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The Hyaluronan Synthesis Inhibitor 4-Methylumbelliferone Prevents Keratinocyte Activation and Epidermal Hyperproliferation Induced by Epidermal Growth Factor

      Since excessive epidermal hyaluronan is associated with hyperproliferative states and disturbed terminal differentiation of the keratinocytes, we hypothesized that 4-methylumbelliferone (4-MU), an inhibitor of hyaluronan synthesis, could counteract these phenotypic features. Cultured epidermal keratinocytes showed a concentration dependent, maximum 83% reduction of hyaluronan in the presence of 0.2–1.0 mM 4-MU, whereas less decline was seen in the synthesis of chondroitin and heparan sulfate. The reduced hyaluronan was associated with no apparent change in its molecular mass. The 4-MU-treated keratinocytes showed an accentuated epithelial morphology with a flat, round cell shape, increased stress fibers and large vinculin-positive adhesion plaques, cytoskeletal changes consistent with the markedly reduced migration rate observed in scratched monolayer cultures. High concentrations of 4-MU also caused a block in keratinocyte proliferation, reversible upon 4-MU withdrawal. In the epidermis of organotypic cultures, 4-MU prevented the hyaluronan accumulation and epidermal hypertrophy induced by epidermal growth factor. The present results concur with earlier data indicating that enhanced cell locomotion and proliferation are associated with hyaluronan synthesis in activated keratinocytes. Cell proliferation, however, was blocked more strongly than expected on the basis of the incomplete hyaluronan synthesis inhibition, and may represent a novel target of 4-MU. At any rate, 4-MU and equivalent hyaluronan synthesis inhibitors might be considered for situations where suppression of epidermal activation and hyperproliferation is warranted.

      Keywords

      Abbreviations

      bHABC
      biotinylated hyaluronan binding complex
      EGF
      epidermal growth factor
      HABC
      hyaluronan binding complex
      KGF
      Keratinocyte growth factor
      4-MU
      4-methylumbelliferone
      REK
      rat epidermal keratinocyte
      TGF-β
      transforming growth factor beta
      Hyaluronan is a unique glycosaminoglycan of very high molecular mass (up to 107 Da), synthesized directly into the extracellular space by an enzyme (hyaluronan synthase) associated with the cytoplasmic side of the plasma membrane (
      • Weigel P.H.
      • Hascall V.C.
      • Tammi M.
      Hyaluronan synthases.
      ;
      • Tammi M.I.
      • Day A.J.
      • Turley E.A.
      Hyaluronan and homeostasis: A Balancing act.
      ). Hyaluronan expression is associated with tissue remodeling during morphogenesis (
      • Camenisch T.D.
      • Spicer A.P.
      • Brehm-Gibson T.
      • et al.
      Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronan-mediated transformation of epithelium to mesenchyme.
      ;
      • Toole B.P.
      Hyaluronan in morphogenesis.
      ), wound healing (
      • Mack J.A.
      • Abramson S.R.
      • Ben Y.
      • et al.
      Hoxb13 knockout adult skin exhibits high levels of hyaluronan and enhanced wound healing.
      ), and cancer (
      • Toole B.P.
      Hyaluronan in morphogenesis.
      ), where it is supposed to facilitate cell division and migration by creating a soft, transient matrix and by activating intracellular signaling cascades (
      • Turley E.A.
      • Noble P.W.
      • Bourguignon L.Y.
      Signaling properties of hyaluronan receptors.
      ). A direct role of hyaluronan in cell proliferation was supported by the finding that transfection of Has2 gene to fibrosarcoma cells lead to accelerated cell proliferation and cell migration (
      • Kosaki R.
      • Watanabe K.
      • Yamaguchi Y.
      Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity.
      ).
      Hyaluronan is actively synthesized by keratinocytes and forms a loose extracellular matrix between adjacent basal and spinous cells of human epidermis (
      • Tuhkanen A.L.
      • Tammi M.
      • Pelttari A.
      • Ågren U.M.
      • Tammi R.
      Ultrastructural analysis of human epidermal CD44 reveals preferential distribution on plasma membrane domains facing the hyaluronan-rich matrix pouches.
      ). The location of hyaluronan between the vital cell layers suggests that as a space filler it contributes to the maintenance of the free interstitial space between keratinocytes, in order to facilitate exchange of metabolites between circulation and keratinocytes, and to maintain the plasticity of the epidermis by allowing keratinocyte movements and changes of cell shape during differentiation (
      • Tuhkanen A.L.
      • Tammi M.
      • Pelttari A.
      • Ågren U.M.
      • Tammi R.
      Ultrastructural analysis of human epidermal CD44 reveals preferential distribution on plasma membrane domains facing the hyaluronan-rich matrix pouches.
      ). Furthermore, hyaluronan synthesis creates signals that control migration and proliferation of epidermal keratinocytes (
      • Rilla K.
      • Lammi M.J.
      • Sironen R.
      • et al.
      Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.
      ). A hyperproliferative and migrating phenotype is typical of epidermal activation. Activation occurs after epidermal injury and is common in many diseases involving epidermis, psoriasis as an example (
      • Nickoloff B.J.
      • Turka L.A.
      Keratinocytes: Key immunocytes of the integument.
      ;
      • Tomic-Canic M.
      • Komine M.
      • Freedberg I.M.
      • Blumenberg M.
      Epidermal signal transduction and transcription factor activation in activated keratinocytes.
      ). Accordingly, the proliferation rate and thickness of epidermis, regulated by growth factors like EGF (
      • Pienimäki J.P.
      • Rilla K.
      • Fülöp C.
      • et al.
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ;
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ), KGF (
      • Karvinen S.
      • Pasonen-Seppanen S.
      • Hyttinen J.M.
      • et al.
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ), and TGFβ (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ), is closely correlated with the synthesis of hyaluronan by keratinocytes. The tight association of keratinocyte hyaluronan synthesis to epidermal activation implies that inhibition of hyaluronan synthesis might suppress these phenotypic features, and modify epidermal differentiation.
      The coumarine derivative 4-methylumbelliferone (4-MU) has been reported to specifically inhibit hyaluronan synthesis in cultured mammalian cells (
      • Nakamura T.
      • Takagaki K.
      • Shibata S.
      • Tanaka K.
      • Higuchi T.
      • Endo M.
      Hyaluronic-acid-deficient extracellular matrix induced by addition of 4-methylumbelliferone to the medium of cultured human skin fibroblasts.
      ,
      • Nakamura T.
      • Funahashi M.
      • Takagaki K.
      • Munakata H.
      • Tanaka K.
      • Saito Y.
      • Endo M.
      Effect of 4-methylumbelliferone on cell-free synthesis of hyaluronic acid.
      ;
      • Kosaki R.
      • Watanabe K.
      • Yamaguchi Y.
      Overproduction of hyaluronan by expression of the hyaluronan synthase Has2 enhances anchorage-independent growth and tumorigenicity.
      ;
      • Sohara Y.
      • Ishiguro N.
      • Machida K.
      • et al.
      Hyaluronan activates cell motility of v-Src-transformed cells via Ras-mitogen-activated protein kinase and phosphoinositide 3-kinase-Akt in a tumor-specific manner.
      ). Although the same compound has been in clinical use as a spasmolytic drug (
      • Stacchino C.
      • Spano R.
      • Pettiti A.
      Spasmolytic activity of some 4-methylumbelliferone derivatives.
      ), as far as we know, its effects on skin or skin diseases has not been studied. In this work, we show that the inhibition of hyaluronan synthesis by 4-MU in epidermal keratinocytes was associated with low cell proliferation, and cytoskeletal changes consistent with markedly reduced migratory activity. Interestingly, the suppression by 4-MU was more potent in a hyperproliferative epidermis created by epidermal growth factor, suggesting that it might be useful in normalizing such conditions. The data stress the importance of hyaluronan synthesis in epidermal activation.

      Results

      4-MU inhibits hyaluronan synthesis and its EGF-induced stimulation in keratinocyte cultures

