Abstract
The suprabasal keratin 6 (K6) is remarkable among the keratins as, in addition to being constitutively expressed in different stratified epithelia, it is induced in epidermis under hyperproliferative conditions, such as benign or malignant tumors, psoriasis, and wound healing. In addition, this keratin is also induced in skin treated with 12-O-tetradecanoylphorbol-13-acetate or retinoic acid (RA). These characteristics make the study of K6 regulatory elements an especially interesting issue, in particular because these elements could be useful in designing gene constructs for the therapy of skin diseases. We have analyzed by mobility shift and footprinting experiments the cell type-specific enhancer of the bovine K6 beta gene (Blessing, M., Jorcano, J. L., and Franke, W. W. (1989) EMBO J. 8, 117-126) and have identified an AP-2-like element, two AP-1 elements (one of them composite), and a retinoic acid-responsive element (RARE). Mutagenesis experiments and cotransfections with retinoic acid receptors show that the RARE mediates enhancer activation by RA. Chloramphenicol acetyltransferase assays show that under normal culture conditions, the AP-1 element retains most of the enhancer transcriptional activity, while the RARE and AP-2 are weakly active. However, following RA treatment, the AP-1 element is repressed and the RARE is activated, resulting in an overall stimulation of the enhancer by RA in the BMGE+H cells used in our study. These results explain in part the complex and sometimes contradictory response of keratin 6 to hyperproliferative stimuli.
Highlights
MATERIALS AND METHODSPlasmid Constructs and DNA Sequencing—The nucleotide sequence of the BK6 5Ј-upstream region has been published previously [15] and can be obtained under GenBank/EMBL accession number X14478
In the last few years, much attention has been drawn toward keratins, since this broad family of cytoskeletal proteins expressed in epithelia has been found to be related to a series of epidermal hereditary diseases
These characteristics make the study of keratin 6 (K6) regulatory elements an especially interesting issue, in particular because these elements could be useful in designing gene constructs for the therapy of skin diseases
Summary
Plasmid Constructs and DNA Sequencing—The nucleotide sequence of the BK6 5Ј-upstream region has been published previously [15] and can be obtained under GenBank/EMBL accession number X14478. All constructs used were made by cutting plasmid pBLCAT2-EcoRI-XmnI (650 bp; called here EXm-CAT2) or pBLCAT3-BglII-BglII (2.2 kilobase pairs) [15], with appropriate restriction enzymes and cloning the desired fragments into the pBLCAT2 vector [16]. The obtained plasmids were designated X-CAT2, where X represents the corresponding restriction fragment from the BK6 enhancer present in the plasmid. All constructs were sequenced [17] to verify the presence of a correct insert and purified twice on a cesium chloride gradient. Chemical DNA sequencing was done according to Chupilo and Kravchenko [18]. Reporter plasmid pRSVLacZ contains the gene for -galactosidase under the
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