Activating Transcription Factor 2 Is Necessary for Maximal Activity and Serum Induction of the Cyclin A Promoter in Chondrocytes

Endochondral bone growth is regulated through the proliferation and differentiation of growth plate chondrocytes. Mice deficient for the activating transcription factor 2 (ATF-2) gene show reduced proliferation of chondrocytes. Here we demonstrate that the cyclin A gene is a target of ATF-2 in chondrocytes. Serum stimulation of chondrogenic rat chondrosarcoma cells induces cyclin A expression. A cyclic AMP response element (CRE) is necessary for optimal activity and serum inducibility of the cyclin A promoter and confers regulation by ATF-2. Phosphorylation and activity of ATF-2 are enhanced dramatically upon serum stimulation of rat chondrosarcoma cells. Mutation of the CRE or overexpression of dominant-negative ATF-2 inhibits serum induction of the cyclin A promoter. Chondrocytes from ATF-2-deficient mice display reduced and delayed induction of cyclin A upon serum stimulation. The ATF-2-related transcription factor CRE-binding protein contributes to the activity of the cyclin A CRE in chondrocytes, whereas c-Jun and c-Fos regulate the promoter independently of the CRE. Our data suggest that the reduction in cyclin A levels in chondrocytes from ATF-2-deficient mice contributes to their phenotype of reduced chondrocyte proliferation and dwarfism. Endochondral bone growth is regulated through the proliferation and differentiation of growth plate chondrocytes. Mice deficient for the activating transcription factor 2 (ATF-2) gene show reduced proliferation of chondrocytes. Here we demonstrate that the cyclin A gene is a target of ATF-2 in chondrocytes. Serum stimulation of chondrogenic rat chondrosarcoma cells induces cyclin A expression. A cyclic AMP response element (CRE) is necessary for optimal activity and serum inducibility of the cyclin A promoter and confers regulation by ATF-2. Phosphorylation and activity of ATF-2 are enhanced dramatically upon serum stimulation of rat chondrosarcoma cells. Mutation of the CRE or overexpression of dominant-negative ATF-2 inhibits serum induction of the cyclin A promoter. Chondrocytes from ATF-2-deficient mice display reduced and delayed induction of cyclin A upon serum stimulation. The ATF-2-related transcription factor CRE-binding protein contributes to the activity of the cyclin A CRE in chondrocytes, whereas c-Jun and c-Fos regulate the promoter independently of the CRE. Our data suggest that the reduction in cyclin A levels in chondrocytes from ATF-2-deficient mice contributes to their phenotype of reduced chondrocyte proliferation and dwarfism. cyclin-dependent kinase(s) cyclic AMP response element CRE-binding protein activating transcription factor 2 rat chondrosarcoma fetal bovine serum Growth of endochondral bone is controlled by the coordination of proliferation and differentiation of growth plate chondrocytes (for recent review, see Refs. 1.Cancedda R. Cancedda F.D. Castagnola P. Int. Rev. Cytol. 1995; 159: 265-358Crossref PubMed Scopus (351) Google Scholar, 2.Mundlos S. Olsen B.R. FASEB J. 1997; 11: 125-132Crossref PubMed Scopus (158) Google Scholar, 3.Mundlos S. Olsen B.R. FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (125) Google Scholar). Disruption of these processes by gene mutations commonly results in chondrodysplasias that are characterized by dwarfism, skeletal deformities, and early onset osteoarthritis (2.Mundlos S. Olsen B.R. FASEB J. 1997; 11: 125-132Crossref PubMed Scopus (158) Google Scholar,3.Mundlos S. Olsen B.R. FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (125) Google Scholar). Although the intracellular mechanisms involved in the regulation of chondrocyte proliferation and differentiation are poorly understood, recent evidence suggests that cell cycle genes play an important role in these processes (for review, see Ref. 4.Beier F. Leask T.A. Haque S. Chow C. Taylor A.C. Ballock R.T. LuValle P. Matrix Biol. 1999; 18: 109-120Crossref PubMed Scopus (42) Google Scholar). Progression through the mammalian cell cycle is controlled by the sequential activation of several complexes of cyclins and cyclin-dependent kinases (CDKs)1 (for review, see Ref.5.Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar). Activation of CDKs is in part controlled by the expression levels of their respective cyclin partners. Therefore, much attention has been paid to the elucidation of the mechanisms controlling cyclin gene expression. The cyclin A gene is induced at the transition from the G1 to the S phase of the cell cycle and is necessary for progression through the cell cycle from this point on, first in a complex with CDK2, later with CDK1 (5.Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4312) Google Scholar, 6.Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1128) Google Scholar). Induction of cyclin A is caused, at least in part, by transcriptional activation. At least twocis-active sites in the cyclin A promoter have demonstrated importance for the induction of cyclin A transcription: a binding site for E2F transcription factors (7.Schulze A. Zerfass K. Spitkovsky D. Middendorp S. Berges J. Helin K. Jansen-Durr P. Henglein B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11264-11268Crossref PubMed Scopus (320) Google Scholar) and a cyclic AMP response element (CRE; 8–10). One of the transcription factors binding to the CRE is ATF-2 (activating transcription factor 2; 11). ATF-2 is a member of the ATF/CREB family of transcription factors capable of forming homodimers and heterodimers with other family members as well as with c-Jun (11.Ivashkiv L.B. Liu H.-C. Kara C.J. Lamph W.W. Verma I.M. Glimcher L.H. Mol. Cell. Biol. 1990; 10: 1609-1621Crossref PubMed Scopus (177) Google Scholar, 12.Benbrook D.M. Jones N.C. Oncogene. 1990; 5: 295-302PubMed Google Scholar, 13.Landshultz W.H. Johnson P.F. McKnight S.L. Science. 1988; 240: 1759-1764Crossref PubMed Scopus (2538) Google Scholar, 14.Maekawa T. Sakura H. Kanei-Ishii C. Sudo T. Yoshimura T. Fujisawa J.-I. Yoshida M. Ishii S. EMBO J. 1989; 8: 2023-2028Crossref PubMed Scopus (292) Google Scholar, 15.Hai T. Liu F. Coukos W. Green M. Genes Dev. 1989; 3: 2083-2090Crossref PubMed Scopus (758) Google Scholar, 16.Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Crossref PubMed Scopus (1114) Google Scholar). ATF-2-deficient mice display reduced chondrocyte proliferation, causing dwarfism and skeletal deformities (17.Reimold A.M. Grusby M.J. Kosaras B. Fries J.W.V. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (241) Google Scholar). We have recently identified the cyclin D1 gene, which is involved in progression through the G1 phase of the cell cycle, as one target of ATF-2 in chondrocytes (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Reduced levels of cyclin D1 in the ATF-2-deficient mice likely contribute to the phenotype of these mice. However, the skeletal phenotype of cyclin D1-deficient mice (19.Sicinski P. Donaher J.L. Parker S.B. Li T. Fazeli A. Gardner H. Haslam S.Z. Bronson R.T. Elledge S.J. Weinberg R.A. Cell. 1995; 82: 621-630Abstract Full Text PDF PubMed Scopus (892) Google Scholar) is much less severe than that of the ATF-2-deficient mice, suggesting that additional target genes of ATF-2 are involved. Here we demonstrate that the cyclin A gene is a second target of ATF-2 in chondrocytes and that reduction in cyclin A levels as a result of the absence of ATF-2 may also be in part responsible for the phenotype of ATF-2-deficient mice. The plasmids pcycAluc707 and pcycAluc707m have been described recently (9.Shimizu M. Nomura Y. Suzuki H. Ichikawa E. Takeuchi A. Suzuki M. Nakamura T. Nakajima T. Oda K. Exp. Cell Res. 1998; 239: 93-103Crossref PubMed Scopus (105) Google Scholar) and were generously provided by K. Oda. The expression plasmids for wild type and dominant-negative ATF-2 have been described recently (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Expression vectors for dominant-negative c-Fos, c-Jun, and CREB were generously provided by C. Vinson (20.Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Scopus (448) Google Scholar). The GAL4-ATF-2 fusion plasmid (pFA-ATF2), the control plasmid encoding the GAL4-DNA binding domain alone (pFC2-dbd), and the GAL4 reporter vector (pFR-Luc) were purchased from Stratagene. ATF-2-deficient mice (17.Reimold A.M. Grusby M.J. Kosaras B. Fries J.W.V. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (241) Google Scholar) were generously provided by L. Glimcher and genotyped as described (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). The cyclin A (SC596-G) and ATF-2 antibodies (SC-187) were from Santa Cruz Biotechnologies, the phospho-specific ATF-2 antibody was from New England Biolabs, and the actin antibody was from Roche Molecular Biochemicals. Western blot analyses and densitometry of blots were performed as described recently (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Cell culture of rat chondrosarcoma (RCS) cells (21.Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. Crombrugghe B.D. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar), isolation of primary mouse chondrocytes, transfections, luciferase assays, and statistical analyses of promoter data were performed as recently described (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Luciferase values are representative of multiple independent experiments, done in triplicate. Cotransfections were performed using 1.0 μg of cyclin A promoter plasmid, 0.3 μg of empty expression vector or expression vectors for wild type or dominant-negative transcription factors, and 0.2 μg of pRlSV40 (Promega) for standardization. Serum induction experiments involved transfections of primary chondrocytes or RCS cells as described (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Cells were serum starved for 3 days following transfection and then incubated in medium containing 10% FBS. Cells were harvested at the time points indicated. For the GAL4 assays, RCS cells were transfected with 0.2 μg of pFA-ATF2 or pFC2-dbd, 1.0 μg of pRF-Luc, and 0.1 μg of pRlSV40. Cells were serum starved for 72 h, and the medium was exchanged for new serum-free medium or medium containing 10% FBS. Cells were harvested for luciferase assays 12 h after the medium change. For protein time course experiments, subconfluent RCS cells or primary chondrocytes were serum starved for 3 days, stimulated with 10% FBS, and harvested for Western blot analyses at the indicated time points. Chondrogenic RCS cells were serum starved for 3 days and restimulated with 10% FBS to examine cyclin A expression and serum inducibility. Cells were harvested at 4-h intervals, and cyclin A protein expression was investigated using Western blot analyses. Whereas cyclin A protein was hardly detectable in serum-starved cells and in the first hours after serum stimulation, cyclin A levels began to rise sharply after 4 h of serum stimulation and reached maximal levels at 12–16 h (Fig.1). This was paralleled by an increase in DNA replication as measured by bromdeoxyuridine incorporation. 2F. Beier, A. C. Taylor, and P. LuValle, unpublished observations. In contrast, medium changed to fresh serum-free medium induced only a very slight increase in cyclin A protein after 24 h (data not shown). We addressed the question of whether increased levels of cyclin A are caused by enhanced transcriptional activity by transfecting the plasmid pcycAluc707, which contains 707 nucleotides of the rat cyclin A promoter fused to the firefly luciferase gene (9.Shimizu M. Nomura Y. Suzuki H. Ichikawa E. Takeuchi A. Suzuki M. Nakamura T. Nakajima T. Oda K. Exp. Cell Res. 1998; 239: 93-103Crossref PubMed Scopus (105) Google Scholar), into RCS cells. After transfection, cells were serum starved for 3 days, restimulated with 10% FBS, and measured for promoter activity at 4-h intervals. The low basal activity of the cyclin A promoter began to increase after 4 h of serum stimulation and reached its maximum at 12 h (Fig. 2). Medium changed to fresh serum-free medium induced only slight activation of the cyclin A promoter, suggesting that the serum inducibility of the cyclin A gene is caused, at least in part, by increased transcription. The 707-base pair cyclin A promoter fragment has been shown to contain a functional CRE that can confer binding of transcription factors of the CREB/ATF family (9.Shimizu M. Nomura Y. Suzuki H. Ichikawa E. Takeuchi A. Suzuki M. Nakamura T. Nakajima T. Oda K. Exp. Cell Res. 1998; 239: 93-103Crossref PubMed Scopus (105) Google Scholar). We used primary mouse chondrocytes (Fig.3 A) and RCS cells (Fig.3 B) to examine the involvement of the CRE in the regulation of the cyclin A promoter in chondrocytes. Cells were transfected with the plasmids pcycAluc707 and pcycAluc707m (in which the CRE has been mutated; 9). Mutation of the CRE caused a reduction of 70–80% in promoter activity in both cell types. The transcription factor ATF-2 is expressed in proliferating chondrocytes and necessary for chondrocyte proliferation (17.Reimold A.M. Grusby M.J. Kosaras B. Fries J.W.V. Mori R. Maniwa S. Clauss I.M. Collins T. Sidman R.L. Glimcher M.J. Glimcher L.H. Nature. 1996; 379: 262-265Crossref PubMed Scopus (241) Google Scholar, 18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). pcycAluc707 and pcycAluc707m were cotransfected with expression vectors for wild type and dominant-negative ATF-2 (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar) or empty expression vectors into RCS cells (Fig. 3 B). Whereas overexpression of wild type ATF-2 caused a 2.8-fold increase in the activity of pcycAluc707, dominant-negative ATF-2 strongly inhibited the activity of the wild type cyclin A promoter. In contrast, pcycAluc707m showed only weak responses to overexpression of both forms of ATF-2. Transcriptional activation by ATF-2 is enhanced by phosphorylation of NH2-terminal threonine residues through several mitogen-activated protein kinases (22.Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar, 23.Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (474) Google Scholar). We tested whether ATF-2 phosphorylation is increased by serum stimulation by Western blot with an antibody recognizing ATF-2 which is phosphorylated on threonine 71 (Fig. 4). After 12 h of serum stimulation (the time point where maximal activation of the cyclin A promoter is observed; Fig. 2), the of phosphorylated ATF-2 is in stimulated cells than in control cells by In contrast, the of ATF-2 is very in stimulated and cells. whether enhanced phosphorylation of ATF-2 in response to serum to increased transcriptional we used a fusion protein of the GAL4-DNA binding domain and the ATF-2 transcriptional activation domain This fusion protein can transcription from containing only the ATF-2 domain is Serum stimulation for 12 h caused a increase in the activity of a promoter by whereas a control plasmid encoding the GAL4-DNA binding domain alone showed response to serum B). whether the CRE is involved in the serum induction of the cyclin A promoter, pcycAluc707 and pcycAluc707m were transfected into RCS and cells were serum starved for 3 days and restimulated with medium containing 10% FBS. type promoter activity was induced 4 and h and reached its maximum at 12 h (Fig. of wild type ATF-2 caused a basal activity of the cyclin A promoter on its inducibility. of dominant-negative ATF-2 caused a in the induction of cyclin A promoter activity and reduced maximal The induction of the CRE a of 4 h with the wild type promoter (Fig. B). In the maximal activity by the promoter reached only of the of the wild type promoter. Although the of ATF-2 on the activity of the CRE were weak with its on the wild type promoter, wild type and dominant-negative ATF-2 showed or on the serum induction of the cyclin A promoter. We addressed the of ATF-2 in the control of cyclin A expression in chondrocytes in by the regulation of the cyclin A gene in primary chondrocytes from ATF-2-deficient mice which display reduced chondrocyte and their which are type cyclin A promoter activity was reduced by in chondrocytes from with promoter activity in chondrocytes from Mutation of the CRE caused a reduction of promoter activity in whereas the the activity of the cyclin A promoter by in cells. We also demonstrated that of ATF-2 caused a in serum induction and a reduction in maximal promoter activity in chondrocytes from ATF-2-deficient mice with (Fig. B). of ATF-2 both of these we cyclin A protein levels in and ATF-2-deficient mice (Fig. Whereas chondrocytes the and of not the recently described T. F. M. Nomura S. M. Y. T. H. M. A. Glimcher L.H. Ishii S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar), of the be in chondrocytes. analyses that cyclin A levels in ATF-2 chondrocytes in the of 10% were 2.8-fold than in ATF-2 cells to actin we the induction of cyclin A protein expression by 10% FBS in serum-starved primary chondrocytes from and ATF-2-deficient mice In maximal induction of cyclin A protein was after h of serum stimulation, whereas in cells this induction was and maximal expression was ATF-2-deficient chondrocytes levels of cyclin A protein as well as activity of the CRE in the cyclin A promoter, we the that other transcription factors be responsible for this The CRE is the target for the transcription factor and CREB with ATF-2 in the regulation of the cyclin D1 CRE in RCS cells (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). In and have been shown to to the cyclin A CRE in other cell (9.Shimizu M. Nomura Y. Suzuki H. Ichikawa E. Takeuchi A. Suzuki M. Nakamura T. Nakajima T. Oda K. Exp. Cell Res. 1998; 239: 93-103Crossref PubMed Scopus (105) Google Scholar, A.M. D. K. V. J. 1998; PubMed Scopus Google Scholar). the of these in the regulation of cyclin A we cyclin A promoter with expression vectors for dominant-negative of c-Fos, c-Jun, and ATF-2 into subconfluent chondrocytes from A) and B) ATF-2-deficient mice. dominant-negative caused a reduction in cyclin A promoter activity in cells. However, the by dominant-negative CREB and ATF-2 was on the whereas dominant-negative c-Fos and c-Jun the of the promoter plasmids with wild type and CRE to a (Fig. ATF-2 in whereas dominant-negative CREB reduced the activity of the wild type promoter to the for the promoter with the CRE B). In contrast, dominant-negative c-Fos and c-Jun caused in activity of wild type and We show that the cyclin A CRE is necessary both for basal cyclin A promoter activity in chondrocytes and for serum induction of the cyclin A promoter. We have shown that ATF-2 is the transcription factors binding to the CRE in chondrocytes, the transcription of the cyclin D1 gene (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). Here we the cyclin A gene as a second target gene of ATF-2 in chondrocytes. wild type ATF-2 can the cyclin A promoter in chondrocytes, whereas dominant-negative ATF-2 inhibits the activity of the promoter. In activity and phosphorylation of ATF-2 are strongly enhanced after 12 h of serum stimulation, cyclin A promoter activity is ATF-2 activity is regulated through phosphorylation by several mitogen-activated protein kinases as well as protein (22.Gupta S. Campbell D. Derijard B. Davis R.J. Science. 