iiilii
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Signalling through CSF receptors Gino Vairo and John A. Hamilton The finely regulated process of blood cell formation is under the control of a family of glycoprotein hormones, known as colony-stimulating factors (CSFs), and their receptors. The complexity of the intracellular mechanisms involved in the action of such factors has been appreciated only recently. In this review, Gino Vairo and John Hamilton discuss the biochemistry of CSF action and its relevance to growth control, and examine the possibility that different CSFs may use common control pathways within the one cell type. Over the last twenty years, much information regarding the identities of stimulatory factors involved in haemopoiesis, including CSFst, has been obtained. The actions of the CSFs are pleiotropic in nature: they are involved in the survival, proliferation, differentiation and activation of haemopoietic cells at various stages of their development, and can also act on nonhaemopoietic cell types. The recent cloning of many of the CSFs and their receptors has facilitated the study of the biochemistry of CSF action. This review examines the signalling processes and biochemical responses involved in the action of three of the better characterized CSFs, namely macrophage CSF (CSF-1 or M-CSF), granulocyte-macrophage CSF (GMCSF) and interleukin-3 (IL-3 or multi-CSF), concentrating on the macrophage as a target cell. It will also examine the relationship of these CSF-induced responses to their mitogenic action.
oL(TNF-o~),which potently downmodulates CSF-1 receptor levels in murine bone-marrow-derived macrophages6, has little effect on CSF-1-stimulated pinocytosis and by itself does not stimulate pinocytosis in these cells7. The lack of inhibition of this CSF-1 response by TNF-(x is consistent with low receptor occupancy being sufficient for biological responses to CSFs1. Phorbol esters can also downmodulate CSF-1 receptor levels in murine macrophages8, probably via the activation of protein kinase C (PKC)9. In c-fms-transfected fibroblasts, the downmodulation of CSF-1 receptor levels by phorbol esters and other PKC activators occurs via a different mechanism from autologous downregulation1°. Since PKC activation by the CSFs is also likely (see later), these findings may be relevant to the receptor transdownmodulation seen between the CSFs (and other agents which downmodulate CSF receptors).
Signal transduction through CSF receptors CSFs and their receptors An overview of the actions of CSF-1, a prototypic CSF, The first of the CSF receptors to be isolated and is illustrated in Fig. 1. The formation of an active ligandcharacterized was the CSF-1 receptor, which was shown receptor complex is the initial event in the growth factor to be the product of the c-fms proto-oncogene and a signal transduction pathway (Fig. l(a)). For the growth member of the tyrosine kinase receptor family2. More factor receptor tyrosine kinases, such as the CSF-1 receprecently, the receptors for GM-CSF and IL-3 have been tor, activation of receptor kinase activity follows ligand cloned (reviewed in Ref. 3); these receptors share certain binding, and results in receptor autophosphorylation folstructural homologies and have been grouped together in lowed by a rapid phosphorylation on tyrosine of an array the haemopoietin receptor superfamity. Unlike the CSF-1 of cytosolic and membrane-associated proteins (Refs receptor, the haemopoietin receptors do not show con- 11,12 and Fig. l(b)), including phosphatidylinositol sensus sequences characteristic of tyrosine kinases. (PI)-3 kinase (Table 1 and Refs 13,14). This enzyme The binding of CSFs to their receptors is accompanied phosphorylates the D-3 hydroxyl position of the inositol by rapid receptor downregulation, internalization of the ring of PI, the physiological role of which is, as yet, ligand-receptor complex and subsequent destruction of unknown. Other candidate substrates for receptor tyrothe CSF. It has been suggested that the intracellular sine kinases, including phospholipase C~/(PLC~) is, the accumulation of CSF-1, as well as accumulation of RAF-1 serine/threonine kinase 16, and the p21ras surface-bound CSF-1, may be involved in the mitogenic GTPase-activating protein (GAP)~4 do not appear to be response 4. Binding of a CSF to its specific receptor can directly phosphorylated by the CSF-1 receptor. Actialso result in the downmodulation of receptors for other vation of some of these enzymes, however, can still occur CSFs. This phenomenon has been termed 'trans- via indirect mechanisms16. The identities of the nudownmodutation', and the relative activities among IL-3, merous other cellular proteins phosphorylated on tyroGM-CSF and CSF-1 follow a distinct hierarchys. sine in response to CSF-1 remains to be determined. For CSF-1 (and other growth factors that have tyroAlthough it has been suggested that transdownmodulation may be accompanied by an activation of signal transduc- sine kinase receptors) the activation of the tyrosine tion through the receptor, 'transactivation', even in the kinase, presumably via the modulation of the activities of absence of its respective ligand, its biological signifi- an array of cellular targets, is the 'master switch' that cance is unclear; transdownmodulation of CSF receptors initiates the cellular response cascade. How the nonalone is insufficient to trigger at least some of the CSF- tyrosine-kinase (haemopoietin) receptors initiate their induced responses. For instance, tumour necrosis factor responses is, as yet, undetermined. A clue may come from © 1991, Elsevier Science Publishers Ltd, UK. 0167--4919/91/$02.00
Immunology Today
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Vol. 12 No. 10 1991
!!i~iiiii~!~iilili~iiiiii~ii~iii!ii!i!iiii{iiiiiiiiiiiiii ~iiIiii~~iiiii!~iiiii!iiiii,~iiil~~iiiiii~i~~iiiii~iiiiii~iiiiiiiiii~i!iiiiiii~il~!iii!iil iii~~i~iiiiii iiiii~i!iiii!iiiii iiiii CSF-1
[a] [c ]
41---
>_-~-
f - -Ill~ . [ ,~'b],~ ,~ " ..,..
\ .,
nucleus
"~_...~ P(rr°ts/G Ap?,
tyr t y'r-(~ receptor substrates " \ e.g. PI-3 kinase
~[g]
I •
/
e.g. u-PA, JE, K C / , 13 actin, fibronectin R (15) hck, fgr fos, jun, myc G1 cyclins
-- " ~ [d] phosphoJi pids I: (e.g. PC)--~--~lon ~phospholipases "! \(e.g. PLC) DAG
other effectors e.g. F ~F-1 kinase
[e]
;'1' o . A synthesis
.
"
.o N a+
Fluid-phase pinocytosis [J] Glucose
K+
Fig. 1. CSF-l-induced responses and their relationship to DNA synthesis are shown. (a) Formation of a CSF-l-receptor complex; (b) stimulation of
receptor tyrosine kinase activity, resulting in receptor autophosphorylation and substrate phosphorylation at tyrosine; (c) activation of a G protein which in turn (d) stimulates phospholipase-mediated hydrolysis of membrane phospholipids with the production of D A G; (e) D A G binding to cytosolic PK C induces its translocation and activation, with subsequent stimulation (f) of a variety of cellular responses with possible relevance to D NA synthesis. Other kinases may also be activated (g), either by direct phosphorylation by the receptor (e.g. PI-3 kinase) or via an indirect mechanism (e.g. RAF-1 kinase), which may also be involved in CSF-1 action (h). Activation of PKC or other effectors stimulates other parameters important for mitogenesis including (i) Na+,H + exchange activity, with the Na + influx in turn resulting in (j) stimulation of Na+,K +-ATPase activity or (k) enhanced expression of a variety of genes, including those encoding growth-related proteins and other structural, enzymatic or secreted proteins. Other rapid responses to CSF-1, including stimulation of glucose uptake, fluid phase pinocytosis and u-PA secretion are also shown. Question marks on the arrows indicate that there is no evidence for a direct relationship between responses.
the finding that the IL-6 receptor, another member of the haemopoietin receptor superfamily, associates, in the presence of IL-6, with a 130 kDa membrane-associated glycoprotein (gp130), possibly forming a signal transducing 'effector' subunit 17. Such effectors may possess intrinsic kinase activity or may modulate other transducing proteins to initiate the cellular response cascade. Competition between different CSF receptors for a limiting 'adaptor' subunit, which is required for highaffinity ligand binding, has also been suggested 18 and Immunology Today
recently the cloning of such an affinity-modulating subunit of the GM-CSF receptor has been described19. Such a mechanism may explain the apparent, but as yet unidentified, crossreactive GM-CSF/IL-3 receptor(s) 2°.
G proteins Inhibition of growth factor responses by pertussis toxin (PT) has been used to investigate the involvement of guanine-nucleotide-binding proteins (G proteins) in growth factor signal transduction. PT can inhibit the
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r o t 12 No. 10 1991
Table 1. Relationship of CSF-induced responses to their mitogenic action Response
Stimuli CSF
Receptor targets PI-3 kinase phosphorylation
Cell Type Nonmitogen
CSF-1 CSF-1 CSF-1
G proteins GTP binding/GTPase CSF-1 activity p21 ras phosphorylation GM-CSF p62 'GAP'-associated CSF-1 protein tyrosine phosphorylation Phospholipid hydrolysis DAG turnover CSF-1/GMCSF/IL-3 CSF-1 Protein kinases PKC (translocation) PKC substrate phosphorylation
RAF-1 kinase
Cyclic nucleotides I" cGMP $ cAMP
Ion fluxes Na+,H + exchange
Refs
Cycling
Poorly-cycling
A31 fibroblasts (c-fms) 3T3 (c-fins) BMM/BAC1.2F5
P388D1 a 13 3T3 (fms 809 F) 59 14 PBM
CFC 3T3/R2C1 fibroblasts (c-fins)
Zymosan
BMM
(not RPM
30,31
PBM
22
FDC-P1 NSF.60 NSF.60.8
32 22 33 33 33
B6SUtA1 BAC1.2F5 FDC-P1 DA3
34 16 42 42
PBM
PMN
GM-CSF CSF-1 GM-CSF
BMM PMN
CSF-1/GMCSHIL-3/G-CSF LPS/TNF-c~ CSF-1/GMCSF/IL-3 CSF-1 PAF GM-CSF GM-CSF IL-3
activity of some (but not all) G proteins by ADPribosylation of the active, % subunit of the heterotrimeric G protein complex21-23. CSF-1 increases GTP binding and elevates GTPase activity in human monocyte membrane preparations, and a variety of responses of these cells to CSF-1 are PT sensitive (Refs 21,22 and Fig. 1(c)). The actions of CSF-1, GM-CSF and 1L-3 on murine haemopoietic cells23,24 appear to be less sensitive to the effects of PT. It therefore appears that PT-sensitive G proteins have only a minor role, if any, in the CSF response in mouse cells, although PT-insensitive G proteins may still be involved. The rasGAP system, which may be functionally related to prototypic G proteins, has also been implicated in the action of both CSF-1 and GM-CSF (Refs 14,25,26 and Table 1).
