The taphonomic fate of isorenieratene in Lower Jurassic shales-controlled by iron?

Fossil derivatives of isorenieratene, an accessory pigment in brown-colored green sulfur bacteria, are often used as tracers for photic zone anoxia through Earth's history, but their diagenetic behavior is still incompletely understood. Here, we assess the preservation of isorenieratene derivatives in organic-rich shales (1.5-8.4 wt.% TOC) from two Lower Jurassic anoxic systems (Bächental oil shale, Tyrol, Austria; Posidonia Shale, Baden-Württemberg, Germany). Bitumens and kerogens were investigated using catalytic hydropyrolysis (HyPy), closed-system hydrous pyrolysis (in gold capsules), gas chromatography-mass spectrometry (GC-MS) and gas chromatography combustion isotope ratio-mass spectrometry (GC-C-IRMS). Petrography and biomarkers indicate a syngenetic relationship between bitumens and kerogens. All bitumens contain abundant isorenieratane, diverse complex aromatized isorenieratene derivatives, and a pseudohomologous series of 2,3,6-trimethyl aryl isoprenoids. In contrast, HyPy and mild closed-system hydrous pyrolysis of the kerogens yielded only minor amounts of these compounds. Given the overall low maturity of the organic matter (below oil window), it appears that isorenieratene and its abundant derivatives from the bitumen had not been incorporated into the kerogens. Accordingly, sulfur cross-linking, the key mechanism for sequestration of functionalized lipids into kerogens in anoxic systems, was not effective in the Jurassic environments studied. We explain this by (i) early cyclization/aromatization and (ii) hydrogenation reactions that have prevented effective sulfurization. In addition, (iii) sulfide was locally removed via anoxygenic photosynthesis and efficiently trapped by the reaction with sedimentary iron, as further indicated by elevated iron contents (4.0-8.7 wt.%) and the presence of abundant pyrite aggregates in the rock matrix. Although the combined processes have hampered the kerogen incorporation of isorenieratene and its derivatives, they may have promoted the long-term preservation of these biomarkers in the bitumen fraction via early defunctionalization. This particular taphonomy of aromatic carotenoids has to be considered in studies of anoxic iron-rich environments (e.g., the Proterozoic ocean).

Novel mechanism of steroid action in skin through glucocorticoid receptor monomers.

Glucocorticoids (GCs), important regulators of epidermal growth, differentiation, and homeostasis, are used extensively in the treatment of skin diseases. Using keratin gene expression as a paradigm of epidermal physiology and pathology, we have developed a model system to study the molecular mechanism of GCs action in skin. Here we describe a novel mechanism of suppression of transcription by the glucocorticoid receptor (GR) that represents an example of customizing a device for transcriptional regulation to target a specific group of genes within the target tissue, in our case, epidermis. We have shown that GCs repress the expression of the basal-cell-specific keratins K5 and K14 and disease-associated keratins K6, K16, and K17 but not the differentiation-specific keratins K3 and K10 or the simple epithelium-specific keratins K8, K18, and K19. We have identified the negative recognition elements (nGREs) in all five regulated keratin gene promoters. Detailed footprinting revealed that the function of nGREs is to instruct the GR to bind as four monomers. Furthermore, using cotransfection and antisense technology we have found that, unlike SRC-1 and GRIP-1, which are not involved in the GR complex that suppresses keratin genes, histone acetyltransferase and CBP are. In addition, we have found that GR, independently from GREs, blocks the induction of keratin gene expression by AP1. We conclude that GR suppresses keratin gene expression through two independent mechanisms: directly, through interactions of keratin nGREs with four GR monomers, as well as indirectly, by blocking the AP1 induction of keratin gene expression.

Disease-activated transcription factor: allergic reactions in human skin cause nuclear translocation of STAT-91 and induce synthesis of keratin K17.

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.

The homeoprotein DLX3 and tumor suppressor p53 co-regulate cell cycle progression and squamous tumor growth.

Epidermal homeostasis depends on the coordinated control of keratinocyte cell cycle. Differentiation and the alteration of this balance can result in neoplastic development. Here we report on a novel DLX3-dependent network that constrains epidermal hyperplasia and squamous tumorigenesis. By integrating genetic and transcriptomic approaches, we demonstrate that DLX3 operates through a p53-regulated network. DLX3 and p53 physically interact on the p21 promoter to enhance p21 expression. Elevating DLX3 in keratinocytes produces a G1-S blockade associated with p53 signature transcriptional profiles. In contrast, DLX3 loss promotes a mitogenic phenotype associated with constitutive activation of ERK. DLX3 expression is lost in human skin cancers and is extinguished during progression of experimentally induced mouse squamous cell carcinoma (SCC). Reinstatement of DLX3 function is sufficient to attenuate the migration of SCC cells, leading to decreased wound closure. Our data establish the DLX3-p53 interplay as a major regulatory axis in epidermal differentiation and suggest that DLX3 is a modulator of skin carcinogenesis.

Rays and arrays: the transcriptional program in the response of human epidermal keratinocytes to UVB illumination.

The epidermis, our first line of defense from ultraviolet (UV) light, bears the majority of photodamage, which results in skin thinning, wrinkling, keratosis, and malignancy. Hypothesizing that skin has specific mechanisms to protect itself and the organism from UV damage, we used DNA arrays to follow UV-caused gene expression changes in epidermal keratinocytes. Of the 6,800 genes examined, UV regulates the expression of at least 198. Three waves of changes in gene expression can be distinguished, 0.5-2, 4-8, and 16-24 h after illumination. The first contains transcription factors, signal transducing, and cytoskeletal proteins that change cell phenotype from a normal, fast-growing cell to an activated, paused cell. The second contains secreted growth factors, cytokines, and chemokines; keratinocytes, having changed their own physiology, alert the surrounding tissues to the UV damage. The third wave contains components of the cornified envelope, as keratinocytes enhance the epidermal protective covering and, simultaneously, terminally differentiate and die, removing a carcinogenic threat. UV also induces the expression of mitochondrial proteins that provide additional energy, and the enzymes that synthesize raw materials for DNA repair. Using a novel skin organ culture model, we demonstrated that the UV-induced changes detected in keratinocyte cultures also occur in human epidermis in vivo.

Structural and biochemical changes underlying a keratoderma-like phenotype in mice lacking suprabasal AP1 transcription factor function.

Epidermal keratinocyte differentiation on the body surface is a carefully choreographed process that leads to assembly of a barrier that is essential for life. Perturbation of keratinocyte differentiation leads to disease. Activator protein 1 (AP1) transcription factors are key controllers of this process. We have shown that inhibiting AP1 transcription factor activity in the suprabasal murine epidermis, by expression of dominant-negative c-jun (TAM67), produces a phenotype type that resembles human keratoderma. However, little is understood regarding the structural and molecular changes that drive this phenotype. In the present study we show that TAM67-positive epidermis displays altered cornified envelope, filaggrin-type keratohyalin granule, keratin filament, desmosome formation and lamellar body secretion leading to reduced barrier integrity. To understand the molecular changes underlying this process, we performed proteomic and RNA array analysis. Proteomic study of the corneocyte cross-linked proteome reveals a reduction in incorporation of cutaneous keratins, filaggrin, filaggrin2, late cornified envelope precursor proteins, hair keratins and hair keratin-associated proteins. This is coupled with increased incorporation of desmosome linker, small proline-rich, S100, transglutaminase and inflammation-associated proteins. Incorporation of most cutaneous keratins (Krt1, Krt5 and Krt10) is reduced, but incorporation of hyperproliferation-associated epidermal keratins (Krt6a, Krt6b and Krt16) is increased. RNA array analysis reveals reduced expression of mRNA encoding differentiation-associated cutaneous keratins, hair keratins and associated proteins, late cornified envelope precursors and filaggrin-related proteins; and increased expression of mRNA encoding small proline-rich proteins, protease inhibitors (serpins), S100 proteins, defensins and hyperproliferation-associated keratins. These findings suggest that AP1 factor inactivation in the suprabasal epidermal layers reduces expression of AP1 factor-responsive genes expressed in late differentiation and is associated with a compensatory increase in expression of early differentiation genes.

