Mutations in the 180-kD bullous pemphigoid antigen (BPAG2), a hemidesmosomal transmembrane collagen (COL17A1), in generalized atrophic benign epidermolysis bullosa.

Junctional epidermolysis bullosa (JEB) is a heterogeneous autosomal recessively inherited blistering skin disorder associated with fragility at the dermal-epidermal junction. Characteristic ultrastructural findings in JEB are abnormalities in the hemidesmosome-anchoring filament complexes. These focal attachment structures, which extend from the intracellular compartment of the basal keratinocytes to the underlying basement membrane, have been shown to be hypoplastic or rudimentary in different forms of JEB. Previously, in different JEB phenotypes, mutations have been found in the three genes for the anchoring filament component laminin 5 (LAMA3, LAMB3, and LAMC2) and in the gene for the hemidesmosome-associated integrin beta 4 subunit. Here, we describe the first mutations in the gene encoding the 180-kD bullous pemphigoid antigen (BPAG2), a transmembranous hemidesmosomal collagen, also known as type XVII collagen (COL17A1). The patient is affected with generalized atrophic benign epidermolysis bullosa (GABEB), a rare variant of JEB, and is a compound heterozygote for premature termination codons on both alleles. These novel findings emphasize the molecular heterogeneity of this group of genodermatoses, and attest to the importance of BPAG2 in maintaining adhesion between the epidermis and the dermis.

Plectin deficiency results in muscular dystrophy with epidermolysis bullosa.

We report that mutation in the gene for plectin, a cytoskeleton-membrane anchorage protein, is a cause of autosomal recessive muscular dystrophy associated with skin blistering (epidermolysis bullosa simplex). The evidence comes from absence of plectin by antibody staining in affected individuals from four families, supportive genetic analysis (localization of the human plectin gene to chromosome 8q24.13-qter and evidence for disease segregation with markers in this region) and finally the identification of a homozygous frameshift mutation detected in plectin cDNA. Absence of the large multifunctional cytoskeleton protein plectin can simultaneously account for structural failure in both muscle and skin.

Increase in actin contents and elongation of apical projections in retinal pigmented epithelial cells during development of the chicken eye.

The structural and biochemical changes of cytoskeletal components of retinal pigmented epithelial cells were studied during the development of chicken eyes. When the cytoskeletal components of the pigmented epithelial cells from various stages of development were examined by SDS PAGE, actin contents in the cells markedly increased between the 15-d-old and hatching stages. Immunofluorescence microscopy showed that chicken pigmented epithelial cells have two types of actin bundles. One is the circumferential bundle associated with the zonula adherens region as previously reported (Owaribe, K., and H. Masuda, 1982, J. Cell Biol., 95:310-315). The other is the paracrystalline bundle forming the core of the apical projections. The increase in actin contents after the 15-d-old stage is accompanied by the formation and elongation of core filaments of apical projections in the cells. During this period the apical projections extend into extracellular space among outer and inner segments of photoreceptor cells. Accompanying this change is an elongation of the paracrystalline bundles of actin filaments in the core of the projection. By electron microscopy, the bundles decorated with muscle heavy meromyosin showed unidirectional polarity, and had transverse striations with approximately 12-nm intervals, as determined by optical diffraction of electron micrographs. Since the shape of these bundles was not altered in the presence or absence of Ca2+, they seemed not to have villin-like proteins. Unlike the circumferential bundles, the paracrystalline bundles did not contract when exposed to Mg-ATP. These observations indicate that the paracrystalline bundles are structurally and functionally different from the circumferential actin bundles.

Isolation and characterization of circumferential microfilament bundles from retinal pigmented epithelial cells.

Chicken retinal pigmented epithelial cells have circumferential microfilament bundles (CMBs) at the zonula adherens region. We have isolated these CMBs in intact form and characterized them structurally and biochemically. Pigmented epithelia obtained from 11-d-old chick embryos were treated with glycerol and Triton. Then, the epithelia were homogenized by passing them through syringe needles. Many isolated CMBs were found in the homogenate by phase-contrast microscopy. They formed polygons, mostly pentagons and hexagons, or fragments of polygons. Polygons were filled with meshwork structures, i.e. they were polygonal plates. Upon exposure to Mg-ATP, isolated CMBs showed clear and large contraction. The contraction was inhibited by treatment with N-ethylmaleimide-modified myosin subfragment-1. After purification by centrifugation with the density gradient of Percoll, CMBs were analyzed by SDS PAGE. The electrophoretic pattern gave three major components of 200, 55, and 42 kdaltons and several minor components. Electron microscopy showed that the polygons were composed of thick bundles of actin-containing microfilaments, and the meshworks were composed primarily of intermediate filaments.

Identification of a new hemidesmosomal protein, HD1: a major, high molecular mass component of isolated hemidesmosomes.

Hemidesmosomes (HDs) mediate cell adhesion to the extracellular matrix and have morphological association with intermediate-sized filaments (IFs) through cytoplasmic plaques. Though several proteins have been located in HDs, most of them have not been well characterized, with the exception of the 230-kD antigen of bullous pemphigoid (BP), an autoimmune skin blistering disease. Only recently we have succeeded in isolating HDs from bovine corneal epithelial cells and in identifying five major components on SDS-PAGE (Owaribe K., Y. Nishizawa, and W. W. Franke. 1991. Exp. Cell Res. 192:622-630). In this study we report on immunological characterization of one of the major components, termed HD1, with an apparent molecular mass of 500 kD. Immunofluorescence microscopy showed colocalization of HD1 with BP antigen at the basement membrane zone of those tissues that have typical HDs, including skin epidermis, corneal and tracheal epithelia, and myoepithelium. In cultured keratinocytes, HD1 demonstrated colocalization with BP antigen in the precise way, while being absent from focal adhesions. Immunoelectron microscopy revealed that an epitope of HD1 was located on the cytoplasmic side of HDs. Taking all these results together, we conclude that HD1 is a new hemidesmosomal component. Interestingly, HD1 also exists in endothelial and glial cells, which lack typical HDs.

Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.

Insulin-induced formation of ruffling membranes of KB cells and its correlation with enhancement of amino acid transport.

Insulin induced the formation of ruffling membranes in cultured KB cells (a cell strain derived from human epidermoid carcinoma) within 1-2 min after its addition. The ruffled regions were stained strongly with antibody to actin but not that to tubulin. Pretreatment of KB cells with agents disrupting microfilaments (cytochalasins), but not with those disrupting microtubules (colcemid, nocodazole, and colchicine) completely inhibited the formation of ruffling membranes. Pretreatment of KB cells with dibutyryl cyclic AMP, but not with dibutyryl cyclic GMP, also inhibited the formation of ruffling membranes. Addition of insulin enhanced Na+-dependent uptake of a system A amino acid (alpha-amino isobutyric acid; AIB) by the cells within 5 min after the addition, and decreased the cyclic AMP content of the cells. Treatments that inhibited insulin-induced formation of ruffling membranes of KB cells also inhibited insulin-induced enhancement of their AIB uptake. From these observations, the mechanism of insulin-induced formation of ruffling membranes and its close correlation with AIB transport are discussed.

Demonstration of contractility of circumferential actin bundles and its morphogenetic significance in pigmented epithelium in vitro and in vivo.

Each pigmented epithelial cell bears circumferential actin bundles at its apical level when the pigmented epithelium is established in eyes in situ or in culture in vitro. Well-differentiated pigmented epithelia in culture were treated with a 50% glycerol solution containing 0.1 M KCl, 5 mM EDTA, and 10 mM sodium phosphate buffer, pH 7.2, for 24 h or more at 4 degrees C. When the glycerinated epithelium was transferred to the ATP solution, each cell constituting the epithelium began to contract. The epithelium was cleaved into many cell groups as a result of contraction of each cell. The periphery of each cell group was lifted to form a cup or vesicle and eventually detached from the substratum. However, those cells that had not adhered tightly and not formed a monolayer epithelium with typical polygonal cellular pattern contracted independently as observed in the glycerinated fibroblasts. Contraction of the glycerinated pigmented epithelial cells was inhibited by N-ethylmaleimide but not by cytochalasin B. ITP and UTP also effected the contraction of the glycerinated cells, but GTP and ADP did not. Ca2+ was not required. This contractile model of pigmented epithelium provides a useful experimental system for analyzing the function of actin in cellular morphogenesis.

Defective expression of plectin/HD1 in epidermolysis bullosa simplex with muscular dystrophy.

Epidermolysis bullosa simplex with muscular dystrophy (MD-EBS) is a disease characterized by generalized blistering of the skin associated with muscular involvement. We report that the skin of three MD-EBS patients is not reactive with antibodies 6C6, 10F6, or 5B3 raised against the intermediate filament-associated protein plectin. Immunofluorescence and Western analysis of explanted MD-EBS keratinocytes confirmed a deficient expression of plectin, which, in involved skin, correlated with an impaired interaction of the keratin cytoskeleton with the hemidesmosomes. Consistent with lack of reactivity of MD-EBS skin to plectin antibodies, plectin was not detected in skeletal muscles of these patients. Impaired expression of plectin in muscle correlated with an altered labeling pattern of the muscle intermediate filament protein desmin. A deficient immunoreactivity was also observed with the monoclonal antibody HD121 raised against the hemidesmosomal protein HD1. Furthermore, immunofluorescence analysis showed that HD1 is expressed in Z-lines in normal skeletal muscle; whereas this expression is deficient in patient muscle. Colocalization of HD1 and plectin in normal skin and muscle, together with their impaired expression in MD-EBS tissues, strongly suggests that plectin and HD1 are closely related proteins. Our results therefore provide strong evidence that, in MD-EBS patients, the defective expression of plectin results in an aberrant anchorage of cytoskeletal structures in keratinocytes and muscular fibers leading to cell fragility.

180-kD bullous pemphigoid antigen (BP180) is deficient in generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a form of nonlethal junctional epidermolysis bullosa characterized by universal alopecia and atrophy of the skin. We report a deficiency of the 180-kD bullous pemphigoid antigen in three patients with GABEB from unrelated families. We screened specimens of clinically normal skin from nine junctional epidermolysis bullosa patients (3 GABEB, 4 lethal, 1 cicatricial, 1 pretibial) by immunofluorescence using monoclonal antibodies to the 180-kD and 230-kD bullous pemphigoid antigens (BP180 and BP230). In the skin of the three GABEB patients there was no reactivity with antibodies to BP180, whereas staining for BP230 was normal. In the skin of the other six, non-GABEB patients, included in this study the expression of BP180 and BP230 was normal. Immunoblot analysis of cultured keratinocytes from one of the GABEB patients also failed to detect BP180 antigen, whereas BP230 was present in normal amounts. The deficient expression of BP180 is reflected in the RNA message, as in Northern blot analysis a reduced amount of BP180 transcripts, although of normal length, were detected. Interestingly, in another GABEB patient there were not-involved areas of skin, in which blistering could not be induced by rubbing. Biopsy material from these areas showed interrupted staining for BP180. There was no staining for BP180 in areas of clinically normal but involved skin of this patient. In conclusion, this study reveals that the BP180 antigen is deficient and the BP180 mRNA is reduced in generalized atrophic benign epidermolysis bullosa.

Some properties of Physarum actinin. A regulatory protein of actin polymerization.

A factor termed Physarum actinin was isolated and partially purified from plasmodia of a myxomycete, Physarum polycephalum. When Physarum actinin was mixed with purified Physarum or rabbit striated muscle G-actin in a weight ratio of about 1 actinin to 9 actin and then the polymerization of G-actin induced, G-actin polymerized to the ordinary F-actin on addition of 0.1 M KCl. However, it polymerized to Mg-polymer on addition of 2 mM MgCl2. The reduced viscosity (etasp/C) of the Mg-polymer was 1.2 dl/g, about one-seventh of that of the F-actin (7.4 dl/g). The sedimentation coefficient of the Mg-polymer was 22.8 S, almost the same as that of the F-actin (29.4 S). The Mg-polymer showed the specific ATPase activity of the order of 1 . 10(-3) mumol ATP/mg actin per min. It was shown that Physarum actinin copolymerized with G-actin to form Mg-polymer on addition of 2 mM MgCl2. The molecular weights of Physarum actinin were about 90 000 in salt-free or slat solutions and 43 000 in a dodecyl sulfate solution. The range of salting out with ammonium sulfate was 50--65% saturation, which was different from that of Physarum actin (15--35% saturation). Physarum actinin did not interact with Physarum myosin or muscle heavy meromyosin. When the weight ratio of actinin to actin increased, the flow birefringence of the formed Mg-polymer decreased, and it became almost zero at the weight ratio of 1 actinin to 5 actin. ATPase activity reached the maximum level (2.2 . 10(-3) mumol ATP/mg actin per min) at the same ratio. On the addition of Physarum actinin to purified Physarum F-actin which had been polymerized on addition of 2 mM MgCl2 the viscosity decreased rapidly, suggesting that the F-actin filaments were broken in the smaller fragments or that they transformed to Mg-polymers. A factor with properties similar to Physarum actinin was isolated from acetone powder of sea urchin eggs.

Altered distribution of plectin/HD1 in dystrophinopathies.

Plectin/HD1 is a high molecular weight protein (approximately 500 kDa) that has been proposed to act as an important and versatile cytoskeletal cross-linker molecule. Mutations of the human plectin gene have recently been associated with the autosomal recessive disorder epidermolysis bullosa simplex with muscular dystrophy. We studied the expression of plectin/HD1 in various neuromuscular disorders by indirect immunofluorescence. In cross sections of normal human muscle, plectin/HD1 showed a checkerboard-like distribution with moderate to intense cytoplasmic and sarcolemmal staining in type 1 fibers and a faint staining of the sarcolemma in type 2 fibers. In longitudinal sections of plectin/HD1-positive fibers a cross-striation staining pattern was noted. This fiber type-related expression was significantly altered in the group of dystrophinopathies, whereas it was maintained in all other myopathies and denervating disorders. In seven dystrophinopathies studied, a markedly increased plectin/HD1 immunoreactivity at the sarcolemmal level of type 2 fibers was observed. Confocal laser microscopy of normal skeletal muscle revealed a colocalization of desmin and plectin/HD1 at the level of the sarcolemma. This suggests that plectin/HD1- in analogy to its demonstrated involvement in cytokeratin-hemidesmosome linkage in epidermis-may mediate the anchorage of desmin to the sarcolemma (i.e. to costameres).

Human autoantibodies against HD1/plectin in paraneoplastic pemphigus.

Paraneoplastic pemphigus (PNP) is an autoimmune blistering disease that occurs in association with underlying neoplasms. PNP patients develop characteristic autoantibodies directed against multiple antigens, mostly identified as members of the plakin family of cytoplasmic proteins (desmoplakin I and II, bullous pemphigoid antigen I, envoplakin, and periplakin). HD1/plectin, another member of the plakin family, has not previously been detected in the characteristic PNP antigen complex, which may relate to practical difficulties associated with its large size (molecular weight approximately 500 kDa). In this study, a combination of immunoprecipitation and immunoblot is used to demonstrate that HD1/plectin is also recognized by sera from PNP patients. Thirteen of 16 PNP sera tested were positive for HD1/plectin compared with none of 43 control sera (11 pemphigus vulgaris, 11 pemphigus foliaceus, 11 bullous pemphigoid, and 10 normal individuals). Combined with our recent finding that desmoglein 3 and desmoglein 1 are cell surface target antigens in PNP, this demonstration of plectin/HD1 as another component of the antigen complex in PNP confirms that PNP is an autoimmune disease against desmoglein and plakin family molecules.

The extracellular domain of BPAG2 has a loop structure in the carboxy terminal flexible tail in vivo.

The 180 kDa bullous pemphigoid antigen is a hemidesmosome-associated transmembranous protein with a molecule length estimated to be 190-230 nm, which is much longer than the transverse length of the lamina lucida and lamina densa. The purpose of this study was to clarify the precise in vivo structure of the 180 kDa bullous pemphigoid antigen in normal human skin. We used three monoclonal antibodies directed to (i) the intracellular globular head of the 180 kDa bullous pemphigoid antigen, (ii) the mid-portion of the flexible tail of the antigen, corresponding approximately to amino acids 1000-1320, and (iii) the carboxyl terminal end, corresponding approximately to amino acids 1320-1500 of the antigen. Using low temperature postembedding immunoelectron microscopy, we quantitated the distribution of immunogold labeling of these monoclonal antibodies in normal human skin. The results showed that the monoclonal antibodies (i) bound to the intracellular portion of the hemidesmosome at a mean distance of 20 nm from the plasma membrane, (ii) bound to the lamina densa beneath the hemidesmosome at a mean distance of 65 nm from the plasma membrane, and (iii) bound to the lamina densa-lamina lucida interface at a mean distance of 39 nm from the plasma membrane. Considering the reported size of the 180 kDa bullous pemphigoid antigen, our results indicate that the extracellular domain of the antigen has at least one loop structure in the lamina densa in vivo. This unique structure of the antigen is thought to contribute to dermo- epidermal adhesion by intertwining with other basement membrane components.

Novel homozygous and compound heterozygous COL17A1 mutations associated with junctional epidermolysis bullosa.

Junctional epidermolysis bullosa is a heritable, heterogeneous blistering skin disease with mechanically induced dermal-epidermal separation, mild skin atrophy, nail dystrophy, and alopecia. Four unrelated junctional epidermolysis bullosa families with different phenotypes were investigated here and four novel mutations associated with the disease were identified. Patients 1, 2, and 3 had generalized atrophic benign epidermolysis bullosa, with nonscarring blistering and varying degree of alopecia. Patient 4 had the localisata variant of junctional epidermolysis bullosa, with predominantly acral blistering and normal hair. All patients had mutations in the COL17A1 gene encoding collagen XVII, a hemidesmosomal transmembrane protein. Patients 1 and 2 carried homozygous deletions 520delAG and 2965delG, respectively. Patient 3 was compound heterozygous for a missense and a deletion mutation (G539E and 2666delTT), and patient 4 was heterozygous for a known mutation R1226X. The deletions led to premature termination codons and to drastically reduced collagen XVII mRNA and protein levels, consistent with the absence of the collagen in generalized atrophic benign epidermolysis bullosa skin. The missense mutation G539E allowed synthesis of immunoreactive collagen XVII in keratinocytes, but prevented its secretion, thus causing lack of the protein in the skin. The data suggest that different COL17A1 mutations and their combinations can result in a spectrum of biologic and clinical phenotypes of not only generalized atrophic benign epidermolysis bullosa, but also localized junctional epidermolysis bullosa.

A homozygous nonsense mutation in type XVII collagen gene (COL17A1) uncovers an alternatively spliced mRNA accounting for an unusually mild form of non-Herlitz junctional epidermolysis bullosa.

In this study we describe six Italian patients presenting an unusually mild variant of non-Herlitz junctional epidermolysis bullosa associated with a reduced expression of type XVII collagen. All patients are homozygous for a novel nonsense mutation (R795X) within exon 33 of COL17A1 and show a common haplotype, attesting propagation of an ancestral allele within the Italian population. Analysis of patients' COL17A1 transcripts showed the presence of two mRNA species: a normal-sized mRNA carrying mutation R795X that undergoes rapid decay, and a transcript generated by in-frame skipping of exon 33. Patients keratinocytes were shown to synthesize minute amounts of type XVII collagen, which appeared correctly localized along the cutaneous basement membrane. We therefore suggest that the exon 33-deleted COL17A1 splice variant encodes for type XVII collagen molecules that maintain a functional role and account for the mild phenotype of our patients.

Expression of plectin in muscle fibers with cytoarchitectural abnormalities.

Plectin is a protein belonging to the cytoskeletal anchoring system, concentrated at sites of mechanical stress in different cell types. In normal skeletal muscle, plectin is located at level of Z-discs, sarcolemma, post-synaptic membrane, and intermyofibrillar network. We investigated plectin immunocytochemistry in lobulated fibers, fibers with tubular aggregates, target fibers, central core disease and centronuclear myopathy. Thirty to forty percent of lobulated fibers had patchy increase of plectin immunoreactivity at sarcolemmal level with focal subsarcolemmal increases. Tubular aggregates revealed a low binding for plectin. Ten percent of central cores exhibited faint focal increase of plectin immunoreactivity. Target formations had a normal plectin pattern. In centronuclear myopathy, plectin immunoreactivity was increased around the centrally located nuclei in 8-12% of the fibers, at the sarcolemma of 50% of type 2 fibers, and at the membrane of small vacuoles located peripherally around the central nuclei. We postulate that plectin may play a role in the subsarcolemmal aggregation of mitochondria in the lobulated fibers, and in the central position of nuclei as well as in shape formation, positioning and moving of the vacuoles in centronuclear myopathy.

Intracellular ionic changes induced by bullous pemphigoid IgG subclasses.

To ascertain whether membrane signal transduction is induced by bullous pemphigoid (BP) antibody and whether cell lysis is induced by its complement activation, we assessed the intracellular Ca2+ concentration ([Ca2+]i), intracellular pH, membrane potential and morphology of living cells by following the time course of fluorescence intensity of Fluo-3/AM, Snaff-1/AM, Dioc-5 and Luciffer yellow, respectively. A transient increase of Fluo-3 fluorescence intensity in DJM-1 cells (a squamous cell carcinoma line) was revealed when the cells were incubated with 2 of five IgG1 BP antibodies. However, no transient increase of Fluo-3 fluorescence intensity was revealed when the cells were incubated with IgG2 and IgG4 BP antibodies. A transient increase of Fluo-3 fluorescence intensity was revealed in DJM-1 cells incubated with 3 of seven IgG1 and 1 of four IgG2 BP antibodies in an EGTA-containing low-Ca2+ medium. On the other hand, the Dioc-5 fluorescence intensity did not change significantly, though the increase of Fluo-3 fluorescence intensity was observed. The increase of Snarf-1 fluorescence intensity was revealed in DJM-1 cells incubated with 2 of five IgG1 BP antibodies, but was not revealed in the cells incubated with IgG2 or IgG4 of BP antibodies. Study of complement activation by BP IgG1 showed a transient increase of Fluo-3 fluorescence intensity of with 3 of five IgG1 BP antibodies when DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. At the same time, however, endocytosis and cell lysis were not observed with 2 IgG1 BP antibodies which did induce an increase of Fluo-3 fluorescence intensity when Lucifer-yellow-loaded DJM-1 cells were incubated with complement-supplemented normal-Ca2+ medium. We examined next whether anti-180 kD BP antigen monoclonal antibodies (mAbs R-223 and 233) induce an increase of Fluo-3 fluorescence intensity. MAb R-223 did not induce any increase of Fluo-3 fluorescence intensity in DJM-1 cells, when incubated with normal- and low-Ca2+ media However, mAb R-223 induced a transient increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with complement-supplemented normal-Ca2+ medium. MAb 233 did not induced an increase of Fluo-3 fluorescence intensity in DJM-1 cells when incubated with normal- and low-Ca2+ media. These results suggest that the BP IgG1 induces Ca2+ release from intracellular storage sites, however, the complement activated by BP IgG1 does not induce cell lysis. It could not be confirmed that anti-180 kD BP antigen antibody induced Ca2+ release from intracellular storage sites.

