title: Macropinocytosis of type XVII collagen induced by bullous pemphigoid IgG is regulated via protein kinase C.
abstract: Macropinocytosis is an endocytic pathway that is involved in the nonselective fluid uptake of extracellular fluid. Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease associated with autoantibodies to type XVII collagen (COL17), which is a component of hemidesmosome. When keratinocytes are treated with BP-IgG, COL17 internalizes into cells by way of the macropinocytosis. We investigated the mechanism of COL17 macropinocytosis using DJM-1 cells, a cutaneous squamous cell carcinoma cell line. First, non-hemidesmosomal COL17 was preferentially depleted by stimulation with the BP-IgG in the DJM-1 cells. To investigate the signaling involved in COL17-macropinocytosis, the inhibition of small GTPase family members Rac1 and Cdc42 was found to strongly repress COL17 internalization; in addition, the Rho inhibitor also partially blocked that internalization, suggesting these small GTPases are involved in signaling to mediate COL17-macropinocytosis. Western blotting using Phostag-SDS-PAGE demonstrated high levels of COL17 phosphorylation in DJM-1 cells under steady-state condition. Treatment with BP-IgG increased the intracellular calcium level within a minute, and induced the overabundant phosphorylation of COL17. The overabundant phosphorylation of COL17 was suppressed by a protein kinase C (PKC) inhibitor. In addition, PKC inhibitor repressed COL17 endocytosis using cell culture and organ culture systems. Finally, the depletion of COL17 was not observed in the HEK293 cells transfected COL17 without intracellular domain. These results suggest that COL17 internalization induced by BP-IgG may be mediated by a PKC pathway. In summary, BP-IgG initially binds to COL17 distributed on the plasma membrane, and COL17 may be internalized by means of a macropinocytic pathway related to the phosphorylation of the intracellular domain by PKC.
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Hiroaki
authors: H
authors: Iwata
authors: Department of Oral Diagnosis and Medicine Hokkaido University, Graduate School of Dental Medicine, Sapporo, Japan.
authors: Mayumi
authors: M
authors: Kamaguchi
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Hideyuki
authors: H
authors: Ujiie
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Machiko
authors: M
authors: Nishimura
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Kentaro
authors: K
authors: Izumi
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Ken
authors: K
authors: Natsuga
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Satoru
authors: S
authors: Shinkuma
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Wataru
authors: W
authors: Nishie
authors: Department of Dermatology, Hokkaido University Graduate School of Medicine, Sapporo, Japan.
authors: Hiroshi
authors: H
authors: Shimizu
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001323:Autoantibodies / Q000494:pharmacology*; D001324:Autoantigens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D045744:Cell Line, Tumor; D057809:HEK293 Cells; D006801:Humans; D007074:Immunoglobulin G / Q000494:pharmacology*; D015603:Keratinocytes / Q000187:drug effects* / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D051379:Mice; D024041:Non-Fibrillar Collagens / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010391:Pemphigoid, Bullous / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D010446:Peptide Fragments; D010766:Phosphorylation / Q000187:drug effects; D010873:Pinocytosis / Q000187:drug effects*; D054730:Protein Interaction Domains and Motifs; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D011994:Recombinant Proteins; D046509:Tissue Culture Techniques; D015854:Up-Regulation / Q000187:drug effects; D000097805:Collagen Type XVII
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D001324:Autoantigens; D007074:Immunoglobulin G; D024041:Non-Fibrillar Collagens; D010446:Peptide Fragments; D011493:Protein Kinase C; D047428:Protein Kinase Inhibitors; D011994:Recombinant Proteins; D000097805:Collagen Type XVII