Protein kinase C-alpha mediates TNF release process in RBL-2H3 mast cells.
1 To clarify the mechanism of mast cell TNF secretion, especially its release process after being produced, we utilized an antiallergic drug, azelastine (4-(p-chlorobenzyl)-2-(hexahydro-1-methyl-1H-azepin-4-yl)-1-(2H)- phthalazinone), which has been reported to inhibit TNF release without affecting its production in ionomycin-stimulated RBL-2H3 cells. 2 Such inhibition was associated with the suppression of an ionomycin-induced increase in membrane-associated PKC activity rather than the suppression of Ca2+ influx, suggesting that PKC might be involved in TNF release process. 3 To see whether conventional PKC family (cPKCs) are involved, we investigated the effects of a selective cPKC inhibitor (Gö6976) and an activator (thymeleatoxin) on TNF release by adding them 1 h after cell stimulation. By this time, TNF mRNA expression had reached its maximum. Gö6976 markedly inhibited TNF release, whereas thymeleatoxin enhanced it, showing a key role of cPKC in TNF post-transcriptional process, possibly its releasing step. 4 To determine which subtype of cPKCs could be affected by azelastine, Western blotting and live imaging by confocal microscopy were conducted to detect the translocation of endogenous cPKC (alpha, betaI and betaII) and transfected GFP-tagged cPKC, respectively. Both methods clearly demonstrated that 1 microM azelastine selectively inhibits ionomycin-triggered translocation of (alpha)PKC without acting on betaI or betaIIPKC. 5 In antigen-stimulated cells, such a low concentration of azelastine did not affect either (alpha)PKC translocation or TNF release, suggesting a functional link between (alpha)PKC and the TNF-releasing step. 6 These results suggest that (alpha)PKC mediates the TNF release process and azelastine inhibits TNF release by selectively interfering with the recruitment of (alpha)PKC in the pathway activated by ionomycin in RBL-2H3 cells.
Abdel-Raheem
Ihab T
IT
Department of Pharmacology, Faculty of Pharmacy, Al-Azhar University, Assiut 71511, Egypt.
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Izumi
I
Yanase
Yuhki
Y
Shigemoto-Mogami
Yukari
Y
Sakai
Norio
N
Shirai
Yasuhito
Y
Saito
Naoaki
N
Hamada
Farid M
FM
El-Mahdy
Nagh A
NA
Elsisy
Alaa El-Din E
Ael-D
Sokar
Samya S
SS
Nakata
Yoshihiro
Y
D000818:Animals; D015153:Blotting, Western; D002227:Carbazoles / Q000494:pharmacology; D045744:Cell Line, Tumor; D004140:Dinitrophenols / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018613:Microscopy, Confocal; D010793:Phthalazines / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012710:Serum Albumin, Bovine / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism*
D002227:Carbazoles; D004140:Dinitrophenols; D004791:Enzyme Inhibitors; D007211:Indoles; D010793:Phthalazines; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; C034638:dinitrophenyl-bovine serum albumin; C081021:Go 6976; D049452:Green Fluorescent Proteins; D012710:Serum Albumin, Bovine; D015759:Ionomycin; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; C020976:azelastine