Reciprocal effects of C/EBPalpha and PKCdelta on JunB expression and monocytic differentiation depend upon the C/EBPalpha basic region.
Monocytic differentiation of 32DPKCdelta cells in response to activation of protein kinase C delta (PKCdelta) by phorbol 12-myristate 13-acetate (PMA) was inhibited by exogenous CCAAT/enhancer binding protein alpha-estradiol receptor (C/EBPalpha-ER), which impeded morphologic maturation and induction of macrosialin mRNA. Inhibition of monopoiesis was also evident in 32DPKCdelta subclones expressing C/EBPalphaLeu12Val-ER, which cannot dimerize or bind DNA because of mutation of the leucine zipper, C/EBPalphaGZ-ER, in which the leucine zipper has been replaced by the GCN4 zipper, or C/EBPalphaDelta3-8-ER, lacking the C/EBPalpha transactivation domains. In contrast, C/EBPalphaBR3-ER, containing a mutant basic region, did not inhibit monocytic differentiation. C/EBPalpha-ER strongly inhibited endogenous AP-1 DNA-binding. Supershift analysis revealed that the major AP-1 complex contains JunB. Activation of C/EBPalpha-ER specifically reduced endogenous JunB RNA and protein and exogenous JunB levels without affecting endogenous or exogenous c-Jun. The stability of PMA-induced JunB was not affected. Thus, C/EBPalpha-ER suppresses both JunB transcription and posttranscriptional protein generation or induction. PU.1 levels and activity were increased. The Leu12Val, GZ, and Delta3-8 mutants also inhibited JunB expression, whereas the BR3 mutant was ineffective, indicating that inhibition of JunB expression and monocytic differentiation by C/EBPalpha-ER depends upon an interaction mediated by its basic region. Exogenous JunB restored AP-1 DNA-binding but did not prevent inhibition of macrosialin expression by C/EBPalpha-ER, indicating that JunB is not the only target relevant to inhibition of monopoiesis by C/EBPalpha.
Liu
Huaitian
H
Division of Pediatric Oncology, Johns Hopkins University, Baltimore, MD, USA.
Keefer
Jeffrey R
JR
Wang
Qian-fei
QF
Friedman
Alan D
AD
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002454:Cell Differentiation; D002460:Cell Line; D002478:Cells, Cultured; D017931:DNA Primers; D016758:Genes, jun / Q000187:drug effects*; D006801:Humans; D016350:Leucine Zippers; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D016297:Mutagenesis, Site-Directed; D011493:Protein Kinase C / Q000502:physiology*; D051745:Protein Kinase C-delta; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic
D022763:CCAAT-Enhancer-Binding Protein-alpha; D017931:DNA Primers; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate