Hyaluronan Synthase 3 Regulates Hyaluronan Synthesis in Cultured Human Keratinocytes

Tetsuya Sayo, Yoshinori Sugiyama, Yoshito Takahashi, Naoko Ozawa, Shingo Sakai, Osamu Ishikawa,* Masaaki Tamura,* and Shintaro Inoue Basic Research Laboratory, Kanebo Ltd, Kanagawa, Japan; *Department of Dermatology, Gunma University School of Medicine, Gunma, Japan

Three human (HAS1, growth factor b downregulated HAS3 transcript HAS2, and HAS3) have been cloned, but the func- levels. The expression of HAS1 mRNA was not sig- tional differences between these HAS genes remains ni®cantly affected by either cytokine, and HAS2 obscure. The purpose of this study was to examine mRNA expression was undetectable under either which of the HAS genes are selectively regulated in basal or cytokine-stimulated conditions by northern epidermis. We examined the relation of changes blot using total RNA. Furthermore, in situ mRNA between hyaluronan production and HAS hybridization showed that mouse epidermal kerati- expression when cytokines were added to cultured nocytes abundantly expressed HAS3 mRNA from the human keratinocytes. Interferon-g increased hyaluro- basal to the granular cell layers, suggesting that nan production whereas transforming growth factor HAS3 functions in epidermis. These ®ndings suggest b decreased it. Both cytokines affected preferentially that HAS3 gene expression plays a crucial role in the high-molecular-mass (> 106 Da) hyaluronan produc- regulation of hyaluronan synthesis in the epidermis. tion. Consistent with the change in hyaluronan syn- Key words: HAS1/HAS2/HAS3/IFN-g/TGF-b. J Invest thesis, we found that interferon-g markedly Dermatol 118:43±48, 2002 upregulated HAS3 mRNA whereas transforming

yaluronan, a high-molecular-weight linear glycosa- (Wells et al, 1991). These ®ndings indicate that hyaluronan is minoglycan consisting of alternating glucuronic acid closely involved in keratinocyte proliferation and differentiation, and N-acetylglucosamine residues, is found in the and hence may participate in epidermal structure and turnover. extracellular matrix (Laurent and Fraser, 1992). In Molecular cloning of genes encoding hyaluronan synthase is one Hthe skin, hyaluronan was previously thought to of the essential steps to investigate the biologic functions of derive exclusively from dermis. Histologic staining techniques, hyaluronan. Recently, several putative mammalian hyaluronan however, later revealed that hyaluronan can also be found around synthases (HAS) have been identi®ed and cloned. These HAS keratinocytes in the epidermis (Tammi et al, 1988). Moreover, we genes, HAS1 (Itano and Kimata, 1996a; 1996b; Shyjan et al, 1996), have recently shown that hyaluronan can be found not only in the HAS2 (Spicer et al, 1996; Watanabe and Yamaguchi, 1996), and basal to the spinous layer, but also in the stratum corneum (Sakai et al, HAS3 (Spicer et al, 1997), encode predicted plasma membrane 2000). These ®ndings strongly suggest that keratinocytes themselves proteins with multiple transmembrane domains. The expression of synthesize the epidermal hyaluronan. any one HAS gene in transfected mammalian cells leads to high Accumulation of hyaluronan is correlated with cell proliferation levels of hyaluronan biosynthesis. In a previous study, our group and migration in several developing tissues and organs (Brecht et al, found that human skin ®broblasts abundantly express HAS1 and 1986; Chen et al, 1989), but the functions of epidermal hyaluronan HAS2 mRNA, and that transforming growth factor b (TGF-b) are not well known. Previous studies showed that retinoic acid, an upregulates the expression of both of these HAS genes (Sugiyama et inhibitor of epidermal differentiation, leads to the accumulation of al, 1998). Whereas the cytokines TGF-b, platelet-derived growth hyaluronan in the epidermal intercellular space (Tammi et al, 1989), factor (Heldin et al, 1989; Suzuki et al, 1995), interferon g (IFN-g), and also that hydrocortisone, an agent widely used in the treatment tumor necrosis factor a (TNF-a) (Elias et al, 1988), interleukin-1 of skin diseases as an antiproliferative or aniti-in¯ammatory, inhibits (IL-1) (Postlethwaite et al, 1989), and epidermal growth factor epidermal hyaluronan synthesis at pharmacologic doses (AÊ gren et al, (EGF) (Hata et al, 1988) have been shown to stimulate ®broblast 1995). In psoriasis, characterized by enhanced epidermal prolifer- hyaluronan synthesis, the cytokines regulating keratinocyte hyalur- ation, hyaluronan accumulates abundantly in the basal cell layer onan synthesis have not been adequately investigated. The purpose of this study was to examine which of the HAS genes are selectively regulated in epidermis by cytokines. We Manuscript received April 8, 2001; revised September 10, 2001; accepted for publication September 13, 2001. examined HAS gene expression and changes in the quality and Reprint requests to: Dr. Shintaro Inoue, Basic Research Laboratory, quantity of the hyaluronan synthesized in cultured human Kanebo Ltd, 5-3-28 Kotobuki-cho, Odawara, Kanagawa 250-0002, Japan. keratinocytes stimulated with cytokines whose receptors are Email: [email protected] expressed in keratinocytes, namely, IFN-g, TGF-b, IL-1a, TNF- Abbreviations: DIG, digoxigenin; HAS, hyaluronan synthase. a, IL-8, and IL-10.

