Distinct Transglutaminase 2-independent and Transglutaminase 2-dependent Pathways Mediate Articular Chondrocyte Hypertrophy
2003; Elsevier BV; Volume: 278; Issue: 21 Linguagem: Inglês
10.1074/jbc.m301055200
ISSN1083-351X
AutoresKristen Johnson, Deborah Van Etten, Nisha Nanda, Robert M. Graham, Robert Terkeltaub,
Tópico(s)Platelet Disorders and Treatments
ResumoAltered chondrocyte differentiation, including development of chondrocyte hypertrophy, mediates osteoarthritis and pathologic articular cartilage matrix calcification. Similar changes in endochondral chondrocyte differentiation are essential for physiologic growth plate mineralization. In both articular and growth plate cartilages, chondrocyte hypertrophy is associated with up-regulated expression of certain protein-crosslinking enzymes (transglutaminases (TGs)) including the unique dual-functioning TG and GTPase TG2. Here, we tested if TG2 directly mediates the development of chondrocyte hypertrophic differentiation. To do so, we employed normal bovine chondrocytes and mouse knee chondrocytes from recently described TG2 knockout mice, which are phenotypically normal. We treated chondrocytes with the osteoarthritis mediator IL-1β, with the all-trans form of retinoic acid (ATRA), which promotes endochondral chondrocyte hypertrophy and pathologic calcification, and with C-type natriuretic peptide, an essential factor in endochondral development. IL-1β and ATRA induced TG transamidation activity and calcification in wild-type but not in TG2 (–/–) mouse knee chondrocytes. In addition, ATRA induced multiple features of hypertrophic differentiation (including type X collagen, alkaline phosphatase, and MMP-13), and these effects required TG2. Significantly, TG2 (–/–) chondrocytes lost the capacity for ATRA-induced expression of Cbfa1, a transcription factor necessary for ATRA-induced chondrocyte hypertrophy. Finally, C-type natriuretic peptide, which did not modulate TG activity, comparably promoted Cbfa1 expression and hypertrophy (without associated calcification) in TG2 (+/+) and TG2 (–/–) chondrocytes. Thus, distinct TG2-independent and TG2-dependent mechanisms promote Cbfa1 expression, articular chondrocyte hypertrophy, and calcification. TG2 is a potential site for intervention in pathologic calcification promoted by IL-1β and ATRA. Altered chondrocyte differentiation, including development of chondrocyte hypertrophy, mediates osteoarthritis and pathologic articular cartilage matrix calcification. Similar changes in endochondral chondrocyte differentiation are essential for physiologic growth plate mineralization. In both articular and growth plate cartilages, chondrocyte hypertrophy is associated with up-regulated expression of certain protein-crosslinking enzymes (transglutaminases (TGs)) including the unique dual-functioning TG and GTPase TG2. Here, we tested if TG2 directly mediates the development of chondrocyte hypertrophic differentiation. To do so, we employed normal bovine chondrocytes and mouse knee chondrocytes from recently described TG2 knockout mice, which are phenotypically normal. We treated chondrocytes with the osteoarthritis mediator IL-1β, with the all-trans form of retinoic acid (ATRA), which promotes endochondral chondrocyte hypertrophy and pathologic calcification, and with C-type natriuretic peptide, an essential factor in endochondral development. IL-1β and ATRA induced TG transamidation activity and calcification in wild-type but not in TG2 (–/–) mouse knee chondrocytes. In addition, ATRA induced multiple features of hypertrophic differentiation (including type X collagen, alkaline phosphatase, and MMP-13), and these