A novel hemidesmosomal plaque component: tissue distribution and incorporation into assembling hemidesmosomes in an in vitro model

The hemidesmosome and its associated structures, such as anchoring fibrils, form a complex structure, the polypeptide composition of which has only recently begun to be elucidated. We describe the characterization of a monoclonal antibody, mAb6A5, directed against a 200-kDa polypeptide found in the cytoplasmic-most area of the hemidesmosomal plaque. This 200-kDa polypeptide is immunologically distinct from the 180- and 230-kDa hemidesmosomal plaque components recognized by bullous pemphigoid (BP) autoantibodies. mAb6A5 recognizes hemidesmosomes of stratified squamous epithelia in a number of species, including human tissue. mAb6A5 also recognizes pseudo-stratified epithelium, but not simple or transitional epithelia. During de novo hemidesmosome assembly in an in vitro model of epithelial wound healing, the 200-kDa polypeptide is in most instances deposited at the epithelial-stromal interface after plaque components recognized by BP autoantibodies, but before the collagen type VII component of anchoring fibrils. We discuss possible mechanisms of hemidesmosomal plaque assembly.     

Formation of hemidesmosomes in vitro by a transformed rat bladder cell line

Two hemidesmosomal plaque components of 230 and 180 kD have recently been characterized using autoantibodies in the serum samples of bullous pemphigoid (BP) patients (Klatte, D. H., M. A. Kurpakus, K. A. Grelling, and J. C. R. Jones. 1989, J. Cell Biol. 109:3377-3390). These BP autoantibodies generate the type of staining patterns that one would predict for formed hemidesmosomes, i.e., a punctate staining pattern towards the substratum; in less than 50% of various primary epithelial and transformed epidermal cell lines even when such cells are maintained in culture for prolonged periods. In contrast, affinity- purified human autoantibodies against the 230-kD hemidesmosomal plaque component generate intense immunofluorescence staining along the region of cell-substratum interaction in the rat bladder tumor cell line 804G maintained on uncoated glass cover-slips. This pattern is distinct from that observed in the 804G cells using an antibody preparation directed against vinculin, a component of adhesion plaques. Ultrastructural analyses of the 804G cells reveals that hemidesmosome-like structures occur along the basal surface of cells where they abut the substratum. These structures are present in 804G cells maintained in culture in reduced levels of Ca2+ and are recognized by autoantibodies directed against the 230-kD hemidesmosomal plaque component as determined by immunogold ultrastructural localization. To study hemidesmosome appearance in this cell line, 804G cells were trypsinized and then allowed to readhere to glass coverslips. In rounded, unattached 804G cells, hemidesmosome-like plaque structures occur along the cell surface. These structures are recognized by the 230-kD autoantibodies. At 1 h after plating, hemidesmosomes are observed along the substratum attached surface of cells. Protein synthesis is not required for the appearance of these hemidesmosomes. Within 4 h of plating, autoantibody staining and hemidesmosomes appear towards the cell periphery. Subsequently, the polypeptide recognized by the BP autoantibodies becomes concentrated in the perinuclear region, where there are numerous hemidesmosomes. We propose that the hemidesmosomes in 804G cells are involved in cell-substratum adhesion. We discuss possible mechanisms of assembly of hemidesmosomes in the 804G cells. Indeed, the 804G cells should prove an invaluable cell line for the biochemical and molecular dissection of hemidesmosome structure, function, and assembly.     

Surface relocation of alpha 6 beta 4 integrins and assembly of hemidesmosomes in an in vitro model of wound healing

A transmembrane extracellular matrix receptor of the integrin family, alpha 6 beta 4, is a component of the hemidesmosome, an adhesion complex of importance in epithelial cell-connective tissue attachment (Stepp, M. A., S. Spurr-Michaud, A. Tisdale, J. Elwell, and I. K. Gipson. 1990. Proc. Natl. Acad. Sci. USA. 87:8970-8974; Jones, J. C. R., M. A. Kurpakus, H. M. Cooper, and V. Quaranta. 1991. Cell Regulation. 2:427-438). Cytosolic components of hemidesmosomes include bullous pemphigoid (BP) antigens while extracellular components include a 125-kD component of anchoring filaments (CAF) and collagen type VII-containing anchoring fibrils. We have monitored the incorporation of the alpha 6 beta 4 integrins into forming hemidesmosomes in an in vitro wound-healing explant model. In epithelial cells recently migrated from the edges of unwounded sites over bare connective tissue, alpha 6 beta 4 first appears along the entire cell surface. At this stage, these cells contain little or no cytosolic hemidesmosomal components, at least as detectable by immunofluorescence using BP autoantibodies, whereas they are already positive for laminin and CAF. At a later stage, as cells become positive for cytosolic hemidesmosome components such as BP antigens as well as collagen type VII, alpha 6 beta 4 becomes concentrated along the basal pole of the epithelial cell where it abuts the connective tissue of the explant. Polyclonal antibodies to beta 4 do not interfere with the migration of epithelial cells in the explant. However, they prevent assembly of hemidesmosomal complexes and inhibit expression of collagen type VII in cells that have migrated over wound areas. In addition, they induce disruption of established hemidesmosomes in nonmigrating cells of the unwounded area of the explant. Monoclonal antibodies to alpha 6 have a more dramatic effect, since they completely detach epithelial cells in the unwounded area of the explant. Antibodies to CAF also detach epithelial cells in unwounded areas, apparently by inducing separation between epithelium and connective tissue at the lamina lucida of the basement membrane zone. These results suggest a model whereby polarization of alpha 6 beta 4 to the basal surface of the cells, perhaps induced by a putative anchoring filament-associated ligand, triggers assembly of hemidesmosome plaques.     

大疱性类天疱疮IgG型抗基底膜带抗体的靶抗原初步分析

目的　研究大疱性类天疱疮 Ｉｇ Ｇ 型抗基底膜带抗体的靶抗原。方法　应用 Ｄ Ｉ Ｆ与 Ｉ Ｉ Ｆ检测的 Ｉｇ Ｇ型抗体阳性 Ｂ Ｐ 血清进行 Ｗ ｅｓｔｅｒｎ 免疫印迹分析。结果　初步研究发现 Ｉｇ Ｇ 阳性的 Ｂ Ｐ 血清能结合抗原１８０ Ｋ Ｄ,１６０ Ｋ Ｄ 以及 １０５ Ｋ Ｄ 与 ７７ Ｋ Ｄ 的多肽。结论　 Ｉｇ Ｇ 型 Ｂ Ｐ自身抗体可能靶向基底膜带不同层出现的不同抗原并提示该病的发生可能涉及不同的免疫机制。     

Molecular heterogeneity of the bullous pemphigoid antigens as detected by immunoblotting

Sera from 28 patients with bullous pemphigoid (BP), four patients with cicatricial pemphigoid (CP), and 24 controls (normal volunteers and patients with pemphigus, systemic lupus erythematosus, or other skin diseases) were tested against extracts of human epidermis by immunoblotting techniques. The extraction buffer included 1% SDS, 5% beta-mercaptoethanol, and six protease inhibitors with various specificities. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total of five bands of 240 kD, 200 kD, 180 kD, 97 kD, and 77 kD reacted with BP sera; the 240-kD band reacted with one CP sera, and none of these bands was detected by the control sera. The 240-kD and 180-kD bands reacted very strongly with some sera and were most frequently observed (43% and 29%, respectively). The 200-kD, 97-kD, and 77-kD bands were less frequently observed (25%, 7%, and 7%, respectively), but when present, their reactions were usually strong. Eleven percent of the BP sera did not react with any bands. Contrary to previous reports, this study shows that BP autoantibodies react with several protein bands, as detected by immunoblotting. We have recently shown by immunoelectron microscopy that BP autoantibodies bind to the basal cell hemidesmosomes. It remains to be determined which of these protein bands represent specific hemidesmosomal proteins and which antibody-antigen interactions are relevant to the pathogenesis of this disease.     

The N terminus of the transmembrane protein BP180 interacts with the N-terminal domain of BP230, thereby mediating keratin cytoskeleton anchorage to the cell surface at the site of the hemidesmosome

In epidermal cells, the keratin cytoskeleton interacts with the elements in the basement membrane via a multimolecular junction called the hemidesmosome. A major component of the hemidesmosome plaque is the 230-kDa bullous pemphigoid autoantigen (BP230/BPAG1), which connects directly to the keratin-containing intermediate filaments of the cytoskeleton via its C terminus. A second bullous pemphigoid antigen of 180 kDa (BP180/BPAG2) is a type II transmembrane component of the hemidesmosome. Using yeast two-hybrid technology and recombinant proteins, we show that an N-terminal fragment of BP230 can bind directly to an N-terminal fragment of BP180. We have also explored the consequences of expression of the BP230 N terminus in 804G cells that assemble hemidesmosomes in vitro. Unexpectedly, this fragment disrupts the distribution of BP180 in transfected cells but has no apparent impact on the organization of endogenous BP230 and alpha6beta4 integrin. We propose that the BP230 N terminus competes with endogenous BP230 protein for BP180 binding and inhibits incorporation of BP180 into the cell surface at the site of the hemidesmosome. These data provide new insight into those interactions of the molecules of the hemidesmosome that are necessary for its function in integrating epithelial and connective tissue types.     

Isolation and characterization of hemidesmosomes from bovine corneal epithelial cells

The hemidesmosome (HD) is a specialized cell-to-substratum junction of stratified and complex epithelia which is characterized by a cytoplasmic plaque to which intermediate filaments (IFs) are anchored. To identify and characterize HD constituents systematically, we have developed a procedure to isolate and fractionate HDs. When bovine corneal epithelium is peeled off from the extracellular matrix stroma, HDs attached to the basal lamina are left behind, together with tufts of cytokeratin IFs attached to the cytoplasmic HD plaques. After rinsing these residual basal cell elements with EDTA, the HDs could be mechanically detached from the stroma and collected by centrifugation. The fraction obtained was examined biochemically and electron microscopically, showing enrichment of HD structures as well as of a prominent 230-kDa polypeptide, the "pemphigoid antigen" known to be located in the HD plaque. In addition, the HD fraction revealed, besides residual amounts of corneal cytokeratins, major polypeptides of Mr 120, 180, 200, 230, and 480 kDa, of which the first three appeared to be glycoproteins. Using the isolated HDs for immunization, we prepared monoclonal antibodies specific for the 230- and 180-kDa polypeptides, respectively, and showed that both were exclusively located in HDs. This method for isolating HDs and the availability of antibodies to HD proteins will be useful in studies of the molecular organization of HDs and make HD research independent from human autoimmune antibodies.     

Hemidesmosome protein dynamics in live epithelial cells

Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin beta4 subunit (GFP-hbeta4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-hbeta4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw-like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw-like clusters of both GFP-hbeta4 and GFP-BP180 are stable over periods of >60 min, other GFP-hbeta4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-hbeta4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-hbeta4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin beta4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions.     

Further analysis of pemphigus autoantibodies and their use in studies on the heterogeneity, structure, and function of desmosomes

Pemphigus is an autoimmune disease that causes blistering of human epidermis. We have recently shown that autoantibodies in the serum of three pemphigus patients bind to desmosomes (Jones, J. C. R., J. Arnn, L. A. Staehelin, and R. D. Goldman, 1984, Proc. Natl. Acad. Sci. USA., 81:2781-2785), and we suggested that pemphigus blisters form, at least in part, from a specific antibody-induced disruption of desmosomes in the epidermis. In this paper, experiments are described that extend our initial observations. 13 pemphigus serum samples, which include four known pemphigus vulgaris (Pv) and four known pemphigus foliaceus (Pf) serum samples, have been analyzed by both immunofluorescence and by immunoblotting using cell-free desmosome preparations. Tissue sections of mouse skin processed for double indirect immunofluorescence using each of the pemphigus serum samples and a rabbit antiserum directed against a component of the desmosomal plaque (desmoplakin) show similar punctate cell surface staining patterns. This suggests that all 13 pemphigus serum samples contain autoantibodies that recognize desmosomes. These autoantibodies appear specific for stratified squamous epithelial cell desmosomes and do not recognize desmosomes of other tissues (e.g., mouse heart and mouse intestine). Cultured mouse keratinocytes, which possess well-defined desmosomes, were processed for indirect immunofluorescence using the pemphigus serum samples. Eight of the 13 sera (including the four known Pv samples but not the known Pf sera) stain desmosomes in these preparations. By double indirect immunofluorescence the desmoplakin antiserum stains a double fluorescent line along the contacting edges of cultured keratinocytes, whereas the positive pemphigus serum samples stain a single fluorescent line along this same border. We believe that these pemphigus autoantibodies recognize extracellular antigens located somewhere within the region between the two apposing membranes that comprise the desmosome. The pemphigus sera exhibit positive immunoblotting reactions with desmosome-enriched fractions obtained from bovine tongue epithelium. Three serum samples (including two of the four known Pf serum samples) react with 160- and 165-kD desmosome-associated polypeptides (Koulu, L., A. Kusimi, M. S. Steinberg, V. Klaus-Kovtun, and J. R. Stanley, 1984, J. Exp. Med., 160:1509-1518). Another eight serum samples (including the four known Pv sera) recognize a 140-kD desmosome-associated polypeptide. We propose that the antigens recognized by these human autoantibodies may play important roles in the adhesion of cells within the epidermis.(ABSTRACT TRUNCATED AT 400 WORDS)     

IFAP 300 is common to desmosomes and hemidesmosomes and is a possible linker of intermediate filaments to these junctions

The distribution of IFAP 300, a protein previously characterized as cross-linking vimentin intermediate filaments (IF), has been investigated in epithelial cells. In frozen sections of bovine tongue epithelium the staining obtained with IFAP 300 antibodies is concentrated in the peripheral cytoplasm of keratinocytes, including the entire peripheral region of basal cells. Further immunofluorescence studies reveal that in primary cultures of mouse keratinocytes the distribution of IFAP 300 is similar to that of the desmosomal protein desmoplakin. In rat bladder carcinoma 804G cells the staining pattern of IFAP 300 antibodies coincides with that obtained with antibodies against the hemidesmosomal protein BP 230. By immunogold electron microscopy IFAP 300 is mainly located at sites where IF appear to attach to desmosomes and hemidesmosomes. Morphometric analyses of the distribution of the gold particles show that IFAP 300 overlaps with desmoplakin and BP 230, but also that it extends deeper into the cytoplasm than these latter two proteins. The staining reaction seen in epithelial cells by immunofluorescence and immunogold is specific for IFAP 300 as shown by immunoblotting. Immunoblotting also reveals that IFAP 300 is present in both cell-free preparations of desmosomes and hemidesmosomes. These morphological and biochemical results are intriguing since, in recent years, the proteins appearing in these two types of junctions have been found to be different. One possible exception is plectin, a protein that has been suggested to be very similar to IFAP 300. However, we show here that IFAP 300 differs from plectin in several respects, including differences at the primary sequence level. We also show that purified IFAP 300 pellets with in vitro polymerized IF prepared from desmosome-associated keratins under conditions in which IFAP 300 alone is not sedimentable. This indicates that IFAP 300 can associate with keratin IF. These data, taken together with the immunogold results, suggest that IFAP 300 functions in epithelial cells as a linker protein connecting IF to desmosomes as well as to hemidesmosomes, possibly through structurally related proteins such as desmoplakin and BP 230, respectively.     

Isolation of a hemidesmosome-rich fraction from a human squamous cell carcinoma cell line

Hemidesmosomes are cell-to-matrix adhesion complexes anchoring keratinocytes to basement membranes. For the first time, we present a method to prepare a fraction from human cultured cells that are highly enriched in hemidesmosomal proteins. Using DJM-1 cells derived from human squamous cell carcinoma, accumulation of hemidesmosomes was observed when these cells were cultured for more than 10 days in a commercial serum-free medium without supplemental calcium. Electron microscopy demonstrated that numerous electron-dense adhesion structures were present along the basal cell membranes of DJM-1 cells cultured under the aforementioned conditions. After removing cellular materials using an ammonia solution, hemidesmosomal proteins and deposited extracellular matrix were collected and separated by electrophoresis. There were eight major polypeptides, which were determined to be plectin, BP230, BP180, integrin α6 and β4 subunits, and laminin-332 by immunoblotting and mass spectrometry. Therefore, we designated this preparation as a hemidesmosome-rich fraction. This fraction contained laminin-332 exclusively in its unprocessed form, which may account for the promotion of laminin deposition, and minimal amounts of Lutheran blood group protein, a nonhemidesmosomal transmembrane protein. This hemidesmosome-rich fraction would be useful not only for biological research on hemidesmosomes but also for developing a serum test for patients with blistering skin diseases.     

Breaking the connection: displacement of the desmosomal plaque protein desmoplakin from cell-cell interfaces disrupts anchorage of intermediate filament bundles and alters intercellular junction assembly

The desmosomal plaque protein desmoplakin (DP), located at the juncture between the intermediate filament (IF) network and the cytoplasmic tails of the transmembrane desmosomal cadherins, has been proposed to link IF to the desmosomal plaque. Consistent with this hypothesis, previous studies of individual DP domains indicated that the DP COOH terminus associates with IF networks whereas NH2-terminal sequences govern the association of DP with the desmosomal plaque. Nevertheless, it had not yet been demonstrated that DP is required for attaching IF to the desmosome. To test this proposal directly, we generated A431 cell lines stably expressing DP NH2-terminal polypeptides, which were expected to compete with endogenous DP during desmosome assembly. As these polypeptides lacked the COOH-terminal IF-binding domain, this competition should result in the loss of IF anchorage if DP is required for linking IF to the desmosomal plaque. In such cells, a 70-kD DP NH2- terminal polypeptide (DP-NTP) colocalized at cell-cell interfaces with desmosomal proteins. As predicted, the distribution of endogenous DP was severely perturbed. At cell-cell borders where endogenous DP was undetectable by immunofluorescence, there was a striking absence of attached tonofibrils (IF bundles). Furthermore, DP-NTP assembled into ultrastructurally identifiable junctional structures lacking associated IF bundles. Surprisingly, immunofluorescence and immunogold electron microscopy indicated that adherens junction components were coassembled into these structures along with desmosomal components and DP-NTP. These results indicate that DP is required for anchoring IF networks to desmosomes and furthermore suggest that the DP-IF complex is important for governing the normal spatial segregation of adhesive junction components during their assembly into distinct structures.     

Is the hemidesmosome a half desmosome? An immunological comparison of mammalian desmosomes and hemidesmosomes

Although the mammalian epidermal basal cell hemidesmosome bears some superficial resemblance to one half of a desmosome at the ultrastructural level, examination of the structure of the electron-dense submembranous plaques of the hemidesmosome and desmosome reveals that they differ with respect to their overall morphology and dimensions. Based on these findings, we wondered whether components of the desmosome are present in the hemidesmosome. In order to determine this we prepared a number of stratified squamous epithelial tissues for indirect immunofluorescence using antibody preparations directed against known desmosome components including desmoplakin and certain glycoproteins. These antibody preparations do not show reaction with hemidesmosomes by indirect immunofluorescence criteria. We have also utilized bullous pemphigoid (BP) autoantibodies that have been shown to recognize hemidesmosomes in mammalian skin cells [Mutasim et al., J. Invest. Derm., 84:47-53, 1985]. Double label indirect immunofluorescence observations of neonatal mouse skin prepared using desmoplakin antibodies and BP autoantibodies reveal that hemidesmosomes that are stained by the BP autoantibodies are not recognized by the desmoplakin antibodies. We confirmed these findings at the ultrastructural level by indirect immunogold localization of desmoplakin antibodies and BP autoantibodies. Therefore, the hemidesmosome does not appear to be one half of a desmosome and may possess a very different molecular organization relative to the desmosome. We raise the possibility that the variability between the hemidesmosome and desmosome that we detect at the morphological and immunological level may reflect the functional differences of these two types of junctions.     

Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly

Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.     

Identification and characterization of epitopes recognized by T lymphocytes and autoantibodies from patients with herpes gestationis

Autoantibodies associated with herpes gestationis (HG), a pregnancy-associated autoimmune skin disease, target the hemidesmosomal protein BP180. It was shown that the major noncollagenous stretch of the BP180 ectodomain (NC16A) harbors epitopes recognized by HG sera. Furthermore, Abs reactive with the homologous domain of murine BP180 are known to trigger a cutaneous blistering disease in mice by passive transfer experiments. The present study was aimed at characterizing the T cell responses and specificities of autoantibodies from two HG patients. Using immunoblotting and T cell proliferation assays, we have identified a 14-amino-acid stretch of the BP180 ectodomain (MCW-1; aa 507-520) that is recognized by both T cells and autoantibodies produced by the HG patients. The neonate born to one of these HG patients showed no signs of skin disease and had no detectable T cell response to the BP180 Ag, but did have a low titer of circulating anti-BP180 autoantibodies, presumably of maternal origin. BP180-specific T cell lines and clones developed from an HG patient specifically reacted with the MCW-1 epitope. The proliferative responses of these clones were restricted to HLA-DR, but not -DQ or -DP. These Ag-specific T cells expressed alpha/beta TCRs and a CD4 memory T cell phenotype and secreted IFN-gamma and IL-2, but not IL-4 or IL-6, suggesting that they are Th1-type lymphocytes. Further characterization of these Ag-specific T cells and autoantibodies will aid in elucidating the autoimmune mechanism(s) leading to the development of HG.     

