Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene.

We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos. However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were down-regulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes.

[1]  E. Wagner,et al.  Structure and chromosomal assignment of the mouse fra-1 gene, and its exclusion as a candidate gene for oc (osteosclerosis) , 1997, Oncogene.

[2]  G. Superti-Furga,et al.  Identification of Fos target genes by the use of selective induction systems , 1992, Journal of Cell Science.

[3]  G. Stein,et al.  Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation. , 1996, Endocrinology.

[4]  B. Spiegelman,et al.  Pleiotropic effects of a null mutation in the c-fos proto-oncogene , 1992, Cell.

[5]  T. Curran,et al.  Cell transformation by c-fos requires an extended period of expression and is independent of the cell cycle , 1994, Molecular and cellular biology.

[6]  N. Copeland,et al.  Limb alterations in brachypodism mice due to mutations in a new member of the TGFβ-superfamily , 1994, Nature.

[7]  E. Wagner,et al.  Osteoblasts are target cells for transformation in c-fos transgenic mice , 1993, The Journal of cell biology.

[8]  Clifford J. Tabin,et al.  Regulation of Rate of Cartilage Differentiation by Indian Hedgehog and PTH-Related Protein , 1996, Science.

[9]  N. Copeland,et al.  The mouse short ear skeletal morphogenesis locus is associated with defects in a bone morphogenetic member of the TGFβ superfamily , 1992, Cell.

[10]  P. Leder,et al.  Fibroblast Growth Factor Receptor 3 Is a Negative Regulator of Bone Growth , 1996, Cell.

[11]  Y. Ikawa,et al.  A chondrogenic cell line derived from a differentiating culture of AT805 teratocarcinoma cells. , 1990, Cell differentiation and development : the official journal of the International Society of Developmental Biologists.

[12]  J. McCluskey,et al.  Control of cell cycle gene expression in bone development and during c-Fos-induced osteosarcoma formation. , 1998, Developmental genetics.

[13]  N. Amizuka,et al.  Parathyroid hormone-related peptide-depleted mice show abnormal epiphyseal cartilage development and altered endochondral bone formation , 1994, The Journal of cell biology.

[14]  R. Sidman,et al.  Chondrodysplasia and neurological abnormalities in ATF-2-deficient mice , 1996, Nature.

[15]  A. McMahon,et al.  Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton. , 1998, Science.

[16]  T. Curran,et al.  Stimulus-transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun. , 1991, Annual review of neuroscience.

[17]  S. Mundlos,et al.  Cbfa1, a Candidate Gene for Cleidocranial Dysplasia Syndrome, Is Essential for Osteoblast Differentiation and Bone Development , 1997, Cell.

[18]  D. Stéhelin,et al.  The Ets Transcription Factors Interact with Each Other and with the c-Fos/c-Jun Complex via Distinct Protein Domains in a DNA-dependent and -independent Manner* , 1997, The Journal of Biological Chemistry.

[19]  M. Greenberg,et al.  Fos Family Members Induce Cell Cycle Entry by Activating Cyclin D1 , 1998, Molecular and Cellular Biology.

[20]  R. Weinberg,et al.  Shared role of the pRB-related p130 and p107 proteins in limb development. , 1996, Genes & development.

[21]  D. Church,et al.  Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia , 1994, Cell.

[22]  F. Luyten,et al.  A human chondrodysplasia due to a mutation in a TGF-β superfamily member , 1996, Nature Genetics.

[23]  Derek W. Abbott,et al.  Finkel-Biskis-Reilly Osteosarcoma Virus v-Fos Inhibits Adipogenesis and Both the Activity and Expression of CCAAT/Enhancer Binding Protein α, a Key Regulator of Adipocyte Differentiation* , 1997, The Journal of Biological Chemistry.

[24]  H. Iba,et al.  Chondrocytes as a specific target of ectopic Fos expression in early development. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  M. Karin,et al.  The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. , 1991, Biochimica et biophysica acta.

[26]  R. Derynck,et al.  Toward a molecular understanding of skeletal development , 1995, Cell.

