title: Retinoic acid-induced normal and tumor-associated aberrant expression of the murine keratin K13 gene does not involve a promotor sequence with striking homology to a natural retinoic acid responsive element.
abstract: The type I keratin K13, normally restricted to suprabasal cells of internal stratified epithelia, is aberrantly expressed in 7,12-dimethylbenz[a]anthracene (DMBA)-12-O-tetradecanoyl-phorbol-13-acetate-induced murine epidermal tumors and constitutes an early marker of malignant progression. Aberrant K13 expression also occurs in epidermal cell lines derived from DMBA-TPA-induced tumors. As in cultured primary keratinocytes from normal internal stratified epithelia, the in vitro expression of K13 in transformed epidermal cell lines can be induced either by Ca2+ or by retinoic acid (RA). We have found that the promoter of the K13 gene contains a sequence element GGTTCA(N)5TGTTCT, in the following referred to as K13-RARE, that is highly related to the natural retinoic acid responsive element (RARE) of the retinoic acid receptor beta 2 gene. Both elements differ only in the second half-motifs, in which the first and sixth position is occupied by thymidines (K13-RARE) instead of adenines (beta 2-RARE), as well as in their pentameric spacer sequences. Despite this striking homology in the receptor binding domains, we show by transfection of reporter gene constructs of the elements into primary fore-stomach keratinocytes and transformed epidermal cell lines that in both cell systems, unlike beta 2-RARE, the wild-type K13-RARE completely lacks transactivating properties in the presence of RA. A recent hypothesis proposes that aberrant gene expression during tumorigenesis may occur through conversion of inactive response elements with high homology to hormone response elements into functional enhancers by carcinogen-induced point mutations at critical positions (Nawaz et al., Mol. Carcinogen., 7, 76-82, 1993). To investigate whether the aberrantly expressed K13 gene falls into the category of those genes, reporter gene constructs of K13-RARE variants in which either the initial or the terminal thymidine of the second half-motif was replaced by adenine were transfected into epidermal cell lines. Neither mutant exhibited RA-dependent transactivating properties. Strong transactivation could only be achieved by a K13-RARE mutant in which both critical thymidines were substituted by adenines. This type of closely spaced base exchange, unlikely to be created during DMBA initiation of mouse epidermis, was not detectable on sequencing genomic DNA of a squamous cell carcinoma and a transformed epidermal cell line. Instead, in both cases, only the wild-type K13-RARE could be demonstrated. A regulatory role of this RARE-like sequence in the promoter of the murine K13 gene for both normal and aberrant expression of the gene can therefore be excluded.
authors.affiliation: Research Program 2, German Cancer Research Center, Heidelberg.
authors.forename: H
authors.initials: H
authors.lastname: Winter
authors.forename: P
authors.initials: P
authors.lastname: Fink
authors.forename: J
authors.initials: J
authors.lastname: Schweizer
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene; D000818:Animals; D001483:Base Sequence; D004817:Epidermis / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D005809:Genes, Regulator; D007633:Keratins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D018168:Receptors, Retinoic Acid / Q000235:genetics*; D014212:Tretinoin / Q000633:toxicity*
chemical_list: D018168:Receptors, Retinoic Acid; D014212:Tretinoin; D015127:9,10-Dimethyl-1,2-benzanthracene; D007633:Keratins