The Raf-1/MEK/ERK Pathway Regulates the Expression of the p21Cip1/Waf1 Gene in Chondrocytes
1999; Elsevier BV; Volume: 274; Issue: 42 Linguagem: Inglês
10.1074/jbc.274.42.30273
ISSN1083-351X
AutoresFrank Beier, Allison C. Taylor, Phyllis LuValle,
Tópico(s)Ubiquitin and proteasome pathways
ResumoThe gene encoding the cyclin-dependent kinase inhibitor p21Cip1/Waf1 is up-regulated in many differentiating cells, including maturing chondrocytes. Since strict control of chondrocyte proliferation is essential for proper bone formation and since p21 is likely involved in this control, we initiated analyses of the mechanisms regulating expression of p21 in chondrocytes. p21 expression and promoter activity was strongly increased during the differentiation of chondrogenic MCT cells. We have identified a 68-base pair fragment conferring transcriptional up-regulation of the p21 gene in chondrocytes. The activity of this fragment required active Raf-1 in MCT cells as well as in primary mouse chondrocytes. Inhibition of downstream factors of Raf-1 (MEK1/2, ERK1/2, and Ets2) also repressed the activity of the 68-base pair fragment in MCT cells. The chemical MEK1/2 inhibitor PD98059 reduced protein levels of p21 in MCTs and primary mouse chondrocytes. These data suggest that signaling through the Raf-1 pathway is necessary for the optimal expression of p21 in chondrocytes and may play an important role in the control of bone formation. The gene encoding the cyclin-dependent kinase inhibitor p21Cip1/Waf1 is up-regulated in many differentiating cells, including maturing chondrocytes. Since strict control of chondrocyte proliferation is essential for proper bone formation and since p21 is likely involved in this control, we initiated analyses of the mechanisms regulating expression of p21 in chondrocytes. p21 expression and promoter activity was strongly increased during the differentiation of chondrogenic MCT cells. We have identified a 68-base pair fragment conferring transcriptional up-regulation of the p21 gene in chondrocytes. The activity of this fragment required active Raf-1 in MCT cells as well as in primary mouse chondrocytes. Inhibition of downstream factors of Raf-1 (MEK1/2, ERK1/2, and Ets2) also repressed the activity of the 68-base pair fragment in MCT cells. The chemical MEK1/2 inhibitor PD98059 reduced protein levels of p21 in MCTs and primary mouse chondrocytes. These data suggest that signaling through the Raf-1 pathway is necessary for the optimal expression of p21 in chondrocytes and may play an important role in the control of bone formation. cyclin-dependent kinase base pair mitogen-activated protein extracellular signal-regulated kinase mitogen-activated protein kinase/extracellular signal-regulated kinase kinase wild type Axial and appendicular skeletal elements are formed by endochondral ossification, a process that involves formation of cartilage precursors and subsequent replacement by bones (reviewed in Ref. 1Cancedda R. Cancedda F.D. Castagnola P. Int. Rev. Cytol. 1995; 159: 265-358Crossref PubMed Scopus (352) Google Scholar). This transition from cartilage to bone occurs within the growth plate, where longitudinal bone growth is controlled. Growth plate chondrocytes undergo a series of rapid cell divisions before they withdraw from the cell cycle and differentiate to large, hypertrophic cells. These hypertrophic chondrocytes undergo apoptosis, their extracellular matrix is degraded, and they are replaced by osteoblasts that produce a bone matrix. Both proliferation and differentiation-associated enlargement of growth plate chondrocytes contribute to bone growth. Analyses of transgenic and knockout mice have demonstrated that disturbance of the fine balance between chondrocyte proliferation and differentiation can cause skeletal defects such as skeletal dysplasias (2Mundlos S. Olsen B.R. FASEB J. 1997; 11: 125-132Crossref PubMed Scopus (158) Google Scholar, 3Mundlos S. Olsen B.R. FASEB J. 1997; 11: 227-233Crossref PubMed Scopus (126) Google Scholar, 4Serra R. Johnson M. Filvaroff E.H. LaBorde J. Sheehan D.M. Derynck R. Moses