[27]  P. Gruss,et al.  Proto-oncogene c-fos expression in growth regions of fetal bone and mesodermal web tissue , 1987, Nature.

[28]  J. Aubin,et al.  Analysis of chondroprogenitor frequency and cartilage differentiation in a novel family of clonal chondrogenic rat cell lines. , 1996, Differentiation; research in biological diversity.

[29]  H. Ito,et al.  Noggin and bone morphogenetic protein-4 coordinately regulate the progression of chondrogenic differentiation in mouse clonal EC cells, ATDC5. , 1999, Biochemical and biophysical research communications.

[30]  V. Lefebvre,et al.  Type X collagen gene expression in mouse chondrocytes immortalized by a temperature-sensitive simian virus 40 large tumor antigen , 1995, The Journal of cell biology.

[31]  A. Pearman,et al.  Parathyroid Hormone Induces c-fos Promoter Activity in Osteoblastic Cells through Phosphorylated cAMP Response Element (CRE)-binding protein Binding to the Major CRE* , 1996, The Journal of Biological Chemistry.

[32]  J. Massagué,et al.  Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage. , 1997, Genes & development.

[33]  M. Tymms,et al.  Down's syndrome-like skeletal abnormalities in Ets2 transgenic mice , 1996, Nature.

[34]  B. Hogan,et al.  Bone morphogenetic proteins: multifunctional regulators of vertebrate development. , 1996, Genes & development.

[35]  T. Atsumi,et al.  Cellular Hypertrophy and Calcification of Embryonal Carcinoma‐Derived Chondrogenic Cell Line ATDC5 In Vitro , 1997, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[36]  S. Nomura,et al.  Maturational disturbance of chondrocytes in Cbfa1‐deficient mice , 1999, Developmental dynamics : an official publication of the American Association of Anatomists.

[37]  J. Feingold,et al.  An autonomously replicating eukaryotic expression vector with a tetracycline-responsive promoter. , 1996, Gene.

[38]  E. Wagner,et al.  Differentiation of F9 teratocarcinoma stem cells after transfer of c-fos proto-oncogenes , 1984, Nature.

[39]  N. Selvamurugan,et al.  Parathyroid Hormone Regulates the Rat Collagenase-3 Promoter in Osteoblastic Cells through the Cooperative Interaction of the Activator Protein-1 Site and the runt Domain Binding Sequence* , 1998, The Journal of Biological Chemistry.

[40]  D. Goltzman,et al.  Regulation of expression of the chondrocytic phenotype in a skeletal cell line (CFK2) in vitro , 1993, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[41]  Makoto Sato,et al.  Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts , 1997, Cell.

[42]  Richard J Smeyne,et al.  Continuous c-fos expression precedes programmed cell death in vivo , 1993, Nature.

[43]  E. Wagner,et al.  Bone and haematopoietic defects in mice lacking c-fos , 1992, Nature.

[44]  M. Gossen,et al.  Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[45]  Peter A. Jones,et al.  Multiple new phenotypes induced in 10T 1 2 and 3T3 cells treated with 5-azacytidine , 1979, Cell.

[46]  J. Aubin,et al.  Effects of dexamethasone and vitamin D3 on cartilage differentiation in a clonal chondrogenic cell population. , 1989, Endocrinology.

[47]  E. Wagner,et al.  Analysis of the differentiation‐promoting potential of inducible c‐fos genes introduced into embryonal carcinoma cells. , 1985, The EMBO journal.

[48]  E. Wagner,et al.  Fos and bone cell development: lessons from a nuclear oncogene. , 1995, Trends in genetics : TIG.

[49]  P. Angel,et al.  Both AP-1 and Cbfa1-like factors are required for the induction of interstitial collagenase by parathyroid hormone , 1999, Oncogene.

[50]  H. Jüppner,et al.  A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. , 1995, Science.

[51]  S. Werner,et al.  Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse. , 1992, Development.

[52]  J. Aubin,et al.  Differentiation of muscle, fat, cartilage, and bone from progenitor cells present in a bone-derived clonal cell population: effect of dexamethasone , 1988, The Journal of cell biology.

