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 μM). 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 μM). 8‐(N,N‐diethylamino) octyl‐3,4,5‐trimethoxy benzoate (TMB‐8; 1.0 μM), mepacrine (220 μM), n‐α‐p‐tosyl‐L‐lysine chloromethylketone (TLCK; 100 μM), phenylmethoxysulphonylfluoride (PMSF; 500 μM), and e‐aminocaproic acid (e‐ACA; 100 μM); however, amiloride (100 μM), A23187 (20 μM), and chloroquine (1 mM) were unable to inhibit this effect. Melittin disruption of vinculin was inhibited by (in order of decreasing effectiveness) mepacrine > TMB‐8 > TFP > leupeptin > 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.