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• W2055294875 abstract "Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS. Genetic mutations of Twist, a basic helix-loop-helix transcription factor, induce premature fusion of cranial sutures in Saethre-Chotzen syndrome (SCS). We report here a previously undescribed mechanism involved in the altered osteoblastogenesis in SCS. Cranial osteoblasts from an SCS patient with a Twist mutation causing basic helix-loop-helix deletion exhibited decreased expression of E3 ubiquitin ligase Cbl compared with wild-type osteoblasts. This was associated with decreased ubiquitin-mediated degradation of phosphatidyl inositol 3 kinase (PI3K) and increased PI3K expression and PI3K/Akt signaling. Increased PI3K immunoreactivity was also found in osteoblasts in histological sections of affected cranial sutures from SCS patients. Transfection with Twist or Cbl abolished the increased PI3K/Akt signaling in Twist mutant osteoblasts. Forced overexpression of Cbl did not correct the altered expression of osteoblast differentiation markers in Twist mutant cells. In contrast, pharmacological inhibition of PI3K/Akt, but not ERK signaling, corrected the increased cell growth in Twist mutant osteoblasts. The results show that Twist haploinsufficiency results in decreased Cbl-mediated PI3K degradation in osteoblasts, causing PI3K accumulation and activation of PI3K/Akt-dependent osteoblast growth. This provides genetic and biochemical evidence for a role for Cbl-mediated PI3K signaling in the altered osteoblast phenotype induced by Twist haploinsufficiency in SCS. Saethre-Chotzen syndrome (SCS), also called acrocephalosyndactyly III (ACS III), is an autosomal dominant hereditary disorder characterized clinically by facial dysmorphism, digit defects, and premature fusion of coronal sutures (craniosynostosis).1Wilkie AO Craniosynostosis: genes and mechanisms.Hum Mol Genet. 1997; 6: 1647-1656Crossref PubMed Scopus (413) Google Scholar, 2el Ghouzzi V Le Merrer M Perrin-Schmitt F Lajeunie E Benit P Renier D Bourgeois P Bolcato-Bellemin AL Munnich A Bonaventure J Mutations of the Twist gene in the Saethre-Chotzen syndrome.Nat Genet. 1997; 15: 42-46Crossref PubMed Scopus (532) Google Scholar, 3Howard TD Paznekas WA Green ED Chiang LC Ma N Ortiz De Luna RI Garcia Delgado C Gonzales-Ramos M Kline AD Jabs EW Mutations in Twist, a basic helix-loop-helix transcription factor, in Saethre-Chotzen syndrome.Nat Genet. 1997; 15: 36-41Crossref PubMed Scopus (531) Google Scholar This disorder is induced by multiple genetic mutations in the gene for Twist, a basic helix-loop-helix (bHLH) factor involved in mesodermal differentiation. Most Twist mutations in SCS are located in the highly conserved bHLH domain.4El Ghouzzi V Lajeunie E Le Merrer M Cormier-Daire V Renier D Munnich A Bonaventure J Mutations within or upstream of the basic helix-loop-helix domain of the Twist gene are specific to Saethre-Chotzen syndrome.Eur J Hum Genet. 1999; 7: 27-33Crossref PubMed Scopus (64) Google Scholar, 5Gripp K Zackai E Stolle C Mutations in the human Twist gene.Hum Mutat. 2000; 15: 150-155Crossref PubMed Scopus (89) Google Scholar Twist mutations in SCS cause Twist protein degradation, resulting in Twist haploinsufficiency, loss of dimerization with E proteins, and reduced binding to DNA canonical sequences in the promoter of target genes.6El Ghouzzi V Legeai-Mallet L Aresta S Benoist C Munnich A de Gunzburg J Bonaventure J Saethre-Chotzen mutations cause Twist protein degradation or impaired nuclear location.Hum Mol Genet. 2000; 9: 813-819Crossref PubMed Scopus (78) Google Scholar, 7El Ghouzzi V Legeai-Mallet L Benoist-Lasselin C Lajeunie E Renier D Munnich A Bonaventure J Mutations in the basic domain and the loop-helix II junction of Twist abolish DNA binding in Saethre-Chotzen syndrome.FEBS Lett. 2001; 492: 112-118Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Despite the important implication of Twist mutations in craniosynostosis in SCS, our knowledge of the molecular mechanisms by which Twist alters the osteoblast phenotype in SCS remains incomplete. Our previous studies showed that Twist haploinsufficiency induced by deletion of the bHLH domain in SCS alters the osteoblast phenotype by affecting signaling molecules that control cell differentiation and apoptosis.8Yousfi M Lasmoles F Lomri A Delannoy P Marie PJ Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in the Saethre-Chotzen syndrome.J Clin Invest. 2001; 107: 1153-1161Crossref PubMed Scopus (76) Google Scholar, 9Yousfi M Lasmoles F Marie PJ TWIST inactivation reduces CBFA1/RUNX2 expression and DNA binding to the osteocalcin promoter in osteoblasts.Biochem Biophys Res Commun. 2002; 297: 641-644Crossref PubMed Scopus (54) Google Scholar, 10Yousfi M Lasmoles F El Ghouzzi V Marie PJ Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvaria osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.Hum Mol Genet. 2002; 11: 359-369Crossref PubMed Google Scholar, 11Guenou H Kaabeche K Mee SL Marie PJ A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre-Chotzen syndrome.Hum Mol Genet. 2005; 14: 1429-1439Crossref PubMed Scopus (58) Google Scholar In addition, Twist was found to inhibit the functional activity of Runx2, a master gene controlling osteoblast differentiation in the developing mouse,12Bialek P Kern B Yang X Schrock M Sosic D Hong N Wu H Yu K Ornitz DM Olson EN Justice MJ Karsenty G A twist code determines the onset of osteoblast differentiation.Dev Cell. 2004; 6: 423-435Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar suggesting that multiple mechanisms may contribute to the altered osteoblast phenotype in SCS. However, the signaling pathways that act downstream of Twist and are involved in the altered osteoblast recruitment in SCS remain primarily unknown. Proteasome degradation of ubiquitin-targeted proteins is an important mechanism that negatively controls activated signaling pathways.13Roos-Mattjus P Sistonen L The ubiquitin-proteasome pathway.Ann Med. 2004; 36: 285-295Crossref PubMed Scopus (104) Google Scholar Cbl is an E3 ubiquitin ligase that targets tyrosine kinase receptors and other signaling proteins, resulting in their ubiquitination and down-regulation.14Sanjay A Horne WC Baron R The Cbl family: ubiquitin ligases regulating signaling by tyrosine kinases.Sci STKE. 2001; 2001: PE40PubMed Google Scholar, 15Schmidt MH Dikic I The Cbl interactome and its functions.Nat Rev Mol Cell Biol. 2005; 6: 907-919Crossref PubMed Scopus (328) Google Scholar In bone, Cbl regulates osteoclast activity by interacting with Src and associated proteins.16Horne WC Sanjay A Bruzzaniti A Baron R The role(s) of Src kinase and Cbl proteins in the regulation of osteoclast differentiation and function.Immunol Rev. 2005; 208: 106-125Crossref PubMed Scopus (132) Google Scholar In osteoblasts, we previously showed that increased Cbl recruitment induced by fibroblast growth factor receptor-2 (FGFR2)-activating mutations in Apert syndrome results in increased ubiquitin-mediated degradation of FGFR2, Src proteins, and α5 integrin subunit, resulting in altered osteoblast differentiation and survival.17Kaabeche K Lemonnier J Le Mee S Caverzasio J Marie PJ Cbl-mediated degradation of Lyn and Fyn induced by constitutive fibroblast growth factor receptor-2 activation supports osteoblast differentiation.J Biol Chem. 2004; 279: 36259-36267Crossref PubMed Scopus (72) Google Scholar, 18Kaabeche K Guenou H Bouvard D Didelot N Listrat A Marie PJ Cbl-mediated ubiquitination of alpha5 integrin subunit mediates fibronectin-dependent osteoblast detachment and apoptosis induced by FGFR2 activation.J Cell Sci. 2005; 118: 1223-1232Crossref PubMed Scopus (72) Google Scholar Among other proteins, Cbl proteins can interact with the p85-regulatory subunit of phosphatidyl inositol 3 kinase (PI3K), resulting in PI3K ubiquitylation and degradation.19Sattler M Salgia R Okuda K Uemura N Durstin MA Pisick E Xu G Li JL Prasad KV Griffin JD The proto-oncogene product p120CBL and the adaptor proteins CRKL and c-CRK link c-ABL, p190BCR/ABL and p210BCR/ABL to the phosphatidylinositol-3′ kinase pathway.Oncogene. 1996; 12: 839-846PubMed Google Scholar, 20Tsygankov AY Teckchandani AM Feshchenko EA Swaminathan G Beyond the RING: CBL proteins as multivalent adapters.Oncogene. 2001; 20: 6382-6402Crossref PubMed Scopus (106) Google Scholar PI3K catalyzes the production of phosphatidylinositol-3,4,5-trisphosphate and thereby contributes to the activation of various signaling components involved in the regulation of gene expression and cell survival.21Cantley LC The phosphoinositide 3-kinase pathway.Science. 2002; 296: 1655-1657Crossref PubMed Scopus (4674) Google Scholar, 22Downward J PI 3-kinase, Akt and cell survival.Semin Cell Dev Biol. 2004; 15: 177-182Crossref PubMed Scopus (679) Google Scholar In bone, PI3K controls osteoblast differentiation and survival23Golden LH Insogna KL The expanding role of PI3-kinase in bone.Bone. 2004; 34: 3-12Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar by interacting with local signaling factors24Debiais F Lefevre G Lemonnier J Le Mée S Lasmoles F Mascarelli F Marie PJ Fibroblast growth factor-2 induces osteoblast survival through a phosphatidylinositol 3-kinase-dependent, -beta-catenin-independent signaling pathway.Exp Cell Res. 2004; 297: 235-246Crossref PubMed Scopus (76) Google Scholar, 25Ghosh-Choudhury N Abboud SL Nishimura R Celeste A Mahimainathan L Choudhury GG Requirement of BMP-2-induced phosphatidylinositol 3-kinase and Akt serine/threonine kinase in osteoblast differentiation and Smad-dependent BMP-2 gene transcription.J Biol Chem. 2002; 277: 33361-33368Crossref PubMed Scopus (267) Google Scholar, 26Radcliff K Tang TB Lim J Zhang Z Abedin M Demer LL Tintut Y Insulin-like growth factor-I regulates proliferation and osteoblastic differentiation of calcifying vascular cells via extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase pathways.Circ Res. 2005; 96: 398-400Crossref PubMed Scopus (103) Google Scholar, 27Almeida M Han L Bellido T Manolagas SC Kousteni S Wnt proteins prevent apoptosis of both uncommitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT.J Biol Chem. 2005; 280: 41342-41351Crossref PubMed Scopus (355) Google Scholar and Runx2.28Fujita T Azuma Y Fukuyama R Hattori Y Yoshida C Koida M Ogita K Komori T Runx2 induces osteoblast and chondrocyte differentiation and enhances their migration by coupling with PI3K-Akt signaling.J Cell Biol. 2004; 166: 85-95Crossref PubMed Scopus (361) Google Scholar In nonskeletal cells, PI3K was found to control cell growth through activation of the downstream Akt signaling pathway.22Downward J PI 3-kinase, Akt and cell survival.Semin Cell Dev Biol. 2004; 15: 177-182Crossref PubMed Scopus (679) Google Scholar, 29Wennstrom S Downward J Role of phosphoinositide 3-kinase in activation of ras and mitogen-activated protein kinase by epidermal growth factor.Mol Cell Biol. 1999; 19: 4279-4288Crossref PubMed Scopus (254) Google Scholar, 30Borgatti P Martelli AM Bellacosa A Casto R Massari L Capitani S Neri LM Translocation of Akt/PKB to the nucleus of osteoblast-like MC3T3–E1 cells exposed to proliferative growth factors.FEBS Lett. 2000; 477: 27-32Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 31Lee YR Park J Yu HN Kim JS Youn HJ Jung SH Up-regulation of PI3K/Akt signaling by 17beta-estradiol through activation of estrogen receptor-alpha, but not estrogen receptor-beta, and stimulates cell growth in breast cancer cells.Biochem Biophys Res Commun. 2005; 336: 1221-1226Crossref PubMed Scopus (104) Google Scholar Consequently, deregulation of PI3K activity may lead to increased cell growth and tumor formation.32Kim D Cheng GZ Lindsley CW Yang H Cheng JQ Targeting the phosphatidylinositol-3 kinase/Akt pathway for the treatment of cancer.Curr Opin Invest Drugs. 2005; 6: 1250-1258PubMed Google Scholar, 33Stephens L Williams R Hawkins P Phosphoinositide 3-kinases as drug targets in cancer.Curr Opin Pharmacol. 2005; 5: 357-365Crossref PubMed Scopus (97) Google Scholar, 34Wymann MP Marone R Phosphoinositide 3-kinase in disease: timing, location, and scaffolding.Curr Opin Cell Biol. 2005; 17: 141-149Crossref PubMed Scopus (179) Google Scholar Nothing is known, however, of the role of PI3K in osteoblast growth and bone pathology. In this study, we investigated the role of Cbl and PI3K in the abnormal osteoblast phenotype induced by Twist haploinsufficiency in SCS. We show here that Twist haploinsufficiency in human calvarial osteoblasts is associated with decreased Cbl expression resulting in PI3K accumulation, increased PI3K/Akt signaling, and osteoblast proliferation, a mechanism that may contribute to the premature cranial ossification in the SCS. Calvaria bone samples at the coronal suture level from three infants (age, 3.5 to 7 months) with SCS were obtained by surgical operation, and normal calvaria bone samples at equivalent areas were obtained from three normal age-matched infants who underwent local reconstruction of the skull unrelated to bone diseases, according to the French ethical committee recommendations.10Yousfi M Lasmoles F El Ghouzzi V Marie PJ Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvaria osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.Hum Mol Genet. 2002; 11: 359-369Crossref PubMed Google Scholar The mutations studied cause Twist haploinsufficiency by inducing either degradation of truncated Twist protein (Y103X, Q109X) or loss of Twist DNA binding capacities (R118C).6El Ghouzzi V Legeai-Mallet L Aresta S Benoist C Munnich A de Gunzburg J Bonaventure J Saethre-Chotzen mutations cause Twist protein degradation or impaired nuclear location.Hum Mol Genet. 2000; 9: 813-819Crossref PubMed Scopus (78) Google Scholar, 7El Ghouzzi V Legeai-Mallet L Benoist-Lasselin C Lajeunie E Renier D Munnich A Bonaventure J Mutations in the basic domain and the loop-helix II junction of Twist abolish DNA binding in Saethre-Chotzen syndrome.FEBS Lett. 2001; 492: 112-118Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar Coronal sutures from patients and controls were fixed in 10% formaldehyde and embedded in paraffin, deparaffinized in xylene, and rehydrated through a graded series of ethanol. Sections were digested with 20 μg/ml proteinase K for 15 minutes at 37°C. Endogenous peroxidase was quenched with 0.3% H2O2 for 1 hour.10Yousfi M Lasmoles F El Ghouzzi V Marie PJ Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvaria osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.Hum Mol Genet. 2002; 11: 359-369Crossref PubMed Google Scholar Sections were permeabilized with 0.1% Triton X-100, at 4°C for 2 minutes, incubated for 1 hour at 37°C with anti-PI3K (1/40; Cell Signaling, Danvers, MA), and the signal was revealed with diaminobenzidine. Negative controls were obtained by omitting the primary antibody from the reaction. Calvaria cell populations obtained by collagenase digestion from coronal sutures in one SCS subject with the Y103X mutation, which leads to deletion of the functional bHLH domain, and from an age-matched normal patient were immortalized and called mutant (M-Tw) and normal (wild-type) immortalized calvaria cell populations, respectively.8Yousfi M Lasmoles F Lomri A Delannoy P Marie PJ Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in the Saethre-Chotzen syndrome.J Clin Invest. 2001; 107: 1153-1161Crossref PubMed Scopus (76) Google Scholar The osteoblast phenotype in these cells is consistent with the phenotype obtained in primary human calvaria cells in vitro and in vivo.8Yousfi M Lasmoles F Lomri A Delannoy P Marie PJ Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in the Saethre-Chotzen syndrome.J Clin Invest. 2001; 107: 1153-1161Crossref PubMed Scopus (76) Google Scholar, 10Yousfi M Lasmoles F El Ghouzzi V Marie PJ Twist haploinsufficiency in Saethre-Chotzen syndrome induces calvaria osteoblast apoptosis due to increased TNFalpha expression and caspase-2 activation.Hum Mol Genet. 2002; 11: 359-369Crossref PubMed Google Scholar The cells were cultured in Dulbecco's modified Eagle's medium supplemented with glutamine (292 mg/L), 10% heat-inactivated fetal calf serum, and antibiotics (100 IU/ml penicillin and 100 μg/ml streptomycin). For analysis of cell growth, M-Tw and wild-type cells were plated at preconfluence and cultured for 72 hours in the presence of the PI3K inhibitor LY294002 (5 μmol/L; Calbiochem VWR, Fontenay, France), the Akt inhibitor SH-5, which does not decrease the phosphorylation of other kinases (20 μmol/L; Calbiochem), or the MAPK kinase 1 (MEK 1) inhibitor PD98059 (20 μmol/L; Calbiochem) or their solvent, and cell replication was measured using the 5-bromo-2′-deoxyuridine (BrdU) enzyme-linked immunosorbent assay (Roche, Neuilly sur Seine, France) according to the manufacturer's instructions. Data were expressed as absorbance in optical density and reported as the mean ± SEM of 8 to 10 replicates. To determine the implication of Twist and Cbl, Twist mutant cells were plated at 2500 cells/cm2 the day before transfection. The cells were co-transfected with the plasmid (2.5 μg/3 cm2 dish) and pSV-β-galactosidase (50 ng of β-gal) control vector (Promega, Charbonnières, France) in Dulbecco's modified Eagle's medium with 1% fetal calf serum. Cells were incubated for 48 hours with empty expression vector (pBK or pcDNA3), pBK-Cbl, or pcDNA3-Twist vector and Exgen (Euromedex, Souffelweyersheim, France) according to the manufacturer's directions. Efficiency of transfection was ∼15 to 20% at 48 hours after transfection as controlled by β-gal activity (β-gal reporter gene assay; Roche). Although the transfection efficiency was low in these cells, the strong promoter (CMV) used in plasmids allowed a high level of expression of exogenous Twist or Cbl in the cell population. To determine whether PI3K levels may be rescued by activation of the proteasome, permeabilized Twist mutant cells were treated with the PA28 proteasome activator (200 μmol/L; Calbiochem). After 48 hours, phosphorylated PI3K (p85) protein levels were determined by Western blot analysis as described below. The mRNA levels of Cbl, PI3K (p85), and the osteoblast marker genes Runx2, type I collagen (COLIA1), and osteocalcin were analyzed by RT-PCR.11Guenou H Kaabeche K Mee SL Marie PJ A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre-Chotzen syndrome.Hum Mol Genet. 2005; 14: 1429-1439Crossref PubMed Scopus (58) Google Scholar Cells were washed with phosphate-buffered saline and lysed with Extract-All (Eurobio, Courtaboeuf, France) reagent according to the manufacturer's instructions. Three μg of total cellular RNA from each sample were reverse-transcribed, and the cDNA samples were then divided and amplified by PCR using specific primers. Primers for PI3K (p85) were for sense 5′-AGCACCGACTTCAAGACTACG-3′ and for anti-sense 5′-GGATGCCAATGAGATTGTCC-3′. Primers for Cbl, Runx2, COLIA1, and GAPDH were as described.11Guenou H Kaabeche K Mee SL Marie PJ A role for fibroblast growth factor receptor-2 in the altered osteoblast phenotype induced by Twist haploinsufficiency in the Saethre-Chotzen syndrome.Hum Mol Genet. 2005; 14: 1429-1439Crossref PubMed Scopus (58) Google Scholar Autoradiographic signals were quantified using a scanner densitometer, and the signal for each gene was related to that of GAPDH. Cell proteins were extracted in RIPA buffer with 1 mmol/L phenylmethyl sulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 10 nmol/L calyculin A, 50 nmol/L microcystin LR, 2 mmol/L Na3VO4.35Maestro R Dei Tos AP Hamamori Y Krasnokutsky S Sartorelli V Kedes L Doglioni C Beach DH Hannon GJ Twist is a potential oncogene that inhibits apoptosis.Genes Dev. 1999; 13: 2207-2217Crossref PubMed Scopus (455) Google Scholar Lysates were clarified by centrifugation at 12,000 × g for 30 minutes at 4°C, and protein content of the supernatants was determined using the DC protein assay (Bio-Rad Laboratories, Hercules, CA). For Western blot analysis, equal aliquots (50 μg of protein lysates were electrophoresed in 4 to 20% sodium dodecyl sulfate-polyacrylamide gradient gels. Electrophoresed proteins were transferred onto polyvinylidene difluoride membranes (Hybond-P; Amersham, Saclay, France). The membranes were then reacted with antibodies for phospho-PI3K (Cell Signaling), total PI3K (1/1000; Cell Signaling), phospho-Akt (1/500; Cell Signaling), total Akt (1/500, Cell Signaling) that recognizes the three Akt forms, or β-actin (1/200; Sigma, St. Louis, MO), incubated for 1 hour with the appropriate affinity-purified anti-rabbit or anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), probed with peroxidase-coupled specific secondary antibodies and visualized using the enhanced chemiluminescence detection kit (Amersham). The levels of proteins were measured by scanning densitometry and corrected for actin. For immunoprecipitation analysis, equal aliquots (100 μg) of protein lysates were immunoprecipitated using 2.5 μg of specific anti-Cbl (Santa Cruz) or anti-PI3K (Cell Signaling) and incubated overnight at 4°C in a rotating device. After 24 hours, 20 μl of protein A/G agarose (Santa Cruz) were added and incubated for 1 hour at 4°C. Immunoprecipitates were then collected by centrifugation at 1200 × g for 3 minutes, and the pellets were washed four times with lysis buffer and resuspended in 25 μl of running buffer. Aliquots were then subjected to electrophoresis as described above, and membranes were reacted with PI3K, phospho-PI3K (Cell Signaling), or ubiquitin (Cell Signaling) antibodies. Immunoblots were probed with peroxidase-coupled specific secondary antibodies and visualized by enhanced chemiluminescence. The data are representative of two to four different experiments. Differences between the mean values ± SEM were analyzed using the statistical package superanalysis of variance (Macintosh; Abacus Concepts, Inc., Berkeley, CA) with a minimal significance of P < 0.05. To analyze the potential role of Cbl in osteoblast pathology in SCS, Cbl expression was determined in wild-type osteoblasts and Twist mutant osteoblasts expressing the natural Twist Y103X mutation causing bHLH deletion and Twist haploinsufficiency.6El Ghouzzi V Legeai-Mallet L Aresta S Benoist C Munnich A de Gunzburg J Bonaventure J Saethre-Chotzen mutations cause Twist protein degradation or impaired nuclear location.Hum Mol Genet. 2000; 9: 813-819Crossref PubMed Scopus (78) Google Scholar, 7El Ghouzzi V Legeai-Mallet L Benoist-Lasselin C Lajeunie E Renier D Munnich A Bonaventure J Mutations in the basic domain and the loop-helix II junction of Twist abolish DNA binding in Saethre-Chotzen syndrome.FEBS Lett. 2001; 492: 112-118Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar, 8Yousfi M Lasmoles F Lomri A Delannoy P Marie PJ Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in the Saethre-Chotzen syndrome.J Clin Invest. 2001; 107: 1153-1161Crossref PubMed Scopus (76) Google Scholar RT-PCR analysis showed that Twist mutant cells expressed decreased Cbl mRNA levels compared with wild-type cells (Figure 1A). Decreased Cbl levels at the protein level were also found in Twist mutant osteoblasts compared with wild-type osteoblasts, as demonstrated by Western blot analysis (Figure 1B). To further determine the role of Twist in the alteration of Cbl expression, mutant osteoblasts were transfected with Twist expression vector, and Cbl protein levels were determined. Western blot analysis showed that forced expression of Twist in mutant osteoblasts corrected Cbl expression at both the RNA and protein levels (Figure 1, C and D). These results show that Twist haploinsufficiency is associated with decreased Cbl expression in Twist mutant osteoblasts, suggesting that Twist may control Cbl at the transcriptional and/or posttranscriptional level. Cbl is an adaptor protein that interacts with PI3K and mediates its ubiquitination and proteasome degradation.14Sanjay A Horne WC Baron R The Cbl family: ubiquitin ligases regulating signaling by tyrosine kinases.Sci STKE. 2001; 2001: PE40PubMed Google Scholar, 15Schmidt MH Dikic I The Cbl interactome and its functions.Nat Rev Mol Cell Biol. 2005; 6: 907-919Crossref PubMed Scopus (328) Google Scholar We therefore hypothesized that Cbl may interact