FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2072506593> ?p ?o ?g. }

• W2072506593 endingPage "4904" @default.
• W2072506593 startingPage "4897" @default.
• W2072506593 abstract "Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCα vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCα signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCα pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells. Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCα vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCα signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCα pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells. Mesenchymal stem cells (MSCs) 3The abbreviations used are: MSC, mesenchymal stem cells; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; ERK1/2, extracellular signal-regulated kinase; PKC, protein kinase C; FGFR, fibroblast growth factor receptor; WT, wild type; MT, mutant; FCS, fetal calf serum; JNK, c-Jun N-terminal kinase; ALP, alkaline phosphatase.3The abbreviations used are: MSC, mesenchymal stem cells; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol 3-kinase; ERK1/2, extracellular signal-regulated kinase; PKC, protein kinase C; FGFR, fibroblast growth factor receptor; WT, wild type; MT, mutant; FCS, fetal calf serum; JNK, c-Jun N-terminal kinase; ALP, alkaline phosphatase. have the potential to differentiate into different lineages, including osteoblasts, chondroblasts, and adipocytes (1Pittenger M.F. Mackay A.M. Beck S.C. Jaiswal R.K. Douglas R. Mosca J.D. Moorman M.A. Simonetti D.W. Craig S. Marshak D.R. Science. 1999; 284: 143-147Crossref PubMed Scopus (17765) Google Scholar, 2Bianco P. Riminucci M. Gronthos S. Robey P.G. Stem Cells. 2001; 19: 180-192Crossref PubMed Scopus (1720) Google Scholar, 3Owen M. Cell Sci. Suppl. 2002; 10: 63-76Google Scholar, 4Aubin J.E. Triffitt J.T. Bilezikian J.P. Raisz L.G. Rodan G.A. Principles of Bone Biology. 1. Academic Press, New York2002: 59-82Crossref Google Scholar, 5Gimble J.M. Zvonic S. Floyd Z.E. Kassem M. Nuttall M.E. J. Cell. Biochem. 2006; 98: 251-266Crossref PubMed Scopus (428) Google Scholar, 6Marie P.J. Fromigué O. Regen. Med. 2006; 1: 539-548Crossref PubMed Scopus (105) Google Scholar, 7Phinney D.G. Prockop D.J. Stem Cells. 2007; 25: 2896-2902Crossref PubMed Scopus (1538) Google Scholar). The osteoblast differentiation program of MSCs is characterized by cell recruitment, which is followed by timely expressed genes including Runx2, alkaline phosphatase (ALP), type I collagen (ColA1), and osteocalcin (OC), which is associated with extracellular matrix mineralization (8Karsenty G. Semin. Cell Dev. Biol. 2000; 11: 343-346Crossref PubMed Scopus (146) Google Scholar, 9Aubin J.E. Rev. Endocr. Metab. Disord. 2001; 2: 81-94Crossref PubMed Scopus (391) Google Scholar, 10Lian J.B. Javed A. Zaidi S.K. Lengner C. Montecino M. van Wijnen A.J. Stein J.L. Stein G.S. Crit. Rev. Eukaryot. Gene Expr. 2004; 14: 1-41Crossref PubMed Google Scholar). The program of MSC osteogenic differentiation can be induced by soluble molecules such as bone morphogenetic proteins (BMPs) or Wnt proteins that activate several signaling pathways to trigger osteoblast differentiation (11Hanada K. Dennis J.E. Caplan A.I. J. Bone Miner. Res. 1997; 12: 1606-1614Crossref PubMed Scopus (317) Google Scholar, 12Gori F. Thomas T. Hicok K.C. Spelsberg T.C. Riggs B.L. J. Bone Miner. Res. 1999; 14: 1522-1535Crossref PubMed Scopus (261) Google Scholar, 13Etheridge S.L. Spencer G.J. Heath D.J. Genever P.G. Stem Cells. 2004; 22: 849-860Crossref PubMed Scopus (313) Google Scholar, 14Gaur T. Lengner C.J. Hovhannisyan H. Bhat R.A. Bodine P.V. Komm B.S. Javed A. van Wijnen A.J. Stein J.L. Stein G.S. Lian J.B. J. Biol. Chem. 2005; 280: 33132-33140Abstract Full Text Full Text PDF PubMed Scopus (894) Google Scholar, 15Osyczka A.M. Leboy P.S. Endocrinology. 