effects required TG2. Significantly, TG2 (–/–) chondrocytes lost the capacity for ATRA-induced expression of Cbfa1, a transcription factor necessary for ATRA-induced chondrocyte hypertrophy. Finally, C-type natriuretic peptide, which did not modulate TG activity, comparably promoted Cbfa1 expression and hypertrophy (without associated calcification) in TG2 (+/+) and TG2 (–/–) chondrocytes. Thus, distinct TG2-independent and TG2-dependent mechanisms promote Cbfa1 expression, articular chondrocyte hypertrophy, and calcification. TG2 is a potential site for intervention in pathologic calcification promoted by IL-1β and ATRA. In physiologic endochondral growth plate mineralization, chondrocytes undergo a multi-step differentiation process in which there is ordered progression from a resting to proliferative state followed by maturation to a terminally differentiated hypertrophic state (1Erlebacher A. Filvaroff E.H. Gitelman S.E. Derynck R. Cell. 1995; 80: 371-378Abstract Full Text PDF PubMed Scopus (611) Google Scholar). Hypertrophic chondrocytes are specialized to remodel and mineralize their matrix (2Alini M. Kofsky Y. Wu W. Pidoux I. Poole A.R. J. Bone Miner. Res. 1996; 11: 105-113Crossref PubMed Scopus (86) Google Scholar). For example, hypertrophic chondrocytes demonstrate up-regulated expression of MMP-13, a mediator of matrix degradation in osteoarthritis (OA) 1The abbreviations used are: OA, osteoarthritis; AP, alkaline phosphatase; ATRA, all-trans form of retinoic acid; Cbfa1, core binding factor alpha 1; CNP, C-type natriuretic peptide; NPP, nucleotide pyrophosphatase phosphodiesterase; RAR, retinoic acid receptor; RXR, retinoid X receptor; TG, transglutaminase; RT, reverse transcription; FXIIIA, factor XIIIA.1The abbreviations used are: OA, osteoarthritis; AP, alkaline phosphatase; ATRA, all-trans form of retinoic acid; Cbfa1, core binding factor alpha 1; CNP, C-type natriuretic peptide; NPP, nucleotide pyrophosphatase phosphodiesterase; RAR, retinoic acid receptor; RXR, retinoid X receptor; TG, transglutaminase; RT, reverse transcription; FXIIIA, factor XIIIA. (3Krane S.M. J. Clin. Invest. 2001; 107: 31-32Crossref PubMed Scopus (13) Google Scholar). In addition, hypertrophic chondrocytes demonstrate altered patterns of collagen subtype generation that include stereotypic up-regulated type X collagen expression, and hypertrophic chondrocytes show markedly increased release of mineralization-competent secretory vesicles (2Alini M. Kofsky Y. Wu W. Pidoux I. Poole A.R. J. Bone Miner. Res. 1996; 11: 105-113Crossref PubMed Scopus (86) Google Scholar). In addition, hypertrophic chondrocytes demonstrate increased alkaline phosphatase (AP) activity and other alterations in the metabolism of Pi and PPi (4Gerstenfeld L.C. Shapiro F.D. J. Cell. Biochem. 1996; 62: 1-9Crossref PubMed Google Scholar, 6Alini M. Carey D. Hirata S. Grynpas M.D. Pidoux I. Poole A.R. J. Bone Miner. Res. 1994; 9: 1077-1087Crossref PubMed Scopus (72) Google Scholar). In this context, Pi and PPi mediate both calcification and growth plate organization (5Hessle L. Johnson K.A. Anderson H.C. Narisawa S. Sali A. Goding J.W. Terkeltaub R. Millan J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9445-9449Crossref PubMed Scopus (660) Google Scholar), and Pi promotes chondrocyte hypertrophy (6Alini M. Carey D. Hirata S. Grynpas M.D. Pidoux I. Poole A.R. J. Bone Miner. Res. 1994; 9: 1077-1087Crossref PubMed Scopus (72) Google Scholar). In contrast to chondrocytes in growth plate cartilage, chondrocytes in normal articular cartilage remain largely in a resting state and do not undergo terminal differentiation or mineralize their matrix (7Goldring M.B. Arthritis Rheum. 