      In the standard culture medium with 5% fetal bovine serum (FBS), slightly preconfluent rat epidermal keratinocyte (REK) cultures secreted approximately 4 ng of hyaluronan per 10,000 cells in 24 h, as measured by the ELSA assay. A concentration-dependent decrease in the amount of secreted hyaluronan was found with 0.2–1.0 mM concentrations of 4-MU (Figure 1a,c). With 0.2 mM 4-MU the relative inhibition was ∼20%, whereas at 1 mM concentration the relative inhibition was ∼35% and ∼60% after treatments of 6 and 24 h, respectively (Figure 1a,c).
      Figure thumbnail gr1
      Figure 1Effect of 4-MU on hyaluronan secreted in the growth medium of epidermal keratinocytes. Subconfluent REK cultures were subjected to the indicated concentrations of 4-MU for 6 h (A, B) and 24 h (C, D), with (B, D) and without 20 ng per mL of EGF (A, C), and the media were analyzed for hyaluronan concentration using the ELSA assays. The data represent means and ranges of two independent experiments. The molecular mass distribution of metabolically labeled hyaluronan secreted into the culture medium during 6 h incubations with different concentrations of 4-MU was analyzed with gel filtration on a 1 × 30 cm column of Sephacryl S-1000 (E).
      A concentration-dependent decrease in the total amount of hyaluronan synthesized in the culture (medium and cell layer combined) was also found using quantitative metabolic labeling (Table I). Furthermore, it appeared that the synthesis of hyaluronan was more sensitive to 4-MU than the synthesis of sulfated glycosaminoglycans (Table I). Thus, 0.2 mM 4-MU had little effect on chondroitin sulfate (CS) or heparan sulfate synthesis. At 0.5 mM and 1 mM of 4-MU the synthesis of heparan sulfate was also decreased, but less than that of hyaluronan, whereas no constant effect was seen in CS (Table I).
      Table IREK cultures were metabolically labeled with 3H-glucosamine and 35S-sulfate for 6 h, and the glycosaminoglycans synthesized were analyzed using Superdex Peptide chromatography
      Glycosaminoglycan (ng per 10,000 cells)
      4-MU concentration (mM)HACSHS
      01.08±0.040.38±0.215.82±2.27
      0.20.46±0.090.54±0.375.13±2.15
      0.50.29±0.030.49±0.333.59±0.83
      10.19±0.010.46±0.341.87±0.21
      The data represent the total amounts of glycosaminoglycans synthesized in the cultures (cells+medium), expressed as means and ranges from two independent experiments.
      REK, rat epidermal keratinocyte; 4-MU, 4-methylumbelliferone; HA, hyaluronan; CS, chondroitin sulfate; HS, heparan sulfate.
      Hyaluronan synthesis is strongly induced in keratinocytes by effectors like EGF (
      • Pienimäki J.P.
      • Rilla K.
      • Fülöp C.
      • et al.
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ;
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ) and KGF (
      • Karvinen S.
      • Pasonen-Seppanen S.
      • Hyttinen J.M.
      • et al.
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ). As compared to its effects on non-stimulated cells (Figure 1a,c), 4-MU caused a relatively more prominent, 65–85% inhibition in cells treated with EGF (Figure 1b,d), i.e., 4-MU was particularly effective in inhibiting the hyaluronan synthesis in activated keratinocytes.
      A 6 h treatment with 4-MU did not change the molecular mass distribution of the secreted hyaluronan (Figure 1e) or that associated with the cell layer (data not shown), when analyzed with gel filtration.

      Hyaluronan localization in REK is changed by 4-MU

      In control cultures, hyaluronan visualized with the biotinylated hyaluronan binding complex (bHABC) probe was typically localized as dense patches (Figure 2a), mostly on the dorsal cell surfaces, and frequently colocalized with CD44 (Figure 2c) (
      • Tammi R.
      • MacCallum D.
      • Hascall V.C.
      • Pienimäki J.-P.
      • Hyttinen M.
      • Tammi M.
      Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides.
      ), whereas the ventral surface showed a lower hyaluronan signal, except close to the edge of the lamellipodia (Figure 2e). Hyaluronan in 4-MU-treated cells had a generally lower staining intensity, and it was more diffusely distributed (Figure 2b). Moreover, the remaining hyaluronan in the 4-MU-treated REK was mostly on the underside of the cells (Figure 2d), often surrounded by a circle of adhesion plaques (Figure 2f).
      Figure thumbnail gr2
      Figure 2Distribution of cell-associated hyaluronan, CD44, actin, and vinculin in 4-MU-treated REK cells. Preconfluent cultures incubated in 0.5 mM of 4-MU for 24 h and stained with bHABC (B, D, F) were compared with controls (A, C, E) to illustrate the changed localization of hyaluronan. The vertical sections of single cells obtained by deconvolution of a stack of horizontal confocal sections (C, D) show the localization of CD44 (red) and hyaluronan (green) on the apical plasma membrane in controls (C), and the shift of hyaluronan to the ventral side in 4-MU-treated cells (D). The plate bottom is shown by the white dashed line. The arrows point the predominant localization of hyaluronan in the cells. Panels (E, F) demonstrate the increased quantity and changed distribution of vinculin-positive adhesion plaques (red) in the undersurface of cells cultured with 4-MU. Hyaluronan (green) resides under the central part of the 4-MU treated cells, surrounded by a belt of adhesion plaques. Panel (H) demonstrates the loss of phalloidin -positive filopodia, present in control cells (G, arrow) and reorganization of actin into stress fibers in the 4-MU-treated cells (H). Scale bars=20 μm (AD) and 10 μm (EH).

      Cytoskeletal rearrangements in 4-MU-treated REK

      In subconfluent REK cells, the lamellipodia usually present in control cultures were largely lost by 4-MU-treatment, and the cells became flat and circular (Figure 2b,d,f,h). These changes in cell shape started already after 1 h in 1 mM 4-MU when monitored by phase contrast microscopy of live cells (data not shown). In control cells, phalloidin staining visualized filamentous actin in the cell cortex and in numerous filopodia (Figure 2g), whereas the filopodia were severely depleted in 4-MU-treated REK. Instead, stress fibers, very rare in control cultures, appeared after a 6 h treatment with 4-MU, often forming a spindle-like structure in the middle of the cell (Figure 2h). The cytoskeletal changes induced by 4-MU included an increased number and size of adhesion plaques under the cells, as demonstrated by confocal microscopy of an immunohistochemical staining for vinculin (Figure 2e,f). The circular arrangement of the vinculin-positive adhesion plaques (Figure 2h) appeared to fix the cell periphery to the substratum and was correlated with a truncation of lamellipodia and filopodia. In addition, microscopic assays showed that all EGF-induced features in the REK phenotype (
      • Pienimäki J.P.
      • Rilla K.
      • Fülöp C.
      • et al.
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ), including accumulation of cell surface hyaluronan, and the lifted, elongated cell shape, were efficiently blocked by 4-MU (data not shown).

      4-MU inhibits REK cell migration

      Removing cells by scratching with a pipette tip from the bottom of the dish induces rapid migration of the REK, completely covering the cleared area in less than 2 d. In the presence of 0.1 mM 4-MU, a 25% reduction in the migration was found in a 24 h follow up, whereas 0.5 mM 4-MU produced 60% inhibition (Figure 3a). The inhibition was obvious already after 3 h (Figure 3b), excluding the possible contribution of cell proliferation in filling the cleared area.
      Figure thumbnail gr3
      Figure 3The effect of 4-MU on proliferation and scratch-induced migration. Cells in REK cultures were scratched off from standardized areas with a pipette tip, the cleared area recorded by videomicroscopy, the cultures incubated in the presence of 0–0.5 mM of 4-MU, and recorded again to calculate the average migration rate of the cell front. Panel (A) shows the migration rate as a function of 4-MU concentration during a 24-h period (means±SE of five experiments with eight wounds in each, *p< 0.05 and **p<0.01 as compared with control, paired t test). Panel (B) shows the early time points of migration in the presence of 0.2 mM 4-MU (means and ranges of two independent cultures). For the proliferation assay, equal numbers of REK cells were plated on day 0 with the standard medium containing 0–1 mM of 4-MU. Cell numbers were counted on the days indicated, and also 4 h after plating to determine the plating efficiency (C). In panel (D) one set of cultures was grown completely without 4-MU (control), another was grown in 1 mM 4-MU for 1 d (days 1–2), then changed back to control medium, and a third set was cultured in 1 mM 4-MU through the experiment (days 1–4). The dashed line indicates the number of cells plated. The error bars represent ranges from two identical but separate experiments.
      High molecular weight hyaluronan added to the culture medium caused ∼10% increase of the migration rate in both control and 4-MU-treated cultures (data not shown), indicating that 4-MU does not block the relatively small migration stimulation shown previously to occur in REK by exogenous, soluble hyaluronan (
      • Rilla K.
      • Lammi M.J.
      • Sironen R.
      • et al.
      Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.
      ).

      4-MU inhibits cell proliferation

      Control REK doubled their numbers approximately every 20 h until confluency slowed down the proliferation. Treatment with 0.2 mM 4-MU reduced the number of cells by 50% on the third day in culture, whereas with 0.5 mM dose, cell proliferation was completely arrested (Figure 3c). The cells were also counted 4 h after plating, resulting in equal numbers for control and 4-MU-treated cultures, which indicates that 4-MU does not disturb cell attachment to substratum. The number of dead, floating cells (on average 2.4% of those bound) was also similar to that in control cultures (data not shown). Addition of high molecular weight hyaluronan (100 μg per mL) did not change the proliferation rate of 4-MU-treated cells (data not shown).
      We also tested the reversibility of the proliferation block by removing 4-MU from cultures after a 24 h treatment. The effect of 4-MU on cell proliferation was fully reversible (Figure 3d) the cell number being duplicated 24 h after 4-MU removal.

      EGF-induced hyaluronan accumulation and epidermal thickening are inhibited by 4-MU in organotypic epidermal cultures

      The REK cells stratify and differentiate into structurally normal epidermis when cultured on a collagen gel in the air–liquid interphase (
      • Tammi R.H.
      • Tammi M.I.
      • Hascall V.C.
      • Hogg M.
      • Pasonen S.
      • MacCallum D.K.
      A preformed basal lamina alters the metabolism and distribution of hyaluronan in epidermal keratinocyte organotypic cultures grown on collagen matrices.
      ;
      • Pasonen-Seppänen S.
      • Suhonen T.M.
      • Kirjavainen M.
      • et al.
      Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier.
      ), allowing prediction of pharmacological impacts on epidermis in vivo. In the organotypic cultures, the effect of 4-MU on the hyaluronan deposited in the control tissue (Figure 4a) or that secreted into culture medium (Figure 4b) was relatively small. Importantly, 4-MU did not affect the normal structure or differentiation pattern of control cultures (Figure 4a).
      Figure thumbnail gr4
      Figure 4Effect of 4-MU on hyaluronan concentration and tissue morphology of normal and EGF-treated organotypic keratinocyte cultures. EGF (20 ng per mL) was present in the indicated 2.5-wk-old organotypic REK cultures during the whole period, and 4-MU (0.6 mM) was added at 1.5 wk. Two and half-wk-old cultures were fixed, embedded in paraffin, sectioned, and double-stained for CD44 (red) and hyaluronan (green). The yellow signal indicates colocalization of CD44 and hyaluronan. Vertical brackets indicate the stratum corneum. Scale bar=10 μm (A). Hyaluronan secretion into growth medium during a 16-h incubation at the end of the culture period was analyzed with the ELSA assay. The error bars represent the range of two independent experiments (B). The thickness of the vital part of the epidermis was analyzed by morphometry. The data represent means±SE of three separate experiments (C).
      As shown previously (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ), EGF causes a dramatic increase in epidermal hyaluronan, increases proliferation, and interferes with the normal tissue structure. 4-MU blocked the EGF-induced hyaluronan synthesis and accumulation in the epidermis (Figure 4a,b). Furthermore, 4-MU normalized the epidermal thickness and tissue architecture disturbed by EGF (Figure 4a,c).

