Increased expression of protein kinase Calpha, interleukin-1alpha, and RhoA guanosine 5'-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 receptor Apert mutation: identification by analysis of complementary DNA microarray.
Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Ca (PKC-alpha), interleukin-1alpha (IL-1alpha), and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-alpha, IL-1alpha, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1alpha and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1alpha and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-alpha, IL-1alpha, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.
Lomri
A
A
Institut National de la Santé et de la Recherche Médicale Unit 349, Lariboisiere Hospital, Paris, France.
Lemonnier
J
J
Delannoy
P
P
Marie
P J
PJ
D000168:Acrocephalosyndactylia / Q000196:embryology / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D019943:Amino Acid Substitution; D002461:Cell Line, Transformed / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005333:Fetus; D005419:Flavonoids / Q000494:pharmacology; D005786:Gene Expression Regulation; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007375:Interleukin-1 / Q000096:biosynthesis* / Q000235:genetics; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009281:Naphthalenes / Q000494:pharmacology; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000378:metabolism*; D017354:Point Mutation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics*; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000235:genetics*; D012886:Skull / Q000196:embryology / Q000473:pathology; D013382:Subtraction Technique; D048051:p38 Mitogen-Activated Protein Kinases; D020742:rhoA GTP-Binding Protein / Q000096:biosynthesis* / Q000235:genetics
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007375:Interleukin-1; D007527:Isoenzymes; D009281:Naphthalenes; D011725:Pyridines; D017468:Receptors, Fibroblast Growth Factor; C496353:FGFR2 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017346:Protein Serine-Threonine Kinases; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D020742:rhoA GTP-Binding Protein; C058819:calphostin C; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one