0022-202X/01/$15.00 ´ Copyright # 2002 by The Society for Investigative Dermatology, Inc. 43 44 SAYO ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

MATERIALS AND METHODS Hyaluronan production in culture supernatants was measured with a Chugai hyaluronan plate. This kit is based on the speci®city of a Materials Normal human foreskin keratinocytes and bovine pituitary hyaluronan-binding protein isolated from bovine cartilage for hyaluronan extract were purchased from Kurabo (Osaka, Japan), and male hairless (Kondo et al, 1991). mice (Hos/HR-1) were from SLC (Shizuoka, Japan). MCDB153 Molecular mass distribution of labeled hyaluronan Pronase-treated medium, calcium ionophore A23187, and insulin were purchased from conditioned media were applied to a DE-52 ion-exchange column Wako Pure Chemical Industries (Osaka, Japan). EGF was purchased (1.0 3 12 cm) equilibrated with 10 mM Tris±HCl (pH 8.4). Each from Sigma (St. Louis, MO), hydrocortisone from Merck (Darmstadt, column was washed with 10 ml of the equilibration buffer and then Germany), 2-aminoethanol from Nacalai Tesque (Kyoto, Japan), and O- eluted with an NaCl gradient (0±0.6 M) in the 10 mM Tris±HCl. phosphorylethanolamine from Tokyo Kasei (Tokyo, Japan). Fractions of 2 ml were collected, and aliquots were measured for Recombinant human IFN-g and TGF-b were obtained from R&D radioactivity. The puri®ed hyaluronan was identi®ed by a change in Systems (Minneapolis, MN), and IL-1a, TNF-a, IL-8, and IL-10 from elution position during Sephadex G-50 gel chromatography after Intergen (Boston, MA). Eagle's minimal essential medium was purchased treatment of the second aliquots with Streptomyces hyaluronidase. The from ICN Biomedicals (Costa Mesa, CA) and fetal bovine serum from fractions of the DE-52 column containing hyaluronan were pooled, a Irvine Scienti®c (Santa Ana, CA). Superscript II was purchased from single aliquot (1.0 ml) from these pooled fractions was used for the Gibco BML (Rockville, MD), and the GeneAmp PCR Kit was from molecular mass determination on a Sepharose CL-2B column Perkin-Elmer (Norwalk, CT). Digoxigenin (DIG) RNA labeling kit, (1.0 3 55 cm) eluted with 1.0 M NaCl, and 2.5 ml fractions were DIG nucleic acid detection kit, anti-DIG alkaline phosphatase conjugate, collected and counted for radioactivity. and positively charged nylon membrane were from Roche (Tokyo, Japan), RNaid Kit was from Biol01 (Vista, CA), and human HAS3 cDNA isolation Human HAS1 and HAS2 cDNA isolations were described previously (Sugiyama et al, 1998). The cDNA of mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was from and human HAS3 genes were generated using reverse transcription Clontech (Palo Alto, CA). Oligotex-dT30 was obtained from Takara polymerase chain reaction (RT-PCR) and a second PCR. The following Shuzou (Kyoto, Japan). Hyaluronidase derived from Streptomyces primers were designed for RT-PCR: for mouse HAS3, sense primer 5¢- hyalurolyticus was purchased from Seikagaku (Tokyo, Japan), and ACTGCCTTCAAGGCCCTTGG-3¢ and antisense primer 5¢- Streptomyces griseus pronase from Calbiochem-Novabiochem (La Jolla, 3 AATGTTCCAGATGCGGCCAC-3¢; for human HAS3, sense primer CA). D-[1,6- H(N)]glucosamine hydrochloride (62 Ci per mmol) was 5¢-GGAAAGCTTGGCATGTACCGCAACAG-3¢ and antisense primer obtained from NEN Research Products (Boston, MA), Sephadex G-50 5¢-AGAGGAGGGAGTAGAGGGAC-3¢ (Spicer et al, 1997). Total and Sepharose CL-2B from Pharmacia (Uppsala, Sweden), and the DE- RNA from human keratinocytes for human HAS3 and mouse skin for 52 from Whatman (Maidstone, England). Chugai hyaluronan plate was mouse HAS3 were used for RT-PCR. PCR consisted of 30 cycles of obtained from Chugai Diagnostics Science (Tokyo, Japan). denaturation at 94°C for 1 min, annealing at 60°C for 2 min, and Cell culture Human epidermal keratinocytes were routinely grown in elongation at 72°C for 2 min. The expected cDNA fractions were used MCDB153 (0.1 mM Ca2+) supplemented with 5 mg per liter insulin, as templates for the second PCR. The following primers were designed 180 mg per liter hydrocortisone, 14.1 mg per liter O- for the second PCR: for mouse HAS3, sense primer 5¢- phosphorylethanolamine, 6.1 mg per liter 2-aminoethanol, 100 ng per GGAAAGCTTGACTCTGACACTGTGC-3¢ and antisense primer 5¢- liter EGF, and 0.4% (vol/vol) bovine pituitary extract. The culture AGGGAATTCTGTAGAGCCATTCCCG-3¢; for human HAS3, sense medium was changed every 2 d. Once the cells had become primer 5¢-GGAAAGCTTGGCATGTACCGCAACAG-3¢ and antisense subcon¯uent, they were subcultured in 24-well plates (1.0 3 104 cells primer 5¢-AGGGAATTCGGAAGCAGGCGTAGGTG-3¢. The under- per well) or in 90 mm dishes (4.0 3 105 cells per dish) in the same lined sequences correspond to the restriction sites for HindIII/EcoRI. medium. After subculturing for 5 d, cells were con¯uent and the The isolated cDNA was digested with HindIII/EcoRI and then ligated medium was exchanged for an identical medium but without EGF and into the HindIII/EcoRI site of pSPT18 (Roche). The nucleotide bovine pituitary extract. At 24 h after the medium change, each cytokine sequences of isolated cDNA were con®rmed to be the authentic HAS3 was added to the medium and cultured. The 18-h conditioned media of cDNA sequences by sequencing. DIG-labeled antisense/sense RNA probes were prepared from isolated cDNA using a DIG RNA labeling the 24-well plates were assayed for hyaluronan synthesis and production, kit. and total RNA extracted from the cells treated with cytokines for 6 h in the 90 mm dishes was examined by northern blot analysis. Northern blot analysis Total RNA or poly(A)+ RNA samples Human skin ®broblast Detroit 551 (ATCC CCL110) cells were seeded prepared from cells were separated by electrophoresis on 0.8% in Eagle's minimal essential medium supplemented with 10% fetal bovine formaldehyde/agarose gel and transferred to a nylon membrane. DIG- serum in 225 cm2 plastic tissue culture ¯asks (2.0 3 106 cells per dish). labeled HAS3 antisense RNA probes were prepared as described above. After culturing for 4 d, cells were con¯uent and poly(A)+ RNA was Hybridization was performed according to the protocol in the DIG extracted from the cells. Nucleic Acid Detection Kit. The chemiluminescent detection was Corni®ed envelope formation of keratinocytes was estimated by the performed as described previously (Engler-Blum et al, 1993). The method described previously (Shimada et al, 1998). Brie¯y, keratinocytes membranes were washed at 95°C for 3 min in 0.1% SDS, cooled to from the 24-well plates were harvested with 0.5 mM ethylenediamine room temperature, and rehybridized with a DIG-labeled GAPDH tetraacetic acid and 0.25% trypsin and then divided into two fractions. antisense RNA probe. One aliquot was resuspended in medium and the total number of cells In situ mRNA hybridization In situ mRNA hybridization was was counted in a hemocytometer. A second aliquot was resuspended in performed as described previously (Shimada et al, 1998). Skin-tissue medium containing 1.2 mM calcium and treated with 20 mM calcium sections from 10-wk-old, male, hairless Hos/HR mice were used. ionophore at 37°C for 2 h followed by boiling in 2% sodium dodecyl DIG-labeled mouse HAS3 antisense RNA probes (®nal concentration, sulfate (SDS)/20 mM dithiothreitol solution at 100°C for 5 min. The 1 mg per ml) were used to detect HAS3 mRNA. In control experiments, number of remaining cells, which were resistant to SDS because of their DIG-labeled sense RNA probes (®nal concentration, 1 mg per ml) were corni®ed envelope formation, was counted. used. Assay of hyaluronan synthesis and content IFN-g (1.0±30 ng per Statistics The Dunnett test was used for comparisons between control ml), TGF-b (0.3±10 ng per ml), IL-1a (10 ng per ml), TNF-a (10 ng and experimental groups (Dunnett, 1964). per ml), IL-8 (10 ng per ml), or IL-10 (10 ng per ml) was added to the cells in the 24-well plates and the cultures were incubated for 18 h with [3H]glucosamine (10 mCi per ml). After incubation, we estimated the RESULTS hyaluronan synthesis in the conditioned media by measuring the Hyaluronan production was increased by IFN-g but radioactivity of the Streptomyces hyaluronidase-sensitive materials as decreased by TGF-b To evaluate whether the cytokines described previously (Sakai et al, 1999). Brie¯y, the culture media were harvested and treated with pronase (30 mg per ml). The pronase-treated IFN-g, TGF-b, IL-1a, TNF-a, IL-8, and IL-10 affect the hyaluronan synthesis of keratinocytes, we added 10 ng per ml of solution was divided into two parts, one for digestion by Streptomyces 3 hyaluronidase (1.5 TRU per ml) and the other left untreated as a each cytokine with [ H]glucosamine to the medium and cultured control. The reaction mixtures were subjected to gel chromatography on them for 18 h. Compared to nontreated cells, IFN-g-treated cells a Sephadex G-50 column (1.5 3 5 cm), and void fractions were exhibited a 3.5-fold increment in hyaluronan synthesis, whereas collected and measured for radioactivity. TGF-b-treated cells exhibited a 50% reduction (Fig 1). None of VOL. 118, NO. 1 JANUARY 2002 REGULATION OF EPIDERMAL HYALURONAN SYNTHESIS 45 the other cytokines, however, had any marked effects on ml TGF-b gave a plateau response (Fig 2B). The results were hyaluronan synthesis. We further examined keratinocyte con®rmed by a direct measurement of accumulated hyaluronan in hyaluronan synthesis at several concentrations of IFN-g and the media (Table I), which corresponded well with the change in TGF-b. The amount of newly synthesized hyaluronan released [3H]glucosamine incorporation into hyaluronan (Fig 2). into the culture medium after IFN-g addition was signi®cantly Under these conditions, the addition of IFN-g or TGF-b at increased in a dose-dependent manner from 1.0 ng per ml to 10 ng concentrations of 1.0 ng per ml and 10 ng per ml did not induce per ml (Fig 2A). In contrast, TGF-b suppressed hyaluronan notable alterations in the total cell count or corni®ed cell count synthesis at concentrations above 0.3 ng per ml, and 1.0 ng per (Table II), indicating that the effects of these cytokines on hyaluronan synthesis were independent of cell proliferation and differentiation. Gel chromatography was used to estimate the effects of IFN-g and TGF-b on the size distribution of hyaluronan synthesized by cultured human keratinocytes. Hyaluronan fractions eluted from a DE-52 anion-exchange column were pooled and passed through a Sepharose CL-2B column (Fig 3). The puri®ed hyaluronan from conditioned media of cytokine-stimulated or nonstimulated keratinocytes was mainly eluted in void fraction (> 1 3 106 Da), indicating that both IFN-g and TGF-b induced the synthesis of high-molecular-mass hyaluronan. HAS3 mRNA level was upregulated by IFN-g but downregulated by TGF-b To investigate which of the known members of the HAS family are regulated by IFN-g or TGF-b, total RNA extracted from keratinocytes stimulated with IFN-g or TGF-b was examined by northern blot analysis. HAS3 mRNA appeared as two differently sized species, i.e., a major transcript of approximately 4.9 kb and a minor transcript of 2.0 kb. When keratinocytes were treated with IFN-g for 6 h, HAS3

Table I. Effects of IFN-g and TGF-b on hyaluronan production

Concentration Hyaluronan productiona Treatment (ng per ml) (ng per well)

Control 0 126 6 9 Figure 1. Effects of cytokines on hyaluronan synthesis in cultured IFN-g 1 185 6 34* human keratinocytes. Con¯uent human keratinocytes were incubated 10 473 6 14* in the absence or presence of IL-1a (10 ng per ml), TNF-a (10 ng per TGF-b 1 64 6 5* ml), IL-8 (10 ng per ml), IL-10 (10 ng per ml), IFN-g (10 ng per ml), 10 64 6 4* or TGF-b (10 ng per ml) with [3H]glucosamine. The incorporation of 3 [ H]glucosamine into hyaluronan during the 18 h incubation period was aKeratinocytes were grown in the same conditions as for Fig 2. Hyaluronan determined as described in Materials and Methods. Each column represents content was measured as described in Materials and Methods. The data represent the mean 6 SD of four separate experiments. *Signi®cantly different means 6 SD of four separate experiments. from the control value; p < 0.01. *Signi®cantly different from the control value; p < 0.01.

Figure 2. The hyaluronan synthesis of human keratinocytes was upregulated by IFN-g but downregulated by TGF-b in a dose- dependent manner. Con¯uent human keratinocytes were incubated with fresh medium for 24 h followed by cultivating with [3H]glucosamine and various concentrations of IFN-g (1±30 ng per ml) or TGF-b (0.3±10 ng per m) for 18 h. The incorporation of [3H]glucosamine into hyaluronan was determined as described in Materials and Methods. Each column represents the mean 6 SD of four separate experiments. *Signi®cantly different from the control value; p < 0.01. 46 SAYO ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

Table II. Effects of IFN-g and TGF-b on total cell and corni®ed cell number

Treatment Concentration (ng per ml) Total cell number (310±5 cells per well) Corni®ed cell numbera (310±5 cells per well)

Control 0 4.86 6 0.34 1.33 6 0.25 IFN-g 1 4.58 6 0.46 1.56 6 0.18 10 4.56 6 0.57 1.18 6 0.18 TGF-b 1 4.48 6 0.71 1.32 6 0.11 10 4.38 6 0.66 1.30 6 0.21

aKeratinocytes were grown in the same conditions as for Fig 2. The number of corni®ed cells formed in response to calcium ionophore were estimated as described in Materials and Methods. The results are the means of four wells 6 SD.

Figure 4. The HAS3 transcript level of human keratinocytes was upregulated by IFN-g but downregulated by TGF-b in a dose-dependent manner. Human keratinocytes were cultured in various concentrations of IFN-g (0±10 ng per ml) (A) or TGF-b (0.3± 3 ng per m) (B) for 6 h and total RNA was extracted. Equal amounts of samples (5 mg per lane) were hybridized with HAS1 and HAS3 probes. (C) The level of HAS2 mRNA in cultured human keratinocytes was lower than that in cultured human skin ®broblasts. Poly(A)+ RNA extracted from con¯uent cells was used for northern blot analysis. Equal amounts of samples (2 mg per lane) from ®broblasts and keratinocytes were hybridized with HAS2 probe and the ®lter was overexposed to X- Figure 3. Human keratinocytes synthesized high-molecular-mass ray ®lms. Blots were stripped and rehybridized with a GAPDH probe to hyaluronan under basal or cytokine-stimulated conditions. control for variation in loading. Similar results were obtained in two Con¯uent human keratinocytes were incubated with [3H]glucosamine independent experiments. only (open circles), [3H]glucosamine and IFN-g (10 ng per ml) (closed triangles), or [3H]glucosamine and TGF-b (10 ng per m) (closed circles) for 18 h. Hyaluronan in media fractions was puri®ed using DE-52 ion- exchange chromatography and chromatographed using a Sepharose CL- 2B column. stronger signals were observed in the stratum granulosum. In dermis, ®broblasts also expressed the HAS3 mRNA. The DIG-labeled sense RNA probe showed a low background (Fig 5B), indicating that the signals derived from the DIG-labeled HAS3 antisense mRNA levels increased markedly and dose dependently (Fig 4A). RNA probe were speci®c. On the other hand, TGF-b decreased HAS3 transcript levels (Fig 4B). DISCUSSION In contrast to the dramatic regulation of HAS3 mRNA by these cytokines, the expression of 2.4 kb HAS1 mRNA was slightly Although a family of hyaluronan synthases has been identi®ed, the upregulated by TGF-b (Fig 4B) and was entirely unaffected by precise regulatory mechanism of each HAS is still unknown. We IFN-g (Fig 4A). We were unable to detect HAS2 mRNA demonstrated that the levels of HAS3 mRNA regulated by IFN-g expression under either basal or cytokine-stimulated conditions and TGF-b corresponded well to the hyaluronan synthesis in by northern blot analysis using total RNA. To con®rm the cultured human keratinocytes. expression of HAS2 mRNA in keratinocytes, poly(A)+ RNA Our in situ hybridization study clearly demonstrated that HAS3 prepared from keratinocytes was subjected to northern blot analysis, mRNA was abundantly expressed in epidermis. Though hyalur- and it was compared with that from human skin ®broblasts, which onan has been thought to reside exclusively in the basal to spinous abundantly express HAS2 mRNA (Sugiyama et al, 1998; Zhang et cell layers, we detected strong signals of HAS3 mRNA in the al, 2000). HAS2 mRNA transcripts of approximately 3.2 and stratum granulosum. This result accords well with our recent ®nding 4.8 kb were detected in cultured skin ®broblasts, but as a trace of that hyaluronan resides not only in the basal to the upper spinous bands in keratinocytes. With overexposure to X-ray ®lm, the cell layers but also in the stratum corneum (Sakai et al, 2000), HAS2 mRNA transcripts became detectable as weak signals in suggesting that synthesized hyaluronan by keratinocytes is trans- keratinocytes (Fig 4C). ferred to the stratum corneum. The signals of HAS3 mRNA were detected in mouse dermis. HAS3 mRNA is expressed in epidermis We evaluated the We con®rmed the expression of HAS1, HAS2, and HAS3 genes constitutive expression of HAS3 mRNA in mouse epidermis by in using an RNase protection assay in mouse dermis separated from situ mRNA hybridization (Fig 5A). In epidermis, HAS3 mRNA skin (data not shown). Recent quantitative study of HAS mRNA was abundantly expressed in the basal to granular cell layers, and levels, however, revealed that no HAS3 mRNA was detected in VOL. 118, NO. 1 JANUARY 2002 REGULATION OF EPIDERMAL HYALURONAN SYNTHESIS 47

Figure 5. HAS3 mRNA is expressed in mouse skin. Skin specimens from 10-wk-old male hairless mice were analyzed by in situ hybridization. The following DIG-labeled mouse HAS3 RNA probes were used: (A) DIG-labeled antisense RNA probe; (B) DIG-labeled sense RNA probe. Results shown are taken from one representative experiment repeated twice.