1995; 267: 389-393Crossref PubMed Scopus (1337) Google Scholar, 23.Livingstone C. Patel G. Jones N. EMBO J. 1995; 14: 1785-1797Crossref PubMed Scopus (474) Google Scholar, H. J. R. H. R. K. Y. Jones N. Genes Dev. 1998; PubMed Scopus Google Scholar). However, the protein phosphorylation site is not in the GAL4 fusion protein used in these is phosphorylation of this site by the phospho-specific ATF-2 antibody Therefore, a role of in the regulation of ATF-2 activity in chondrocytes be to the intracellular and kinases ATF-2 activity in chondrocytes are performed in The of ATF-2 on the cyclin A promoter is by the of the CRE not the of ATF-2 on cyclin A promoter activity data suggest that ATF-2 the cyclin A promoter through a second element in to the CRE. The cyclin A gene is also a target of the cell transcription factors of the E2F family (7.Schulze A. Zerfass K. Spitkovsky D. Middendorp S. Berges J. Helin K. Jansen-Durr P. Henglein B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11264-11268Crossref PubMed Scopus (320) Google Scholar). E2F activity is regulated by the phosphorylation of which in is controlled by the activity of CDKs and by cyclin ATF-2 the expression of the cyclin D1 gene in chondrocytes (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar), is likely that also E2F activity and cyclin A promoter activity through we suggest a in which ATF-2 the cyclin A promoter in a through the CRE and through the E2F site (Fig. 11). are in to examine whether the E2F site contributes to the of ATF-2 on the cyclin A promoter. The ATF-2 and cyclin A in as shown by the in cyclin A protein cyclin A promoter and serum induction of cyclin A expression in ATF-2-deficient chondrocytes. data also suggest that the reduction in cyclin A protein levels is at least responsible for the phenotype of the ATF-2-deficient mice. However, of the CRE a in promoter activity than of ATF-2. In CRE activity is in ATF-2-deficient chondrocytes, suggesting that other factors cyclin A transcription from this The ATF-2-deficient mice used in this are to one of ATF-2 T. F. M. Nomura S. M. Y. T. H. M. A. Glimcher L.H. Ishii S. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). However, expression of this not be in chondrocytes from or ATF-2-deficient mice. Therefore, is that this the activity of the CRE in chondrocytes. In contrast, overexpression of dominant-negative CREB in the ATF-2-deficient the activity of the wild type cyclin A promoter to that of the CRE promoter, suggesting that CREB for this the of We have a of CREB and ATF-2 in the regulation of the cyclin D1 gene in chondrocytes (18.Beier F. Lee R.J. Taylor A.C. Pestell R.G. LuValle P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1433-1438Crossref PubMed Scopus (146) Google Scholar). In the phenotype of mice D. A. P. B. G. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar) suggests a role for CREB in skeletal The transcription factors c-Jun and c-Fos have also been shown to to the cyclin A CRE in other cell (9.Shimizu M. Nomura Y. Suzuki H. Ichikawa E. Takeuchi A. Suzuki M. Nakamura T. Nakajima T. Oda K. Exp. Cell Res. 1998; 239: 93-103Crossref PubMed Scopus (105) Google Scholar, A.M. D. K. V. J. 1998; PubMed Scopus Google Scholar). of dominant-negative forms of these inhibited cyclin A promoter activity in these were independent of the suggesting that through promoter both c-Fos and c-Jun are necessary for maximal activity of the cyclin D1 is that their through regulation of E2F Our data show that the CRE and ATF-2 are important for serum induction of the cyclin A promoter. of the as well as of both a and in serum However, cyclin A promoter activity is to serum in both suggesting that additional promoter are involved in serum A likely for this is the E2F site E2F activity can likely be induced by that not ATF-2 or In data the cyclin A gene as a second target of ATF-2 in chondrocytes. cyclin D1 and cyclin A are involved in the control of progression through the G1 and S of the cell cycle, suggesting that ATF-2 its on chondrocyte proliferation and endochondral bone growth through the regulation of cell cycle The of these processes contribute to of the mechanisms controlling and of the with skeletal We are to B. Crombrugghe and V. Lefebvre for RCS C. Vinson for dominant-negative expression vectors for CREB and and to K. Oda for the cyclin A promoter We also L. Glimcher and A. for the ATF-2-deficient mice.

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