Conventional protein kinase C mediates phorbol-dibutyrate-induced cytoskeletal remodeling in a7r5 smooth muscle cells

Exp Cell Res. 2002 Oct 15;280(1):64-74. doi: 10.1006/excr.2002.5592.


Phorbol dibutyrate (PDBu) induced the formation of podosome-like structures together with partial disassembly of actin stress fibers in A7r5 smooth muscle cells. These podosomes contained alpha-actinin, F-actin, and vinculin and exhibit a tubular, column-like structure arising perpendicularly from the bottom of PDBu-treated cells. The conventional protein kinase C (PKC) antagonist, GO6976, inhibited PDBu-induced cytoskeletal remodeling at 0.1 microM, whereas the novel PKC antagonist, rottlerin, was ineffective at 10 microM. PDBu induced the translocation of the conventional PKC-alpha but not the novel PKC-delta to the sites of podosome formation in A7r5 cells. Although partial disassembly of actin stress fibers was observed in both Y-27632- and PDBu-treated cells, focal adhesions were much reduced in number and size only in Y-27632-treated cells. Furthermore, PDBu restored focal adhesions in Y-27632-treated cells. Live video fluorescence microscopy of alpha-actinin GFP revealed a lag phase of about 20 min prior to the rapid formation and dynamic reorganization of podosomes during PDBu treatment. These findings suggest that conventional PKCs mediate PDBu-induced formation of dynamic podosome-like structures in A7r5 cells, and Rho-kinase is unlikely to be the underlying mechanism. The podosome columns could represent molecular scaffolds where PKC-alpha phosphorylates regulatory proteins necessary for Ca(2+) sensitization in smooth muscle cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetophenones / pharmacology
  • Actinin / metabolism
  • Actins / metabolism
  • Amides / pharmacology
  • Animals
  • Benzopyrans / pharmacology
  • Carbazoles / pharmacology
  • Cells, Cultured
  • Cytoskeleton / drug effects
  • Cytoskeleton / physiology
  • Cytoskeleton / ultrastructure*
  • Enzyme Inhibitors / pharmacology
  • Green Fluorescent Proteins
  • Indoles / pharmacology
  • Isoenzymes / metabolism
  • Luminescent Proteins / metabolism
  • Muscle, Smooth, Vascular / drug effects
  • Muscle, Smooth, Vascular / physiology
  • Muscle, Smooth, Vascular / ultrastructure*
  • Phorbol 12,13-Dibutyrate / pharmacology*
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • Protein Kinase C / physiology*
  • Protein Kinase C-alpha
  • Protein Kinase C-delta
  • Protein Transport / drug effects
  • Pyridines / pharmacology
  • Rats
  • Recombinant Fusion Proteins / metabolism
  • Transfection
  • Vinculin / metabolism


  • Acetophenones
  • Actins
  • Amides
  • Benzopyrans
  • Carbazoles
  • Enzyme Inhibitors
  • Indoles
  • Isoenzymes
  • Luminescent Proteins
  • Pyridines
  • Recombinant Fusion Proteins
  • Actinin
  • Vinculin
  • Go 6976
  • Y 27632
  • Green Fluorescent Proteins
  • Phorbol 12,13-Dibutyrate
  • rottlerin
  • Prkcd protein, rat
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Protein Kinase C-delta