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Human Molecular Genetics Pages 2223-2231

Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia
Introduction
Results
   In vitro TPA-inducible promoter activity of the 2.5 kb 5' upstream region of the human TGase 1 gene
   Generation of transgenic mice expressing the TGM1-lacZ fusion gene
   TGM1-lacZ transgene expression in adult tissues of transgenic mice
   The expression of the TGM1-lacZ transgene in the epidermis of fetal and neonatal transgenic mice
   The induction of the TGM1-lacZ transgene expression in the epidermis by TPA
Discussion
Materials And Methods
   Materials
   Cell culture
   Construction of plasmids
   Transient expression assay
   DNA microinjection and screening of transgenic mice
   [beta]-Gal histochemistry
   In situ hybridization
Acknowledgements
References

Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia

Activation of the human transglutaminase 1 promoter in transgenic mice: terminal differentiation-specific expression of the TGM1-lacZ transgene in keratinized stratified squamous epithelia Keiko Yamada, Masato Matsuki, Yohichi Morishima, Eiichiro Ueda, Kohich Tabata, Hirokazu Yasuno, Misao Suzuki1 and Kiyofumi Yamanishi*

Department of Dermatology, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602, Japan and 1Institute of Molecular Embryology and Genetics, Kumamoto University School of Medicine, 4-24-1 Kuhonji, Kumamoto 862, Japan

Received June 13, 1997; Revised and Accepted September 23, 1997

Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes [epsilon]-([gamma]-glutamyl) lysine cross-links of proteins to form the cell envelope at the periphery of cornified cells. A transient expression assay using a luciferase reporter gene linked to the 2.5 kb 5' upstream region of the human TGase 1 gene (TGM1) showed phorbol ester-responsive promoter activity in cultured normal human keratinocytes. To assess its promoter activity in vivo, we generated transgenic mice expressing the Escherichia coli[beta]-galactosidase gene (lacZ) directed by the 5' upstream region. [beta]-Galactosidase histochemistry revealed that the TGM1-lacZ transgene was expressed in terminally differentiating keratinocytes in upper layers of stratified squamous epithelia in embryonic, neonatal and adult transgenic mice. The expression pattern was similar to that of endogenous TGase 1 mRNA detected by in situ hybridization. Furthermore, topical application of a phorbol ester to adult tail skin enhanced expression of the transgene as well as TGase 1 mRNA in the epidermis. Thus, the 2.5 kb 5' upstream sequence of TGM1 includes elements regulating tissue- and terminal differentiation-specific gene expression in stratified squamous epithelia.

INTRODUCTION

Transglutaminases (EC 2.3.2.13) are Ca2+-dependent enzymes that catalyze protein cross-linking reactions between [gamma]-carboxamide groups of glutamine residues as acyl donors and primary amino groups of protein-bound lysines or small molecular weight amines as acceptors (1 ). When polypeptides participate in such cross-linking reactions, [epsilon]-([gamma]-glutamyl) lysine linkages are formed between the molecules. This cross-bridging produces stable structures composed of polymerized proteins, which are essential in a wide variety of biological processes to maintain cell and tissue integrity (2 ).

Transglutaminase 1 (TGase 1; keratinocyte TGase), a member of the transglutaminase family (3 ), is expressed primarily in stratified squamous epithelia (4 ), the homeostasis of which is maintained by proliferation and terminal differentiation of keratinocytes with the ordered expression of specialized genes (5 ). In the epidermis of skin, TGase 1 is predominantly expressed in the upper spinous and granular layers (6 -8 ), possibly to form the cell envelope at the periphery of cornified cells (3 ). The cell envelope is a highly insoluble structure, composed of [epsilon]-([gamma]-glutamyl) lysine cross-linked proteins including involucrin (9 ), cystatin-[alpha]/keratolinin (10 ,11 ), loricrin (12 ,13 ), elafin/SKALP (14 -16 ), cornifins/small proline-rich proteins (17 ,18 ), keratin intermediate filaments (15 ,19 ), filaggrin (15 ), envoplakin (20 ), annexin I and various other proteins (21 ). The cell envelope retards water loss through the epidermis and protects the internal milieu of the body against external mechanical stimuli, chemical injury and biological invasion.

The human TGase 1 gene (TGM1), which encodes a 92 kDa protein consisting of 816 amino acid residues, is located on 14q11.2 (22 ,23 ). A linkage study (24 ) has revealed that the TGM1 locus is linked to the autosomal recessive skin disorder lamellar ichthyosis (LI; MIM number 242100). LI often presents as `collodion baby' at birth, a condition of generalized erythroderma in which babies are encased with a translucent membrane and suffer from dehydration and infection due to impairment of skin barrier function (5 ,25 ). Later, LI develops with large brownish plate-like desquamations and epidermal hyperkeratosis (26 ). Several studies describing mutations of TGM1 in LI patients suggest that the TGase 1 gene is involved in this skin disorder (27 -29 ), although LI is genetically heterogeneous since another locus for LI has been mapped on 2q33-q35 (30 ) and several cases of LI have been reported which have no mutation in the TGase 1 gene (29 ,31 ).

Analysis of TGase 1 gene regulation should lead not only to a more complete understanding of molecular mechanisms operating in the terminal differentiation of stratified squamous epithelia but also to new methods of treatment of LI, including gene therapy, based on the molecular pathogenesis of the disorder. TGase 1 gene expression can be induced by Ca2+ (32 -34 ), 12-O-tetradecanoylphorbol-13-acetate (TPA), (23 ,35 ,36 ), ganglioside GQ1b (34 ), and interferon-[gamma] (37 ) in cultured keratinocytes or in rabbit tracheal epithelial cells. Our group (38 ) and Saunders et al. (39 ) have reported that the 5' upstream region of TGM1 has promoter activity and is responsive to TPA, an activator of protein kinase C. However, it remains unclear whether the 5' upstream promoter of TGM1 actually directs gene expression in vivo as well as in vitro. Therefore, in this study we generated transgenic mice in which the 2.5 kb 5' upstream region of TGM1 was linked to the Escherichia coli [beta]-galactosidase ([beta]-gal) gene (lacZ) as a reporter in order to analyze its in vivo promoter activity.

RESULTS

In vitro TPA-inducible promoter activity of the 2.5 kb 5' upstream region of the human TGase 1 gene

To examine the promoter activity of the 2.5 kb 5' upstream region of TGM1, we constructed a reporter plasmid, termed pTG1-2.5Luc, which contains this upstream region linked to a luciferase reporter gene, and have used it for a transient transfection assay in normal human epidermal keratinocytes (NHEK) and HT-1080 fibrosarcoma cells. Treatment of NHEK cells transfected with pTG1-2.5Luc, with 1 nM TPA, an inducer of TGase 1 mRNA, increased the luciferase activity ~2-fold at 24 h (Fig. 1 A). On the other hand, when pTG1-2.5Luc was transfected into non-keratinocytic HT-1080 cells, luciferase activity was only slightly inducible by TPA (Fig. 1 B), although PGV-C, a positive control vector containing an SV40 promoter/enhancer, effectively induced luciferase activity (data not shown). The difference in luciferase induction between these two cell types implies that the activation of the TGase 1 promoter is specific to keratinocytes, the major cell lineage of stratified squamous epithelia, but not to fibroblastic cells. However, this in vitro transfection assay is not sufficient to characterize the tissue specific regulation of the promoter, and thus we established transgenic mouse lines and analyzed the in vivo regulation of the human TGase 1 promoter.


Figure 1.The in vitro promoter activity of the 2.5 kb 5' upstream region of the human TGase 1 gene. pTG1-2.5Luc, a reporter plasmid containing the 2.5 kb 5' upstream region of human TGase 1 gene linked to a luciferase gene, was transfected into NHEK cells (A) or into HT-1080 fibrosarcoma cells (B). The cells were treated in the presence or absence of 1 nM TPA for 24 h, then the luciferase activity was determined as described under Materials and Methods. The luciferase activity was normalized to [beta]-gal activity, an internal control for variation in transfection efficiencies. Error bars represent one standard error for triplicate experiments. PGV-B is a basic luciferase reporter vector used for construction of pTG1-2.5Luc.

Generation of transgenic mice expressing the TGM1-lacZ fusion gene

We generated transgenic mice using the transgene, TGM1-lacZ, which contains a 2.5 kb upstream sequence of the human TGase 1 gene, an SV40 intron, a [beta]-gal gene and an SV40 polyadenylation sequence (Fig. 2 A). Seventeen founder mice were obtained, four of which failed to generate lines. The transgene structure of the remaining 13 lines were examined by Southern hybridization (Fig. 2 B). Lines A1, F5, B4, F3, D2 and H6 showed some novel rearranged bands of the transgene. The transgene copy numbers of the transgenic lines were estimated at 1-24 copies per haploid genome (Fig. 2 B). We excluded lines C5, H2, D3 and H6 from further analysis because their fertility was low.A


Figure 2.The transgene TGM1-lacZ and Southern analysis of transgenic mice DNA. (A) The structure of TGM1-lacZ. The transgene, TGM1-lacZ was isolated from pTG1-2.5lacZ by digestion with KpnI and SalI. TGM1-lacZ consists of the 2.5 kb 5' upstream sequence of TGM1, an SV40 intron with splice donor/splice acceptor sites (*), the Escherichia coli [beta]-gal gene (lacZ) and an SV40 polyadenylation sequence (black box). K, KpnI; Bg, BglII; S, SalI. (B) Southern hybridization to integrated transgenes in F1 offspring of the established transgenic lines. 10 µg of BamHI-digested F1 tail genomic DNA was hybridized with a 32P-labeled 3.6 kb BamHI fragment of the [beta]-gal gene isolated from pTK[beta]. The copy number of integrated transgenes in F1 transgenic mice (indicated at the bottom of each lane) was estimated as described in Materials and Methods.

TGM1-lacZ transgene expression in adult tissues of transgenic mice

TGM1-lacZ transgene expression was examined in 6-14 week old F1 transgenic mice by [beta]-gal histochemistry. [beta]-Gal staining was detectable in all nine transgenic lines examined, but its intensity varied. Lines A1, B1 and B5 showed higher levels of [beta]-gal expression compared with the other transgenic lines. Table 1 summarizes the expression of the TGM1-lacZ transgene in adult transgenic mice of these lines. In these lines, skin, tongue, oral mucosa, esophagus, forestomach and vagina were stained blue using [beta]-gal histochemistry, but these same tissues taken from non-transgenic mice showed no [beta]-gal staining.

The localization of TGM1-lacZ expression was then examined in sections of these tissues (Fig. 3 ). Transgene expression was evident in the upper spinous and granular layers of the foot pad epidermis (Fig. 3 a), which was hyperplastic and hyperkeratotic. The stratified tail epidermis was likewise [beta]-gal-positive in the line B5 and only focally positive in lines A1 and B1. In contrast to the thickened epidermis of adult mice, TGM1-lacZ expression was undetectable in other parts of the skin, such as dorsal skin, where the epidermis was too thin to discriminate each stratified layer. The staining of hair follicles was most clearly observed in the tail of line B1 and it was localized to the inner root sheath (Fig. 3 b). In the tongue, TGM1-lacZ was expressed in the granular layers of the filiform papillae (Fig. 3 c). A part of the oral mucosa near the tongue and lips (data not shown) and the stratified squamous epithelia of the esophagus, forestomach and vagina also showed similar transgene expression patterns in the spinous and granular layers (Fig. 3 d, f and h). In the basal layers of these tissues, [beta]-gal staining was hardly detectable. All these [beta]-gal staining patterns in keratinized stratified squamous epithelia were reproducible for these lines and for different litters at the same age within each line.

Table 1 . Tissue-specific [beta]-gal expression in adult TGM1-lacZ transgenic mice
  [beta]-Gal expression
  Line A1 Line B1 Line B5
Skin
tail skin + + +++
foot pads + +++ +
hair follicles ++ +++ +
Tongue +++ +++ +++
Oral mucosa + + +
Esophagus +++ +++ +++
Forestomach +++ +++ +++
Vagina +++ +++ +++
Other tissuesa -b -b -b
+++, Strong staining; ++, staining in most mice; +, focal staining in most mice; -, no staining.
aBrain, eye, trachea, thymus, heart, lung, stomach, intestine, liver, spleen, kidney, uterus, bladder, bone and muscle were examined.
bEctopic expression was observed in some hepatocytes of line B1 and in some cells of brain, bone and blood vessels of line B5.


Figure 3. Expression of the TGM1-lacZ transgene in the keratinized stratified squamous epithelia of adult transgenic mice. The expression of the TGM1-lacZ transgene in tissues from adult transgenic mice was examined by [beta]-gal histochemistry. The sections show the skin of the foot pad (a), the hair follicles of the tail (b), and the epithelia of the tongue (c), the esophagus (d), the trachea (e), the forestomach (f), the stomach (g) and the vagina (h). Note that in (a), (d), (f) and (h), [beta]-gal staining was observed from the spinous to the granular layers of the keratinized stratified squamous epithelium; in (b), the inner root sheath cells were [beta]-gal-positive; in (c), [beta]-gal expression was localized in the granular layers of the filiform papilla; in (e) and (g), [beta]-gal staining was negative in the mucosa. Ectopic [beta]-gal staining was observed in some nerve cells in the brain of line B5 (I), in several periportal hepatocytes of line B1 (j), and in chondrocytes in part of the bone in line B5 (k). Bar, 50 µm in (a)-(d), (f)-(h) and (j); 25 µm in (e), (i) and (k).

The localization of the TGM1-lacZ transgene expression in these tissues was compared with the expression of endogenous TGase 1 mRNA, which was assessed by in situ hybridization using a digoxigenin (DIG)-labeled mouse TGase 1 cRNA. Representative results are shown in Figure 4 . Endogenous TGase 1 mRNA was expressed within the spinous and granular layers of the hyperplastic epidermis of foot pads (Fig. 4 A). In the thin epidermis of dorsal skin, where epidermal layers are not evident, TGase 1 mRNA was hardly detectable beneath the stratum corneum (data not shown). In the filiform papilla of the tongue, TGase 1 mRNA was localized in the granular layers (Fig. 4 B). The expression pattern of the TGM1-lacZ transgene was similar to that of TGase 1 mRNA in the esophagus (Fig. 4 C), forestomach and vagina (data not shown). TGase 1 mRNA was not evident in the basal layer of these stratified squamous epithelia.

Ectopic expression of the transgene in tissues other than stratified squamous epithelia was observed in the liver of line B1 and in parts of the brain, bone and blood vessels of line B5 (Fig. 3 i-k). Since it was not detected in at least two or more of the transgenic lines examined, we considered that the ectopic expression was a variation in the transgenic experiment. Indeed, in situ hybridization for TGase 1 mRNA was negative in these tissues.

The expression of the TGM1-lacZ transgene in the epidermis of fetal and neonatal transgenic mice

The TGM1-lacZ transgene expression in the embryonic stages at 12.5, 16.5, 17.5 and 18.5 days post conception (p.c.) was analyzed in lines A1, B1 and B5 by [beta]-gal histochemistry. At 16.5 days p.c. as well as at 12.5 days p.c., transgene expression was not detected except for ectopic expression in these lines (Fig. 5 A). At 17.5 days p.c., [beta]-gal staining in the epidermis of dorsal skin was only faintly detectable (Fig. 5 B). After 18.5 days p.c. (Fig. 5 C) and during the neonatal periods (Fig. 5 D), TGM1-lacZ expression was visible in the upper spinous and granular layers of the epidermis, the stratified layers of which were more evident than in adult dorsal skin. The staining intensity was prominent in the lower granular layers, and it lessened near the cornified layers. In neonatal skin, endogenous TGase 1 mRNA was also evident in these layers (Fig. 5 E). The localization of transgene expression was similar to each other in these transgenic lines, and the intensity of [beta]-gal stain was highest in line B5 embryos and neonates. The tongue, oral cavity and forestomach were also clearly [beta]-gal-positive in neonates (data not shown).


Figure 4. The localization of endogenous TGase 1 mRNA in keratinized stratified squamous epithelia. Expression of TGase 1 mRNA in the foot pad (A), tongue (B) and esophagus (C) was examined by in situ hybridization with a DIG-labeled mouse TGase 1 cRNA probe. TGase 1 mRNA was localized in upper layers of these stratified epithelia but not in the basal layer. The TGase 1 mRNA expression pattern was similar to that of the transgene shown in Figure 3. Arrowheads indicate the basement membrane. Bar, 50 µm.

The induction of the TGM1-lacZ transgene expression in the epidermis by TPA

To assess whether the human TGase 1 promoter is inducible in the stratified squamous epidermis in vivo, TPA was applied topically on the adult tail of transgenic lines B1, B5 and E3. After application of 100 µg TPA on the tail, a time-dependent expression of the TGM1-lacZ transgene was observed in these three lines. Even in a portion of line B1 tail epidermis in which the expression of [beta]-gal was hardly detectable, a marked induction of transgene expression was evident 48 h after the treatment (Fig. 6 A and B). Transgene expression was localized in the spinous and granular layers of the hyperplastic epidermis. Regardless of the intensity of staining in outer stratified layers, the transgene was not induced in the basal layer. TPA treatment induced the expression of TGase 1 mRNA in the suprabasal layers with a hyperplasia of the epidermis (Fig. 6 C and D), the localization of which was almost identical to that of TGM1-lacZ expression.


Figure 5. The expression of the TGM1-lacZ transgene in the epidermis of embryonic and neonatal transgenic mice. The dorsal skin from 16.5 days p.c. (A), 17.5 days p.c. (B) and 18.5 days p.c. (C) embryos and a neonate (D) of transgenic mice (line B5) was stained by [beta]-gal histochemistry, and the localization of TGase 1 mRNA in the neonatal skin (E) was examined by in situ hybridization with a DIG-labeled mouse TGase 1 cRNA probe. At 17.5 days p.c., transgene expression was only weakly visible in the epidermis. After 18.5 days p.c., it was evident in the upper spinous and granular layers. TGase 1 mRNA was also localized in the upper spinous and granular layers of the neonate epidermis. The arrowhead indicates the basement membrane. Bar, 50 µm.


Figure 6. The induction of the TGM1-lacZ transgene expression by TPA in the tail skin of transgenic mice. The tails of adult transgenic mice (line B1) were treated topically with 50 µl ethanol (vehicle) (A) and (C) or with 100 µg TPA in ethanol (B) and (D). After 48 h, expression of the TGM1-lacZ transgene (A) and (B) or endogenous TGase 1 mRNA (C) and (D) was examined. A marked induction of the TGM1-lacZ transgene and TGase 1 mRNA expression was observed in the epidermis 48 h after TPA treatment. Even during strong induction, the basal layer was still negative for expression. The arrowhead indicates the basement membrane. Bar, 50 µm.

DISCUSSION

In this transgenic study, we showed that the 2.5 kb 5' upstream sequence of human TGase 1 confers a tissue- and terminal differentiation-specific promoter activity in vivo. Although ectopic expression of the transgene was noted in some of the transgenic lines, the human TGase 1 promoter directs gene expression predominantly in upper layers of stratified squamous epithelia, suggesting that the regulation is operated in the late stage of keratinization. This pattern agrees closely with that of endogenous TGase 1 mRNA detected by in situ hybridization and TGase 1 immunoreactivity (4 ,6 ). Thus, the 2.5 kb 5' upstream sequence seems sufficient to direct appropriate expression of the transgene in these tissues. Recently, van Bokhoven et al. (40 ) reported that the TGase 1 gene is located only 1.4 kb downstream from the 3' end of the Rab geranylgeranyl transferase [alpha]-subunit gene. Therefore, the upstream sequence used for this transgenic study probably contains almost all of the 5'-regulatory elements required for human TGase 1 gene expression. Interestingly, based on TGase 1 promoter analysis by Mariniello et al. (41 ), it is likely that the 3' regulatory elements of this gene overlap the 5' regulatory elements of the TGase 1 gene.

The process of epidermal gene expression is organized sequentially during the embryonic stage of mice (42 ). The stratified squamous epidermis is completed during the last 4 days of development (43 ). Embryonic keratinocytes produce the differentiation-specific keratins, K1 and K10, from 15 days p.c. (44 ). Assembly of the cell envelope just beneath the plasma membrane occurs in the final stage of epidermal maturation. Bickenbach et al. have shown that loricrin, a major cell envelope precursor, is expressed at 16 days p.c. and is then cross-linked into the cell envelope (45 ). Since the TGM1-lacZ transgene is expressed in the epidermis from 17.5 days p.c., the schedule of human TGase 1 promoter regulation seems well-timed for production of the cell envelope according to the program of epidermal maturation.

The primary location of TGM1-lacZ transgene expression is the outer differentiating layers of the stratified squamous epithelia in transgenic mice. Transgene expression in dorsal skin was detectable in the thickened epidermis of neonatal mice but, unexpectedly, not in the thin epidermis of adult mice. It is probable that transgene expression might be below the sensitivity of [beta]-gal histochemistry that we used, since the levels of endogenous TGase 1 mRNA expression there were much lower than in neonatal epidermis and other thickened epidermis of adult foot pads and tail skin. We noticed that the [beta]-gal staining of keratinocytes often became less intense near the uppermost granular layers and was undetectable in the cornified layers. We speculate that reduction of [beta]-gal activity is associated with an event at the final stage of keratinization in which cellular components unnecessary to construct the cornified cells are digested by lysosomal enzymes (46 ). This may not be the case when an overexpression of [beta]-gal is achieved to overcome this degradation process. Indeed, in some sections of transgenic mice, an intense [beta]-gal staining was observed even in cornified layers.

The transgenic lines that we generated in this study may be useful to assess the in vivo effects of topically applied chemicals or drugs on the activation of the human TGase 1 promoter since TPA applied to adult tail skin up-regulates the promoter activity in the epidermis. Even following strong induction by TPA, the activation is restricted to the suprabasal layers, suggesting that the regulatory system of the TGase 1 promoter is functional only after the keratinocytes commit to terminal differentiation. We have shown that the human TGase 1 promoter is activated by nPKC[eta], a novel isoform of protein kinase C, in cultured keratinocytes (47 ). The in vivo expression pattern of nPKC[eta] is closely related to that of TGase 1 mRNA in normal and in hyperplastic psoriatic epidermis (48 -51 ), implying that nPKC[eta] is involved in TGase 1 promoter activation in the stratified squamous epithelia. With respect to the elements involved in TGase 1 gene regulation, Mariniello et al. has found an AP2-like response element from -516 to -507 in the 5' upstream sequence of the human TGase 1 gene, which binds an 85 kDa nuclear protein (41 ). In contrast, we have proposed that the TGase 1 5' upstream sequence from -95 to -67 in which two Sp1 consensus elements exist, is essential for TPA-responsive TGase 1 gene activation. We have shown that exogenous Sp1 activates the TGase 1 promoter regardless of cell type, and that Sp1 actually binds these Sp1 sites of the TGase 1 gene (52 ). Sp1 is also involved in the gene regulation of TGase 3, another keratinocyte-specific TGase (53 ). We have reported previously that exogenous c-JUN activates the TGase 1 promoter as well as an involucrin promoter (38 ,54 ), suggesting that c-JUN is also involved in the regulation of these genes. However, since the localization of these transcription factors in the epidermis does not necessarily coincide with the expression of TGase 1 mRNA (unpublished data) (55 ), the signaling system of nPKC[eta] and perhaps other unknown mechanisms might play important roles in TGase 1 gene regulation.

Wang et al. have recently reported that the human keratin 14 promoter is useful for expressing a foreign gene in keratinocytes and for transporting the gene product to the bloodstream using a K14 promoter-human growth hormone fusion gene (56 ). A vector based on the human keratin K1 promoter, established by Greenhalgh et al. (57 ,58 ), is also useful to direct gene expression exclusively in the epidermis. The human TGase 1 promoter, as for the involucrin promoter (59 ,60 ), confines gene expression to the upper layers of the stratified squamous epithelia. Therefore, these promoters may be suitable for keratinocyte gene therapy without affecting basal cells, the proliferation of which is essential to renewal and stratification of the epidermis. In addition, the activation level of the human TGase 1 promoter is controlled by topical application of an inducer, such as TPA. For gene therapy of homozygous gene defects or mutations, including LI (27 -29 ), the introduced gene must be expressed in the appropriate sites where the endogenous gene is activated. In contrast with vector systems for constitutive gene expression, such as the cytomegalovirus promoter (61 ), a vector using the TGase 1 promoter should provide an inducible gene therapy system, similar to a keratin 6a promoter vector (62 ), targeting terminally differentiating keratinocytes of the stratified squamous epithelia.

MATERIALS AND METHODS

Materials

Normal human epidermal keratinocytes (NHEK) and KGM keratinocyte culture medium were purchased from Clonetics. HT-1080 fibrosarcoma cells (63 ) were supplied by the Japanese Cancer Research Resources Bank (Foundation for Promotion of Cancer Research, Tokyo, Japan). The luciferase reporter plasmids PGV-B and PGV-C were purchased from TOYO INK MFG. Co., Ltd; TPA was from Sigma; and [[alpha]-32P]dCTP was from ICN. Oligonucleotides were synthesized using a DNA synthesizer Model 392 (Applied Biosystems, Inc.). All other reagents and chemicals were purchased from commercial sources and were either of reagent grade or the highest purity available.

Cell culture

NHEK cells were cultured in KGM according to the manufacturer's instructions. HT-1080 cells were cultured in Eagle's minimum essential medium supplemented with 10% fetal calf serum and non-essential amino acids (ICN Biochemicals Inc.) at 37°C under 5% CO2 in air. HT-1080 cells were subcultured every 3-4 days until they reached 80% confluence.

Construction of plasmids

The 2.5 kb 5' upstream region was derived from pcHETG, the cosmid clone including an entire structure of the gene (23 ). The fragment was inserted into a KpnI-BglII site of a basic luciferase reporter vector PGV-B to obtain the plasmid pTG1-2.5Luc. The plasmid pTG1-2.5lacZ was constructed by insertion of the fragment into a KpnI-SalI site of pBluescript II KS(+) (Stratagene) with a BglII-SalI fragment of pTK[beta] vector (Clontech Laboratories, Inc.) containing an SV40 intron with splice donor/splice acceptor sites, lacZ and an SV40 polyadenylation site. The transgene TGM1-lacZ was isolated from pTG1-2.5lacZ by digestion with KpnI and SalI and was used for microinjection.

Transient expression assay

NHEK and HT-1080 cells were seeded at a density of 1 × 105 and 6 × 104 cells, respectively, per 2 ml of medium in 35 mm dishes and incubated at 37°C under 5% CO2 in air. Two days later, DNA was transfected into the cells by lipofection using LipofectACE (Life Technologies, Inc.), according to the manufacturer's instructions. Transfected DNA consisted of 1.8 µg of reporter plasmid, 0.4 µg of pCMV[beta] vector (Clontech Laboratories, Inc.) as an internal control for variation in transfection efficiencies, and the appropriate amount of pGEM3Z (Promega Corp.) to standardize the total amount of DNA. After incubation at 37°C for 24 h, the medium was changed, and the cells were incubated with or without TPA for 24 h, then harvested and tested for luciferase and [beta]-gal assays. Luciferase activity was measured with a PicaGene Kit (PGK-L500, TOYO INK MFG. Co., Ltd) using a luminometer MONOLIGHT 2010 (Analytical luminescence laboratory). [beta]-Gal activity was assayed using a [beta]-gal Enzyme Assay System (Promega Corp.) and a microplate reader MODEL 450 (Bio-Rad). Each experiment was repeated at least three times, and representative results in which the luciferase activities from triplicate experiments were normalized with [beta]-gal activities are shown in the figures.

