Epidermal Growth Factor Activates Hyaluronan Synthase 2 in Epidermal Keratinocytes and Increases Pericellular and Intracellular Hyaluronan

Csaba Fülöp,Mikko J Lammi,Sanna Pasonen,Juha-Pekka Pienimäki,Kirsi Rilla,Reijo K Sironen,Vincent C Hascall,Raija Tammi,Susanna Karvinen,Markku I Tammi

doi:10.1074/jbc.m007601200

Abstract

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.

Highlights

Hyaluronan is a large glycosaminoglycan found in the extracellular space of most animal tissues
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
The importance of the strikingly high concentration and turnover of hyaluronan in the multilayered squamous epithelia is not completely understood, but we have hypothesized that the former is necessary to maintain an extracellular space for the nutritional needs of the more superficial cell layers, whereas the latter allows the dramatic modulation of cell shape that occurs during differentiation and for the high migratory potential of keratinocytes that is activated, e.g. in wound healing [16]

Summary

The abbreviations used are

Hyaluronan synthase;EGF, epidermal growth factor; EGF-R, epidermal growth factor receptor; REK, rat epidermal keratinocyte; RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; bHABC, biotinylated hyaluronan binding complex; DAB, 3,3Ј-diaminobenzidine. The migration induced by EGF requires the actin binding domain of EGF-R [46], and recent studies have shown an important role of matrix metalloproteinases (MMPs) in keratinocyte migration as well as in their ability to invade other tissues [47, 48]. Whereas both increased hyaluronan and EGF signaling have been observed in migrating cells and in wound healing, neither activation of Has nor production of hyaluronan in response to EGF has been investigated in keratinocytes. A large proportion of the newly synthesized hyaluronan of EGF-treated cells resides in intracellular vesicle-like structures, suggesting that a significant proportion of Has2-directed hyaluronan is endocytosed immediately and recycled back into the cell

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION