title: 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.
abstract: The hemidesmosome is an adhesion structure of the epidermal-dermal junction in keratinocytes. When keratinocytes migrate laterally or upward to differentiate, they must control the formation and disintegration of the hemidesmosomes. When keratinocytes are cultured in low-calcium (below 0.1 mM) medium, all cells behave like basal cells, adhere to the culture dish, and proliferate without differentiation. The calcium addition induces the differentiation. A bullous pemphigoid antigen, 180-kDa BPA, has been shown to be a component of the hemidesmosome. Using a monoclonal antibody to the 180-kDa BPA and a human squamous cell carcinoma cell line (DJM-1 cells), the fate of hemidesmosomes was studied after the addition of calcium to low-calcium-grown cells and 12-tetradecanoylphorbol-13-acetate (TPA) to high-calcium (1.87 mM) grown cells by immunofluorescence and immunoelectron microscopy. The antigen was distributed evenly as fine dots on the entire ventral surface of low-calcium cells, whereas they formed a peculiar, concentric ring or arch arrangement on the ventral surface of high-calcium cells. Immunoelectron microscopy revealed the deposits of gold particles at sites on the membrane surface, where some filamentous or electron-dense materials were associated, although the complete structure of hemidesmosomes was not formed. They deposited directly onto the membrane surface in low-calcium cells and with a distance of 20-50 nm from the membrane surface in high-calcium cells. The calcium addition caused a profound reduction of the 180-kDa BPA-positive area for 30 to 120 min and then formed the high-calcium-ring pattern after 4 to 6 h. A similar calcium response was seen in normal human keratinocytes. TPA (16 nM) treatment caused disintegration of the ring pattern in high-calcium DJM-1 cells. This was inhibited with a protein kinase C (PKC) inhibitor. H7 (20 microM). These results suggest that the hemidesmosome is a dynamic structure and PKC can be one of the major factors in controlling the hemidesmosome, since it is known that the low-high calcium shift induces a calcium influx and a PKC activation, and TPA activates PKC in keratinocytes.
authors.affiliation: Department of Dermatology, Jichi Medical School, Tochigiken, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kitajima
authors.forename: K
authors.initials: K
authors.lastname: Owaribe
authors.forename: Y
authors.initials: Y
authors.lastname: Nishizawa
authors.forename: Y
authors.initials: Y
authors.lastname: Jokura
authors.forename: H
authors.initials: H
authors.lastname: Yaoita
mesh_terms: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001324:Autoantigens / Q000032:analysis / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D002294:Carcinoma, Squamous Cell; D002352:Carrier Proteins; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D003094:Collagen; D003598:Cytoskeletal Proteins; D003896:Desmosomes / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000071821:Dystonin; D005455:Fluorescent Antibody Technique; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D015603:Keratinocytes / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D016253:Microscopy, Immunoelectron; D008970:Molecular Weight; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010391:Pemphigoid, Bullous / Q000473:pathology*; D010879:Piperazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012878:Skin Neoplasms; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D000097805:Collagen Type XVII
chemical_list: D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D001324:Autoantigens; D002118:Calcium; D002352:Carrier Proteins; D003094:Collagen; D003598:Cytoskeletal Proteins; D000071821:Dystonin; D007546:Isoquinolines; D009419:Nerve Tissue Proteins; D024041:Non-Fibrillar Collagens; D010879:Piperazines; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D000097805:Collagen Type XVII; C486268:DST protein, human