Fibroblast Growth Factor Inhibits Chondrocytic Growth through Induction of p21 and Subsequent Inactivation of Cyclin E-Cdk2

Abstract

Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G1 phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb. Fibroblast growth factor (FGF) and its receptor (FGFR) are thought to be negative regulators of chondrocytic growth, as exemplified by achondroplasia and related chondrodysplasias, which are caused by constitutively active mutations in FGFR3. To understand the growth-inhibitory mechanisms of FGF, we analyzed the effects of FGF2 on cell cycle-regulating molecules in chondrocytes. FGF2 dramatically inhibited proliferation of rat chondrosarcoma (RCS) cells and arrested their cell cycle at the G1 phase. FGF2 increased p21 expression in RCS cells, which assembled with the cyclin E-Cdk2 complexes, although the expression of neither cyclin E nor Cdk2 increased. In addition, the kinase activity of immunoprecipitated cyclin E or Cdk2, assessed with retinoblastoma protein (pRb) as substrate, was dramatically reduced by FGF-2. Moreover, FGF2 shifted pRb to its underphosphorylated, active form in RCS cells. FGF2 not only induced p21 protein expression in proliferating chondrocytes in mouse fetal limbs cultured in vitro but also decreased their proliferation as assessed by the expression of histone H4 mRNA, a marker for cells in S phase. Furthermore, inhibitory effects of FGF2 on chondrocytic proliferation were partially reduced in p21-null limbs, compared with those in wild-type limbs in vitro. Taken together, FGF's growth inhibitory effects of chondrocytes appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb. fibroblast growth factor fibroblast growth factor receptor signal transducers and activators of transcription cyclin-dependent kinase CDK inhibitor rat chondrosarcoma wild type FGFs1 are a large family of at least 23 related polypeptides that bind to and activate a family of four tyrosine kinase receptors, FGFRs. They play important roles in regulating proliferation and differentiation of various types of cells, including those involved in limb development and long bone formation (1Szebenyi G. Fallon J.F. Int. Rev. Cytol. 1999; 185: 45-106Crossref PubMed Google Scholar, 2Xu X. Weinstein M. Li C. Deng C.X. Cell Tissue Res. 1999; 296: 33-43Crossref PubMed Scopus (127) Google Scholar). Long bones form by endochondral ossification, which is characterized by mesenchymal condensation, chondrogenic differentiation, chondrocytic proliferation, synthesis of cartilage matrix, hypertrophic differentiation, and replacement by bone. These sequential growth and differentiation processes are regulated by numerous growth factors and their receptors, such as parathyroid hormone-related protein, Indian hedgehog, and insulin-like growth factor-I (3Karp S.J. Schipani E. St-Jacques B. Hunzelman J. Kronenberg H. McMahon A.P. Development. 2000; 127: 543-548Crossref PubMed Google Scholar, 4Liu J.P. Baker J. Perkins A.S. Robertson E.J. Efstratiadis A. Cell. 1993; 75: 59-72Abstract Full Text PDF PubMed Scopus (2533) Google Scholar). Recently, achondroplasia, thanatophoric dysplasia, and hypochondroplasia have been shown to be caused by constitutively active mutations in the FGFR3 gene (5Webster M.K. Donoghue D.J. Trends Genet. 1997; 13: 178-182Abstract Full Text PDF PubMed Scopus (269) Google Scholar). Also, FGFR3-deficient mice displayed overgrowth of long bones (6Deng C. Wynshaw-Boris A. Zhou F. Kuo A. Leder P. Cell. 1996; 84: 911-921Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar, 7Colvin J.S. Bohne B.A. Harding G.W. McEwen D.G. Ornitz D.M. Nat. Genet. 1996; 12: 390-397Crossref PubMed Scopus (742) Google Scholar), and mice carrying dominant active FGFR3 genes exhibited dwarfism similar to that in patients with achondroplasia and thanatophoric dysplasia (8Wang Y. Spatz M.K. Kannan K. Hayk H. Avivi A. Gorivodsky M. Pines M. Yayon A. Lonai P. Givol D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4455-4460Crossref PubMed Scopus (191) Google Scholar, 9Li C. Chen L. Iwata T. Kitagawa M. Fu X.Y. Deng C.X. Hum. Mol. Genet. 1999; 8: 35-44Crossref PubMed Scopus (192) Google Scholar, 10Iwata T. Chen L. Li C. Ovchinnikov D.A. Behringer R.R. Francomano C.A. Deng C.X. Hum. Mol. Genet. 