Basic Helix-loop-helix Protein DEC1 Promotes Chondrocyte Differentiation at the Early and Terminal Stages
2002; Elsevier BV; Volume: 277; Issue: 51 Linguagem: Inglês
10.1074/jbc.m206771200
ISSN1083-351X
AutoresMing Shen, Eri Yoshida, Weiqun Yan, Takeshi Kawamoto, Ketut Suardita, Yasuhiko Koyano, Katsumi Fujimoto, Mitsuhide Noshiro, Yukio Kato,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-β or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-β and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation. The mRNA level of basic helix-loop-helix transcription factor DEC1 (BHLHB2)/Stra13/Sharp2 was up-regulated during chondrocyte differentiation in cultures of ATDC5 cells and growth plate chondrocytes, and in growth plate cartilage in vivo. Forced expression of DEC1 in ATDC5 cells induced chondrogenic differentiation, and insulin increased this effect of DEC1 overexpression. Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) suppressed DEC1 expression and the differentiation of ATDC5 cells, but DEC1 overexpression antagonized this inhibitory action of PTH/PTHrP. Transforming growth factor-β or bone morphogenetic protein-2, as well as insulin, induced DEC1 expression in ATDC5 cultures where it induced chondrogenic differentiation. In pellet cultures of bone marrow mesenchymal stem cells exposed to transforming growth factor-β and insulin, DEC1 was induced at the earliest stage of chondrocyte differentiation and also at the hypertrophic stage. Overexpression of DEC1 in the mesenchymal cells induced the mRNA expressions of type II collagen, Indian hedgehog, and Runx2, as well as cartilage matrix accumulation; overexpression of DEC1 in growth plate chondrocytes at the prehypertrophic stage increased the mRNA levels of Indian hedgehog, Runx2, and type X collagen, and also increased alkaline phosphatase activity and mineralization. To our knowledge, DEC1 is the first transcription factor that can promote both chondrogenic differentiation and terminal differentiation. The development of the vertebrate long bones occurs through the process of endochondral ossification, which is initiated in the embryo with the condensation of mesenchymal cells and then progresses with their commitment and differentiation into chondrogenic cells. By the late embryonic stage, the epiphyseal growth plate has developed with distinguishable, well organized and spatially distinct zones of resting, proliferating, and post-proliferative hypertrophic chondrocytes. The hypertrophic cartilage calcifies and is invaded by capillaries, and is subsequently replaced by new bone (1Mundlos S. Olsen B.R. FASEB J. 1997; 11: 125-132Crossref PubMed Scopus (155) Google Scholar). Recent studies have identified several transcription factors involved in endochondral ossification. 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On the other hand, little is known about the role of the basic helix-loop-helix (bHLH) 1The abbreviations used are: bHLH, basic helix-loop-helix; BMP, bone morphogenetic protein; FBS, fetal bovine serum; Ihh, Indian hedgehog; m.o.i., multiplicity of infection; MSC, mesenchymal stem cells; PTH, parathyroid hormone; PTHrP, PTH-related peptide; RA, Retinoic acid; TGF-β, transforming growth factor-β; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase1The abbreviations used are: bHLH, basic helix-loop-helix; BMP, bone morphogenetic protein; FBS, fetal bovine serum; Ihh, Indian hedgehog; m.o.i., multiplicity of infection; MSC, mesenchymal stem cells; PTH, parathyroid hormone; PTHrP, PTH-related peptide; RA, Retinoic acid; TGF-β, transforming growth factor-β; RT, reverse transcriptase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase transcription protein family in endochondral ossification, although many bHLH proteins play a critical role in neurogenesis, myogenesis, and hematopoiesis (14Weintraub H. 