1mrnunology Today
26 14
FDC-P1
IL-3 CSF-1 IL-3 IL-3/G-CSF IL-3/G-CSF (not GM-CSF) IL-3/GM-CSF CSF-1 IL-3 GM-CSF/IL-3
21
52 50 52
CFC
36
BMM
37,55, 6
PBM BMM AML 193 FDC-P1
21 30 56 35
Phospholipid hydrolysis The activation of a phosphatidylinositol 4,5-bisphosphate (HP2)-specific phospholipase C (PLC), which hydrolyses membrane inositol phospholipids to yield the so-called second messengers, inositol-l,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), could play an important role in the action of various growth factors9. However, studies involving a variety of CSF-responsive cell types have so far failed to detect enhanced inositol phosphate turnover in response to CSFs22,27-29. The hydrolysis of phosphatidylcholine (PC) by a PC-specific PLC or phospholipase-D (PLD) is a major alternative pathway for DAG production9. Elevated DAG turnover, possibly via PLC-mediated PC hydrolysis, has been described in human monocytes in response
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Table 1. Relationship of CSF-induced responses to their mitogenic action Response
Na+,K+-ATPase
Stimuli Cycling
Poorly-cycling
CSF-1/GMCSF/IL-3 CSF-1
LPS/ConA/ TNF-c~
BMM
RPM
37, b
PBM
21
CSF-1/GMCSF/IL-3 CSF-1 CSF-1/GM-CSF c-myc mRNA CSF-1/GMCSF/IL-3 CSF-1/GM-CSF c-jun mRNA CSF-1 CSF-1 c-fgr mRNA CSF-1/GM-CSF hck mRNA CSF-1 Ornithine decarboxylase GM-CSF mRNA
u-PA mRNA G1 cyclin mRNA
Refs
Nonmitogen
Gene expression c-fos mRNA
Fbn.R (13)mRNA [3-actin mRNA JE mRNA KC mRNA
Cell Type
CSF
LPS/zymosan/ TNF-c~
Zymosan
LPS/IFN-y
28,57, b
3T3 (c-fms) BAC1.2F5 BMM
3T3 (fins 809 F) 59 Aut.l a 58 28,57
BAC1.2F5 3T3 (fins)
NSF.60.8
Aut.l a 58 3T3 (fms 809 F) 59 PBM 48 47 PBM 48 53
BAC1.2F5 BAC1.2F5
PBM PBM Aut.1 a Aut.1 a
BMM
CSF-1 CSF-1 CSF-1/GM-CSF CSF-I (not GM-CSF) CSF-1/GMConA CSF/IL-3 CSF-1
Other responses Ornithine decarboxylase GM-CSF/ G-CSF/IL-3 G1 cyclin expression CSF-1 Protein synthesis CSF-1 RNA synthesis CSF-1 Glucose uptake CSF-1/GMCSF/IL-3 IL-3 Pinocytosis CSF-1/GMCSF/IL-3 u-PA production CSF-1/GMCSF/IL-3
BMM
48 48 58 58
BMM
24
BAC1.2F5
62
NSF.60.8
53
LPS/ConA
BAC1.2F5 BMM BMM BMM
62 66 55 38
LPS/zymosan
FDC-P2 BMM
ConA
BMM
RPM
60 7,39, c RPM
24
The relationshipof CSF-inducedresponsesto their mitogenicactionwas examined.The responsesof the CSFs,when examinedin the one study, were compared with other nonmitogenicstimuli. The responses of cells that progress through the cell cyclein response to CSFs (cycling)were also comparedwith cellsthat showa poor proliferativeresponse (poorlycycling).DAG: 1,2-diacylglycerol;PI: phosphatidyl inositol; Fbn.R ([3): fibronectinreceptor [3subunit; GAP: GTPase-activatingprotein; ConA: concanavalinA; IFN-y: gamma-interferon; LPS: lipopolysaccharide;PAF: platelet-activatingfactor; TNF-(x:turnout necrosis factor ~; BMM: bone-marrow-derivedmacrophages; CFC: colony-formingprogenitor cells; c-fins: cells transfectedwith wild type CSF-1 receptor; fins 809 F: cells transfectedwith human CSF-I receptor containing phenylalanine substitution at position 809; PBM: human peripheral blood monocytes; PMN: human polymorphonuclearcells; RPM: murine residentperitoneal macrophages, aConstitutivelygrowingcell lines for which CSFsdo not act as mitogens, bG. Vairo, A.K. Royston and J.A. Hamilton, (unpublished).CK.R.Knight,G. Vairo and J.A. Hamilton, (unpublished). to CSF-1 (Ref. 22 and Table 1). CSF-1 has also been found to increase DAG turnover in bone-marrowderived macrophages but not in the poorly cycling murine resident peritoneal macrophage population 3°, suggesting that such enhanced DAG turnover may represent an important early signal for macrophage proliferation (Fig. l(d)). Enhanced DAG turnover also occurs in
Immunology Today
bone-marrow-derived macrophages in response to both GM-CSF and IL-3 (Ref. 31 and Table 1), Further evidence suggesting a role for PLC in CSF action comes from the findings that exogenous, nonspecific PLC is itself a weak mitogen for bone-marrow-derived macrophages and elicits a variety of responses common to CSF-1, GMCSF and IL-3 (Refs 24,28). The nonspecific PLC also
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elevates DAG turnover in these cells3° without a detectable increase in inositol phosphate levels28. Protein kinases Protein kinases play an important role in regulating the activities of many of the key enzymes and other proteins involved in growth factor responses. The serine/ threonine kinase, PKC, which is activated by DAG, has been implicated in the action of many growth factors 9. CSF-1, GM-CSF and IL-3 have been reported to induce a translocation and/or activation of PKC in a number of haemopoietic cell types (Refs 22,32-34 and see Table 1 and Fig. l(e)). Evidence for a role of PKC in CSF action also comes from the findings that phorbol esters and other direct PKC activators elicit a similar series of early responses to CSFs24,28,3°,35-39, and are themselves weakly mitogenic 33-37 and synergize with CSFs for stimulation of DNA synthesis 33,4°. The inhibition of PKC with pharmacological agents or by chronic phorbol ester pretreatment inhibits responses to different CSFs, including Na+,H + exchange, activation and proliferation =,35,41. The possible involvement of PKC in these CSF-1 responses is shown in Fig. l(f). Although there is increasing evidence that PKC activation may be involved in CSF action, at least in some cell types, PKC-independent mechanisms may also be involved. PKC-independent serine phosphorylation has been described in response to IL-3 or GM-CSF 34, and CSFs stimulate RAF-1 kinase activity in several factordependent cell lines (Refs t6, 42 and see Table 1). Whereas the known receptors for IL-3 and GM-CSF do not contain a consensus sequence for tyrosine kinase activity 3, these CSFs can induce the rapid tyrosine phosphorylation of a variety of cellular substrates in a number of cell types 34,43. Whether this activity is due to an uncharacterized receptor(s) for these CSFs, which possesses tyrosine kinase activity, or whether the nontyrosine kinase receptors activate a nonreceptor tyrosine kinase remains to be determined. Evidence for a regulatory role for tyrosine phosphorylation in the proliferative response to such CSFs comes from the findings that the protein tyrosine phosphatase inhibitor, vanadate, which enhances tyrosine phosphorylation in response to GMCSF or IL-3 in a factor-dependent cell line, also enhances the proliferative response to these CSFs44. In addition, introduction and expression of nonreceptor tyrosine kinase oncogenes, such as v-src or v-abl, into CSFdependent cell lines induces factor independence via a nonautocrine mechanism 4s,46. Increased mRNA levels for the src-related genes, c-fgr and hck, in response to CSF-1 or GM-CSF have been reported (Refs 47, 48 and see Table 1). The activation of other nonreceptor 'effector' kinases by CSF-1 is shown in Fig. l(g). Possible roles for such kinases in mediating the numerous responses to CSF-1 are represented in Fig. l(h). cAMP-dependent protein kinases (PKA), which mediate the second messenger function of cAMP, have also been implicated in modulation of growth factor action. The pleiotropic role of cAMP which is seen in many cellular systems is also evident in the haemopoietic system: increased levels enhance IL-3- and phorbol-esterinduced megakaryocyte colony formation, have a weaker effect on erythroid colony formation and inhibit granulo-
Immunology Today
cyte colony formation from murine bone marrow 49. Recent reports indicate that elevation of cAMP levels inhibits CSF-stimulated macrophage proliferation 5°,51. For bone-marrow-derived macrophages, CSF-1 reverses the inhibition of DNA synthesis caused by agents that activate adenylyl cyclase but not direct PKA activators. This suggests that CSF-1 inhibits cAMP accumulation, perhaps by inhibiting adenylyl cyclase and/or increasing cAMP phosphodiesterase activities s°. Interestingly, GMCSF inhibits adenylyl cyclase activity and enhances guanylyl cyclase activity and cGMP levels in human neutrophils (Ref. 52 and Table 1). The inhibition of bone-marrow-derived macrophage DNA synthesis by cAMP elevation occurs, even quite late, in the G1 phase of the cell cycle without affecting a variety of other CSF- 1 responses s°, suggesting that cAMP acts at some late G1 control point to inhibit S phase progression. Consistent with this, cAMP inhibition of GM-CSF-stimulated proliferation of a factor-dependent cell line is associated with an inhibition of the late induction in ornithine decarboxylase gene expression and enzyme activity 53. cAMP elevation has been found to inhibit CSF-t-, GM-CSF- and IL-3-stimulated urokinasetype plasminogen activator (u-PA) mRNA levels and enzyme production in bone-marrow-derived macrophages, although this response does not appear to be necessary for their proliferation 24. Ion fll~txes
Stimulation of the amiloride-sensitive Na+,H + exchanger is a ubiquitous early response to growth factors. Inhibition of this intracellular pH (pHi) regulator often abrogates the proliferative response, suggesting that antiport activation is an important early mitogenic signal (reviewed in Ref. 54). Certainly, in the absence of HCO 3- this antiporter can be the major pH i regulator, and as such is required to maintain a sufficiently alkaline pH i to allow DNA synthesis. However, in the presence of HCO3 , other HCO3--dependent pH i regulatory processes can also contribute to pH i homeostasis 54. Under these more physiological conditions the role of the Na+,H + exchange-mediated increase in pH i as a signal for growth factor action has been questioned 54. CSF-I, GM-CSF and IL-3 have all been found to activate Na+,H + exchange activity in a number of cell types (see Table 1). Inhibition of CSF-stimulated proliferation by amiloride analogues in the absence 29,35,36,ss or presence 55,56 of HCO3- indicates an important role for Na+,H + exchange in CSF action under these conditions. The inhibition of CSF-stimulated proliferation in the presence of HCO 3- suggests that the Na + influx, rather than any pH i increase, may be the more important consequence of Na+,H + exchange activation s5,56. The stimulation of Na+,H + exchange activity by CSF-1 is shown in Fig. 1(i). The Na + influx accompanying Na+,H + exchange activation has been shown in turn to activate the Na-,K+-ATPase (Na+-pump) (see Fig. l(j)), which is responsible for maintaining intracellular Na + ([Na+]i) levels21,37,55. This co-ordinated activation of both of these ion transporters has been referred to as a 'Na + cycle '37,55. The Na +, K+-ATPase, via its effects on intracellular Na +, K + and ATP levels, may also be important for growth factor action.
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The inhibition of CSF-l-stimulated bone-marrowderived macrophage DNA synthesis by an amiloride analogue occurs even if added several hours after the growth factor, suggesting that Na + cycle activity late in the G1 stage of their cell cycle is required for S phase progression ss. Consistent with this proposal are the reports that CSF-1 and GM-CSF result in a persistent stimulation of Na+,H + exchange activity in these cells29,s5. Although Na ÷ cycle activity appears necessary for CSF-l-stimulated bone-marrow-derived macrophage proliferation, this is not so for a variety of other early CSF-1 responses 24,3s,55, suggesting that Na ÷ cycle activation itself is unlikely to be a general mediator of CSF action. The lack of effect of CSFs on intracellular Ca 2+ levels is consistent with a lack of production of the CaZ+-mobilizing inositol phosphates in the same systems 27-29,36. Furthermore, for bone-marrow-derived macrophages at least, a lack of Ca 2+ involvement in CSF action is suggested by the findings that Ca p+ ionophores are not mitogenic 28 and do not elicit many of the responses induced by CSFs 24,28.
sequent DNA synthesis. As shown in Table 1, CSFinduced responses are not specific (indeed they can also be elicited by nonmitogens) and many of the CSFs elicit similar responses in both cycling and poorly cycling cells. Thus, the early events alone are not sufficient to induce a proliferative response and it remains to be determined whether or not many of the events play a direct role in the mitogenic action of the CSFs. Some of the parameters listed have even been dissociated from the proliferative response to CSFs; for instance, stimulation of macrophage proliferation can occur in the absence of u-PA production 24 or KC gene expression s8. On the other hand, inhibition of some parameters, including Na+,H + exchange 29,3s,36,ss,s6 and PKC 3s,41 activities, prevents CSF-stimulated proliferation. Induction of these early events, although not sufficient, is necessary for the proliferative response to CSFs. The observation that CSF-1 is required throughout the G1 phase of the bone-marrowderived macrophage cell cycle for S phase progression 61 is consistent with the notion that the early transient responses are necessary, but not sufficient on their own, to induce DNA synthesis. Clearly, events later in the G1 phase of the cell cycle play an important role in S phase Gene expression progression, as evidenced by the ability of inhibitors of Binding of growth factor to its receptor results in the CSF-stimulated proliferation, such as cAMP, to exert transmission of a 'signal' to the nucleus, presumably via their effect late in G1 (Refs 50, 55 and Fig. 2). The recent the action of pre-existing cytosolic effectors, whereupon report of CSF-l-induced expression of novel G1 cyclins a programmed process of gene expression is initiated. (putative S phase regulators) in the BAC1.2F5 macroCSFs induce the expression of a variety of genes (see phage cell line 62 is also relevant to this proposal. Table 1) that encode kinases (for example c-fgr, hck) 47,48 Under certain conditions the proliferative actions of and transcription factors (for example c-fos, c-jun and CSFs can be inhibited without affecting CSF-mediated c-myc) ~s,48,57-59, which can further amplify the growth survival 7,s°,51,ss or certain functional responses ~4. These factor signal; housekeeping proteins with enzymatic or results imply that distinct signalling pathways may mestructural activities, including metabolic enzymes (for diate the pleiotropic actions of the CSFs. example ornithine decarboxylase) s3, matrix and cytoskeletal proteins (for example fibronectin receptor, Comparison of the actions of the CSFs [3-actin) 48, which are required to prepare for the proDo the different CSFs which show mitogenic actions liferative or functional response of the cell; and secreted for the one cell type use a common signalling pathway? proteins, which are involved in intercellular communi- For bone-marrow-derived macrophages, which repcation (for example the products of the KC and JE resent a homogeneous normal haemopoietic cell type, genes) ss, or functional activation (for example u-PA) 24. there is potent synergism between CSF-1 and GM-CSF or The stimulation of gene expression by CSF-1 is represen- IL-3 for DNA synthesis 4°. The simplest explanation for ted in Fig. l(k). the synergism is that there is a co-operative interaction via common post-receptor pathways initiated by the Glucose transport different CSFs (see Fig. 2). The common pattern of early The stimulation of cell metabolism accompanying events in response to the different CSFs also suggests that growth factor action places an increased demand for activation of common effector mechanisms, such as tyroenergy on the cell. Increases in glucose uptake and sine kinases, PKC and RAF-1 kinase, are likely. Simiglycolytic flux are, therefore, common responses to many larities in the negative modulation of CSF-I-, GM-CSFgrowth factors. CSF-1, GM-CSF and IL-3 have all been and IL-3-stimulated bone-marrow-derived macrophage found to increase glucose uptake in bone-marrow- DNA synthesis by cAMP 5° and other proliferation derived macrophages and murine resident peritoneal inhibitors, such as TNF-% gamma-interferon and macrophages 38. In contrast to the factor-dependent lipopolysaccharide 7, also suggests a common level of FDC-P2 cell line 6°, increased glucose uptake alone was control. The synergism of all three CSFs with phorbol not sufficient to maintain bone-marrow-derived macro- esters 4° suggests a convergence at, or prior to, PKC phage survival, suggesting that the relationship between activation, with the negative control by cAMP occurring enhanced glucose uptake and cell survival in normal later (Ref. 50 and Fig. 2). haemopoietic cell types may differ from those of There are some differences in the responses among haemopoietic cell lines. CSFs even within the same cell type. For instance, GMCSF, unlike CSF-1, does not elevate KC mRNA levels in Relationship of CSF responses to DNA synthesis the BAC1.2F5 macrophage cell line58; GM-CSF primes It is not clear whether there is a causal relationship bone-marrow-derived macrophages for subsequent trigbetween the early response triggered by CSFs and sub- gering of the respiratory burst whereas CSF-1 actually Immunology Today
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12 No. 10 1991
IL-3
CSF-1 Phorbol esters
GM-CSF
F•2
1
Adenylyl~ cyclase IL-3R ~1~
CSF-1R (c
Plasma membrane
-fms)
cAMP
/ RESPONSE I
v
G1 phase I Sphase
GO
Fig. 2. interactions between signalling pathways for CSF-I, GM-CSF and IL-3 are shown. Each CSF is proposed to initiate multiple pathways. Activation of putative common effectors results in a convergence or overlap in early responses, some of which may be involved (+) in the subsequent proliferative response (see Table 1). Such a scheme may account for the synergism between CSF-1 and GM-CSF or IL-3 for D NA synthesis 4°. It may be that synergism could also involve other mechanisms, such as autocrine growth factor production, increased CSF receptor affinity and~or numbers, or inhibition of CSF internalization or destruction (but see Refs 5 and 65 for evidence against these proposals in the murine system). Inhibition of CSFinduced S phase progression (-) by agents that elevate cAMP levels occurs later.
inhibits such priming of these cells63; and GM-CSF stimulates major histocompatibility complex (MHC) class II expression in these cells whereas CSF-1 inhibits it 64. The differences in responses between CSFs may reflect unique pathways for each CSF (Fig. 2). This may explain why GM-CSF and IL-3 show a weaker mitogenic effect for bone-marrow-derived macrophages than CSF-1 while inducing other responses to similar levels: perhaps GM-CSF and IL-3 lack the ability to fully activate some critical event(s) (possibly later in the cell cycle?) for the proliferative response. Conclusion The three major concepts presented in this review are: (1) CSFs initiate a series of rapid responses, some of which are necessary, but alone are not sufficient, to induce a proliferative response; (2) later events in the G1 stage of the cell cycle have an important role in determining S phase progression and need further analysis; (3) different CSFs, which can act upon the one cell type, Immunology Today
initiate multiple signalling pathways, one or more of which converge or overlap, resulting in stimulation of many common responses. The synergism between CSFs for stimulation of DNA synthesis may reflect cooperative interactions of these post-receptor signalling pathways. The characterization of receptors for most of the CSFs will undoubtedly be followed by further identification of CSF receptor targets and/or effectors. The use of techniques allowing specific inhibition ('knockout') of these and other cellular proteins by strategies such as genetic manipulation, antisense oligonucleotides or the use of more specific pharmacological inhibitors will establish the importance of individual components of the signalling cascades for the various effects mediated by different CSFs.
Our work described in this review was supported by the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria. We would like to
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thank Sharon Wong for typing the manuscript and Nurin Veis and Wayne Phillips for helpful discussions.
32 Farrar, W.L., Thomas, T.P. and Anderson, W.B. (1985) Nature 315, 235-237 33 Evans, S.W., Renick, D. and Farrar, W.L. (1987) Biochem. J. 244, 683-691 Gino Vairo and John Hamilton are at The University of 34 Sorensen, P.H.B., Mui, A.L-F., Murthy, S.C. and Krystal, Melbourne, Dept of Medicine, Royal Melbourne Hospital, G. (1989) Blood 73,406-418 Parkville, Australia 3050. 35 Whetton, A.D., Vallance, S.J., Monk, P.N. et al. (1988) Biochem. J. 256, 585-592 References 36 Cook, N., Dexter, T.M., Lord, B.I. et al. (1989) EMBOJ. i Metcalf, D. (1989) Nature 339, 27-30 8, 2967-2974 2 Sherr, C.J. (1990) Blood 75, 1-12 37 Vairo, G. and Hamilton, J.A. (1988) J. Cell. Physiol. 134, 13-24 3 Cosman, D., Lyman, S.D., Idzerda, R.L. et al. (1990) Trends Biochem. Sci. 15,265-270 38 Hamilton, J.A., Vairo, G. and Lingelbach, S.R. (1988) 4 Guilbert, L.J. and Stanley, E.R. (1986) J. Biol. Chem. 261, J. Cell. Physiol. 134, 405-412 4024-4032 39 Racoosin, E.L. and Swanson, J.A. (1989) J. Exp. Med. 5 Walker, F., Nicola, N.A., Metcalf, D. and Burgess, A.W. 170, 1635-1648 (1985) Cell 43,269-276 40 Hamilton, J.A., Vairo, G., Nicola, N. et al. (1988) Blood 6 Branch, D.R., Turner, A.R. and Guilbert, L.J. (1989) Blood 71, 1574-1580 73,307-311 41 Katayama, N., Nishikawa, M., Minami, N. and 7 Vairo, G., Argyriou, S., Knight, K.R. and Hamilton, J.A. Shirakawa, S. (1989) Blood 73,123-130 (1991) J. Immunol. 146, 3469-3477 42 Carrol, M.P., Clark-Lewis, I., Rapp, U.R. and May, W.S. 8 Guilbert, L.J., Nelson, D.J., Hamilton, J.A. and Williams, (1990) J. Biol. Chem. 265, 19812-19817 N. (1983) J. Cell. Physiol. 115,276-282 43 Morla, A.O., Schreurs, J., Miyajima, A. and Wang, J.Y.J. 9 Bishop, W.R. and Bell, R.M. (1988) Oncogene Res. 2, (1988) Mol. Cell. Biol. 8, 2214-2218 205-218 44 Kanakura, Y., Druker, B, Cannistra, S.A. et al. (1990) 10 Downing, J.R., Roussel, M.F. and Sherr, C.J. (1989) Mol. Blood 76, 706-715 Cell. Biol. 9, 2890-2896 45 Cook, W.D., Metcalf, D., Nicola, N.A. et al. (1985) Cell 11 Downing, J.R., Rettenmier, C.W. and Sherr, C.J. (1988) 41,677-683 Mol. Cell. Biol. 8, 1795-1799 46 Watson, J.D., Le Gros, G.S., Overell, R. et al. (1987) 12 Sengupta, A., Liu, W-K., Yeung, Y.G. et al. (1988) Proc. J. Immunol. 139, 123-129 Natl Acad. Sci. USA 85, 8062-8066 47 Yi, T-L. and Willman, C.L. (1989) Oncogene 4, 13 Varticovski, L., Druker, B., Morrison, D. et al. (1989) 1081-1087 Nature 342, 699-702 48 Mufson, R.A. (1990) J. Immunol. 145, 2333-2339 14 Reedijk, M., Liu, X. and Pawson, T. (1990) Mol. Cell. 49 Long, M.W., Heffner, C.H. and Gragowski, L.L. (1988) Biol. 10, 5601-5608 Exp. Haematol. 16, 195-200 15 Downing, J.R., Margolis, B.L., Zilberstein, A. et al. 50 Vairo, G., Argyriou, S., Bordun, A-M. et al. (1990) (1989) EMBO J. 8, 3345-3350 J. Biol. Chem. 265, 2692-2701 16 Baccarini, M., Sabatini, D.M., App, H. et al. (1990) 51 Jackowski, S., Rettenmier, C.W. and Rock, C.O. (1990) EMBO J. 9, 3649-3657 J. Biol. Chem. 265, 6611-6616 17 Taga, T., Hibi, M., Hirata, Y. et al. (1989) Cell 58, 52 Coffey, R.G., Davis, J.S. and Djeu, J.Y. (1988) 573-581 J. Immunol. 140, 2695-2701 53 Farrar, W.L. and Harel-Beltan, A. (1989) Blood 73, 18 Gearing, D.P., King, J.A., Gough, N.M. and Nicola, N.A. 1468-1475 (1989) EMBO J. 8, 3667-3676 19 Hayashida, K., Kitamura, T., Gorman, D.M. et al. (1990) 54 Grinstein, S., Rotin, D. and Mason, M.J. (1989) Biochim. Proc. Natl Acad. Sci. USA 87, 9655-9659 Biophys. Acta 988, 73-97 55 Vairo, G., Argyriou, S., Bordun, A-M. et al. (1990) 20 Park, L.S., Friend, D., Price, V. etal. (1989)]. Biol. Chem. 264, 5420-5427 J. Biol. Chem. 265, 16929-16939 21 Imamura, K. and Kufe, D. (1988) J. Biol. Chem. 263, 56 Caracciolo, D., Pannocchia, A., Treves, S. et al. (1990) 14093-14098 J. Cell. Physiol. 143, 133-139 22 Imamura, K., Dianoux, A., Nakamura, T. and Kufe, D. 57 Muller, R., Curran, T., Muller, D. and Guilbert, L. (1985) Nature 314, 546-548 (1990) EMBO J. 9, 2423-2429 23 He, Y., Hewlett, E., Terneles, D. and Quesenberry, P. 58 Orlofsky, A. and Stanley, E.R. (1987) EMBO J. 6, 2947-2952 (1988) Blood 5, 1187-1195 24 Hamilton, J.A., Vairo, G., Knight, K.R. and Cocks, B.G. 59 Roussel, M.F., Shurtleff, S.A., Downing, J.R. and Sherr, (1991) Blood 77, 616-627 C.J. (1990) Proc. Natl Acad. Sci. USA 87, 6738-6742 60 Whetton, A.D., Bazill, G.W. and Dexter, T.M. (1984) 25 Smith, M.R., DeGudicibus, S.J. and Stacey, D.W. (1986) EMBO J. 3,409-413 Nature 320, 540-543 26 Stanley, I.J., Nicola, N.A. and Burgess, A.W. (1989) 61 Tushinski, R.J. and Stanley, E.R. (1985) J. Cell. Physiol. 122, 221-228 Growth Factors 2, 53-59 27 Whetton, A.D., Monk, P.N., Consalvey, S.D. and 62 Matsushime, H., Roussel, M.F., Ashmun, R.A. and Sherr, Downes, C.P. (1986) EMBO J. 5, 3281-3286 C.J. (1991) Cell 65,701-713 63 Phillips, W.A. and Hamilton, J.A. (1990) J. Cell. Physiol. 28 Hamilton, J.A., Veis, N., Bordun, A-M. et al. (1989) 144, 190-196 J. Cell. Physiol. 141,618-626 29 Vallance, S.J., Dowries, C.P., Cragoe, E.J. and Whetton, 64 Willman, C.L., Stewart, C.C., Miller, V. et al. (1989) J. Exp. Med. 170, 1559-1567 A.D. (1990) Biochem. J. 265,359-364 30 Veis, N. and Hamilton, J.A. (1991) J. Cell. Physiol. 147, 65 Hamilton, J.A. and Dientsman, S.R. (1981) J. Cell. Physiol. 106, 445-450 298-305 66 Tushinski, R.J. and Stanley, E.R. (1983) J. Cell. Physiol. 31 Veis, N. and Hamilton, J.A. Biochem. Biophys. Res. 116, 67-75 Commun. (in press)
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Signalling through CSF receptors Gino Vairo and John A. Hamilton The finely regulated process of blood cell formation is under the control of a family of glycoprotein hormones, known as colony-stimulating factors (CSFs), and their receptors. The complexity of the intracellular mechanisms involved in the action of such factors has been appreciated only recently. In this review, Gino Vairo and John Hamilton discuss the biochemistry of CSF action and its relevance to growth control, and examine the possibility that different CSFs may use common control pathways within the one cell type. Over the last twenty years, much information regarding the identities of stimulatory factors involved in haemopoiesis, including CSFst, has been obtained. The actions of the CSFs are pleiotropic in nature: they are involved in the survival, proliferation, differentiation and activation of haemopoietic cells at various stages of their development, and can also act on nonhaemopoietic cell types. The recent cloning of many of the CSFs and their receptors has facilitated the study of the biochemistry of CSF action. This review examines the signalling processes and biochemical responses involved in the action of three of the better characterized CSFs, namely macrophage CSF (CSF-1 or M-CSF), granulocyte-macrophage CSF (GMCSF) and interleukin-3 (IL-3 or multi-CSF), concentrating on the macrophage as a target cell. It will also examine the relationship of these CSF-induced responses to their mitogenic action.
oL(TNF-o~),which potently downmodulates CSF-1 receptor levels in murine bone-marrow-derived macrophages6, has little effect on CSF-1-stimulated pinocytosis and by itself does not stimulate pinocytosis in these cells7. The lack of inhibition of this CSF-1 response by TNF-(x is consistent with low receptor occupancy being sufficient for biological responses to CSFs1. Phorbol esters can also downmodulate CSF-1 receptor levels in murine macrophages8, probably via the activation of protein kinase C (PKC)9. In c-fms-transfected fibroblasts, the downmodulation of CSF-1 receptor levels by phorbol esters and other PKC activators occurs via a different mechanism from autologous downregulation1°. Since PKC activation by the CSFs is also likely (see later), these findings may be relevant to the receptor transdownmodulation seen between the CSFs (and other agents which downmodulate CSF receptors).
