Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein—Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells

Tamura, Yasuhiro; Takeuchi, Yasuhiro; Suzawa, Miyuki; Fukumoto, Seiji; Kato, Mitsuyasu; Miyazono, Kohei; Fujita, Toshiro

doi:10.1359/jbmr.2001.16.10.1772

Cited by 104 publications

(93 citation statements)

References 25 publications

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“…In fact, we previously showed that surface chemistry modulates focal adhesion assembly and site-specific focal adhesion kinase (FAK) phosphorylation (14). FAK plays a central role in integrating adhesion and growth factor signals to direct Runx2 transcriptional activity, osteoblast-specific gene expression, and matrix mineralization in this cell line (26,27).…”

Section: Discussionmentioning

confidence: 99%

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Keselowsky

Collard

Garcı́a

2005

Proc. Natl. Acad. Sci. U.S.A.

618

466

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Biomaterial surface chemistry has profound consequences on cellular and host responses, but the underlying molecular mechanisms remain poorly understood. Using self-assembled monolayers as model biomaterial surfaces presenting well defined chemistries, we demonstrate that surface chemistry modulates osteoblastic differentiation and matrix mineralization independently from alterations in cell proliferation. Surfaces were precoated with equal densities of fibronectin (FN), and surface chemistry modulated FN structure to alter integrin adhesion receptor binding. OH-and NH 2-terminated surfaces up-regulated osteoblast-specific gene expression, alkaline phosphatase enzymatic activity, and matrix mineralization compared with surfaces presenting COOH and CH 3 groups. These surface chemistry-dependent differences in cell differentiation were controlled by binding of specific integrins to adsorbed FN. Function-perturbing antibodies against the central cell binding domain of FN completely inhibited matrix mineralization. Furthermore, blocking antibodies against ␤1 integrin inhibited matrix mineralization on the OH and NH 2 surfaces, whereas function-perturbing antibodies specific for ␤3 integrin increased mineralization on the COOH substrate. These results establish surface-dependent differences in integrin binding as a mechanism regulating differential cellular responses to biomaterial surfaces. This mechanism could be exploited to engineer materials that control integrin binding specificity to elicit desired cellular activities to enhance the integration of biomaterials and improve the performance of biotechnological culture supports.cell adhesion ͉ signaling ͉ osteoblast ͉ mineralization B iomaterial surface chemistry modulates in vitro and in vivo cellular responses, including adhesion, survival, cell cycle progression, and expression of differentiated phenotypes (1-8). These cell-material interactions regulate cell and host responses to implanted devices, biological integration of biomaterials and tissue-engineered constructs, and the performance of cell arrays and biotechnological cell culture supports (9-12). For instance, anionic and neutral hydrophilic surfaces increase macrophage͞ monocyte apoptosis and reduce macrophage fusion to modulate inflammatory responses to implanted materials (8). The effects of biomaterial surface properties on cellular responses are generally attributed to material-dependent differences in adsorbed protein species, concentration, and͞or biological activity. Nonetheless, the molecular mechanisms modulating these substrate-dependent, complex higher-order cellular activities remain poorly understood. This lack of a fundamental understanding of cell-material interactions hinders progress toward the development of synthetic materials that elicit desired cellular responses. Using self-assembled monolayers (SAMs) presenting well defined chemistries as model biomaterial surfaces, we previously showed that surface chemistry modulates the structure and activity of adsorbed fibronectin (FN) (13)...

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Section: Discussionmentioning

confidence: 99%

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Keselowsky

Collard

Garcı́a

2005

Proc. Natl. Acad. Sci. U.S.A.

618

466

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“…Integrin signaling could modulate Smad transcriptional activity. For example, when focal adhesion kinase is inhibited by antisense, Smad transcriptional activity is reduced in the presence of BMP (34). Thus a link between integrins and growth factors pathways (Smads) could be influenced by additional ECM signals.…”

Section: Discussionmentioning

confidence: 99%

Extracellular Matrix-mediated Signaling by Dentin Phosphophoryn Involves Activation of the Smad Pathway Independent of Bone Morphogenetic Protein

Jadlowiec

Zhang

et al. 2006

Journal of Biological Chemistry

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Cells have ingenious mechanisms for interpreting complex signals from their external microenvironment. Previously, we have shown that phosphophoryn (PP) regulates the expression of bone/ dentin marker genes via the integrin/MAPK signaling pathway (Jadlowiec, J., Koch, H., Zhang, X., Campbell, P. G., Seyedain, M., and Sfeir, C. (2004) J. Biol. Chem. 279, 53323-53330). We hypothesize that other signaling pathways important for mineralized tissue morphogenesis such as the Smad pathway could be involved in PP signaling. We determined activation of the Smad pathway in human adult mesenchymal stem cells following treatment with recombinant PP (rPP). We observed that PP enhanced phosphorylation of Smad1 within 30 min and Smad1 translocation to the nucleus within 1 h. PP up-regulated the expression of Smad1 target genes, Smad6, Dlx5, and Runx2. The timing of PP activation of Smad1 implies this is a direct effect; however, we also investigated the possible involvement of bone morphogenetic proteins in PP stimulation of the Smad pathway. PP was shown to up-regulate Bmp-2 gene expression 12 h post-treatment with PP, which is much later than initial detection of Smad1 phosphorylation at 30 min. Furthermore, addition of Noggin did not block Smad1 phosphorylation by PP. We propose that PP could signal via the Smad pathway by either directly stimulating the phosphorylation of Smad1 via integrins or other mechanisms. These might include integrin/bone morphogenetic protein receptor interactions or involvement of PP with other growth factors leading to the modulation of intracellular signaling. It is noteworthy that a non-transforming growth factor-␤ family member activates the Smad pathway. The role of PP in regulating the Smad pathway raises very interesting questions regarding the role of PP during bone and tooth development.The ECM relays complex signals from the microenvironment to cells. This directs proliferation, differentiation, and apoptosis for proper tissue development and remodeling. During formation of mineralized tissues such as bone and dentin, the cell utilizes several intracellular signaling pathways representing an interconnected network of proteins with the goal to decipher cues from the ECM. We focused our current work on the role of the non-collagenous proteins of dentin such as phosphophoryn (PP) 2 in cell signaling and differentiation. We have determined the involvement of the MAPK pathway in PP's signaling (1). In the present study, our hypothesis is that the Smad pathway might be involved since many of the genes up-regulated by PP are critical to bone differentiation.The Smad pathway is one such pathway important in signal interpretation from the ECM during osteogenesis/dentinogenesis. Smad proteins are intracellular signaling molecules that are activated by members of the transforming growth factor (TGF-␤) family of growth factors which includes the bone morphogenetic proteins (BMPs). Smads are the initial responders to receptor activation of the TGF-␤ family and have been studied as transcriptional a...

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“…FAK is an intracellular signaling molecule involved in integrin-mediated signal transduction and the osteogenic differentiation pathway [23,24]. We used phosphotyrosine-specific antibodies to examine the activation state of three important tyrosines in FAK: Y397 (autophosphorylation site), Y576 (essential for maximal kinase activity), and Y861 (major Src phosphorylation site) (Fig.…”

Section: Bio-functionalized Implant Coatings To Convey Integrin Bindimentioning

confidence: 99%

The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration

et al. 2008

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Implant osseointegration, defined as bone apposition and functional fixation, is a requisite for clinical success in orthopaedic and dental applications, many of which are restricted by implant loosening. Modification of implants to present bioactive motifs such as the RGD cell-adhesive sequence from fibronectin (FN) represents a promising approach in regenerative medicine. However, these biomimetic strategies have yielded only marginal enhancements in tissue healing in vivo. In this study, clinical-grade titanium implants were grafted with a non-fouling oligo(ethylene glycol)-substituted polymer coating functionalized with controlled densities of ligands of varying specificity for target integrin receptors. Biomaterials presenting the α 5 β 1 -integrin-specific FN fragment FNIII 7-10 enhanced osteoblastic differentiation in bone marrow stromal cells compared to unmodified titanium and RGD-presenting surfaces. Importantly, FNIII 7-10 -functionalized titanium significantly improved functional implant osseointegration compared to RGD-functionalized and unmodified titanium in vivo. This study demonstrates that bioactive coatings that promote integrin binding specificity regulate marrow-derived progenitor osteoblastic differentiation and enhance healing responses and functional integration of biomedical implants. This work identifies an innovative strategy for the rational design of biomaterials for regenerative medicine.

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Focal Adhesion Kinase Activity Is Required for Bone Morphogenetic Protein—Smad1 Signaling and Osteoblastic Differentiation in Murine MC3T3-E1 Cells

Cited by 104 publications

References 25 publications

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Integrin binding specificity regulates biomaterial surface chemistry effects on cell differentiation

Extracellular Matrix-mediated Signaling by Dentin Phosphophoryn Involves Activation of the Smad Pathway Independent of Bone Morphogenetic Protein

The effect of integrin-specific bioactive coatings on tissue healing and implant osseointegration

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