EGF Upregulates, Whereas TGF-β Downregulates, the Hyaluronan Synthases Has2 and Has3 in Organotypic Keratinocyte Cultures: Correlations with Epidermal Proliferation and Differentiation
2003; Elsevier BV; Volume: 120; Issue: 6 Linguagem: Inglês
10.1046/j.1523-1747.2003.12249.x
ISSN1523-1747
AutoresSanna Pasonen‐Seppänen, Susanna Karvinen, Kari Törrönen, Juha M. T. Hyttinen, Tiina Jokela, Mikko J. Lammi, Markku Tammi, Raija Tammi,
Tópico(s)Skin and Cellular Biology Research
ResumoHyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor β, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2′-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor β reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor β suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor β through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation. Hyaluronan, a major extracellular matrix molecule in the vital cell layers of skin epidermis, has been suggested to support proliferation and migration of keratinocytes, during challenges like wounding and inflammation. An organotypic keratinocyte culture originated from continuous rat epidermal keratinocyte cell line was subjected to the proliferative and antiproliferative growth factors epidermal growth factor and transforming growth factor β, respectively, to study their influence on hyaluronan synthesis and epidermal morphology. Epidermal growth factor induced a 4-fold increase of epidermal hyaluronan concentration. This was associated with upregulation of the hyaluronan synthases Has2 and Has3, and the hyaluronan receptor CD44. 5-Bromo-2′-deoxyuridine labeling, basal cell height, and the thickness of vital epidermis were increased, reflecting the hyperplastic effects of epidermal growth factor. The expression of keratin 10 and the maturation of filaggrin were inhibited, and epidermal permeability barrier became less efficient, indicating compromised terminal differentiation by epidermal growth factor. In contrast, transforming growth factor β reduced the content of hyaluronan and the mRNA of Has2 and Has3. At the same time, transforming growth factor β suppressed keratinocyte proliferation and epidermal thickness, but retained intact differentiation. The results suggest that epidermal hyaluronan synthesis, controlled by epidermal growth factor and transforming growth factor β through changes in the expression of Has2 and Has3, correlates with epidermal proliferation, thickness, and differentiation. biotinylated hyaluronan binding complex The structure and function of epidermis depend on a controlled balance between keratinocyte proliferation and differentiation, disturbed in pathologic situations like wounding (Stoscheck et al., 1992Stoscheck C.M. Nanney L.B. King Jr., L.E. Quantitative determination of EGF-R during epidermal wound healing.J Invest Dermatol. 1992; 99: 645-649Crossref PubMed Scopus (73) Google Scholar) and psoriasis (King et al., 1990King Jr., L.E. Gates R.E. Stoscheck C.M. Nanney L.B. Epidermal growth factor/transforming growth factor α receptors and psoriasis.J Invest Dermatol. 1990; 95: 10S-12SAbstract Full Text PDF Scopus (61) Google Scholar). Both in the normal and diseased epidermis, keratinocyte growth and differentiation are regulated by autocrine and paracrine signaling molecules such as members of the epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor β (TGF-β) families (Eckert et al., 1997Eckert R.L. Crish J.F. Robinson N.A. The epidermal keratinocyte as a model for the study of gene regulation and cell differentiation.Physiol Rev. 1997; 77: 397-424Crossref PubMed Scopus (343) Google Scholar;Hashimoto, 2000Hashimoto K. Regulation of keratinocyte function by growth factors.J Dermatol Sci. 2000; 24: S46-S50Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). For example, by stimulation of the human EGF receptor, keratinocytes proliferate faster, degrade extracellular matrix components, and become migratory (Nickoloff et al., 1988Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors.Am J Pathol. 1988; 132: 543-551PubMed Google Scholar;Chen et al., 1993Chen J.D. Kim J.P. Zhang K. Sarret Y. Wynn K.C. Kramer R.H. Woodley D.T. Epidermal growth factor (EGF) promotes human keratinocyte locomotion on collagen by increasing the α2 integrin subunit.Exp Cell Res. 1993; 209: 216-223Crossref PubMed Scopus (147) Google Scholar), whereas TGF-β inhibits keratinocyte proliferation (Alexandrow and Moses, 1995Alexandrow M.G. Moses H.L. Transforming growth factor β1 inhibits mouse keratinocytes late in G1 independent of effects on gene transcription.Cancer Res. 1995; 55: 3928-3932PubMed Google Scholar). The expression patterns, and regulation during keratinocyte growth and differentiation, are well documented for many cytoskeletal proteins (Presland et al., 2001Presland R.B. Kuechle M.K. Lewis S.P. Fleckman P. Dale B.A. Regulated expression of human filaggrin in keratinocytes results in cytoskeletal disruption, loss of cell-cell adhesion, and cell cycle arrest.Exp Cell Res. 2001; 270: 199-213Crossref PubMed Scopus (54) Google Scholar) but less is known on the role of cell surface and extracellular molecules in these processes. Hyaluronan, a large unsulfated glycosaminoglycan, is expressed by many cell types and contributes to cellular functions such as adhesion, proliferation, migration, and differentiation by its unique physicochemical properties and interactions with specific cell surface receptors (for a recent review, seeTammi et al., 2002Tammi M.I. Day A.J. Turley E.A. Hyaluronan and homeostasis: a balancing act.J Biol Chem. 2002; 277: 4581-4584Crossref PubMed Scopus (385) Google Scholar). Hyaluronan staining is abundant on the surface and between the basal and spinous cell layers of normal human epidermis, but virtually absent in the terminally differentiated layers (Tammi et al., 1988Tammi R. Ripellino J.A. Margolis R.U. Tammi M. Localization of epidermal hyaluronic acid using the hyaluronate binding region of cartilage proteoglycan as a specific probe.J Invest Dermatol. 1988; 90: 412-414Abstract Full Text PDF PubMed Scopus (186) Google Scholar;Ågren et al., 1997Ågren U.M. Tammi M. Ryynänen M. Tammi R. Developmentally programmed expression of hyaluronan in human skin and its appendages.J Invest Dermatol. 1997; 109: 219-224Abstract Full Text PDF PubMed Scopus (31) Google Scholar). Other stratified epithelia like that of mouth (Tammi et al., 1990Tammi R. Tammi M. Häkkinen L. Larjava H. Histochemical localization of hyaluronate in human oral epithelium using a specific hyaluronate-binding probe.Arch Oral Biol. 1990; 35: 219-224Crossref PubMed Scopus (42) Google Scholar) and esophagus (Wang et al., 1996Wang C. Tammi M. Guo H. Tammi R. Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers.Am J Pathol. 1996; 148: 1861-1869PubMed Google Scholar) show a hyaluronan distribution similar to that in epidermis, whereas hyaluronan is not detected in most simple epithelia (Wang et al., 1996Wang C. Tammi M. Guo H. Tammi R. Hyaluronan distribution in the normal epithelium of esophagus, stomach, and colon and their cancers.Am J Pathol. 1996; 148: 1861-1869PubMed Google Scholar). This distribution suggests that hyaluronan is important for the stratified epithelia and their differentiation. Accordingly, large alterations in hyaluronan synthesis rate have been observed in human skin organ cultures by retinoic acid (Tammi et al., 1989Tammi 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.J Invest Dermatol. 1989; 92: 326-332Abstract Full Text PDF PubMed Scopus (126) Google Scholar) and corticosteroids (Ågren et al., 1995Ågren U.M. Tammi M. Tammi R. Hydrocortisone regulation of hyaluronan metabolism in human skin organ culture.J Cell Physiol. 1995; 164: 240-248Crossref PubMed Scopus (39) Google Scholar), both known for their powerful influences on keratinocyte proliferation and differentiation. Recently, direct evidence on the role of hyaluronan in epidermal keratinocytes emerged by the finding that hyaluronan synthesis (Has2 expression) controls the migration rate of keratinocytes in scratch-wounded monolayer cultures (Rilla et al., 2002Rilla 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.J Cell Sci. 2002; 115: 3633-3643Crossref PubMed Scopus (59) Google Scholar). Growth factors are known to control hyaluronan synthesis; however, the influence seems to be specific for each cell type, and may also depend on the stage of the cellular differentiation. Thus, TGF-β stimulates hyaluronan production in fibroblasts (Sugiyama et al., 1998Sugiyama Y. Shimada A. Sayo T. Sakai S. Inoue S. Putative hyaluronan synthase mRNA are expressed in mouse skin and TGF-β upregulates their expression in cultured human skin cells.J Invest Dermatol. 1998; 110: 116-121Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), but was recently reported to inhibit hyaluronan synthesis in human keratinocytes (Sayo et al., 2002Sayo T. Sugiyama Y. Takahashi Y. et al.Hyaluronan synthase 3 regulates hyal-uronan synthesis in cultured human keratinocytes.J Invest Dermatol. 2002; 118: 43-48Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Studies on rat epidermal keratinocytes cultured as monolayers have suggested that elevated hyaluronan synthesis is an important feature of the EGF response (Pienimäki et al., 2001Pienimäki J.P. Rilla K. Fulop C. et al.Epidermal growth factor activates hyaluronan synthase 2 in epidermal keratinocytes and increases pericellular and intracellular hyaluronan.J Biol Chem. 2001; 276: 20428-20435Crossref PubMed Scopus (165) Google Scholar). There are no previous reports on the influences of EGF and TGF-β on hyaluronan synthesis in the keratinocytes of structurally normal epidermis. This work explores the effects of a mitogenic growth factor, EGF, and that of an antiproliferative growth factor, TGF-β, on hyaluronan metabolism and epidermal morphology in an organotypic keratinocyte culture with normal epidermal tissue architecture. The results show that hyaluronan concentration is closely correlated with the proliferative activity and volume of the vital part of the epidermis, and inversely related with the markers of differentiation, suggesting that hyaluronan synthesis regulated by Has2 and Has3 is an important component in the proliferative reactions of epidermis and is also involved in the epidermal differentiation process. A keratinocyte cell line derived from newborn rat skin (REK, rat epidermal keratinocyte) was used (Baden and Kubilus, 1983Baden H.P. Kubilus J. The growth and differentiation of cultured newborn rat keratinocytes.J Invest Dermatol. 1983; 80: 124-130Abstract Full Text PDF PubMed Scopus (80) Google Scholar). The stock cultures were grown in minimum essential medium (Life Technologies, Paisley, U.K.) with 10% fetal bovine serum (HyClone, Logan, UT), 4 mM L-glutamine (Sigma, St. Louis, MO), 50 μg per ml streptomycin sulfate, and 50 U per ml penicillin (Sigma) at 37°C in humidified 95% air/5% CO2. REKs were passaged twice a week at a 1:5 split ratio using 0.05% trypsin (wt/vol), 0.02% ethylenediamine tetraacetic acid (EDTA) (wt/vol) in phosphate-buffered saline (PBS). For organotypic cultures, REKs were cultured at the air–liquid interface on type I collagen support (from rat tail) as described inPasonen-Seppänen et al., 2001Pasonen-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.Histochem Cell Biol. 2001; 116: 287-297Crossref PubMed Scopus (62) Google Scholar. The culture medium was supplemented with EGF (2–20 ng per ml; Sigma) or TGF-β1 (1–4 ng per ml; Life Technologies) from the day after the culture was raised to the air–liquid interface. The cultures for these studies were grown for 2–3 wk, with the medium changed every 2 d for the first week and daily thereafter. The organotypic cultures were fixed in 2% buffered