Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5.
Human mesenchymal stem cell (hMSC) differentiation into osteoblasts and the signaling events involved are poorly understood. We recently established that contact with specific extracellular matrix (ECM) proteins, in particular laminin-5, is sufficient to induce an osteogenic phenotype in hMSC through an extracellular signal-related kinase (ERK)-dependent pathway. Activation of ERK 1/2 by laminin-5 induces phosphorylation of the runx2/cbfa-1 transcription factor that controls osteogenic gene expression. We hypothesized that focal adhesion kinase (FAK) mediated signaling pathways supply a link between cell surface integrin-ECM binding and activation of ERK 1/2, and that laminin-5 promotes its osteogenic effects through this pathway. To test this hypothesis, we plated hMSC on a laminin-5 matrix in the presence or absence of FAK-specific small inhibitory RNAs (siRNA), and assayed for phosphorylation of runx2/cbfa-1 as well as expression of established osteogenic differentiation markers (bone sialoprotein, osteocalcin, alkaline phosphatase, calcium deposition, and mineral:matrix ratio). We found that siRNA treatment reduced total endogenous FAK protein by approximately 40%, and reduced FAK phosphorylation on Y397 by approximately 33% in cells plated on laminin-5 for 30 min. SiRNA treated cells exhibited a decrease in ERK 1/2 phosphorylation after 1 h, and reduced serine/threonine phosphorylation of Runx2/Cbfa-1 after 8 days. Finally, FAK inhibition blocked 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 establish FAK as an important mediator of laminin-5-induced osteogenic differentiation of hMSC.
Salasznyk
Roman M
RM
Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180-3596, USA.
Klees
Robert F
RF
Boskey
Adele
A
Plopper
George E
GE
D015415:Biomarkers; D002113:Calcification, Physiologic; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D004789:Enzyme Activation; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D006801:Humans; D007797:Laminin / Q000494:pharmacology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D010012:Osteogenesis / Q000187:drug effects; D010766:Phosphorylation; D034741:RNA, Small Interfering / Q000235:genetics; D015398:Signal Transduction; D013234:Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D014162:Transfection
D015415:Biomarkers; D050659:Core Binding Factor Alpha 1 Subunit; D007797:Laminin; D034741:RNA, Small Interfering; C493709:RUNX2 protein, human; D051416:Focal Adhesion Protein-Tyrosine Kinases; D020928:Mitogen-Activated Protein Kinases