Rho-mediated assembly of stress fibers is differentially regulated in corneal fibroblasts and myofibroblasts.
Corneal keratocytes (stromal cells) are activated to fibroblasts and myofibroblasts during wound healing. Myofibroblast transdifferentiation is accompanied by the expression of alpha-smooth muscle actin (alpha-SMA) and the assembly of a robust stress fiber network and larger focal adhesions (FAs). The regulation of the assembly of stress fibers was evaluated in cultured corneal fibroblast and myofibroblast phenotypes. In both cell types, the inhibition of Rho GTPase activity by microinjecting C3 transferase into the cells resulted in the disassembly of stress fibers and FAs. However, the inhibition of the Rho-associated kinases ROKalpha and ROKbeta with their inhibitor, Y27632, or by overexpression of their mutant kinase-dead forms resulted in only a partial loss of the stress fibers and FAs in myofibroblasts but a total loss in fibroblasts. ROK inhibitor-sensitive and -resistant stress fibers in myofibroblasts contained alpha-SMA, nonmuscle myosin II, tropomyosin, and calponin. The ROK inhibition-resistant stress fibers and FAs were lost upon the overexpression of the dominant-negative form of mDia1 (a mammalian homolog of Drosophila diaphanous protein). These findings indicated that while the assembly of stress fibers in fibroblasts critically involves both ROK and mDia1, in myofibroblasts, the assembly of alpha-SMA-containing stress fibers also occurs independently of ROK and involves Rho/mDia1.
Anderson
Susan
S
Ophthalmology Department, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, USA.
DiCesare
Lisa
L
Tan
Ivan
I
Leung
Thomas
T
SundarRaj
Nirmala
N
D036002:ADP Ribose Transferases; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D022001:Focal Adhesions / Q000187:drug effects / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D009154:Mutation / Q000235:genetics; D024462:Myosin Type II / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D022502:Stress Fibers / Q000378:metabolism* / Q000648:ultrastructure; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D014335:Tropomyosin / Q000378:metabolism; D014945:Wound Healing / Q000502:physiology; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases; D000096985:Calponins
D036002:ADP Ribose Transferases; D000199:Actins; D001905:Botulinum Toxins; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D024462:Myosin Type II; D017346:Protein Serine-Threonine Kinases; D014335:Tropomyosin; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases; D000096985:Calponins; C063007:exoenzyme C3, Clostridium botulinum