the RNA from human skin ®broblasts (Zhang et al, 2000), and we (Ignotz and Massagu, 1986; Roberts et al, 1986). In studies using also obtained the same results by northern blot in our culture cultured human skin ®broblasts, we have shown that TGF-b system. One possible explanation for the difference could be a upregulates HAS1 and HAS2 mRNA (Sugiyama et al, 1998) and divergence between species. also increases the synthesis of hyaluronan (data not shown). In When we probed the factors modulating hyaluronan synthesis contrast, we demonstrated here that TGF-b decreased hyaluronan from cytokines to evaluate the regulation of the HAS mRNA level synthesis and downregulated the expression of HAS3 mRNA in in keratinocytes, we discovered that IFN-g increased the cultured human keratinocytes. These results indicate that HAS keratinocyte hyaluronan synthesis, whereas TGF-b decreased it. genes differentially respond to TGF-b in the dermis and epidermis, This assay was carried out with con¯uent cells under a serum-free respectively. TGF-b often has opposite effects on cell types within a and low-Ca2+ (0.1 mM) condition in which the keratinocytes were given organ. In the skin, in vitro studies have shown that TGF-b already in a growth-arrest stage or an early stage of differentiation. induces proliferation of dermal ®broblasts (Soma and Grotendorst, The effects of these cytokines on hyaluronan production were 1989; Jutley et al, 1993) but arrests growth of epidermal independent of cell number or corni®ed envelope formation. IFN- keratinocytes (Shipley et al, 1986). TGF-b is constitutively g and TGF-b have opposite effects on diverse cellular functions expressed in the basal layer and under basal proliferating conditions (Czarniecki et al, 1988; Bauvois et al, 1992; Letterio and Roberts, in cultured keratinocytes (Fowlis et al, 1992; Escherick et al, 1993), 1998). The addition of IFN-g to keratinocyte cultures resulted in a suggesting the involvement of TGF-b in a negative feedback dramatic upregulation of HAS3 mRNA. Conversely, the addition growth regulatory loop. Thus, we postulate that TGF-b may of TGF-b resulted in a downregulation of HAS3 transcript levels. regulate the proliferation of keratinocytes directly, and also IFN-g failed to regulate the HAS1 mRNA level, but TGF-b indirectly by modulating hyaluronan synthesis through HAS3 slightly upregulated this expression. Our previous study showed mRNA expression. that the TGF-b-induced accumulation of HAS1 mRNA in We examined the HAS2 mRNA expression in cultured cultured keratinocytes was rapid but transient (Sugiyama et al, keratinocytes, but it was so low that the transcripts were dif®cult 1998). Thus, we assumed that HAS1 does not contribute to the to estimate by northern blot. Our previous study, however, has hyaluronan accumulation in the culture medium. shown that HAS2 mRNA was abundantly expressed in mouse The distinct functional roles of each mammalian HAS protein are epidermis using in situ mRNA hybridization (Sugiyama et al, 1998), not fully understood. A recent analysis of the size of hyaluronan and a more recent study showed that HAS2 mRNA was expressed molecules generated in an in vitro system by various types of HAS- constitutively in a rat keratinocyte cell line and was induced by expressed COS-1 cells showed that membrane preparations of EGF (Pienimaki et al, 2001). We cultured the keratinocytes in the HAS2 protein synthesized hyaluronan of signi®cantly higher presence of hydrocortisone (180 ng per ml), as in subcultures it molecular mass than that synthesized using HAS3 (3 3 105 Da makes the colony morphology more orderly and distinctive to > 2 3 106 Da vs < 2 3 105 Da to »3 3 105 Da) (Spicer and (Rheinwald and Green, 1975). Hydrocortisone is known to be a McDonald, 1998). We showed that human keratinocytes synthe- suppression factor of hyaluronan synthesis in keratinocytes and in sized high-molecular-mass hyaluronan (> 1 3 106 Da) under other cells (Wells et al, 1992; Asplund and Heldin, 1994; AÊ gren et either basal or cytokine-stimulated conditions. The polymer size of al, 1995), and HAS2 gene expression was strongly suppressed in synthesized hyaluronan differs between cell-free and cell-cultured response to glucocorticoids by mesothelial cells, ®broblasts, and systems. This size can be regulated not only by HAS proteins but osteosarcoma (Jacobson et al, 2000; Zhang et al, 2000). When also by other regulatory proteins such as a hyaluronan degradation hydrocortisone was depleted from the culture media, HAS1 and protein, as well accessory proteins. HAS3 transcript levels were increased, but upregulation of HAS2 IFN-g, a major cytokine released by activated T lymphocytes mRNA was not observed by northern blot using poly(A)+ RNA found at elevated levels in hyperproliferative diseases, upregulated (T. Sayo, unpublished data). On this basis, in cultured human the keratinocyte hyaluronan synthesis. IFN-g is known to stimulate keratinocytes, HAS2 does not contribute substantially to the keratinocyte proliferation by inducing the expression of TGF-a hyaluronan accumulation regardless of the presence of hydro- (Nickoloff et al, 1989). cortisone under the experimental conditions. TGF-b, a cytokine with signi®cant potential for modulation of In conclusion, we found that IFN-g and TGF-b had opposite connective tissue wound healing, has been reported to stimulate the effects on hyaluronan production in cultured human keratinocytes, production of matrix proteins such as ®bronectin and collagen which corresponded to the pattern of keratinocyte HAS3 mRNA 48 SAYO ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY expression. The expression of HAS3 mRNA may play a crucial transforming growth factor-alpha production in keratinocytes cultured from role in the regulation of hyaluronan synthesis in the epidermis. psoriatic lesions. Br J Dermatol 121:161±174, 1989 Pienimaki JP, 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 276:20428±20435, 2001 We thank Ms. Y. Ota and Mr. K. Onuma for skillful technical assistance. 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