Molecular complexity of the cutaneous basement membrane zone

Ultrastructural examination of the cutaneous basement membrane zone (BMZ) reveals the presence of several attachment structures, which are critical for integrity of the stable association of epidermis and dermis. These include hemidesmosomes which extend from the intracellular compartment of the basal keratinocyte to the underlying basement membrane where they complex with anchoring filaments, thread-like structures traversing the lamina lucida. At the lower portion of dermal-epidermal attachment zone, anchoring fibrils extend from the lamina densa to the papillary dermis, where they associate with basement membrane-like structures, known as anchoring plaques. Molecular cloning of the cutaneous BMZ components has allowed elucidation of the structural features of the proteins which constitute these attachment structures. Specifically, hemidesmosomes have been shown to consist of at least four distinct proteins. The intracellular hemidesmosomal inner plaque is comprised of the 230-kD bullous pemphigoid antigen (BPAG1), and plectin, a high-molecular weight cytomatrix protein, encoded by the corresponding gene, PLEC1. The transmembrane component of the hemidesmosomes consists of the 180-kD bullous pemphigoid antigen (BPAG2), a collagenous protein also known as type XVII collagen (COL17A1), as well as of the basal keratinocyte-specific integrin 64. The anchoring filaments consist predominantly of laminin 5 with three constitutive subunit polypeptides, the 3, 3 and 2 chains, which is associated with laminin 6 with the chain composition 3, 1 and 1. Also associated with anchoring filaments is a novel protein, ladinin, which serves as autoantigen in the linear IgA disease, and the corresponding gene, LAD1, has been mapped to human chromosome 1. Finally, the major, if not the exclusive, component of anchoring fibrils is type VII collagen, encoded by the gene (COL7A1) which consists of 118 distinct exons, the largest number of exons in any gene published thus far. Collectively, the cutaneous basement membrane zone is a complex continuum of macromolecules which form a network providing the stable association of the epidermis to the underlying dermis. Thus, genetic lesions resulting in abnormalities in any part of this network could result in a blistering skin disease, such as epidermolysis bullosa.Abbreviations BMZ basement membrane zone - EB epidermolysis bullosa - JEB junctional EB - GABEB generalized atrophic benign EB - EB-MD epidermolysis bullosa with muscular dystrophy - EB-PA epidermolysis bullosa with pyloric atresia     

Role of FcRs in animal model of autoimmune bullous pemphigoid

Bullous pemphigoid (BP) is a bullous dermatosis associated with autoantibodies directed against the hemidesmosomal Ags BP180 and BP230. Lesional skin is characterized by detachment of the epidermis from the dermis with an intense inflammatory cell infiltrate in the upper dermis. In experimental BP, subepidermal blistering is triggered by rabbit anti-murine BP180 (mBP180) IgG and depends upon complement activation, mast cell degranulation, and neutrophil infiltration. In this study, we determined the role of Fc gammaRs on neutrophils in experimental BP. Mice deficient in Fc gammaRIII (Fc gammaRIII-/-) and those deficient in both Fc gammaRI and Fc gammaRIII (Fc gammaRI&III-/-) but not in Fc gammaRII (Fc gammaRII-/-) were resistant to BP. Pathogenic IgG activated wild-type neutrophils, but not Fc gammaRIII-deficient neutrophils, to secrete proteolytic enzymes. The function of anti-mBP180 IgG depended entirely on its Fc domain; F(ab')2 of IgG had no pathogenic activities. In wild-type mice injected with pathogenic IgG, an Fc gammaR blocker abolished the BP phenotype and inhibited activation of wild-type neutrophils stimulated by pathogenic IgG. Results from this study establish that Fc gammaRIII plays a critical role in the activation of infiltrating neutrophils and the subsequent blistering in experimental BP.     

Localization of the 230-kilodalton bullous pemphigoid antigen in cultured keratinocytes: formation of a prehemidesmosome.

