Faculty Bibliography

Mapping studies of functional keratin genes in the human genome have localized most of the acidic keratin genes to chromosome 17q12-q21 and the basic keratin genes to chromosome 12q11-q13. Within the acidic keratin locus two clusters were identified, one containing the genes for K15 and K19, the other the genes for K14, K16, and K17. The relative positions and the distance between the two clusters have not been determined previously. In this paper we describe our analysis of P1 clones containing multiple acidic keratin genes, which were studied using restriction analysis and Southern blot hybridization with PCR-amplified probes specific for functional human keratin genes 15, 17, and 19. Our results show that the two clusters are very closely linked to each other, within a 55-kb region in the human genome. The genes are organized 5' to 3' in the following order: 5'-K19-K15-K17-K16-K14. Between K15 and K17 at least one additional, unidentified keratin gene is present

Keratin K17, while not present in healthy skin, is expressed under various pathological conditions, including psoriasis and cutaneous allergic reactions. The regulatory circuits involved in transcription of the human keratin K17 gene are poorly understood. To begin an analysis of the molecular mechanisms that regulate K17 gene transcription, we have studied the interactions between the nuclear proteins and the promoter region of the human K17 gene. That promoter region comprised 450 bp upstream from the translation initiation site. For these studies, we used electrophoretic mobility-shift assays, computer analysis, site-directed mutagenesis, and DNA-mediated cell transfection. In addition to the previously characterized interferon-gamma-responsive elements, we identified eight protein binding sites in the promoter. Five of them bind the known transcription factors NF1, AP2, and Sp1 and three others bind still unidentified proteins. Using site-directed mutagenesis, we have demonstrated the importance of the protein binding sites for the promoter function involved in both constitutive and interferon-induced expression of the K17 keratin gene

Keratinocyte growth factor (KGF) is a stromally derived paracrine mitogen that belongs to the fibroblast growth factor (FGF) family. It is secreted by dermal fibroblasts and specifically promotes keratinocyte proliferation. We have recently shown that epidermal growth factor (EGF) and transforming growth factor beta (TGF beta), modulators of keratinocyte proliferation, regulate expression of specific keratin genes. However KGF, unlike EGF and TGF beta, allows keratinocytes to differentiate normally. With this in mind, we sought to determine whether KGF may be involved in keratinocyte differentiation through a mechanism that does not involve regulation of keratin gene expression. We transfected human epidermal keratinocytes with ten different keratin gene promoters linked to a reporter gene, and grew the transfected cells in the presence or absence of KGF. Interestingly, no significant change in keratin gene regulation was observed in the presence of KGF relative to control. The possibility that KGF influences the induction of keratin gene expression by other keratinocyte modulators, such as EGF, TGF beta and gamma interferon (IFN gamma), was also explored. In these experiments, the transformed keratinocytes were exposed simultaneously to KGF and another modulator. KGF did not significantly change the effects of EGF, TGF beta or IFN gamma on keratin gene expression. KGF's lack of ability to directly regulate keratin gene expression suggests that KGF affects keratinocyte growth and differentiation through a pathway independent of keratin gene regulation. These results illustrate that regulation of keratinocyte proliferation can be separated from the regulation of keratin gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)

TGFbeta is an important regulator of epidermal keratinocyte function because it suppresses cell proliferation, while it induces synthesis of extracellular matrix proteins and their cells surface receptors. To examine whether TGFbeta affects synthesis of intracellular proteins as well, specifically the transcription of keratin genes, we transfected a series of DNA constructs that contain keratin gene promoters into human epidermal keratinocytes. The transfected cells were grown in the presence and absence of TGFbeta. We found that TGFbeta specifically induces transcription controlled by the promoters of K:5 and K:14 keratin genes, markers of basal cells. No other keratin gene promoters were induced. The effect of TGFbeta is concentration-dependent, can be demonstrated in HeLa cells, does not depend on keratinocyte growth conditions and can be elicited by both TGFbeta1 and TGFbeta2. We conclude that TGFbeta promotes the basal cell phenotype in stratified epithelia such as the epidermis

Keratinocytes are known to produce, store, and release IL-1 alpha and therefore we suspected that the DNA-mediated cell transfection procedure may release the stored IL-1 alpha from keratinocytes into the medium. Using enzyme-linked immunosorbent assay, we determined the IL-1 alpha concentration in culture supernatants during keratinocyte transfection. The following transfection methods were compared: lipofection with lipofectACE and lipofectAMINE (GIBCO), Ca3(PO4)2 co-precipitation, and polybrene-dimethylsulfoxide (DMSO). The supernatants were collected immediately prior to transfection, after 5-h incubation with lipofectin or Ca3(PO4)2, and 24 and 48 h after transfection. In the polybrene-DMSO method, the supernatant was also collected immediately before and after DMSO shock. LipofectAMINE caused the highest release of IL-1 alpha, whereas the lipofectACE and polybrene-DMSO mediated transfection with confluent cells released the least. The other two methods released intermediate levels of IL-1 alpha. Our data indicate that a substantial amount of IL-1 alpha is released during the keratinocyte transfection procedure, which can affect the results of transfection in studies of gene expression

Epidermal keratinocytes have important immunologic functions, which is apparent during wound healing, in psoriasis, and in allergic and inflammatory reactions. In these processes, keratinocytes not only produce cytokines and growth factors that attract and affect lymphocytes but also respond to the polypeptide factors produced by the lymphocytes. Gamma interferon (IFN-gamma) is one such signaling polypeptide. Its primary molecular effect is activation of specific transcription factors that regulate gene expression in target cells. In this work, we present a molecular mechanism of lymphocyte-keratinocyte signaling in the epidermis. We have induced cutaneous delayed-type hypersensitivity reactions that are associated with an accumulation of lymphocytes. These resulted in activation and nuclear translocation of STAT-91, the IFN-gamma-activated transcription factor, in keratinocytes in vivo and subsequent induction of transcription of keratin K17. Within the promoter of the K17 keratin gene, we have identified and characterized a site that confers the responsiveness to IFN-gamma and that binds the transcription factor STAT-91. Other keratin gene promoters tested were not induced by IFN-gamma. These results characterize at the molecular level a signaling pathway produced by the infiltration of lymphocytes in skin and resulting in the specific alteration of gene expression in keratinocytes

In most malignant and benign skin diseases, the normal pattern of keratin expression is altered. Among other phenotypic changes, the expression of hyperproliferation- and activation-associated keratins K:16 and K:6 is induced. Because the molecular mechanisms and the nuclear regulators involved in this induction are unknown, we have characterized the transcriptional regulators of expression of the keratin K:16 promoter. Our previous studies have shown that the transcription of K:16 is strongly and specifically induced in epidermal keratinocytes by epidermal growth factor (EGF), through the EGF-responsive element (RE). In the present work, using an electrophoretic mobility-shift assay, we have found several nuclear protein binding sites that have been identified as an Sp1 site, an AP2 site, the EGF-RE, and an enhancer element. The function of each site was assessed in transfection assays using specific deletions. Both the Sp1 and EGF-RE sites are essential for K:16 promoter activity. The site that functions as an independent enhancer, E, was found adjacent to and interacting with a sequence recognized by the AP2 transcription factor. This knowledge of the nuclear regulators of expression of the disease-associated K:16 keratin provides insight into the molecular parameters that might be important in skin diseases