Smooth muscle actin determines mechanical force-induced p38 activation.
The mitogen-activated protein kinase p38 is activated by mechanical force, but the cellular elements that mediate force-induced p38 phosphorylation are not defined. As alpha-smooth muscle actin (SMA) is an actin isoform associated with force generation in fibroblasts, we asked if SMA participates in the activation of p38 by force. Tensile forces (0.65 pn/mum(2)) generated by magnetic fields were applied to collagen-coated magnetite beads bound to Rat-2 cells. Immunoblotting showed that p38alpha was the predominant p38 isoform. Analysis of bead-associated proteins demonstrated that SMA enrichment of collagen receptor complexes required the alpha2beta1 integrin. SMA was present almost entirely as filaments. Swinholide depolymerized SMA filaments and blocked force-induced p38 phosphorylation and force-induced increases of SMA. Knockdown of SMA (70% reduction) using RNA interference did not affect beta-actin but inhibited force-induced p38 phosphorylation by 50%. Inhibition of Rho kinase blocked SMA filament assembly, force-induced increases of SMA, and force-induced p38 activation. Force application increased SMA content and 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 SMA negative cells, there was no force-induced p38 phosphorylation. We conclude that force-induced p38 phosphorylation is dependent on an SMA filament-dependent pathway that uses a feed-forward amplification loop to synergize force-induced SMA expression with p38 activation.
Wang
Jiaxu
J
Canadian Institutes of Health, Group in Matrix Dynamics, Faculty of Dentistry, University of Toronto, Ontario, Canada.
Fan
Jennie
J
Laschinger
Carol
C
Arora
Pamela D
PD
Kapus
Andras
A
Seth
Arun
A
McCulloch
Christopher A
CA
D000199:Actins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000818:Animals; D001696:Biomechanical Phenomena; D002460:Cell Line; D038982:Integrin alpha2beta1 / Q000378:metabolism; D009130:Muscle, Smooth / Q000737:chemistry* / Q000166:cytology; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D051381:Rats; D038981:Receptors, Collagen; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism
D000199:Actins; D038982:Integrin alpha2beta1; D020033:Protein Isoforms; D038981:Receptors, Collagen; D048051:p38 Mitogen-Activated Protein Kinases; D020741:rho GTP-Binding Proteins; C498862:Acta2 protein, rat