title: Transcriptional regulation of a contractile gene by mechanical forces applied through integrins in osteoblasts.
abstract: We examined mechanotranscriptional regulation of the contractile gene, alpha-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 beta-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 beta-galactosidase or green fluorescent protein coding sequences, SMA promoter activity was increased by approximately 60% after 4 h of force, whereas control (Rous sarcoma virus) promoter activity was unaffected. Transfections with beta-galactosidase or green fluorescent protein reporter constructs showed that force-loaded cells exhibited higher beta-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 approximately 70% 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.
authors.affiliation: Canadian Institutes for Health Research Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Toronto, Ontario M5S 3E8, Canada.
authors.forename: Jiaxu
authors.initials: J
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Su
authors.forename: Jennie
authors.initials: J
authors.lastname: Fan
authors.forename: Arun
authors.initials: A
authors.lastname: Seth
authors.forename: Christopher A
authors.initials: CA
authors.lastname: McCulloch
mesh_terms: D000199:Actins / Q000096:biosynthesis* / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D015152:Blotting, Northern; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002645:Chickens; D015638:Cytochalasin D / Q000378:metabolism; D017353:Gene Deletion; D049452:Green Fluorescent Proteins; D015151:Immunoblotting; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009130:Muscle, Smooth / Q000378:metabolism*; D009154:Mutation; D010006:Osteoblasts / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species; D013314:Stress, Mechanical; D013844:Thiazoles / Q000378:metabolism; D053778:Thiazolidines; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D000199:Actins; D019086:Bridged Bicyclo Compounds, Heterocyclic; D008164:Luminescent Proteins; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D013844:Thiazoles; D053778:Thiazolidines; D049452:Green Fluorescent Proteins; D015638:Cytochalasin D; D001616:beta-Galactosidase; C037068:latrunculin B