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I M Freedberg

Publications and source records attributed to I M Freedberg.

At least 37 records · Page 2Linked to original sources
PubMed1995

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)

Cell Division↗
PubMed1995

Message of nexin 1, a serine protease inhibitor, is accumulated in the follicular papilla during anagen of the hair cycle.

A group of specialized mesenchymal cells located at the root of the mammalian hair follicle, known as the follicular or dermal papillary cells, are involved in regulating the hair cycle, during which keratinocytes of the lower follicle undergo proliferation, degeneration and regrowth. Using the arbitrarily primed-PCR approach, we have identified a 1.3 kb messenger RNA that is present in large quantities in cultured rat follicular papillary cells, but not in skin fibroblasts. This mRNA encodes nexin 1, a potent protease inhibitor that can inactivate several growth-modulating serine proteases including thrombin, urokinase and tissue plasminogen activator. In situ hybridization showed that nexin 1 message is accumulated in the follicular papilla cells of anagen follicles, but is undetectable in keratinocytes or other skin mesenchymal cells. In addition, nexin 1 message level varies widely among several immortalized rat vibrissa papillary cell lines, and these levels correlate well with the reported abilities of these cell lines to support in vivo follicular reconstitution. These results suggest a possible role of nexin 1 in regulating hair follicular growth.

Amyloid beta-Protein Precursor↗
PubMed1994

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.

Cation Exchange Resins↗
PubMed1994

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.

Base Sequence↗
PubMed1994

Molecular effects of T lymphocytes on the regulation of keratin gene expression.

The interactions between lymphocytes and the epidermis are very important in autoimmune skin diseases. Mutual interaction between keratinocytes and T cells is effected both by soluble peptides and by direct cell-to-cell contact. We investigated the possibility that direct cell-to-cell contact with T cells may also play a role in the regulation of keratin gene expression. We have transfected human epidermal keratinocytes with the constructs containing promoters of keratin genes and then cocultured them with the HUT78 strain of human T cells. We found that T cells induce transcription of K5, K6, K14 and K16 genes, as well as the RSV viral promoter, but not K17, K10 or the SV40 viral promoter controls.

Cell Communication↗
PubMed1993

Epidermal growth factor and transforming growth factor alpha specifically induce the activation- and hyperproliferation-associated keratins 6 and 16.

Epidermal injury results in activation of keratinocytes which produce and respond to growth factors and cytokines and become migratory. Activated keratinocytes express a specific pair of keratin proteins, K6 and K16, distinct from the keratins in the healthy epidermis. Keratinocytes can be activated, for example, by binding of the appropriate ligands to the epidermal growth factor receptor (EGFR). We have analyzed the effects of EGFR activation on keratin gene transcription by transfecting DNAs containing keratin promoters linked to a reporter gene into primary cultures of human epidermal keratinocytes in the presence or absence of EGF or transforming growth factor alpha (TGF alpha), two growth factors that activate EGFR. The activation of EGFR had no effect on the promoters of simple epithelial, basal-layer-specific, or differentiation-specific keratins. In contrast, the expression of K6 and K16 was strongly and specifically induced. A 20-bp DNA segment of the K16 gene promoter conveyed the EGF regulation, functioned in a heterologous construct, and therefore constituted an EGF-responsive element. A nuclear protein specifically bound to this element and to the analogous sequence of the K6 promoter. Thus, EGF specifically induces K6 and K16, markers of activated keratinocytes, via nuclear proteins that bind to EGF-responsive elements in the promoters of these keratin genes.

Base Sequence↗
PubMed1993

Retinoid hepatitis.

A 65-year-old woman treated with etretinate for pityriasis rubra pilaris developed chronic active hepatitis. The elevated transaminases were noted 2 months after initiation of therapy and peaked 2 months after discontinuation of etretinate. The spectrum of liver toxicity induced by etretinate is reviewed. We suggest that reported cases of retinoid-induced liver disease can be divided into four distinct categories: nonspecific reactive hepatitis, acute hepatitis, chronic active hepatitis, and severe fibrosis or cirrhosis.

Aged↗
PubMed1993

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.

Base Sequence↗
PubMed1993

Human hair keratins.

Human hair keratins were among the first to be studied but it is only recently that sufficient information has been obtained to gain a basic biologic perspective of these proteins. Hair keratins are members of the intermediate filament family of proteins, yet are sufficiently divergent from epidermal keratins to warrant separate classification: type Ia and IIa ("hard"/hair keratins) and type Ib and IIb (epidermal and other "soft" keratins). As with hair keratins from other species, the human proteins may be distinguished from their epidermal counterparts by a relatively higher cysteine content, 7.6% versus 2.9%, respectively. This feature reflects utilization of disulfide bonding in producing a tougher, more durable structure in the tissues in which the hair keratins are distributed. Although prominent in hair, their distribution is not strictly limited to this tissue. A number of molecular characteristics have been elucidated from human hair keratin gene studies including amino acid sequence data for a type Ia hair keratin. Studies of various pedigrees has revealed a fairly wide latitude of variation in human hair keratin expression that is tolerated without associated obvious hair phenotypic change. Thus, a foundation of knowledge regarding these proteins has emerged and continues to evolve.

