title: Up-regulation of cell surface insulin receptor by protein kinase C-alpha in adrenal chromaffin cells: involvement of transcriptional and translational events.
abstract: Our previous study showed that treatment of cultured bovine adrenal chromaffin cells with phorbol 12, 13-dibutyrate (PDBu) or 12-O-tetradecanoylphorbol 13-acetate (TPA) caused a rapid (<15 min) and persistent (>15 h) translocation of both conventional (c) protein kinase C-alpha (PKC-alpha) and novel PKC-epsilon (but not atypical PKC-zeta) from cytosol to membranes, whereas thymeleatoxin (TMX) increased the similar but selective membrane association of only cPKC-alpha. In the present study, chronic (>/=12 h) treatment of chromaffin cells with PDBu raised cell surface (125)I-insulin binding without altering the K(D) value; it developed in a concentration (EC(50) = 1.9 nM)-and time (t(1/2) = 14.6 h)-dependent manner, reaching its maximum 115% increase at 48 h. Either TPA (30 nM) or TMX (EC(50) = 6.4 nM) also increased (125)I-insulin binding by 97 or 88%, whereas the biologically inactive 4alpha-TPA had no effect. The increasing effect of PDBu (30 nM for 24 h) on (125)I-insulin binding was significantly blocked, even when H7, an inhibitor of PKC, was added at 8 h after the initiation of PDBu treatment. Concurrent treatment with brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, cycloheximide, an inhibitor of protein synthesis, or 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA synthesis, abolished the PDBu-induced increment of (125)I-insulin binding. Western blot analysis, using antibody against the beta-subunit of the insulin receptor, showed that treatment with PDBu (30 nM) or TMX (EC(50) = 2.3 nM) increased levels of insulin receptor precursor (approximately 190 kDa; t(1/2) = 7.1 h) and insulin receptor beta-subunit (t(1/2) = 15.4 h), causing their almost maximum 52 and 59% rises, respectively, at 24 h. Northern blot analysis revealed that PDBu or TMX increased levels of insulin receptor mRNAs by approximately 35% as soon as 3 h, producing its monophasic peak approximately 76% increases at 24 h. All of these increasing effects of PDBu and TMX on (125)I-insulin binding and insulin receptor beta-subunit and insulin receptor mRNA levels were entirely prevented by simultaneous treatment with Gö6976, a selective inhibitor of cPKC. These results suggest that long-term activation of cPKC-alpha up-regulates the density of the cell surface insulin receptor via transcriptional/translational events.
authors.affiliation: Department of Pharmacology, Miyazaki Medical College, Miyazaki, Japan.
authors.forename: R
authors.initials: R
authors.lastname: Yamamoto
authors.forename: H
authors.initials: H
authors.lastname: Kobayashi
authors.forename: T
authors.initials: T
authors.lastname: Yanagita
authors.forename: H
authors.initials: H
authors.lastname: Yokoo
authors.forename: T
authors.initials: T
authors.lastname: Kurose
authors.forename: S
authors.initials: S
authors.lastname: Shiraishi
authors.forename: S
authors.initials: S
authors.lastname: Minami
authors.forename: S
authors.initials: S
authors.lastname: Matsukura
authors.forename: A
authors.initials: A
authors.lastname: Wada
mesh_terms: D000313:Adrenal Medulla / Q000166:cytology / Q000502:physiology*; D000818:Animals; D002227:Carbazoles / Q000494:pharmacology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000166:cytology / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007211:Indoles / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D010703:Phorbol Esters / Q000494:pharmacology; D014176:Protein Biosynthesis; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D011972:Receptor, Insulin / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D014158:Transcription, Genetic; D015854:Up-Regulation
chemical_list: D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D007328:Insulin; D007527:Isoenzymes; D010703:Phorbol Esters; C081021:Go 6976; D015240:Phorbol 12,13-Dibutyrate; C080287:thymeleatoxin; D011972:Receptor, Insulin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha