The calreticulin-binding sequence of thrombospondin 1 regulates collagen expression and organization during tissue remodeling.
Amino acids 17-35 of the thrombospondin1 (TSP1) N-terminal domain (NTD) bind cell surface calreticulin to signal focal adhesion disassembly, cell migration, and anoikis resistance in vitro. However, the in vivo relevance of this signaling pathway has not been previously determined. We engineered local in vivo expression of the TSP1 calreticulin-binding sequence to determine the role of TSP1 in tissue remodeling. Surgical sponges impregnated with a plasmid encoding the secreted calreticulin-binding sequence [NTD (1-35)-EGFP] or a control sequence [mod NTD (1-35)-EGFP] tagged with enhanced green fluorescent protein were implanted subcutaneously in mice. Sponges expressing NTD (1-35)-EFGP formed a highly organized capsule despite no differences in cellular composition, suggesting stimulation of collagen deposition by the calreticulin-binding sequence of TSP1. TSP1, recombinant NTD, or a peptide of the TSP1 calreticulin-binding sequence (hep I) increased both collagen expression and matrix deposition by fibroblasts in vitro. TSP1 stimulation of collagen was inhibited by a peptide that blocks TSP1 binding to calreticulin, demonstrating the requirement for cell surface calreticulin. Collagen stimulation was independent of TGF-β activity and Smad phosphorylation but was blocked by an Akt inhibitor, suggesting that signaling through the Akt pathway is important for regulation of collagen through TSP1 binding to calreticulin. These studies identify a novel function for the NTD of TSP1 as a mediator of collagen expression and deposition during tissue remodeling.
Sweetwyne
Mariya T
MT
Departments of Cell Biology, University of Alabama, Birmingham, Birmingham, Alabama 35294-0019, USA.
Pallero
Manuel A
MA
Lu
Ailing
A
Van Duyn Graham
Lauren
L
Murphy-Ullrich
Joanne E
JE
D000818:Animals; D001011:Aorta / Q000166:cytology / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D037282:Calreticulin / Q000378:metabolism*; D002417:Cattle; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism*; D004622:Embryo, Mammalian / Q000166:cytology* / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051899:Smad2 Protein / Q000235:genetics / Q000378:metabolism; D019700:Thrombospondin 1 / Q000235:genetics / Q000378:metabolism*; D054457:Tissue Scaffolds; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
D037282:Calreticulin; D010446:Peptide Fragments; D012333:RNA, Messenger; D051899:Smad2 Protein; D019700:Thrombospondin 1; D016212:Transforming Growth Factor beta; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D003094:Collagen; D051057:Proto-Oncogene Proteins c-akt