physically with PI3K in osteoblasts to mediate ubiquitination of this protein. As shown in Figure 2A, Cbl was found to co-immunoprecipitate with PI3K (p85) in Twist mutant osteoblasts and wild-type cells, indicating physical interactions between the two proteins. The levels of total and phosphorylated PI3K associated with Cbl were much lower in Twist mutant cells compared with wild-type cells (Figure 2, A and B) as a result of the decreased amount of Cbl protein in mutant osteoblasts (Figure 1A). Further immunoprecipitation studies showed decreased PI3K polyubiquitination in mutant osteoblasts compared with control cells (Figure 2C). Overall, these results show that the decreased Cbl level in Twist mutant cells is associated with decreased PI3K protein ubiquitination and degradation. To assess whether this effect results in alteration of PI3K levels and signaling, we determined total and phosphorylated PI3K levels in Twist mutant osteoblasts. As shown in Figure 3A, Western blot analysis revealed a significant increase in total PI3K (p85) protein level in Twist mutant cells compared with wild-type cells. In addition, the amount of phosphorylated PI3K (p85) was increased in Twist mutant osteoblasts compared with wild-type cells (Figure 3A). In contrast, RT-PCR analysis showed a similar amount of p85-PI3K transcripts in Twist mutant cells compared with wild-type cells (Figure 3B). Because Akt/PKB acts as a downstream effector of PI3K, we analyzed the alteration of this signaling pathway in Twist mutant osteoblasts. As shown in Figure 3C, phosphorylated Akt levels were increased in Twist mutant cells compared with wild-type osteoblasts. These results indicate that PI3K accumulation induced by Twist haploinsufficiency is associated with increased PI3K/Akt signaling in mutant osteoblasts. The increased PI3K protein levels may arise from change in translational efficiency as well as increased protein stability. Because Cbl interacts with PI3K to control its stability, we investigated whether the increased PI3K signaling in mutant cells may result from reduced proteasome degradation. Permeabilized Twist mutant cells were treated with the PA28 proteasome activator and phosphorylated PI3K levels were determined by Western blot analysis. Activation of proteasome with PA28 slightly decreased phosphorylated PI3K levels in Twist mutant cells (Figure 2D). The effect observed, however, was not striking most likely because of the low penetration within cells of PA28 resulting from the large size of the proteasome activator. However, collectively the results suggest that the increased PI3K protein levels and activity in Twist mutant osteoblasts results, at least in part, from decreased degradation by the proteasome. Because we used human osteoblasts directly derived from affected coronal tissues, the observed in vitro phenotype is likely to reflect the in vivo situation in SCS. To confirm that the increased PI3K expression in vitro also occurs in vivo, we examined PI3K expression in histological sections of coronal sutures in a 3-month-old SCS patient with the Y103X mutation compared with age-matched controls. The immunohistochemical analysis of coronal sutures in this SCS patient showed increased PI3K expression in osteoblasts and mesenchymal cells compared with normal sutures from age-matched controls (Figure 4). Similar findings were observed in two patients with Twist nonsense mutation (Q109X) or missense mutation (R118C) compared with normal sutures from age-matched controls (Figure 4). These results indicate that Twist haploinsufficiency in SCS i" @default.
• W2055294875 created "2016-06-24" @default.
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• W2055294875 date "2006-10-01" @default.
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• W2055294875 title "Down-Regulation of Ubiquitin Ligase Cbl Induced by Twist Haploinsufficiency in Saethre-Chotzen Syndrome Results in Increased PI3K/Akt Signaling and Osteoblast Proliferation" @default.
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