2005; 146: 3428-3437Crossref PubMed Scopus (163) Google Scholar). Although various downstream signals are known to promote osteogenic differentiation (16Jaiswal R.K. Jaiswal N. Bruder S.P. Mbalaviele G. Marshak D.R. Pittenger M.F. J. Biol. Chem. 2000; 275: 9645-9652Abstract Full Text Full Text PDF PubMed Scopus (657) Google Scholar, 17Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 18Fujita T. Azuma Y. Fukuyama R. Hattori Y. Yoshida C. Koida M. Ogita K. Komori T. J. Cell Biol. 2004; 166: 85-95Crossref PubMed Scopus (351) Google Scholar, 19Kratchmarova I. Blagoev B. Haack-Sorensen M. Kassem M. Mann M. Science. 2005; 308: 1472-1477Crossref PubMed Scopus (481) Google Scholar, 20Ge C. Xiao G. Jiang D. Franceschi R.T. J. Cell Biol. 2007; 176: 709-718Crossref PubMed Scopus (407) Google Scholar), the molecular mechanisms that control the early stages of MSC osteoblast differentiation are not fully elucidated.Fibroblast growth factors (FGFs) play an important major role in the control of cell proliferation, differentiation, and survival in several tissues including bone (21Powers C.J. McLeskey S.W. Wellstein A. Endocr. Relat. Cancer. 2000; 7: 165-197Crossref PubMed Scopus (1110) Google Scholar, 22Dailey L. Ambrosetti D. Mansukhani A. Basilico C. Cytokine Growth Factor Rev. 2005; 16: 233-247Crossref PubMed Scopus (523) Google Scholar, 23Ornitz D.M. Cytokine Growth Factor Rev. 2005; 16: 205-213Crossref PubMed Scopus (294) Google Scholar, 24Marie P.J. Gene. 2003; 316: 23-32Crossref PubMed Scopus (232) Google Scholar). Notably, FGF2 was found to promote cell growth and osteoblast differentiation in bone marrow-derived mesenchymal cells (25Martin I. Muraglia A. Campanile G. Cancedda R. Quarto R. Endocrinology. 1997; 138: 4456-4462Crossref PubMed Scopus (372) Google Scholar, 26Pri-Chen S. Pitaru S. Lokiec F. Savion N. Bone. 1998; 23: 111-117Crossref PubMed Scopus (83) Google Scholar). Consistent with an important role of FGF signaling in the control of osteoprogenitor cells, deletion of FGF2 in mice results in decreased bone marrow stromal cell osteogenic differentiation and altered bone formation (27Montero A. Okada Y. Tomita M. Ito M. Tsurukami H. Nakamura T. Doetschman T. Coffin J.D. Hurley M.M. J. Clin. Investig. 2000; 105: 1085-1093Crossref PubMed Scopus (400) Google Scholar). The actions of FGFs are highly dependent on high affinity FGF receptors (FGFRs) (28Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1464) Google Scholar). FGF binding to FGFRs leads to receptor dimerization and phosphorylation of intrinsic tyrosine residues, which leads to activation of several signal transduction pathways including phospholipase Cγ (PLCγ), mitogen-activated protein kinases (MAPK), and phosphatidylinositol 3-kinase (PI3K) (29Mohammadi M. Dikic I. Sorokin A. Burgess W.H. Jaye M. Schlessinger J. Mol. Cell. Biol. 1996; 16: 977-989Crossref PubMed Scopus (341) Google Scholar, 30Ong S.H. Hadari Y.R. Gotoh N. Guy G.R. Schlessinger J. Lax I. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 6074-6079Crossref PubMed Scopus (263) Google Scholar). In bone, activation of extracellular-related kinase (ERK1/2) MAPK and protein kinase C (PKC) was found to enhance osteoblast gene expression (31Xiao G. Jiang D. Gopalakrishnan R. Franceschi R.T. J. Biol. Chem. 2002; 277: 36181-36187Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 32Kim H.J. Kim J.H. Bae S.C. Choi J.Y. Kim H.J. Ryoo H.M. J. Biol. Chem. 2003; 278: 319-326Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The important role of FGFR signaling in bone formation is highlighted by the finding that gain-of-function mutations in FGFRs results in premature cranial osteogenesis (33Ornitz D.M. Marie P.J. Genes Dev. 2002; 16: 1446-1465Crossref PubMed Scopus (713) Google Scholar, 34Wilkie A.O. Patey S.J. Kan S.H. van den Ouweland A.M. Hamel B.C. Am. J. Med. Genet. 2002; 112: 266-278Crossref PubMed Scopus (160) Google Scholar). FGFR1 was recently shown to be an important transducer of FGF signals in proliferating osteoblasts (35Ling L. Murali S. Dombrowski C. Haupt L.M. Stein G.S. van Wijnen A.J. Nurcombe V. Cool S.M. J. Cell. Physiol. 2006; 209: 811-825Crossref PubMed Scopus (55) Google Scholar). In contrast, activated FGFR2 was shown to enhance osteoblast differentiation in Apert syndromic craniosynostosis (36Lomri A. Lemonnier J. Hott M. de Parseval N. Lajeunie E. Munnich A. Renier D. Marie P.J. J. Clin. Investig. 1998; 101: 1310-1317Crossref PubMed Google Scholar, 37Fragale A. Tartaglia M. Bernardini S. Di Stasi A.M. Di Rocco C. Velardi F. Teti A. Battaglia P.A. Migliaccio S. Am. J. Pathol. 1999; 154: 1465-1477Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 38Lemonnier J. Hay E. Delannoy P. Lomri A. Modrowski D. Caverzasio J. Marie P.J. J. Bone Miner. Res. 2001; 16: 832-845Crossref PubMed Scopus (66) Google Scholar, 39Kaabeche K. Lemonnier J. Le Mée S. Caverzasio J. Marie P.J. J. Biol. Chem. 2004; 279: 36259-36267Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 40Tanimoto Y. Yokozeki M. Hiura K. Matsumoto K. Nakanishi H. Matsumoto T. Marie P.J. Moriyama K. J. Biol. Chem. 2004; 279: 45926-45934Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 41Baroni T. Carinci P. Lilli C. Bellucci C. Aisa M.C. Scapoli L. Volinia S. Carinci F. Pezzetti F. Calvitti M. Farina A. Conte C. Bodo M. J. Cell. Physiol. 2005; 202: 524-535Crossref PubMed Scopus (35) Google Scholar). However, the role of FGFR2 signaling in osteogenic differentiation of mesenchymal stem cells is yet to be elucidated.In the present study, we investigated the specific role of FGFR2 signaling on osteoblast commitment and differentiation in murine mesenchymal progenitor cells. Our results indicate that wild-type and activated FGFR2 induce osteogenic differentiation in mesenchymal cells through distinct activation of ERK1/2 and PKCα signaling. These data indicate that FGFR2-induced activation of specific downstream signaling pathways mediates osteogenic differentiation of murine mesenchymal cells.EXPERIMENTAL PROCEDURESCells and Materials—Murine pluripotent mesenchymal C3H10T1/2 cells and MC3T3-E1, a recognized osteoblastic cell line, were obtained from the ATCC. Cells were routinely cultured in Dulbecco's Modified Eagles Medium (DMEM; Invitrogen, Paisley, Scotland) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% l-glutamine, and penicillin/streptomycin (10,000 units/ml and 10,000 μg/ml, respectively), at 37 °C in a humidified atmosphere containing 5% CO2 in air. Culture media were changed every 2 days. Anti-FGFR2, anti-phosphotyrosine (p-Tyr), anti-ERK, and anti-p-ERK were from Santa Cruz Biotechnology (Santa Cruz, CA). Other antibodies (anti-AKT, anti-p-AKT, anti-JNK, anti-p-JNK, anti-PI3K p85, and anti-p-PI3K p85 were from Cell Signaling (Sigma-Aldrich). The β-actin antibody, U0126 (an inhibitor of MAPK kinase 1 and 2 (MEK1/2) that blocks phosphorylation and activation of ERK1/2 (42Favata M.F. Horiuchi K.Y. Manos E.J. Daulerio A.J. Stradley D.A. Feeser W.S. Van Dyk D.E. Pitts W.J. Earl R.A. Hobbs F. Copeland R.A. Magolda R.L. Scherle P.A. Trzaskos J.M. J. Biol. Chem. 1998; 273: 18623-18632Abstract Full Text Full Text PDF PubMed Scopus (2738) Google Scholar) and Gö6976, a specific PKCα inhibitor were from Sigma.Plasmids and Transfections—For stable transfection, C3H10T1/2 cells were grown in tissue culture plates and transfected at 80% confluence with 2.5 μg of the empty vector (EV), FGFR2IIIc-CMV13 (WT), or FGFR2IIIcS252W-CMV13 (MT) (40Tanimoto Y. Yokozeki M. Hiura K. Matsumoto K. Nakanishi H. Matsumoto T. Marie P.J. Moriyama K. J. Biol. Chem. 2004; 279: 45926-45934Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar) using Exgen 500 (Euromedex, Mundolsheim, France). After 48 h, the transfectants were selected in DMEM medium containing 10% FCS and 1 mg/ml G418 (Geneticin, Sigma) for 4 weeks. To analyze whether FGFR2 activation may affect more mature osteoprogenitor cells, we also performed transient transfection experiments with WT or MT FGFR2 in MC3T3-E1 mouse calvaria cells. In parallel experiments, EV, WT, and MT C3H10T1/2 cells were transiently transfected with a dominant-negative ERKp44-MAPK kinase-deficient mutant vector (DN-ERK) (43Pagés G. Lenormand P. L'Allemain G. Chambard J.C. Meloche S. Pouysségur J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8319-8323Crossref PubMed Scopus (923) Google Scholar), a dominant-negative PKCα vector (DN-PKCα) or empty vectors and Exgen (Euromedex) according to the manufacturer's instructions.Cell Proliferation and Apoptosis Assays—For the cell proliferation assay, stably transfected C3H10T1/2 cells were cultured in 6-well plates (105 cells per well), and cell number was evaluated by cell counting. For the apoptosis assay, stably transfected C3H10T1/2 cells were cultured in the presence of 1 or 5% FCS, and apoptosis was detected using the Apopercentage apoptosis assay (Biocolor, Belfast, Northern Ireland). Briefly, 20 μl of dye (1/100) were added to the medium. After 30 min, the cells were washed twice with phosphate-buffered saline. Cells were lysed in DMSO, and absorbance at 550 nm was determined. In some wells, H2O2 (5 mmol/liter) was added to the medium for 2 h before harvesting the cells to induce apoptosis, thereby serving as a positive control.Protein Kinase C Activity—PKC activity was determined using a PKC Activity Assay kit (Assay Designs, Ann Arbor, MI) based on a solid phase enzyme-linked immuno-absorbent assay (ELISA), according to the manufacturer's instructions. The assay utilizes a specific synthetic peptide as a substrate for PKC, and a polyclonal antibody that recognizes the phosphorylated form of the substrate.Quantitative RT-PCR Analysis—Total RNAs were isolated using Trizol reagent (Invitrogen). 3 μg of total RNA from each sample were reverse-transcribed using MMLV reverse transcriptase and oligodT primers at 37 °C for 90 min. The relative mRNA levels were evaluated by quantitative PCR (LightCycler, Roche Applied Science, Indianapolis, OH) using a SYBR Green PCR kit (ABGen, Courtabœuf, France) and specific primers (supplemental Table S1). Signals were normalized to glyceraldehyde-3-phosphate dehydrogenase as internal control.Western Blot Analysis—Cell lysates were prepared as described (38Lemonnier J. Hay E. Delannoy P. Lomri A. Modrowski D. Caverzasio J. Marie P.J. J. Bone Miner. Res. 2001; 16: 832-845Crossref PubMed Scopus (66) Google Scholar). Briefly, proteins (30 μg) were resolved on 4–12% SDS-PAGE and transferred onto polyvinylidene difluoride nitrocellulose membranes (Millipore Corp., Bedford, MA). Filters were incubated for 2 h in 50 mm Tris/HCl, pH 7.4, 150 mm NaCl, 0.1% (v/v) Tween-20, 0.5% (w/v), bovine serum albumin (TBST/BSA), then overnight at 4 °C on a shaker with specific primary antibodies (1/500–1/1000 in TBST/BSA). Membranes were washed twice with TBST and incubated for 2 h with appropriate horseradish peroxidase-conjugated secondary antibody (1:10,000–1:20,000 in TBST/BSA). After final washes, the signals were visualized with enhanced chemiluminescence Western blotting detection reagent (ECL, Amersham Biosciences, Piscataway, NJ) and autoradiographic film (X-OMAT-AR, Eastman Kodak Company, Rochester, NY). Densitometric analysis using ImageQuant software was performed following digital scanning (Agfa). Representative images of immunoblots are shown. For immunoprecipitation analysis, cell lysates were prepared as for Western blot analysis, and aliquots of total protein (150 μg) were incubated overnight under weak agitation at 4 °C with 2 μg of specific anti-FGFR2 and 20 μl of Dynabeads protein G (Invitrogen). Components of the bound immune complex (both antigen and antibody) were eluted from the Dynabeads and analyzed by SDS-PAGE according to the manufacturer's recommendations.Alkaline Phosphatase Staining—ALP staining was performed using Sigma FAST kit according to the manufacturer's recommendations (Sigma). Briefly, cells were fixed in 75% ethanol, rinsed in phosphate-buffered saline, and incubated with the substrate buffer at 37 °C.Osteogenic, Chondrogenic, and Adipogenic Assays—Cell culture medium was supplemented with 50 μmol/liter ascorbic acid and 3 mm inorganic phosphate (NaH2PO4) to allow matrix synthesis and mineralization. At the indicated time points, cells were fixed in 4% paraformaldehyde in phosphate-buffered saline. Matrix mineralization was evaluated by alizarin red and von Kossa staining as described (44Ahdjoudj S. Lasmoles F. Oyajobi B.O. Lomri A. Delannoy P. Marie P.J. J. Cell. Biochem. 2001; 81: 23-38Crossref PubMed Scopus (100) Google Scholar) and microphotographed using an Olympus microscope (Japan). Glycoprotein synthesis was determined by alcian blue staining (44Ahdjoudj S. Lasmoles F. Oyajobi B.O. Lomri A. Delannoy P. Marie P.J. J. Cell. Biochem. 2001; 81: 23-38Crossref PubMed Scopus (100) Google Scholar). For adipogenesis, the accumulation of lipid droplets, a hallmark of functional adipogenesis, was detected by oil red staining (44Ahdjoudj S. Lasmoles F. Oyajobi B.O. Lomri A. Delannoy P. Marie P.J. J. Cell. Biochem. 2001; 81: 23-38Crossref PubMed Scopus (100) Google Scholar) and quantified at 14 days of culture, as described (45Laughton C. Anal. Biochem. 1986; 156: 307-314Crossref PubMed Scopus (31) Google Scholar). Briefly, the stain solution was removed, and cells were rinsed in 500 μl of 60% isopropyl alcohol for 5 s. To extract dye, 700 μl of 60% isopropyl alcohol was added per well, and sealed plates were shaken for 2 h. The extracted dye was quantitated spectrophotometrically at 510 nm.Statistical Analysis—The data are the mean ± S.D. of an average of six samples and are representative of at least three distinct experiments. The data were analyzed by Student's t test, and a minimal level of p < 0.05 was considered significant.RESULTSExpression of FGFRs in C3H10T1/2 Cells—We first determined the expression levels of FGFR1–4 in growing C3H10T1/2 cells. Quantitative PCR analysis showed that mRNA expression levels of the four FGFRs differed markedly at 3 days of culture (Fig. 1A). FGFR1 was higher than FGFR2 mRNA levels. In contrast, FGFR3 mRNA expression was weak, and FGFR4 level was almost undetectable (Fig. 1A), suggesting that FGFR1 and FGFR2 are major FGF receptors expressed in these mesenchymal cells. The kinetics analysis of change of the two main FGFRs expressed in growing C3H10T1/2 cells showed that the FGFR1 mRNA level rose and then declined in confluent cells whereas the FGFR2 mRNA level increased in confluent cells (Fig. 1, B and C), suggesting that FGFR2 may be an important transducer of FGF signals in post-proliferating osteoblasts.Expression and Activation of FGFR2 in Stably Transfected C3H10T1/2 Cells—We then analyzed the effect of FGFR2 signaling in C3H10T1/2 cells. To this goal, the cells were stably transfected with wild-type (WT) FGFR2 or a constitutively activating FGFR2 due to Apert S252W mutation (MT). As expected, quantitative PCR analysis showed that transfection with the two vectors increased FGFR2 mRNA levels in C3H10T1/2 cells (data not shown). Western blot analysis showed a 2–3-fold increase in FGFR2 protein levels in WT- and MT-transfected cells, indicating that FGFR2 protein levels increased similarly in WT and MT FGFR2-transfected cells (Fig. 2A). FGFR activation is characterized by phosphorylation of the cytoplasmic domain that transduces intracellular signals. We analyzed tyrosine phosphorylation of FGFR2 in WT and MT FGFR2-transfected cells. As shown in Fig. 2, B and C, WT