Increased expression of protein kinase C alpha, interleukin-1 alpha, and RhoA guanosine 5 '-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 Apert mutation: Identification by analysis of complementary DNA microarray

Apert (Ap) syndrome is a craniofacial malformation characterized by prematu re fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap s yndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we us ed the emerging technology of complementary DNA (cDNA) microarray to identi fy genes that are involved in osteoblast abnormalities induced by the Ser25 2Trp FGFR-2 mutation. To identify the signaling pathways involved in this s yndrome, we used radioactively labeled cDNAs derived from two sources of ce llular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expr essed, protein kinase C alpha (PKC-alpha), interleukin-1 alpha (IL-1 alpha) , and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridi zation array was confirmed by Northern blot analysis using mRNAs derived fr om different cultures. Furthermore, immunochemical and Western blot analyse s showed that mutant Ap cells displayed increased PKC-alpha, IL-1 alpha, an d RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1 alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-la and Rh oA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated prote in kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced TL-1 alpha and RhoA expression in Ap cells. These data show t hat the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overe xpression of PKC-alpha, IL-1 alpha, and small GTPase RhoA, suggesting a rol e for these effectors in osteoblast alterations induced by the mutation. Th e cDNA microarray technology appears to be a useful tool to gain informatio n on abnormal gene expression and molecular pathways induced by genetic mut ations in bone cells.