The Krü ppel-like family of transcription factors comprises genes that appear to have tissue-restricted functions. Expression of gut-enriched Krü ppel-like factor (GKLF) may be important in the switch from proliferation to differentiation in the squamous epithelium. We sought to determine transcriptionally mediated effects of GKLF on two promoters active in the esophageal squamous epithelium, namely the Epstein-Barr virus ED-L2 and human keratin 4 promoters. Both promoters contain a CACCC-like motif previously shown to bind GKLF. To determine whether GKLF regulates genes containing this element, we first demonstrated expression and then cloned the full-length human GKLF from an esophageal squamous carcinoma cell line. In a transient transfection system, GKLF increased the activity of both promoters >25-fold, localized to regions containing the CACCC-like element. Recombinant GKLF specifically binds the CACCC-like motif in both promoters. GKLF epitope-tagged protein leads to the formation of two proteins of 65 and 34 kDa. The chromatographically purified 65-kDa protein binds the CACCC-like element from both Epstein-Barr virus ED-L2 and keratin 4 promoters, which is not attenuated by the 34-kDa protein. In summary, GKLF is expressed in esophageal squamous epithelial cells and transcriptionally activates two esophageal epithelial promoters important at the transition toward differentiation.

Keratins are intermediate filaments of epithelial cells.Mutations in keratin genes expressed in skin lead to human disorders, including epidermolysis bullosa simplex and epidermolytic hyperkeratosis. We examined the role of keratin 4 (K4) in maintaining the integrity of internal epithelial linings by using gene targeting to generate mice containing a null mutation in the epithelial K4 gene. Homozygous mice that do not express K4 develop a spectrum of phenotypes that affect several organs which express K4 including the esophagus, tongue, and cornea. The cellular phenotypes include basal hyperplasia, lack of maturation, hyperkeratosis, atypical nuclei, perinuclear clearing, and cell degeneration. These results are consistent with the notion that K4 is required for internal epithelial cell integrity. As mutations in K4 in humans lead to a disorder called white sponge nevus, the K4-deficient mice may serve as models for white sponge nevus and for understanding the role of K4 in cellular proliferation and differentiation.

Zf9/CPBP/KLF6 is a widely expressed member of the Kru «ppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that Zf9 was highly expressed in human esophageal squamous cancer cell lines. Additionally, Zf9 localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, Zf9 transactivates the human keratin 4 (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of Zf9 and GKLF/KLF4, which is also a member of the Kru «ppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between GKLF and Zf9, a novel finding for Kru «ppel-like family members.z 2000 Federation of European Biochemical Societies.

We previosuly described the oral-esophageal tissuespeci®c expression of cyclin D1 with the Epstein ± Barr virus ED-L2 promoter in transgenic mice, and resulting dysplasia. Given the evidence for an interplay between environmental and genetic factors in esophageal squamous carcinogenesis, the aim of this study was to determine the potential cooperation of the nitrosamine compound N-nitrosomethylbenzylamine (NMBA), an esophageal speci®c carcinogen, in the cyclin D1 transgenic mice. NMBA was ®rst demonstrated to induce dysplasia in two strains of inbred mice, C57BL/ 6 and FVB/N. Subcutaneous NMBA was then administrated to wild type and transgenic mice beginning at 4 weeks of age. Mice were monitered for the duration of the study for general appearance, activity and weight, and were euthanized at 12 and 15 months. Histopathologic analysis revealed increased severity of dysplasia in cyclin D1 mice treated with NMBA compared with treated age-matched wild-type mice and untreated mice. There was also increased proliferating cell nuclear antigen (PCNA) expression in the esophagi of NMBA treated cyclin D1 mice. Taken together, these ®ndings suggest that a genetic alteration, speci®cally cyclin D1 overexpression and a chemical carinogen, NMBA, may cooperate to increase the severity of esophageal squamous dysplasia, a prominent precursor to carcinoma.

Protein carriers vary in their ability to increase the immunogenicity of poorly immunogenic or T-lymphocyte-independent antigens. We examined one such carrier, the outer membrane protein complex derived from Neisseria meningikidis serogroup B strain Bli, in an attempt to determine why this outer membrane protein complex was more immunogenic in young infants and in relevant animal models than two other carriers used in coqjugates made with Haemophilus influenzae type b polysaccharide, a T-cell-independent antigen.