CHARACTERIZATION OF THE MOLECULES INVOLVED IN THE HEMATOPOIETIC MICROENVIRONMENT PROVIDED BY MOUSE STROMAL CELL-LINE MC3T3-G2 PA6 USING A UNIQUE REPORTER SYSTEM THAT ANALYZES THE DIRECT CELL-TO-CELL INTERACTION/

As an approach to characterizing the molecules involved in the hematop oietic microenvironment provided by a murine clonal preadipose cell li ne MC3T3G2/PA6 (PA6), we developed a unique system to detect the early phase of signal transduction caused by the direct cell-to-cell intera ction using the reporter plasmid pfosluc2 with the c-fos enhancer/prom oter linked with the Photinus pyralis luciferase gene. The plasmid pfo sluc2 was genetically introduced into a mouse myeloid leukemia cell li ne NFS-60 which showed a growth dependency on contact with PA6 cells, and the mechanism by which stromal PA6 cells promote the proliferation of NFS-60 cells through the direct cell-to-cell interaction was analy zed. The direct cell-to-cell interaction with PA6 cells was found to c ause a significant c-fos induction to NFS-60 cells within 1 h. Approxi mately 10(5) cDNA clones prepared from PA6 cells were screened for the ir activity to promote the c-fos expression in NFS-60 cells through th e direct cell-to-cell interaction, and 13 positive clones were obtaine d. Of these positive clones, five clones encoded the stem cell factor, and the others encoded the hepatocyte growth factor (HGF). The c-fos induction caused by the contact with PA6 cells in NFS-60 was completel y inhibited by addition of both antagonistic anti-c-kit and anti-HGF a ntibodies. These results represent direct evidence for the action of H GF on the proliferation of hematopoietic cells through direct cell-to- cell interaction with stromal cells. Thus, our developed reporter syst em can be useful in investigating the direct cell-to-cell interaction between stromal and hematopoietic cells.