No abstract

Epidermal keratinocytes undergo a program of differentiation that results in assembly of the epidermis (1). This differentiation process involves a series of morphological and biochemical changes that are tightly controlled and involve specific temporal and spatial changes in gene expression (1-3). These changes include activation of the gene that encodes human involucrin (hINV).1 Involucrin is not expressed in the basal epidermal layer, but expression is activated in the late spinous layer and continues in the granular layer (4, 5). Involucrin is an ␣-helical, rod-shaped, 68 kilodalton, glutamine-and glutamic acid-rich structural protein that is an efficient transglutaminase substrate (6 -9). During the final stages in keratinocyte differentiation, involucrin is incorporated, via the formation of interprotein ⑀-(␥-glutamyl)lysine cross-links with other proteins, into the keratinocyte cornified envelope (10). This envelope provides an essential protective barrier (11)(12)(13)(14). Involucrin play a similar role in other stratifying epithelia, including esophagus, cornea, ectocervix, vagina, etc. (15, 16). In each tissue, expression is confined to the suprabasal layers. We are interested in the regulation of hINV expression and identification of the regulatory elements responsible for the tissuespecific and differentiation-appropriate expression in these tissues. Recent studies indicate that a 6-kb segment of the hINV gene, including the hINV upstream regulatory region, the intron, and the coding sequence, targets expression to the appropriate tissues (16). Targeted expression is also observed when the upstream regulatory region is linked to the SV40 intron and ␤-galactosidase (17). In vitro studies show that the promoter is expressed in cells derived from stratifying epithelia (18,19), is regulated by agents that modulate differentiation (18 -20), and is not expressed in fibroblasts (18). Deletion mapping and point mutation studies identify two regions of the upstream regulatory region, the proximal regulatory region (PRR) and the distal regulatory region (DRR), that are required for optimal expression in cultured cells (18,19). Both the DRR and PRR contain AP1 sites, AP1-5 and AP1-1, respectively, that are required for optimal promoter activity (18). In addition, the PRR contains an Sp1 site that synergistically activates expression in conjunction with the AP1-5 site (19). In the present study, we evaluate the role of the DRR in mediating in vivo expression using a transgenic mouse model. Among other findings, we demonstrate that the DRR is necessary and sufficient for expression of the transgene in epidermis and other stratifying epithelia. MATERIALS AND METHODS Construction of hINV Transgenes-E13E was constructed byEcoRI digestion of phage, Charon 4AI-3 (21). A 13-kb EcoRI fragment was then subcloned into pBKS(ϩ) to yield pBKS-E13E. The EcoRI insert from this plasmid is shown in Fig. 1. The H6B transgene is a 6-kb HindIII/BamHI fragment that was derived by restricting Charon 4AI-3 with HindIII/BamHI and subcl...

In these mice, DNA synthesis is activated in suprabasal keratinocytes, leading to acanthosis, parakeratosis and enhanced desquamation. The full-length E6/E7 transcript and two alternately spliced products are produced and in most lines the predominant product is E6*. In the present study, we examine the effects of E6/E7 on cell cycle regulatory protein expression. E6/E7 expression in mouse epidermis is correlated with increased levels of the p53, p21, p27, cdk2, cdk4, cdk6, cyclin D1 and cyclin E regulatory proteins. Hyperproliferation is also observed in the buccal mucosa and the tongue epithelia of E6/E7 mice, and p53 levels are markedly increased in these epithelia. These results suggest that the major changes in cell cycle regulatory protein expression are in response to the presence of E7 and that E6 has a lesser impact.

No abstract