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

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Abstract
As an approach to characterizing the molecules involved in the hematopoietic microenvironment provided by a murine clonal preadipose cell line MC3T3-G2/PA6 (PA6), we developed a unique system to detect the early phase of signal transduction caused by the direct cell-to-cell interaction using the reporter plasmid pfosluc2 with the c-fos enhancer/promoter linked with the Photinus pyralis luciferase gene. The plasmid pfosluc2 was genetically introduced into a mouse myeloid leukemia cell line 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 analyzed. The direct cell-to-cell interaction with PA6 cells was found to cause a significant c-fos induction to NFS-60 cells within 1 h. Approximately 10(5) cDNA clones prepared from PA6 cells were screened for their activity to promote the c-fos expression in NFS-60 cells through the direct cell-to-cell interaction, and 13 positive clones were obtained. 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 completely inhibited by addition of both antagonistic anti-c-kit and anti-HGF antibodies. These results represent direct evidence for the action of HGF on the proliferation of hematopoietic cells through direct cell-to-cell interaction with stromal cells. Thus, our developed reporter system can be useful in investigating the direct cell-to-cell interaction between stromal and hematopoietic cells.

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