Cultured keratinocytes from plectin/HD1-deficient epidermolysis bullosa simplex showed altered ability of adhesion to the matrix.

Epidermolysis bullosa simplex associated with late onset of muscular dystrophy has been found to show defective expression of plectin, an intracytoplasmic protein in hemidesmosomes. In this report, we examined ability of cell-to-matrix attachment of cultured keratinocytes derived from a case with this disease by various cell biological methods, and compared it to that of normal keratinocytes. In cell adhesion assay, the patient keratinocytes showed more prominent short-time cell adhesion than normal keratinocytes. In contrast, the patient keratinocytes could be detached much easier than normal keratinocytes in cell detachment assay by treatment with dispase. In phagokinetic track assay, no apparent difference of cell migration was observed between the patient and normal keratinocytes. These results indicate that plectin-deficiency may up-regulate short-term cell contact and reduce stable cell-matrix adhesion at the epidermal basement membrane zone.

Transient translocation of hemidesmosomal bullous pemphigoid antigen 1 from cytosol to membrane fractions by 12-O-tetradecanoylphorbol-13-acetate treatment and Ca2+-switch in a human carcinoma cell line.

We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) and Ca2+-switch from low (0.07 mM) to normal (1.87 mM) concentration in culture medium, which were also linked to activation of protein kinase C (PKC), lead to phosphorylation of 180 kDa-bullous pemphigoid antigen (BPAG) 2, but not of 230 kDa-BPAG1, and possibly to its disassembly from hemidesmosomes in a human squamous cell carcinoma cell line (DJM-1). In this study, we examined the effects of TPA and Ca2+-switch on intracellular localization of BPAG1 by immuno-blotting and immuno-fluorescence microscopy with monoclonal antibodies to the antigen after sub-cellular fractionation. In DJM-1 cells cultured in low Ca2+ medium, BPAG1 was detected as phosphate buffered saline-soluble (cytosolic), Triton X-100 soluble (roughly membrane-associated) and Triton X-100 insoluble (cytoskeleton-bound) forms, whereas in normal Ca2+-grown cells only as cytosolic and cytoskeleton-bound forms. In normal Ca2+-cultured cells, TPA (50 nM) caused a complete translocation of BPAG1 from cytosol to membrane fractions within 10 min, that was inhibited by pretreatment with H7 (a selective PKC inhibitor) at 40 microM. After 30 min and 4 h of TPA-treatment, BPAG1 was exclusively detected in cytoskeleton fractions. Morphologically, immuno-fluorescence microscopy showed that treatment caused a marked reduction of BPAG1 from the cytoplasm and generated a linear pattern at cell-cell contacts, suggesting translocation of BPAG1 from the cytosol to the plasma membrane. In contrast, the Ca2+-switch from low to normal caused a prominent increase of BPAG1, both in cytosolic and membrane-associated forms after 4 h, that was inhibited both with H7 and cycloheximide (an inhibitor of protein synthesis) at 70 microM, suggesting a role for PKC and BPAG1 synthesis in these Ca2+-induced effects. These results suggest that TPA and Ca2+-switch induced BPAG1 translocation to membrane fractions possibly mediated by PKC-activation. Furthermore, whereas TPA affects the redistribution of BPAG1 among their pools without inducing their synthesis, Ca2+-switch induces both membrane translocation and synthesis of BPAG1, suggesting involvement of signaling other than PKC pathways in control of BPAG1 synthesis.