Regulation of C/EBPβ mRNA expression and C/EBPβ promoter activity by protein kinases A and C in a myelomonocytic cell line (HD11)

Abstract.

Objective:

The transcription factor CCAAT/enhancer- binding protein (C/EBP) β is involved in inflammatory responses in immune cells, including myelomonocytic cells. In this study, signal transduction pathways regulating C/EBPβ expression were investigated.

Methods:

The expression of C/EBPβ mRNA in cells treated with various activators and inhibitors of PKA and PKC was analyzed by Northern blot hybridization. C/EBPβ promoter activity was investigated by transient transfection assays with C/EBPβ promoter CAT constructs.

Results:

Phorbol 12-myristate 13-acetate (PMA), forskolin and 3-isobutyl-1-methyl-xanthine (IBMX), an inhibitor of cAMP and cGMP phosphodiesterases, but not cGMP, when added to chicken myelomonocytic HD11 cells, markedly stimulated the C/EBPβ mRNA expression. However, transfection experiments using HD11 cells showed that CAT constructs controlled by the 5′ flanking sequence from –704 to +24 of chicken C/EBPβ gene were activated by PMA, but not by forskolin. In contrast to forskolin, IBMX was able to activate the C/EBPβ promoter CAT constructs. Further transient transfection experiments using other cell lines demonstrated that the chicken C/EBPβ promoter was responsive to forskolin in mouse fibroblasts NIH3T3, but not in human hepatoma HepG2 cells. Increase in C/EBPβ mRNA stability in HD11 cells was induced by forskolin and PMA.

Conclusion:

The results indicate that the C/EBPβ gene is regulated transcriptionally as well as post-transcriptionally in response to forskolin and PMA, and the forskolin responsiveness of the C/EBPβ promoter seems to depend on cellular cAMP turnover.