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In this article, we refer to them as DEC1irrespective of species, unless otherwise specified.2The Human Gene Nomenclature Committee (www.gene.ucl.ac.uk/nomenclature/) has assigned the name ofBHLHB2 to DEC1. Stra13, orSharp2, is the mouse, or rat, ortholog of humanDEC1. In this article, we refer to them as DEC1irrespective of species, unless otherwise specified. a novel bHLH transcription factor, was identified in human chondrocytes by the subtraction method (20Shen M. Kawamoto T. Yan W. Nakamasu K. Tamagami M. Koyano Y. Noshiro M. Kato Y. Biochem. Biophys. Res. Commun. 1997; 236: 294-298Crossref PubMed Scopus (141) Google Scholar). A mouse ortholog (Stra13) and a rat ortholog (Sharp2) of DEC1 were cloned from P19 embryonic carcinoma cells and rat brain, respectively (21Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (215) Google Scholar,22Rossner M.J. Dorr J. Gass P. Schwab M.H. Nave K.A. Mol. Cell Neurosci. 1997; 10: 460-475Crossref PubMed Scopus (107) Google Scholar). DEC1/Stra13 works as a transcriptional repressor, decreasing its own transcription, as well as that of c-myc, through the histone deacetylase-dependent and general transcription factor-dependent mechanisms, respectively (23Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (158) Google Scholar). In P19 cells, DEC1/Stra13 overexpression promoted neuronal differentiation when the cells were exposed to retinoic acid in monolayer culture, or promoted it after aggregation in the absence of retinoic acid (21Boudjelal M. Taneja R. Matsubara S. Bouillet P. Dolle P. Chambon P. Genes Dev. 1997; 11: 2052-2065Crossref PubMed Scopus (215) Google Scholar). In NIH3T3 cells, DEC1/Stra13 expression was associated with growth arrest, and overexpression suppressed proliferation (23Sun H. Taneja R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4058-4063Crossref PubMed Scopus (158) Google Scholar). Recently, DEC1/Stra13-deficient mice have shown defects in several phases of T cell activation, resulting in lymphoid organ hyperplasia and chronic systemic lupus-like autoimmune disease (24Sun H. Lu B. Li R.Q. Flavell R.A. Taneja R. Nat. Immunol. 2001; 11: 1040-1047Crossref Scopus (143) Google Scholar). To explore the role of DEC1 in chondrocyte differentiation, we overexpressed human DEC1 in mouse ATDC5 cells, rabbit mesenchymal stem cells (MSC), and rabbit chondrocytes. ATDC5 cells can mimic chondrocyte differentiation processes from chondroprogenitors to fully differentiated hypertrophic chondrocytes in response to insulin or insulin-like growth factor-1 (25Atsumi T. Miwa Y. Kimata K. Ikawa Y. Cell Differ. Dev. 1990; 30: 109-116Crossref PubMed Scopus (331) Google Scholar, 26Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (342) Google Scholar). Bone marrow MSC can differentiate into chondrocytes, osteoblasts, tenocytes, adipocytes, muscle cells, and nerve cells in vitro and/or in vivo (27Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (17647) Google Scholar, 28Deans R.J. Moseley A.B. Exp. Hematol. 2000; 28: 875-884Abstract Full Text Full Text PDF PubMed Scopus (1300) Google Scholar, 29Tsutsumi S. Shimazu A. Miyazaki K. Pan H. Koike C. Yoshida E. Takagishi K. Kato Y. Biochem. Biophys. Res. Commun. 2001; 288: 413-419Crossref PubMed Scopus (486) Google Scholar). MSC in pellet, but not in monolayer cultures, undergo chondrogenic differentiation in response to insulin and TGF-β. We show here that forced expression of DEC1 promotes chondrogenic differentiation, hypertrophy, and/or mineralization in the cultures of ATDC5 cells, MSC, and chondrocytes. Furthermore, TGF-β, bone morphogenetic protein-2 (BMP-2), and insulin all induced DEC1 expression, whereas PTH/PTHrP suppressed this expression. DEC1 may play an important role in the control of chondrocyte differentiation from the early to the terminal stage. Chondrocytes were isolated from growth plates of the rib cartilage of 4-week-old male Japanese white rabbits, as previously described (30Shimomura Y. Yoneda T. Suzuki F. Calcif. Tissue Res. 1975; 19: 179-187Crossref PubMed Scopus (169) Google Scholar). The experimental procedures on animal care and treatment were performed with permission, and following the rules and guidelines of Hiroshima University. Chondrocytes were seeded at a density of 2 × 104 or 7 × 104 cells in a 16- or 23-mm plastic tissue culture dish, respectively, and maintained in α-minimal essential medium (Sanko Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), 32 units/ml penicillin, 60 μg/ml kanamycin (Meiji Seika Co., Tokyo, Japan), and 250 ng/ml amphotericin B (Dainippon Pharmaceutical Co., Osaka, Japan) at 37 °C in a humidified atmosphere of 5% CO2 in air. ATDC5 cells (Riken, Tsukuba, Japan) were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium (Flow Laboratories) containing 5% FBS, 10 μg/ml human transferrin (Roche Molecular Biochemicals, Mannheim, Germany), 3 × 10−8m sodium selenite, 32 units/ml penicillin, 60 μg/ml kanamycin, and 250 ng/ml amphotericin B in the absence (medium A) or presence of 10 μg/ml bovine insulin (Sigma) (26Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (342) Google Scholar). Inoculum density of the cells was 3 × 104 cells/23 mm in 12-multiwell plates or 6 × 104 cells/36 mm in 6-multiwell plates (Corning, New York, NY). The medium was replaced every other day. In some studies, ATDC5 cells were incubated with a medium containing 0.5% FBS for 3 days. Insulin (10 μg/ml) was added at 72 h, and BMP-2 (100 ng/ml), TGF-β1 (5 ng/ml), or bone extracts (2.5 μg/ml) were added 48 h before the end of the incubation. Bone extracts (Sangi BMP Mixture) were purchased from Wako Pure Chemical Industries, Ltd., Osaka Japan. BMP mixture induces alkaline phosphatase activity in osteogenic cells in vitro and induces bone formationin vivo. Marrow aspirates were obtained from three 4-week-old male Japan White rabbits. The cells were seeded at 2 × 108 cells per 100-mm tissue culture dish and maintained in 10 ml of Dulbecco's modified Eagle's medium supplemented with 10% FBS, 32 units/ml penicillin, and 60 μg/ml kanamycin at 37 °C under 5% CO2 in air (29Tsutsumi S. Shimazu A. Miyazaki K. Pan H. Koike C. Yoshida E. Takagishi K. Kato Y. Biochem. Biophys. Res. Commun. 2001; 288: 413-419Crossref PubMed Scopus (486) Google Scholar). Three days after seeding, floating cells were removed and the medium was replaced by fresh medium. Thereafter, attached cells were fed with fresh medium every 3 days and used as MSC. Passages were performed when cells were reaching confluence. Cells were seeded at 5 × 103 cells/cm2 in 100-mm dishes. For chondrogenic differentiation, cells were seeded at 2 × 105 cells/15-ml plastic centrifuge tube, and maintained in 0.5 ml of serum-free α-minimal essential medium (high glucose) supplemented with 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenite, 5.33 μg/ml linolate, 1.25 mg/ml bovine serum albumin, 10 ng/ml TGF-β1, 100 nm dexamethasone, and 50 μg/ml ascorbic acid-2-phosphate (Wako). The cultures were fed with 0.5 ml of the medium for 4 days after seeding. Thereafter, the cultures were fed with 1 ml of medium every other day (29Tsutsumi S. Shimazu A. Miyazaki K. Pan H. Koike C. Yoshida E. Takagishi K. Kato Y. Biochem. Biophys. Res. Commun. 2001; 288: 413-419Crossref PubMed Scopus (486) Google Scholar). Full-length human DEC1 cDNA was cloned into the HindIII-XbaI site of the expression vector pcDNA3.1/Zeo(+) (Invitrogen) to yield pcDNA3.1/Zeo-DEC1. Stable transfection for pcDNA3.1/Zeo-DEC1 or pcDNA3.1/Zeo(+) was carried out using SuperFect transfection reagent (Qiagen, Crawley, UK). After transfection, the cells were incubated in medium A containing 0.15 mg/ml zeocin (Invitrogen), and several individual clones were isolated. Parental ATDC5 cells and clones transfected with pcDNA3.1/Zeo-DEC1 or pcDNA3.1/Zeo (+) were cultured in 1 ml of medium A/23-mm dish in 12-multiwell plates for 20 days. The cells were exposed to [35S]sulfate (0.5 μCi/culture) for 8 h before the end of the incubation. We estimated the level of proteoglycan synthesis by measuring incorporation of [35S]sulfate into material precipitated with cetylpyridinium chloride after digestion with 2 mg/ml Pronase E (31Kato Y. Nomura Y. Tsuji M. Ohmae H. Nakazawa T. Suzuki F. J. Biochem. (Tokyo). 