Effects of wortmannin and rapamycin on CSF-1-mediated responses in macrophages

Abstract

There are differing views regarding the roles of phosphatidylinositol 3-kinases (PI3-kinases) and p70 S6 kinase (p70^s6k) in growth factor-induced cellular responses. One approach that is widely employed to investigate these roles is to use the inhibitors, wortmannin and rapamycin, respectively. This approach is used here to study the responses in macrophages to colony stimulating factor-1 (CSF-1). Wortmannin (≥30 nM) and rapamycin (≥3 nM) both weakly inhibited CSF-1-stimulated DNA synthesis in murine bone marrow-derived macrophages (BMM), suggesting that there are PI3-kinase- and p70^s6k-independent pathways required for the onset of S phase; interestingly the combination of the drugs gave dramatic suppression. Inhibition of DNA synthesis by rapamycin on the BMM was much less than that observed with the CSF-1-dependent cell line, BAC1.2F5. In BMM, wortmannin suppressed CSF-1-stimulated increase in p70^s6k activity indicating that PI3-kinase activity may lie upstream. In contrast to some other growth factor/cell systems, no evidence was obtained using the inhibitors for the involvement of PI3-kinase or p70^s6k in CSF-1-mediated induction of c-fos mRNA expression or Erk-1 activity; in addition, no evidence was found for an involvement in the CSF-1-mediated increase in cyclin D1 expression or STAT activation. The findings reinforce the need to study the signal transduction cascades relevant to each individual growth factor and preferably not in cell lines.

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. p70^s6k is the physiological S6 kinase activity in mammalian cells[19]. There are disagreements on the relationship of p70^s6k 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 p70^s6k activation. For the case of the CSF-1 response, rapamycin, which can prevent activation of p70^s6k 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 p70^s6k, 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 p70^s6k 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 p70^s6k 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.