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 the vital cell layers of the epidermis. In this study, epidermal growth factor (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of 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-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has2 mRNA increased 2-3-fold in less than 2 h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has2 mRNA internal standard. The average level of Has2 mRNA increased from approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately 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 synthesis and its transcriptional regulation.
Department of Anatomy, University of Kuopio, FIN-70211 Kuopio, Finland.
J P
JP
Pienimaki
K
K
Rilla
C
C
Fulop
R K
RK
Sironen
S
S
Karvinen
S
S
Pasonen
M J
MJ
Lammi
R
R
Tammi
V C
VC
Hascall
M I
MI
Tammi
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004705:Endocytosis; D004789:Enzyme Activation; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism*; D000076002:Hyaluronan Synthases; D006820:Hyaluronic Acid / Q000378:metabolism*; D015603:Keratinocytes / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D017931:DNA Primers; D004815:Epidermal Growth Factor; D006820:Hyaluronic Acid; D014453:Glucuronosyltransferase; C491097:Has2 protein, rat; D000076002:Hyaluronan Synthases