2000; 43: 1916-1926Crossref PubMed Scopus (615) Google Scholar). But in OA, foci of articular chondrocyte hypertrophy develop, typically near sites of cartilage surface lesions (8Poole A.R. Matsui Y. Hinek A. Lee E.R. Anat. Rec. 1989; 224: 167-179Crossref PubMed Scopus (139) Google Scholar, 9von der Mark K. Kirsch T. Nerlich A. Kuss A. Weseloh G. Gluckert K. Stoss H. Arthritis Rheum. 1992; 35: 806-811Crossref PubMed Scopus (395) Google Scholar). Chondrocyte hypertrophy in OA may function partly to modulate matrix remodeling and repair (10Vignon E. Arlot M. Hartmann D. Moyen B. Ville G. Ann. Rheum. Dis. 1983; 42: 82-88Crossref PubMed Scopus (124) Google Scholar) and partly to promote calcification, as hypertrophic chondrocytes are commonly co-localized with deposits of hydroxyapatite and calcium pyrophosphate dihydrate crystals in the disease (11Ishikawa K. Masuda I. Ohira T. Yokoyama M. J. Bone Joint Surg. Am. 1989; 71: 875-886Crossref PubMed Scopus (65) Google Scholar). Significantly, deposits of hydroxyapatite and calcium pyrophosphate dihydrate crystals crystals can stimulate intra-articular inflammation and further damage to cartilage in OA (12McCarthy G.M. Curr. Rheumatol. Rep. 1999; 1: 101-106Crossref PubMed Scopus (7) Google Scholar, 13Terkeltaub R.A. J. Rheumatol. 2002; 29: 411-415PubMed Google Scholar). One of the shared features of hypertrophic chondrocytes in growth plate and articular cartilages is up-regulated expression of two transglutaminase (TG) isoenzymes (14Nurminskaya M. Linsenmayer T.F. Dev. Dyn. 1996; 206: 260-271Crossref PubMed Scopus (78) Google Scholar, 15Aeschlimann D. Wetterwald A. Fleisch H. Paulsson M.J. Cell Biol. 1993; 120: 1461-1470Crossref PubMed Scopus (166) Google Scholar). These are factor XIIIA (FXIIIA), a distinct tissue homodimeric form of the circulating heterotetrameric coagulation factor XIII, and TG2 (also termed tissue TG and Gh), which is unique among the human TG isoenzymes in being a dual function TG and GTPase/ATPase (16Chen J.S. Mehta K. Int. J. Biochem. Cell Biol. 1999; 31: 817-836Crossref PubMed Scopus (176) Google Scholar). TGs catalyze a calcium-dependent transamidation reaction that produces covalent cross-linking of available substrate glutamine residues to a primary amino group (EC 2.3.2.13). TGs thereby can modify the matrix through effects including protein cross-linking and stabilization (16Chen J.S. Mehta K. Int. J. Biochem. Cell Biol. 1999; 31: 817-836Crossref PubMed Scopus (176) Google Scholar), and OA cartilage contains a variety of potential TG substrates including several collagen subtypes, fibronectin, and the mineralization-regulatory protein osteopontin (17Kaartinen M.T. El-Maadawy S. Rasanen N.H. McKee M.D. J. Bone Miner. Res. 2002; 17: 2161-2173Crossref PubMed Scopus (109) Google Scholar). Furthermore, TG transamidation catalytic activity has been shown to increase in both an OA severity-dependent and age-dependant manner in joint cartilages (18Rosenthal A.K. Derfus B.A. Henry L.A. Arthritis Rheum. 1997; 40: 966-970Crossref PubMed Scopus (66) Google Scholar, 19Johnson K. Hashimoto S. Lotz M. Pritzker K. Terkeltaub R. Am. J. Pathol. 2001; 159: 149-163Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We recently observed that certain mediators implicated in OA, including IL-1β (20Attur M.G. Patel I.R. Patel R.N. Abramson S.B. Amin A.R. Proc. Assoc. Am. Physicians. 1998; 110: 65-72PubMed Google Scholar) stimulates chondrocyte matrix calcification (19Johnson K. Hashimoto S. Lotz M. Pritzker K. Terkeltaub R. Am. J. Pathol. 