The effect of nickel sulphate, potassium dichromate, cobalt nitrate and cadmium sulphate on the proteins of cellular contacts and actin skeleton of cultivated human keratinocytes.

UNLABELLED: The authors tested the effects of known allergens, namely nickel sulphate, potassium dichromate, cobalt nitrate and cytotoxic cadmium sulphate on the proteins of cellular contacts (vinculin, talin, E-cadherin, desmoplaktin) and actin cytoskeleton (actin filaments) of cultivated human keratinocytes. The localisation of proteins of cellular contacts was detected by means of direct immunofluorescence. The authors have detected a decrease in, and destruction of cellular contact proteins and actin cytoskeleton after testing the effect of all allergens, while the most significant changes were detected in E-cadherin, vunkulin and actin filaments. Desmoplaktin and talin were less damaged. Potassium dichromate caused damage already in concentration of 1 microg/ml. A similar effect of the other two tested haptanes was brought about in concentration being 100-fold higher. CONCLUSION: the gained results indicate that the investigation of cellular contact proteins and actin cytoskeleton of cultivated human keratinocytes can possibly become a part of the testing of allergy-triggering potential of chemical substances. (Tab. 1, Fig. 4, Ref. 18.)

Polarity, protrusion-retraction dynamics and their interplay during keratinocyte cell migration.

Keratinocyte migration on a two-dimensional substrate can be split into four distinct phases: cell extension, attachment, contraction, and detachment. It is preceded by polarization of the cell which leads to a functional asymmetry observable by the formation of a leading lamella. In this work variation of fibronectin coating concentrations and competitive inhibition with RGD peptides are used to investigate the dependency of polarization, migration, lamella dynamics, and ruffling on substrate adhesiveness. Looking at migrating human epidermal keratinocytes with a well-defined polarity we find that a fibronectin-coating concentration of 10 microg/cm(2) stimulates migration and ruffling speed twofold, whereas protrusion speed increases only by 20% (compared to 2.5 microg/cm(2) fibronectin). Nonpolar cells show a constant migration and ruffling speed independent of the amount of fibronectin. In contrast protrusion speeds of polar and nonpolar cells are equal. Treatment of cells on 10 microg/cm(2) fibronectin with 1 mg/ml GRGDS reduces the characteristic migration, protrusion, and ruffling speed of polar cells which corresponds to lowering the effective coating concentration to under 5 microg/cm(2). The probability of being polarized (quantified by a polarity index) increases with increasing fibronectin concentration. However, addition of soluble RGD on 10 microg/cm(2) fibronectin does not simply reduce the polarity index like one would expect from the corresponding changes in the other motility parameters, but it remains unchanged.

Skin-specific expression of ank-3(93), a novel ankyrin-3 splice variant.

Ankyrins represent a protein family whose members are associated with membrane proteins and the actin cytoskeleton. The principal ankyrin domain structure comprises an amino-terminal membrane-binding, a spectrin-binding, and a regulatory domain, and can be modulated by alternative splicing. In order to investigate the role of ankyrin-3 in skin, we have isolated three complete ankyrin-3 cDNA clones of 5.8 kb, 5.2 kb, and 2.5 kb by reverse transcription-polymerase chain reaction of mouse skin RNA. DNA sequencing confirmed the isolated clones to be splice variants of ankyrin-3. Of these, the smallest cDNA represents a novel ankyrin named ankyrin-3(93). Surprisingly, this novel ankyrin subtype lacks not only all ankyrin repeats, but also the first 75 amino acids of the spectrin-binding domain. Immuno-fluorescence analysis of mouse skin showed that ankyrin-3 is expressed in all living layers of mouse epidermis. Here, it predominates along the basal and lateral membranes of the basal layer in addition to an even cytoplasmic distribution. In primary mouse keratinocytes grown at elevated Ca2+ levels, ankyrin-3(93) was localized along the plasma membrane and throughout the cell in a Golgi-like fashion. Depending on fixation conditions, nuclear staining became apparent in many cells. In agreement with previous data, northern blotting revealed a widespread distribution of the two larger ankyrin splice variants. In contrast, the mRNA coding for ankyrin-3(93)was restricted to mouse skin. Reverse transcription-polymerase chain reaction of mouse skin RNA strongly suggested additional ankyrin isoforms in skin. Our data on ankyrin-3(93), which lacks a part of the spectrin-binding domain that regulates the affinity to spectrin, suggests a new function for this member of the ankyrin family.

