Identification of the cellular mechanisms responsible for platelet-derived growth factor induced alterations in cytoplasmic vinculin distribution.
Exposure of quiescent density arrested BALB/c-3T3 cells (clone A31) to platelet-derived growth factor (PDGF; 6-12 ng/ml) results in a rapid, reversible, time- and dose-dependent removal of vinculin from adhesion plaques (Herman and Pledger, 1985). Potential cellular mechanisms involved in PDGF-induced removal of vinculin from adhesion plaques were examined. Removal of vinculin from adhesion plaques following exposure of cells to PDGF was temperature dependent, occurred in many fibroblast cell lines, and could be mimicked by 12-tetradecanoyl phorbol-13-acetate (TPA; 5-125 nM) or melittin (0.35 microM). Unlike the effect of PDGF, TPA- or melittin-induced vinculin disruption was not reversible. The removal of vinculin from adhesion plaques was inhibited by trifluoroperazine (TFP; 2.5 microM). 8-(N,N-diethylamino) octyl-3,4,5-trimethoxy benzoate (TMB-8; 1.0 microM), mepacrine (220 microM), n-alpha-p-tosyl-L-lysine chloromethylketone (TLCK; 100 microM), phenylmethoxysulphonylfluoride (PMSF; 500 microM), and epsilon-aminocaproic acid (epsilon-ACA; 100 microM); however, amiloride (100 microM), A23187 (20 microM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine greater than TMB-8 greater than TFP greater than leupeptin greater than PMSF, whereas A23187 and amiloride had no effect. The return of vinculin to adhesion plaques following PDGF treatment required de novo mRNA transcription and protein synthesis and was associated with PDGF-stimulated synthesis of vinculin. The observation that both PDGF- and melittin-induced removal of vinculin from adhesion plaques is inhibited by mepacrine suggests that phospholipase activation may be an early and important step in PDGF-induced disruption of vinculin from adhesion plaques. In addition, TFP, TMB-8 and protease inhibitor inhibition of both the PDGF and melittin effects on vinculin distribution, coupled with the finding that TPA can mimic the PDGF or melittin response, suggests that Ca2+, calmodulin, protein kinase C, and/or proteolysis may play an important role(s) in the removal of vinculin from adhesion plaques following PDGF addition. The lack of effect of A23187 addition on vinculin distribution suggests that alterations in cellular Ca2+ is necessary but not sufficient for vinculin removal from adhesion plaques.
Herman
B
B
Harrington
M A
MA
Olashaw
N E
NE
Pledger
W J
WJ
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000502:physiology; D002448:Cell Adhesion; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D051379:Mice; D008807:Mice, Inbred BALB C; D009124:Muscle Proteins / Q000378:metabolism*; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology*; D011493:Protein Kinase C / Q000032:analysis; D012964:Sodium / Q000378:metabolism; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014268:Trifluoperazine / Q000494:pharmacology; D016596:Vinculin
D009124:Muscle Proteins; D010982:Platelet-Derived Growth Factor; D016596:Vinculin; D014268:Trifluoperazine; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D000584:Amiloride; D004247:DNA; D012964:Sodium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium