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 ∼40%, and reduced FAK phosphorylation on Y397 by ∼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. J. Cell. Biochem. 100: 499–514, 2007. © 2006 Wiley‐Liss, Inc.