      Discussion

      This study shows that 4-MU causes a marked inhibition of hyaluronan production in epidermal keratinocytes and that the inhibition is particularly effective in the epidermis activated by EGF. The decline in hyaluronan synthesis is associated with a strong inhibition of cell proliferation, to the extent of a complete but reversible block of growth in keratinocyte monolayer cultures. Considering the potential of clinical applications, it is interesting that in the organotypic epidermis, the hyperproliferation and hypertrophy caused by EGF was normalized by 4-MU.
      These results fit into an emerging pattern that keratinocyte hyaluronan synthesis is tightly connected with keratinocyte proliferation and epidermal thickness. Thus, epidermal hyaluronan synthesis is specifically upregulated by retinoic acid (
      • Tammi R.
      • Ripellino J.A.
      • Margolis R.U.
      • Maibach H.I.
      • Tammi M.
      Hyaluronate accumulation in human epidermis treated with retinoic acid in skin organ culture.
      ), EGF (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ), and KGF (
      • Karvinen S.
      • Pasonen-Seppanen S.
      • Hyttinen J.M.
      • et al.
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ), factors known to stimulate keratinocyte proliferation and increase the number of vital cell layers in the epidermis. Furthermore, the effect of 4-MU on keratinocytes resembles the biological activities of TGFβ (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ), and hydrocortisone (
      • Ågren U.M.
      • Tammi M.
      • Tammi R.
      Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture.
      ), both of which suppress hyaluronan synthesis, keratinocyte proliferation, and epidermal thickening. It can be concluded that 4-MU is a novel chemical effector that confirms the association between keratinocyte hyaluronan metabolism and epidermal proliferation. The inhibition levels, however, of hyaluronan synthesis and proliferation rate are not correlated in absolute units. Furthermore, since the mechanism of action of 4-MU is unknown, it is still possible that the two inhibitions are independent of each other.
      The inhibition of hyaluronan synthesis by 4-MU is also associated with changes in the phenotype of single keratinocytes observed in monolayer cultures. A striking effect was inhibition of cell migration. As expected, there were distinct cytoskeletal alterations consistent with this functional transition, including the increase of stress fibers, the reduction of dynamic lamellipodia and filopodia, and the appearance of large, vinculin positive focal adhesions. Vinculin is known to be downregulated in migrating keratinocytes; during wound healing in human epidermis, vinculin is absent at the leading edge (
      • Kubler M.D.
      • Watt F.M.
      Changes in the distribution of action-associated proteins during epidermal wound healing.
      ). Similar cytoskeletal and morphological alterations are displayed by keratinocytes in which hyaluronan synthesis is inhibited by transfection of a Has2 antisense gene (
      • Rilla K.
      • Lammi M.J.
      • Sironen R.
      • et al.
      Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.
      ). Furthermore, the Has2 antisense inhibition also reduces keratinocyte migration (
      • Rilla K.
      • Lammi M.J.
      • Sironen R.
      • et al.
      Changed lamellipodial extension, adhesion plaques and migration in epidermal keratinocytes containing constitutively expressed sense and antisense hyaluronan synthase 2 (Has2) genes.
      ).
      4-MU thus suppresses migration and proliferation, two phenotypic features typical of epidermal activation, a response common in injury, irritation, inflammation, transformation, and many diseases involving epidermis, psoriasis as an example. In general, the present findings: (1) strengthen conclusions on the phenotypic features that follow changes in hyaluronan production, (2) indicate that 4-MU reproduces many of the effects of specific antisense inhibition of Has2 expression, (3) suggest that 4-MU is a readily available means to suppress hyaluronan synthesis, useful in exploring the cellular functions associated with hyaluronan, and (4) offers a potentially useful drug for the treatment of excessive epidermal activation.

      Materials and Methods

      Cell culture

      A newborn REK cell line (
      • Baden H.P.
      • Kubilus J.
      The growth and differentiation of cultured newborn rat keratinocytes.
      ) was cultured in minimum essential medium, (MEM, Life Technologies, Paisley, Scotland, UK) supplemented with 5% fetal bovine serum (FBS, HyClone, Logan, Utah), 4 mM glutamine (Sigma, St Louis, Missouri) and 50 μg per mL streptomycin sulfate and 50 U per mL penicillin (Sigma). Keratinocytes were passaged twice a week at a 1:10 split ratio using 0.05% trypsin (w/v), and 0.02% EDTA (w/v) in phosphate-buffered saline (PBS, Reagena, Kuopio, Finland).
      4-MU (sodium salt, Sigma) at 10 or 100 mM stock solution in Hank's balanced salt solution (HBSS, Euroclone, Milano, Italy) was added into the culture medium to reach the concentrations indicated.

      Organotypic cultures

      For organotypic cultures–cells were cultured on type I collagen support, and lifted to the air-liquid interface just after having reached confluence as described previously (
      • Pasonen-Seppänen S.
      • Suhonen T.M.
      • Kirjavainen M.
      • et al.
      Vitamin C enhances differentiation of a continuous keratinocyte cell line (REK) into epidermis with normal stratum corneum ultrastructure and functional permeability barrier.
      ). EGF (20 ng per mL) was present in culture medium for the whole culture period (2.5 wk), and 4-MU (0.6 mM) was added after 1.5 weeks and kept until the end of the experiment (i.e., for 1 wk). The secretion of hyaluronan was assayed from a medium kept for 16 h with the cultures at the end of the whole period. A part of the cultures was fixed in Histochoice® (Amresco, Solon, Ohio), and embedded in paraffin. Hematoxylin/eosin-stained sections were morphometrically analysed to measure epidermal thickness as described previously (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ). Antigen retrieval using TUF (Monosan, Uden, the Netherlands) was done before the double-staining for hyaluronan and CD44 as described (
      • Pasonen-Seppänen S.
      • Karvinen S.
      • Törrönen K.
      • et al.
      EGF upregulates, whereas TGF-beta downregulates, the hyaluronan synthases Has2 and Has3 in organotypic keratinocyte cultures: Correlations with epidermal proliferation and differentiation.
      ).

