The International Journal of Biochemistry & Cell Biology
Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages
Introduction
Colony stimulating factor-1 (CSF-1) or macrophage-CSF (M-CSF) is involved in the survival, proliferation, differentiation and activation of cells of the monocyte/macrophage lineage[1]. Its receptor is encoded by the c-fms protooncogene and is structurally related to a family of growth factor receptors which include the α and β platelet-derived growth factor receptors, c-Kit and Flt 3/Flk2[2]. Because of these receptor similarities, it has sometimes been assumed that the signal transduction pathways activated by these growth factors will be quite similar. However, there are differences meaning that signal transduction pathways in response to growth factors, such as CSF-1, need to be examined for each ligand/receptor couple, preferably in normal cells rather than in cell lines and preferably in cells which normally express the receptor in question.
Phosphatidylinositol 3-kinases (PI3-kinases) are enzymes which phosphorylate inositol phospholipids in the D-3 position of the inositol ring, but which also have serine kinase activity[3]. The receptor tyrosine kinase-activated PI3-kinase consists of a heterodimer of an 85 kDa regulatory subunit (p85), which can bind directly to specific phosphorylated tyrosine residues of receptors via its SH2 domains, and a 110 kDa catalytic subunit (p110). The role(s) of PI3-kinase in growth factor-regulated signal transduction cascades is unclear. For example, there are conflicting data regarding its role in the regulation of DNA synthesis4, 5, 6, 7, and MAP kinase activation8, 9, 10, 11; in addition, it has been reported to be involved in c-fos expression5, 12. For CSF-1-stimulated DNA synthesis, one report using 3T3 cells transfected with human c-fms showed that PI3-kinase was not required ([13]) while another report using the same system found a correlation between PI-3 kinase activity and the level of DNA synthesis observed[14]. In macrophages, PI3-kinase has been shown to be activated following CSF-1 stimulation15, 16and forms a complex with Grb2.Sos in human monocytes[17]. Recently we have found in CSF-1-stimulated murine macrophages that PI-3 kinase is tyrosine phosphorylated and is stably associated with several tyrosine–phosphorylated proteins, including c-fms itself[18].
Phosphorylation of the S6 protein of the 40S ribosomal subunit is a highly conserved response of animal cells to treatment with a wide range of stimuli, including growth factors. p70s6k is the physiological S6 kinase activity in mammalian cells[19]. There are disagreements on the relationship of p70s6k activity to growth factor-mediated DNA synthesis, fos expression and MAP kinase activity4, 20, 21, 22, 23, 24, 25, 26, 27. In addition, there is both supporting4, 28, 29, 30and conflicting31, 32evidence for a role of PI3-kinase in p70s6k activation. For the case of the CSF-1 response, rapamycin, which can prevent activation of p70s6k in many cells via its ultimate target mTOR/RAFT/FRAP[33], inhibits CSF-1-stimulated DNA synthesis in BAC1.2F5 cells[34]and cell proliferation in murine bone marrow-derived macrophages (BMM)[35].
In general, an inhibitor of an intracellular target, even though it may not be exquisitely specific, can provide evidence which is consistent with a role of its target in a particular cellular response, for example, to a growth factor — if its action is very effective on a target but does not modulate a particular cellular response then it could be that the target of the inhibitor is not required for that response. Wortmannin and rapamycin are widely used as inhibitors to delineate the cellular functions of PI3-kinase and p70s6k, respectively36, 37. At concentrations up to 100 nM, wortmannin irreversibly inhibits both the lipid kinase and the serine kinase activities of PI3-kinase, through covalent interaction with the p110 catalytic site[38]. With the possible exception of a soluble PI4-kinase[39]and phospholipase A2[40], this is the only high affinity target for wortmannin in mammalian cells described so far.
As mentioned above, there is a variety of conclusions about the roles of PI3-kinase and p70s6k in growth factor-mediated responses, including their possible relationship. In this study, in order to gain some idea of their roles in CSF-1-mediated signalling, we analysed the effects of wortmannin and rapamycin in CSF-1-treated murine macrophages. Especially for CSF-1-treated BMM, the data indicate that significant reductions in PI3-kinase and p70s6k activities are not critical for the induction of DNA synthesis; however, the combination of wortmannin and rapamycin gave dramatic suppression of this induction. In this system at least, the activities of the two kinases also do not seem to be critical for the stimulation of c-fos mRNA, cyclin D1 expression and Erk-1 activity.
Section snippets
Cell culture
Bone marrow cells were obtained from femurs of CBA mice by aspiration. Bone marrow-derived macrophages (BMM) were prepared essentially as described[41]. Briefly, bone marrow cells were cultured in 175 cm2 tissue culture flasks (Nunc, Roskilde) for 3 days at 1×106 cells/ml in 40 ml RPMI-1640 supplemented with 5×10−5 M 2-mercaptoethanol, 20 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS) and 30% L-cell conditioned medium (LCM) as a source of murine CSF-1. The non-adherent population was
Effect of wortmannin and rapamycin on CSF-1-stimulated macrophage DNA synthesis
In Fig. 1 the dose response curves are presented for the effect of wortmannin on CSF-1-stimulated DNA synthesis in both BMM and BAC1.2F5 cells, respectively. For both murine macrophage populations, wortmannin (100 nM) had some effect (5–30% inhibition) (n=42 for BMM; n=21 for BAC1.2F5 cells); lower concentrations were usually inactive. Independently we have shown that wortmannin (>50 nM) completely abolished the CSF-1-stimulated P13-kinase activity in BMM, measured as described previously[16], 5
Discussion
We showed above that wortmannin at relatively high concentrations is a weak inhibitor of CSF-1-induced BMM and BAC1.2F5 DNA synthesis and that there was no suppression of this DNA synthesis at lower concentrations (<30 nM) at which there was significant inhibition of PI3-kinase activity (Fig. 1). However, it should be borne in mind that DNA synthesis was monitored several hours after CSF-1-stimulated PI3-kinase activity was measured, viz. 5 min — it is therefore difficult to be sure of the
Acknowledgements
We would like to thank V. Kanagasundaram and U. Novak for PI3-kinase activity and STAT measurements, respectively, and R. Sallay for typing the manuscript. The work was supported by a Program Grant from the National Health and Medical Research Council of Australia.
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