2000; 9: 1603-1613Crossref PubMed Scopus (175) Google Scholar, 11Chen L. Adar R. Yang X. Monsonego E.O. Li C. Hauschka P.V. Yayon A. Deng C.X. J. Clin. Invest. 1999; 104: 1517-1525Crossref PubMed Scopus (210) Google Scholar, 12Naski M.C. Colvin J.S. Coffin J.D. Ornitz D.M. Development. 1998; 125: 4977-4988Crossref PubMed Google Scholar, 13Segev O. Chumakov I. Nevo Z. Givol D. Madar-Shapiro L. Sheinin Y. Weinreb M. Yayon A. Hum. Mol. Genet. 2000; 9: 249-258Crossref PubMed Scopus (89) Google Scholar). Moreover, targeted overexpression of FGF9 in cartilage results in dwarfism in mice similar to the dwarfism in achondroplasia (14Garofalo S. Kliger-Spatz M. Cooke J.L. Wolstin O. Lunstrum G.P. Moshkovitz S.M. Horton W.A. Yayon A. J. Bone Miner. Res. 1999; 14: 1909-1915Crossref PubMed Scopus (80) Google Scholar). Although these findings support the hypothesis that FGF and its receptors are negative regulators of endochondral bone development, little is known about the mechanisms by which FGF inhibits chondrocytic growth. Sahni et al. recently reported that FGF inhibited chondrocytic growth through activating the signal transducer and activator of transcription 1 (STAT1) pathway and suggested that p21 might be a factor responsible for inhibiting chondrocytic proliferation (15Sahni M. Ambrosetti D.C. Mansukhani A. Gertner R. Levy D. Basilico C. Genes Dev. 1999; 13: 1361-1366Crossref PubMed Scopus (317) Google Scholar). Cell cycle progression is known to be governed by the assembly of cyclins, cyclin-dependent kinases (CDKs), and CDK inhibitors (CKIs) and subsequent regulation of retinoblastoma protein (pRb) activity. In the early G1 phase of the cell cycle, for example, cyclin D-Cdk4 phosphorylates pRb, and in the late G1 phase cyclin E-Cdk2/cyclin D-Cdk4 phosphorylates pRb. Hyperphosphorylated pRb then releases E2F family transcription factors, which activate genes that are necessary for S phase entry (16Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1954) Google Scholar). The CKI p21cip1, waf-1 belongs to a Cip/Kip family with p27kip1 and p57kip2 and broadly inhibits the activity of cyclin D-, E-, and A-dependent kinases (17Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5071) Google Scholar, 18Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Crossref PubMed Scopus (371) Google Scholar). In contrast, the second class of CKIs (i.e. Ink4 (inhibitors for Cdk4) proteins (p16INK4a, p15INK4b, p18INK4c, and p19INK4d), bind to and inactivate only Cdk4 and Cdk6 but not other CDKs or D-type cyclins (17Sherr C.J. Roberts J.M. Genes Dev. 1999; 13: 1501-1512Crossref PubMed Scopus (5071) Google Scholar, 18Vidal A. Koff A. Gene (Amst.). 2000; 247: 1-15Crossref PubMed Scopus (371) Google Scholar). p21 has been shown to be expressed in postmitotic, hypertrophic chondrocytes but not in chondrocytes in the proliferating zone (19Parker 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 (1016) Google Scholar), and activation of STAT1 and increased expression of p21 in cartilage have been demonstrated in thanatophoric dysplasia human embryos (20Su W.C. Kitagawa M. Xue N. Xie B. Garofalo S. Cho J. Deng C. Horton W.A. Fu X.Y. Nature. 1997; 386: 288-292Crossref PubMed Scopus (268) Google Scholar). Based on these data, p21 has been suggested to be involved in FGF's growth inhibition of cartilage. Interestingly, in some breast cancer cells, FGF has been shown to inhibit their proliferation through activation of STAT1 and induction of p21 expression (21Wang H. Rubin M. Fenig E. DeBlasio A. Mendelsohn J. Yahalom J. Wieder R. Cancer Res. 1997; 57: 1750-1757PubMed Google Scholar, 22Johnson M.R. Valentine C. Basilico C. Mansukhani A. Oncogene. 1998; 16: 2647-2656Crossref PubMed Scopus (53) Google Scholar). In this study, we first examine the effects of FGF2 on cell cycle progression and p21 expression in rat chondrosarcoma (RCS) cells, whose proliferation is potently inhibited by FGF2. Second, we analyze how p21 induction by FGF2 leads to cell cycle arrest in RCS cells by examining the binding of FGF2-induced p21 to cyclins and CDKs as well as by assessing the changes in CDK- and cyclin-dependent kinase activities after FGF2 treatment. Finally, we examine the functional involvement of p21 in FGF's growth inhibition by comparing the effects of FGF2 on cartilaginous growth in vitro in limbs isolated from p21-deficient and wild-type mice. RCS cells were grown in high glucose Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (HyClone), 100 units/ml penicillin, and 100 µg/ml streptomycin (23Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. de Crombrugghe B. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Cells were passaged using trypsin-EDTA (Life Technologies) and were split at a 1:15 ratio on a 100-mm dish (Falcon; Becton and Dickinson). Cell proliferation was assessed by [3H]thymidine incorporation assay as well as by measuring DNA contents. RCS cells were plated at a density of 3 × 104 cells/24-well plate (Falcon), and growing cells (70% confluent) were treated with vehicle (0.5 m NaCl, 0.1% bovine serum albumin/phosphate-buffered saline) or the indicated dose of FGF2 (R&D Systems) for 24 h. Cells were labeled with 0.5 µCi/ml [3H]thymidine for the last 2 h of culture and dissolved with 0.1 n NaOH, 0.01% bovine serum albumin, 1% Triton X-100 (Sigma). Samples were mixed with the same volume of ice-cold 10% trichloroacetic acid for 10 min at 4 °C and then centrifuged at 14,000 rpm at 4 °C for 10 min. After washing with 5% trichloroacetic acid, pellets were dissolved in 0.1 n NaOH, 1% Triton X-100, and their radioactivity was measured by a scintillation counter (Beckman). DNA content was measured by a fluorometric method. Briefly, RCS cells were plated at a density of 1 × 104 cells/24-well plate and treated with vehicle or indicated doses of FGF2. Cells were digested with 20 µg/ml of papain (Sigma) in 0.1 m sodium acetate, 50 mm EDTA, 10 mm cysteine-HCl (pH 5.53) at 55 °C for 16 h. The DNA content in digested lysates were measured using PicoGreen DNA quantitation kit (Molecular Probes, Inc., Eugene, OR). RCS cells were plated at a density of 8 × 105 cells/60-mm culture dishes, and subconfluent cells were treated with 3 nm of FGF2 for 6, 12, and 24 h. Cells were then trypsinized, washed twice with phosphate-buffered saline, fixed in 70% ethanol, and stained with propidium iodide (50 µg/ml; Sigma), RNase A (200 µg/ml; Sigma), 0.1% glucose in phosphate-buffered saline for 4 h at room temperature. Samples were analyzed on an Epics Alta flow cytometer (Beckman Coulter). For Western blotting, cells were lysed in 2× SDS buffer containing 125 mm Tris-HCl (pH 6.8), 4% SDS containing phosphatase inhibitor mixtures I and II (Sigma), 2 mmphenylmethylsulfonyl fluoride (Sigma), and 132 µg/ml aprotinin (Sigma). Protein concentration of the lysates was determined using protein assay kit DC (Bio-Rad). Samples were then diluted by adding the same volume of 2× dye containing 20% glycerol, 4% 2-mercaptoethanol (Sigma), and equal amounts of protein were resolved on 6.5% SDS gel for pRb or 12.5% gel for all other proteins. Proteins were then transferred onto ImmobilonTM-P membranes (Millipore Corp.). After being washed twice with 0.1% Tween 20, 10 mmTris-HCl, 150 mm NaCl (pH 8.0) were with buffer in for 1 h at room and then with diluted in buffer for 1 h at room or at 4 for Western were diluted as A E from Inc., and were and diluted in buffer at Western were with In some were with mm Tris-HCl (pH 6.8), 100 mm 2-mercaptoethanol at 50 °C for min and with other RCS cells growing on 100-mm were treated with vehicle or 3 nm FGF2 for h. Cells were then lysed in 50 mm Tris-HCl (pH 150 mm NaCl, 0.1% Triton X-100 containing 10 phosphatase inhibitor mixtures I and 1 mm and µg/ml of aprotinin for min on were with 0.5 µg/ml for min at 4 °C and then with of protein (Life Technologies) for min at 4 °C with were then by and of protein of the was with of to p21 cyclin Cdk2 cyclin E and or mouse to Cdk4 for 1 h at 4 °C and then with of protein for Cdk4 or protein (Life Technologies) for all other at 4 °C with Samples were then washed four with dissolved in and resolved on 12.5% SDS by Western For CDK kinase were washed four with buffer and then twice with kinase buffer (50 10 mm 1 The kinase was in of kinase buffer containing 2 of pRb and 10 of for min at were by after the of the same volume of 2× SDS Samples were resolved on 10% SDS gel and analyzed using a were in with the for the and of mice for p21 J. C. D. T. Nature. 