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, the myoepithelial keratin, is expressed in psoriasis but is not present in healthy skin. Psoriasis is associated with production of gamma interferon (IFN gamma), which induces the expression of keratin K17 by activating transcription factor STAT1. Our hypothesis states that the induction of K17 is specific for the inflammatory reactions associated with high levels of IFN gamma and activation of STAT1. One of the corollaries of the hypothesis is that the STAT1-activating cytokines should induce the expression of keratin K17, whereas those cytokines that work through other mechanisms should not. Furthermore, because the STAT activation pathway is dependent upon protein phosphorylation events, phosphorylation inhibitors should attenuate the induction of keratin K17, whereas protein phosphatase inhibitors should augment it. To test this hypothesis, we analyzed lesional samples of inflammatory diseases using immunofluorescence, transfected keratinocytes with K17 gene promoter DNAs in the presence of various cytokines, and followed nuclear translocation of STAT1 in keratinocytes using specific antibodies. Confirming the hypothesis, we found that K17 is induced in psoriasis and dermatitis caused by delayed type hypersensitivity, which are associated with high levels of IFN gamma, but not in samples of atopic dermatitis, which is not. Two cytokines, interleukin-6 and leukemia inhibitory factor, which can induce phosphorylation of STAT1, can also induce K17 expression, whereas interleukin-3, interleukin-4, interleukin-10, and granulocyte macrophage colony stimulating factor have no effect on K17 expression. As expected, staurosporine and genistein inhibited, whereas okadaic acid augmented, the induction of K17 by IFN gamma. Our data indicate that in inflammatory skin diseases, lymphocytes, through the cytokines they produce, differently regulate not only each other, but also keratin gene expression in epidermis one of their target tissues

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

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

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

The normal pattern of keratin expression in epidermis is altered in carcinomas as well as in nonmalignant diseases such as psoriasis and wound healing. Under these circumstances, the transcription of differentiation-specific keratins K1 and K10 is suppressed, whereas the activation- and hyperproliferation-associated keratins K6 and K16 are induced. Very little is known regarding transcriptional regulators involved in this switch. To investigate the nuclear factors that participate in regulation of expression of the K6 gene, we have characterized the binding sites for nuclear proteins on the promoter DNA of the K6 gene by gel retardation assays and site-specific deletion mutagenesis. We found four nuclear protein binding sites in the K6 gene promoter. Two are near the TATA box, but their ability to bind HeLa or keratinocyte nuclear extracts is independent of the TATA box-binding protein complex. The third binding site is a large palindrome. The sequences of these three sites do not correspond to any described target sequences for characterized transcriptional factors. The fourth is an AP-1 site, the target sequence for the proto-oncoproteins fos and jun. All four sites are independent of the previously characterized epidermal growth factor-responsive element, EGF-RE. These findings suggest that there may be two parallel pathways of induction of K6 transcription. One proceeds through the EGF-RE, which may be involved in nonmalignant hyperproliferation processes; the other, through the AP-1 site and the fos-jun proto-oncoproteins, may be related to induction in malignant processes