      Assay of hyaluronan

      A total of 80,000 cells were seeded on 24-well plates and cultured for 24 h. Next day, a fresh medium (5% FBS) with different concentrations of 4-MU and sometimes EGF (20 ng per mL, Sigma) was added for 6 to 24 h before counting the cells and harvesting the media for ELISA of hyaluronan. The sandwich type assay was performed as described previously (
      • Hiltunen E.L.
      • Anttila M.
      • Kultti A.
      • et al.
      Elevated hyaluronan concentration without hyaluronidase activation in malignant epithelial ovarian tumors.
      ). Briefly, maxisorp 96-well plates (Nunc, Roskilde, Denmark) were precoated with (non-biotinylated) HABC (hyaluronan binding region of aggrecan and link protein), to catch hyaluronan from the samples and standards (range 0–50 ng per mL). After washes, biotinylated HABC was added to detect the bound hyaluronan using horseradish peroxidase streptavidin and TMB substrate solution (0.5% 3,3′,5,5′-tetramethylbenzidine in dimethyl sulfoxide (Sigma) diluted 1:50 with 0.1 M sodium acetate, 1.5 mM citric acid and 0.005% H2O2) for spectrophotometric quantitation.
      The HABC preparations contained the hyaluronan binding regions of aggrecan and link protein purified from a trypsin digest of bovine cartilage using sequential hydroxyl apatite chromatography and Sephacryl S-1000 gel filtration (
      • Tammi R.
      • Ågren U.M.
      • Tuhkanen A.-L.
      • Tammi M.
      Hyaluronan metabolism in skin.
      ). Part of the material was biotinylated. Both HABC and bHABC were chromatographed on Sephacryl S400 gel filtration under dissociative conditions to remove hyaluronan.

      Metabolic labeling and analysis of glycosaminoglycan synthesis

      REK cultures were incubated for 6 h in the presence of different concentrations of 4-MU and 20 μCi per mL of [3H]-glucosamine and 100 μCi of [35S]-sulfate (Amersham, Little Chalfont, UK). The culture media, cell trypsinization solution and cells were collected, and analyzed for labeled glycosaminoglycans with Superdex gel filtration of specific disaccharides as described previously (
      • Tammi R.
      • MacCallum D.
      • Hascall V.C.
      • Pienimäki J.-P.
      • Hyttinen M.
      • Tammi M.
      Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides.
      ).

      Molecular mass distribution of hyaluronan

      To analyze the size of secreted hyaluronan, REK cultures were incubated for 6 h in the presence of different concentrations of 4-MU and 20 μCi per mL of [3H]-glucosamine (Amersham). The culture media were collected, precipitated with ethanol and dissolved in 150 mM sodium acetate buffer, pH 6.8 containing 0.1% CHAPS. The samples were chromatographed on Sephacryl S-1000 (Pharmacia, Sweden) as described previously (
      • Karvinen S.
      • Pasonen-Seppanen S.
      • Hyttinen J.M.
      • et al.
      Keratinocyte growth factor stimulates migration and hyaluronan synthesis in the epidermis by activation of keratinocyte hyaluronan synthases 2 and 3.
      ). Two aliquots were taken from each chromatography fraction, one incubated at 60°C for 2 h with 0.5 turbidity reducing units of Streptomyces hyaluronidase (Seikagaku Kogyo, Tokyo, Japan), and the other in buffer only. 2% cetyl pyridinium chloride was added 1:1 to all samples, and the precipitates recovered with centrifugation at 13,000 ×g for 15 min. The content of hyaluronan in each fraction was calculated by subtracting the radiolabel in the Streptomyces hyaluronidase-treated samples (resistant to hyaluronidase) from those incubated in buffer only.

