Interaction of epidermal growth factor, Ca2+, and matrix metalloproteinase-9 in primary keratinocyte migration

Elevations of epidermal growth factor (EGF) and Ca2+ concentrations in the wound site are associated with reepithelialization during wound healing. In addition, Ca2+ and EGF can both induce increases in matrix metalloproteinase-9 (MMP-9) synthesis. However, little is known about the interplay of these events in regulating the migration properties of primary keratinocytes on collagen I, the most abundant extracellular matrix component in the skin. We found that EGF stimulated both chemokinetic and chemotactic migration of primary keratinocytes on collagen I; however, MMP-9 was required for EGF-stimulated chemotaxis but not EGF-stimulated chemokinesis. Calcium at 0.5 mM stimulated chemokinetic migration of keratinocytes. Together, Ca2+ and EGF stimulated higher levels of chemokinesis than either stimulus alone. Furthermore, Ca2+ could restore the ability of keratinocytes from MMP-9 null mice to undergo EGF-stimulated chemotaxis. The phosphatidylinositol-3 kinase inhibitor LY294002 inhibited both EGF- and Ca2+-stimulated chemokinetic migration. In contrast, the MEK inhibitor PD98059 blocked Ca2+- but not EGF-stimulated chemokinetic migration of keratinocytes. A combination of PD98059 and LY294002 was required to inhibit Ca2+ enhancement of EGF-stimulated migration completely. Calcium-stimulated chemokinesis was completely blocked by either the protein kinase C-alpha inhibitor Go6976 or the src/fyn inhibitor PP2. Using primary keratinocytes, our results showed how the combined action of Ca2+, EGF, and MMP-9 regulated the contributions of extracellular-regulated kinase and phosphatidylinositol-3 kinase toward chemokinetic and chemotactic migration of keratinocytes.