Is thyrotropin-releasing hormone a novel neuroendocrine modulator of keratin expression in human skin?

BACKGROUND: Hair and epithelial keratins constitute the major structural components of the skin and its appendages, including the hair fibre. While it is appreciated that selected steroid hormones regulate specific keratins, little is known about the neuroendocrine control of human hair keratin expression. Preliminary evidence had suggested that thyrotropin-releasing hormone (TRH) may regulate keratin gene transcription. OBJECTIVES: To clarify whether TRH operates as a novel neuroendocrine regulator of human hair and epithelial keratin expression under physiologically relevant conditions in situ. METHODS: Microdissected human female scalp hair follicles (HFs) and female scalp skin were treated in serum-free organ culture for 12 h to 6 days with 100 ng mL(-1) TRH or vehicle. Both quantitative immunohistomorphometry and quantitative real-time polymerase chain reaction were utilized to assess expression of selected keratins. RESULTS: TRH significantly increased expression of the hair keratins K31 and K32, while that of K85 and K86, and of the epithelial keratins K14 and K17, was reduced. In the interfollicular epidermis, TRH stimulated expression of K6, K14 and K17, both at the mRNA and protein levels. Stimulation of the same keratins was also evident in the eccrine sweat and sebaceous glands. CONCLUSIONS: Selected human hair and epithelial keratins are modulated in situ. This may be relevant to explain hair shaft growth-promoting effects of TRH. Our pilot study suggests that the neuroendocrine controls that regulate the expression of human keratins deserve more systematic exploration and that these may be harnessed therapeutically.

Mutations in the hair cortex keratin hHb6 cause the inherited hair disease monilethrix.

Pathogenic mutations in a large number of human epithelial keratins have been well characterized. However, analogous mutations in the hard alpha-keratins of hair and nail have not yet been described. Monilethrix is a rare autosomal dominant hair defect with variable expression. Hairs from affected individuals show a beaded structure of alternating elliptical nodes and constrictions (internodes). These internodes exhibit a high prospensity to weathering and fracture. Strong evidence that trichocyte keratin defects might underlie this hair disorder was provided by genetic linkage analyses that mapped this disease to the type-II keratin gene cluster on 12q13. All affected individuals from a four-generation British family with monilethrix, previously linked to the type-II keratin gene cluster, as well as three unrelated single monilethrix patients, exhibited a heterozygous point mutation in the gene for type-II hair cortex keratin hHb6, leading to lysine substitution of a highly conserved glutamic acid residue in the helix termination motif (Glu 410 Lys). In a three-generation French family with monilethrix of a milder and variable phenotype, we detected another heterozygous point mutation in the same glutamic acid codon of hHb6, which resulted in a conservative aspartic acid substitution (Glu 410 Asp). These mutations provide the first direct evidence for involvement of hair keratins in hair disease.

Epidermolytic palmoplantar keratoderma cosegregates with a keratin 9 mutation in a pedigree with breast and ovarian cancer.

Epidermolytic palmoplantar keratosis (EPPK) cosegregates with breast and ovarian cancers in a large French pedigree, raising the possibility that a single genetic mutation might cause these conditions and offering a potential lead to the identification of a hereditary breast/ovarian cancer gene. We have performed linkage analysis and show that the EPPK locus lies on the long arm of chromosome 17 near the type I keratin gene cluster and the proposed breast cancer gene (BRCA1). The type I keratin 9 gene has been partially sequenced in four affected individuals. A single base mutation within the rod domain of the protein cosegregates with EPPK in all affected individuals tested. Although inheritance of this mutation is likely responsible for EPPK, it is unlikely to be the cause of the breast and ovarian cancer.

Keratin 9 gene mutations in epidermolytic palmoplantar keratoderma (EPPK).

We have isolated the gene for human type I keratin 9 (KRT9) and localised it to chromosome 17q21. Patients with epidermolytic palmoplantar keratoderma (EPPK), an autosomal dominant skin disease, were investigated. Three KRT9 mutations, N160K, R162Q, and R162W, were identified. All the mutations are in the highly conserved coil 1A of the rod domain, thought to be important for heterodimerisation. R162W was detected in five unrelated families and affects the corresponding residue in the keratin 14 and keratin 10 genes that is also altered in cases of epidermolysis bullosa simplex and generalised epidermolytic hyperkeratosis, respectively. These findings provide further evidence that mutations in keratin genes may cause epidermolysis and hyperkeratosis and that hyperkeratosis of palms and soles may be caused by different mutations in the KRT9 gene.

The 'melanocyte-keratin' mystery revisited: neither normal human epidermal nor hair follicle melanocytes express keratin 16 or keratin 6 in situ.

BACKGROUND: Keratin family proteins are generally accepted as being restricted to epithelial cells. However, several studies have challenged this paradigm by reporting, for example, that melanoma cells can express keratins and that normal human epidermal melanocytes, which derive from the neural crest, express keratin 16 (K16) in situ. OBJECTIVES: We wished to confirm or refute that K16 and/or its intermediate filament partner, keratin 6 (K6), are expressed in normal human epidermal and/or hair follicle melanocytes in situ. METHODS: Cryosections of normal human scalp skin were subjected to highly sensitive double immunohistochemistry with specific antibodies against K16 or K6 and against the melanocyte-specific marker NKI/beteb (gp100). Immunoreactivity (IR) was visualized by conventional light microscopy and confocal fluorescence microscopy. RESULTS: Despite the use of different, high-sensitivity immunostaining methods, stringent positive and negative controls, and monospecific, well-characterized antikeratin antibodies, we could detect neither K16 nor K6 IR within intraepidermal or intrafollicular pigment cells of normal human scalp skin. Instead, NKI/beteb+ cells were found to be intimately embedded in foci of K16+ and/or K6+ keratinocytes, which might create the illusion of keratin expression by these cells. CONCLUSIONS: Human epidermal or hair follicle melanocytes do not express K16 and/or K6 while residing in their natural habitat.

Characterization and expression analysis of the hair keratin associated protein KAP26.1.

BACKGROUND: Human hair follicle keratin-associated proteins (KAPs) comprise a large multigene family of proteins thought to be responsible for the bundling of keratin intermediate filaments. Recently, four new KAP family members KAP24.1, KAP25.1, KAP26.1 and KAP27.1 were identified from the genome, but the expression of only one, KAP24.1, was investigated and shown in hair follicles. OBJECTIVES: In the current study, the expression of the remaining members of the family were analysed. METHODS: Reverse transcriptase-polymerase chain reaction analysis of samples from numerous human organs was used. RESULTS: Only KAP26.1 showed expression, which was limited to the hair follicle. By in situ hybridization and immunohistochemistry using a specific antiserum, KAP26.1 was localized to the differentiated portion of the hair cuticle. CONCLUSIONS: As well as KAP24.1 in hair follicles, expression of KAP26.1 was shown and is found in the differentiated part of the hair cuticle.

New concepts on the histogenesis of eccrine neoplasia from keratin expression in the normal eccrine gland, syringoma and poroma.

BACKGROUND: Peripheral and luminal layers of eccrine sweat gland ducts are self-renewing structures. Proliferation is restricted to the lowermost luminal layer, but randomly scattered in the peripheral layer. Each layer exhibits differential expression of keratins K5/K14 and K6/K16. Keratin K1 occurs only in peripheral cells and the novel keratin K77 is specific for luminal cells. OBJECTIVES: To investigate the expression of luminal (K77), peripheral (K1) and further discriminatory keratins in two eccrine sweat gland tumours: syringoma, thought to show differentiation towards luminal cells of intraepidermal sweat ducts and eccrine poroma, considered to arise from poroid cells, i.e. peripheral duct cells; and keratinocytes of the lower acrosyringium/sweat duct ridge differentiating towards cells of intradermal/intraepidermal duct segments. METHODS: Paraffin-embedded sections were examined by immunohistochemistry using several keratin, smooth muscle actin and Ki-67 antibodies. RESULTS: We confirmed the ductal nature of syringomas. Despite drastic morphological alterations in both layers, their keratin patterns remained almost undisturbed compared with normal ducts. In eccrine poroma epidermal keratins K5/K14 were ubiquitously expressed in all poroid cells. Cell islands deviating morphologically from poroid cells contained epidermal keratins K1/K10. K77 expression was limited to luminal cells of intact duct structures within the tumours. CONCLUSIONS: Syringomas are benign tumours of luminal cells of the lowermost intraglandular sweat duct. Poroid precursor cells of poromas do not comprise peripheral duct cells nor do poromas differentiate towards peripheral or luminal duct cells. Instead, poroid cells consist only of keratinocytes of the lowermost acrosyringium and the sweat duct ridge and poromas tend to differentiate towards the cells of the upper acrosyringium.

Drebrin particles: components in the ensemble of proteins regulating actin dynamics of lamellipodia and filopodia.

Drebrin, an actin-binding 70-kDa protein with an unusually slow SDS-PAGE mobility corresponding to approximately 120 kDa, containing a proline-rich, profilin-binding motif, had originally been reported from neuronal cells, but recently has also been found in diverse other kinds of tissues and cell lines. In biochemical analyses of various cells and tissues, employing gel filtration, sucrose gradient centrifugation, immunoprecipitation and -blotting, we have identified distinct states of soluble drebrin: a approximately 4S monomer, an 8S, ca. 217-kDa putative trimer, a 13S and a > 20S oligomer. In the 8S particles only [35S]methionine-labelled drebrin but no other actin-binding protein has been detected in stoichiometric amounts. By immunofluorescence and immunoelectron microscopy, drebrin-positive material often appeared as "granules" up to 400 nm in diameter, in some cell types clustered near the Golgi apparatus or in lamellipodia, particularly at leading edges, or in dense-packed submembranous masses at tips (acropodia) or ruffles of leading edges, in filopodia and at plaques of adhering junctions. We conclude that these drebrin complexes and drebrin-rich structures allow the build-up and maintenance of high local drebrin concentrations in strategic positions for the regulation of actin filament assembly, thereby contributing to cell motility and morphology, in particular local changes of plasticity and the formation of protrusions.

Genomic characterization of the human type I cuticular hair keratin hHa2 and identification of an adjacent novel type I hair keratin gene hHa5.

Hair keratins, a subset of the keratin multigene family expressed in hard keratinizing structures, previously have been thought to comprise four members of each subfamily, designated Ha1-4 (type I) and Hb1-4 (type II), which are differentially expressed in the cuticle and cortex of the hair follicle. This report describes the genomic cloning and sequencing of the human type I cuticular hair keratin hHa2, as well as the identification of a previously unknown human type I hair keratin gene. The 12.5-kilobase pair genomic clone ghkI2.12, obtained by hybridization of a human genomic deoxyribonucleic acid library with a 3'-complementary deoxyribonucleic acid probe of hHa2, as well as the partially overlapping 14.4-kilobase pair genomic clone ghkI2.17, isolated using a 5'-fragment of clone ghkI2.12, allowed the characterization of the entire hHa2 gene. The gene displays the same exon/intron structure as two previously characterized type I mouse and sheep hair/wool keratin genes with strict positional conservation of the six introns in the region coding for the central alpha-helix. At the 5'-extremity of clone ghkI2.17, i.e., approximately 8.0 kilobase pairs upstream of the hHa2 gene and oriented in the same transcriptional direction, lies the gene for a hitherto unknown human type I hair keratin. Clone ghkI2.17 contains partial sequence information for this gene beginning with intron 5 and extending to the end of the gene. Screening of a human scalp complementary deoxyribonucleic acid library with a 3'-fragment of the gene yielded a full length complementary deoxyribonucleic acid clone of the new hair keratin, which in continuation of the current nomenclature for hair keratins was termed hHa5. Remarkably, the hHa5 gene, which contains an additional 7th intron in its 3'-noncoding region, is expressed mainly in supramatricial cells and lowermost cortical cells of the hair bulb and thus constitutes a very early component of hair morphogenesis. Our results confirm the type specific clustering of keratin genes and indicate that the human type I hair keratin subfamily contains more members than previously assumed.

The distribution of the desmosomal protein, plakophilin 1, in human skin and skin tumors.

Desmosomes are predominant among the types of plaque-bearing adhering junctions found in human skin. These structures contain a set of desmosomal cadherins and cytoplasmic plaque proteins, the synthesis of which is differentiation dependent. As plakophilin 1, a member of the armadillo gene family, is an important accessory desmosomal plaque protein, we raised several monoclonal antibodies specific for this protein and applied immunohistochemical and immunoblotting procedures to study the distribution of plakophilin 1 in desmosomes in adult and fetal skin, psoriatic epidermis, various epithelial skin tumors, and keratinocyte sheets grown in culture. In epidermis, the spinous layers were prominently immunostained by plakophilin 1 antibodies, whereas the basal cell layer was only weakly stained and the stratum corneum was entirely unstained. The staining observed in psoriatic epidermis was somewhat heterogeneous. In hair follicles, the outer root sheath (ORS) was delineated in its suprabasal cell layers, with variable staining in its upper and lower parts. All basal cells of the ORS remained unstained, as did upper inner root sheath (IRS) and matrix cells of lower bulb. In eccrine sweat glands, the reaction was confined to inner dermal ductal cells, with the acini remaining unstained. The desmosomal immunostaining observed in basal cell carcinomas (BCCs) and squamous cell carcinomas (SCCs) was very heterogeneous: In general, junctions in well-differentiated stratified tumor regions were more intensely stained than sections of poorly differentiated and invasively growing BCCs and SCCs. Plakophilin 1 was also prominent in the desmosomes of keratinocyte sheets grown in culture. The cell type-specific, i.e., differentiation-dependent, distribution of desmosomal plakophilin 1 is discussed in relation both to the stratification of the cutaneous epithelia and to tumor differentiation and growth.

Characterization of a 300 kbp region of human DNA containing the type II hair keratin gene domain.

Screening of an arrayed human genomic P1 artificial chromosome DNA library by means of the polymerase chain reaction with a specific primer pair from the human type II hair keratin hHb5 yielded two P1 artificial chromosome clones covering approximately 300 kb of genomic DNA. The contig contained six type II hair keratin genes, hHb1-hHb6, and four keratin pseudogenes psihHbA-psihHbD. This hair keratin gene domain was flanked by type II epithelial keratins K6b/K6hf and K7, respectively. The keratin genes/pseudogene are 5-14 kbp in size with intergenic distances of 5-19 kbp of DNA and do not exhibit a single direction of transcription. With one exception, type II hair keratin genes are organized into nine exons and eight introns, with strictly conserved exon-intron boundaries. The functional hair keratin genes are grouped into two distinct subclusters near the extremities of the hair keratin gene domain. One subcluster encodes the highly related hair keratins hHb1, hHb3, and hHb6; The second cluster encodes the structurally less related hair keratins hHb2, hHb4, and hHb5. Reverse transcription-polymerase chain reaction shows that all hair keratin genes are expressed in the hair follicle. Pseudogene psihHbD is also transcriptionally expressed, albeit with alterations in splicing and frameshift mutations, leading to premature stop codons in the splice forms analyzed. Evolutionary tree analysis revealed a divergence of the type II hair keratin genes from the epithelial keratins, followed by their segregation into the members of the two subclusters over time. We assume that the approximately 200 kbp DNA domain contains the entire complement of human type II hair keratin genes.

A novel human type II cytokeratin, K6hf, specifically expressed in the companion layer of the hair follicle.

In an attempt to identify new members of the human type II hair keratin family by means of 3'- and 5'-RACE methods and cDNA from anagen hair follicles, we detected a sequence that encoded a hitherto unknown type II cytokeratin. The novel cytokeratin comprises 251 amino acids and exhibits the highest sequence homology with K5. Comparative one- and two-dimensional western blots of keratins from anagen hair bulbs, containing or not containing the outer and inner root sheaths (ORS/IRS), and from footsole epidermis with an antibody against the new cytokeratin, revealed its comigration with K6 and its expression in the ORS/IRS complex. We have therefore named the new cytokeratin K6hf, to distinguish it from the various K6 isoforms and to indicate its expression in the hair follicle. Both in situ hybridization with a K6hf-specific cRNA probe and indirect immunofluorescence with the K6hf antibody showed that K6hf is exclusively expressed in the so-called "companion layer" of the hair follicle, a single layered band of flat and vertically oriented cells between the cuboidal ORS cells and the IRS that stretches from the lowermost bulb region to the isthmus of the follicle. Concomitant K17 and K16 expression studies showed that besides suprabasal ORS cells, these cytokeratins are sequentially expressed subsequent to K6hf in companion cells above the hair bulb. Our study confirms the view of a vertically oriented companion layer differentiation. The clearly delayed K17 and K16 expression relative to that of K6hf in companion cells most probably excludes these keratins as possible type I partners of K6hf and suggests the existence of a still unknown type I partner of its own. Thus, not only morphologically but also biochemically, the companion layer is different from the ORS and can therefore be regarded as an independent histologic compartment of the hair follicle.

Laminin matrix formation and S-100 protein and/or desmin-positive cells in malignant fibrous histiocytoma (MFH).

16/30 human storiform-pleomorphic malignant fibrous histiocytomas (MFH) showed a focal pericellular immunostaining for laminin. 14/16 of these laminin-positive tumours additionally revealed an atypical cellular differentiation (desmin- and/or S-100 protein-positive cells). The significance of focal laminin positivity along with atypical, non-entity specific differentiated cells is discussed before the background of laminin as being an indicator and promoter of differentiation in MFH. The limited value of a laminin detection in MFH for solving differential diagnostic questions (MFH versus other poorly differentiated sarcomas with basement membrane formation) has been pointed out.

Molecular diversity of plaques of epithelial-adhering junctions.

In biochemical and immunocytochemical comparisons of adhering junctions of different epithelia, we have observed differences in molecular composition not only between the intermediate filament-attached desmosomes and the actin filaments-anchoring adherens junctions but also between desmosomes of different tissues and of different strata in the same stratified epithelium. In addition we now report cell type-specific differences of molecular composition and immunoreactivity in both desmosomes and adherens junctions of certain simple epithelia. Whereas the zonula adhaerens of human intestinal and colonic epithelial cells, and of carcinomas derived therefrom, contains the additional armadillo-type plaque protein ARVCF, this protein has not been detected in the zonula adhaerens of hepatocytes. Similarly, plakophilin 3 is present in the desmosomal plaques of intestinal and colonic cells but appears to be absent from the hepatocytic desmosomes. We suggest that these profound compositional differences in the junctions of related simple epithelia are correlated to functional differences of the specific type of epithelium.

Lectin histochemistry of human testicular germ cell tumors.

The lectin binding pattern (WGA, UEA-I, PNA, PSA, Con A, RCA and LCA) of 28 human testicular germ cell tumors (pure or combination tumors) was investigated. Lectin binding sites could be demonstrated in all germ cell tumor types. In classic and spermatocytic seminomas as well as seminomas with high mitotic index an equal distribution of lectin binding sites was observed. In embryonal carcinomas the lectin binding of UEA-I, PNA, WGA, LCA and RCA correlated to histological differentiation. A polarized staining of WGA, PNA, RCA and UEA-I, typical for embryonal carcinomas, yolk sac tumors and teratomas, was never seen in seminomatous tumors and may be of importance in differential diagnosis. By means of Con A decoration it was possible to distinguish cytotrophoblastic and syncytiotrophoblastic differentiation. Aspects of lectin histochemistry in tumor biology in general and in differential diagnosis of germ cell tumors in particular are discussed.