Amino Acid Sequence↗
PubMed1993

Keratin: a journey of three decades.

During the past three decades, major progress has been made in our understanding of the processes which lead to the formation of a keratinized epidermis in normal and pathologic situations. Stimulated by clinical studies of exfoliative dermatitis and related diseases, a series of investigations have been performed which proved initially that the pathways and controls of epidermal protein synthesis were equivalent to protein synthetic pathways in all other tissues of the body. Keratin was identified as not only an insoluble protein which makes up the vast majority of the intracellular protein in the stratum corneum, but as a member of the intermediate filament family of cytoskeletal proteins. Of all such proteins, the keratins are most complex, occurring in two families separable on the basis of size, structure and isoelectric point. The keratin intermediate filaments are heteropolymers of two paired components, one from each family. The pairs of keratins which form the intermediate filaments in basal and differentiated layers of epidermis and other epithelia have been defined and antibodies to specific keratins are now being used for diagnostic purposes. Sophisticated biochemical, physicochemical, and molecular biologic studies have led to complete definitions of almost all the epithelial keratin molecules and to cloning of their genes. These genes are currently being used in analyses of control of keratin expression and definition of the specific abnormalities associated with "keratinopathies" including epidermolysis bullosa simplex and epidermolytic hyperkeratosis.

Animals↗
PubMed1993

Solitary mastocytoma in an adult. Treatment by excision.

BACKGROUND: In approximately 65% of patients, mastocytosis presents between birth and 15 years of age. Although solitary mastocytomas usually appear within the first 3 months of life, in unusual circumstances they may appear in adulthood. OBJECTIVE: The rare entity of solitary mastocytoma in adulthood and the simple treatment method of excision are discussed. METHODS: Simple surgical excision without manipulation of the lesion was performed. RESULTS: The lesion was surgically excised without recurrence. CONCLUSION: Solitary mastocytoma is a rare lesion in adulthood. The differential diagnosis includes a melanocytic nevus, xanthogranuloma and leukemia cutis. Surgical excision offers a rapid, relatively simple and effective mode of treatment.

Adult↗
PubMed1993

Expression of the carcinoma-associated keratin K6 and the role of AP-1 proto-oncoproteins.

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.

Animals↗
PubMed1993

A cluster of five nuclear proteins regulates keratin gene transcription.

A common feature of all epithelial cells is the presence of keratin proteins that assemble into an intermediate filament cytoskeletal network. Whereas other cell types often use a specific master transcription factor to coordinate cell type-specific transcription, analysis of transcriptional regulation of keratin genes suggests that specific groupings of widely expressed transcription factors, acting on clusters of recognition elements in the promoter regions, confer epithelia-specific transcription. We define such a cluster of three sites that binds five transcription factors in the human K5 keratin gene. Within this cluster, an unusual Sp1 site binds the Sp1 transcription factor and two additional proteins. Flanking the Sp1 site are an AP2 site and another sequence, Site A; each binds a transcription factor. Similar clusters of recognition sites for the same five transcription factors have been also identified in other keratin genes. Such clusters may play a role in epithelia-specific expression of keratins.

Base Sequence↗
PubMed1993

On the role of AP2 in epithelial-specific gene expression.

Transcription factor AP2 plays an important role in transcription of keratin genes, and it has been suggested that AP2 confers epithelial specificity. Promoters of keratin genes contain AP2 sites, usually within tight clusters of binding sites for other nuclear transcription factors. The role of AP2 was examined by in vitro gel shift analysis, AP2 binding site mutagenesis, and stable and transient transfection experiments. Nonepithelial cells, such as GM10 fibroblasts and melanocytes, neither express keratin nor become phenotypically epithelial when transfected with an AP2-expressing vector. However, in 3T3 and HeLa cells, co-transfection of an AP2-expressing vector increases the level of transcription from keratin gene promoters. This increase requires an intact AP2 binding site. Thus, the role of AP2 in keratin gene expression is quantitative rather than qualitative. AP2 interacts with other transcription factors and may convey extracellular regulatory signals to the transcription complex in the promoters of keratin genes.

3T3 Cells↗
PubMed1992

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.

Animals↗
PubMed1992

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.

Base Sequence↗
PubMed1992

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.

Base Sequence↗