Mechanical forces represent fundamental regulators of cell growth and pattern formation as well as key contributing factors to many prevalent diseases, including hypertension, atherosclerosis, and osteoporosis. Many of the changes in cell function and tissue development that are induced mechanically result from specific stress-dependent changes in gene expression. While stress-sensitive regulatory elements have been identified in the promoters of certain genes, little is known about how application of a mechanical stress to the cell's surface membrane leads to transcriptional activation. Controlled mechanical stresses were applied directly to surface receptors on living cells by magnetically twisting bound ligand-coated microbeads. Briefly, 4.5 um ferromagnetic microbeads coated with either integrin or non-integrin binding ligands and were allowed to bind capillary endothelial cells in 1% BSA/DMEM for 15 min, and the unbound beads were washed away using 1% BSA/DMEM. Using, a magnetic twisting device, controlled forces were applied. Magnetic twisting cytometry involves the application of a brief (lOusec) but strong (1000 gauss) magnetic field that magnetizes the beads in the horizontal direction, followed by the application of a longer (1-15 min) and weaker (30 gauss) vertical magnetic field 90 degrees to the original orientation: the result is that the bead twists in place. With removal of the vertical magnetic field, the bead """"""""untwists"""""""" toward its original direction Twisting integrin receptors resulted in a stress-dependent rise in intracellular cyclic AMP (cAMP), enh anced translocation of the catalytic subunit of protein kinase A (PKA) into the nucleus, and a concomitant increase in phosphorylation of the transcriptional regulator, CREB. Cells expressing gene reporter constructs driven by the cAMP response element (CRE) also exhibited stress-dependent induction of gene transcription. In contrast, mechanical force application through a metabolic transmembrane receptor failed to activate these cAMP-dependent signaling events. Thus, integrins appear to regulate mechanochemical transduction by providing a preferred pathway for transmembrane transfer of mechanical forces as well as a mechanism to translate these signals into changes in intracellular biochemistry and gene expression.
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