Supplementation of a mutant keratin by stable expression of desmin in cultured human EBS keratinocytes.

Mutations in keratin genes give rise to a number of inherited skin fragility disorders, demonstrating that the intermediate filament cytoskeleton has an essential function in maintaining the structural integrity of epidermis and its appendages. Epidermolysis bullosa simplex (EBS) is an autosomal dominant disorder caused by mutations in keratins K5 or K14, which are expressed in the basal layer of stratified epithelia. Using a keratinocyte cell line established from an EBS patient, we investigated whether the muscle-specific intermediate filament protein desmin would be able to functionally complement a mutant keratin 14 in cultured keratinocytes. We show that in stably transfected EBS cells, desmin forms an extended keratin-independent cytoskeleton. Immunogold-EM analysis demonstrated that in the presence of numerous keratin filaments attached to desmosomes, desmin could nevertheless interact with desmosomes in the same cell, indicating the dynamic nature of the filament-desmosome association. When desmin-transfected cells were subjected to heat shock, the mutant keratin filaments showed a transient collapse while desmin filaments were maintained. Thus the defective keratin filaments and the wild-type desmin filaments appear to coexist in cells without interference. Expression of a type III intermediate filament protein like desmin may offer a strategy for the treatment of patients suffering from epidermal keratin mutations.

Annular erythema caused by impending pacemaker extrusion.

A 72-year-old patient with an implanted cardiac pacemaker presented with a circumscribed erythematous area on his chest. It was only several months later, after he had developed positional, localized pain in this area, that the diagnosis of an impending pacemaker extrusion became evident. This case illustrates the diagnostic difficulties in patients with pacemaker-associated skin lesions. Regular follow-up examinations and close co-operation of dermatologists, cardiologists and cardiothoracic surgeons are of major importance in view of the potentially life-threatening complications.

Quantifying lamella dynamics of cultured cells by SACED, a new computer-assisted motion analysis.

Formation of lamellipodia and the retraction of ruffles are essential activities during motility and migration of eukaryotic cells. We have developed a computer-assisted stroboscopic method for the continuous observation of cell dynamics (stroboscopic analysis of cell dynamics, SACED) that allows one to analyze changes in lamellipodia protrusion and ruffle retraction with high resolution in space and time. To demonstrate the potential of this method we analyzed keratinocytes in culture, unstimulated or stimulated with epidermal growth factor (EGF), which is known to induce cell motility and migration. Keratinocytes stimulated with EGF exhibited a 2.6-fold increase in their migration velocity, which coincided with enhanced ruffle retraction velocity (144%) and increased ruffle frequency (135%) compared to control cells. We also recorded an enhanced frequency of lamellipodia (135%), whereas the velocity of lamellipodia protrusion remained unchanged. These results on ruffle and lamellipodia dynamics in epidermal cells show that SACED is at least equal to established methods in terms of accuracy. SACED is, however, advantageous concerning resolution in time and therefore allows one to analyze the activity of lamellipodia and ruffles in as yet unknown detail. Moreover, SACED offers two opportunities that render this technique superior to established methods: First, several parameters relevant to cell motility can be analyzed simultaneously. Second, a large number of cells can conveniently be examined, which facilitates the compilation of statistically significant data.

Localization and quantification of the cytoskeleton-associated protein adducin in the kidneys of normal and Milan hypertensive rats.

Hypertension and kidney dysfunction in sodium transport observed in the Milan hypertensive strain (MHS) of rats are genetically associated with point mutations of adducin, an actin- and spectrin-binding protein of the membrane cytoskeleton. Polymorphism in the adducin locus has been reported to occur also in cases of human primary hypertension. In this study we show by immunostaining that adducin is localized along the basolateral epithelial membrane surface of the entire proximal and distal tubule with no detectable differences between MHS rats and the normotensive control strain (MNS). However, the total amount of adducin in kidney homogenates is reduced by about 45% in MHS rats as determined by quantitative immunoblotting. In erythrocyte membranes of MHS rats, adducin is reduced approximately 10%. The reduction of renal adducin in MHS rats is mainly caused by a reduction of the adducin pool that is loosely associated with kidney membranes and can be released by the non-ionic detergent, Triton X-100. The Triton-resistant, tightly membrane-bound pool of renal adducin differed by approximately 10% between MHS and MNS rats. Since several ion transporters have been shown to be tethered to the membrane cytoskeleton, we suppose that the reduction of the dynamic, loosely bound pool of adducin in MHS rats might interfere with the normal turnover and incorporation of yet unknown transporters involved in kidney sodium transport. However, the Na+,K+-ATPase appears to be not involved, as indicated by normal distribution and amounts of NA+,K+-ATPase in the kidney of MHS rats revealed by immunostaining and immunoblotting.

Biosynthesis, processing, and intracellular transport of GM2 activator protein in human epidermal keratinocytes. The lysosomal targeting of the GM2 activator is independent of a mannose-6-phosphate signal.

The processing, intracellular transport, and endocytosis of the GM2 activator protein (GM2AP), an essential cofactor of beta-hexosaminidase A for the degradation of ganglioside GM2, was investigated in human epidermal keratinocytes. The GM2AP precursor is synthesized as an 18-kDa peptide, which is singly glycosylated, resulting in 22-kDa high mannose and 24-27-kDa complex glycoforms. A small portion of the 22-kDa form bears phosphomannosyl residues. About 30% of the GM2AP precursor is secreted during 12 h after synthesis, consisting almost exclusively of complex glycoforms. In a post-Golgi compartment, the intracellular remainder is converted to a 20-kDa mature form within 24 h, bearing a heavily trimmed N-glycan on a 17-kDa backbone. Interestingly, even nonglycosylated GM2AP is delivered to the lysosome, as shown by tunicamycin treatment and subcellular fractionation. Also, its endocytosis is independent of carbohydrate-linked signals and is even more effective for nonglycosylated GM2AP. We conclude that a mannose-6-phosphate-independent pathway for the lysosomal delivery of GM2AP exists in cultured human keratinocytes.

Interkeratinocyte adherens junctions: immunocytochemical visualization of cell-cell junctional structures, distinct from desmosomes, in human epidermis.

Vinculin and beta-catenin are intracellular attachment proteins linking transmembrane adhesion molecules (E-cadherin) to the actin microfilament cytoskeleton, thus participating in formation of cell-cell adherens junctions, or zonulae adherentes. This type of junction was only recently described in human epidermis due to the imprecise morphological criteria for its recognition. In this study, we investigated the relationship between the expression of the zonula adherens-associated proteins vinculin, beta-catenin, E-cadherin, and actin, on the one hand, and the presence of electron microscopically discernable structures in normal human epidermis on the other. Mouse jejunal epithelium with its orderly arrangement of various junctional structures served as a positive control. Simple and dual post-embedding immunogold labeling was performed on ultrathin sections of Lowicryl K4M and Lowicryl K11M embedded tissues. The overall distribution of the antigens in human epidermis was evaluated on frozen tissue sections using immunofluorescence and laser confocal scanning microscopy. Antibodies against proteins associated with desmosomes (i.e., keratins, desmoglein 1, and plakoglobin) were used as controls. Vinculin and beta-catenin were localized to junctional structures distinct from desmosomes, thus defining the presence of zonulae adherentes. Labeling of actin and E-cadherin was less clearly restricted to the junctions, but these two proteins were also co-expressed at zonulae adherentes and not at desmosomes. In human epidermis, zonula adherens-associated labeling was consistently detected near desmosomes, indicating the possibility of a functional relationship between the two types of junctions.