Interleukin-1 alpha is released during transfection of 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.

Comparison of methods for transfection of human epidermal keratinocytes.

Several methods for DNA-mediated cell transfection were tested to determine the optimal conditions for transfection of human epidermal keratinocytes. The following methods were compared: electroporation, lipofection, Ca3(PO4)2 co-precipitation, DEAE-dextran, and polybrene-mediated transfection. The transfected DNA included human keratinocyte-specific promoter for keratin K14 as well as SV40 and RSV viral promoters. Enzyme assays and in situ staining were used to evaluate both quantitative and qualitative aspects of transfection, and both subconfluent and post-confluent, stratifying keratinocytes were examined. Lipofection, Ca3(PO4)2 co-precipitation, and polybrene methods transfect very efficiently, but lipofection is expensive and Ca++ in the co-precipitation procedure induces keratinocytes to differentiate. We have found that polybrene-mediated transfection followed by a 27% DMSO shock is optimal for introducing DNA into human epidermal keratinocytes.

Regulation of epidermal expression of keratin K17 in inflammatory skin diseases.

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.

Regulation of keratin gene expression: the role of the nuclear receptors for retinoic acid, thyroid hormone, and vitamin D3.

Keratinization, the orderly process of differentiation of epidermal keratinocytes from stratum basale to stratum corneum, is influenced by hormones and vitamins. We have used expression of epidermal keratins as a paradigm of keratinization processes and analyzed the effects of retinoic acid, thyroid hormone, and vitamin D3 on keratin gene expression. DNA constructs in which keratin gene promoters drive expression of reporter genes were co-transfected with vectors expressing nuclear receptors for the above molecules into various cell types. The keratin promoters studied included K3, K5, K10, K14, and K16. The recipient cell types were HeLa and primary cultures of rabbit corneal and esophageal epithelial cells and of human epidermal keratinocytes. We found that retinoic acid, via its nuclear receptor, suppresses expression of all the above-listed keratin genes. Thyroid hormone and its receptor similarly suppressed those genes. The site of interaction between these two receptors and the promoter sequences of K10 and K14 genes has been identified. Surprisingly, vitamin D3 and its receptor had no direct effect on keratin promoters. Our results suggest that a retinoic acid has a twofold effect on keratin gene expression: by regulating keratinocyte differentiation it determines which keratins are expressed, basal cell specific or differentiation specific; by direct interaction between its receptor and keratin genes, retinoic acid determines the total amount of keratin protein within the cell. Vitamin D3, on the other hand, also regulates keratinocyte differentiation, but does not directly interact with the keratin genes.

Identification of the retinoic acid and thyroid hormone receptor-responsive element in the human K14 keratin gene.

The promoter of human K14 keratin gene, specific for the basal layer of stratified epithelia, is regulated by nuclear receptors for retinoic acid and thyroid hormone. However, the DNA sequences responsible for this regulation have not yet been identified. To identify the retinoic acid-responsive site, we have devised a simple site-specific mutagenesis method and introduced mutations into the K14 keratin gene promoter. These mutations identify the retinoic acid-responsive site. The site consists of a cluster of consensus palindrome half-sites in various orientations. As shown previously, retinoic acid and thyroid hormone receptors can recognize and bind common sequences in regulated genes. Here, we describe mutations that abolish regulation by both receptors. Interestingly, the hormone-dependent and -independent regulatory sites of the thyroid hormone nuclear receptor can be separated. Clusters of half-sites that share structural organization with the K14 regulatory site were found in the K5 and K10 keratin gene promoters. Similar clusters may be responsible for retinoic acid-mediated transcription regulation in epidermis.

Keratins and the keratinocyte activation cycle.

In wound healing and many pathologic conditions, keratinocytes become activated: they turn into migratory, hyperproliferative cells that produce and secrete extracellular matrix components and signaling polypeptides. At the same time, their cytoskeleton is also altered by the production of specific keratin proteins. These changes are orchestrated by growth factors, chemokines, and cytokines produced by keratinocytes and other cutaneous cell types. The responding intracellular signaling pathways activate transcription factors that regulate expression of keratin genes. Analysis of these processes led us to propose the existence of a keratinocyte activation cycle, in which the cells first become activated by the release of IL-1. Subsequently, they maintain the activated state by autocrine production of proinflammatory and proliferative signals. Keratins K6 and K16 are markers of the active state. Signals from the lymphocytes, in the form of Interferon-gamma, induce the expression of K17 and make keratinocytes contractile. This enables the keratinocytes to shrink the provisional fibronectin-rich basement membrane. Signals from the fibroblasts, in the form of TGF-beta, induce the expression of K5 and K14, revert the keratinocytes to the healthy basal phenotype, and thus complete the activation cycle.

Nuclear proteins involved in transcription of the human K5 keratin gene.

Keratin K5 is expressed in the basal layer of stratified epithelia in mammals and its synthesis is regulated by hormones and vitamins such as retinoic acid. The molecular mechanisms that regulate K5 expression are not known. To initiate analysis of the protein factors that interact with the human K5 keratin gene upstream region, we have used gel-retardation and DNA-mediated cell-transfection assays. We found five DNA sites that specifically bind nuclear proteins. DNA-protein interactions at two of the sites apparently increase transcription levels, at one decrease it. The importance of the remaining two sites is, at present, unclear. In addition, the location of the retinoic acid and thyroid hormone nuclear receptor action site has been determined, and we suggest that it involves a cluster of five sites similar to the consensus recognition elements. The complex constellation of protein binding sites upstream from the K5 gene probably reflects the complex regulatory circuits that govern the expression of the K5 keratin in mammalian tissues.

Functional comparison of the upstream regulatory DNA sequences of four human epidermal keratin genes.

The promoters of epidermal keratin genes, K5, K6, and K10 were cloned and their functions compared with that of the previously described promoter of the K14 keratin gene in non-epithelial and transformed epithelial cell lines, as well as in primary cultures of cells derived from simple and stratified epithelia. The four promoters were functional only in epithelial cells. Although the promoter for the basal cell-specific, acidic-type K14 gene was active in all epithelial cells tested, its basic-type partner, K5, and the promoter for the hyper-proliferation-associated K6 were active only in primary cultures of stratified epithelia. The promoter for the epidermal differentiation-specific K10 keratin gene was active at a low level in primary cultures of stratified epithelial cells on non-epidermal origin. Thus, the K14 gene promoter is functional in all epithelial cells, but the upstream regions of the K5 and K6 keratin genes restrict their expression to stratified epithelia, whereas the epidermal determinants of the K10 gene are not in the proximal upstream sequences.

Interleukin-1 induces transcription of keratin K6 in human epidermal keratinocytes.

Keratinocytes respond to injury by releasing the proinflammatory cytokine interleukin-1, which serves as the initial "alarm signal" to surrounding cells. Among the consequences of interleukin-1 release is the production of additional cytokines and their receptors by keratinocytes and other cells in the skin. Here we describe an additional effect of interleukin-1 on keratinocytes, namely the alteration in the keratinocyte cytoskeleton in the form of the induction of keratin 6 expression. Keratin 6 is a marker of hyperproliferative, activated keratinocytes, found in wound healing, psoriasis, and other inflammatory disorders. Skin biopsies in organ culture treated with interleukin-1 express keratin 6 in all suprabasal layers of the epidermis, throughout the tissue. In cultured epidermal keratinocytes, the induction of keratin 6 is time and concentration dependent. Importantly, only confluent keratinocytes respond to interleukin-1, subconfluent cultures do not. In the cells starved of growth factors, epidermal growth factor or tumor necrosis factor-alpha, if added simultaneously with interleukin-1, they synergistically augment the effects of interleukin-1. Using DNA-mediated cell transfection, we analyzed the molecular mechanisms regulating the keratin 6 induction by interleukin-1, and found that the induction occurs at the transcriptional level. We used a series of deletions and point mutations to identify the interleukin-1 responsive DNA element in the keratin 6 promoter, and determined that it contains a complex of C/EBP binding sites. The transcription factor C/EBPbeta binds this element in vitro, and the binding is augmented by pretreatment of the cells with interleukin-1. The interleukin-1 responsive element is clearly distinct from the epidermal growth factor responsive one, which means that the proinflammatory and proliferative signals independently regulate the expression of keratin 6. Thus, interleukin-1 initiates keratinocyte activation not only by triggering additional signaling events, but also by inducing directly the synthesis of keratin 6 in epidermal keratinocytes, and thus changing the composition of their cytoskeleton.