DNA microinjection and screening of transgenic mice

BDF1 (C57BL/6 × DBA/2) mice were used to obtain fertilized eggs, and several hundred molecules of TGM1-lacZ were microinjected into the pronucleus of fertilized eggs, as described previously (64 ). When the mice were 4 weeks of age, total nucleic acid was extracted from a piece of each mouse tail and used for screening by PCR and Southern hybridization. The PCR primers were U703, 5'-GCTCCCTCCCTAGCATCTTCT-3', and L1079, 5'-AACGACATGGTGACTTCTTTT-3'. Southern blots of BamHI-digested tail DNA were hybridized with a 32P-labeled 3.6 kb [beta]-gal gene isolated by BamHI digestion of pTK[beta]. Transgenic founders were subsequently mated to BDF1 mice, and transgene-positive offspring from this cross were likewise mated. Thus, the lines were propagated through successive generations. To estimate the copy number of integrated transgenes in F1 mice of each line, the radioactivity hybridized to tail DNA was compared with that of a copy number standard using a BAS2000 bio-imaging analyzer (FUJIX). Gestational days were counted from the presence of a vaginal plug in the morning referred to as 0.5 days post conception (p.c.) (65 ). The experimental procedures used for mice in this study were approved by the Committee for Animal Research, Kyoto Prefectural University of Medicine.

[beta]-Gal histochemistry

Tissues were removed from transgenic mice and fixed in 4% (w/v) paraformaldehyde in phosphate-buffered saline (100 mM sodium phosphate, 150 mM NaCl, pH 7.5) at 4°C for 1 h. The pre-fixed tissues were rinsed six times for 10 min each with rinse buffer, 100 mM sodium phosphate (pH 7.5), 2 mM MgCl2, 0.01% sodium deoxycholate and 0.02% (v/v) NP-40. They were then stained overnight with rinse buffer containing 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 1 mg/ml 5-bromo-4-chloro-3-indolyl-[beta]-D-galactoside at 30°C. The stained tissues were post-fixed in 10% formalin at 4°C and stored in 70% ethanol at 4°C until preparation of paraffin-embedded sections and counter-staining with nuclear fast red.

In situ hybridization

A 1.1 kb cDNA for mouse TGase 1 was amplified by RT-PCR of 1 µg total RNA from CD-1 mouse epidermis using the primers U205 5'-CCTTCTGGGCTCGCTGTGG-3' and L1353 5'-CAGAATCATGGTTCAGGTGCTC-3', which were designed on the basis of the homology between the human and rat TGase 1 cDNA sequences. The amplified cDNA was cloned in the pGEM-T vector (Promega Corp.) to obtain pMTG1-1.1. The insert cDNA hybridizes specifically to the 2.9 kb TGase 1 mRNA in northern blot analysis of 18.5 days p.c. mouse skin poly(A)+ RNA. After linearizing the plasmid pMTG1-1.1 by SalI digestion, antisense cRNA was synthesized with T7 RNA polymerase, using a digoxigenin (DIG) RNA Labeling Kit (Boehringer Mannheim). The cRNA was fragmented by limited alkaline hydrolysis and then used as a probe. For in situ hybridization of paraffin-embedded sections, we modified our previous protocol for frozen-sections (51 ,66 ). Mouse tissue sections were deparaffinized in xylene and rehydrated through a graded ethanol series. After proteinase K digestion (18 µg/ml), the sections were post-fixed with 4% (w/v) paraformaldehyde in phosphate-buffered saline for 10 min and treated with 0.1 M triethanolamine-HCl (pH 8.0) for 1 min. Following acetylation for 10 min, the sections were dehydrated, air-dried and then incubated overnight at 50°C in hybridization buffer composed of 50% formamide, 10 mM Tris-HCl (pH 7.5), 1 mg/ml yeast tRNA (Sigma), 1* Denhardt's solution (Sigma), 10% PEG6000, 600 mM NaCl, 0.25% SDS, 1 mM EDTA, and 0.2 µg/ml probe. After hybridization, the sections were washed at 45°C for 1 h in 50% formamide and 2* SSC, and digested with 20 µg/ml RNase (Sigma) in 10 mM Tris-HCl (pH 8.0) and 500 mM NaCl at 37°C for 30 min. Hybridized DIG-labeled probes were visualized with a Nucleic Acid Detection Kit (Boehringer Mannheim).

ACKNOWLEDGEMENTS

We would like to thank Prof. Mitsuhiro Kawata, Department of Anatomy, Kyoto Prefectural University of Medicine, for helpful discussions. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture of Japan.

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*To whom correspondence should be addressed. Tel/Fax: +81 75 251 5587; Email: kyamanis@koto.kpu-m.ac.jp

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