[53]  E. Wang,et al.  Cells en route to apoptosis are characterized by the upregulation of c‐fos, c‐myc, c‐jun, cdc2, and RB phosphorylation, resembling events of early cell‐cycle traverse , 1995, Journal of cellular biochemistry.

[54]  B. Lanske,et al.  PTH/PTHrP Receptor in Early Development and Indian Hedgehog--Regulated Bone Growth , 1996, Science.

[55]  J Glowacki,et al.  Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. , 1994, Genes & development.

[56]  S. Elledge,et al.  Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith–Wiedemann syndrome , 1997, Nature.

[57]  M. Takigawa,et al.  Expression of c‐fos gene inhibits proteoglycan synthesis in transfected chondrocyte , 1996, FEBS letters.

[58]  G. Stein,et al.  Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. , 1995, Experimental cell research.

[59]  Véronique Lefebvre,et al.  A new long form of Sox5 (L‐Sox5), Sox6 and Sox9 are coexpressed in chondrogenesis and cooperatively activate the type II collagen gene , 1998, The EMBO journal.

[60]  U. Möhle-Steinlein,et al.  A novel target cell for c‐fos‐induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras. , 1991, The EMBO journal.

[61]  P. Verde,et al.  Heterodimerization of c-Jun with ATF-2 and c-Fos is required for positive and negative regulation of the human urokinase enhancer. , 1995, Oncogene.

[62]  Sakae Tanaka,et al.  Bcl-2 Lies Downstream of Parathyroid Hormone–related Peptide in a Signaling Pathway That Regulates Chondrocyte Maturation during Skeletal Development , 1997, The Journal of cell biology.

[63]  E. Wagner,et al.  Fra‐1 potentiates osteoclastic differentiation in osteoclast‐macrophage precursor cell lines , 1999, Journal of cellular physiology.

[64]  P. Sawchenko,et al.  Detection of fos protein during osteogenesis by monoclonal antibodies , 1988, Molecular and cellular biology.

[65]  N. Amizuka,et al.  Inactivating mutation in the human parathyroid hormone receptor type 1 gene in Blomstrand chondrodysplasia. , 1998, Endocrinology.

[66]  R. Baron,et al.  Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation , 2005 .

[67]  G. Superti-Furga,et al.  Hormone-dependent transcriptional regulation and cellular transformation by Fos-steroid receptor fusion proteins. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[68]  J. Roume,et al.  Absence of functional receptors for parathyroid hormone and parathyroid hormone-related peptide in Blomstrand chondrodysplasia. , 1998, The Journal of clinical investigation.

[69]  C. Tickle,et al.  Vertebrate limb development. , 1995, Current opinion in genetics & development.

[70]  E. Wagner,et al.  c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. , 1994, Science.

[71]  R. Pestell,et al.  Identification of the cyclin D1 gene as a target of activating transcription factor 2 in chondrocytes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[72]  G. Segre,et al.  Parathyroid hormone induces sequential c-fos expression in bone cells in vivo: in situ localization of its receptor and c-fos messenger ribonucleic acids. , 1994, Endocrinology.

[73]  H. Iba,et al.  C-Jun and JunD suppress maturation of chondrocytes. , 1997, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[74]  Andy Greenfield,et al.  The Sry-related gene Sox9 is expressed during chondrogenesis in mouse embryos , 1995, Nature Genetics.

[75]  T. Atsumi,et al.  Chondrogenic differentiation of clonal mouse embryonic cell line ATDC5 in vitro: differentiation-dependent gene expression of parathyroid hormone (PTH)/PTH-related peptide receptor , 1996, The Journal of cell biology.

[76]  D. Ornitz,et al.  Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. , 1998, Development.

[77]  B. Lanske,et al.  Targeted expression of constitutively active receptors for parathyroid hormone and parathyroid hormone-related peptide delays endochondral bone formation and rescues mice that lack parathyroid hormone-related peptide. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[78]  E. Wagner,et al.  Stable murine chondrogenic cell lines derived from c‐fos‐induced cartilage tumors , 1993, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.