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
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1
Masato Matsuki
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1
Yohichi Morishima
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1
Eiichiro Ueda
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1
Kohich Tabata
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1
Hirokazu Yasuno
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1
Misao Suzuki
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1
Kiyofumi Yamanishi
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1
0
Molecular Embryology and Genetics, Kumamoto University School of Medicine
,
4-24-1 Kuhonji, Kumamoto 862
,
Japan
1
Department of Dermatology, Kyoto Prefectural University of Medicine
,
Kamigyo-ku, Kyoto 602
,
Japan
Transglutaminase 1 (TGase 1) is a tissue-specific enzyme which is expressed in the keratinized stratified squamous epithelia and which catalyzes e -(g -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 b -galactosidase gene (lacZ) directed by the 5 upstream region. b -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.
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Transglutaminases (EC 2.3.2.13) are Ca2+-dependent enzymes
that catalyze protein cross-linking reactions between
g -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, e -(g -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 (68), possibly to form the cell
envelope at the periphery of cornified cells (3). The cell envelope
is a highly insoluble structure, composed of e -(g -glutamyl) lysine
cross-linked proteins including involucrin (9), cystatin-a
/keratolinin (10,11), loricrin (12,13), elafin/SKALP (1416), 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
(2729), although LI is genetically heterogeneous since another
locus for LI has been mapped on 2q33q35 (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+ (3234),
12-O-tetradecanoylphorbol-13-acetate (TPA), (23,35,36), ganglioside GQ1b (34),
and interferon-g (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 t (...truncated)