Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy

Kitajima, Yasuo; Owaribe, Katsushi; Jokura, Yoji; Yaoita, Hideo

doi:10.1016/0014-4827(92)90034-6

Cited by 35 publications

(48 citation statements)

References 23 publications

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“…Thus if structural change in the cytoplasmic domain cannot cause detachment of its extracellular domain from its ligand, a capacity for cleavage would be necessary to allow differentiation and movement away from the basement membrane. Kitajima et al (42,43) observed disappearance of BP180 in DJM-1 cells during rearrangement of hemidesmosomes induced by the treatment with 12-O-tetradecanoylphorbol-13-acetate. However, the possibility that the fragment plays a more direct role in cell-matrix adhesion distinct from the intact molecule, cannot be excluded.…”

Section: Figmentioning

confidence: 99%

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Hirako¹,

Usukura²,

Uematsu³

et al. 1998

Journal of Biological Chemistry

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114

124

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The hemidesmosome (HD) is a cell-to-substrate adhesion apparatus found in stratified and complex epithelia. One of the putative cell-matrix adhesion molecules present in the HD is the 180-kDa bullous pemphigoid antigen (BP180), also termed type XVII collagen. In our previous study, using a monoclonal antibody (mAb) 1337, we have detected a 120-kDa collagenase-sensitive polypeptide in the HD fraction (Uematsu, J. and Owaribe, K. (1993) Cell Struct. Funct. 18, 588 (abstr.)). The present study was undertaken to assess the relation of the 120-kDa polypeptide to this BP180. Immunofluorescence microscopy of bovine skin revealed the basement membrane zone of skin to be stained clearly with mAb 1337, whereas the lateral surfaces of basal cells, which were decorated by typical antibodies against BP180, were not. The antibody did not detect HDs in cultured cells but rather in the culture medium. These results indicate a localization of mAb 1337 antigen distinct from BP180. However, the same polypeptide was also recognized by monoclonal antibodies to the extracellular but not the cytoplasmic part of BP180, and found to react with a polyclonal antibody against the non-collagenous 16A domain of BP180. Therefore, the polypeptide was identified as an extracellular fragment of BP180. mAb 1337 immunoprecipitated the 120-kDa fragment from the medium, but not the 180-kDa molecule of BP180 extracted from cultured cells, indicating that the antibody specifically recognizes the fragment. The mAb 1337 apparently recognizes a unique epitope that is exposed or formed by the cleavage. Hence, the staining pattern observed for bovine skin demonstrated the presence of the 120-kDa extracellular fragment. Rotary shadow electron microscopy of affinity-purified 120-kDa fragments demonstrated that they have the unique molecular shape consisting of a central rod and a flexible tail, without the globular head that is present in the BP180 molecule. From these results, we conclude that mAb 1337 shows unique epitope specificity, recognizing only the 120-kDa extracellular fragment of BP180, which is constitutively cleaved on the cell surface as a 120-kDa fragment both in in vivo and in vitro.

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Section: Figmentioning

confidence: 99%

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Hirako¹,

Usukura²,

Uematsu³

et al. 1998

Journal of Biological Chemistry

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114

124

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“…2ϩ -induced Redistribution of Plectin 1a in Differentiating Keratinocytes-In the early stages of keratinocyte differentiation (as well as of wound healing) hemidesmosomal components, such as integrin ␣6␤4 and BPAG-2, show a redistribution from HDs to other cell compartments (24,33). To examine the fate of HD-associated plectin 1a under similar conditions, confluent cultures of primary mouse keratinocytes were subjected to differentiation by exposure to 1.3 mM CaCl 2 for up to 8 h (Fig.…”

Section: Camentioning

confidence: 99%

Plectin Isoform-dependent Regulation of Keratin-Integrin α6β4 Anchorage via Ca2+/Calmodulin

Košťan

Gregor

Walko

et al. 2009

Journal of Biological Chemistry

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The detachment of epithelial cells from the basal matrix during wound healing and differentiation of keratinocytes requires the disassembly of the hemidesmosomal multiprotein adhesion complex. Integrin ␣6␤4-plectin interaction plays a major role in the formation of hemidesmosomes, and thus the mechanisms regulating this interaction should be critical also for the disassembly process. Here we show that a particular plectin isoform (1a) interacts with the Ca 2؉ -sensing protein calmodulin in a Ca 2؉ -dependent manner. As a result of this interaction, binding of the hemidesmosome-associated plectin isoform 1a to integrin ␤4 is substantially diminished. Calmodulin-binding inhibits also the interaction of plectin with F-actin. Further, we found that, during Ca 2؉ -induced keratinocyte differentiation, plectin 1a is first relocated within the cell and later down-regulated, suggesting that Ca 2؉ affects the fate of plectin 1a upon its release from hemidesmosomes. We propose a novel model for the disassembly of hemidesmosomes during keratinocyte differentiation, where both, binding of calmodulin to plectin 1a and phosphorylation of integrin ␤4 by protein kinases, are required for disruption of the integrin ␣6␤4-plectin complex.Basal cell layer keratinocytes of the skin are firmly attached to the underlying basement membrane through hemidesmosomes (HDs), 2 protein complexes that mediate stable anchoring by providing a tight link between the intracellular intermediate filament network (IF) system and the extracellular matrix (1, 2). Plectin, a universal and functionally versatile cytolinker protein, has been implicated in HD functions since early on (3, 4). Binding of plectin to the ␤4 subunit of integrin ␣6␤4 turned out to be a critical step in the formation of HDs, and various regions of plectin have been shown to be involved in this interaction. One of them is a canonical actin-binding domain (ABD) of the tandem calponin homology (CH) domain type (5-7), which binds to the second fibronectin type III (FnIII) domain of the ␤4 subunit. Residues crucial for this binding are clustered on the surface of the first N-terminal calponin homology domain. They partially overlap with one of the essential F-actin-binding sequences, providing a structural basis for the competitive nature of plectin binding to integrin ␤4 and F-actin (2,8). A second integrin ␤4-binding site, located in the plakin domain of plectin (9), interacts with sequences downstream of the second FnIII domain of integrin ␤4 (10). In addition, interactions of the C-terminal repeat domain R6 of plectin with the segment connecting the second and third FnIII domains of integrin ␤4 (C-segment), and with integrin ␤4 tail domain, have been reported (9).In accordance with plectin being a prominent constituent of HDs, abnormalities in its expression in humans lead to epidermolysis bullosa simplex associated with muscular dystrophy, manifesting as severe skin blistering and muscle disorders (for review see Ref. 11); an autosomal dominant form of the disease (epidermolysis ...

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“…An isolated cell line (DJM-1) from human skin squamous cell carcinoma was used in the present study (Kitajima et al, 1988(Kitajima et al, , 1992. DJM-1 cells were cultured in MEM (Life Technologies, Inc., Rockville, Maryland) containing 10% FCS, 0.4 mg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 84 ng/ml cholera toxin, 100 mg/ml kanamycin, and 1.8 mM Ca ϩϩ .…”

Section: Cell Culturesmentioning

confidence: 99%

Assembly Pathway of Desmoglein 3 to Desmosomes and Its Perturbation by Pemphigus Vulgaris-IgG in Cultured Keratinocytes, as Revealed by Time-Lapsed Labeling Immunoelectron Microscopy

Sato

Aoyama

Kitajima

2000

Laboratory Investigation

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SUMMARY:To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or 10-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes. (Lab Invest 2000, 80:1583-1592.

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Phorbol ester- and calcium-induced reorganization of 180-kDa bullous pemphigoid antigen on the ventral surface of cultured human keratinocytes as studied by immunofluorescence and immunoelectron microscopy

Cited by 35 publications

References 23 publications

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Cleavage of BP180, a 180-kDa Bullous Pemphigoid Antigen, Yields a 120-kDa Collagenous Extracellular Polypeptide

Plectin Isoform-dependent Regulation of Keratin-Integrin α6β4 Anchorage via Ca2+/Calmodulin

Assembly Pathway of Desmoglein 3 to Desmosomes and Its Perturbation by Pemphigus Vulgaris-IgG in Cultured Keratinocytes, as Revealed by Time-Lapsed Labeling Immunoelectron Microscopy

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