paraformaldehyde containing 0.5% glutaraldehyde, or in Histochoice® (Amresco, Solon, OH) overnight, washed with 0.1 M sodium phosphate buffer (PB), pH 7.4, dehydrated in graded ethanol, embedded in paraffin, and cut into 3 μm thick vertical sections. The morphometric measurements were performed on hematoxylin/eosin stained sections with the NIH-Image 1.62/fat software for Macintosh. The cultures were systematically sampled by taking 10 digital images with a CoolSNAP camera (Photometrics) from each culture at constant distances in each section using a 20× objective and an 1.25× intermediate lens (Nikon Microphot FXA microscope, Japan). The thickness of the vital cell layers, the thickness of the stratum corneum, and the height of the basal cells were recorded. In stratum corneum measurements, thresholding of areas exhibiting background intensity was used to exclude the space between separated corneocytes. Histochoice®-fixed, deparaffinized sections were first incubated in target unmasking fluid (TUF™, Monosan, Uden, The Netherlands) at 95°C and then for 5 min with 1% H2O2 to block endogenous peroxidase, washed with PB, and incubated in 1% bovine serum albumin (BSA) in PB for 30 min to block nonspecific binding. Thereafter the sections were incubated overnight at 4°C with monoclonal antikeratin 10 (1:10 dilution in 1% BSA) (Monosan), polyclonal antifilaggrin (1:5000 dilution) (a generous gift of Dr. Beverly Dale-Crunk, University of Washington, Seattle, WA), or monoclonal anti-CD44 antibody (OX50; Biosource, Camarillo, CA), followed by a 1 h incubation with biotinylated antimouse antibody (dilution 1:50) (Vector Laboratories, Burlingame, CA) or biotinylated antirabbit antibody (dilution 1:70) (Vector Laboratories), respectively. The bound antibodies were visualized with the avidin-biotin peroxidase method (1:200 dilution, Vectastain Kit, Vector Laboratories). The sections were incubated for 5 min in 0.05% 3,3′-diaminobenzidine (Sigma) and 0.03% hydrogen peroxide in PB. After washes, the sections were counterstained with Mayer's hematoxylin for 1 min, washed, dehydrated, and mounted in DPX (Gurr®, BDH Laboratory Supplies, Poole, U.K.). The controls included sections treated in the same way but with the primary antibody omitted. Hyaluronan staining was done to paraformaldehyde (2%)-glutaraldehyde (0.5%) fixed cultures with a specific probe, biotinylated hyaluronan binding complex (bHABC), purified from a 4 M guanidine-HCl extract of bovine articular cartilage after dialysis and trypsin digestion as described previously (Tammi et al., 1994Tammi R. Ågren U.M. Tuhkanen A.L. Tammi M. Hyaluronan metabolism in skin.Prog Histochem Cytochem. 1994; 29: 1-81Crossref PubMed Scopus (138) Google Scholar). The specificity of the staining was controlled by preincubating the sections with Streptomyces hyaluronidase to remove hyaluronan from the tissue (Tammi et al., 1989Tammi 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.J Invest Dermatol. 1989; 92: 326-332Abstract Full Text PDF PubMed Scopus (126) Google Scholar). Intracellular hyaluronan was detected using confocal microscopy. Deparaffinized, TUF-treated sections were incubated for 30 min with 50 mM glycine to block autofluorescence, washed with PB, and incubated in 1% BSA in PB for 30 min to block nonspecific binding. For double staining, the anti-CD44 monoclonal antibody (1:20) was added to the bHABC solution (5 μg per ml), and sections were incubated overnight at 4°C, washed, and incubated simultaneously with Texas-Red-labeled antimouse antibody (1:50) and fluorescein isothiocyanate-avidin (1:1000) for 1 h at room temperature. The sections were coverslipped using Vectashield (Vector) mounting medium. The micrographs were obtaind with an Ultraview® confocal scanner (Perkin Elmer Life Sciences, Wallac-LSR, Oxford, U.K.) on a Nicon Eclipse TE300 microscope using a 100× oil immersion objective. Two-week-old REK cultures were incubated with 5-bromo-2′-deoxyuridine (BrdU) for 1 h, washed with PBS, and fixed overnight in Histochoice®. Deparaffinized, TUF-treated sections were incubated at 95°C, immunostained with the anti-BrdU antibody, and counterstained with propidium iodide (0.01 μg per ml; Sigma) according to the instructions of the manufacturer (5-Bromo-2′-deoxyuridine Labeling and Detection Kit I, Roche Diagnostics Corporation, IN). Approximately 10 fields per section were counted for the labeled cells in six separate experiments with a Nikon Microphot FXA microscope using a 10× objective lens. Serum-free medium (1.5 ml) otherwise identical with that used in the organotypic cultures was changed 24 h before the assay to exclude the hyaluronan introduced by serum. Medium, epidermis, and collagen support were each analyzed, the last two after separation with fine tweezers. Epidermis and collagen were extracted twice with 2 ml of acetone at 4°C for 1 d. The lipid extracts were discarded and the samples were dried in air before weighing. The epidermis was digested with 200 μl of a solution containing 250 μg per ml papain (Sigma) in 5 mM cysteine and 5 mM EDTA at 60°C overnight, and the collagen matrix with 550 μl of the same solution but with 400 μg per ml papain. After digestion, the samples were boiled for 10 min to inactivate the enzyme, centrifuged, and the precipitates were discarded. Medium (1.5 ml) and the digests were diluted (1:100–1:300) with 1% BSA in PBS before the assay. Ninety-six-well Maxisorp Plates (Nunc, Roskilde, Denmark) were coated with 1 μg per ml of the HABC overnight at 4°C. After incubation, the plates were washed 3×3 min with PBS containing 0.5% Tween (Tween-PBS) and blocked with 1% BSA for 1 h at 37°C. Standard hyaluronan (1–50 ng per ml; Provisc®, Algon Laboratories, Fort Worth, TX) and samples were diluted into 1% BSA in PBS, and 100 μl aliquoted to the wells. After 1 h incubation at 37°C, the plates were washed with Tween-PBS, incubated with 1 μg per ml bHABC for 1 h at 37°C, and washed 3×3 min with Tween-PBS. Then 100 μl of horseradish peroxidase streptavidin complex (Vector) was added and allowed to stand for 1 h at 37°C. After washing 5×3 min with Tween-PBS, 100 μl of 1 mg per ml of O-phenylenediamine dihydrochloride (Sigma) 0.03% H2O2 in 0.1 M phosphate citrate buffer, pH 5, was added and kept at 37°C. The reaction was stopped after 15 min by adding 50 μl of 8 N H2SO4 and the absorbances were read at 490 nm. Each sample and standard were analyzed in triplicate. For RT-PCR, total RNA was isolated from 2-wk-old organotypic REK cultures with TRIzol®-reagent (Gibco) according to the instructions of the manufacturer. The samples were DNase treated, and equal amounts of RNA were taken for the RT-PCR done with the GeneAmp® Gold RNA PCR Reagent Kit (Applied Biosystems, CA). The primers used for the RT-PCR of rat Has1, Has2, Has3, CD44, profilaggrin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), the annealing temperatures, and the number of PCR cycles are shown in Table I. The RT-PCR products separated on 1.5% agarose gels were digitized by BioDocII™ Video Documentation system (Biometra, Göttingen, Germany), and the band densities were compared with the NIH-Image 1.62/fat software. The pixel density of the electrophoretic band obtained by image analysis is not linearly correlated with the actual level of mRNA or cDNA in the sample. Still, the values, when divided by that from GAPDH of the same mRNA isolate, can be used for normalization to indicate whether there is an increase or decrease in the level of expression.Table IPrimers, annealing temperatures, and number of cycles used in the RT-PCRHas15′-GCT CTA TGG GGC GTT CCT C-3′ (left)57°C35–37×5′-CAC ACA TAA GTG GCA GGG TCC-3′ (right)Has25′-TCG GAA CCA CAC TGT TTG GAG TG-3′62°C33–35×5′-CCA GAT GTA AGT GAC TGA TTT GTC CC-3′Has35′-ACT CTG CAT CGC TGC CTA CC-3′66°C33×5′-ACA TGA CTT CAC GCT TGC