H.L. J. Cell Biol. 1997; 139: 541-552Crossref PubMed Scopus (418) Google Scholar). Deregulation of chondrocyte proliferation may also be involved in the pathogenesis of chondrosarcomas (5Aigner T. Frischholz S. Dertinger S. Beier F. Girkontaite I. von der Mark K. Histochem. Cell Biol. 1997; 107: 435-440Crossref PubMed Scopus (29) Google Scholar). However, the molecular processes regulating chondrocyte proliferation and differentiation remain largely unknown. Disruptions of several mouse genes involved in growth control have been shown to cause severe deformities and growth abnormalities of the skeleton with frequently lethal consequences. Examples are the inactivation of the genes encoding the transcription factors ATF-2 and c-Fos (6Reimold 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 (243) Google Scholar, 7Wang Z.-Q. Ovitt C. Grigoriadis A.E. Moehle-Steinlein U. Ruether U. Wagner E.F. Nature. 1992; 360: 741-745Crossref PubMed Scopus (817) Google Scholar), the antiapoptosis protein Bcl-2 (8Amling M. Neff L. Tanaka S. Inoue D. Kuida K. Weir E. Philbrick W.M. Broadus A.E. Baron R. J. Cell Biol. 1997; 136: 205-213Crossref PubMed Scopus (275) Google Scholar), the thyroid hormone α and vitamin D receptors (9Yoshizawa T. Handa Y. Uematsu Y. Takeda S. Sekine K. Yoshihara Y. Kawakami T. Arioka K. Sato H. Uchiyama Y. Masushige S. Fukamizu A. Matsumoto T. Kato S. Nat. Genet. 1997; 16: 391-396Crossref PubMed Scopus (1012) Google Scholar, 10Li Y.C. Pirro A.E. Amling M. Delling G. Baron R. Bronson R. Demay M.B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9831-9835Crossref PubMed Scopus (842) Google Scholar, 11Fraichard A. Chassande O. Plateroti M. Roux J.P. Trouillas J. Dehay C. Legrand C. Gauthier K. Kedinger M. Malaval L. Rousset B. Samarut J. EMBO J. 1997; 16: 4412-4420Crossref PubMed Scopus (279) Google Scholar), the fibroblast growth factor receptor 3 (12Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Abstract Full Text Full Text PDF PubMed Scopus (933) Google Scholar), the parathyroid hormone-related peptide (13Amizuka N. Warshawsky H. Henderson J.E. Goltzman D. Karaplis A.C. J. Cell Biol. 1994; 126: 1611-1623Crossref PubMed Scopus (424) Google Scholar,14Karaplis A.C. Luz A. Glowacki L. Bronson R.T. Tybulewicz V.L.J. Kronenberg H.M. Mulligan R.C. Genes Dev. 1994; 8: 277-289Crossref PubMed Scopus (1014) Google Scholar), the p57Kip2 gene (15Yan Y. Friesen J. Lee M.-H. Massague J. Barbacid M. Genes Dev. 1997; 11: 973-983Crossref PubMed Scopus (439) Google Scholar), and the double knockout of the p107 and p130 genes (16Cobrinik D. Lee M.-H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (380) Google Scholar). The p57, p107, and p130 genes belong to the large group of genes controlling progression through the cell cycle. Our current knowledge of the role and regulation of cell cycle genes in chondrocytes has been reviewed recently (17Beier F. Leask T.A. Haque S. Chow C. Taylor A.C. Ballock R.T. LuValle P. Matrix Biology. 1999; 18: 104-120Crossref Scopus (42) Google Scholar). p107 and p130 are closely related to the retinoblastoma protein (pRb) (reviewed in Ref. 18Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4352) Google Scholar). These proteins, called pocket proteins, are capable of inhibiting cell cycle progression when they are present in their hypophosphorylated forms. The cyclin-dependent kinases (CDKs)1 phosphorylate (and thereby inactivate) the pocket proteins. CDK activity is regulated by at least three mechanisms. The CDKs must associate with their partners, the cyclins, to be active. In addition, activation of CDKs requires phosphorylation of some amino acid residues and dephosphorylation of others. As well, CDK activity can be inhibited by members of two groups of CDK inhibitors: the Cip/Kip group, consisting of p21Waf1/Cip1, p27Kip1, and p57Kip2, and the Ink group, consisting of p15Ink4b, p16Ink4a, p18Ink4c, and p19Ink4d(reviewed in Ref. 19Harper J.W. Elledge S.J. Curr. Biol. 1996; 6: 56-64Google Scholar). Simultaneous inactivation of the p107 and p130 genes results in delayed chondrocyte differentiation, leading to deformed bones, shortened limbs, and perinatal death (16Cobrinik D. Lee M.