Mitogen-activated protein kinase activates human placental lactogen-B enhancer by an NF-IL6-dependent pathway

Computer analysis of the human placental lactogen-beta (hPL-B) enhancer rev eals two putative binding sites for the transcription factor NF-IL6, but th e role of NF-IL6 in the regulation of the enhancer is unknown. Using gel mo bility shift and supershift assays, we demonstrated that NF-IL6 binds to bo th enhancer sites. Transient transfection studies indicated that the transc ription factor NF-IL6 stimulates hPL-B enhancer activity by 4.4-fold in pri mary cultures of human trophoblast cells and by 32.0- and 8.4-fold in JAR a nd BeWo choriocarcinoma cells, respectively. Overexpression of MEK (mitogen -activated protein [MAP] kinase kinase), which is known to stimulate phosph orylation of NF-IL6, induced a 3.6-fold increase in hPL-B enhancer activity . The induction by MEK was completely inhibited by an expression plasmid fo r a dominant/negative mutant of NF-IL6 or by mutation of the NF-IL6 binding sites on the enhancer. PD98059, an inhibitor of MEK, inhibited hPL release from cultured trophoblast cells by about 50%. Taken together, these result s indicate that MAP kinase stimulates the hPL-B enhancer by an NF-IL-6-depe ndent pathway.