Cholesterol Sulfate Activates Transcription of Transglutaminase 1 Gene in Normal Human Keratinocytes

Cholesterol Sulfate Activates Transcription of Transglutaminase 1 Gene in Normal Human Keratinocytes Shoko Kawabe,1 Togo Ikuta,2 Motoi Ohba,3 Kazuhiro Chida,4 Eichiro Ueda,* Kiyofumi Yamanishi,* and Toshio Kuroki The Institute of Molecular Oncology, Showa University, Hatanodai, Shinagawa-ku, Tokyo, Japan; *Department of Dermatology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto, Japan

Cholesterol sulfate and transglutaminase 1 are essential for the process of keratinization. Cholesterol sulfate is formed during keratinization and activates the η isoform of protein kinase C. Transglutaminase 1 is a key enzyme for formation of the cornified envelope in terminally differentiated keratinocytes. In this study, we demonstrated that cholesterol sulfate acts as a transcriptional activator of the transglutaminase 1 gene in normal human keratinocytes. Growth of normal human keratinocytes was inhibited by cholesterol sulfate, but not by its parental cholesterol. Treatment of normal human keratinocytes with cholesterol sulfate induced activity of transglutaminase 1 in a dose- and time-dependent manner. Activation of transcription of transglutaminase 1 by cholesterol sulfate was demonstrated by northern blotting analysis,

whereas that by cholesterol was not. In order to identify a cholesterol sulfate responsive region in the transglutaminase 1 gene, plasmids were constructed containing a luciferase reporter gene ligated to deletion fragments of the 59 upstream region of the tranglutaminase 1 gene and were transfected into normal human keratinocytes. Transfected cells were treated with cholesterol sulfate, the phorbol ester 12-O-tetradecanoylphorbol-13-acetate and a high concentration of Ca2F. Our results indicate that the responsive element(s) for cholesterol sulfate and phorbol ester is located upstream of the human transglutaminase 1 gene at a position(s) between –819 and –549, whereas the responsive element for Ca2F is located at a position between –79 and –49. Key words: differentiation/phorbol ester/promoter region. J Invest Dermatol 111:1098–1102, 1998

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proteins (Rice and Green, 1977b; Michel et al, 1987) such as loricrin (Hohl et al, 1991), involucrin (Eckert and Green, 1986), filaggrin (Steinert and Marekov, 1995), and SPR protein (Kartasova et al, 1988, 1996). These cross-linking reactions are catalyzed by transglutaminase 1 (TGase 1), a 92 kDa calcium-ion dependent enzyme (Rice and Green, 1977b; Folk, 1980; Ichinose et al, 1990; Yamanishi et al, 1991). TGase 1 is associated with plasma membrane through postsynthetic esterification at the cluster region of cysteine near its amino terminus (Chakravarty and Rice, 1989; Rice et al, 1990). It is expressed in the granular layer of the epidermis, implying that it functions during the late stage of epidermal differentiation (Michel et al, 1992; Schroeder et al, 1992). Cholesterol sulfate (CS) is a membrane lipid formed during the process of keratinization (Elias et al, 1984; Yen et al, 1987) by the action of cholesterol sulfotransferase. It is hydrolyzed by steroid sulfatase, recycling during metabolic turnover (Epstein et al, 1984). The activity of cholesterol sulfotransferase is highly inducible by inducers of keratinization (Rearick et al, 1987). The upper layers of the epidermis in vivo are rich in CS (Jetten et al, 1989), with cholesterol:CS ratios of 5:1–10:1 (Williams and Elias, 1981); however, the ratio in gastrointestinal epithelia is µ500:1 (Lin and Horowitz, 1980). We have reported elsewhere that tumor promotion of mouse skin carcinogenesis is inhibited by CS (Chida et al, 1995). The importance of TGase 1 and CS in keratinization has been shown in human hereditary diseases. Mutation in the TGase 1 gene leads to autosomal recessive lamellar ichthyosis (Huber et al, 1995, 1997; Russell et al, 1995). In recessive X-linked ichthyosis, CS is concentrated in the uppermost cornifield layer due to a lack of steroid sulfatase (Webster et al, 1978; Kubilus et al, 1979; Yen et al, 1987). Inducers of keratinization, such as 12-O-tetradecanoylphorbol-13-

he epidermis provides an essential barrier between organism and environment. The protective function of skin is a consequence of the programmed process of keratinocyte differentiation. During the course of differentiation, keratinocytes leave the basal layer and migrate upward through the spinous, granular, and cornified layers of the epidermis. This process includes reorganization of intermediate filaments, production of lipids, expression of differentiation-specific proteins, and formation of a cornified envelope. All of these events are tightly regulated. A cornified envelope is assembled beneath the plasma membrane during the later stage of keratinization (Sun and Green, 1976; Rice and Green, 1977a). This chemically stable and highly insoluble structure is formed by the ε-(γ-glutamyl) lysine cross-linking of the precursor