-H. Hannon G. Mulligan G. Bronson R.T. Dyson N. Harlow E. Beach D. Weinberg R.A. Jacks T. Genes Dev. 1996; 10: 1633-1644Crossref PubMed Scopus (380) Google Scholar). A similar phenotype was observed in p57Kip2 null mice (15Yan Y. Friesen J. Lee M.-H. Massague J. Barbacid M. Genes Dev. 1997; 11: 973-983Crossref PubMed Scopus (439) Google Scholar). These data suggest that loss of these negative regulators of cellular proliferation delays the exit from the cell cycle that normally occurs during chondrocyte differentiation. A second member of the Cip/Kip group of CDK inhibitors, p21Waf1/Cip1, was recently shown to be up-regulated in differentiated, hypertrophic chondrocytes (20Stewart M.C. Farnum C.E. MacLeod J.N. Calcif. Tissue Int. 1997; 61: 199-204Crossref PubMed Scopus (32) Google Scholar), consistent with its putative role in exit from the cell cycle during differentiation. p21Waf1/Cip1 was originally cloned as an inhibitor of cyclin-dependent kinases, as a gene up-regulated in cellular senescence, and as a p53-responsive gene that was able to confer cell cycle arrest in response to activation of p53 upon DNA damage and other stresses (21el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7989) Google Scholar, 22Noda A. Ning Y. Venable S.F. Pereira-Smith O.M. Smith J.R. Exp. Cell Res. 1994; 211: 90-98Crossref PubMed Scopus (1320) Google Scholar, 23Harper J.W. Adami G.R. Wei N. Keyomarsi K. Elledge S.J. Cell. 1993; 75: 805-816Abstract Full Text PDF PubMed Scopus (5271) Google Scholar, 24Gu Y. Turek C.W. Morgan D.O. Nature. 1993; 366: 707-710Crossref PubMed Scopus (711) Google Scholar, 25Xiong Y. Hannon G.J. Zhang H. Casso D. Kobayashi R. Beach D. Nature. 1993; 366: 701-704Crossref PubMed Scopus (3197) Google Scholar). p21 expression during development is often high in differentiated, postmitotic cells, and appears to be largely independent of p53 (26Parker S.B. Eichele G. Zhang P. Rawls A. Sands A.T. Bradley A. Olson E.N. Harper J.W. Elledge S.J. Science. 1995; 267: 1024-1027Crossref PubMed Scopus (1028) Google Scholar). On the other hand, p53, a tumor suppressor, is involved, for example, in the nerve growth factor-induced activation of the p21 promoter during differentiation of PC12 cells (27Poluha W. Schonhoff C.M. Harrington K.S. Lachyankar M.B. Crosbie N.E. Bulseco D.A. Ross A.H. J. Biol. Chem. 1997; 272: 24002-24007Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Although p21−/− mice show normal development, indicating that the role of p21 in terminal differentiation is redundant, fibroblasts from p21-deficient mice are deficient in their ability to arrest in the G1 phase of the cell cycle (28Deng C. Zang P. Harper J.W. Elledge S.J. Leder P. Cell. 1995; 82: 675-684Abstract Full Text PDF PubMed Scopus (1961) Google Scholar). In addition, keratinocytes from p21−/− mice have been shown to display enhanced proliferative capacity, reduced expression of differentiation markers, and enhanced tumorigenesis in vivo(29Missero C. Cunto F.D. Kiyokawa H. Koff A. Dotto G.P. Genes Dev. 1996; 10: 3065-3075Crossref PubMed Scopus (286) Google Scholar). These results imply that p21 plays an important role in the terminal differentiation of certain cell types. We have investigated the expression of p21 in the differentiating chondrogenic cell line MCT (30Lefebvre V. Garofalo S. de Crombrugghe B. J. Cell Biol. 1995; 128: 239-245Crossref PubMed Scopus (60) Google Scholar) as well as in primary mouse chondrocytes in order to identify the mechanisms regulating p21 expression in chondrocytes. Our data indicate that a 68-base pair fragment of the p21 promoter, containing binding sites for Ets family transcription factors and p53, is responsible for the up-regulation of p21 in these cells. ERK1/2 phosphorylation increases in parallel with p21 induction, suggesting that the Raf-1 pathway (which activates Ets family transcription factors), rather than p53, is necessary for this up-regulation of p21. Inhibition of Raf-1, MEK1/2, ERK1/2, or c-Ets2 activity obstructs the induction of