Decreased Proliferation and Altered Differentiation in Osteoblasts from Genetically and Clinically Distinct Craniosynostotic Disorders
1999; Elsevier BV; Volume: 154; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65401-6
ISSN1525-2191
AutoresAlessandra Fragale, Marco Tartaglia, Silvia Bernardini, Anna Maria Michela Di Stasi, Concezio Di Rocco, Francesco Velardi, Anna Teti, Piero A. Battaglia, Silvia Migliaccio,
Tópico(s)dental development and anomalies
ResumoCraniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Cα and protein kinase Cε isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Cζ levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype. Craniosynostoses are a heterogeneous group of disorders characterized by premature fusion of cranial sutures. Mutations in fibroblast growth factor receptors (FGFRs) have been associated with a number of such conditions. Nevertheless, the cellular mechanism(s) involved remain unknown. We analyzed cell proliferation and differentiation in osteoblasts obtained from patients with three genetically and clinically distinct craniosynostoses: Pfeiffer syndrome carrying the FGFR2 C342R substitution, Apert syndrome with FGFR2 P253R change, and a nonsyndromic craniosynostosis without FGFR canonic mutations, as compared with control osteoblasts. Osteoblasts from craniosynostotic patients exhibited a lower proliferation rate than control osteoblasts. P253R and nonsyndromic craniosynostosis osteoblasts showed a marked differentiated phenotype, characterized by high alkaline phosphatase activity, increased mineralization and expression of noncollagenous matrix proteins, associated with high expression and activation of protein kinase Cα and protein kinase Cε isoenzymes. By contrast, the low proliferation rate of C342R osteoblasts was not associated with a differentiated phenotype. Although they showed higher alkaline phosphatase activity than control, C342R osteoblasts failed to mineralize and expressed low levels of osteopontin and osteonectin and high protein kinase Cζ levels. Stimulation of proliferation and inhibition of differentiation were observed in all cultures on FGF2 treatment. Our results suggest that an anticipated proliferative/differentiative switch, associated with alterations of the FGFR transduction pathways, could be the causative common feature in craniosynostosis and that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous differentiative phenotype. Craniosynostosis, the premature ossification of one or more sutures of the flat bones of the developing skull, is a relatively common defect of the cranial morphogenetic program, with a prevalence at birth of approximately 1:3000. It results in a wide spectrum of craniofacial anomalies, including abnormal head shape, protruding eyes, and midface underdevelopment.1Cohen Jr, MM Craniosynostosis: diagnosis, evaluation and management. Raven Press, New York1986Google Scholar Surgical treatment of craniosynostosis is frequently required to alleviate the skull deformity; however, in most cases reconstructive craniotomy is also directed to prevent its most severe consequences, ie, increased intracranial pressure, severe exorbitism, and obstructive apnea.2Di Rocco C Velardi F Surgical management of craniosynostosis.in: Galli G Craniosynostosis. CRC Press, Boca Raton, FL1984: 181-248Google Scholar Craniosynostoses can occur as isolated cranial defect or as a feature of more than 100 syndromes, which are clinically distinguished on the basis of the suture(s) involved, the progression of their closure over time, the resulting craniofacial profile, and the pattern of cerebral, cardiac, genital, and limb involvement.1Cohen Jr, MM Craniosynostosis: diagnosis, evaluation and management. Raven Press, New York1986Google Scholar, 3McKusick VA Mendelian inheritance in man.Catalogs of Human Genes and Genetic Disorders. Johns Hopkins University Press, Baltimore1994Google Scholar In about half of these conditions a genetic cause has been established or suggested; most of them are monogenic and are inherited in an autosomal dominant manner, with complete penetrance and variable expressivity.3McKusick VA Mendelian inheritance in man.Catalogs of Human Genes and Genetic Disorders. Johns Hopkins University Press, Baltimore1994Google ScholarConsiderable advances have been made recently in the understanding of the molecular basis of craniosynostotic diseases. Mutations in three members of the fibroblast growth factor receptor (FGFR) family have been recently associated with a number of clinically distinct craniosynostotic conditions.4Bonaventure J Rousseau F Legeai-Mallet L Le Merrer M Munnich A Maroteaux P Common mutations in the fibroblast growth factor receptor 3 (FGFR3) gene account for achondroplasia, hypochondroplasia, and thanatophoric dwarfism.Am J Med Genet. 1996; 63: 148-154Crossref PubMed Scopus (105) Google Scholar, 5Wilkie AOM Genes and mechanisms.Hum Mol Genet. 1997; 6: 1647-1656Crossref PubMed Scopus (407) Google Scholar The FGFR family includes four cell surface tyrosine kinase receptors with a structure consisting of a glycosylated extracellular region characterized by three immunoglobulin-like (Ig-like) motifs, a single membrane-spanning segment, and an intracellular portion containing a split tyrosine kinase domain.6Jaye M Schlessinger J Dionne CA Fibroblast growth factor receptor tyrosine kinases: molecular analysis and signal transduction.Biochem Biophys Acta. 1992; 1135: 185-190Crossref PubMed Scopus (596) Google Scholar FGFRs bind to fibroblast growth factors (FGFs), which are known to regulate proliferation, survival, differentiation, and migration of a wide variety of cells.7Basilico C Moscatelli D The FGF family of growth factors, and oncogenes.Adv Cancer Res. 1992; 59: 115-165Crossref PubMed Scopus (1049) Google Scholar, 8Fernig DJ Gallager JT Fibroblast growth factors and their receptor: an information network controlling tissue growth, morphogenesis and repair.Prog Growth Factor Res. 1994; 5: 353-377Abstract Full Text PDF PubMed Scopus (178) Google Scholar Transduction of the FGF signals is mediated by receptor dimerization, followed by autophosphorylation in the dimer and phosphorylation of cellular substrates regulating the ras/mitogen-activated protein (MAP) kinases pathway,9Nakafuku M Satoh T Kaziro Y Differentiation factors, including nerve growth factor, fibroblast growth factor and interleukin-6, induce an accumulation of an active Ras GTP complex in rat pheochromocytoma PC12 cells.J Biol Chem. 1992; 267: 19448-19454Abstract Full Text PDF PubMed Google Scholar, 10Wang JK Xu H Li H-C Goldfarb M Broadly expressed SNT-like proteins link FGF receptor stimulation to activators of RAS.Oncogene. 1996; 12: 721-729Google Scholar the phospholipid turnover, and activation of protein kinase Cs (PKCs).11Burgess WH Dionne CA Kaplow J Mudd R Friesel R Zilberstein A Schlessinger J Jaye M Characterization and cDNA cloning of phospholipase C-γ, a major substrate for heparin growth factor 1 (acidic fibroblast growth factor) activated tyrosine kinase.Mol Cell Biol. 1990; 10: 4770-4777Crossref PubMed Google Scholar The great majority of craniosynostosis-associated FGFR mutations are spotted in two contiguous extracellular domains involved in FGF binding.5Wilkie AOM Genes and mechanisms.Hum Mol Genet. 1997; 6: 1647-1656Crossref PubMed Scopus (407) Google Scholar More precisely, two adjacent amino acidic changes (Ser252Trp and Pro253Arg) in the linker stretch between the second and third Ig-like domains account for the vast majority of cases of Apert syndrome. Homologous substitutions in FGFR1 (Pro252Arg) and FGFR3 (Pro250Arg) have been reported in the Pfeiffer syndrome and in a heterogeneous group of craniosynostotic conditions, respectively. Different mutations located in the third Ig-like domain of FGFR2 are associated with the Crouzon, Pfeiffer, and Jackson-Weiss syndromes. Among them, substitution of Cys-342 is the most recurrent mutation; the presence of an unpaired cysteine residue in this domain (by loss of Cys-278 or Cys-342 or by the introduction of an additional cysteine residue) has been observed in more than 50% of all cases.5Wilkie AOM Genes and mechanisms.Hum Mol Genet. 1997; 6: 1647-1656Crossref PubMed Scopus (407) Google Scholar All of the FGFR mutations are dominantly acting, and at least two distinct ways in which the FGF transduction pathway may be altered have been proposed. Functional studies focused on the role of mutations spotted in the third Ig-like domain in FGFR2 support that these mutations act by disrupting the intradomain disulfide bond formation, leading to constitutive receptor activation due to homodimerization between mutated receptors.12Neilson KM Friesel RE Constitutive activation of fibroblast growth factor receptor-2 by a point mutation associated with Crouzon syndrome.J Biol Chem. 1995; 270: 26037-26040Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 13Mangasarian K Li Y Mansukhani A Basilico C Mutation associated with Crouzon syndrome causes ligand-independent dimerization and activation of FGF receptor-2.J Cell Physiol. 1997; 172: 117-125Crossref PubMed Scopus (75) Google Scholar, 14Robertson SC Meyer AN Hart KC Galvin BD Webster MK Donoghue DJ Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulphide bond in the third immunoglobulin-like domain.Proc Natl Acad Sci USA. 1998; 95: 4567-4572Crossref PubMed Scopus (152) Google Scholar By contrast, FGFR mutations located in the linker seem to affect ligand binding stability, and they could affect either FGF binding affinity or specificity.15Anderson J Burns HD Enriquez-Harris P Wilkie AOM Heath JK Apert syndrome mutations in fibroblast growth factor receptor 2 exhibit increased affinity for FGF ligand.Hum Mol Genet. 1998; 7: 1475-1483Crossref PubMed Scopus (216) Google ScholarThe FGF signaling plays a pivotal role in the control of intramembranous and endochondral ossification.16Mundolos S Olsen BR Heritable diseases of the skeleton. Part I: molecular insights into skeletal development-transcription factors and signalling pathways.FASEB J. 1997; 11: 125-132PubMed Google Scholar In particular, FGFs are involved in cranial bones' normal growth as well as in their maintenance as discrete individual structures, separated from one another, during the intramembranous ossification of the skull.16Mundolos S Olsen BR Heritable diseases of the skeleton. Part I: molecular insights into skeletal development-transcription factors and signalling pathways.FASEB J. 1997; 11: 125-132PubMed Google Scholar, 17Canalis E The hormonal and local regulation of bone formation.Endocrin Rev. 1983; 4: 62-67Crossref PubMed Scopus (170) Google Scholar Much evidence suggests that FGFs regulate both in vitro pre-osteoblast cell proliferation and osteoblast differentiation.18Hurley MM Kessler M Gronowicz G Raisz LG The interaction of heparin and basic fibroblast growth factor on collagen synthesis in 21-day fetal ray calvariae.Endocrinology. 1992; 130: 2675-2682Crossref PubMed Scopus (51) Google Scholar, 19Tang K-S Capparelli C Stein JL Stein GS Lian JB Huber AC Braverman LE DeVito WJ Acidic fibroblast growth factor inhibits osteoblast differentiation in vitro: altered expression of collagenase, cell growth-related, and mineralization-associated genes.J Cell Biochem. 1996; 61: 152-166Crossref PubMed Scopus (49) Google Scholar, 20Martin I Muraglia A Campanile G Cancedda R Quarto R Fibroblast growth factor-2 supports ex vivo expansion and maintenance of osteogenic precursors from human bone marrow.Endocrinology. 1997; 138: 4456-4462Crossref PubMed Scopus (372) Google Scholar, 21Debiais F Hott M Graulet AM Marie PJ The effects of fibroblast growth factor-2 on human neonatal calvaria osteoblastic cells are differentiation stage specific.J Bone Miner Res. 1998; 13: 645-654Crossref PubMed Scopus (149) Google Scholar However, only a few data are available on the role of the FGFs/FGFRs network in the progression of undifferentiated cells toward differentiated osteoblasts, as well as in the control of suture growth and closure.22Iseki S Wilkie AOM Heath JK Ishimaru T Eto K Morris-Kay GM Fgfr2, and osteopontin domains in the developing skull vault are mutually exclusive, and can be altered by locally applied FGF2.Development. 1997; 124: 3357-3384Google Scholar, 23Kim H-J Rice DPC Kettunen PJ Thesleff I FGF-, BMP-, and Shh-mediated signalling pathways in the regulation of cranial suture morphogenesis, and calvarial bone development.Development. 1998; 125: 1241-1251Crossref PubMed Google Scholar, 24Most D Levine JP Chang J Sung J McCarthy JG Schendel SA Longaker MT Studies in cranial suture biology: up-regulation of transforming growth factor-β 1 and basic fibroblast growth factor mRNA correlates with posterior frontal cranial suture fusion in the rat.Plast Reconstr Surg. 1998; 101: 1431-1440Crossref PubMed Scopus (104) Google Scholar For this reason, the mechanisms leading to the premature closure of cranial sutures in craniosynostosis and underlying interference of FGFR mutations with cell proliferation or differentiation remain unclear.The direct study of primary osteoblast cultures from patients affected by craniosynostosis is one approach to investigating the relationships between altered FGFR function and onset of the craniosynostotic condition. In vitro osteoblast cultures represent a useful tool to characterize possible alterations in cell function, differentiation, and metabolism. By such an approach, it has recently been shown that osteoblasts isolated from prematurely fused sutures are characterized by an increased maturation rate compared to osteoblasts from unaffected sutures of the same patient.25De Pollack C Renier D Hott M Marie PJ Increased bone formation and osteoblastic cell phenotype in premature cranial suture ossification (craniosynostosis).J Bone Min Res. 1996; 11: 401-407Crossref PubMed Scopus (79) Google Scholar However, only a single study has focused on the proliferative/differentiative relationships in osteoblasts carrying a specific FGFR2 mutation26Lomri A Lemonnier J Hott M de Parseval N Lajeunie E Munnich A Renier D Marie PJ Increased calvaria cell differentiation and bone matrix formation induced by fibroblast growth factor receptor 2 mutations in Apert syndrome.J Clin Invest. 1998; 101: 1310-1317Crossref PubMed Google Scholar and no data are available on potential relationships between distinct mutational events in the FGFR or other genes and the cellular and molecular anomaly.In this study, we analyzed possible heterogeneity in cell growth and differentiation among primary cultures of osteoblasts obtained from patients affected by three genetically and clinically distinct craniosynostotic disorders: the Pfeiffer type 2 syndrome associated with the Cys342Arg substitution in FGFR2, the Apert syndrome characterized by the Pro253Arg change in the same receptor, and a nonsyndromic craniosynostotic condition apparently not associated with mutations in the canonic FGFRs' hot spots. Our results indicate that an alteration of the proliferative/differentiative pattern, induced by mutation of the FGFR transduction pathways, could be the causative common feature in craniosynostoses. Our data also suggest that mutations in distinct FGFR2 domains are associated with an in vitro heterogeneous osteoblastic differentiative phenotype.Materials and MethodsMaterialsDulbecco's modified Eagle's medium (DMEM) nutrient MIX F-12, fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco BRL (Grand Island, NY). [Methyl-3H]-thymidine was purchased from DuPont-New England Nuclear (Boston, MA). Collagenase type IV, trypsin, ascorbic acid, β-glycerophosphate, dexamethasone, and heparin were obtained from Sigma Chemical Co. (St. Louis, MO). Micro BCA Protein Assay was obtained from Pierce (Rockford, IL). Goat or rabbit antibodies anti-β-actin and anti-protein kinase C isoforms were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated anti-rabbit and anti-goat IgG and ECL were obtained from Amersham (Arlington Heights, IL). Human recombinant FGF-2 (hr-FGF2) was obtained from Becton Dickinson Labware (Bedford, MA). AmpliTaq polymerase was purchased from Perkin-Elmer (Branchburg, NJ), T7 Sequenase 2.0 DNA sequencing kit from USB (Cleveland, OH), and BstUI and BsaAI endonucleases from New England Biolabs (Beverly, MA). Anti-osteopontin (LF-123), anti-bone sialoprotein (LF-83), and anti-osteonectin (Bon-I) antisera were generously provided by Dr. Larry Fisher of the Craniofacial and Skeletal Disease Branch, National Institute of Dental Research, National Institutes of Health (Bethesda, MD).Clinical EvaluationThree clinically distinct craniosynostotic conditions were considered in the study. The first patient (male, 3 months old) had severe craniosynostosis resulting in a “cloverleaf skull” malformation, midfacial hypoplasia, and severe ocular proptosis. Typical digit anomalies, ie, broad and medially deviated thumbs and great toes and partial bilateral cutaneous syndactily of the second and third toes, were also present. Such clinical features fit the Pfeiffer syndrome type 2 phenotype.27Cohen Jr, MM Pfeiffer syndrome update, clinical subtypes and guideline for differential diagnosis.Am J Med Genet. 1993; 45: 300-307Crossref PubMed Scopus (190) Google Scholar The patient affected by Apert syndrome (male, 2 months old) showed acrocephaly, proptosis, mid-facial hypoplasia, and severe bilateral bony and cutaneous syndactily of both hands and feet. The third patient (male, 9 months old) had a clinically unclassified nonsyndromic craniosynostosis, initially described as Crouzon syndrome, characterized by stenosis of the coronal suture with plagiocephaly, hypertelorism, and invaginated occipital bone. All cases were sporadic.Bone SamplesSkull bone samples were obtained from the three infants during cranial surgery and consisted of bone fragments close to the sutures involved. Bone samples were also obtained from a control subject who underwent local bone surgery for an unrelated disease (male, 3 months old). Bone samples were obtained with parents' signed consent. Samples were placed in cold Hanks' solution supplemented with 100 μg/ml streptomycin and 100 units/ml penicillin and immediately processed.Mutation AnalysisGenomic DNAs were extracted from peripheral blood leukocytes. Molecular screening was carried out by single strand conformational analysis on the FGFR2 third Ig-like domain (exons IIIa, IIIb, and IIIc) and transmembrane segment (TM) coding sequences and exons IIIa of both FGFR1 and FGFR3 genes. Polymerase chain reaction (PCR) conditions for amplifications of the FGFR2 exons coding the third Ig-like domain and exons IIIa of both FGFR1 and FGFR3 were performed as previously described.28Tartaglia M Valeri S Velardi F Di Rocco C Battaglia PA Trp290Cys mutation in the exon IIIa of the fibroblast growth factor receptor 2 (FGFR2) gene is associated with Pfeiffer syndrome.Hum Genet. 1997; 99: 602-606Crossref PubMed Scopus (41) Google Scholar, 29Tartaglia M Di Rocco C Lajeunie E Valeri S Velardi F Battaglia PA Jackson-Weiss syndrome: identification of two novel FGFR2 missense mutations shared with Crouzon and Pfeiffer craniosynostotic disorders.Hum Genet. 1997; 101: 47-50Crossref PubMed Scopus (41) Google Scholar, 30Tartaglia M Saulle E Bordoni V Battaglia PA Polymorphism at position 882 of the fibroblast growth factor receptor 3 (FGFR3) gene detected by SSCP analysis.Mol Cell Probes. 1998; 12: 335-337Crossref PubMed Scopus (4) Google Scholar PCR conditions for FGFR2 exons IIIb and TM were the same as those for FGFR2 exon IIIc amplification, by the oligonucleotide pairs previously reported.31Przylepa KA Paznekas W Zhang M Golabi M Bias W Bamshad MJ Carey JC Hall BD Stevenson R Orlow SJ Cohen Jr, MM Jabs EW Fibroblast growth factor receptor 2 mutations in Beare-Stevenson cutis gyrata syndrome.Nat Genet. 1996; 13: 492-494Crossref PubMed Scopus (163) Google Scholar The purified FGFR2 exon IIIa and exon IIIc PCR products were directly sequenced on both strands, by using the same oligonucleotides utilized during PCR. Mutations were confirmed by endonuclease digestion of the unpurified PCR products with BstUI (exon IIIa) and BsaAI (exon IIIc) according to the manufacturer's specifications.Osteoblast CulturesSamples were processed by a modification of the sequential collagenase/trypsin digestion method.32Robey Gehron P Termine JD Human bone cells in vitro.Calcif Tissue Int. 1985; 37: 453-460Crossref PubMed Scopus (635) Google Scholar Briefly, bone fragments were washed in sterile phosphate-buffered saline (PBS), minced, and treated with 1 mg/ml collagenase type IV and 0.25% trypsin for 30 minutes at 37°C with gentle agitation. The procedure was repeated three times; cells from the second and third digestions were collected by centrifugation and plated in 25-cm2 flasks. At the end of digestions, fragments were also plated in 35-mm Petri dishes; cells proliferated from the bone fragments within 10 days of culture. At confluence, cells were trypsinized and amplified for characterization of the osteoblast phenotype and experiments. Osteoblasts were maintained in DMEM nutrient MIX F-12, supplemented with 100 μg/ml streptomycin and 100 units/ml penicillin and 10% heat-inactivated FBS at 37°C in a humidified incubator with 5% CO2. Medium was changed twice a week.Cell ProliferationOsteoblasts were plated at a density of 8 × 103 cells/well into 24-well plates and allowed to proliferate for 2 days. Cells were incubated with 1 μCi/ml 3H-thymidine for 12 hours. Osteoblasts were then washed twice in PBS, extracted with 1 ml of 1% sodium dodecyl sulfate (SDS) and precipitated with 10% thricloracetic acid. After centrifugation, pellets were dissolved in 1% SDS and aliquots transferred to vials containing 5 ml of scintillation fluid for counting the radioactivity in a β-counter Beckman LS 6500. To assay the effects of hr-FGF2 on cell growth, osteoblasts were seeded at 5 × 103 cells/well in 24-well plates, cultured in basal conditions until preconfluence, washed 3 times, and cultured for 24 hours in serum-free medium. Osteoblasts were then treated with hr-FGF2 (20 ng/ml) and heparin (50 μg/ml) for another 24 hours. Untreated cells were incubated in serum-free medium for 24 hours and processed under identical conditions.Alkaline Phosphatase DetectionAlkaline phosphatase (ALP) activity was detected histochemically in monolayers cultured for 1 week in standard conditions. Osteoblasts were washed twice in 0.1 mol/L cacodylate buffer, pH 7.4, fixed for 10 minutes in 4% paraformaldehyde in 0.1 mol/L cacodylate buffer, and processed using the Fast Blue RR Salt and Mayer's Hematoxylin (Sigma kit no. 85). Quantitative determinations of ALP were performed in cells lysated with 0.1% SDS in PBS, using a commercially available procedure and following the manufacturer's instructions (Sigma kit no. 104). To study the effect of hr-FGF2 on ALP activity, osteoblasts after confluence were rinsed three times, cultured for 24 hours in serum-free medium, and treated for another 48 hours with or without hr-FGF2 (20 ng/ml) and heparin (50 μg/ml).Analysis of in Vitro MineralizationCells were seeded in 24-well plates at a density of 104 cells/well and cultured in mineralization medium (DMEM-F12 with 10% FBS, supplemented with 100 μg/ml ascorbic acid, 10 mmol/L β-glycerophosphate) in presence or absence of 10−8 mol/L dexamethasone (DEX), for 6 weeks. Cells became confluent after 1 week of culture. Mineralization medium was changed twice a week. Cultures were fixed in 4% PFA in 0.1 mol/L cacodylate buffer, pH 7.4, for 10 minutes and washed twice in distilled water. For von Kossa's staining, silver nitrate (5%) was added,33Bills CE Eisenberg H Pallante SL Complexes of organic acids with calcium phosphate: the von Kossa stain as clue to the composition of bone mineral.Johns Hopkins Med J. 1971; 128: 194-207PubMed Google Scholar and plates were placed under an UV lamp (20 cm distance) for 60 minutes. Cultures were rinsed twice in distilled water, treated for 2 minutes with 5% sodium thiosulfate, rapidly rinsed twice in ethanol, and allowed to dry. When osteoblasts were treated for 2 weeks with hr-FGF2 (20 ng/ml) and heparin (50 μg/ml), after confluence cultures were washed twice and grown as described above except that FBS concentration was lowered to 0.1%.Western Blot AnalysisOsteoblasts were grown in 90-mm culture Petri dishes in standard condition until confluence, then washed twice with cold PBS