| Intercellular communication is critical for the survival and function of cells in multicellular organisms, and this research area has become one of the hot points in life science. The abnormal intercellular communication is closely related to human diseases. Though classical mechanism of intercellular communication, based on one-directional signal transduction, has been deeply investigated and the concept of bidirectional signal has been established, the knowledge of reverse signal is far from elucidation. The membrane form of macrophage colony-stimulating factor (mM-CSF) is an alternative splicing variant of this cytokine. As the proteolytic cleavage sites used to release the secreted isoforms have been spliced out, mM-CSF is stably expressed at the cell surface. Our previous research showed that mM-CSF could play adhesion molecule-like roles and partly replaced M-CSF functions by juxtacrine mechanism. High expression of mM-CSF was detected in leukemia and lymphoma, which suggested mM-CSF might play important roles in hematopoietic malignancies. Basing on our previous studies on mM-CSF, we try to explore the roles of mM-CSF in hematopoietic malignancies and the mechanisms of reverse signal mediated by mM-CSF.In this study, stable transfectant clones expressing mM-CSF (Namalwa-M and Ramos-M) were obtained. Namalwa-M and Ramos-M were verified by RT-PCR, Western blot and immunofluorescence methods. Namalwa-M and Ramos-M showed reduced proliferation potential in vitro. Moreover, the in vivo study showed that Namalwa-M and Ramos-M exhibited enhanced oncogenicity in tumor size in nude mice model, which could be inhibited by M-CSF monoclonal antibody. A remarkable increase in infiltrating macrophage and the vessel densities was found in tumor tissues formed by Namalwa-M or Ramos-M cells and the expression of murine origin VEGF, bFGF and HGF was elevated in Namalwa-M formed tumor tissues, which suggested the involvement of macrophages in this process. The in vitro results using coculture system demonstrated that macrophages could promote Namalwa-M and Ramos-M proliferation and activate ERK MAPK signal pathway. Above results suggested that macrophages recruited in tumor tissue might be abnormally activated by hematopoietic malignant cells expressing mM-CSF, which could in turn promote tumor development by stimulating proliferation of tumor cells expressing mM-CSF and angiogenesis.Using mM-CSF positive K562 cells and stably transfected clones (Namalwa-M/ Namalwa-30 and Ramos-M/Ramos-30), we studied the reverse signal by mM-CSF and its biological functions. The tyrosine phosphorylation level of several proteins with the molecular masses of approximately 45kD and 30kD was increased by sM-CSFR stimulation. Furthermore, using immunoprecipitation, we demonstrated that the ERK MAPK signal pathway might be involved in this reverse signal. The in vivo results showed that reverse signal through mM-CSF might play roles in the process of enhancement of tumor development by macrophages.To further explore the mechanisms of reverse signal mediated by mM-CSF, expressing vectors, carrying mM-CSF truncated 10 or 20 residues, or point mutation on serine or threonine residues located in the intracellular region of mM-CSF, were constructed. Then, those vectors were transfected into leukemia cell line Namalwa. The in vitro charactertistics of these cells were studies.In conclusions, we deeply explored the biological roles of mM-CSF in leukemia cells and first found that mM-CSF promoted the development of hematopoietic malignances by abnormally activated macrophages. We first proved by experiments that mM-CSF can transmit reverse signal, which is associated with tumor development. Expression vectors carrying either truncated or point mutational mM-CSF were constructed and stably transfected clones were obtained, which provide solid platform for further work toward the elucidation of the reverse signal transduction by mM-CSF. |
