SPARC fusion protein induces cellular adhesive signaling

PLoS One. 2013;8(1):e53202. doi: 10.1371/journal.pone.0053202. Epub 2013 Jan 21.

Abstract

Secreted protein, acidic and rich in cysteine (SPARC) has been described as a counteradhesive matricellular protein with a diversity of biological functions associated with morphogenesis, remodeling, cellular migration, and proliferation. We have produced mouse SPARC with a FLAG-tag at the N-terminus of SPARC (Flag-SPARC, FSP) in a Bac-to-Bac baculoviral expression system. After affinity purification, this procedure yields SPARC of high purity, with an electrophoretic mobility of ∼44 kDa under reducing conditions, and ∼38-39 kDa under non-reducing conditions. Unexpectedly, FSP adsorbed to plastic supported cell attachment and spreading, in a calcium-dependent manner. The adhesive activity of native FSP was inhibited by prior incubation with anti-SPARC IgG. Cell adhesion to FSP induced the formation of filopodia and lamellipodia but not focal adhesions that were prominent on cells that were attached to fibronectin. In addition, FSP induced the tyrosine phosphorylation of FAK and paxillin in attached epithelial cells. Erk1/2 and Rac were also activated in cells attached to FSP, but at a lower level in comparison to cells on fibronectin. This study provides new insight into the biological functions of SPARC, a matricellular protein with important roles in cell-extracellualr matrix interactions.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Calcium / pharmacology
  • Cattle
  • Cell Adhesion / drug effects
  • Cell Line
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects
  • Enzyme Activation / drug effects
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • Extracellular Matrix / drug effects
  • Extracellular Matrix / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Focal Adhesion Kinase 1 / metabolism
  • Focal Adhesions / drug effects
  • Glycoproteins / genetics
  • Glycoproteins / pharmacology*
  • Mice
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Mitogen-Activated Protein Kinase 3 / metabolism
  • Osteonectin
  • Paxillin / metabolism
  • Pseudopodia / drug effects
  • Pseudopodia / metabolism
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / pharmacology*
  • Signal Transduction / drug effects*
  • Tumor Suppressor Proteins / genetics
  • Tumor Suppressor Proteins / pharmacology*
  • rac1 GTP-Binding Protein / metabolism

Substances

  • Glycoproteins
  • Osteonectin
  • Paxillin
  • Recombinant Fusion Proteins
  • SPARC protein, mouse
  • Tumor Suppressor Proteins
  • Focal Adhesion Kinase 1
  • Ptk2 protein, mouse
  • Mitogen-Activated Protein Kinase 1
  • Mitogen-Activated Protein Kinase 3
  • rac1 GTP-Binding Protein
  • Calcium