Transcriptional Regulation of a Contractile Gene by Mechanical Forces Applied through Integrins in Osteoblasts

Abstract

We examined mechanotranscriptional regulation of the contractile gene, ␣-smooth muscle actin (SMA), in osteoblastic cells. Tensile forces were applied through collagen-coated magnetite beads to ROS17/2.8 cells. These cells were desmin؊, vimentin؉ and expressed low levels of SMA. After force application (480 piconewton/ cell), SMA protein and mRNA were increased but ␤-actin was unchanged. Beads coated with bovine serum albumin or poly-L-lysine produced no change of SMA. In cells transiently transfected with plasmids containing the SMA promoter fused to ␤-galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by ϳ60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with ␤-galactosidase or green fluorescent protein reporter constructs showed that forceloaded cells exhibited higher ␤-galactosidase activity than cells without force. Cytochalasin D and latrunculin B inhibited force-induced increases of SMA promoter activity. Deletion analyses showed that SMA promoter activity was increased ϳ70% after force with a minimal construct containing 155 bp upstream of the translation start site. The force effect on the SMA promoter was abrogated in cells transfected with CArG-B box mutants. Gel mobility shift analyses of nuclear extracts showed strong binding to the CArG-B motif after force. We conclude that the CArG-B box is a force-responsive element in the SMA promoter.

Force activates smooth muscle α-actin promoter activity through the Rho signaling pathway

“…Rho kinase, actin assembly and MRTF regulate forceinduced SMA promoter activity SMA promoter activity is increased by tensile forces in osteoblastic cells (Wang et al, 2002). In the fibroblasts used here, application of force caused a 3.5-fold increase of SMA promoter activity up to 3 hours (P<0.01), which was completely blocked by transfection of cells with dominantnegative MRTF A, by inhibition of Rho kinase with Y27632 or by actin filament disassembly with latrunculin B (Fig.…”

“…Role of MRTF in regulating SMA expression in response to force Application of exogenous force can increase SMA expression in fibroblasts through activation of the promoter of the gene encoding SMA (Wang et al, 2002). The regulation of SMA expression is dependent on CArG elements in the SMA promoter (Mack and Owens, 1999;Wang et al, 2002).…”

“…The regulation of SMA expression is dependent on CArG elements in the SMA promoter (Mack and Owens, 1999;Wang et al, 2002). The CArG element is a core sequence of the SRE and plays a pivotal role in SMA transcription (Mack and Owens, 1999).…”

“…ROCK-induced phosphorylation of LIM kinase can thus regulate actin dynamics through phosphorylation of cofilin (Arber et al, 1998). The relative concentration of actin filaments and actin monomers can control serum response factor (SRF) activity (Miralles et al, 2003), an important determinant of forceinduced SMA expression (Wang et al, 2002). Myocardinrelated transcription factor (MRTF, also termed MAL, BSAC or MKL) is a broadly expressed, actin monomer-associated SRF co-activator which relocates from the cytoplasm to the nucleus in response to Rho-induced actin assembly (Miralles et al, 2003).…”

Regulation of Tension-induced Mechanotranscriptional Signals by the Microtubule Network in Fibroblasts

“…Cellular responses to tensile forces applied through integrins require an intact actin cytoskeleton (7,8), but the role of the microtubule network in mediating mechanical signaling is currently not defined. Microtubules can modulate contraction and can also affect cell attachment to the extracellular matrix through regulation of turnover at adhesion sites (12)(13)(14)(15).…”