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

Open AccessPublished:January 01, 2001DOI:https://doi.org/10.1074/jbc.M007601200
      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 vitroin 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 ∼6 copies/cell in cultures before change of fresh medium, up to ∼54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of ∼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.
      Has
      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
      3,3′-diaminobenzidine
      Hyaluronan is a large glycosaminoglycan found in the extracellular space of most animal tissues. It forms a loose, highly hydrated, gel-like matrix that contributes to the maintenance of the extracellular space and facilitates nutrient diffusion. Furthermore, hyaluronan is involved in cell proliferation and differentiation, produces an environment favorable for migration (
      • Toole B.P.
      ), and stimulates cell locomotion (
      • Turley E.A.
      ,
      • Hall C.L.
      • Lange L.A.
      • Prober D.A.
      • Zhang S.
      • Turley E.A.
      ). Elevated tissue levels of hyaluronan occur during embryonic growth of tissues and organs (
      • Toole B.P.
      ), wound healing (
      • Chen W.Y.
      • Abatangelo G.
      ,
      • Oksala O.
      • Salo T.
      • Tammi R.
      • Häkkinen L.
      • Jalkanen M.
      • Inki P.
      • Larjava H.
      ), inflammation (
      • Lesley J.
      • Hyman R.
      • English N.
      • Catterall J.B.
      • Turner G.A.
      ), and invasion of certain cancers (
      • Knudson W.
      ,
      • Anttila M.A.
      • Tammi R.H.
      • Tammi M.I.
      • Syrjänen K.J.
      • Saarikoski S.V.
      • Kosma V.M.
      ,
      • Auvinen P.
      • Tammi R.
      • Parkkinen J.
      • Tammi M.
      • Ågren U.
      • Johansson R.
      • Hirvikoski P.
      • Eskelinen M.
      • Kosma V.M.
      ,
      • Ropponen K.
      • Tammi M.
      • Parkkinen J.
      • Eskelinen M.
      • Tammi R.
      • Lipponen P.
      • Ågren U.
      • Alhava E.
      • Kosma V.M.
      ).
      In skin epidermis, the narrow extracellular space surrounding keratinocytes contains a high concentration of hyaluronan (
      • Ågren U.M.
      • Tammi M.
      • Tammi R.
      ,
      • Tammi R.
      • Ripellino J.A.
      • Margolis R.U.
      • Maibach H.I.
      • Tammi M.
      ), as do other stratifying squamous epithelia (
      • Tammi R.
      • Tammi M.
      • Häkkinen L.
      • Larjava H.
      ,
      • Wang C.
      • Tammi M.
      • Guo H.
      • Tammi R.
      ). The half-life of labeled epidermal hyaluronan in human skin organ culture is ∼1 day (
      • Tammi R.
      • Säämänen A.M.
      • Maibach H.I.
      • Tammi M.
      ), indicating fast local turnover by keratinocytes. 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 (
      • Tammi R.
      • Ågren U.
      • Tuhkanen A.-L.
      • Tammi M.
      ).
      Unlike other glycosaminoglycans, hyaluronan is synthesized at the inner surface of the plasma membrane by hyaluronan synthase (Has)1 and is extruded through the plasma membrane into the extracellular space simultaneously with the ongoing synthesis (for review, see Ref.
      • Weigel P.H.
      • Hascall V.C.
      • Tammi M.
      ). Currently, three different Has genes have been identified in mammalian cells:Has1 (
      • Shyjan A.M.
      • Heldin P.
      • Butcher E.C.
      • Yoshino T.
      • Briskin M.J.
      ,
      • Itano N.
      • Kimata K.
      ), Has2 (
      • Fülöp C.
      • Salustri A.
      • Hascall V.C.
      ,
      • Spicer A.P.
      • Augustine M.L.
      • McDonald J.A.
      ,
      • Watanabe K.
      • Yamaguchi Y.
      ), and Has3(
      • Spicer A.P.
      • Olson J.S.
      • McDonald J.A.
      ). The three Has genes are highly homologous but appear to differ from each other in kinetic properties and product size (
      • Brinck J.
      • Heldin P.
      ,
      • Itano N.
      • Sawai T.
      • Yoshida M.
      • Lenas P.
      • Yamada Y.
      • Imagawa M.
      • Shinomura T.
      • Hamaguchi M.
      • Yoshida Y.
      • Ohnuki Y.
      • Miyauchi S.
      • Spicer A.P.
      • McDonald J.A.
      • Kimata K.
      ). Limited data are available on the factors that regulate the expression level and enzymatic activity of the different Has enzymes in various cells and tissues (
      • Fülöp C.
      • Salustri A.
      • Hascall V.C.
      ,
      • Zhang W.
      • Watson C.E.
      • Liu C.
      • Williams K.J.
      • Werth V.P.
      ,
      • Kaback L.A.
      • Smith T.J.
      ,
      • Feusi E.
      • Sun L.
      • Sibalic A.
      • Beck-Schimmer B.
      • Oertli B.
      • Wuthrich R.P.
      ,
      • Nishida Y.
      • Knudson C.B.
      • Eger W.
      • Kuettner K.E.
      • Knudson W.
      ), but a number of studies have suggested that the overall synthesis rate of hyaluronan is stimulated by some growth factors and cytokines (
      • Ellis I.
      • Banyard J.
      • Schor S.L.
      ,
      • Carley W.W.
      • Szczepanski A.
      • Gerritsen M.E.
      ,
      • Hamerman D.
      • Wood D.D.
      ,
      • Haubeck H.D.
      • Kock R.
      • Fischer D.C.
      • Van de Leur E.
      • Hoffmeister K.
      • Greiling H.
      ,
      • Heldin P.
      • Laurent T.C.
      • Heldin C.H.
      ,
      • Kawakami M.
      • Suzuki K.
      • Matsuki Y.
      • Ishizuka T.
      • Hidaka T.
      • Konishi T.
      • Matsumoto M.
      • Kataharada K.
      • Nakamura H.
      ,
      • Sampson P.M.
      • Rochester C.L.
      • Freundlich B.
      • Elias J.A.
      ,
      • Tiedemann K.
      • Malmström A.
      • Westergren-Thorsson G.
      ,
      • Tirone E.
      • D'Alessandris C.
      • Hascall V.C.
      • Siracusa G.
      • Salustri A.
      ).
      Epidermal growth factor (EGF) is one of the most powerful agents that influences the behavior of keratinocytes. EGF transmits its information through the EGF receptor (EGF-R), which belongs to the erbB receptor tyrosine kinase family (
      • Tzahar E.
      • Yarden Y.
      ,
      • Carraway K.L. r.
      • Cantley L.C.
      ). Signaling through EGF-R regulates many cellular processes, including cell adhesion, expression of matrix-degrading proteinases, and cell locomotion; these phenomena are all important in skin wound healing. Keratinocyte EGF-R expression is transiently elevated 5–7-fold within 2 days after wounding and returns nearly to baseline within 4 days (
      • Stoscheck C.M.
      • Nanney L.B.
      • King Jr., L.E.
      ). It is also important for keratinocyte proliferation and migration during reepithelization (for review, see Ref.
      • Hudson L.G.
      • McCawley L.J.
      ). Exogenously added EGF and overexpression of EGF-R result in enhanced ligand-mediated migration of keratinocytes and faster reepithelialization (
      • McCawley L.J.
      • O'Brien P.
      • Hudson L.G.
      ).
      Aberrant expression or activation of the EGF-R is common and has been proposed to have a role in epithelial tumor progression (
      • Kolettas E.
      • Lymboura M.
      • Khazaie K.
      • Luqmani Y.
      ,
      • Lichtner R.B.
      • Kaufmann A.M.
      • Kittmann A.
      • Rohde-Schulz B.
      • Walter J.
      • Williams L.
      • Ullrich A.
      • Schirrmacher V.
      • Khazaie K.
      ). As in wound healing, the EGF-R may provide an important contribution to the migratory and invasive potential of carcinomas. The migration induced by EGF requires the actin binding domain of EGF-R (
      • van der Heyden M.A.
      • Van Bergen en Henegouwen P.M.
      • de Ruiter N.
      • Verdaasdonk M.A.
      • van den Tweel J.G.
      • Rijksen G.
      • Boonstra J.
      • Joling P.
      ), 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 (
      • Martin P.
      ,
      • McCawley L.J.
      • O'Brien P.
      • Hudson L.G.
      ).
      Whereas both increased hyaluronan and EGF signaling have been observed in migrating cells and in wound healing, neither activation ofHas nor production of hyaluronan in response to EGF has been investigated in keratinocytes. In this study, we establish that EGF specifically increases the mRNA level of Has2, resulting in enhanced synthesis of hyaluronan that correlates with enhanced keratinocyte migration in a wounding assay. Interestingly, 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.

      Acknowledgments

      We thank Alpo Pelttari for the facilities of the Dept. of Electron Microscopy. Expert technical help by Arja Venäläinen, Riikka Tiihonen, and Päivi Perttula is gratefully acknowledged.

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