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 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.
Satoh
M
M
Tokyo Research Laboratories, Kyowa Hakko Kogyo, Japan.
Mioh
H
H
Konishi
N
N
Takahashi
I
I
Tamaoki
T
T
D000818:Animals; D001854:Bone Marrow Cells; D002450:Cell Communication / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D017930:Genes, Reporter; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D008811:Mice, Inbred DBA; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D017154:Stromal Cells / Q000166:cytology
D016760:Proto-Oncogene Proteins c-fos