1981; 90: 1377-1386Crossref PubMed Scopus (25) Google Scholar). The glycosaminoglycan content was determined as described previously (32Farndale R.W. Sayersm C.A. Barrett A.J. Connect. Tissue Res. 1982; 9: 247-248Crossref PubMed Scopus (1143) Google Scholar). In some experiments, the amount of proteoglycan accumulation in the cell layer was estimated by toluidine blue staining. DNA was determined using bisbenzimidazole (Hoechst 33258) (33Labarca C. Paigen K. Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4537) Google Scholar). Total RNA was extracted by the guanidine thiocyanate/cesium trifluoroacetate method (34Smale G. Sasse J. Anal. Biochem. 1992; 203: 352-356Crossref PubMed Scopus (69) Google Scholar). Poly(A)+ RNA (2 μg) that had been enriched using Oligotex(dT)30 (Nippon Roche Ltd., Tokyo, Japan) was electrophoresed on 1% agarose gel containing 2.2 mformaldehyde, and transferred to Nytran nylon membrane (Schleicher & Schuell). Hybridization was carried out with 32P-labeled specific cDNA probes. The membranes were washed at 65 °C for 30 min with 0.1× SSC containing 0.5% SDS, and exposed to BioMax x-ray film (Eastman Kodak Co.) at −70 °C with an intensifying screen. The first-strand cDNA was synthesized from 1 μg of total RNA using the SuperScript II preamplification system (Invitrogen). Pairs of oligonucleotides: 5′-AGAGACGTGACCGGATTAAA-3′ and 5′-CCATAGCCACTGTCTGTGTC-3′ for rabbit DEC1; 5′-AGAGACGTGACCGGATTAAC-3′ and 5′-CGGTATCTTGTCTGGGTTCA-3′ for mouse DEC1; 5′-ATGATCCGCCTCGGGGCTCC-3′ and 5′-TCTGGGCACCACCACCAGCCTTC-3′ for rabbit type II collagen; 5′-CAGGAAAACCTGGACAGCAG-3′ and 5′-ACCCTTAGGACCATTGAGAC-3′ for mouse type X collagen; 5′-GCTTGATGACTCTAAACCTA-3′ and 5′-AAAAAGGGCCCAGTTCTGAA-3′ for Runx2; and 5′-CAAGCAGTTCAGCCCCAACG-3′ and 5′-ACGTGGGCCTTGGACTCGTA-3′ for Indian hedgehog (Ihh), were used as amplification primers. Other gene-specific primers were as previously described (35Kawamoto T. Pan H. Yan W. Ishida H. Usui E. Oda R. Nakamasu K. Noshiro M. Kawashima-Ohya Y. Fujii M. Shintani H. Okada Y. Kato Y. Eur. J. Biochem. 1998; 256: 503-509Crossref PubMed Scopus (28) Google Scholar, 36Kawashima-Ohya Y. Kuruta Y. Yan W. Kawamoto T. Noshiro M. Kato Y. Endocrinology. 1999; 140: 1075-1081Crossref PubMed Scopus (6) Google Scholar, 37Nakamasu K. Kawamoto T. Shen M. Gotoh O. Teramoto M. Noshiro M. Kato Y. Biochim. Biophys. Acta. 1999; 1447: 258-264Crossref PubMed Scopus (31) Google Scholar). PCR reactions were performed using an aliquot of the first-strand cDNA as a template, under standard conditions with KlenTaq polymerase (Clontech Laboratories Inc.) for 22 cycles, which proved optimal for comparison of the amplified products. The amplified products were separated on 1% agarose gels and stained by ethidium bromide, or subcloned into the pGEM T-easy vector (Promega, Madison, WI) to determine the cDNA sequences. Hybridizations were performed with 32P-labeled specific cDNA probes under the same conditions as above described. Quantitative real time PCR analysis was performed using the ABI PRISM 7700 Sequence Detection System instrument and software (PE Appled Biosystems, Inc., Foster City, CA). First-strand cDNA prepared by RT-PCR reaction was amplified using 5′-GCAAGGAAACTTACAAACTGCC-3′ and 5′-CAATGCACTCGTTAATCCGGT-3′ for mouse DEC1, 5′-GAAAGGATCGGCGCAATTAA-3′ and 5′-CATCATCCGAAAGCTGCATC-3′ for human DEC1, 5′-ACGGCCAGGTCATCATCACTATTG-3′ and 5′-CAAGAAGGAAGGCTGGAAAAGA-3′ for β-actin, and 5′-AACTCACTGGCATGGCCTT-3′ and 5′-GCTTCACCACCTTCTTGATG-3′ for GAPDH. The amplified cDNAs were quantified using 6FAM-CACCGGCTGATTGAGAAAAAGAGACGT-TAMRA for mouse, DEC1, 5′-6FAM-CAAGAGTCCGAAGAACCCCCCACAAAA-TAMRA-3′ for human, DEC1, 5′-VIC-CAACGAGCGGTTCCGATGCCC-TAMRA-3′ for β-actin, and 5′-VIC-TGCCGCCTGGAGAAAGCTGCTAAGTA-TAMRA-3′ for GAPDH. DEC1-overexpressing, empty vector integrated and wild-type ATDC5 cells were maintained in 36-mm dishes in the presence or absence of 10 μg/ml insulin. On days 4, 7, and 10, the growth was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, using CellTiter 96 Aqueous One solution cell proliferation assay (Promega). The recombinant adenovirus was constructed as described (38Miyake S. Makimura M. Kanegae Y. Harada S. Sato Y. Takamori K. Tokuda C. Saito I. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1320-1324Crossref PubMed Scopus (786) Google Scholar). Briefly, human DEC1 cDNA was subcloned into the I-CeuI and PI-SceI sites of pAdeno-X (Clontech), which is defective in adenovirus E1A, E1B, and E3 regions. Each cosmid bearing the expression unit and adenovirus DNA-terminal protein complex was cotransfected into the E1 transcomplementing cell line HEK293. Adenovirus carrying human DEC1 was grown in HEK293 cells and purified. Infection of the recombinant adenoviruses into cells was performed at a multiplicity of infection (m.o.i.) of 1–100. Adenovirus carrying LacZ was generously supplied by Dr. Kohei Miyazono (The University of Tokyo) (3Fujii M. Takeda K. Imamura T. Aoki H. Sampath T.K. Enomoto S. Kawabata M. Kato M. Ichijo H. Miyazono K. Mol. Biol. Cell. 1999; 10: 3801-3813Crossref PubMed Scopus (366) Google Scholar). Alkaline phosphatase activity was determined by the method of Bessey et al. (39Bessey O.A. Lowry O.H. Brock M.J. J. Biol. Chem. 1946; 164: 321-329Abstract Full Text PDF PubMed Google Scholar), and calcium content was determined by the method of Gitelman (40Gitelman H.J. Anal. Biochem. 1967; 18: 521-531Crossref Scopus (539) Google Scholar). To examine the changes in the DEC1 mRNA level during chondrocyte differentiation, we incubated rabbit growth plate chondrocytes in high density cultures. The chondrocytes underwent proliferation (day 6), cartilage-matrix synthesis (days 10–14), prehypertrophy (day 18), and hypertrophy (day 22) (41Jikko A. Murakami H. Yan W. Nakashima K. Ohya Y. Satakeda H. Noshiro M. Kawamoto T. Nakamura S. Okada Y. Suzuki F. Kato Y. Endocrinology. 1996; 137: 122-128Crossref PubMed Scopus (30) Google Scholar, 42Yoshida E. Noshiro M. Kawamoto T. Tsutsumi S. Kuruta Y. Kato Y. Exp. Cell Res. 2001; 265: 64-72Crossref PubMed Scopus (38) Google Scholar). Chondrocyte differentiation was associated with the sequential expressions of types II and X collagen mRNA, which are the markers of cartilage-matrix synthesis and hypertrophy, respectively (Fig.1 A). In this system, PTHrP receptor mRNA was expressed at the highest level on day 18 (42). The DEC1 mRNA level was low during the proliferating stage, increased during the matrix-forming/prehypertrophic stages, and reached a maximum in the hypertrophic (terminal) stage. The mRNA level of GAPDH was consistent throughout the culture period (Fig.1 A). We also sliced the growth plate cartilage to identify the expression of DEC1 mRNA in vivo (S1, the proliferating zone; S2, the matrix forming zone; and S3, the hypertrophic zone) (43Kawashima-Ohya Y. Satakeda H. Kuruta Y. Kawamoto T. Yan W. Akagawa Y. Hayakawa T. Noshiro M. Okada Y. Nakamura S. Kato Y. Endocrinology. 1998; 139: 2120-2127Crossref PubMed Google Scholar). Aggrecan mRNA was expressed in all zones, with the level decreasing in the hypertrophic zone. Type X collagen mRNA was undetectable in the proliferating zone, whereas it was abundant in the hypertrophic zone. DEC1 mRNA was expressed in the proliferating, matrix-forming and hypertrophic zones at low, moderate, and high levels, respectively (Fig. 1 B). Mouse embryo cell line ATDC5 cells in confluent cultures exposed to a high concentration of insulin undergo chondrogenic differentiation. In ATDC5 cultures, DEC1 mRNA was barely detectable before the addition of insulin, increased during the earliest stage of chondrogenic differentiation in response to insulin, when the expressions of type II collagen and aggrecan were initiated (6 days after adding insulin), and further increased during the hypertrophic stage (14–18 days after adding insulin) (Fig. 1 C). These findings obtained with primary chondrocytes, growth plate slices, and ATDC5 cells suggest that DEC1 expression starts at the early stage of chondrocyte differentiation and reaches a maximum at the hypertrophic stage. To determine whether the bHLH protein DEC1 is functionally involved in chondrogenic differentiation, we isolated several zeocin-resistant clones (D1–D7) expressing human DEC1. Northern blot analysis showed that human DEC1 mRNA was expressed in D1, D2, and D7 cultures at high levels, and in D3, D4, D5, and D6 cultures at low or moderate levels (Fig.2). The endogenous mouse DEC1 mRNA level in undifferentiated wild-type ATDC5 cultures was very low compared with the human DEC1 mRNA levels in D1, D2, and D7 cultures on day 5 (Fig. 2), but it markedly increased during chondrogenic differentiation (Fig. 1 C). Thus, the human DEC1 mRNA level in mouse D1, D2, and D7 cultures may be comparable with the endogenous DEC1 level in differentiated chondrocytes. In pilot studies, D1, D2, and D7 cells showed prominent chondrogenic differentiation even in the absence of insulin, estimated under a phase-contrast microscope, whereas D3, D4, D5, and D6 cells showed low or moderate levels of differentiation. The degree of chondrogenic differentiation correlated with the human DEC1 mRNA levels. Thus, subsequent studies used the D1, D2, and D7 clones as DEC1-overexpressing cells unless otherwise specified. In the absence of insulin, D1, D2, and D7 cells mimicked the insulin-inducible cell changes, including cellular condensation, cartilage nodule formation, and the growth of cartilage nodules, after the cultures became confluent. The cell shape changes in D1, D2, and D7 cultures were observed from day 10, and most D1, D2, and D7 cells were morphologically altered from fibroblast-like cells to spherical cells with a refractile extracellular matrix by day 20. In the absence of insulin, such cell shape changes were rarely observed with parental ATDC5 cells (AT) or vector-integrated cells (Pc1 and Pc2) throughout the culture period (Fig. 3 A). Accordingly, the intensity with which toluidine blue stained cartilage proteoglycan was much greater in D1, D2, and D7 cultures than in AT, Pc1, and Pc2 cultures (Fig. 3 B). Proteoglycan synthesis by these cells was estimated by measuring the incorporation of [35S]sulfate into glycosaminoglycans precipitated with the cetylpyridinium chloride on day 20. The level of proteoglycan synthesis was higher in D1, D2, and D7 cultures than in AT, Pc1, and Pc2 cultures (Fig. 3 C). Northern blot analysis showed that DEC1 overexpression markedly increased the expression of aggrecan, type II collagen, and type X collagen mRNAs, whereas these mRNAs were barely detectable in the control cultures (Fig. 3 D). These findings indicate that the expression of DEC1 initiates chondrogenic differentiation from the early to the terminal stage. In other studies, D1, Pc1, and AT cells were seeded and maintained in 36-mm dishes in the presence or absence of insulin, and on days 4, 7, and 10, the growth was estimated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay. The cells proliferated at similar growth rates under these conditions (Fig. 3 E). In addition, we repeatedly observed, using a phase-contrast microscope, that DEC1 overexpression had little effect on the proliferation of ATDC5 cells. Because insulin also induces chondrogenic differentiation, the effect of DEC1 overexpression was compared with that of insulin. The intensity with which toluidine blue stained the cell-matrix layer in human DEC1-expressing cultures (D1 and D2) without insulin was similar to that in the control cultures (Pc1 and Pc2) maintained with insulin on days 10 and 18 (Fig.4 B, left andmiddle lanes). Furthermore, DEC1 overexpression plus insulin elicited a synergistic enhancement in proteoglycan accumulation on days 10 and 18 (Fig. 4 B, middle lanes). Because PTH/PTHrP suppresses the cellular condensation process and subsequent chondrogenic differentiation of ATDC5 cells (26Shukunami C. Shigeno C. Atsumi T. Ishizeki K. Suzuki F. Hiraki Y. J. Cell Biol. 1996; 133: 457-468Crossref PubMed Scopus (342) Google Scholar), we examined the effect of PTH/PTHrP on chondrogenic differentiation in DEC1-overexpressing cells. PTH abolished both the cell shape changes, from fibroblast-like cells to spherical cells (Fig. 4 A,right lane), and proteoglycan accumulation (Fig.4 B, right lanes) in insulin-exposed Pc1 and Pc2 cultures. In contrast, PTH had little, if any, effect on chondrogenic differentiation or proteoglycan accum
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