[Skin cultivation in treatment of severely burned patients]. / Die Hautzüchtung bei der Behandlung Schwerstverbrannter.

In severe burns a total body surface area (TBSA) of more than 60% restricts possible donor areas for autologous STS coverage. Additional wound surfaces may further harm the patient. From a skin biopsy of 5 cm2 the total body surface can be covered using keratinocyte cultures to multiply cells by 1000 up to 10,000. The cultured keratinocytes may be used as "sheets" (CEA) or suspended in fibrin glue (KFGS), which must be covered with meshed allogenic STS graft. Long culture times from 14 (KFGS) up to 28 days (CEA), infection of the culture and the woundbed, mechanical instability in the first period after grafting, restoring the dermal equivalent in full thickness burns and high costs are the problems of this new means of burn wound covering. Technical details of cultivation and coverage procedures using CEA and KFGS are discussed.

Cutaneous angiomyxoma: a benign neoplasm distinct from cutaneous focal mucinosis.

We describe a cutaneous angiomyxoma on the head of a 38-year-old man without evidence of Carney's complex. Complete excision of the tumor appeared to be curative. Histologic examination revealed fibroblast-like cells embedded in a well-demarcated, lobulate, mucinous, and vascularized stroma with a delicate reticulin network. Immunohistologically, the stromal cells were consistently positive for vimentin and focally positive for smooth muscle A-actin but were negative for desmin, KP1, MAC387, factor XIIIa, CD34, Leu-7, and S-100. Cutaneous angiomyxoma appears to represent a myofibroblastic neoplasm that should be distinguished from cutaneous focal mucinosis.

Patterns of spontaneous motility in videomicrographs of human epidermal keratinocytes (HEK).

The subject of our observations was the spontaneous behaviour of normal and transfected human epidermal keratinocytes. Cell movements were recorded on video micrographs and analyzed by a mathematical approach, using new methods of image processing and statistical correlation analysis. Protrusive activity of single lamellae was examined using one-dimensional analysis of phase-contrast image sequences along section lines transversal to the cell edge. This method revealed high periodicity and correlation in the motility patterns of lamellae and ruffles. Two-dimensional correlation analysis of automatically digitized cell outlines was applied to detect spatiotemporal patterns and coordination of lamellar extension and retraction. Most cells showed regularly alternating pulsations of lamellar protrusions. In some extreme cases, extension waves rotating around the cell periphery were observed. The results were compared with computer simulations of two simple models for lamellar dynamics and shape deformation, based on few assumptions about chemical kinetics of F-actin and cytomechanical properties of the actin network, neglecting regulatory effects of actin-associated proteins or extracellular stimulations. The simulation results reproduced the main dynamical features of the observed real cells, indicating the possibility that the basic universal mechanism for lateral coordination of lamellipodial protrusion is the interplay between hydrostatic pressure and viscocontractile tension in the cortical F-actin-plasma membrane complex.

[Sclerotic fibroma]. / Das sklerotische Fibrom.

The clinical and histological diagnosis of sclerotic fibroma is important because of its potential association with Cowden's syndrome. Despite its distinctive histological appearance the lesion is often mis-diagnosed. We therefore present five of our own cases (2F, 3M) in which the tumor was located on the head (n = 2), arm (n = 2) and leg (n = 1), respectively. Clinically, the lesions were white to flesh-colored firm nodules ranging in size from 0.5 to 1.2 cm. None of our patients revealed any clinical evidence of Cowden's disease. Simple surgical excision seems to be curative. Histologically, they were well-circumscribed but not encapsulated dermal nodules composed of stori-form-arranged sclerosing collagen bundles and vimentin-positive fibroblastlike cells interspersed in three cases by a number of alpha-smooth-muscle actin-positive myofibroblasts. Approximately 50% of cells (dermal dendritic cells (DD)) also reacted for factor XIIIa evenly scattered throughout the lesion in contrast to the very few (< 5%) CD34+ DD found predominantly at the lower border, thus possibly reflecting the distribution of these cells in normal skin. Sclerotic fibroma expands the spectrum of fibrous lesions that may express alpha-smooth-muscle actin.

