Differential requirement for MEK/ERK and SMAD signaling in PAI-1 and CTGF expression in response to microtubule disruption.
Colchicine and nocodazole, both established microtubule disruptors, are useful tools to investigate cytoskeletal-dependent signaling cascades and the associated downstream transcriptional targets. Since cytoskeletal events impact pathophysiologic consequences in the vascular system, the signaling requirements underlying colchicine-stimulated expression of PAI-1 and CTGF, two prominent cell deformation-sensitive fibrosis-initiating proteins, were evaluated in vascular smooth muscle cells. Microtubule disruption rapidly induced EGFR transactivation (at the src kinase-sensitive EGFR(Y845) site) in a ROS-dependent manner. Genetic deficiency of EGFR, inhibition of EGFR signaling with AG1478 or introduction of a kinase-deficient EGFR construct effectively blocked colchicine-stimulated PAI-1 and CTGF expression. MEK/ERK involvement downstream of ROS generation was critical for PAI-1, but not CTGF, expression following cytoskeletal perturbation suggesting bifurcation of signaling pathways downstream of EGFR activation. Colchicine also stimulated SMAD2/3 phosphorylation by a Rho/ROCK-dependent mechanism independent of TGF-beta1 release or receptor activity. Rho/ROCK signaling initiated by tubulin network collapse was required for both CTGF and PAI-1 induction. Colchicine-initiated SMAD3 phosphorylation, however, was essential for PAI-1, but not CTGF, expression further highlighting divergence of signaling events downstream of Rho/ROCK that mediate microtubule deformation-associated changes in profibrotic gene transcription.
Samarakoon
Rohan
R
Center for Cell Biology and Cancer Research, Albany Medical College, 47 New Scotland, Albany, NY 12208, USA.
Higgins
Craig E
CE
Higgins
Stephen P
SP
Higgins
Paul J
PJ
D000818:Animals; D003078:Colchicine / Q000494:pharmacology; D055513:Connective Tissue Growth Factor / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020935:MAP Kinase Signaling System / Q000187:drug effects*; D051379:Mice; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D015739:Nocodazole / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D051785:Smad Proteins / Q000378:metabolism*; D015533:Transcriptional Activation / Q000187:drug effects; D050257:Tubulin Modulators / Q000494:pharmacology*; D054460:rho-Associated Kinases / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D017395:Plasminogen Activator Inhibitor 1; D017382:Reactive Oxygen Species; D051785:Smad Proteins; D050257:Tubulin Modulators; D055513:Connective Tissue Growth Factor; D066246:ErbB Receptors; D016392:Proto-Oncogene Proteins pp60(c-src); D054460:rho-Associated Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D020742:rhoA GTP-Binding Protein; D015739:Nocodazole; D003078:Colchicine