SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin-like region and the Ca(2+)-binding EF-hand

J Cell Biochem. 1995 Feb;57(2):341-50. doi: 10.1002/jcb.240570218.

Abstract

SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy-Ullrich et al. (1991): J Cell Biol 115:1127-1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose-dependent loss of focal adhesion-positive cells, that was maximal at approximately 1 microgram/ml (0.03 microM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F-actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin alpha v beta 3 remained clustered in a plaque-like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin-extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine-rich peptide 2.1 (amino acids 54-73) and the Ca(2+)-binding EF-hand-containing peptide 4.2 (amino acids 254-273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion-labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor-mediated event.

Publication types

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

MeSH terms

  • Actins / metabolism
  • Amino Acid Sequence
  • Animals
  • Antibodies
  • Aorta
  • Binding Sites
  • Calcium / metabolism*
  • Cattle
  • Cell Adhesion / drug effects*
  • Cells, Cultured
  • Dose-Response Relationship, Drug
  • Extracellular Matrix Proteins / chemistry*
  • Extracellular Matrix Proteins / metabolism
  • Extracellular Matrix Proteins / pharmacology*
  • Fluorescent Antibody Technique
  • Follistatin
  • Glycoproteins / chemistry*
  • Integrins / metabolism
  • Kinetics
  • Mice
  • Microscopy, Interference
  • Molecular Sequence Data
  • Osteonectin / chemistry
  • Osteonectin / pharmacology*
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Protein Conformation
  • Receptors, Cytoadhesin / metabolism
  • Receptors, Vitronectin
  • Vinculin / metabolism

Substances

  • Actins
  • Antibodies
  • Extracellular Matrix Proteins
  • Follistatin
  • Glycoproteins
  • Integrins
  • Osteonectin
  • Peptide Fragments
  • Receptors, Cytoadhesin
  • Receptors, Vitronectin
  • Vinculin
  • Calcium