23014Abstract Full Text PDF PubMed Google Scholar, 5Diab M. Wu J.J. Eyre D. Biochem. J. 1996; 314: 327-332Crossref PubMed Scopus (84) Google Scholar). There is a kink between COL2 and COL3 domains making the COL3 domain project into the perifibrillar space, whereas the COL1 and COL2 domains are arranged on the surface of the fibril. Many but not all collagen IX molecules are proteoglycans because a glycosaminoglycan (GAG) chain may be attached to NC3 domain via a serine residue in the α2(IX) chain (6Bruckner P. Vaughan L. Winterhalter K.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2608-2612Crossref PubMed Scopus (72) Google Scholar). Collagen IX is essential for the normal structure and function of cartilage. Its mutations in man cause multiple epiphyseal dysplasia (7Muragaki Y. Mariman E.C. van Beersum S.E. Perälä M. van Mourik J.B. Warman M.L. Olsen B.R. Hamel B.C. Nat. Genet. 1996; 12: 103-105Crossref PubMed Scopus (172) Google Scholar, 8Holden P. Canty E.G. Mortier G.R. Zabel B. Spranger J. Carr A. Grant M.E. Loughlin J.A. Briggs M.D. Am. J. Hum. 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For example, integrins may participate in regulation of collagen fibril formation (30Velling T. Risteli J. Wennerberg K. Mosher D.F. Johansson S. J. Biol. Chem. 2002; 277: 37377-37381Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 31Li S. Van Den Diepstraten C. D'Souza S.J. Chan B.M. Pickering J.G. Am. J. Pathol. 2003; 163: 1045-1056Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 32Jokinen J. Dadu E. Nykvist P. Käpylä J. White D.J. Ivaska J. Vehviläinen P. Reunanen H. Larjava H. Häkkinen L. Heino J. J. Biol. Chem. 2004; 279: 31956-31963Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). In this report we demonstrate that based on their expression pattern all four collagen receptor integrins are putative receptors for type IX and that they, and their corresponding recombinant αI domains, each bind to human recombinant collagen IX. For every integrin collagen IX was among the best collagenous ligands. This makes collagen IX exceptional among all collagens tested so far and emphasizes the role of FACITs as cell adhesion proteins. Our results indicate that α1I and α2I domains recognize only one site in collagen IX that is in COL3 very close to the kink formed by NC3 domain. The binding mechanism does not resemble any of the previously described I domain-collagen interactions. Cell Lines and Reagents—Human osteosarcoma cell lines HOS MNNG and KHOS-240, Chinese hamster ovary (CHO) cells, and human chondrosarcoma cell line HTB-99 were obtained from American Type Culture Collection (ATCC, Manassas, VA). The human primary fetal chondrocyte cultures were initiated as described previously (33Vuorio E. Elima K. Pulkkinen J. Viitanen A.M. FEBS Lett. 1984; 174: 238-242Crossref PubMed Scopus (5) Google Scholar). The cell cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mm glutamine, 100 IU/ml penicillin-G, and 100 μg/ml streptomycin. Generation of Stable Integrin-expressing CHO Cell Line—CHO-α1β1 and CHO-α2β1 cell lines were created as described previously (27Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Full-length α10 cDNA corresponding to nucleotides 19-3525 of the published sequence (19Camper L. Hellman U. Lundgren-Åkerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar) (GenBank™ accession no. AF074015) was generated with the Access RT-PCR kit (Promega) using RNA purified from SAOS-2 cells (ATCC). Primers were designed to introduce BglII and BamHI restriction sites at the 5′ and 3′ ends, correspondingly. Digested cDNA was ligated to pcDNA3 expression vector (Invitrogen) containing cytomegalovirus promoter and the gene for neomycin resistance. The sequence was verified by sequencing. CHO cells (ATCC) were stably transfected with FuGENE 6 transfection reagent (Roche Applied Science). Isolated cell clones were selected with a neomycin analog G418 (400 μg/ml, Invitrogen) for 3 weeks. Clones were analyzed for the expression of the α10 integrin with RT-PCR using the Gene Amp PCR kit (PerkinElmer Life Sciences) and immunoprecipitation. Immunoprecipitation—Polyclonal rabbit antisera against human β1 (34Heino J. Ignotz R.A. Hemler M.E. Crouse C. Massagué J. J. Biol. Chem. 1989; 264: 380-388Abstract Full Text PDF PubMed Google Scholar), α2 (35Santala P. Larjava H. Nissinen L. Riikonen T. Määttä A. Heino J. J. Biol. Chem. 1994; 269: 1276-1283Abstract Full Text PDF PubMed Google Scholar), α10 (a kind gift from Dr. Evy Lundgren-Åkerlund, Lund, Sweden) (36Camper L. Holmvall K. Wangnerud C. Aszodi A. Lundgren-Åkerlund E. Cell Tissue Res. 2001; 306: 107-116Crossref PubMed Scopus (81) Google Scholar), and α11 (a kind gift from Dr. Donald Gullberg, Uppsala, Sweden) (37Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) integrin subunits were used in immunoprecipitation assays. Cell cultures were metabolically labeled with 50 μCi/ml [35S]methionine (Tran35S-label, ICN Biomedicals Inc., Irvine, CA) for 18 h in methionine-free minimum essential medium (Sigma). Cell monolayers were rinsed on ice with a solution containing 150 mm NaCl, 1 mm CaCl2, 1 mm MgCl2, and 25 mm Tris-HCl (pH 7.4) and then detached by scraping. Cell pellets obtained by centrifugation at 500 × g for 5 min were solubilized in 200 μl of the same buffer containing 100 mmn-octyl-β-d-glucopyranoside (Sigma) on ice with occasional vortexing. Insoluble material was removed by centrifugation at 10,000 × g for 5 min at 4 °C. Radioactivity in cell lysates was counted, and an equal amount of radioactivity was used in immunoprecipitation assays. Triton X-100 (0.5%, v/v) and bovine serum albumin (0.5 mg/ml) were added to the supernatants, which were then precleared by incubation with 50 μl of packed protein A-Sepharose® (Amersham Biosciences). Supernatants were immunoprecipitated with anti-integrin antibodies for 12 h at 4 °C. Immune complexes were recovered by binding to protein A-Sepharose and washing the beads four times with 25 mm Tris-buffered isotonic saline (pH 7.4) containing 0.5% Triton X-100 and 1 mg/ml bovine serum albumin and twice with 0.5 m NaCl and 25 mm Tris-HCl (pH 7.4). The immunoprecipitates were analyzed by electrophoresis on sodium dodecyl sulfate-containing 6% polyacrylamide gels under nonreducing conditions followed by autoradiography. Detection of mRNA for Specific Integrin Subunits by Reverse Transcription-Polymerase Chain Reaction—Total cellular RNA from cell cultures was isolated using the RNeasy minikit (Qiagen). For extraction and purification of total RNA from human fetal tissues, the samples were immediately frozen and pulverized in liquid nitrogen, homogenized in guanidinium isothiocyanate, and sedimented through 5.7 m CsCl (38Chirgwin J.M. Przybyla A.E. MacDonald R.J. Rutter W.J. Biochemistry. 1979; 18: 5294-5299Crossref PubMed Scopus (16608) Google Scholar). RT-PCR was done using the Gene Amp PCR kit (PerkinElmer Life Sciences). All oligonucleotides were designed to recognize a unique sequence exclusive for each cDNA. Integrins were amplified using the following primers: α10: sense (s) 5′-CAGGGATCCCCAACATACATGGATGTTGTC-3′ and antisense (as) 5′-GGCTGAATTCCCCTTCAAGGCCAAAAATCCG-3′; α11: s 5′-CAGACCTACATGGACATCG-3′ and as 5′-CATCTCCAGCCCAAAGGAG-3′; α1: s 5′-CACAGGGATCCGTCAGCCCCACATTT-3′ and as 5′-GTGGCTGTCGACAGCTGTGGCTTCCAG-3′; α2: s 5′-CACAGGGATCCCCTGATTTTCAGCTC-3′ and as 5′-GTGGCTGAATTCAACAGTACCTTCAATG-3′. COL9A1 was amplified with the following primers: s 5′-ACAGCAGGACTCCCTGGA-3′ and as 5′-TGATCACCAGGTGCACCAG-3′. Glyceraldehyde-3-phosphate dehydrogenase (a housekeeping gene used as a control) (39Herouy Y. May A.E. Pomschlegel G. Stetter C. Grenz H. Preissner E. J. J. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar) was amplified with the following primers: s and as of PCR were done in 2 mm using the following for α10 and for 1 min at for 1 min at and for 2 min at for α11: for 1 min at for 1 min at and for 2 min at for and α2 for 1 min at for 1 min at and for 2 min at and for for 1 min at for 1 min at and for 2 min at °C. The generated were to electrophoresis on a and were by of Integrin αI α1I and α2I domains were generated by PCR as described J. Ivaska J. Riikonen R. Nykvist P. Pentikäinen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Tulla M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar) using human integrin and α2 as and (Amersham were used to recombinant glutathione of human α1I and α2I domains, The α10I domain cDNA was generated by RT-PCR from RNA isolated from cells cellular RNA was isolated by using the RNeasy minikit (Qiagen). RT-PCR was done using the Gene Amp PCR kit (PerkinElmer Life Sciences). for the were described M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The amplified α10I domain cDNA was with expression vector (Amersham using the BamHI and restriction To the vector the α10I cDNA was ligated with the kit (Amersham Biosciences). The was into the Escherichia for The was and compared with the published α10 sequence (19Camper L. Hellman U. Lundgren-Åkerlund E. J. Biol. Chem. 1998; 273: 20383-20389Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). integrin α11 cDNA (37Velling T. Kusche-Gullberg M. Sejersen T. Gullberg D. J. Biol. Chem. 1999; 274: 25735-25742Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) was used as a when α11I domain was generated by The PCR BamHI and was into and the sequence was by the W.-M. Käpylä J. Puranen S. Knight C.G. Tiger C.-F. Pentikäinen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). and of αI E. cells were with the for protein 500 of medium containing 100 μg/ml was with 50 of of or and the cultures were at the of the were with and to for an h typically at by cells were in (pH 7.4) and then by followed by of Triton X-100 to a of incubation for min on were and were (Amersham was added to the which was at for min with The was then the was and with protein was into The were with PBS, and were using mm recombinant and αI domains were analyzed by and were with the Biochem. PubMed Scopus Google Scholar). The recombinant α1I domain produced was in corresponding to of the the α2I domain was corresponding to of the α2 The of the α1I and α2I domains and J. Ivaska J. Riikonen R. Nykvist P. Pentikäinen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Tulla M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The recombinant α10I domain produced was in corresponding to of the α10 The two and the of α10I M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). The recombinant α11I domain in total corresponding to in the were two and in the were α11I domain as an to the expression and purification W.-M. Käpylä J. Puranen S. Knight C.G. Tiger C.-F. Pentikäinen O.T. Johnson M.S. Farndale R.W. Heino J. Gullberg D. J. Biol. Chem. 2003; 278: 7270-7277Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). αI domains were used as for collagen binding of the αI domain cDNA in a or vector was using PCR to kit The of mutations was by sequencing. were then into E. for of recombinant protein J. Ivaska J. Riikonen R. Nykvist P. Pentikäinen O. Johnson M. Heino J. J. Biol. Chem. 2000; 275: 3348-3354Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 28Tulla M. Pentikäinen O.T. Viitasalo T. Käpylä J. Impola U. Nykvist P. Nissinen L. Johnson M.S. Heino J. J. Biol. Chem. 2001; 276: 48206-48212Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Collagen IX and collagen I and mouse membrane of mouse IV collagens were from II IV and were obtained from Collagen was from human collagen IX was produced as described previously T. Perälä M. M. M. T. E. R. J. Ala-Kokko L. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). cells Invitrogen) in at were at × in II medium (Invitrogen) and supplemented with fetal bovine The cells were with three for the α1(IX), α2(IX), and and a promoter for the and of human M. T. J. H. Pihlajaniemi T. Kivirikko Matrix Biol. 1998; PubMed Scopus Google Scholar) with of of was added to the h of the cells were from the medium by were from the medium by of to and the on ice for 1 h with The was by centrifugation at × g for 1 h at 4 and the was in m mm m NaCl, 2 m buffer with (Roche Applied The recombinant protein was to 200 (Amersham in the same buffer and purified with or (Amersham in 50 mm PIPES, mm NaCl, 2 m with an m collagen IX was analyzed by and to analysis in an Applied collagen IX was in 50 mm at °C. In collagen IX was modified to in as described in J.A. Golbik R. K. Kuhn K. J. 1993; 12: PubMed Scopus (174) Google of using we used (Sigma). Cell were as described previously (27Nykvist P. Tu H. Ivaska J. Käpylä J. Pihlajaniemi T. Heino J. J. Biol. Chem. 2000; 275: 8255-8261Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). were μg/ml in with collagen bovine collagen human collagens and and recombinant human collagen IX collagen II, or was on the the conditions were so that collagens were in triple helical we