Manuscript received February 2, 1998; revised August 12, 1998; accepted for publication September 2, 1998. Reprint requests to: Dr. Toshio Kuroki, Institute of Molecular Oncology, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. Abbreviations: CS, cholesterol sulfate; PKC, protein kinase C; TGase 1, transglutaminase 1. 1Current address: Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194-8511, Japan. 2Current address: Department of Biochemistry, Saitama Cancer Center Research Institute, Inamachi, Saitama 362-0800, Japan. 3Current address: Department of Microbiology, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan. 4Current address: Department of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

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acetate (TPA) (Lichti and Yuspa, 1988; Liew and Yamanishi, 1992; Saunders et al, 1993), a most potent activator of protein kinase C (PKC), and TNF-α (Bikle et al, 1991) can also induce the enzymatic activity of TGase 1. Transcription of the TGase 1 is activated by Ca21 (Floyd and Jetten, 1989; Polakowska et al, 1991; Yada et al, 1993), ganglioside GQ1b (Yada et al, 1993), and interferon (Saunders and Jetten, 1994). Studies using PKC inhibitors (Liew and Yamanishi, 1992; Dlugosz and Yuspa, 1994; Stanwell et al, 1996) suggest that PKC is involved in the transcriptional activation of the TGase 1 gene. We have shown that the η isoform of PKC performs this function (Ueda et al, 1996). To demonstrate this, the luciferase reporter gene under the control of the 59 upstream promoter region of the human TGase 1 gene was transfected into FRSK rat keratinocytes together with the expression vectors for the PKC isoforms. In this study, we report that CS is a transcriptional activator of the TGase 1 gene in normal human keratinocytes. To the best of our knowledge, this is the first demonstration of CS functioning in this capacity. MATERIALS AND METHODS Cell culture Normal human keratinocytes were isolated from skin sections discarded during plastic surgery (Tsunenaga et al, 1994). Skin was cut into small pieces and treated with 0.25% trypsin/0.02% ethylenediamine tetraacetic acid for 24 h at 4°C. The epidermal sheets were separated from the dermis and keratinocytes were collected from both layers. Keratinocytes were grown in a modified serum-free KGM (Kyokuto Seiyaku, Tokyo, Japan) that consists of MCDB153 with high concentrations of amino acids, holo-transferrin (final concentration, 10 µg per ml), insulin (5 µg per ml), epidermal growth factor (10 ng per ml), hydrocortisone (0.5 µg per ml), phosphorylethanolamine (14.1 mg per ml), and bovine pituitary extract (40 µg per ml) (Wille et al, 1984). The final concentration of Ca21 in the medium was 0.03 mM. Subconfluent cultures of keratinocytes were passaged by dispersing with 0.025% trypsin and 0.02% ethylenediamine tetraacetic acid in phosphate-buffered saline. The action of trypsin was stopped by adding soybean trypsin inhibitor to a final concentration of 2 mg per ml. Second or third passages of primary keratinocyte cultures were used for all experiments. Assay of TGase 1 Normal human keratinocytes grown in 100 mm dishes were washed with ice-cold phosphate-buffered saline and were scraped into 2 mM HEPES buffer pH 7.2 containing 2 mM ethylenediamine tetraacetic acid. After sonication and centrifugation (40,000 3 g for 1 h), the pellets were suspended for 1 h in 2 mM HEPES buffer containing 2 mM ethylenediamine tetraacetic acid, 2 mM dithiothreitol, and 0.3% Triton X-100 at 0°C. This was followed by centrifugation at 100,000 3 g for 1 h. The TGase 1 activity of the supernatant was assayed by measuring incorporation of [3H]putrescine into methylated α-casein in 250 mM Tris-HCl pH 8.3, 5 mM CaCl2, 5 mM dithiothreitol, 0.3% Triton X-100. Samples were incubated for 30 min in a final volume of 0.25 ml at 35°C. After acidification with 10% trichloroacetic acid, the precipitated casein was recovered on Whatmann GF/A filters, rinsed, and counted in a liquid scintillation counter (Thacher and Rice, 1985). The activity was defined by incorporation of [3H]putrescine (cpm) into casein per mg protein. Northern blotting Total cellular RNA was isolated by the acid guanidium thiocyanate phenol-chloroform extraction method (Chomozynski and Sacchi, 1987). Fifteen micrograms total RNA per lane was electrophoresed in 1.5% formaldehyde-agarose gel. The RNA was then transferred to Hybond N filters (Amersham International, Amersham, U.K.) and prehybridized in 50% formamide solution containing 200 µg denatured salmon sperm DNA per ml, 5 3 SSPE (750 mM sodium chloride, 50 mM sodium phosphate, 5 mM ethylenediamine tetraacetic acid), 5 3 FBP (0.1% polyvinylpyrrolidone, 0.1% Ficoll 400, 0.1% bovine serum albumin), and 0.5% sodium dodecyl sulfate for 2 h at 42°C. Following this, the filters were hybridized for 16–24 h at 42°C in the same solution containing full-length human TGase 1 cDNA (Yamanishi et al, 1991) that had been labeled with [α-32P]dCTP using a nick translation labeling kit (Amersham International). After hybridization, the filters were washed for 5 min in each of three changes of 2 3 sodium citrate/chloride buffer (1 3 sodium citrate/chloride buffer: 150 mM sodium chloride, 15 mM sodium citrate) containing 0.1% sodium dodecyl sulfate at room temperature, and for 15 min in each of two changes of 0.2 3 sodium citrate/chloride buffer containing 0.1% sodium dodecyl sulfate at 42°C. The filters were exposed to Fuji RX films with enhancing screens for 24–72 h at –80°C. Transient expression assay Normal human keratinocytes were cultured at a density of 2 3 104 cells per 2 ml of medium in 35 mm dishes. On the next

Figure 1. Inhibition of cell growth by CS. Normal human keratinocytes were treated on day 1 with CS at concentrations of 5 µM (r), 10 µM (d), 25 µM (m), and 50 µM (j). Control (u) was treated with 0.25% methanol used as a solvent of CS. Medium-containing CS was changed on days 3 and 5.

day, the DNA was transfected into the cells by lipofection using N-[1-(2,3dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulfate (Boehringer, Mannheim, Germany), according to the manufacturer’s instruction. After incubation at 37°C for 6 h, the medium was changed and the cells were incubated further in the presence of 25 µM CS, 10 nM TPA, or 0.12 mM Ca21 for 48 h. The cells were then harvested and assayed for luciferase and βgalactosidase activities. Luciferase activity was assayed using a PicaGene Kit (PGK-L500, TOYO INK, Tokyo, Japan). β-galactosidase activity was measured using a β-galactosidase assay system (Promega, Madison, WI). The luciferase activities were normalized relative to β-galactosidase activities and protein concentrations. The experiments were repeated at least three times. Construction of reporter plasmids Briefly, deletion fragments of the 59 upstream region of the human TGase 1 gene between –819 and –49 were generated by the polymerase chain reaction using the plasmid pd1L as the template (Ueda et al, 1996). These polymerase chain reaction products and the synthesized oligonucleotide corresponding to the DNA sequence from position – 20 to zero were inserted into the SmaI site of PGV-B, a luciferase reporter plasmid containing neither enhancer nor promoter sequences. For a more detailed description of the method, see Ueda et al (1996).

RESULTS Growth inhibition by CS In this study we used second and third passages of normal human keratinocytes in which keratinization can be induced under appropriate conditions. We first examined effects of CS on the growth of keratinocytes (Fig 1). At concentrations greater than 25 µM, CS inhibited markedly the growth but was not cytocidal as judged by cell morphology. At a concentration of 50 µM, the cells were firmly attached to each other and formed a confluent monolayer. Marginal inhibition was observed in cells treated with a concentration of 5 µM CS. These observations are similar to those of Denning et al (1995) for mouse primary keratinocytes grown in a medium containing a low concentration of Ca21. As compared with the action of CS, parental cholesterol at a concentration of 50 µM had no effect on either the growth or the morphology of keratinocytes. In all subsequent experiments, we used concentrations of CS ranging from 5 to 50 µM. Activation and expression of TGase 1 CS is formed as a consequence of keratinization and accumulates in the upper layer of the epidermis in parallel with an increase in TGase 1 activity (Jetten et al, 1989), thus suggesting a functional link between CS and TGase 1. Treatment of normal human keratinocytes with CS resulted in a doseand time-dependent induction of TGase 1 activity (Fig 2). It had increased significantly (more than 3-fold) by 48 h in cells treated with a concentration of 25 µM and continued to increase from 48 to 96 h.

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Figure 2. Activation of TGase 1 by CS in normal human keratinocytes. (A) Dose-dependent activation of TGase 1 when the cells were treated with the indicated concentrations for 48 h. (B) Time-dependent activation by treatment with 25 µM CS (d). Control (u) was treated with 0.25% methanol. Solid column, control treated with 25 µM cholesterol. The activity was defined by incorporation of [3H]putrescine (cpm) per mg protein. Significantly different from the control at *p , 0.05, **p , 0.01, ***p , 0.001.

By contrast, there was no increase of TGase 1 activity in cells treated with parental cholesterol. Activation of TGase 1 transcription Expression of TGase 1 gene was examined further by northern blotting analysis. As shown in Fig 3, CS activated the transcription of TGase 1 in normal human keratinocytes in a dose- and time-dependent manner. An increase of 2.8 kb mRNA of TGase 1 was evident in cells treated with 10 µM for 48 h. When treated with 25 µM CS, the transcription increased with time of exposure from 24 to 96 h. By contrast, no induction of transcription of TGase 1 was recorded in cells exposed to 25 µM cholesterol for as long as 96 h. Transcriptional activation of the 59 upstream regions of the TGase 1 gene Transcriptional activation of the TGase 1 gene by CS was confirmed by the luciferase assay. For this, the luciferase reporter gene was ligated to deletion fragments of the 59 promoter region of the TGase 1 gene. The constructs were transfected into normal human keratinocytes by lipofection using N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium methylsulfate with a transfection efficiency of 1.0%–1.5%. On the next day, the cells were

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Figure 3. Northern blotting of the TGase 1 mRNA of normal human keratinocytes treated with CS. (A) Dose-dependent expression of the TGase 1 mRNA when the cells were treated with CS for 48 h at 0 µM (lane 1), 5 µM (lane 2), 10 µM (lane 3), 25 µM (lane 4), 30 µM (lane 5), 50 µM (lane 6). (B) Time-dependent expression when treated with 25 µM CS. Lanes 1–4, untreated control group cultured for 24 h (lane 1), 48 h (lane 2), 72 h (lane 3), 96 h (lane 4); lanes 5–8, 25 µM CS-treated group cultured for 24 h (lane 5), 48 h (lane 6), 72 h (lane 7), 96 h (lane 8); lane 9, cultured with 25 µM cholesterol for 96 h.

treated for 48 h with either 25 µM CS, 10 nM TPA, or 0.12 mM Ca21, all of which induce terminal differentiation of keratinocytes. The results are shown in Fig 4. When the luciferase reporter plasmid containing the 819 bp upstream region of TGase 1 gene was transfected into the cells, substantial activity was induced by treatment with CS, TPA, or Ca21. To assess the sequence required for transcritional activation, we used deletion mutants of the 59 upstream region between –819 and –20. Deletion from –819 to –549 caused a total loss of luciferase activity induced by CS and TPA. For Ca21, the major loss of activity was observed when the region between –79 and –49 was deleted. The data presented here clearly indicate that CS, as well as TPA and Ca21, activates transcription of the TGase 1 gene and that the responsive element(s) for CS and TPA are located in the 59 upstream region of the gene between –819 and –549, whereas that for Ca21 is located between –79 and –49.

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Figure 4. Transcriptional activation of luciferase reporter gene under the control of 59 upstream regions from –819 to –20 in normal human keratinocytes. The TGase 1-luciferase reporter genes were transfected into the cells, followed by treatment with 25 µM CS (A), 10 nM TPA (B), and 0.12 mM Ca21 (C) for 48 h. Data represent the means of three samples normalized to β-galactosidase activity and protein concentrations.

