Abstract. A number of cytoskeletal-associated proteins that are concentrated in focal contacts, namely ot-actinin, vinculin, talin, and integrin, have been shown to interact in vitro such that they suggest a potential link between actin filaments and the membrane. Because some of these interactions are of low affinity, we suspect that additional linkages also exist. Therefore, we have used a synthetic peptide corresponding to the cytoplasmic domain of/3~ integrin and affinity chromatography to identify additional integrinbinding proteins. Here we report our finding of an interaction between the cytoplasmic domain of/3~ integrin and the actin-binding protein ot-actinin. /3~-integrin cytoplasmic domain peptide columns bound several proteins from Triton extracts of chicken embryo fibroblasts. One protein at ~100 kD was identified by immunoblot analysis as t~-actinin. Solid phase binding assays indicated that ot-actinin bound specifically and directly to the/3~ peptide with relatively high affinity. Using purified heterodimeric chicken smooth muscle integrin (a/3t integrin) or the platelet integrin glycoprotein l/b/RIa complex (a/33 integrin), binding of a-actinin was also observed in similar solid phase assays, albeit with a lower affinity than was seen using the/3~ peptide, ot-Actinin also bound specifically to phospholipid vesicles into which glycoprotein IIb/IIIa had been incorporated. These results lead us to suggest that this integrin-t~-actinin linkage may contribute to the attachment of actin filaments to the membrane in certain locations.

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of β1-integrins and α-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of α-actinin that inhibits α-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the α-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.

Osteoblasts subjected to fluid shear increase the expression of the early response gene, c-fos, and the inducible isoform of cyclooxygenase, COX-2, two proteins linked to the anabolic response of bone to mechanical stimulation, in vivo. These increases in gene expression are dependent on shear-induced actin stress fiber formation. Here, we demonstrate that MC3T3-E1 osteoblast-like cells respond to shear with a rapid increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that we postulate is important to subsequent cellular responses to shear. To test this hypothesis, MC3T3-E1 cells were grown on glass slides coated with fibronectin and subjected to laminar fluid flow (12 dyn/cm(2)). Before application of shear, cells were treated with two Ca(2+) channel inhibitors or various blockers of intracellular Ca(2+) release for 0. 5-1 h. Although gadolinium, a mechanosensitive channel blocker, significantly reduced the [Ca(2+)](i) response, neither gadolinium nor nifedipine, an L-type channel Ca(2+) channel blocker, were able to block shear-induced stress fiber formation and increase in c-fos and COX-2 in MC3T3-E1 cells. However, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM, an intracellular Ca(2+) chelator, or thapsigargin, which empties intracellular Ca(2+) stores, completely inhibited stress fiber formation and c-fos/COX-2 production in sheared osteoblasts. Neomycin or U-73122 inhibition of phospholipase C, which mediates D-myo-inositol 1,4,5-trisphosphate (IP(3))-induced intracellular Ca(2+) release, also completely suppressed actin reorganization and c-fos/COX-2 production. Pretreatment of MC3T3-E1 cells with U-73343, the inactive isoform of U-73122, did not inhibit these shear-induced responses. These results suggest that IP(3)-mediated intracellular Ca(2+) release is required for modulating flow-induced responses in MC3T3-E1 cells.

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The skeleton's response to mechanical force, or load, has significance to space travel, the treatment of osteoporosis, and orthodontic appliances. How bone senses and processes load remains largely unknown. The cellular basis of mechanotransduction, however, likely involves the integration of diffusion-controlled signaling pathways with a solid-state scaffold linking the cell membrane to the genes. Here, we integrate various concepts from models of connective membrane skeleton proteins, cellular tensegrity, and nuclear matrix architectural transcription factors, to describe how a load-induced deformation of bone activates a change in the skeletal genetic program. We propose that mechanical information is relayed from the bone to the gene in part by a succession of deformations, changes in conformations, and translocations. The load-induced deformation of bone is converted into the deformation of the sensor cell membrane. This, in turn, drives conformational changes in membrane proteins of which some are linked to a solid-state signaling scaffold that releases protein complexes capable of carrying mechanical information, "mechanosomes", into the nucleus. These mechanosomes translate this information into changes in the geometry of the 5' regulatory region of target gene DNA altering gene activity; bending bone ultimately bends genes. We identify specific candidate proteins fitting the profile of load-signaling mechanosomes.