p21 promoter activity during chondrocyte differentiation. Cell culture media, sera, antibiotics, glutamine, and Lipofectin were purchased from Life Technologies, Inc. The Dual Luciferase Assay Kit was from Promega. The p21 (C19) and ERK (D-2) antibodies were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), the actin antibody was from Roche Molecular Biochemicals, and the phosphospecific ERK antibody (number 9105) was from New England Biolabs. The MEK inhibitor PD98059 was purchased from Calbiochem. Raf-1 expression plasmids have been described recently and were provided by Drs. U. Rapp and S. Ludwig (31Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (397) Google Scholar). The dominant negative ERK expression plasmid pCMV-p41mapk (K/A) was kindly provided by Dr. R. Davis (32Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5070) Google Scholar). c-Ets2 expression constructs were obtained from Dr. R. Pestell (33Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar). Construction of pGl p21 H2320 employed a HindIII fragment from WWPluc (a plasmid containing the human p21 promoter; Ref. 21el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7989) Google Scholar) cloned into theHindIII site of pGl2 basic (Promega). Digestion of pGl p21 H2320 with SacI or SmaI and religation yielded the plasmids pGl p21 S2260 and pGl p21 Sm60, respectively. Digestion of pGl p21 H2320 or pGl p21 S2260 with NsiI and PstI and religation yielded the plasmids pGl p21 H −N/P and pGl p21 S −N/P, respectively. Numbering of nucleotides of the p21 promoter was done according to Ref. 21el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7989) Google Scholar. MCT cells were cultured as described (30Lefebvre V. Garofalo S. de Crombrugghe B. J. Cell Biol. 1995; 128: 239-245Crossref PubMed Scopus (60) Google Scholar) in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mml-glutamine, penicillin (50 units/ml), and streptomycin (50 μg/ml) at 32 °C under 5% CO2. The evening before transfections, 1 × 105 cells were seeded into each well of a 24-well plate. The next morning, transfections were performed with Lipofectin according to the manufacturer's protocol. Briefly, each well was transfected with 1.5 μg of reporter gene construct and 0.3 μg of pRlSV40 (Promega) to standardize for transfection efficiency using 1.5 μl of Lipofectin for 4 h at 32 °C. For cotransfections, 1.5 μg of reporter plasmid was cotransfected with 1.0 μg of Raf-1 or ERK expression plasmid or with empty expression vector and 0.3 μg of pRlSV40. After transfections, cells were cultured for further 24 h at 32 or 37 °C and then lysed with Passive Lysis Buffer (Promega) according to the manufacturer's protocol. For inhibition of MEK1/2 activity, cells were transfected as above and cultured for 6 h after transfection in the presence of 10% fetal bovine serum prior to the addition of 20 μm PD98095 or an equivalent volume of Me2SO (for controls) for the last 18 h of incubation. Primary chondrocytes were isolated from newborn mice as described (34Beier 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 (148) Google Scholar, 35Lefebvre V. Garofalo S. Zhou G. Metsaeranta M. Vuorio E. de Crombrugghe B. Matrix Biol. 1994; 14: 329-335Crossref PubMed Scopus (136) Google Scholar). Chondrocytes were plated at a density of 3 × 106cells/10-cm plate, incubated over night to a density of 40%, and treated with Me2SO or 20 μm PD98059 for 18 h in order to analyze p21 protein expression. Cells were then harvested for Western blot analyses as described below. Transfections were performed as described above. Luciferase assays were performed with the Dual Luciferase Assay Kit (Promega) according to the manufacturer's instructions in a Turner TD-20e luminometer (Promega). 10 μl of lysate was assayed first for firefly luciferase and then forRenilla luciferase activity. Firefly luciferase activity was normalized to Renilla luciferase activity. Data represent the average and S.D. of three independent experiments, each done in triplicate. Statistical analyses of all luciferase values were performed using Student's t test. Significant differences were established as p < 0.05. MCT cells were cultured at 32 °C to 40% confluence and then cultured for a further 24 h at 32 or 37 °C and lysed in SDS loading buffer for analyses of p21 expression. MCT cells were transferred to 37 °C at 40% confluence and incubated for 18 h in the presence of 20 μmPD98059 or an equal volume of Me2SO. Primary chondrocytes were cultured to 40% confluence and then incubated for 18 h in the presence or absence of PD98059 as above. The amount of protein corresponding to 5 × 105 cells was separated on SDS gels and transferred to Hybond-C membranes (Amersham Pharmacia Biotech). Proteins binding to the primary antibodies were detected with a horseradish peroxidase-conjugated secondary anti-rabbit antibody (Santa Cruz Biotechnology) and visualized with the enhanced chemiluminescense detection kit (ECL; Amersham Pharmacia Biotech). MCT cells were cultured as above and harvested for isolation of RNA as described (36Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63398) Google Scholar). 10 μg of total RNA were separated on 0.8% denaturing agarose gels, blotted onto Hybond-N+ membranes (Amersham Pharmacia Biotech), and hybridized to a digoxigenin-labeled p21 probe. Digoxigenin labeling of the mouse p21 cDNA (37Huppi K. Siwarski D. Dosik J. Michieli P. Chedid M. Reed S. Mock B. Givol D. Mushinski J.F. Oncogene. 1994; 9: 3017-3020PubMed Google Scholar), hybridization, washing, and detection (using CDP-Star) were performed with reagents from Roche Molecular Biochemicals according to the manufacturer's instructions. Expression of p21 in growth plate chondrocytes has recently been shown to be linked to maturing and hypertrophic chondrocytes (20Stewart M.C. Farnum C.E. MacLeod J.N. Calcif. Tissue Int. 1997; 61: 199-204Crossref PubMed Scopus (32) Google Scholar). In order to identify possible cell culture models for the study of the regulation of p21 gene expression in chondrocytes, we analyzed the levels of p21 protein during differentiation of MCT cells. MCT cells are derived from murine chondrocytes immortalized with a temperature-sensitive SV40 large T antigen. At 32 °C they show moderate expression of several markers of maturing and differentiated, hypertrophic chondrocytes. Expression of these markers strongly increases upon transfer to 37 °C and is paralleled by an immediate stop in proliferation and subsequent cell death within 96 h (Ref. 30Lefebvre V. Garofalo S. de Crombrugghe B. J. Cell Biol. 1995; 128: 239-245Crossref PubMed Scopus (60) Google Scholar; data not shown). Levels of p21 increased strongly after 24 h at 37 °C (Fig.1 A). p21 mRNA expression also increased significantly upon transfer to 37 °C (Fig.1 B). Transient transfections of a plasmid encoding the firefly luciferase reporter gene driven by a 2320-bp (base pair) fragment of the human p21 promoter (plasmid pGl p21 H2320) showed a similar pattern of activity. Whereas the p21 promoter conferred promoter activity similar to the herpes simplex thymidine kinase promoter in MCT cells cultured at 32 °C, it increased 6-fold after transfer to 37 °C (Fig. 1 C). We transfected several deletion constructs of the 2320-base pair (bp) fragment of the p21 promoter (Fig.2 A) into MCT cells at 37 °C in order to analyze activities in differentiating chondrocytes. Deletion of a 68-bp fragment at the 5′-end of the construct (plasmid pGl p21 S2260) resulted in a 75% decrease in promoter activity, compared with plasmid pGl p21 H2320 (Fig. 2 B). Deletion of an internal fragment of 1874 bp (plasmid pGl p21 H −N/P) had almost no effect on promoter activity. However, deletion of the 5′ 68-bp fragment and the internal fragment together (plasmid pGl p21 S −N/P) reduced promoter activity to less then 10% of that of the 2320-bp promoter fragment. The activity of pGl p21 S −N/P was very similar to that conferred by a basal 68-bp fragment of the p21 promoter (plasmid pGl p21 SmH 60) (Fig. 2 B). We investigated the role of the 68-bp fragment on p21 promoter up-regulation at 37 °C during MCT differentiation by determining the activities of plasmids pGl