DISCUSSION This study clearly demonstrated that CS activates transcription of the TGase 1 gene and that this results in induction of enzyme activity. Furthermore, by use of the luciferase reporter system, it has been possible to locate the responsive element(s) for CS at a position between –891 and –549 upstream of the TGase 1 gene. As a result of these findings, possible roles for CS and TGase 1 in the development of skin diseases with scaling disorder can be suggested. It is well known, for example, that in recessive X-linked ichthyosis, CS accumulates in the uppermost cornified layer of the skin due to a lack of steroid sulfatase (Webster et al, 1978; Kubilus et al, 1979; Yen et al, 1987). Although no plausible explanation has been put forward, this observation suggests that accumulated CS activates transcription of the TGase 1 gene resulting in scaling. To our knowledge, however, overexpression of the TGase 1 gene at the mRNA or protein levels or stimulation of its enzyme activity has not been reported for cases of recessive X-linked ichthyosis. In lamellar ichthyosis, another hereditary skin disease with scaling disorder, affected individuals have homozygous or heterozygous mutations in the TGase 1 gene, resulting in reduction in its expression and activity. In a previous study (Ikuta et al, 1994) we demonstrated that CS activates the η isoform of PKC, an isoform of PKC that is expressed

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in epithelial tissues in close association with differentiation (Osada et al, 1990, 1993; Koizumi et al, 1993). In the presence of CS, phorbol ester activated the η isoform only marginally, suggesting that CS itself acts as a second messenger for the η isoform. Denning et al (1995) confirmed and extended our observation by finding that CS activates the α, δ, ε, and ζ isoforms along with the η isoform. Furthermore, we have found that overexpression of the η isoform in normal human keratinocytes resulted in transcriptional activation of the TGase 1 gene (Ohba et al, 1998). These results suggest the possibility that transcriptional activation of TGase 1 by CS is mediated by the η isoform of PKC. The results of the luciferase assay using deletion constructs of the 59 upstream of the TGase 1 gene suggest that a putative responsive region(s) to CS and TPA is situated between positions –819 and –549. At position –679 there is a KTF-1-binding sequence, CCCCGAGGCT, which is closely related to the AP-2-binding site (Yamanishi et al, 1992). The KTF-1 was first identified as a Xenopus embryo nuclear factor that binds to epidermis-specific keratin gene (Snap et al, 1990, 1991). The KTF-1/AP-2 site is one of the candidates for transcriptional activation of the TGase 1 gene by CS and TPA. Other transcriptionfactor binding sequences within the region from –819 to –1 include AP-1 at –536; KER-1, a keratinocyte-specific transcription factor in the human K14 keratin gene (Leask et al, 1990), at –516 and –266; AP-2-like sites at –477, –430, and –400; and FP-1, a transcriptional element of human papilloma virus 18 (Vassar et al, 1989), at –25. In a previous study (Ueda et al, 1996), we reported the presence of a putative responsive region to the η isoform of PKC located between positions –95 to –67 of the TGase 1 gene. This is different from the findings reported here and may be attributable to the type of cells used. FRSK, the fetal rat skin keratinocyte cell line used in our previous study, showed higher basal and lower induced luciferase activities as compared with the normal human keratinocytes employed in this study. Using rat bladder epithelial cells, Mariniello et al (1995) demonstrated that three putative AP-2-like responsive elements between – 516 and –430 may be responsible for transcription of TGase 1 in the absence of transcriptional activators. These results suggest that the transcriptional regulation of TGase 1 is largely dependent on a cellular machinery(ies) mediating signals and achieving terminal differentiation. This work affords a new insight into the signal transduction pathway mediating keratinization. Examination of existing data leads to the conclusion that CS activates the η isform which in turn activates transcription of TGase 1, leading to keratinization. There may, however, be other processes involved in this pathway: firstly, the δ isoform of PKC may also take part in transcriptional activation because its overexpression by adenovirus vector similarly activates the TGase 1 gene in normal human keratinocytes (Ohba et al, 1998). Secondly, additional step(s) may intervene in the pathway from CS to TGase 1. Further studies are needed to elucidate the molecular mechanisms underlying keratinization.

This work is supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (#06281216).

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