title: Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells.
abstract: The intracellular signaling events controlling human mesenchymal stem cells (hMSC) differentiation into osteoblasts are not entirely understood. We recently demonstrated that contact with extracellular matrix (ECM) proteins is sufficient to induce osteogenic differentiation of hMSC through an ERK-dependent pathway. We hypothesized that FAK signaling pathways provide a link between activation of ERK1/2 by ECM, and stimulate subsequent phosphorylation of the Runx2/Cbfa-1 transcription factor that controls osteogenic gene expression. We plated hMSC on purified collagen I (COLL-I) and vitronectin (VN) in the presence or absence of FAK-specific siRNA, and assayed for phosphorylation of Runx2/Cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein-2, osteocalcin, alkaline phosphatase, calcium deposition, and spectroscopically determined mineral:matrix ratio). We found that siRNA treatment reduced FAK mRNA levels by >40% and decreased ECM-mediated phosphorylation of FAK Y397 and ERK1/2. Serine phosphorylation of Runx2/Cbfa-1 was significantly reduced after 8 days in treated cells. Finally, FAK inhibition blocked osterix transcriptional activity and the osteogenic differentiation of hMSC, as assessed by lowered expression of osteogenic genes (RT-PCR), decreased alkaline phosphatase activity, greatly reduced calcium deposition, and a lower mineral:matrix ratio after 28 days in culture. These results suggest that FAK signaling plays an important role in regulating ECM-induced osteogenic differentiation of hMSC.
authors.affiliation: Department of Biology, Rensselaer Polytechnic Institute, 110th 8th Street, Troy, NY 12180-3596, USA.
authors.forename: Roman M
authors.initials: RM
authors.lastname: Salasznyk
authors.forename: Robert F
authors.initials: RF
authors.lastname: Klees
authors.forename: William A
authors.initials: WA
authors.lastname: Williams
authors.forename: Adele
authors.initials: A
authors.lastname: Boskey
authors.forename: George E
authors.initials: GE
authors.lastname: Plopper
mesh_terms: D001483:Base Sequence; D002448:Cell Adhesion; D002454:Cell Differentiation; D002478:Cells, Cultured; D024042:Collagen Type I / Q000378:metabolism; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D051417:Focal Adhesion Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000201:enzymology*; D010012:Osteogenesis / Q000502:physiology*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D019096:Vitronectin / Q000378:metabolism
chemical_list: D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D017931:DNA Primers; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; D019096:Vitronectin; D051417:Focal Adhesion Kinase 1; C496050:PTK2 protein, human