Pma, Pge <sub> 2 </ Sub> Mouse Tnf-�� Gene Transcriptional Regulation

Tumor necrosis factor-alpha (TNF-α) is a cytokine with pleiotropic biological activity participating in many physiological and pathological processes in vivo, such as: immunoregulation, inflammation, endotoxic shock, killing of tumor cells. TNF-α is mainly produced by activated macrophages. Many inducers can activate macrophages and induce expression of TNF-α gene transcription. Production of TNF-α needs two steps: priming and triggering. Signal transduction pathways associated with TNF-α production by activated macrophages can be divided into two types: one is PKC-dependent pathway mainly induced by PMA, another is non-PKC-dependent pathway. The molecular mechanisms involving in the two signal transduction pathways and their relationship are unclear. Perhaps different transactivator may be responsible for different signal transduction pathways. Previous studies in our laboratory focussed on the effect of LPS inducing mouse TNF-α gene expression and effect of PGE₂ on TNF-α gene expression stimulated with LPS. The expression of TNF-α gene induced by PMA and inhibition effect of PGE₂ on PMA at the level of transcription was further studied. Our experiments demonstrated: PMA could induce P388D1 cell line to secrete TNF-α, PGE₂ was able to inhibit PMA-induced TNF-α gene expression in a dosage-dependent manner. The results of gel retardation assay and CAT assay showed that PMA-induced a DNA-binding protein which specifically bond to the TNF-α 5' flanking region. The cis-element Involved was found located in the region -196— +138. PGE2 might induce an inhibitor which combines with the transactivator induced by PMA to form a complex which could no more bind to the motif in TNF-α 5' flanking region. As reported in the literature, there are four locifor binding of NF-kB between -850------210 in the TNF-α 5'flanking region responsive to LPS. Our results indicate that transcriptional expression of TNF-α gene induced by PMA may not be related to NF-κB.