p21 H2320 and pGl p21 2260 at both temperatures (Fig. 2 C). Whereas the full-length promoter displayed 6-fold higher activity at 37 °C, the promoter fragment missing the 68 nucleotides showed only a small increase in activity at the higher temperature. Two transcription factor binding sites have been described in the 3′-half of the 68-bp fragment: a p53 response element (21el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7989) Google Scholar) and an overlapping binding site for Ets family transcription factors (38Funaoka K. Shindoh M. Yoshida K. Hanzawa M. Hida K. Nishikata S. Totsuka Y. Fujinaga K. Biochem. Biophys. Res. Commun. 1997; 236: 79-82Crossref PubMed Scopus (33) Google Scholar). These sites are conserved among the human, mouse, and rat p21 genes (Fig. 3; Refs. 21el-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (7989) Google Scholar, 31Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (397) Google Scholar, and 32Xia Z. Dickens M. Raingeaud J. Davis R.J. Greenberg M.E. Science. 1995; 270: 1326-1331Crossref PubMed Scopus (5070) Google Scholar). In contrast, the 5′-half of the 68-bp fragment shows much less conservation; computer analyses revealed no potential binding sites for transcription factors conserved among all three species (data not shown). Since Ets transcription factors are common targets of mitogen-activated protein (MAP) kinase pathways and since Raf-1, a central component of one MAP kinase pathway, has recently been shown to be up-regulated during chondrocyte differentiation (40Kaneko Y. Tanzawa H. Sato K. Calcif. Tissue Int. 1994; 54: 426-430Crossref PubMed Scopus (11) Google Scholar) and to be necessary for maximal expression of the hypertrophic chondrocyte-specific collagen X gene (41Beier F. Taylor A.C. LuValle P. J. Cell. Biochem. 1999; 72: 549-557Crossref PubMed Scopus (17) Google Scholar), we cotransfected expression constructs for wild type, constitutively active, and dominant negative Raf-1 (31Bruder J.T. Heidecker G. Rapp U.R. Genes Dev. 1992; 6: 545-556Crossref PubMed Scopus (397) Google Scholar) with p21 reporter constructs into MCT cells at 37 °C (Fig. 4). Coexpression of wild type (WT) or constitutively active Raf-1 (BXB) enhanced promoter activity of pGl p21 H2320 2.2- and 3.3-fold, respectively, whereas dominant negative Raf-1 (C4B) caused a 64% decrease in promoter activity. In contrast, the activity of pGl p21 S2260 was only marginally affected by overexpression of the different versions of Raf-1.Figure 4The 68-bp fragment is regulated by Raf-1 activity. pGl p21 H2320 and pGl p21 S2260 were transfected (together with pRlSV40) into MCT cells, together with empty expression vector (vector) or expression vectors for wild type Raf-1 (wt c-Raf), constitutively active Raf-1 (BXB), or dominant negative Raf-1 (C4B). After 24 h at 37 °C, cells were harvested, and cytosolic firefly luciferase activity was measured and standardized to Renilla luciferase activity to yield relative luciferase activity. The effects of the Raf-1 expression constructs on pGl p21 S2260 activity are of statistical significance (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Raf-1 induction of transcription factor activity is mediated by the downstream kinases MEK1/2 and ERK1/2. We performed transfections of pGl p21 H2320 in the presence of Me2SO or PD98059, a chemical inhibitor of MEK1/2, in order to determine whether MEK1/2 activity is necessary. PD98059 reduced the activity of this promoter fragment by more then 60% (Fig.5 A). Cotransfection of pGl p21 H2320 with an expression vector for a dominant negative version of ERK caused a similar reduction in promoter activity (Fig.5 B). We next investigated whether inhibition of the Raf-1/MEK/ERK pathway also causes reduced levels of p21 protein. MCT cells were incubated for 24 h at 37 °C in the presence of Me2SO or PD98059. Western blot analyses showed that inhibition of MEK1/2 with PD98059 induced a dramatic decrease in p21 protein levels (Fig. 6). Our results implied that the activity of the Raf-1/MEK/ERK pathway increases during MCT differentiation. To verify this, the