Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.
Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by > or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.
Monslow
James
J
Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio 44195, USA.
Sato
Nobuyuki
N
Mack
Judith A
JA
Maytin
Edward V
EV
D000818:Animals; D002460:Cell Line; D004285:Dogs; D004817:Epidermis / Q000293:injuries* / Q000378:metabolism*; D066246:ErbB Receptors / Q000502:physiology*; D066257:Heparin-binding EGF-like Growth Factor; D006801:Humans; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D036341:Intercellular Signaling Peptides and Proteins / Q000502:physiology*; D051379:Mice; D008810:Mice, Inbred C57BL; D051381:Rats; D015398:Signal Transduction; D014945:Wound Healing; D014947:Wounds and Injuries / Q000378:metabolism
C587883:HBEGF protein, human; C587884:Hbegf protein, mouse; C587885:Hbegf protein, rat; D066257:Heparin-binding EGF-like Growth Factor; D036341:Intercellular Signaling Peptides and Proteins; D006820:Hyaluronic Acid; D066246:ErbB Receptors