title: Antofine-induced connexin43 gap junction disassembly in rat astrocytes involves protein kinase Cβ.
abstract: Antofine, a phenanthroindolizidine alkaloid derived from Cryptocaryachinensis and Ficusseptica in the Asclepiadaceae milkweed family, is cytotoxic for various cancer cell lines. In this study, we demonstrated that treatment of rat primary astrocytes with antofine induced dose-dependent inhibition of gap junction intercellular communication (GJIC), as assessed by scrape-loading 6-carboxyfluorescein dye transfer. Levels of Cx43 protein were also decreased in a dose- and time-dependent manner following antofine treatment. Double-labeling immunofluorescence microscopy showed that antofine (10ng/ml) induced endocytosis of surface gap junctions into the cytoplasm, where Cx43 was co-localized with the early endosome marker EEA1. Inhibition of lysosomes or proteasomes by co-treatment with antofine and their respective specific inhibitors, NH4Cl or MG132, partially inhibited the antofine-induced decrease in Cx43 protein levels, but did not inhibit the antofine-induced inhibition of GJIC. After 30min of treatment, antofine induced a rapid increase in the intracellular Ca(2+) concentration and activation of protein kinase C (PKC)α/βII, which was maintained for at least 6h. Co-treatment of astrocytes with antofine and the intracellular Ca(2+) chelator BAPTA-AM prevented downregulation of Cx43 and inhibition of GJIC. Moreover, co-treatment with antofine and a specific PKCβ inhibitor prevented endocytosis of gap junctions, downregulation of Cx43, and inhibition of GJIC. Taken together, these findings indicate that antofine induces Cx43 gap junction disassembly by the PKCβ signaling pathway. Inhibition of GJIC by antofine may undermine the neuroprotective effect of astrocytes in CNS.
authors: Institute of Anatomy and Cell Biology, School of Medicine, National Yang-Ming University, Taipei 11221, Taiwan.
authors: Yu-Fang
authors: YF
authors: Huang
authors: Chih-Kai
authors: CK
authors: Liao
authors: Jau-Chen
authors: JC
authors: Lin
authors: Guey-Mei
authors: GM
authors: Jow
authors: Hwai-Shi
authors: HS
authors: Wang
authors: Jiahn-Chun
authors: JC
authors: Wu
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D002118:Calcium / Q000378:metabolism; D002450:Cell Communication / Q000187:drug effects*; D002478:Cells, Cultured; D002614:Chelating Agents / Q000494:pharmacology; D018031:Connexin 43 / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004705:Endocytosis / Q000187:drug effects; D004789:Enzyme Activation; D005260:Female; D017629:Gap Junctions / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D007211:Indoles / Q000633:toxicity*; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D010618:Phenanthrolines / Q000633:toxicity*; D046988:Proteasome Endopeptidase Complex / Q000187:drug effects / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D064546:Protein Kinase C beta; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: D002614:Chelating Agents; D018031:Connexin 43; C580592:Gja1 protein, rat; D007211:Indoles; D010618:Phenanthrolines; D061988:Proteasome Inhibitors; D047428:Protein Kinase Inhibitors; D033921:Vesicular Transport Proteins; C473343:antofine; C093846:early endosome antigen 1; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D046988:Proteasome Endopeptidase Complex; D002118:Calcium