Clinicians' decision making about involuntary commitment.

OBJECTIVE: Clinicians' decision making about involuntary commitment was examined, with a focus on the effects of patient and clinician characteristics and bed availability on decisions to detain patients, the first step in involuntary commitment. METHODS: Eighteen psychologists and social workers in the emergency service of a community mental health center completed the Risk Assessment Questionnaire for 169 consecutive patients they deemed to present some degree of risk. Forty-two patients were detained. RESULTS: Three underlying constructs were significantly associated with a patient's overall risk rating, which in turn predicted the decision to detain. Two were clinician characteristics: the clinician detention ratio, which reflects the proportion of patients detained by the clinician in the past three months, and the setting in which the evaluation occurred, either an in-house emergency service or a mobile crisis unit. The availability of detention beds in the community was also a significant predictor of whether a patient would be detained. No patient characteristic, including diagnosis, sex, age, or insurance status, was significantly related to the detention decision. CONCLUSIONS: The findings suggest that the decision-making process is influenced by multiple factors, such as setting, the clinician's tendency to detain patients, and the availability of detention beds.

Simple devices for the preparation of specimens for scanning and transmission electron microscopy.

A simple and inexpensive equipment especially for the dehydration of cells grown on coverslips in a continuous linear gradient is described. On this way it is possible to handle many objects simultaneously. The coverslips, even irregularly formed, are fixed vertically in simple silicon-rubber-holders. The device including a described special holder can also be used for thorough rinsing of EM-grids during the immunocytochemical labelling.

Modulation of expression of intermediate filaments during the development of established rat liver cell lines.

A number of clear epithelial-like cell lines were established from the liver of fetal and neonatal rats. The intermediate filaments of these cells were investigated using polyclonal prekeratin antisera, monoclonal antibodies against a cytokeratin subfamily and vimentin by means of the indirect immunofluorescence technique and SDS-PAGE analysis. Changes in the expression of cytokeratin filaments were found during the evolution of permanent cell lines. Cells positively stained for cytokeratins could be seen near to other cells which were negative. In early passages (up to the 40th) nearly all cells were strongly stained by the different keratin antibodies. During the following subcultivation the pattern of staining considerably changed. In FRL and NP-RL cell lines keratin-negative cells could already be observed in the early passages, rapidly increasing in the later passages. Compared to this, vimentin-staining of all cells remained constant in its morphological expression. The keratin filaments were seen in thick fiber bundles arranged particularly in the perinuclear ring as well as in finer networks throughout the cytoplasm. Every cell in the established lines showed their very individual staining pattern. The vimentin filaments extended to the whole cytoplasm up to the cell margin. Our observations demonstrate the variability of the system of keratin filaments in established epithelial-like liver cells under cultural conditions.

Lectin histochemistry of experimental murine rhabdomyosarcomas.

Methylcholanthrene-induced murine rhabdomyosarcomas and skeletal muscle of 10 and 18 d old murine embryos were investigated by lectin histochemistry (WGA, RCA-I, LCA, Con-A, PSA, UEA-I, PNA) and by immunohistochemistry (vimentin, desmin, myoglobulin). In rhabdomyosarcomas as well as in the developing skeletal muscle a clear trend was visible. A decrease of vimentin positivity and an increase of desmin positivity were associated with a diminution of binding sites for WGA, RCA, and LCA. No binding moieties for these lectin could be demonstrated in myoglobin positive normal and neoplastic rhabdomyomatous cells at all. The homologous expression or absence of markers reflected the cellular variability in rhabdomyosarcomas and may be explained as a phenomenon of different tumor cell maturation. The results show that rhabdomyosarcomatous cells are imitating the normal skeletal muscle development.