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Biomedical subjects

E Fuchs

Publications and source records attributed to E Fuchs.

At least 19 recordsLinked to original sources
PubMed1995

Gene targeting of BPAG1: abnormalities in mechanical strength and cell migration in stratified epithelia and neurologic degeneration.

BPAG1 is the major antigenic determinant of autoimmune sera of bullous pemphigoid (BP) patients. It is made by stratified squamous epithelia, where it localizes to the inner surface of specialized integrin-mediated adherens junctions (hemidesmosomes). To explore the function of BPAG1 and its relation to BP, we targeted the removal of the BPAG1 gene in mice. Hemidesmosomes are otherwise normal, but they lack the inner plate and have no cytoskeleton attached. Though not affecting cell growth or substratum adhesion, this compromises mechanical integrity and influences migration. Unexpectedly, the mice also develop severe dystonia and sensory nerve degeneration typical of dystonia musculorum (dt/dt) mice. We show that in at least one other strain of dt/dt mice, BPAG1 gene is defective.

Animals↗
PubMed1995

Lymphoid enhancer factor 1 directs hair follicle patterning and epithelial cell fate.

T cell-specific transcription factor (TCF-1) and lymphoid enhancer factor 1 (LEF-1) have been implicated exclusively in the regulation of T cell-specific genes. The only adult tissue other than thymus known to express these factors is spleen and lymph node, which contain low levels of LEF-1 and no TCF-1. We noticed that genes involved in hair-specific gene expression possess LEF-1/TCF-1 consensus motifs located in similar positions relative to their TATA box. We show that of the two factors only LEF-1 is expressed in hair follicles; it can be cloned in both splice forms from human skin keratinocytes and it can bind to these sites in the hair promoters. We show that LEF-1 mRNA is present in pluripotent ectoderm, and it is up-regulated in a highly restricted pattern just before the formation of underlying mesenchymal condensates and commitment of overlying ectodermal cells to invaginate and become hair follicles. New waves of ectodermal LEF-1 spots appear concomitant with new waves of follicle morphogenesis. To test whether LEF-1 patterning might be functionally important for hair patterning and morphogenesis, we used transgenic technology to alter the patterning and timing of LEF-1 over the surface ectoderm. Striking abnormalities arose in the positioning and orientation of hair follicles, leaving a marked disruption of this normally uniform patterning. This provides the first direct evidence that ectodermal cues are critical in establishing these developmental processes, which at later stages are known to be influenced by underlying mesenchyme. Remarkably, elevated LEF-1 in the lip furrow epithelium of developing transgenic animals triggered these cells to invaginate, sometimes leading to the inappropriate adoption of hair follicle and tooth cell fates. Collectively, our findings demonstrate that ectodermal expression of LEF-1 plays a central role in gene expression, pattern formation, and other developmental processes involving epithelial-mesenchymal associations.

Alternative Splicing↗
PubMed1995

Inhibition of skin development by targeted expression of a dominant-negative retinoic acid receptor.

Although pharmacological doses of retinoic acid (RA) have a wide variety of actions in vivo, experimental difficulties have prevented a definitive assignment of its physiological functions. We recently made a dominant-negative retinoic acid receptor (RAR) by a single amino-acid substitution which creates a dominant-negative thyroid hormone receptor. The mutated RAR efficiently inhibited the endogenous activities of RARs (alpha, beta, gamma). Thus, targeted expression of the mutated receptor should reveal RA functions during organogenesis by blocking RA signalling in the tissues concerned. To address this possibility, we expressed the dominant-negative RAR in the epidermis, a potential target organ of RA. We report here that the resultant transgenic mice exhibited dramatic suppression of epidermal maturation, demonstrating the requirement of RA in normal skin development.

Animals↗
PubMed1995

Chronic psychosocial stress induces morphological alterations in hippocampal pyramidal neurons of the tree shrew.

The effect of sustained psychosocial stress on the morphology of hippocampal pyramidal neurons was analysed in male tree shrews after 14, 20, and 28 days of social confrontation. A variety of physiological changes such as constantly elevated levels of urinary cortisol and norepinephrine and reduced body weight, which are indicative of chronic stress were observed in the subordinate, but not in the dominant males. Light microscopic analysis of Nissl-stained hippocampal sections showed that the staining intensity of the nucleoplasm in the CA1 and CA3 pyramidal neurons was increased after prolonged psychosocial stress, indicating a change in the nuclear chromatin structure. These alterations were observed only in subordinate animals and increased in a time dependent manner in accordance with the length of the stress period. There was, however, neither a reduction in density nor a degeneration of pyramidal neurons in chronically stressed animals. Mechanisms which may possibly account for the observed alterations are discussed.

Age Factors↗
PubMed1995

The importance of intramolecular ion pairing in intermediate filaments.

Nuclear and cytoskeletal networks of 10-nm intermediate filaments (IFs) are probably ubiquitous in multicellular eukaryotes. They likely play a role in maintaining the mechanical integrity of a cell. With the exception of the nuclear lamins, IF proteins can form IFs in vitro in the absence of cofactors or associated proteins. Below we present data suggesting that the large alpha-helical "rod" domains of IF proteins are stabilized by large numbers (up to 50) of intra-helical ion pairs formed by residues of opposite charge situated four residues apart. These many ion pairs, sometimes involving up to 30% of the residues within a coiled-coil IF segment, can potentially contribute as much as 10-25 kcal/mol (1 kcal = 4.18 kJ) to the stability of a single alpha-helical rod. Such stabilization is likely to play a major role in the chemical and physical stability of IF networks in vitro and in vivo. An investigation of other coiled-coil proteins shows that selection for intrahelical ion pairing is not simply a property intrinsic to coiled-coil proteins. Rather, there is a correlation between the degree to which there is selection for intrahelical ion pairs and the extent to which a coiled-coil protein participates in highly ordered multimolecular interactions--e.g., as in IFs and myosin thick filaments. The propensity of putative ion pairs in some IF proteins--e.g., epidermal keratins--suggests that an underlying structural stability at the level of the monomer may play an important role in the extraordinary stability of dimers and higher ordered structures in cytoplasmic IFs.

Amino Acid Sequence↗
PubMed1995

Modulation of binding sites for corticotropin-releasing hormone by chronic psychosocial stress.

This study was conducted to determine whether long lasting psychosocial stress would affect corticotropin-releasing hormone (CRH) binding sites in the brain, the pituitary, and the adrenal gland. As a model for sustained emotional stress we used chronic psychosocial conflict in male tree shrews. In subordinate tree shrews, repeated confrontation with a dominant conspecific results in constant hyperactivity of the HPA-axis and an elevated neurosympathetic tone. After 24 days of psychosocial conflict, CRH binding sites were quantified by in vitro-autoradiography with 125I-ovine CRH in 23 discrete brain regions, the pituitaries, and the adrenal glands of subordinate and control animals. Chronic stress significantly reduced the number of binding sites (Bmax) in the anterior lobe of the pituitary, the dentate gyrus, the CA1-CA3 areas of the hippocampus, and in both the stratum griseum superficiale and the stratum opticum of the superior colliculus. In cortical area 17, the reduction of Bmax was counterbalanced by an increase in the affinity (Kd) of the radioligand for the binding sites. A significant stress-induced enhancement of Bmax was observed in the frontal cortex, cingulate cortex, claustrocortex, the central and lateral nucleus of the amygdala, and in the choroid plexus. This increase was accompanied by a significant decrease of Kd-values in the frontal and cingulate cortex, the lateral nucleus of the amygdala, and the choroid plexus. These findings represent the first in vivo demonstration of a modulation of extrahypothalamic CRH receptors by a naturally occurring form of stress. The different response patterns of the central CRH binding sites reflect distinct neuroendocrine processes which are presumed to coordinate behavioral, autonomic, endocrine, and immune responses to long-lasting psychosocial conflict.

Adrenal Glands↗
PubMed1995

The basal keratin network of stratified squamous epithelia: defining K15 function in the absence of K14.

Keratin 5 and keratin 14 have been touted as the hallmarks of the basal keratin networks of all stratified squamous epithelia. Absence of K14 gives rise to epidermolysis bullosa simplex, a human blistering skin disorder involving cytolysis in the basal layer of epidermis. To address the puzzling question of why this disease is primarily manifested in skin rather than other stratified squamous epithelia, we ablated the K14 gene in mice and examined various tissues expressing this gene. We show that a key factor is the presence of another keratin, K15, which was hitherto unappreciated as a basal cell component. We show that the levels of K15 relative to K14 vary dramatically among stratified squamous epithelial tissues, and with neonatal development. In the absence of K14, K15 makes a bona fide, but ultrastructurally distinct, keratin filament network with K5. In the epidermis of neonatal mutant mice, K15 levels are low and do not compensate for the loss of K14. In contrast, the esophagus is unaffected in the neonatal mutant mice, but does appear to be fragile in the adult. Parallel to this phenomenon is that esophageal K14 is expressed at extremely low levels in the neonate, but rises in postnatal development. Finally, despite previous conclusions that the formation of suprabasal keratin filaments might depend upon K5/K14, we find that a wide variety of suprabasal networks composed of different keratins can form in the absence of K14 in the basal layer.

Amino Acid Sequence↗
PubMed1995

Expression of vascular endothelial growth factor and its receptors in human renal ontogenesis and in adult kidney.

Vascular endothelial growth factor (VEGF) may modulate vascular permeability, chemotaxis for monocytes, and protease activity. In addition, VEGF may play a role in embryonic and tumor angiogenesis. In fetal mouse kidney, VEGF mRNA and protein expression have been demonstrated. This finding led to the hypothesis that VEGF might be involved in renal growth and development. To further elucidate the role of VEGF in human kidney, expression of VEGF and its receptors, the specific tyrosine kinase receptors, fit-1 and KDR, were studied. In fetal (6-24 gestational wk; mesonephros and metanephros) and adult kidney, VEGF mRNA and protein could be colocalized in glomerular epithelia and collecting duct cells by in situ hybridization and immunohistology. By reverse transcription-polymerase chain reaction, mRNA of three VEGF isoforms, VEGF121, VEGF165, and VEGF189, were found in fetal kidney and cortex, isolated glomeruli, and medulla of adult human kidney. KDR and flt-1 mRNA were coexpressed in endothelia of glomeruli and in peritubular capillaries in fetal and adult kidney. These data support the assumption that VEGF and its receptors may influence renal ontogenesis. We speculate that the constitutive expression of VEGF in adult kidney may be required for the function of VEGF receptor positive-fenestrated endothelia in glomeruli and postglomerular vessels. The expression of VEGF in collecting duct and of its receptors in medullary capillaries may in addition be relevant for maintaining medullary osmolality.

Aged↗
PubMed1995

The A/B domain of truncated retinoic acid receptors can block differentiation and promote features of malignancy.

Recently, we discovered that stable introduction of a carboxyl-terminally truncated retinoic acid receptor gamma (tRAR gamma) into an epidermal keratinocyte line blocked the ability of these cells to differentiate, as judged by their failure to express late markers of squamous differentiation. We now demonstrate a correlation between the level of residual endogenous RAR activity of tRAR gamma-expressing keratinocyte lines and degree of terminal differentiation. Mutagenesis studies localize the effects to the A/B subdomain of the truncated receptor. Despite tRAR gamma's capacity to interfere with RAR-mediated transactivation of retinoic acid response elements (RAREs) in keratinocytes, the effects of the truncated receptor are independent of its ability to bind DNA and directly interact with endogenous RARs. tRAR alpha also inhibits RARE-mediated gene expression in keratinocytes, even though its full-length counterpart enhances RARE activity in these cells. Intriguingly, both tRAR gamma and RAR gamma suppress keratin promoter activity in epidermal cells, although for tRAR gamma, the effect is mediated through the A/B domain whereas for RAR gamma, the effects require DNA binding. Taken together, these findings suggest that the truncation allows for new and aberrant interactions with transcriptional proteins/cofactors that participate in governing RARE activity. This discovery may have relevance in tumorigenesis, where genetic lesions can result in mutant RARs or in loss of receptor expression.

Base Sequence↗
PubMed1994

Genetic and clinical mosaicism in a type of epidermal nevus.

BACKGROUND: Many skin disorders are characterized by a mosaic pattern, often with alternating stripes of affected and unaffected skin that follow the lines of Blaschko. These nonrandom patterns may be caused by a postzygotic mutation during embryogenesis. We studied the genetic basis of one such disorder, epidermal nevus of the epidermolytic hyperkeratotic type. Epidermolytic hyperkeratosis is an autosomal dominant blistering skin disease arising from mutations in the genes for keratin (K) 1 and 10. The offspring of patients with epidermal nevi may have generalized epidermolytic hyperkeratosis. METHODS: We studied the K1 and K10 genes in blood and in the keratinocytes and fibroblasts of lesional and nonlesional skin from three patients with epidermal nevi and four of their offspring with epidermolytic hyperkeratosis. RESULTS: In the patients with epidermal nevi, point mutations in 50 percent of the K10 alleles of epidermal cells were found in keratinocytes from lesional skin; no mutations were detected in normal skin. This mutation was absent or underrepresented in blood and skin fibroblasts. In the offspring with epidermolytic hyperkeratosis, the same mutations as those in the parents were found in 50 percent of the K10 alleles from all cell types examined. CONCLUSIONS: Epidermal nevus of the epidermolytic hyperkeratotic type is a mosaic genetic disorder of suprabasal keratin. The correlation of mutations in the K10 gene with lesional skin and the correlation of the normal gene with normal skin provide evidence that genetic mosaicism can cause clinical mosaicism.

Alleles↗
PubMed1994

A human keratin 14 "knockout": the absence of K14 leads to severe epidermolysis bullosa simplex and a function for an intermediate filament protein.

Since their discovery, the function of intermediate filaments (IFs) has remained obscure. In skin, epidermal cells have extensive cytoskeletal architectures of IFs, composed of type I and type II keratin heterodimers. Clues to possible functions of these proteins have come from recent studies showing that several autosomal-dominant, blistering skin disorders are caused by defects in genes that encode epidermal keratins. These diseases all exhibit cell degeneration and keratin network perturbations in cells that express the particular mutant keratin gene. However, it is not clear from these studies whether cytolysis arises from the presence of large insoluble keratin aggregates that compromise cellular physiology or from the absence of an extensive keratin filament network, which jeopardizes mechanical integrity. We report here the analysis of an extremely rare case of severe recessive epidermolysis bullosa simplex (EBS), where the patient lacks a discernible keratin filament network in basal epidermal cells. Genetic analyses revealed a homozygous point mutation that yielded a premature termination codon in the major basal type I keratin gene and caused complete ablation of K14. The consanguineous parents were normal, each harboring one copy of the null K14 mutation. Analysis of cultured keratinocytes enabled us to document that the loss of K14 is not compensated for by the up-regulation of any other type I keratin. When taken together with the in vivo studies showing the presence of cell fragility generated from the lack of an extensive basal keratin network, these findings provide the first clear demonstration of loss of function associated with the absence of an IF protein in vivo.

Amino Acid Sequence↗
PubMed1994

Monoamine receptors in the amygdaloid complex of the tree shrew (Tupaia belangeri).

Although it is well known that the mammalian amygdala comprises a heterogeneous complex of cytoarchitectonically and histochemically distinct nuclei, the association of these nuclei with different monoamine systems has not been described in detail. We therefore investigated the pattern of receptors for monoamines in the amygdala of the tree shrew (Tupaia belangeri). Binding sites for the alpha 2-adrenoceptor ligand (3H)rauwolscine, the alpha 1-adrenoceptor ligand (3H)prazosin, the beta-adrenoceptor ligand (125I)iodocyanopindolol, and the serotonin1A-receptor ligand (3H)8-hydroxy-2(di-n-propylamino)tetralin were visualized by in vitro autoradiography, and anatomically localized by comparing the autoradiograms to Nissl- and acetylcholinesterase-stained sections. To characterize binding of the radioligands pharmacologically, displacement experiments with different specific competitors were performed. Whereas the highest number of alpha 2-adrenergic binding sites was detected in the medial and the central nucleus as well as in the intercalated nuclei, the majority of serotonin1A binding sites was found in the magnocellular basal nucleus and the accessory basal nucleus, demonstrating a clear difference in the anatomy of the alpha 2-adrenergic and the serotonin1A receptor systems. In contrast, the pattern of alpha 1-adrenoceptor binding partially overlaps with that of both former receptor types. While the number of alpha-adrenergic and serotonin1A binding sites is relatively high in the tree shrew amygdala, there is a low number of beta-adrenergic binding sites in most nuclei. However, in the cortical nuclei, moderate to high numbers of binding sites for all radioligands are present. Therefore, according to our data on the tree shrew amygdala, which is anatomically similar to the amygdala of cats and primates, alpha 2-adrenoceptors cover primarily the medial part of the amygdaloid formation and serotonin1A-receptors predominantly occupy the basal nuclei, whereas alpha 1-adrenoceptors are present in both parts of the formation.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1994

Overexpression of parathyroid hormone-related protein in the skin of transgenic mice interferes with hair follicle development.

Parathyroid hormone-related peptide (PTHrP) was initially discovered as the cause of the syndrome of humoral hypercalcemia of malignancy. Subsequently, the PTHrP gene has been shown to be expressed in a wide variety of normal tissues, including skin. Because the biological function of PTHrP in skin remains unknown, we used the human keratin 14 promoter to target overexpression of PTHrP to the skin of transgenic mice. We achieved a 10-fold level of overexpression in skin, and human keratin 14 promoter-PTHrP transgenic mice displayed a disturbance in normal hair follicle development. These mice either failed to initiate follicle development or showed a delay in the initiation of follicles. These findings suggest that PTHrP normally plays a role in the early stages of hair follicle development and support previous speculation that the peptide may function in regulating cellular differentiation.

Animals↗
PubMed1994

Hippocampal glucocorticoid receptor expression in the tree shrew: regulation by psychosocial conflict.

1. This study was conducted to determine whether chronic psychosocial conflict alters the expression of glucocorticoid receptor (GR) mRNA in the hippocampus of male tree shrews (Tupaia belangeri). 2. To generate probes for the in situ hybridization, the tree shrew GR gene was partly cloned. There was a 90% homology between the deduced amino acid sequence of the cloned tree shrew GR and that of the corresponding human GR sequence. 35S-Labeled riboprobes which had been transcribed from the tree shrew GR clone hybridized to pyramidal neurons in all subregions of the tree shrew hippocampal formation and to granule neurons in the dentate gyrus. 3. After in situ hybridization, the expression of GR mRNA was semiquantitatively determined by counting silver grains over single neurons of the hippocampal formation of psychosocially stressed tree shrews and control animals. After 12 days of social conflict, the number of silver grains in the CA1 and CA3 pyramidal neurons of stressed animals was significantly lower than in controls. No statistically significant differences in mRNA expression were observed in the pyramidal neurons of the subiculum and in the granule neurons of the dentate gyrus. 4. The present results suggest that psychosocial stress leads to a site-specific down-regulation of hippocampal GR via modification of mRNA expression.

Amino Acid Sequence↗
PubMed1994

The epidermis: rising to the surface.

At the skin surface, the epidermis serves an important protective function which it manifests by building an extensive cytoskeletal architecture of keratin filaments, spanning from the nuclear envelope to hemidesmosomes and desmosomes. Recent studies on epidermal proteins and their interactions have provided insights into human skin diseases, including genetic disorders of keratins, laminins, and collagen. Explorations into the regulatory mechanisms underlying epidermal genes have underscored the importance of transcription factors AP-1 and AP-2, retinoic acid receptors, and POU proteins. Transgenic and gene ablation experiments on TGF-alpha and TGF-beta genes have yielded clues as to how the epidermis maintains a balance of growing and differentiating cells.

Animals↗
PubMed1994

Making a connection: direct binding between keratin intermediate filaments and desmosomal proteins.

In epidermal cells, keratin intermediate filaments connect with desmosomes to form extensive cadherin-mediated cytoskeletal architectures. Desmoplakin (DPI), a desmosomal component lacking a transmembrane domain, has been implicated in this interaction, although most studies have been conducted with cells that contain few or no desmosomes, and efforts to demonstrate direct interactions between desmoplakin and intermediate filaments have not been successful. In this report, we explore the biochemical nature of the connections between keratin filaments and desmosomes in epidermal keratinocytes. We show that the carboxy terminal "tail" of DPI associates directly with the amino terminal "head" of type II epidermal keratins, including K1, K2, K5, and K6. We have engineered and purified recombinant K5 head and DPI tail, and we demonstrate direct interaction in vitro by solution-binding assays and by ligand blot assays. This marked association is not seen with simple epithelial type II keratins, vimentin, or with type I keratins, providing a possible explanation for the greater stability of the epidermal keratin filament architecture over that of other cell types. We have identified an 18-amino acid residue stretch in the K5 head that is conserved only among type II epidermal keratins and that appears to play some role in DPI tail binding. This finding might have important implications for understanding a recent point mutation found within this binding site in a family with a blistering skin disorder.

Amino Acid Sequence↗