FGFR2 induced a slight increase in tyrosine-phosphorylated FGFR2 level under basal conditions (i.e. in the absence of exogenous FGF) compared with EV-transfected cells. In contrast, a 2-fold increase in the tyrosine-phosphorylated FGFR2 level was found in MT FGFR2-transfected cells (Fig. 2, B and C). These data indicate that forced expression of WT FGFR2 slightly activated the receptor in the absence of exogenous ligand, whereas MT FGFR2 induced a marked activation of the receptor.FIGURE 2FGFR2 promotes mesenchymal cell proliferation but not survival. Western blot analysis (A) showing increased FGFR2 protein levels in C3H10T1/2 cells stably transfected with WT FGFR2 or S252W FGFR2 MT compared with the empty vector (EV). Immunoprecipitation analysis (B) and quantification of blots (C) show increased tyrosine-phosphorylated (p)FGFR2 levels in WT and MT FGFR2 cells. a indicates a significant difference with EV-transfected cells; b indicates a significant difference with WT FGFR2-transfected cells (p < 0.05). Time course analysis showed that WT and MT FGFR2 increased cell number compared with control cells transfected with EV (D). * indicates a significant difference with EV-transfected cells (p < 0.05). WT or MT had no effect on cell survival determined by the Apopercentage assay in the presence of 5% serum or in serum-deprived (1%) conditions (E).View Large Image Figure ViewerDownload Hi-res image Download (PPT)FGFR2 Increases C3H10T1/2 Cell Proliferation but Not Survival—Activation of FGFR signaling induces multiple and complex effects on cell proliferation and survival (28Eswarakumar V.P. Lax I. Schlessinger J. Cytokine Growth Factor Rev. 2005; 16: 139-149Crossref PubMed Scopus (1464) Google Scholar). To determine the cellular effect of FGFR2 activation in mesenchymal cells, we first investigated the changes in cell proliferation in WT and MT FGFR2 stably transfected C3H10T1/2 cells. Overexpression of WT or MT FGFR2 increased cell proliferation compared with EV-transfected cells at all times of culture before and after confluence (Fig. 2D). Using an assay that detects early events involved in cell apoptosis, we then examined whether WT or MT FGFR2 may affect cell survival in C3H10T1/2 cells. As shown in Fig. 2E, MT or WT FGFR2 did not affect cell apoptosis when cultured in optimal survival conditions (5% FCS) or in culture condition inducing apoptosis (1% FCS) after 3 days of culture. These data indicate that WT or MT FGFR2 overexpression promotes cell proliferation but not cell survival in C3H10T1/2 cells under these experimental conditions.FGFR2 Promotes Osteogenic Differentiation in MSCs—We then investigated the effects of WT and MT FGFR2 on osteogenic differentiation of C3H10T1/2 cells. Quantitative PCR analysis showed that both WT and MT FGFR2 induced a 3–4-fold increase in mRNA expression of Runx2, a specific osteoblast transcription factor, at 4 and 6 days of culture (Fig. 3, A and B). In contrast, osterix mRNA level was not affected by WT or MT FGFR2. WT and MT FGFR2 induced a similar 2-fold increase in Col1A1 expression at 4 and 6 days (Fig. 3, A and B). Alkaline phosphatase (ALP) and osteocalcin (OC) mRNA levels were also increased by WT and MT FGFR2 at 6 days. However, the rise in ALP and OC was greater in MT than in WT FGFR2 (Fig. 3, A and B). Consistently, MT FGFR2 induced a greater increase in ALP activity and staining at day 2 than WT FGFR2 (Fig. 3, C and D). To confirm this finding in more mature cells, calvaria-derived MC3T3-E1 osteoblastic cells were transiently transfected with WT or MT FGFR2. Consistent with our data in C3H10T1/2 cells, MT FGFR2 increased ALP staining and osteoblast genes (Runx2, ALP, Col1A1) in MC3T3-E1 cells at day 2, and the effect was greater than WT FGFR2, except for osteocalcin expression (Fig. 3E), indicating that MT FGFR2 can promote osteoblast differentiation at