Specific organization of the negative response elements for retinoic acid and thyroid hormone receptors in keratin gene family.

Retinoic acid and thyroid hormone are important regulators of epidermal growth, differentiation, and homeostasis. Retinoic acid is extensively used in the treatment of many epidermal disorders ranging from wrinkles to skin cancers. Retinoic acid and thyroid hormone directly control the transcription of differentiation-specific genes including keratins. Their effect is mediated through nuclear receptors RAR and T3R. We have previously identified the response element in the K14 gene, K14RARE/TRE, to which these receptors bind, and found that it consists of a cluster of five half-sites with variable spacing and orientation. To determine whether this specific structure is found in other keratin genes, we have mapped and analyzed the RARE/TRE elements in three additional epidermal keratin genes: K5, K6, and K17. We used three different approaches to identify these elements: co-transfection of promoter deletion constructs, gel-shift assays, and site-specific mutagenesis. We localized the RARE/TRE elements relatively close to the TATA box in all three promoters. All three RARE/TRE elements have a similar structural organization: they consist of clusters of 3-6 half-sites with variable spacing and orientation. This means that the clustered structure of the RARE/TREs is a common characteristic for keratin genes. RARE and TRE in the K5 promoter are adjacent to each other whereas in the K17 promoter they overlap. All three keratin REs bind specifically both RAR and T3R in gel-shift assays. Interestingly, addition of ligand to the receptor changes the binding pattern ofthe T3R from homodimer to monomer, reflecting the change in regulation from induction to inhibition.

Keratinocyte growth factor and keratin gene regulation.

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)

Epidermal signal transduction and transcription factor activation in activated keratinocytes.

In the area of biology, many laboratories around the world are dissecting and characterizing signal transduction mechanisms and transcription factors responsive to various growth factors and cytokines, in various cell types. However, because of the differences in systems used, it is not clear whether these systems coexist, whether they interact meaningfully, and what their relative roles are. Epidermal keratinocytes are the perfect cell type in which to integrate this knowledge, because in these cells these mechanisms are known to be relevant. Keratinocytes both produce and respond to growth factors and cytokines, especially in pathological conditions and during wound healing, when the physiology of keratinocytes is altered in a way specified by the presence of a subset growth factors and cytokines. In fact, growth factors and cytokines cause the major changes in gene expression and keratinocyte behavior in various cutaneous diseases. In some cases, such as in wound healing, these responses are highly beneficial; in others, such as in psoriasis, they are pathological. It is not clear at present which are operating in which conditions, which are important for the healing process and which are harmful. Growth factors and cytokines affect keratinocytes sometimes simultaneously, at other times individually. In this manuscript we describe the signal transduction pathways responsible for the effects of interferons, the EGF/TGF alpha family and the TNF alpha/IL-1 family of signaling molecules. We also describe the important transcription factors known to be functional in epidermis, with particular emphasis on those factors that are activated by growth factors and cytokines. Finally, we describe what is known about transcriptional regulation of keratin genes, especially those specifically expressed in pathological processes in the epidermis. We expect that the enhanced understanding of the pathways regulating gene expression in keratinocytes will identify the pharmacological targets, the signal transducing proteins and the corresponding transcription factors, used by growth factors and cytokines. This research will led to development of compounds precisely aimed at those targets, allowing us to isolate and inhibit the harmful side effects of growth factors and cytokines. Such compounds should lead to highly specific and therefore more effective treatments of the cutaneous disorders in which these pathways play significant roles.

Characterization of nuclear protein binding sites in the promoter of keratin K17 gene.

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.

Transcriptional regulators of expression of K#16, the disease-associated keratin.

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.