The hemidesmosome is the major attachment structure of the epidermal basal cell visible ultrastructurally in skin. The importance of its components to cultured cell attachment to substratum is not understood, however. A component of the hemidesmosome, the 230-kDa bullous pemphigoid antigen (p230), has been shown to be present in an insoluble or particulate fraction of cultured cells. In order to more fully characterize its potential importance for cell-matrix adhesion in cultured keratinocytes, specific antibodies were raised to the C-terminal region of p230 expressed as a bacterial fusion protein. Such antibodies recognize the hemidesmosome of epidermis, binding on the cytoplasmic region of its plaque. In addition, keratinocytes cultured in a 0.15 mM Ca(2+)-defined medium contain a detergent-resistant pool of p230 which appears to lie in the same focal plane as the culture substrate and has a patchy or irregular distribution by indirect immunofluorescence. Treatment of cultured cells at 4 degrees C with trypsin or pronase sufficient to release keratinocytes from the culture dish does not affect the electrophoretic migration of p230 on SDS-gels, suggesting that p230 is not exposed to the extracellular space. In cells cultured in 0.15 mM Ca2+, 230-kDa BP antigen is localized to discrete clusters resting near the basal plasma membrane of the cell by immunogold staining following brief detergent treatment and fixation. These clusters are approximately 0.1 micron in diameter, which is similar in size to the in vivo hemidesmosome. Fully formed electron dense hemidesmosomal plaques are not observed under the same culture conditions, however. It appears that these clusters are early precursors of the hemidesmosome.     

Integrin alpha 6 beta 4 forms a complex with the cytoskeletal protein HD1 and induces its redistribution in transfected COS-7 cells.

The integrin alpha 6 beta 4 is a major component of hemidesmosomes, in which it is linked to intermediate filaments. Its presence in these structures is dependent on the beta 4 cytoplasmic domain but it is not known whether beta 4 interacts directly with keratin filaments or by interaction with other proteins. In this study, we have investigated the interaction of GST-cyto beta 4A fusion proteins with cellular proteins and demonstrate that a fragment of beta 4A, consisting of the two pairs of fibronectin type III repeats, separated by the connecting segment, forms a specific complex containing a 500-kDa protein that comigrates with HD1, a hemidesmosomal plaque protein. A similar protein was also bound by a glutathione S-transferase fusion protein containing the cytoplasmic domain of a variant beta 4 subunit (beta 4B), in which a stretch of 53 amino acids is inserted in the connecting segment. Subsequent immunoblot analysis revealed that the 500-kDa protein is in fact HD1. In COS-7 cells, which do not express alpha 6 beta 4 or the hemidesmosomal components BP230 and BP180, HD1 is associated with the cytoskeleton, but after transfecting the cells with cDNAs for human alpha 6 and beta 4, it was, instead, colocalized with alpha 6 beta 4 at the basal side of the cells. The organization of the vimentin, keratin, actin, and tubulin cytoskeletal networks was not affected by the expression of alpha 6 beta 4 in COS-7 cells. The localization of HD1 at the basal side of the cells depends on the same region of beta 4 that forms a complex containing HD1 in vitro, since the expression of alpha 6 with a mutant beta 4 subunit that lacks the four fibronectin type III repeats and the connecting segment did not alter the distribution of HD1. The results indicate that for association of alpha 6 beta 4 with HD1, the cytoplasmic domain of beta 4 is essential. We suggest that this association may be crucial for hemidesmosome assembly.     

A function for the integrin alpha 6 beta 4 in the hemidesmosome.

Many epithelial cells appear to use cell-substratum adhesion complexes known as hemidesmosomes as the main means of anchorage to the connective tissue. Initially recognized as distinctive electron-dense images, hemidesmosomes are still poorly understood at the biochemical level. The regulation and mode of their assembly, which is disrupted in certain blistering diseases and is critical to proper wound repair, also remains to be elucidated. The integrin alpha 6 beta 4 is expressed along the basal surface of various epithelial cells. We show here that this integrin localizes to hemidesmosomes as determined by immunoelectron microscopy using antibodies directed against both the extra- and intracytoplasmic domains of alpha 6 beta 4. This result, which agrees with a recent study, suggests a functional role for the alpha 6 beta 4 integrin in the hemidesmosomes. We therefore investigated such a potential role for this integrin using the cultured rat bladder carcinoma cell line 804G, which has the uncommon ability to form hemidesmosomes in vitro when maintained on uncoated glass substrates. By immunoprecipitation and immunofluorescence, we show that 804G cells express alpha 6 beta 4 along their basal surface in a punctate pattern that overlaps with the distribution of hemidesmosomal plaque antigens. However, this pattern is altered when cells are plated in the presence of an antiserum directed against alpha 6 beta 4. Furthermore, no hemidesmosomes are detectable at the ultrastructural level in the alpha 6 beta 4 antibody-treated cells compared with control cells. These results indicate that integrins may play a critical role in assembly and adhesive functions of the hemidesmosome.