different stages of development. We also determined the effects of WT and MT FGFR2 on in vitro matrix calcification in long term culture. As shown in Fig. 3F, MT FGFR2 induced a more rapid (7 versus 14 days) and greater extracellular matrix calcification at 21 days than WT FGFR2 (Fig. 3F). To further determine the effect of WT and MT FGFR2 at a terminal differentiation state, we analyzed the changes in SOST, a specific osteocyte marker (46van Bezooijen R.L. ten Dijke P. Papapoulos S.E. Löwik C.W. Cytokine Growth Factor Rev. 2005; 16: 319-327Crossref PubMed Scopus (286) Google Scholar) and DMP1 that is expressed by osteocytes (47Rios H.F. Ye L. Dusevich V. Eick D. Bonewald L.F. Feng J.Q. J. Musculoskelet. Neuronal Interact. 2005; 5: 325-327PubMed Google Scholar). Fig. 3G shows that SOST mRNA expression was increased by MT FGFR2 at 14 days and by WT FGFR2 at 21 days of culture, whereas DMP1 expression remained unchanged. Overall, these results reveal that both WT and MT FGFR2 increase osteoblast marker expression and osteogenic potential in vitro in C3H10T1/2 cells. However, MT FGFR2 is more effective in promoting early and terminal osteoblast differentiation and in vitro osteogenesis than WT FGFR2 in these cells.FIGURE 3FGFR2 promotes the osteogenic differentiation of mesenchymal cells. Quantitative PCR analysis showing that WT FGFR2 and S252W FGFR2 MT increased the expression of osteoblast markers in C3H10T1/2 cells compared with empty vector (EV) transfected cells (A and B). Biochemical analysis showing that MT FGFR2 enhanced ALP activity more than MT FGFR2 in C3H10T1/2 cells (C). Histochemical staining showing that MT FGFR2 induced a greater stimulatory effect on ALP activity than WT FGFR2 in C3H10T1/2 mesenchymal cells and MC3T3-E1 osteoblastic cells at day 2 (D) compared with WT FGFR2. Transient transfection with WT and MT FGFR2 greatly increased mRNA levels of osteoblast markers in MC3T3-E1 osteoblastic cells, as determined by qPCR analysis (E). MT FGFR2 induced a more rapid and greater stimulatory effect on matrix calcification compared with WT FGFR2 in C3H10T1/2 mesenchymal cells (F). Analysis of gene expression by qPCR at late stages of differentiation showing that MT FGFR2 induced a more rapid increase in SOST mRNA expression compared with WT FGFR2 in C3H10T1/2 mesenchymal cells (G). a indicates a significant difference with EV-transfected cells; b indicates a significant difference with WT FGFR2-transfected cells (p < 0.05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)We then investigated whether MT and WT FGFR2 overexpression may modulate MSC differentiation toward the adipogenic lineage. Quantitative PCR analysis showed that MT FGFR2 decreased mRNA expression of the main adipocyte transcription factor PPARγ2 at 4, 6, and 14 days of culture (supplemental Fig. S2A). Furthermore, MT FGFR2 decreased the mRNA levels of C/EBPα (at 6 days) and C/EBPβ (at 4 and 6 days) that are transcription factors controlling adipogenesis, as well as aP2 (at 4–14 days), which is expressed in more mature adipocytes. Both WT and MT FGFR2 decreased mRNA levels of LPL, a late adipocyte differentiation marker, at 4 and 6 days (supplemental Fig. S2A). Consistent with these data," @default.
• W2072506593 created "2016-06-24" @default.
• W2072506593 creator A5007406185 @default.
• W2072506593 creator A5014174179 @default.
• W2072506593 creator A5024183660 @default.
• W2072506593 creator A5029582174 @default.
• W2072506593 creator A5060545233 @default.
• W2072506593 creator A5064414660 @default.
• W2072506593 date "2009-02-01" @default.
• W2072506593 modified "2023-10-01" @default.
• W2072506593 title "Fibroblast Growth Factor Receptor 2 Promotes Osteogenic Differentiation in Mesenchymal Cells via ERK1/2 and Protein Kinase C Signaling" @default.
• W2072506593 cites W10779754 @default.
• W2072506593 cites W126207357 @default.
• W2072506593 cites W1527082449 @default.
• W2072506593 cites W1537898230 @default.
• W2072506593 cites W1965574057 @default.
• W2072506593 cites W1972714130 @default.
• W2072506593 cites W1974973994 @default.
• W2072506593 cites W1982352590 @default.
• W2072506593 cites W1989073601 @default.
• W2072506593 cites W1993640536 @default.
• W2072506593 cites W1995352016 @default.
• W2072506593 cites W2000706451 @default.
• W2072506593 cites W2001853125 @default.
• W2072506593 cites W2002128589 @default.
• W2072506593 cites W2003358724 @default.
• W2072506593 cites W2006966529 @default.
• W2072506593 cites W2012549032 @default.
• W2072506593 cites W2013977089 @default.
• W2072506593 cites W2015121313 @default.
• W2072506593 cites W2026288162 @default.
• W2072506593 cites W2030475521 @default.
• W2072506593 cites W2038675898 @default.
• W2072506593 cites W2042315393 @default.
• W2072506593 cites W2042545863 @default.
• W2072506593 cites W2047410473 @default.
• W2072506593 cites W2048948189 @default.
• W2072506593 cites W2051180982 @default.
• W2072506593 cites W2053425459 @default.
• W2072506593 cites W2056983875 @default.
• W2072506593 cites W2059494342 @default.
• W2072506593 cites W2064129448 @default.
• W2072506593 cites W2067872631 @default.
• W2072506593 cites W2070345664 @default.
• W2072506593 cites W2070466054 @default.
• W2072506593 cites W2074325221 @default.
• W2072506593 cites W2078277031 @default.
• W2072506593 cites W2086262311 @default.
• W2072506593 cites W2086532049 @default.
• W2072506593 cites W2087124418 @default.
• W2072506593 cites W2089016492 @default.
• W2072506593 cites W2089032162 @default.
• W2072506593 cites W2091514824 @default.
• W2072506593 cites W2092273695 @default.
• W2072506593 cites W2093339971 @default.
• W2072506593 cites W2094907909 @default.
• W2072506593 cites W2097928320 @default.
• W2072506593 cites W2101027724 @default.
• W2072506593 cites W2102300005 @default.
• W2072506593 cites W2102894828 @default.
• W2072506593 cites W2108281583 @default.
• W2072506593 cites W2109882510 @default.
• W2072506593 cites W2111096971 @default.
• W2072506593 cites W2123080551 @default.
• W2072506593 cites W2128406041 @default.
• W2072506593 cites W2136363425 @default.
• W2072506593 cites W2137137973 @default.
• W2072506593 cites W2138178479 @default.
• W2072506593 cites W2145470200 @default.
• W2072506593 cites W2148917087 @default.
• W2072506593 cites W2152150200 @default.
• W2072506593 cites W2153720415 @default.
• W2072506593 cites W2159999899 @default.
• W2072506593 cites W2161392800 @default.
• W2072506593 cites W2166578267 @default.
• W2072506593 cites W2167091901 @default.
• W2072506593 doi "https://doi.org/10.1074/jbc.m805432200" @default.
• W2072506593 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/19117954" @default.
• W2072506593 hasPublicationYear "2009" @default.
• W2072506593 type Work @default.
• W2072506593 sameAs 2072506593 @default.
• W2072506593 citedByCount "134" @default.
• W2072506593 countsByYear W20725065932012 @default.
• W2072506593 countsByYear W20725065932013 @default.
• W2072506593 countsByYear W20725065932014 @default.
• W2072506593 countsByYear W20725065932015 @default.
• W2072506593 countsByYear W20725065932016 @default.
• W2072506593 countsByYear W20725065932017 @default.
• W2072506593 countsByYear W20725065932018 @default.
• W2072506593 countsByYear W20725065932019 @default.
• W2072506593 countsByYear W20725065932020 @default.
• W2072506593 countsByYear W20725065932021 @default.
• W2072506593 countsByYear W20725065932022 @default.
• W2072506593 countsByYear W20725065932023 @default.
• W2072506593 crossrefType "journal-article" @default.
• W2072506593 hasAuthorship W2072506593A5007406185 @default.
• W2072506593 hasAuthorship W2072506593A5014174179 @default.
• W2072506593 hasAuthorship W2072506593A5024183660 @default.

• next