CC-3′CD445′-TTG GGG ACT ACT TTG CCT CTT A-3′55°C33×5′-CCA CTG CTG ACA TCC TCA TCT A-3′Profilaggrin5′-CTC AGG GCA TCG CTC GTC A-3′64°C34×5′-GCT GGT GGC GGT CTT CGT G-3′GAPDH5′-TGA TGC TGG TGC TGA GTA TG-3′60°C32×5′-GGT GGA AGA ATG GGA GTT GC-3′ Open table in a new tab The organotypic REK cultures (epidermis without collagen matrix) were rinsed with ice-cold PBS, homogenized in 8 M urea, 50 mM Tris pH 7.6, 100mM dithiothreitol, 0.13 M 2-mercaptoethanol, 100μg per ml phenylmethylsulfonyl fluoride, and 100 μg per ml aprotinin (Haydock et al., 1993Haydock P.V. Blomquist C. Brumbagh S. Dale B.A. Holbrook K.A. Fleckman P. Antisense profilaggrin RNA delays and decreases profilaggrin expression and alters in vitro differentiation of rat keratinocytes.J Invest Dermatol. 1993; 101: 118-126Abstract Full Text PDF PubMed Scopus (22) Google Scholar) with Ultra Turrax (Ystral, Germany), and centrifuged at 13,000g for 5 min, and the pellets were discarded. From the supernatant, equal amounts of protein (Bradford, 1976Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto Immobilon™-NC membranes (Millipore, Bedford, MA) by 3.5 mA per cm2 constant current with Sammy™ semidry blotter (Schleicher & Schuell, Dassel, Germany). Nonspecific binding was blocked by 5% fat-free milk powder and 0.2% Tween 20 in 10 mM Tris, 150 mM NaCl, pH 7.4 (Tris-saline blocking buffer) overnight at 4°C. The membranes were incubated for 2 h with the polyclonal antifilaggrin (1:9000) and the monoclonal antikeratin 10 (1:100) antibodies described above, both diluted with the blocking buffer. The membranes were washed four times for 15 min with 0.2% Tween 20 in the Tris-saline buffer, incubated for 1 h with 1:20,000 dilution of horseradish peroxidase conjugated secondary antibody - antirabbit IgG (Zymed Laboratories, CA) for filaggrin and antimouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA) for keratin 10 - and washed four times with 0.2% Tween 20 in Tris-saline buffer. The immune complexes were visualized using the NEN™ chemiluminescence detection kit according to the manufacturer's instructions (Life Science Products, Boston, MA). The permeability of the organotypic cultures was tested in two-chamber diffusion cells (Side-by-Side; Crown Glass, Somerville, NJ) with a 3 ml volume in each compartment and an effective diffusional area of 0.64 cm2, as described previously (Pasonen-Seppänen et al., 2001Pasonen-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.Histochem Cell Biol. 2001; 116: 287-297Crossref PubMed Scopus (62) Google Scholar). Briefly, the culture was clamped between the chambers filled with PBS and equilibrated at 37°C. The tritiated corticosterone (NEN Life Science Products; 20,000–40,000 dpm per 10 μl) was added to the donor chamber, and aliquots were withdrawn repeatedly from the acceptor side for liquid scintillation counting. The permeability coefficient (p; cm per s) for the probe permeant was calculated at steady state under sink conditions by dividing the steady state flux (dpm per s cm2) through the skin by the concentration of the test substance (dpm per cm3) in the donor phase. The significances of the differences between the groups in the morphometric and hyaluronan assay measurements and in RT-PCR were tested using the paired samples t test. The data from proliferation and permeability assays were analyzed with the nonparametric Mann–Whitney U test. A difference was considered statistically significant when the associated p-value was less than 0.05. After 2 wk, REKs seeded on a collagen lattice and lifted to the air–liquid interface formed a histologically normal looking epidermis with a basal cell layer, two to four spinous cell layers, and one granular cell layer under a well developed stratum corneum (Figure 1a, Control), as described previously (MacCallum and Lillie, 1990MacCallum D.K. Lillie J.H. Evidence for autoregulation of cell division and cell transit in keratinocytes grown on collagen at an air–liquid interface.Skin Pharmacol. 