      Proliferation assay

      REK cells were seeded in 24-well culture plates at 60,000 cells per well. Different concentrations of 4-MU were added immediately after plating to the culture media. Cell numbers were counted with a hemocytometer after 4 h to determine plating efficiency, and after 2–6 d to determine the proliferation rate. Cells from duplicate wells were trypsinized at each time point. The numbers of detached cells in media were also counted.

      Migration analysis

      The REK cells were seeded at 1 × 105 cells per well in 24-well plates and grown approximately 24 h until confluence. A cell-free area was introduced by scraping the monolayer in each well crosswise with a sterile 300 μL pipette tip. The detached cells were washed off with HBSS, and fresh medium with different concentrations of 4-MU was added. The areas covered by the cells were measured immediately, and 3, 6, and 24 h later by phase contrast microscopy with a videocamera and quantitated by NIH Image software (http://rsb.info.nih.gov/nih-image/). The mean distance traveled by the cell front was calculated by converting the pixel values into micrometers (
      • Pienimäki J.P.
      • Rilla K.
      • Fülöp C.
      • et al.
      Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.
      ).

      bHABC-staining

      Keratinocytes were seeded at 20,000 cells per well on 8-well chamber slides (Nalge Nunc, Naperville, Illinois) precoated with FBS, and grown at 37°C for 48 h. The slides were washed, fixed with 2% paraformaldehyde (vol/vol), washed, permeabilized with 0.3% Triton X-100 in 3% BSA, and probed with 5 μg per mL of bHABC in 1% BSA overnight at 4°C. The slides were washed and incubated with avidin–biotin peroxidase (ABC Standard Kit, Vector Laboratories) for 1 h, and the color was developed with 0.05% 3,3′-diaminobenzine (DAB) and 0.03% H202. The nuclei were stained with hematoxylin, and the preparations were mounted in Supermount (BioGenex, San Ramon, California) and photographed using a Microphot-FXA microscope (Nikon, Tokyo, Japan). The specificity of the staining for hyaluronan was controlled by removing hyaluronan with Streptomyces hyaluronidase (Seikagaku, Kogyo, Tokyo, Japan), and by pretreating the probe with hyaluronan oligosaccharides (
      • Tammi R.
      • MacCallum D.
      • Hascall V.C.
      • Pienimäki J.-P.
      • Hyttinen M.
      • Tammi M.
      Hyaluronan bound to CD44 on keratinocytes is displaced by hyaluronan decasaccharides and not hexasaccharides.
      ).

      Immunofluorescence stainings

      For double staining of hyaluronan and vinculin or CD44, keratinocytes were cultured for 24 h on chamber slides, fixed, permeabilized and blocked as described above, and incubated overnight at 4°C with the anti-vinculin mAB (7.1 μg per mL, clone hvin-1, Sigma) or anti-CD44 monoclonal antibody (OX50, 1:1000, Biosource, Camarillo, California) and bHABC (5 μg per mL) in 1% BSA. After washing, the cells were incubated for 1 h with Texas Red-labeled anti-mouse secondary antibody (Vector, 1:50) and fluorescein isothiocyanate-labeled avidin (Vector, 1:500). For the visualization of the actin filaments, the cells were washed, fixed, permeabilized and blocked as described above and incubated with Bodipy FL Phalloidin (4 U per mL, Molecular Probes, Eugene, Oregon) for 20 min. After washing, the slides were mounted in Vectashield (Vector). The micrographs were obtained with an Ultraview confocal scanner (Perkin-Elmer Life Sciences, Wallac-LSR, Oxford, UK), on a Nikon Eclipse TE300 microscope with a 100 × NA 1.3 oil immersion objective (Nikon). For the three-dimensional imaging, a series of horizontal optical sections were captured through the cell at every 450 nm. The images were deconvoluted using Microtome Deconvolution 7.0 software (Vaytek, Fairfield, Iowa) and rendered with Voxblast software (Vaytek).

      ACKNOWLEDGMENTS

      Special thanks are due to Eija Kettunen, Kari Kotikumpu, Eija Rahunen and Riikka Tiihonen for technical assistance. This work was supported by the Academy of Finland, grant #40807 and #54062 (M.T.), and by grants from The North Savo Cancer Foundation (Pohjois-Savon Syöpärahasto) (K.R.), Paavo Koistinen Foundation (K.R.), Emil Aaltonen Foundation (K.R.) and Finnish Cancer Foundation (R.T.).

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