Human keratinocytes adhere to two distinct heparin-binding synthetic peptides derived from fibronectin.
Fibronectin is present at the dermal-epidermal junction in normal skin and is increased in skin tissues in inflammatory diseases, skin cancers, and wound repair. The present studies focused on further characterizing the interaction between fibronectin and keratinocytes, specifically addressing whether human keratinocytes utilize multiple adhesion promoting sequences within fibronectin. Initially, direct cell-binding assays were utilized in which keratinocyte adhesion to plastic substrata coated with fibronectin or proteolytic fragments of fibronectin was quantified. Intact fibronectin, a 75-kD proteolytic fragment containing the RGD sequence, and 33/66-kD cell adhesion/heparin binding fragments lacking the RGD sequence derived from the A and B chains of fibronectin, all promoted keratinocyte adhesion in a concentration-dependent manner. To further define putative cell-binding domains within the 33/66-kD fibronectin fragments, we studied three chemically synthesized peptides derived from the amino acid sequence of the 33-kD fragment of the fibronectin A chain: FN-C/H-I (YEKPGSPPREVVPRPRPGV), FN-C/H-II (KNNQKSEPLIGRKKT), and CS1 (DELPQLVTLPHPNLHGPEILDVPST). Substrata coated with either FN-C/H-I or FN-C/H-II promoted keratinocyte adhesion in a concentration-dependent and saturable manner, whereas peptide CS1 promoted no significant keratinocyte adhesion. In solution, both exogenous FN-C/H-I and FN-C/H-II partially inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. Furthermore, antibodies prepared against these peptides also inhibited keratinocyte adhesion to the 33/66-kD fibronectin fragments. These data indicate that keratinocyte adhesion to fibronectin is mediated by multiple distinct amino acid sequences, at least two of which are localized to the carboxy-terminal heparin binding domain of fibronectin.
Wilke
M S
MS
Department of Laboratory Medicine and Pathology, University of Minnesota Medical School, Minneapolis 55455-0315.
Skubitz
A P
AP
Furcht
L T
LT
McCarthy
J B
JB
D000906:Antibodies; D001667:Binding, Competitive; D002448:Cell Adhesion / Q000187:drug effects; D005353:Fibronectins / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D015603:Keratinocytes / Q000166:cytology*; D010446:Peptide Fragments / Q000378:metabolism*; D011485:Protein Binding
D000906:Antibodies; D005353:Fibronectins; D010446:Peptide Fragments; D006493:Heparin