Macrophage stimulating protein (MSP) is a hepatocyte-derived serum protein that has high amino acid sequence similarity to hepatocyte growth factor/scatter factor (HGF/SF). MSP was originally purified as a protein that made mouse resident peritoneal macrophages capable of responding to the chemoattractant C5a. However, recent study shows that MSP itself is a chemoattractant for mouse resident macrophages. The receptor for MSP is a receptor tyrosine kinase, Ron/Stk. Identification of MSP receptor revealed that keratinocytes are another target cells of MSP and MSP appears to be involved in the migration of keratinocytes during the healing process after skin injury. We and others previously reported that MSP mRNA was predominantly and constitutively expressed in the liver, probably in hepatic cells. The transcriptional mechanisms of genes specifically expressed in hepatic cells such as albumin, transthyretin, 1-antitrypsin or fibrinogen have been previously investigated. In most of the cases, transcription of these genes is positively regulated by liver-specific transcriptional factors such as CCAAT/enhancer binding protein (C/EBP ), hepatocyte nuclear factor (HNF)-1, HNF-3, or HNF-4. Last year, we reported the transcription of the human MSP gene was positively regulated by the binding of NF-Y, also known as CAATT-binding factor, to the "CAATT" sequence in the promoter region of this gene. This year, we further characterized the MSP promoter. Luciferase assay with multiple deletion constructs showed the importance of the region (+32 to +39) for the promoter activity in Hep3B cells. Two nuclear protein-DNA probe (+15 to +40) complexes, C1 and C2, were detected by electrophoretic mobility shift assay. C2 was specific to hepatoma cells and contained HNF-4. DNase I footprinting with recombinant HNF-4 located another HNF-4 binding site in the distal region (-89 to -54). Mutations in the "CAATT" or the proximal HNF-4 binding site significantly reduced the promoter activity in Hep3B cells and HNF-4-transfected Hela cells, whereas mutations in the distal HNF-4 binding site had no effect. Insertion of 11-bp spacer but not 6, 28, or 33-bp spacer between the "CAATT" and the proximal HNF-4 binding site completely retained the promoter activity, suggesting that a direct contact between NF-Y and HNF-4 was important. Protein-protein interaction between the A-subunit of NF-Y and HNF-4 was detected by an yeast two-hybrid system. The binding of in vitro translated HNF-4 to immobilized NF-YA, and in vitro translated NF-YA to immobilized HNF-4 were also detected. These results suggest the binding of HNF-4 to the proximal HNF-4-binding site directs the basal transcription of the MSP gene and the maximal promoter activity depends on the direct association between HNF-4 and NF-Y. This is the first example of direct cooperation between HNF-4 and NF-Y in the transcription of genes.