Cytoprotection against Mechanical Forces Delivered through β1 Integrins Requires Induction of Filamin A

Mario D'Addario,Christopher A.G Mcculloch,Jennie Fan,Pamela D Arora,Bernhard Ganss,Richard P Ellen

doi:10.1074/jbc.m102715200

Abstract

Cells in mechanically active environments can activate cytoprotective mechanisms to maintain membrane integrity in the face of potentially lethal applied forces. Cytoprotection may be mediated by expression of membrane-associated cytoskeletal proteins including filamin A, an actin-binding protein that increases the rigidity of the subcortical actin cytoskeleton. In this study, we tested the hypotheses that applied forces induce the expression of filamin A specifically and that this putative protective response inhibits cell death. Magnetically generated forces were applied to protein-coated magnetite beads bound to human gingival fibroblasts, cells with constitutively low basal levels of filamin A mRNA and protein. Forces applied through collagen or fibronectin, but not bovine serum albumin or poly-l-lysine-coated beads, increased mRNA and protein content of filamin A by 3-7-fold. Forces had no effect on the expression of other filamin isotypes or other cytoskeletal proteins. This effect was dependent on the duration of force and was blocked by anti-beta(1) integrin antibodies. Force also stimulated a 60% increase in expression of luciferase under the control of a filamin A promoter in transiently transfected Rat2 fibroblasts and was dependent on Sp1 transcription factor binding sites located immediately upstream of the transcription start site. Experiments with actinomycin D-treated cells showed that the increased filamin A expression after force application was due in part to prolongation of mRNA half-life. Antisense filamin oligonucleotides blocked force-induced filamin A expression and increased cell death by >2-fold above controls. The force-induced regulation of filamin A was dependent on intact actin filaments. We conclude that cells from mechanically active environments can couple diverse signals from forces applied through beta-integrins to up-regulate the production of cytoprotective cytoskeletal proteins, typified by filamin A.

Highlights

Cells in biomechanically stressful environments can respond to pericellular changes in mechanical force by transducing physical stimuli into chemical signals that subsequently regu
Effect of Force on Filamin A Expression—We examined forceinduced filamin A mRNA expression in human gingival fibroblasts by Northern analysis (Fig. 1A)
Our results demonstrate that filamin A mRNA and protein are induced in human gingival fibroblasts following application of mechanical forces through ␤1 integrins and actin filaments

Summary

Cytoprotection by Filamin A

In view of the dampening of calcium responses conferred by filamin A on cells exposed to high amplitude applied forces [5], we determined if force could directly regulate filamin A gene expression in human gingival fibroblasts. These cells express low basal levels of filamin A but are periodically exposed to high amplitude forces in vivo. Our data show that mechanical forces applied through ␤1 integrins regulate filamin A transcription and that inhibition of filamin A expression by microinjected antisense oligonucleotides increases force-induced cell death These findings have implications for the design and choice of cells in bioengineered tissues that are designed to withstand high amplitude physical loads

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION

TABLE I Potential transcription factor binding sites