Cultured autologous keratinocytes in fibrin glue suspension, exclusively and combined with STS-allograft (preliminary clinical and histological report of a new technique).

The use of cultured epidermal cell sheets has become a recognized method for the coverage of extensive burns. The disadvantages are a long time-lag until the cells are available, the fragility and difficult handling of the grafts, an unpredictable 'take' and extremely high costs. In three patients with deep partial and full skin thickness burns we have applied cultured autologous keratinocytes suspended in fibrin glue. In two of these patients the keratinocyte culture in the fibrin matrix (KFGS) was overgrafted with allogeneic, glycerine-preserved split thickness cadaver skin. The area thus covered ranged from 3 to 15 per cent TBSA. Cultured grafts were available between 2.5 and 3 weeks. The non-confluent cells developed a continuous epithelial layer within the 4 days until the first dressing change. Histological examination showed a stratified neoepidermis. Clinically the new skin had satisfactory stability and mechanical quality. The epidermis of the allogeneic overgrafts desquamated within a few days without signs of inflammation, but there are indications that the STS-allograft dermis is at least partly integrated into the new skin and may serve as a scaffold for the grafted cell culture. The fibrin glue matrix seems to give sufficient adherence stability to keratinocytes that are grafted in an actively proliferating state. Further advantages are the easy repetition and application, as well as a reduction in operating time and costs in these severely injured patients.

Cologne Burn Centre experience with glycerol-preserved allogeneic skin: Part II: Combination with autologous cultured keratinocytes.

Autologous keratinocytes cultured in vitro from skin biopsies of patients with deep partial and full skin thickness burns were grafted onto nine necrectomized wound surfaces between 17 and 25 days after injury. The cells were applied as nonconfluent single cells suspended in fibrin glue. In four wounds, this cell-fibrin suspension was used to attach an additional glycerolized allogeneic split thickness skin graft (STSG). Re-epithelialization was very rapid as demonstrated clinically and histologically. Keratinocyte grafted areas without cadaver skin overgraft showed less mechanical stability than when the keratinocyte-fibrin glue suspension was combined with allogeneic STSG. There is clinical and histological evidence that the allodermis may be partially integrated into the new skin.

Localization of adducin in epidermis.

On the cytoplasmic side of the plasma membrane of erythrocytes there is a dense protein filament matrix that maintains the shape of the cells. The main constituents of this system, actin and spectrin, which have also been detected in keratinocytes and fibroblasts, are known to be linked in erythrocytes in a network structure by additional proteins such as band 4.1 and adducin. The interaction between actin and spectrin, mediated by adducin, is regulated by calmodulin and protein kinase C. Because we have previously found adducin in cultured keratinocytes, we investigated epidermis by immunochemical techniques. We found adducin to be localized at cell-cell contact sites in epidermis using affinity-purified antibodies against human erythrocyte adducin. Immunofluorescence of epidermis revealed an intense fluorescence in the basal layer, whereas stratum spinosum and stratum granulosum showed moderate staining. There was intense staining at sites of cell-cell contact in cultured human keratinocytes. Immunoblot analysis indicated the presence of adducin polypeptides of 103 kd and 97 kd in epidermis, but in cultured keratinocytes only the higher molecular weight form could be detected. This study indicates adducin, a regulatory protein in erythrocytes, is also present in epidermis. Its localization suggests that it may be involved in the formation of the microfilament matrix of the membrane skeleton at cell-cell contact sites.

Talin: adherens junction protein is localized at the epidermal-dermal interface in skin.

