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E Fuchs

Publications and source records attributed to E Fuchs.

At least 127 records · Page 7Linked to original sources
PubMed1993

Probing keratinocyte and differentiation specificity of the human K5 promoter in vitro and in transgenic mice.

Keratins K5 and K14 form the extensive intermediate filament network of mitotically active basal cells in all stratified epithelia. We have explored the regulatory mechanisms governing cell-type-specific and differentiation stage-specific expression of the human K5 gene in transiently transfected keratinocytes in vitro and in transgenic mice in vivo. Six thousand base pairs of 5' upstream K5 sequence directed proper basal cell-specific expression in all stratified epithelia. Surprisingly, as few as 90 bp of the K5 promoter still directed expression to stratified epithelia, with expression predominantly in epidermis, hair follicles, and tongue. Despite keratinocyte-preferred expression, the truncated K5 promoter displayed departures from basal to suprabasal expression in epidermis and from outer root sheath to inner root sheath expression in the follicle, with some regional variations in expression as well. To begin to elucidate the molecular controls underlying the keratinocyte specificity of the truncated promoter, we examined protein-DNA interactions within this region. A number of keratinocyte nuclear proteins bind to a K5 gene segment extending from -90 to +32 bp and are functionally involved in transcriptional regulation in vitro. Interestingly, several of these factors are common to both the K5 and K14 promoters, although they appear to be distinct from those previously implicated in keratinocyte specificity. Mutagenesis studies indicate that factors binding in the vicinity of the TATA box and transcription initiation are responsible for the cell type specificity of the truncated K5 promoter.

3T3 Cells↗
PubMed1993

Visualization of 125I-endothelin-1 binding sites in human placenta and umbilical vessels.

Endothelin-1 (ET-1) has potent vasoconstrictor effects and thus may be involved in regulating fetoplacental vascular resistance. By using quantitative in vitro autoradiography, the 125I-ET-1 binding sites in human placentas and umbilical vessels were localized and quantified. A high density of specific 125I-ET-1 binding sites was found in the placental villi and vessels. In the media of umbilical vessels, affinity for the peptide was higher in arteries than in veins. In all structures, 125I-ET-1 binding was inhibited with unlabeled ET-1 in the picomolar range. Unrelated peptides such as oxytocin or atrial natriuretic peptide failed to compete for 125I-ET-1 binding. The localization of binding sites points to a regulation of hemodynamic functions of ET-1 on the fetal side of the placental circulation and supports evidence regarding ET-1 as a paracrine-acting vasoconstrictor.

Binding Sites↗
PubMed1993

Epidermal differentiation and keratin gene expression.

The epidermis of the skin is a stratified squamous epithelium, which plays an important protective role. It manifests this role by building an extensive cytoskeletal architecture, the unique feature of which is the presence of keratin filaments. There are two major pairs of keratins in the epidermis: one pair is expressed in dividing cells and the other expressed in terminally differentiating cells. As such, keratins provide useful biochemical markers to explore the molecular mechanisms underlying the balance between growth and differentiation in the epidermis. Here, I review what is currently known about epidermal growth and differentiation, and how an understanding of keratin gene expression has been useful in elucidating regulatory pathways in the skin.

Animals↗
PubMed1993

Epidermolysis bullosa simplex.

Epidermolysis Bullosa Simplex (EBS) is a genetic disorder usually characterized by an autosomal dominant mode of transmission in which the skin blisters in response to trivial mechanical trauma. There are several clinical variants of EBS, ranging from clinically mild to very severe and even lethal, but in all cases the primary lesion responsible for the blistering is trauma-induced lysis of the epidermal basal layer. Epidermal basal cells normally feature an extensive cytoplasmic network of 10 nm filaments made of keratins K5 and K14, and the architecture of this network is often perturbed in the epidermis of EBS patients. The recent advent of a variety of molecular genetic techniques has allowed us to study the effects of perturbing the keratin filament network in epidermal cells in situ, and test the possible implications for EBS. Thus, targeted expression of K14 mutants which disrupt 10 nm-filament assembly in the epidermal basal layer of transgenic mice causes a phenotype mimicking EBS remarkably well, suggesting that at least some cases of EBS might arise as a result of mutations in basal-specific keratin genes. Indeed, point mutations in either the K5 or K14 coding sequence have recently been discovered in several incidences of EBS, and compelling evidence that these mutations are indeed responsible for the disease has been provided. These recent findings and their implication for the function of 10 nm keratin filaments in epidermis are discussed in this article.

Animals↗
PubMed1992

[3H]Rauwolscine binding sites in the brains of male tree shrews are related to social status.

The aim of the present study was to investigate the influence of social status on central nervous alpha 2-adrenoceptors. Using the specific alpha 2-adrenoceptor antagonist [3H]rauwolscine, binding sites in the brains of dominant and subordinate male tree shrews were quantified by in vitro autoradiography. In 5 of the 14 brain structures investigated, subordinates had significantly lower numbers of binding sites than dominants. These structures were the solitary tract nucleus, the dorsal motor nucleus of the vagus, the periaqueductal gray, the perifornical region of the hypothalamus and the medial nucleus of the amygdala. These brain areas are all intimately involved in the regulation of autonomic functions and of emotional behavior. Also the affinities for [3H]rauwolscine differed between the groups. In 3 nuclei, the solitary tract nucleus, the periaqueductal gray and the medial nucleus of the amygdala, dominants had significantly higher Kd-values than subordinates. This demonstrates the presence of low affinity binding sites in dominants which do not exist in subordinates. It is suggested that the low number of [3H]rauwolscine binding sites in subordinates results from down-regulation of alpha 2-adrenoceptors by high levels of noradrenaline and/or adrenaline. The disappearance of low affinity [3H]rauwolscine binding sites may play an important role in the etiology of psychosocial stress.

Animals↗
PubMed1992

The genetic basis of epidermolytic hyperkeratosis: a disorder of differentiation-specific epidermal keratin genes.

Epidermolytic hyperkeratosis (EH) is a skin disease characterized by keratin filament clumping and degeneration in terminally differentiating epidermal cells. We have discovered that the genetic basis for EH resides in mutations in differentiation-specific keratins. Two of six distinct incidences of EH had a keratin 10 (K10) point mutation in a highly conserved arginine. Remarkably, this same residue is mutated in the basal epidermal K14 in three incidences of another skin disease, epidermolysis bullosa simplex (EBS). By genetic engineering, gene transfection, and 10 nm filament assembly, we show that this mutation is functionally responsible for the keratin filament clumping that occurs in basal (EBS) or suprabasal (EH) cells. These studies strengthen the link between filament perturbations, cell fragility, and degeneration first established with EBS. They also suggest a correlation between filament disorganization and either cytokinesis or nuclear shape, giving rise to the seemingly binucleate cells typical of EH.

Amino Acid Sequence↗
PubMed1992

Transgenic mice expressing a mutant keratin 10 gene reveal the likely genetic basis for epidermolytic hyperkeratosis.

Epidermolytic hyperkeratosis (EH; previously called bullous congenital ichthyosiform erythroderma) is an autosomal dominant skin disease of unknown etiology, affecting approximately 1 out of 300,000 people. It is typified by hyperkeratotic scaliness, blistering due to cytolysis within suprabasal epidermal cells, and hyperproliferation in basal cells. Histologically, EH epidermis exhibits a thickened stratum corneum and granular layer, with enlarged and irregular-shaped cells. Ultrastructurally, only suprabasal layers are affected, with three major aberrancies: (i) tonofilament clumping, (ii) nuclei and keratohyalin granules of irregular shape and size, and (iii) cell degeneration. We have discovered that transgenic mice expressing a mutant keratin 10 gene have the EH phenotype, thereby suggesting that a genetic basis for human EH residues in mutations in genes encoding suprabasal keratins K1 and K10. In addition, we show that (i) stimulation of basal cell proliferation can arise from a defect in suprabasal cells, and (ii) distortion of nuclear shape or aberrations in cytokinesis can occur when an intermediate filament network is perturbed.

Animals↗
PubMed1992

Interleukin 6: insights to its function in skin by overexpression in transgenic mice.

Interleukin 6 (IL-6) is a cytokine that mediates a wide range of inflammatory and immune responses. Its expression is elevated in inflammatory or immunodeficient diseases, including psoriasis, rheumatoid arthritis, and AIDS. To explore the role of IL-6 in skin, we utilized a human keratin 14 (K14) promoter to express IL-6 in the basal cells of stratified squamous epithelia of transgenic mice. Mice expressing the K14-IL-6 transgene were smaller than normal and exhibited retarded hair growth. Surprisingly, IL-6 expression did not lead to enhanced epidermal proliferation, but it did result in a thicker stratum corneum, with an otherwise seemingly normal program of differentiation. IL-6 expression did not lead to leukocytic infiltration, making it unlikely that it has direct proinflammatory activity in skin. Based on this study, one role of IL-6 relevant to host defense may be to enhance the stratum corneum, thereby providing increased protection from injurious stimuli or infection. If IL-6 plays additional roles in the skin, it is likely to act synergistically with factors that IL-6 alone cannot induce.

Animals↗
PubMed1992

Progressive high frequency hearing loss: an additional feature in the syndrome of congenital adrenal hypoplasia and gonadotrophin deficiency.

In an earlier report, we found that X-linked congenital adrenal hypoplasia may be associated with gonadotrophin deficiency. This combination has since been confirmed by many others. At the last examination, our patients were 22.4, 19.9 and 17.5 years old. They were doing well on replacement therapy with hydrocortisone, fluorohydrocortisone, and long-acting testosterone, but in all of them, a progressive hearing loss had appeared, starting at high frequencies at about 14 years of age. The loss progressed with age to lower frequencies, and the oldest patient had some remaining hearing capacity at 125-500 Hz only with a perceptive hearing loss of -95 dB at frequencies above 500 Hz. It is concluded that patients with this syndrome should be examined for hearing loss. X-linked adrenal hypoplasia may also be associated with glycerol kinase deficiency and myopathy. A molecular XP-deletion has suggested a locus for hypogonadotrophic hypogonadism distal to the glycerol kinase and adrenal hypoplasia loci. The observations in our patients suggest that the locus for at least this type of X-linked deafness may be in the same area.

Adolescent↗
PubMed1992

Distribution of 125I-endothelin-1,-2,-3 binding sites in mammalian kidneys.

1. Binding sites for 125I-labeled endothelin (ET) isopeptides ET-1, ET-2 and ET-3 were visualized by autoradiography in the kidneys of man, baboon, rhesus monkey, tree shrew, pig and rat. 2. Highest levels of binding for the three isoforms were observed in the glomeruli, cortical and medullary vessels of baboon, rhesus monkey, pig and rat, whereas there was no noticeable labeling of glomeruli in human and tree shrew kidneys. 3. The enrichment of binding sites depended on the species and peptide investigated, suggesting different affinities and/or densities of a heterogenous population of renal ET receptors.

Animals↗
PubMed1992

Do the ends justify the mean? Proline mutations at the ends of the keratin coiled-coil rod segment are more disruptive than internal mutations.

Intermediate filament (IF) assembly is remarkable, in that it appears to be self-driven by the primary sequence of IF proteins, a family (40-220 kd) with diverse sequences, but similar secondary structures. Each IF polypeptide has a central 310 amino acid residue alpha-helical rod domain, involved in coiled-coil dinner formation. Two short (approximately 10 amino acid residue) stretches at the ends of this rod are more highly conserved than the rest, although the molecular basis for this is unknown. In addition, the rod is segmented by three short nonhelical linkers of conserved location, but not sequence. To examine the degree to which different conserved helical and nonhelical rod sequences contribute to dimer, tetramer, and higher ordered interactions, we introduced proline mutations in residues throughout the rod of a type I keratin, and we removed existing proline residues from the linker regions. To further probe the role of the rod ends, we introduced more subtle mutations near the COOH-terminus. We examined the consequences of these mutations on (a) IF network formation in vivo, and (b) 10-nm filament assembly in vitro. Surprisingly, all proline mutations located deep in the coiled-coil rod segment showed rather modest effects on filament network formation and 10-nm filament assembly. In addition, removing the existing proline residues was without apparent effect in vivo, and in vitro, these mutants assembled into 10-nm filaments with a tendency to aggregate, but with otherwise normal appearance. The most striking effects on filament network formation and IF assembly were observed with mutations at the very ends of the rod. These data indicate that sequences throughout the rod are not equal with respect to their role in filament network formation and in 10-nm filament assembly. Specifically, while the internal rod segments seem able to tolerate considerable changes in alpha-helical conformation, the conserved ends seem to be essential for creating a very specific structure, in which even small perturbations can lead to loss of IF stability and disruption of normal cellular interactions. These findings have important implications for the disease Epidermolysis Bullosa Simplex, arising from point mutations in keratins K5 or K14.

Amino Acid Sequence↗
PubMed1992

The roles of K5 and K14 head, tail, and R/K L L E G E domains in keratin filament assembly in vitro.

Type I and type II keratins form obligatory heterodimers, which self-assemble into 10-nm intermediate filaments (IFs). Like all IF proteins, they have a central alpha-helical rod domain, flanked by nonhelical head and tail domains. The IF rod is more highly conserved than head and tail, and within the rod, the carboxy R/K L L E G E sequence is more highly conserved than most other regions. Mutagenesis studies have shed some light on the roles of the head, tail, and R/K L L E G E sequence in 10-nm filament structure. However, interpretations have often been complicated in part because many of these studies have focused on transfected cells, where filament structure cannot be evaluated. Of the few in vitro assembly studies thus far conducted, comparison of keratin mutants with other IF mutants have often been difficult, due to the obligatory heteropolymeric nature of keratin IFs. In this report, we describe in vitro filament assembly studies on headless, tailless, headless/tailless, and R/K L L E G E truncated mutants of keratin 5 and its partner keratin 14. Using varying conditions of ionic strength and pH, we examine effects of analogous K5 and K14 mutations on the stability of 10-nm filament structure. Using EM, we examine effects of mutations on the ability of subunits/protofibrils to (a) elongate and (b) laterally associate. Our results demonstrate that (a) tails of K5 and K14 are required for filament stabilization; (b) the head of K5, but not of K14, is required for filament elongation and lateral alignments; and (c) the R/K L L E G E domains are required for lateral alignments, but not for filament elongation.

Amino Acid Sequence↗
PubMed1992

Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice.

Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.

Animals↗