Regulation of TNF-alpha-induced reorganization of the actin cytoskeleton and cell-cell junctions by Rho, Rac, and Cdc42 in human endothelial cells

J Cell Physiol. 1998 Jul;176(1):150-65. doi: 10.1002/(SICI)1097-4652(199807)176:1<150::AID-JCP17>3.0.CO;2-B.


We have investigated the role of the small guanosine-trisphosphate (GTP)-binding proteins, Rho, Rac, and Cdc42, in the early responses of human umbilical vein endothelial cells (HUVECs) to TNF-alpha (tumor necrosis factor-alpha). Quiescent confluent HUVECs incubated with TNF-alpha for 5-30 min showed an increased formation of membrane ruffles, filopodia, and actin stress fibres followed by cell retraction and formation of intercellular gaps. This process was accompanied by the dispersion of cadherin-5 from intercellular junctions. TNF-alpha also induced a transient increase in polymerized F-actin, as determined both by measuring G-actin content and by quantifying fluorescent emission from fluorescein isothiocyanate (FITC)-phalloidin-labelled F-actin. Microinjection of cells with activated RhoA protein led to an increase in polymerized actin, formation of stress fibres, cell retraction as well as dispersion of cadherin-5. The proteins Cdc42 and Rac induced qualitatively similar effects to Rho, although not as dramatic and in addition induced formation of filopodia and lamellipodia. Microinjection of cells with a Rho inhibitor, C3 transferase, prevented gap formation caused by TNF-alpha. Similar effects were observed in cells microinjected with the dominant inhibitory proteins N17Cdc42 and N17Rac1. Cell retraction and gap formation were also prevented by inhibitors of myosin light chain kinase (MLCK). Our data suggest that Cdc42, Rac, and Rho are activated in a hierarchical cascade following stimulation with TNF-alpha leading to actomyosin-mediated cell retraction and formation of intercellular gaps.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ADP Ribose Transferases / pharmacology
  • Actins / metabolism*
  • Antigens, CD
  • Botulinum Toxins*
  • Cadherins / metabolism
  • Cell Cycle Proteins / physiology
  • Cells, Cultured
  • Cytoskeleton / drug effects*
  • Cytoskeleton / ultrastructure
  • Endothelium, Vascular / drug effects*
  • Enzyme Inhibitors / pharmacology
  • Fluorescent Antibody Technique
  • GTP-Binding Proteins / physiology*
  • Humans
  • Microinjections
  • Muscle, Smooth, Vascular / drug effects
  • Myosin-Light-Chain Kinase / antagonists & inhibitors
  • Recombinant Proteins / metabolism
  • Signal Transduction / physiology
  • Tumor Necrosis Factor-alpha / pharmacology*
  • cdc42 GTP-Binding Protein
  • rac GTP-Binding Proteins
  • rhoA GTP-Binding Protein


  • Actins
  • Antigens, CD
  • Cadherins
  • Cell Cycle Proteins
  • Enzyme Inhibitors
  • Recombinant Proteins
  • Tumor Necrosis Factor-alpha
  • cadherin 5
  • ADP Ribose Transferases
  • exoenzyme C3, Clostridium botulinum
  • Myosin-Light-Chain Kinase
  • Botulinum Toxins
  • GTP-Binding Proteins
  • cdc42 GTP-Binding Protein
  • rac GTP-Binding Proteins
  • rhoA GTP-Binding Protein