The interaction between cells of the epidermis and the basal lamina is important for the integrity of the skin. Several hereditary and acquired diseases show changes at the dermal-epidermal interface due to loss of adhesion between basal cells and the basement membrane. The structures mediating this interaction are hemidesmosomes, which have been extensively characterized by biochemical, molecular biologic, and morphologic techniques. Recently, however, a group of adhesion molecules that are distinct from hemidesmosomes and that mediate cell-matrix interactions was described in cultured fibroblasts, keratinocytes, and skin. These adhesion molecules, beta 1 integrins, have been shown to be present in the focal adhesion, a cell-matrix contact associated with microfilaments rather than intermediate filaments characteristic of hemidesmosomes. In cultured cells, integrins of the beta 1 family have been shown to be linked by a protein complex to actin filaments. In this study we describe the localization of talin, the binding protein for beta 1 integrins, and vinculin at the dermal-epidermal interface in skin with immunofluorescence and immunoblotting techniques. These data suggest the presence of a link between the cytoplasmic actin filament system in basal keratinocytes and the extracellular matrix.

Adherens junctions: demonstration in human epidermis.

Adherens junctions are intercellular and cell-matrix junctions that, like desmosomes and hemidesmosomes, mediate adhesion of cells to each other or to matrix structures. These junctions have been detected recently in cultured human keratinocytes, indicating that they may be of importance in epidermis. To investigate the localization of adherens junctions in normal epidermis, we examined human epidermis, human oral mucosa, and monkey esophagus for the presence of vinculin, a major protein of the intracellular plaques of adherens junctions that is thought to be present in all adherens junctions. Western blot analysis demonstrated vinculin in extracts of epidermis. Immunohistochemistry of vinculin in these tissues displayed two distinct locations for adherens junctions: i) at the dermal-epidermal junction, and ii) in the region of cell-cell contacts in all layers of the epidermis. The location of vinculin in the region of the epidermal-dermal junction is reminiscent of the distribution of vinculin-containing focal contacts in cultured keratinocytes, and the intercellular staining of vinculin in epidermis is consistent with the presence of vinculin in adherens junctions in cultured keratinocytes at sites of cell-cell contact. These results demonstrate that adherens junctions are present in human epidermis, oral mucosa, and monkey esophagus. Vinculin-containing junctions in epidermis may be important in the pathogenesis of skin diseases involving alterations in intercellular integrity.

Adducin: Ca++-dependent association with sites of cell-cell contact.

Adducin is a protein recently purified from erythrocytes and brain that has properties in in vitro assays suggesting a role in assembly of a spectrin-actin lattice. This report describes the localization of adducin to plasma membranes of a variety of tissues and the discovery that adducin is concentrated at sites of cell-cell contact in the epithelial tissues where it is expressed. Adducin in tissues and cultured cells always was observed in association with spectrin and actin, although spectrin and actin were evident in the absence of adducin. In sections of intestinal epithelial cells spectrin was present on all plasma membrane surfaces while adducin was restricted to the lateral cell borders. Adducin also was not detected in association with actin stress fibers in cultured cells. The presence of adducin at cell-cell contact sites of cultured epithelial cells requires extracellular Ca++ and occurs within 15 min of addition of 0.3 mM Ca++. Redistribution of adducin after addition of extracellular Ca++ is independent of formation of desmosomal and adherens junctions since assembly of adducin at contact sites requires lower concentrations of Ca++ and occurs more rapidly than redistribution of desmoplakin or vinculin. Treatment of keratinocytes and MDCK cells with nanomolar concentrations of 12-O-tetradecanoylphorbol-13-acetate (TPA) induces redistribution of adducin away from contact sites. The effect of TPA may be a direct consequence of phosphorylation of adducin, since adducin is phosphorylated in TPA-treated cells and the phosphorylation of adducin occurs before disassembly of adducin from sites of cell-cell contact. Spectrin and adducin are both present in a detergent-insoluble form at cell-cell contact sites of cultured cells. These observations are consistent with the idea that adducin recognizes and associates with specific "receptors" localized at regions of cell-cell contact and promotes assembly of spectrin into a more stable structure, perhaps analogous to the highly organized spectrin-actin network of erythrocyte membranes.