and scraped into 200 μl of ice-cold lysis buffer (20 mmol/L Tris, 1 mmol/L dithiothreitol, 1 mmol/L MgCl2, 0.1 mmol/L Na3VO4, 1 mmol/L NaF, 1% SDS, 0.3 mol/L Urea) containing 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 μg/ml benzamidin, 0.1 mmol/L phenylmethylsulfonyl fluoride. Samples were solubilized in 4× loading buffer (10% glycerol, 8% SDS, 0.05% bromophenol blue, 10% β-mercaptoethanol) and boiled at 90°C for 3 minutes. Fifty micrograms of proteins, determined using a Micro BCA Protein assay, were resolved by SDS polyacrylamide gel electrophoresis and transferred to nitrocellulose at 35 V overnight. Blots were saturated with 5% nonfat dry milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (TTBS) for 2 hours at room temperature. Filters were probed with the appropriated antibody diluted in TTBS containing 1% milk overnight at 4°C. Rabbit polyclonal anti-osteopontin (LF-123), anti-osteonectin (Bon-1), and anti-bone sialoprotein (LF-83) and goat anti-β-actin antibodies were used at 1:500 dilution.34Fisher LW Stubb 3rd, JT Young MF Antisera and cDNA probes to human and certain animal model bone matrix noncollagenous proteins.Acta Orthop Scand (suppl). 1995; 266: 61-65PubMed Google Scholar Blots were extensively washed in TTBS and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-goat antibodies for 1 hour at room temperature and washed 3 times in TTBS. Protein bands were visualized by enhanced chemiluminescence (ECL) kit. Densitometric analysis was performed using Multianalyst software with a BioRad GS-700 Imaging Densitometer. To study the effects of FBS, osteoblasts after confluence were incubated for 72 hours with or without 10% FBS. To study the effect of hr-FGF2, osteoblasts were washed twice, cultured in serum-free medium for 24 hours, and treated with or without hr-FGF2 (20 ng/ml) and heparin (50 μg/ml) for an additional 48 hours.PKC Isoenzymes AnalysisPreparation of cytosol or membrane fractions was carried out as previously described.35Wodgett JR Hunter T Immunological evidence for two physiological forms of protein kinase C.Mol Cell Biol. 1987; 7: 85-96PubMed Google Scholar Briefly, cells were scraped in lysis buffer (20 mmol/L Tris-HCl, pH 7.5, 1 mmol/L EDTA, pH 8.0, 1 mmol/L EGTA, 2 mmol/L dithiothreitol, 2 mmol/L phenylmethylsulfonyl fluoride, 25 μg/ml leupeptin, 6 μg/ml aprotinin, 10 mmol/L benzamidin) on ice and transferred to Eppendorf tubes, then sonicated on ice for two bursts at setting 5 of a heat system sonicator. Cell lysates were centrifuged in a TL-100 Beckman ultracentrifuge at 200,000 × g for 50 minutes at 4°C and supernatants (cytosolic fraction) were recovered and precipitated in 10 volumes of cold acetone at −20°C overnight. Pellets (membrane fractions) were gently resuspended in 1 ml of lysis buffer containing 1% Triton-X 100, incubated on ice for 20 minutes, sonicated, and centrifuged for 50 minutes at 200,000 × g to eliminate nonprotein components. Supernatants were then precipitated overnight in cold acetone. The following day samples were centrifuged in a Sorvall centrifuge at 10,000 × g for 30 minutes at 4°C and pellets were resuspended in 50 μl of 10 mmol/L Tris-HCl, pH 7.5, and stored at −80°C until analysis. Twenty micrograms of cytosolic and membrane fractions were resolved by SDS polyacrylamide gel electrophoresis followed by immunoblotting according to the procedure described for the Western blot analysis of matrix proteins. Specific rabbit antibodies anti-protein kinase Cα, ε, and ζ isoenzymes were used.Statistical AnalysisData are expressed as means ± SE. Statistical significance between data points was determined using the two-tailed Student's t-test. P <0.05 was considered statistically significant. Experiments were performed in triplicate at least 3 times.ResultsParameters of osteoblast proliferation and differentiation were investigated in primary osteoblast cultures from skull bone fragments of three genetically and phenotypically distinct craniosynostotic disorders and from an unaffected sex- and age-
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