1995; PubMed Scopus Google and wild-type mice were from for mouse fetal limb culture were A. K. B. Kronenberg C.J. Science. 1996; PubMed Scopus Google Scholar). In limbs of were of and and on a by Corp.). were cultured with replacement of medium (Life Technologies, Inc.) containing 0.1% bovine serum albumin, 100 units/ml penicillin, 100 µg/ml and B. After the of were fixed in 10% for histone H4 were using DNA of the F. G. J. Res. PubMed Scopus Google from of of were from using the and In was as K. B. J.D. H. Kronenberg 1996; PubMed Scopus Google Scholar). were and at 4 °C for After development, were with and and After were and was by twice in 10 mm buffer (pH for min in a was using mouse and mouse kit of the were at least RCS cells numerous chondrocytic including expression of type II and type and of cartilaginous (23Mukhopadhyay K. Lefebvre V. Zhou G. Garofalo S. Kimura J.H. de Crombrugghe B. J. Biol. Chem. 1995; 270: 27711-27719Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In we that RCS cells expressed and which were at tyrosine with FGF2 not we assessed the effects of FGF2 on proliferation by measuring [3H]thymidine incorporation as well as DNA shown in FGF2 incorporation in a from 10 to inhibition by FGF2 was by of DNA content in RCS cells treated with FGF2 we analyzed the effects of FGF2 on cell cycle of RCS cells. shown in cells an of cells the cycle the for a in the G1 at 24 h. cells first in phase with a in cells in S phase at h. cells in G1 phase at and 24 h and which was with a of cells in S phase at 24 The of cells decreased dramatically and h and at this at 24 h. The of cells at 24 h at least to h not To understand the mechanisms of G1 arrest of RCS cells, we the effects of FGF2 on expression of cell cycle proteins. shown in FGF2 induced p21 protein expression by and its expression at h. In contrast, was in the or of FGF2 not Also, expression was at and 24 FGF2 not the of its then the of pRb by Western blotting, using an to pRb that in and pRb in FGF2 shifted pRb from to underphosphorylated, active by h. h and the of pRb was in the underphosphorylated, active form in cells. the increased p21 protein expression as well as the of cells in A of pRb to was in cells also at and 24 which might be related to the in G1 cells in RCS at 24 h. to the of pRb to its active expression of cyclin whose expression is regulated by was dramatically reduced at and 24 h. In contrast, cyclin expression was dramatically increased by cyclin E expression was the Furthermore, FGF2 not the expression of Cdk2 or we which were with treated RCS cells with vehicle or FGF2 for h and then immunoprecipitated with to cyclin cyclin Cdk2, and by Western with to cyclin Cdk2, and shown in FGF2-induced p21 with cyclin cyclin Cdk2, and p21 expressed in cells was with at Although cyclin was with Cdk4 in cells, with Cdk4 and Cdk2 after FGF2 treatment. Also, the cyclin E and Cdk2 was by FGF2 the cyclin assembled only after FGF2 treatment. In these that FGF2 a cyclin and cyclin and cyclin FGF2 not the expression of or its with cyclin cyclin or then which cyclin-dependent kinase activity is regulated by FGF2. To the kinase activity of cell lysates were first immunoprecipitated with to cyclin cyclin Cdk2, or and proteins were then with and shown in h of FGF not the activity of Cdk4 or cyclin kinase but dramatically reduced the activity of Cdk2 and cyclin as assessed by the of pRb in the activity of Cdk2 and cyclin kinases was also using histone as not Taken together, these that FGF2 inhibited of cell cycle through the induction of p21 and subsequent inactivation of cyclin E-Cdk2 in RCS cells. then FGF2 p21 in chondrocytes in the growth plate in limbs isolated from mouse shown in p21 protein expression was to hypertrophic chondrocytes and some proliferating chondrocytes in the of the of the proliferating zone in limbs In contrast, after with p21 was in all of the chondrocytes of p21 in growth plate chondrocytes was also at 16 and 24 h after FGF2 not we inhibitory effects of FGF2 on chondrocytic proliferation in the growth plate of p21 compared with by comparing expression for H4 histone a marker for cells in S phase K. B. J.D. H. Kronenberg 1996; PubMed Scopus Google Scholar). To the caused by we treated limb with vehicle and the limb with the indicated amounts of FGF2. shown in H4 expression was in cells in the proliferating zone but not in the hypertrophic zone in from and p21 mice. In and p21 FGF2 decreased the of cells in the proliferating nm FGF2 dramatically decreased the of chondrocytes in the proliferating zone in limbs, the of chondrocytes in the proliferating zone in p21 limbs was Moreover, and 24 nm FGF2 the chondrocytic proliferation in as by the of H4 chondrocytic proliferation was only partially inhibited in p21 at these doses of FGF2. shown in were treated with nm FGF2 or vehicle for 2 FGF2 dramatically reduced the of the growth plate and the of the hypertrophic zone in compared with the In contrast, neither the growth plate nor the of the hypertrophic zone in p21 was as dramatically as 2 of 24 nm FGF2 decreased of these in p21 as in not with the effects on H4 Although FGF and its receptor are known to cartilage proliferation, little is known about the mechanisms In this study, we the effects of FGF2 on the cell cycle in cell and fetal limb to understand the growth-inhibitory pathway of FGF in chondrocytes. the effects of FGF2 on chondrocytes and cells, FGF2 inhibits growth of RCS cells, its effects in In this study, we of the of RCS cells to analyze the growth-inhibitory mechanisms of FGF in chondrocytes. FGF2 the cell cycle of RCS cells at a known to be by pRb activity Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). pRb is and its in and of pRb by cyclin D-Cdk4 and cyclin E-Cdk2 at the the cell to S phase S. V. A. Cell. Full Text PDF PubMed Scopus Google Scholar, T. T. S. K. K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, J. H. C.J. Genes Dev. 1993; PubMed Scopus Google Scholar, M. Genes Dev. 8: PubMed Scopus Google Scholar). of that the cell cycle arrest at to the decreased kinase activity of cyclin D-Cdk4 or cyclin E-Cdk2 as well as to the of pRb M. Genes Dev. 8: PubMed Scopus Google Scholar, K. C.J. J. Roberts J.M. Koff A. Genes Dev. 8: PubMed Scopus Google Scholar, M. H. M.C. Cancer Res. 1999; Google Scholar, S.J. G. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, H. T. Cell. Full Text PDF PubMed Scopus Google Scholar). with the of and from S phase in RCS cells after FGF2 was to the of pRb and in cyclin whose expression is regulated by E2F family through inactivation of pRb (16Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1954) Google Scholar). Moreover, all of these (i.e. of pRb, and in cyclin were with p21 expression but not with cyclin cyclin Cdk2, and Cdk4 2 and the other a in G1 and of pRb also was in RCS cells. These were with the expression of p21 and at and 24 h RCS cells with the 16 and their growth is and we the cells were 70% is that the G1 arrest and the in p21 and expression at were caused by The involvement of p21 in G1 cell cycle arrest of RCS cells was by and cyclin-dependent kinase that FGF2 induced the assembly of cyclin cyclin and cyclin Also, we demonstrated that FGF2 dramatically reduced the activity of Cdk2 and cyclin Although FGF2 increased the expression of cyclin in RCS cells, cyclin kinase activity was not by FGF2. findings with limb that the in p21 expression induced by FGF2 in vitro also is in growth Moreover, doses of FGF2 are to inhibit cartilaginous growth in limbs from p21 mice compared with those from mice. the of p21 in the mechanisms by which FGF2 inhibition of cartilage growth. In to data, Li et al. C. Chen L. Iwata T. Kitagawa M. Fu X.Y. Deng C.X. Hum. Mol. Genet. 1999; 8: 35-44Crossref PubMed Scopus (192) Google have reported that Ink4 family proteins but not p21 were in growth of mice carrying the FGFR3 gene not examine Ink4 protein expression in this study, FGF2 not the activity of Cdk4 or cyclin kinase in RCS cells. The in and their in be by a in in activation of and activation of FGFR3 or FGF's cell cycle regulation cell cycle inhibitors in fetal and growth In that FGF's inhibitory effects on chondrocytic proliferation appear to be mediated at least partially through p21 induction and the subsequent inactivation of cyclin E-Cdk2 and activation of pRb. M. Kronenberg for of the and and for with flow and