Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan

Hyaluronan is an abundant and rapidly turned over matrix molecule between t he vital cell layers of the epidermis. In this study, epidermal growth fact or (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate o f synthesis in a rat epidermal keratinocyte cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associ ated hyaluronan was most abundant in elongated cells that were stimulated t o migrate by EGF, as determined in vitro in a wound healing assay. Large fl uctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precu rsor of hyaluronan, correlated with medium glucose concentrations but not w ith EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRN A increased 23-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standa rd. The average level of Has2 mRNA increased from similar to6 copied cell i n cultures before change of fresh medium, up to similar to 54 copies/cell a fter 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of similar to 21 copies/cell at 6 h. The change in the Has2 mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward 24 h , a finding in line with a predominantly Has2-dependent hyaluronan synthesi s and its transcriptional regulation.