Transforming Growth Factor-β–induced Mobilization of Actin Cytoskeleton Requires Signaling by Small GTPases Cdc42 and RhoA

Abstract

Transforming growth factor-β (TGF-β) is a potent regulator of cell growth and differentiation in many cell types. The Smad signaling pathway constitutes a main signal transduction route downstream of TGF-β receptors. We studied TGF-β–induced rearrangements of the actin filament system and found that TGF-β1 treatment of PC-3U human prostate carcinoma cells resulted in a rapid formation of lamellipodia. Interestingly, this response was shown to be independent of the Smad signaling pathway; instead, it required the activity of the Rho GTPases Cdc42 and RhoA, because ectopic expression of dominant negative mutant Cdc42 and RhoA abrogated the response. Long-term stimulation with TGF-β1 resulted in an assembly of stress fibers; this response required both signaling via Cdc42 and RhoA, and Smad proteins. A known downstream effector of Cdc42 is p38MAPK; treatment of the cells with the p38MAPK inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(pyridyl)1H-imidazole (SB203580), as well as ectopic expression of a kinase-inactive p38MAPK, abrogated the TGF-β–induced actin reorganization. Moreover, treatment of cells with the inhibitors of the RhoA target-protein Rho-associated coiled-coil kinase (+)-R-trans-4-(aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632) and 1-5(-isoquinolinesulfonyl)homopiperazine (HA-1077), as well as ectopic expression of kinase-inactive Rho coiled-coil kinase-1, abrogated the TGF-β1–induced formation of stress fibers. Collectively, these data indicate that TGF-β–induced membrane ruffles occur via Rho GTPase-dependent pathways, whereas long-term effects require cooperation between Smad and Rho GTPase signaling pathways.

ALK5 promotes tumor angiogenesis by upregulating matrix metalloproteinase-9 in tumor cells

“…JNK may regulate motility by modulating the dynamics of microtubules (Huang et al, 2004) or by affecting the assembly/ stability of focal adhesion complexes via phosphorylation of paxillin and Spir, a WASP family protein (reviewed by Huang et al (2004)). The role of JNK in TGF-b-mediated cell motility, however, is less defined, given that inhibition of JNK does not affect TGF-bmediated actin remodeling and epithelial to mesenchymal transition (EMT) (Bhowmick et al, 2001a;Bakin et al, 2002Bakin et al, , 2004Edlund et al, 2002).…”

TGF-β-mediated activation of RhoA signalling is required for efficient V12HaRas and V600EBRAF transformation

“…TGF-b may activate RhoA signalling in a biphasic manner involving a rapid activation within minutes of ligand addition followed by a second, potentially Smad-dependent wave of activation after hours of TGF-b stimulation (Bhowmick et al, 2001(Bhowmick et al, , 2003Shen et al, 2001;Edlund et al, 2002;Vardouli et al, 2005;Chen et al, 2006). Rapid TGF-bdriven activation of RhoA has been described in Swiss-3T3 and mouse embryonic fibroblast (MEFs) (Shen et al, 2001;Vardouli et al, 2005), and we first investigated whether this also takes place in NIH3T3 cells.…”

“…TGF-b can activate RhoA signalling in several cell types (Bhowmick et al, 2001(Bhowmick et al, , 2003Shen et al, 2001;Edlund et al, 2002;Vardouli et al, 2005;Chen et al, 2006). Rho GTPases are molecular switches that are inactive when GDP bound and active when GTP bound, where they propagate their signals through interaction with numerous downstream signalling effectors (Jaffe and Hall, 2005).…”

Critical role for complement receptor 3 (CD11b/CD18), but not for Fc receptors, in killing of Streptococcus pyogenes by neutrophils in human immune serum

“…The neutrophils were then lysed as described [27]. A GST fusion protein expressing the Rac/Cdc42-binding domain of PAK1B [43] was added and the samples were incubated on ice for 5 min and then centrifuged (14,000Âg, 10 min, 4 C). The cleared lysates were transferred to new tubes containing glutathione-Sepharose 4B beads (40 ll) and incubated for 60 min at 4 C. Following three washes with ice-cold washing buffer (25 mM Tris-HCl pH 7.5, 30 mM MgCl 2 , 500 mM NaCl, 1% Triton X-100, 1 mM DTT, 2 mM Na 3 VO 4 ), bound proteins were eluted by boiling the beads in 40 ll of sample buffer for 10 min.…”