Focal adhesion kinase (FAK) is a ubiquitously expressed signaling protein that links extracellular matrix (ECM)adhesion with activation of intracellular signaling pathways and gene expression. Human mesenchymal stem cells (hMSC) are a source of osteoblast precursors potentially suitable for use in tissue engineering applications to repair damaged bone. Controlled induction and maintenance of osteoblast differentiation is a significant hurdle in the quest to engineer bone. The role of FAK signaling in controlling osteogenic gene expression in hMSC is entirely unknown. Our long range objective is to devise a strategy for transplanting hMSC into sites of skeletal injury under conditions that maximally favor osteogenesis. The goal of this project is to define the role of FAK signaling during hMSC osteogenic differentiation and to establish optimal conditions for this differentiation. Our hypothesis is that FAK is a key signal transducing element in the pathway linking ECM binding and applied tensile strain to expression of osteoblast-specific genes and matrix mineralization in hMSC.
Aims : (1) Quantify FAK phosphorylation/activation states, osteogenic gene expression levels, and matrix mineralization; in cultured hMSC cultured on purified ECM, and develop predictive modelslinking these parameters. (2) Assess the role of FAK phosphorylation in controlling osteogenic gene expression and matrix mineralization using mutant FAK constructs lacking tyrosine residues (Y397, Y576, Y925) important in FAK signaling, and (3) Quantify the effect of tensile strain on FAK phosphorylation, osteogenic gene expression, and matrix mineralization on cells plated on purified ECM proteins. Our rationale is that, once we establish a mechanistic understanding of FAK signaling pathways during hMSC osteogenesis, it may be possible to control hMSC osteogenesis by controlling FAK activity directly. We expect two significant outcomes: (i) quantitative models linking specific extracellular stimuli with FAK activity, osteogenic gene expression, and matrix mineralization; and (ii) definition of optimal extracellular stimuli that will control FAK activity and, ultimately, osteogenic differentiation of hMSC.

Agency
National Institute of Health (NIH)
Institute
National Institute of Biomedical Imaging and Bioengineering (NIBIB)
Type
Research Project (R01)
Project #
5R01EB002197-03
Application #
6935374
Study Section
Special Emphasis Panel (ZRG1-SSS-M (57))
Program Officer
Wang, Fei
Project Start
2003-09-30
Project End
2007-07-31
Budget Start
2005-08-01
Budget End
2006-07-31
Support Year
3
Fiscal Year
2005
Total Cost
$644,936
Indirect Cost
Name
Rensselaer Polytechnic Institute
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
002430742
City
Troy
State
NY
Country
United States
Zip Code
12180
Klees, Robert F; Salasznyk, Roman M; Ward, Donald F et al. (2008) Dissection of the osteogenic effects of laminin-332 utilizing specific LG domains: LG3 induces osteogenic differentiation, but not mineralization. Exp Cell Res 314:763-73
Salasznyk, Roman M; Klees, Robert F; Williams, William A et al. (2007) Focal adhesion kinase signaling pathways regulate the osteogenic differentiation of human mesenchymal stem cells. Exp Cell Res 313:22-37
Salasznyk, Roman M; Klees, Robert F; Boskey, Adele et al. (2007) Activation of FAK is necessary for the osteogenic differentiation of human mesenchymal stem cells on laminin-5. J Cell Biochem 100:499-514
Ward Jr, Donald F; Salasznyk, Roman M; Klees, Robert F et al. (2007) Mechanical strain enhances extracellular matrix-induced gene focusing and promotes osteogenic differentiation of human mesenchymal stem cells through an extracellular-related kinase-dependent pathway. Stem Cells Dev 16:467-80
Bennett, Kristin P; Bergeron, Charles; Acar, Evrim et al. (2007) Proteomics reveals multiple routes to the osteogenic phenotype in mesenchymal stem cells. BMC Genomics 8:380
Ward Jr, Donald F; Williams, William A; Schapiro, Nicole E et al. (2007) Focal adhesion kinase signaling controls cyclic tensile strain enhanced collagen I-induced osteogenic differentiation of human mesenchymal stem cells. Mol Cell Biomech 4:177-88
Klees, Robert F; Salasznyk, Roman M; Vandenberg, Scott et al. (2007) Laminin-5 activates extracellular matrix production and osteogenic gene focusing in human mesenchymal stem cells. Matrix Biol 26:106-14
Salasznyk, Roman M; Klees, Robert F; Westcott, Aaron M et al. (2005) Focusing of gene expression as the basis of stem cell differentiation. Stem Cells Dev 14:608-20
Klees, Robert F; Salasznyk, Roman M; Kingsley, Karl et al. (2005) Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway. Mol Biol Cell 16:881-90
Batorsky, Anna; Liao, Jiehong; Lund, Amanda W et al. (2005) Encapsulation of adult human mesenchymal stem cells within collagen-agarose microenvironments. Biotechnol Bioeng 92:492-500

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