2001; 159: 149-163Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). IL-1β also induces TG transamidation catalytic activity in cultured chondrocytes (19Johnson K. Hashimoto S. Lotz M. Pritzker K. Terkeltaub R. Am. J. Pathol. 2001; 159: 149-163Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Furthermore, selective "gain-of-function" of either chondrocyte TG2 or FXIIIA TG transamidation activity via transfection was associated with potent up-regulation of the capacity of chondrocytes to calcify their matrix (19Johnson K. Hashimoto S. Lotz M. Pritzker K. Terkeltaub R. Am. J. Pathol. 2001; 159: 149-163Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). TG2 was recently observed to regulate vascular smooth muscle cell differentiation (21Vincan E. Neylon C.B. Jacobsen A.N. Woodcock E.A. Mol. Cell. Biochem. 1996; 157: 107-110Crossref PubMed Scopus (8) Google Scholar). In mononuclear phagocytes and fibroblasts, TG2 regulates adhesion and migration (22Fesus L. Piacentini M. Trends Biochem. Sci. 2002; 27: 534-539Abstract Full Text Full Text PDF PubMed Scopus (483) Google Scholar). Thus, we directly tested the role of TG2 in IL-1β-induced calcification by chondrocytes. In addition, we tested the hypothesis that TG2 is not simply a marker of chondrocyte hypertrophy but also a direct mediator of the development of chondrocyte hypertrophic differentiation. To directly probe TG2 functions in chondrocyte differentiation and calcification we have taken advantage of the recent generation of TG2 knockout mice, which are phenotypically normal (23Nanda N. Iismaa S.E. Owens W.A. Husain A. Mackay F. Graham R.M. J. Biol. Chem. 2001; 276: 20673-20678Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 24De Laurenzi V. Melino G. Mol. Cell. Biol. 2001; 21: 148-155Crossref PubMed Scopus (301) Google Scholar). To specifically assess for direct TG2 involvement in articular chondrocyte hypertrophy, we studied the effects on primary chondrocytes of CNP (25Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (365) Google Scholar) and of the vitamin A-derived metabolite all-trans retinoic acid (ATRA) (26Enomoto H. Enomoto-Iwamoto M. Iwamoto M. Nomura S. Himeno M. Kitamura Y. Kishimoto T. Komori T. Biol. Chem. 2000; 275: 8695-8702Abstract Full Text Full Text PDF Scopus (340) Google Scholar, 27Jimenez M.J. Balbin M. Alvarez J. Komori T. Bianco P. Holmbeck K. Birkedal-Hansen H. Lopez J.M. Lopez-Otin C. J. Cell Biol. 2001; 155: 1333-1344Crossref PubMed Scopus (100) Google Scholar). CNP is expressed in developing long bones, and the stimulatory effects of CNP on chondrocyte proliferation and hypertrophy are essential for normal growth plate development (25Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (365) Google Scholar). ATRA promotes maturation to hypertrophy and calcification by chick sternal chondrocytes in vitro (26Enomoto H. Enomoto-Iwamoto M. Iwamoto M. Nomura S. Himeno M. Kitamura Y. Kishimoto T. Komori T. Biol. Chem. 2000; 275: 8695-8702Abstract Full Text Full Text PDF Scopus (340) Google Scholar) in a manner that requires the transcription factor Cbfa1 (27Jimenez M.J. Balbin M. Alvarez J. Komori T. Bianco P. Holmbeck K. Birkedal-Hansen H. Lopez J.M. Lopez-Otin C. J. Cell Biol. 2001; 155: 1333-1344Crossref PubMed Scopus (100) Google Scholar). In addition, hypervitaminosis A and toxicity of certain other retinoids is associated with accelerated terminal differentiation of endochondral chondrocytes and premature epiphyseal closure in vivo (28Cuny J.F. Schmutz J.L. Terver M.N. Aussedat R. Cointin M. Weber M. Beurey J. Ann. Dermatol. Venereol. 1989; 116: 95-102PubMed Google Scholar, 29Mork N.J. Kolbenstvedt A. Austad J. Acta Derm. Venereol. 1992; 72: 445-448PubMed Google Scholar). Moreover, retinoid treatment of cultured chondrocytes is known to increase expression and activity of TGs (30Gionti E. Sanchez M. Arcella A. Pontarelli G. Tavassi S. Gentile V. Cozzolino A. Porta R. Cell Biol. Int. 1999; 23: 41-49Crossref PubMed Scopus (3) Google Scholar) in vitro. Chondrocyte hypertrophic differentiation in both the growth plate and in OA articular cartilage has been heretofore linked closely with a state in which matrix calcification is up-regulated (2Alini M. Kofsky Y. Wu W. Pidoux I. Poole A.R. J. Bone Miner. Res. 1996; 11: 105-113Crossref PubMed Scopus (86) Google Scholar, 4Gerstenfeld L.C. Shapiro F.D. J. Cell. Biochem. 1996; 62: 1-9Crossref PubMed Google Scholar, 8Poole A.R. Matsui Y. Hinek A. Lee E.R. Anat. Rec. 1989; 224: 167-179Crossref PubMed Scopus (139) Google Scholar). However, it has not been clarified if there is an obligate linkage of up-regulated calcification with chondrocyte hypertrophic differentiation. In this study, we demonstrate, using IL-1β and CNP-stimulated cells, that chondrocyte hypertrophy and up-regulated matrix calcification are dissociable states. We also establish TG2 to be an essential mediator of IL-1β-induced calcification, as well as ATRA-induced Cbfa1 expression, hypertrophic differentiation, and calcification in articular chondrocytes. In contradistinction, we establish that CNP-induced Cbfa1 expression and hypertrophic differentiation do not require TG2 in articular chondrocytes. Reagents—Human recombinant IL-1β was obtained from R&D Systems (Minneapolis, MN). ATRA, CNP, and all other reagents were obtained from Sigma, unless otherwise indicated. TG2 Null Mice—We established a breeding colony of TG2 (+/+) and TG2 (–/–) mice as previously described (23Nanda N. Iismaa S.E. Owens W.A. Husain A. Mackay F. Graham R.M. J. Biol. Chem. 2001; 276: 20673-20678Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). The C57BL6/129SVJ heterozygote TG2 (–/–) mice were bred to generate homozygous TG2 (–/–) and congenic wild-type founders. Each set of founders was used for only 5 generations to avoid breeding artifacts. Cell Isolation, Culture Conditions, and Assessment of Matrix Calcification—Primary articular chondrocytes from normal bovine knees (Animal Technologies, Tyler, TX) were isolated after dissection by collagenase digestion of the tibial plateau and femoral condyle articular cartilage (58Johnson K. Farley D. Hu S.H. Terkeltaub R. Arthritis Rheum. 2003; (in press)Google Scholar). Primary mouse articular chondrocytes were isolated by dissection of the tibial plateaus and femoral condyles of the TG2 wild-type and knockout mice at two months of age. The articular cartilage was carefully peeled off with a scalpel under a dissecting microscope to avoid disruption of the subchondral bone. The cartilage was subsequently digested with 2 mg/ml clostridial collagenase at 37 °C for 2 h and then plated in monolayer culture. Subconfluent chondrocytes were shown to express type II collagen in > 95% of the cells by immunocytochemistry. Approximately 2500 primary chondrocytes were obtained initially from a pair of knee joints from each two-month-old mouse. Mouse chondrocytes were allowed to proliferate for 5 days, which yielded ∼10,000 cells per pair of knees from an individual mouse. For the described experiments, knees from 30 mice of each genotype were harvested, and the chondrocytes of each genotype were pooled upon isolation and plated at 70% confluency. Primary chondrocytes were cultured in Dulbecco's modified Eagle's medium high glucose supplemented with 10% fetal calf serum, 1% glutamine, 100 units/ml penicillin, 50 g/ml Streptomycin (Omega Scientific, Tarzana, CA) and maintained at 37 °C in the presence of 5% CO2 for 7 days prior to the initiation of each experiment. Functional studies on chondrocytes were performed in either Medium A (Dulbecco's modified Eagle's medium high glucose supplemented with 1% fetal calf serum, 1% glutamine, 100 units/ml penicillin, 50 g/ml streptomycin, 1 mm sodium phosphate, and 50 g/ml of ascorbic acid) or Medium B (which had the same composition as Medium A except for a lack of sodium phosphate), unless otherwise indicated. To quantify matrix calcification by the primary articular chondrocytes, we used a previously described Alizarin Red S binding assay, which was further validated in each experiment by direct visual observation of Alizarin Red S staining in each plate (5Hessle L. Johnson K.A. Anderson H.C. Narisawa S. Sali A. Goding J.W. Terkeltaub R. Millan J.L. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9445-9449Crossref PubMed Scopus (660) Google Scholar). Here, aliquots of mouse chondrocytes (1 × 103 cells/well) were plated in individual wells of 96-well plates in a volume of 0.2 ml of Medium A. SDS-PAGE/Western Blotting and RT-PCR—For SDS-PAGE/Western blotting, conditioned media was collected at the designated time points and concentrated with cold tricholoracetic acid (final concentration of 15%) for 15 min on ice. Cell lysates and protein pellets from the concentrated media were resuspended in 4% SDS, 0.2 m Tris, pH 6.8, and 40% glycerol, and the protein was determined with the bicinchoninic acid protein assay (Pierce). Aliquots of 0.01 mg protein from each sample were separated by SDS-PAGE under reducing conditions and transferred to nitrocellulose (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar). Anti-MMP-13 (Chemicon, Temecula, CA), anti-type X collagen (Calbiochem, San Diego, CA), anti-FXIIIA (Calbiochem), anti-TG2 (Upstate Biotechnology, Lake Placid, NY), and anit-tubulin were used at a 1:1000 dilution in Western blotting (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar). Total RNA was extracted with Trizol, and RT-PCR was performed as previously described (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar). Primers that amplified the ribosomal gene L30 were used as a loading control. We used previously described primers to amplify L30 (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar), CD38 (32Dogan S. White T.A. Deshpande D.A. Murtaugh M.P. Walseth T.F. Kannan M.S. Biol. Reprod. 2002; 66: 596-602Crossref PubMed Scopus (26) Google Scholar), TG5 (33Candi E. Oddi S. Terrinoni A. Paradisi A. Ranalli M. Finazzi-Agro A. Melino G. J. Biol. Chem. 2001; 276: 35014-35023Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and mouse Cbfa1 (34Ducy P. Zhang R. Geoffroy V. Ridall A. Karsenty G. Cell. 1997; 89: 747-754Abstract Full Text Full Text PDF PubMed Scopus (3578) Google Scholar). TG1 sense (5′-TCAGATGCTGGAGGTGACAG-3′) and TG1 antisense (5′-CCCAGTCTTCCTGTCTGAGC-3′) primers amplified a 171-bp product (positions 2542–2712 in the published sequence). TG2 sense (5′-TGCTCCTATTGGCCTGTACC-3′) and TG2 antisense (5′-CCAAAGTTCCAAGGCACACT-3′) primers amplified a 222-bp product (positions 360–581). TG3 sense (5′-AGCCTGTGAACGTGCAGATGCTCTTC-3′) and antisense (5′-TGATTGCAGGAAACTTGTTGCAGG-3′) primers amplified a 225-bp product (positions 1867–2091). FXIIIA sense (5′-CCTGCGTACTCGAAGAGACC-3′) and FXIIIA antisense (5′-CTTCGAACTGGCCATAGC tc-3′) amplified a 188-bp product (positions 993–1180). TG Activity, PPi, Nucleotide Pyrophosphohydrolase (NPP), AP, and Cellular DNA Assays—Cell-associated TG activity was determined by a modification of a previously described method (35Slaughter