Kristin E. Michael

Dynamic Compression Regulates the Expression and Synthesis of Chondrocyte-Specific Matrix Molecules in Bone Marrow Stromal Cells

The overall objective of the present study was to investigate the mechanotransduction of bovine bone marrow stromal cells (BMSCs) through the interactions between transforming growth factor β1 (TGF‐β1), dexamethasone, and dynamic compressive loading. Overall, the addition of TGF‐β1 increased cell viability, extracellular matrix (ECM) gene expression, matrix synthesis, and sulfated glycosaminoglycan content over basal construct medium. The addition of dexamethasone further enhanced extracellular matrix gene expression and protein synthesis. There was little stimulation of ECM gene expression or matrix synthesis in any medium group by mechanical loading introduced on day 8. In contrast, there was significant stimulation of ECM gene expression and matrix synthesis in chondrogenic media by dynamic loading introduced on day 16. The level of stimulation was also dependent on the medium supplements, with the samples treated with basal medium being the least responsive and the samples treated with TGF‐β1 and dexamethasone being the most responsive at day 16. Both collagen I and collagen II gene expressions were more responsive to dynamic loading than aggrecan gene expression. Dynamic compression upregulated Smad2/3 phosphorylation in samples treated with basal and TGF‐β1 media. These findings suggest that interactions between mechanical stimuli and TGF‐β signaling may be an important mechanotransduction pathway for BMSCs, and they indicate that mechanosensitivity may vary during the process of chondrogenesis.

Focal adhesion kinase modulates cell adhesion strengthening via integrin activation.

Focal adhesion kinase (FAK) is an essential nonreceptor tyrosine kinase regulating cell migration, adhesive signaling, and mechanosensing. Using FAK-null cells expressing FAK under an inducible promoter, we demonstrate that FAK regulates the time-dependent generation of adhesive forces. During the early stages of adhesion, FAK expression in FAK-null cells enhances integrin activation to promote integrin binding and, hence, the adhesion strengthening rate. Importantly, FAK expression regulated integrin activation, and talin was required for the FAK-dependent effects. A role for FAK in integrin activation was confirmed in human fibroblasts with knocked-down FAK expression. The FAK autophosphorylation Y397 site was required for the enhancements in adhesion strengthening and integrin-binding responses. This work demonstrates a novel role for FAK in integrin activation and the time-dependent generation of cell-ECM forces.

Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions.

Actin–myosin contractility modulates focal adhesion assembly, stress fiber formation, and cell migration. We analyzed the contributions of contractility to fibroblast adhesion strengthening using a hydrodynamic adhesion assay and micropatterned substrates to control cell shape and adhesive area. Serum addition resulted in adhesion strengthening to levels 30–40% higher than serum‐free cultures. Inhibition of myosin light chain kinase or Rho‐kinase blocked phosphorylation of myosin light chain to similar extents and eliminated the serum‐induced enhancements in strengthening. Blebbistatin‐induced inhibition of myosin II reduced serum‐induced adhesion strength to similar levels as those obtained by blocking myosin light chain phosphorylation. Reductions in adhesion strengthening by inhibitors of contractility correlated with loss of vinculin and talin from focal adhesions without changes in integrin binding. In vinculin‐null cells, inhibition of contractility did not alter adhesive force, whereas controls displayed a 20% reduction in adhesion strength, indicating that the effects of contractility on adhesive force are vinculin‐dependent. Furthermore, in cells expressing FAK, inhibitors of contractility reduced serum‐induced adhesion strengthening as well as eliminated focal adhesion assembly. In contrast, in the absence of FAK, these inhibitors did not alter adhesion strength or focal adhesion assembly. These results indicate that contractility modulates adhesion strengthening via FAK‐dependent, vinculin‐containing focal adhesion assembly. J. Cell. Physiol. 223:746–756, 2010.

Focal adhesion kinase-dependent regulation of adhesive forces involves vinculin recruitment to focal adhesions

Background information. FAK (focal adhesion kinase), an essential non‐receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK‐dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood.

Cell adhesion strengthening : Measurement and analysis

Cell adhesion to the extracellular matrix is a dynamic process involving numerous focal adhesion components, which act in coordination to strengthen and optimize the mechanical anchorage of cells over time. A method for systematically analyzing the cell adhesion strengthening process and the components involved in this process is described here. The method combines an adhesion strength assay based on applying fluid shearing to a population of cells and quantitative biochemical analyses.

Conformational changes in fibronectin due to adsorption to well-defined surface chemistries

Protein adsorption to surfaces is a complex phenomenon resulting in partial unfolding of the protein thereby changing its structure or conformation. We have shown that changes in the structure of fibronectin (Fn) upon adsorption to surfaces alter the integrin /spl alpha//sub 5//spl beta//sub 1/ binding affinity and modulate subsequent cellular events. This study investigates adsorption-induced conformational changes of a well-characterized recombinant Fn fragment both kinetically and at equilibrium. The work presented here provides a mechanistic understanding of how conformational changes influence integrin binding and subsequent cell function.