Regulation of [alpha]-smooth muscle actin by mechanical force.
Published: 2005
Language: eng
Number of pages: 149
Description: Mechanotransduction is a process by which cells transduce physical force-induced signals into biochemical responses. Currently, there are at least four general models of mechanotransduction that are believed to operate in mammalian cells, all of which invoke cytoskeletal proteins. Actin is one of the major cytoskeletal proteins in mammalian cells and may be an important mediator of mechanical signal transduction. We hypothesized that alpha-smooth muscle actin (SMA), an actin isoform strongly associated with cell-generated mechanical tension, is a critical element in mechanical signaling networks. Initially we studied how mechanical forces regulate SMA expression. We used an in vitro model system that applies static tensile forces (0.65pN/mum2) to integrins via collagen-coated magnetite beads. We examined force-effects on SMA expression when the basal levels of SMA were either high or low. The results indicated that tensile force-induced regulation of SMA protein content is dependent on baseline levels of SMA and by the selective activation of different MAP kinase pathways. Second, we examined mechanotranscriptional regulation of SMA. Cells were transiently transfected with SMA promoter constructs containing the full-length SMA promoter or deletion mutants. SMA promoter activity was increased by &sim;60% after 4 h force. Deletion analyses showed that SMA promoter activity was increased &sim;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. EMSA analyses of nuclear extracts showed strong binding to the CArG-B motif after force that co-migrated with a serum response factor probe. Finally, we asked if SMA is a structural element in the mechanotransduction circuit that activates the p38 MAP kinase by force. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. The actin depolymerizing agent swinholide A or knockdown of SMA by RNA interference, strongly inhibited force-induced p38 phosphorylation. Inhibition of Rho kinase blocked SMA filament assembly and force-induced p38 activation. Force application enhanced the association of phosphorylated p38 with SMA filaments. Blockade of p38 phosphorylation by SB203586 abrogated force-induced increases of SMA. In cells transfected with SMA promoter-beta-galactosidase fusion constructs, co-transfection with constitutively active p38 or MKK6 increased SMA promoter activity by 2.5--3 fold. Dominant negative p38 blocked force-induced activation of the SMA promoter.In summary, the induction of SMA by mechanical forces is dependent on the basal level of SMA, activation of p38 and serum response factor binding to the CArG-B box of the SMA promoter. We conclude that SMA is both an agent of contractile force generation and a critical element in the tensile force mechanotransduction circuit.
Notes: Thesis (Ph.D.)--University of Toronto, 2005.

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Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 3034.