T.F. Achyuthan K.E. Lai T.S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (154) Google Scholar). In brief, 0.02 mg of cell lysate was extracted and sonicated for 10 s in 5 mm Tris-HCl, pH 7.4, 0.25 m sucrose, 0.2 mm MgSO4, 2 mm dithiothreitol, 0.4 mm phenylmethylsulfonyl fluoride, and 0.4% Triton X-100, and was then loaded to Nunc-Immuno Module plates previously coated with 20 mg/ml N,N′-dimethylcasein. The lysates were incubated for 1 h in 100 mm Tris, pH 8.5, 20 mm CaCl2, 40 mm dithiothreitol, and 2 mm 5-(biotinamido)pentylamine (BP) (Pierce), and detection of the bound BP was performed as described (35Slaughter T.F. Achyuthan K.E. Lai T.S. Greenberg C.S. Anal. Biochem. 1992; 205: 166-171Crossref PubMed Scopus (154) Google Scholar). Extracellular PPi was determined radiometrically and equalized for the DNA concentration in each well, as described (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar). We determined specific activity of NPP and AP as described, with units of NPP and AP designated as moles of substrate hydrolyzed/h/g protein in each sample (31Johnson K. Vaingankar S. Chen Y. Moffa A. Goldring M.B. Sano K. Jin-Hua P. Sali A. Goding J. Terkeltaub R. Arthritis Rheum. 1999; 42: 1986-1997Crossref PubMed Scopus (106) Google Scholar). Statistical Analyses—Where indicated, error bars represent S.D. Statistical analyses were performed using the Student's t test (paired 2-sample testing for means). Expression and Activation of TG2 in Articular Cartilage Chondrocytes—To determine which TG isozymes other than TG2 might modulate stimulated changes in articular chondrocyte TG activity, we first tested for expression of TG2 relative to other TG isozymes in situ in mouse knee articular cartilages, using RT-PCR (Fig. 1). In normal cartilages, TG2 and FXIIIA mRNA expression were detectable in this manner, but not the mRNAs for the fibroblast-expressed TG isozymes TG1, TG3, and TG5. Even in the presence of IL-1β, ATRA, or CNP, no expression of these isozymes was found (data not shown). Absent expression of TG2 was confirmed in the TG2 (–/–) mouse knee cartilages (Fig. 1). Qualitative expression of FXIIIA mRNA was detected in the TG2 (–/–) mouse knee cartilages (Fig. 1). There was a small reduction of the total FXIIIA mRNA level seen in the TG2 (–/–) cartilages, but no apparent adaptive changes in expression of the other TG isozymes tested. Next, we assessed and compared TG catalytic activity in articular chondrocytes in response to ATRA, IL-1β, and CNP. In unstimulated primary knee chondrocytes from the TG2 (+/+) and TG2 (–/–) mice we observed a ∼50% reduction in basal TG activity (Fig. 2A). IL-1β and ATRA treatment, but not CNP treatment, induced doubling of TG activity in primary TG2 (+/+) mouse articular chondrocytes (Fig. 2A). The respective findings for chondrocyte TG activity in response to the same agonists were confirmed when primary normal bovine knee articular chondrocytes were studied (Fig. 2B). In articular chondrocytes from TG2 (–/–) mice, IL-1β and ATRA failed to significantly increase TG activity (Fig. 2A). Thus, TG2 accounted for the majority of up-regulated TG2 catalytic activity in knee articular chondrocytes in response to both IL-1β and ATRA. Essential Role of TG2 in IL-1β- and ATRA-induced Chondrocyte Matrix Calcification—We confirmed that IL-1β and ATRA induced matrix calcification by chondrocytes, first using normal bovine knee cells carried for 14 days in a mineralization-promoting culture medium (Fig. 3A). Under the same conditions, CNP failed to significantly induce matrix calcification in bovine knee chondrocytes. To assess if the differential changes in matrix calcification were the result of altered generation of PPi, we measured extracellular PPi, which has been described to rise in association with chondrocyte hypertrophy (36Rosenthal A.K. Henry L.A. J. Rheumatol. 