phosphorylation status of ERK1/2 (which reflects the activity of MEK1/2) in MCT cells cultured at 32 and 37 °C was investigated using Immunoblot analyses with a phosphospecific ERK1/2 antibody. The amount of phosphorylated ERK1 increases 3.2-fold after 24 h at 37 °C (Fig. 7). Immunoblot analyses with a nonphosphospecific ERK1/2 antibody revealed that equal amounts of ERK1 protein were present at both temperatures. With both antibodies, ERK2 could only be detected after prolonged exposure of the blots with either of the antibodies (data not shown). The transcription factor c-Ets2 has been shown to be expressed in chondrocytes (42Maroulakou I.G. Papas T.S. Green J.E. Oncogene. 1994; 9: 1551-1565PubMed Google Scholar) and to be a target of the Raf-1/MEK1/2/ERK1/2 pathway (43Yang B.-S. Hauser C.A. Henkel G. Colman M.S. Beveren V.V. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (319) Google Scholar). We cotransfected the plasmid pGl p21 H2320 with expression constructs for different forms of c-Ets2 (33Albanese C. Johnson J. Watanabe G. Eklund N. Vu D. Arnold A. Pestell R.G. J. Biol. Chem. 1995; 270: 23589-23597Abstract Full Text Full Text PDF PubMed Scopus (764) Google Scholar, 43Yang B.-S. Hauser C.A. Henkel G. Colman M.S. Beveren V.V. Stacey K.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (319) Google Scholar) into MCT cells at 32 °C (data not shown) and at 37 °C (Fig.8 A). Overexpression of WT c-Ets2 at 37 °C increased the activity of the promoter 2.7-fold, whereas overexpression of a mutant that cannot be phosphorylated by ERK1/2 (Ets-2 A72) had no effect. Overexpression of a dominant negative version of c-Ets2 (EtslacZ) caused a reduction of 56% in promoter activity. p21 promoter activity was stimulated by c-Ets2 at 32 °C to a similar degree. We next examined whether c-Ets2 is necessary for Raf-1 activation of the p21 promoter (Fig. 8 B) The plasmid pGl p21 H2320 was cotransfected with expression vectors for activated Raf-1 (BXB) and the different forms of c-Ets2. Stimulation of the p21 promoter by activated Raf-1 was enhanced by WT c-Ets2, but the nonphosphorylatable mutant (Ets-2 A72) and the dominant negative form of c-Ets2 inhibited this effect by 47 and 69%, respectively. We next wanted to determine whether the effects of the Raf-1 pathway on p21 expression that we had observed in the MCT cell line were also present in primary chondrocytes. We transfected pGl p21 H2320 and pGl p21 S2260 together with wild type Raf-1, constitutively active Raf-1 (BXB), and dominant negative Raf-1 (C4B) (Fig. 9). Overexpression of wild type or activated Raf-1 caused a 2.3- and 2.9-fold increase in p21 pGl H2320 activity, respectively, whereas overexpression of the dominant negative form of Raf-1 inhibited promoter activity of this fragment by 65%. In contrast, pGl p21 S2260 displayed only marginal responses to ectopic Raf-1. To verify the role of the Raf-1 pathway in p21 protein expression in primary chondrocytes, we incubated primary mouse chondrocytes with Me2SO or 20 μm PD98059 for 18 h and harvested for Western blot analyses (Fig.10). PD98059 caused a 3.8-fold reduction in p21 protein levels in primary chondrocytes. We have identified a 68-base pair fragment of the p21Cip1/Waf1 promoter that is responsible for the regulation of p21 promoter activity in both the chondrogenic cell line MCT (30Lefebvre V. Garofalo S. de Crombrugghe B. J. Cell Biol. 1995; 128: 239-245Crossref PubMed Scopus (60) Google Scholar) and primary mouse chondrocytes. Deletion of this fragment resulted in an approximately 70% decrease in promoter activity in both cell types (Figs. 2 and 9). In contrast, deletion of the internal 1874-bp NsiI/PstI fragment alone had very little effect on the activity of the p21 promoter in MCT cells. However, deletion of this fragment clearly enhances the effect of the deletion of the 68-bp fragment. These data suggest that response elements for the same pathways or transcription factors are present in both fragments. Binding sites for both Ets family transcription factors and p53
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