1990; 3: 86-96Crossref PubMed Scopus (19) Google Scholar;Pasonen-Seppänen et al., 2001Pasonen-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.Histochem Cell Biol. 2001; 116: 287-297Crossref PubMed Scopus (62) Google Scholar). Cultures grown in the presence of EGF exhibited a dose-dependent stimulation of proliferation as indicated by BrdU labeling (Figure 1b). Accordingly, EGF-treated cultures showed taller basal cells and hypertrophy of the whole vital part of the epidermis, whereas stratum corneum was relatively thin (Figure 1a, c). In the EGF-treated cultures BrdU-labeled nuclei were also found in the suprabasal compartment, whereas in control cultures the cells in the S-phase of the cell cycle were located in the basal cell layer (data not shown). Conversely, the cultures containing 4 ng per ml TGF-β showed significantly fewer labeled cells than control cultures (Figure 1b). These results, indicating a lower proliferation rate, are in line with a decline in the thickness of the epidermis (Figure 1a, c), and with previous reports on TGF-β (Nickoloff et al., 1988Nickoloff B.J. Mitra R.S. Riser B.L. Dixit V.M. Varani J. Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors.Am J Pathol. 1988; 132: 543-551PubMed Google Scholar;Alexandrow and Moses, 1995Alexandrow M.G. Moses H.L. Transforming growth factor β and cell cycle regulation.Cancer Res. 1995; 55: 1452-1457PubMed Google Scholar). In 2-wk-old control cultures, the concentration of hyaluronan in the epidermis was ≈150 ng per mg of tissue dry weight. The collagen matrix and medium, into which a part of the hyaluronan escapes (Tammi et al., 2000Tammi 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.Histochem Cell Biol. 2000; 113: 265-277Crossref PubMed Scopus (46) Google Scholar), contained ≈2800 ng per mg of tissue dry weight. EGF caused a dose-dependent increase of hyaluronan in the epidermis (Figure 2a). A large deposition of hyaluronan was also found in the collagen and medium (Figure 2a). At 2 ng per ml, EGF caused an approximately equal increase in both compartments (1.7 × control), whereas at the higher concentration (20 ng per ml) the increase in the matrix compartment was more obvious than in the epidermis (4.9× and 3.7×, respectively), suggesting enhanced outflow from the epidermis when the synthesis rate is elevated. In the TGF-β-treated cultures (4 ng per ml) the epidermal hyaluronan concentration was similar to that in control cultures (Figure 2a) the hyaluronan content of the matrix compartment was significantly lower than in the control cultures. However (Figure 2a). Thus, less hyaluronan escaped from the tissue, a situation obviously resulting from reduced synthesis in the epidermis or increased degradation. RNA isolated from 2-wk-old organotypic cultures was subjected to RT-PCR of Has1, Has2, Has3, and CD44, and their signals were compared with that of GAPDH. The expression level of Has1 was very low in organotypic REK cultures and in three out of five experiments the band densities were too weak to measure. Those available showed no change with either EGF or TGF-β, however (data not shown). EGF increased the levels of Has2 and Has3 mRNA significantly at both 2 and 20 ng per ml concentrations (Figure 2b). In contrast, the expression of both Has2 and Has3 isoenzymes was significantly reduced at the higher TGF-β concentration (Figure 2b). The size of the RT-PCR product suggested that REKs in organotypic culture mainly expressed the standard form of CD44, both under control conditions and after the growth factor treatments (data not shown). The level of CD44 mRNA was significantly stimulated with the higher dose of EGF. Although the changes in the levels of CD44 appeared modest in the TGF-β-treated cultures, there was a tendency for decrease with the higher dose of T
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