1999; 26: 395-401PubMed Google Scholar). We also quantified cell-associated PPi-generating NPP activity (36Rosenthal A.K. Henry L.A. J. Rheumatol. 1999; 26: 395-401PubMed Google Scholar), which modulates growth plate chondrocyte differentiation in vivo (37Boskey A.L. Curr. Rheumatol. Rep. 2002; 4: 245-251Crossref PubMed Scopus (17) Google Scholar) (Fig. 3, B–C). Under these conditions, CNP failed to significantly alter either PPi levels or NPP activity in bovine chondrocytes (Fig. 3, B–C). We confirmed (38Rosenthal A.K. Henry L.A. Calcif. Tissue Int. 1996; 59: 128-133Crossref PubMed Scopus (15) Google Scholar) that ATRA increased extracellular PPi and cell-associated NPP activity by 2-fold in normal bovine chondrocytes (Fig. 3, B–C). We also confirmed (39Hashimoto S. Ochs R.L. Rosen F. Quach J. McCabe G. Solan J. Seegmiller J.E. Terkeltaub R. Lotz M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3094-3099Crossref PubMed Scopus (225) Google Scholar) that IL-1β significantly decreased both extracellular PPi and NPP activity in the normal bovine chondrocytes. Thus, opposite changes in PPi generation occurred as a component of chondrocyte differentiation in response to ATRA and IL-1β. Parallel experiments using TG2 (+/+) articular chondrocytes treated with ATRA and IL-1β gave comparable results to bovine chondrocytes for stimulation of calcification, NPP-specific activity, and extracellular PPi (Fig. 4, A–C). But in the absence of TG2, mouse chondrocytes stimulated with IL-1β or ATRA failed to carry out significantly increased matrix calcification relative to wild-type cells (Fig. 4A). ATRA increased extracellular PPi by more than 3-fold and NPP activity by more than 4-fold basal levels, and IL-1β significantly decreased both extracellular PPi and NPP activity in TG2 (+/+) mouse chondrocytes (Fig. 4, B–C). TG2 (–/–) mouse chondrocytes had a less pronounced up-regulation of PPi and NPP in response to ATRA than in wild-type cells (Fig. 4, B–C). But the TG2 (–/–) mouse chondrocytes significantly responded to ATRA and IL-1β with regulatory changes in extracellular PPi and NPP activity in the same respective directions as in wild-type cells (Fig. 4, B–C). Because changes in PPi generation appeared less likely than TG2 to be a major force in driving calcification in response to ATRA and IL-1β, we next tested for potential effects of TG2 in modulating altered states of chondrocyte maturation differentially linked to calcification. TG2-dependent and -independent Chondrocyte Hypertrophy—We determined that ATRA-induced type X collagen expression, the cardinal marker for chondrocyte hypertrophy (40Pacifici M. Golden E.B. Iwamoto M. Adams S.L. Exp. Cell Res. 1991; 195: 38-46Crossref PubMed Scopus (94) Google Scholar), was markedly reduced in primary TG2 (–/–) mouse chondrocytes (Fig. 5A). Induction by ATRA of additional markers of chondrocyte hypertrophy (i.e. AP activity (41Iwamoto M. Yagami K. Shapiro I.M. Leboy P.S. Adams S.L. Pacifici M. Microsc. Res. Tech. 1994; 28: 483-491Crossref PubMed Scopus (68) Google Scholar) and MMP-13 expression (42Iwamoto M. Kitagaki J. Tamamura Y. Gentili C. Koyama E. Enomoto H. Komori T. Pacifici M. Enomoto-Iwamoto M. Osteoarthritis Cartilage. 2003; 11: 6-15Abstract Full Text PDF PubMed Scopus (71) Google Scholar)) also became attenuated in TG2 (-/-) chondrocytes (Figs. 5B and 6A). Yet TG2 null cells had the capacity
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