Specific inhibition of skeletal alpha-actin gene transcription by applied mechanical forces through integrins and actin

Skeletal alpha-actin (skA), a prominent fetal actin isoform that is reexpre ssed by adult cardiac myocytes after chronic overload in vivo, provides a m odel for studying cytoskeletal gene regulation by mechanical forces in vitr o. We have determined the mechanisms by which perpendicular applied forces acting through integrins and the actin cytoskeleton regulate the expression of skA, Rat-2 fibroblasts were transiently transfected with plasmids conta ining 5'-regulatory regions of the skA gene fused to luciferase coding sequ ences. A constant, perpendicular force (0.2 pN/mu m(2)) was applied by usin g a collagen-magnetic bead model; a 25 % deformation was obtained on the do rsal cell surface. In this system, force is applied through focal adhesion integrins and strongly induces actin assembly [Glogauer, Arora, Yao, Sokhol ov, Ferrier and McCulloch(1997) J. Cell Sci. 110, 11-21]. skA promoter acti vity was inhibited by 68 % in cells subjected to 4 h of applied force, wher eas Rous sarcoma virus promoter activity was unaffected. In cells transient ly transfected with a skA expression vector there was also a parallel 40 % decrease in skA protein levels by force, as shown by Western blotting. In L 8 cells, constitutive skA expression was decreased by more than 50 %. Analy ses of specific motifs in the skA promoter revealed that transcriptional en hancer factor 1 and Yin and Yang 1 sites, but not serum response factor and Spl sites, mediated inhibitory responses to force. In cells treated with c ycloheximide the force-induced inhibition was abrogated, indicating a depen dence on new protein synthesis. Inhibition of actin filament assembly with either cytochalasin D or Ca2+-depleted medium blocked the inhibitory effect induced by the applied force, suggesting that actin filaments are required for the regulation of skA promoter activity. Western blot analysis showed that p38 kinase, but not Jun N-terminal kinase or extracellular signal-regu lated protein kinase 1/2, was activated by force; indeed, the p38 kinase in hibitor SB203580 relieved the force-induced inhibition of skA. We conclude that the force-induced inhibition of skA promoter activity requires an inta ct actin cytoskeleton and can be map, ed to two different response elements . This inhibition might be mediated through the p38 kinase.