Signal transduction through CSF receptors CSFs and their receptors An overview of the actions of CSF-1, a prototypic CSF, The first of the CSF receptors to be isolated and is illustrated in Fig. 1. The formation of an active ligandcharacterized was the CSF-1 receptor, which was shown receptor complex is the initial event in the growth factor to be the product of the c-fms proto-oncogene and a signal transduction pathway (Fig. l(a)). For the growth member of the tyrosine kinase receptor family2. More factor receptor tyrosine kinases, such as the CSF-1 receprecently, the receptors for GM-CSF and IL-3 have been tor, activation of receptor kinase activity follows ligand cloned (reviewed in Ref. 3); these receptors share certain binding, and results in receptor autophosphorylation folstructural homologies and have been grouped together in lowed by a rapid phosphorylation on tyrosine of an array the haemopoietin receptor superfamity. Unlike the CSF-1 of cytosolic and membrane-associated proteins (Refs receptor, the haemopoietin receptors do not show con- 11,12 and Fig. l(b)), including phosphatidylinositol sensus sequences characteristic of tyrosine kinases. (PI)-3 kinase (Table 1 and Refs 13,14). This enzyme The binding of CSFs to their receptors is accompanied phosphorylates the D-3 hydroxyl position of the inositol by rapid receptor downregulation, internalization of the ring of PI, the physiological role of which is, as yet, ligand-receptor complex and subsequent destruction of unknown. Other candidate substrates for receptor tyrothe CSF. It has been suggested that the intracellular sine kinases, including phospholipase C~/(PLC~) is, the accumulation of CSF-1, as well as accumulation of RAF-1 serine/threonine kinase 16, and the p21ras surface-bound CSF-1, may be involved in the mitogenic GTPase-activating protein (GAP)~4 do not appear to be response 4. Binding of a CSF to its specific receptor can directly phosphorylated by the CSF-1 receptor. Actialso result in the downmodulation of receptors for other vation of some of these enzymes, however, can still occur CSFs. This phenomenon has been termed 'trans- via indirect mechanisms16. The identities of the nudownmodutation', and the relative activities among IL-3, merous other cellular proteins phosphorylated on tyroGM-CSF and CSF-1 follow a distinct hierarchys. sine in response to CSF-1 remains to be determined. For CSF-1 (and other growth factors that have tyroAlthough it has been suggested that transdownmodulation may be accompanied by an activation of signal transduc- sine kinase receptors) the activation of the tyrosine tion through the receptor, 'transactivation', even in the kinase, presumably via the modulation of the activities of absence of its respective ligand, its biological signifi- an array of cellular targets, is the 'master switch' that cance is unclear; transdownmodulation of CSF receptors initiates the cellular response cascade. How the nonalone is insufficient to trigger at least some of the CSF- tyrosine-kinase (haemopoietin) receptors initiate their induced responses. For instance, tumour necrosis factor responses is, as yet, undetermined. A clue may come from © 1991, Elsevier Science Publishers Ltd, UK. 0167--4919/91/$02.00
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!!i~iiiii~!~iilili~iiiiii~ii~iii!ii!i!iiii{iiiiiiiiiiiiii ~iiIiii~~iiiii!~iiiii!iiiii,~iiil~~iiiiii~i~~iiiii~iiiiii~iiiiiiiiii~i!iiiiiii~il~!iii!iil iii~~i~iiiiii iiiii~i!iiii!iiiii iiiii CSF-1
[a] [c ]
41---
>_-~-
f - -Ill~ . [ ,~'b],~ ,~ " ..,..
\ .,
nucleus
"~_...~ P(rr°ts/G Ap?,
tyr t y'r-(~ receptor substrates " \ e.g. PI-3 kinase
~[g]
I •
/
e.g. u-PA, JE, K C / , 13 actin, fibronectin R (15) hck, fgr fos, jun, myc G1 cyclins
-- " ~ [d] phosphoJi pids I: (e.g. PC)--~--~lon ~phospholipases "! \(e.g. PLC) DAG
other effectors e.g. F ~F-1 kinase
[e]
;'1' o . A synthesis
.
"
.o N a+
Fluid-phase pinocytosis [J] Glucose
K+
Fig. 1. CSF-l-induced responses and their relationship to DNA synthesis are shown. (a) Formation of a CSF-l-receptor complex; (b) stimulation of
receptor tyrosine kinase activity, resulting in receptor autophosphorylation and substrate phosphorylation at tyrosine; (c) activation of a G protein which in turn (d) stimulates phospholipase-mediated hydrolysis of membrane phospholipids with the production of D A G; (e) D A G binding to cytosolic PK C induces its translocation and activation, with subsequent stimulation (f) of a variety of cellular responses with possible relevance to D NA synthesis. Other kinases may also be activated (g), either by direct phosphorylation by the receptor (e.g. PI-3 kinase) or via an indirect mechanism (e.g. RAF-1 kinase), which may also be involved in CSF-1 action (h). Activation of PKC or other effectors stimulates other parameters important for mitogenesis including (i) Na+,H + exchange activity, with the Na + influx in turn resulting in (j) stimulation of Na+,K +-ATPase activity or (k) enhanced expression of a variety of genes, including those encoding growth-related proteins and other structural, enzymatic or secreted proteins. Other rapid responses to CSF-1, including stimulation of glucose uptake, fluid phase pinocytosis and u-PA secretion are also shown. Question marks on the arrows indicate that there is no evidence for a direct relationship between responses.
the finding that the IL-6 receptor, another member of the haemopoietin receptor superfamily, associates, in the presence of IL-6, with a 130 kDa membrane-associated glycoprotein (gp130), possibly forming a signal transducing 'effector' subunit 17. Such effectors may possess intrinsic kinase activity or may modulate other transducing proteins to initiate the cellular response cascade. Competition between different CSF receptors for a limiting 'adaptor' subunit, which is required for highaffinity ligand binding, has also been suggested 18 and Immunology Today
recently the cloning of such an affinity-modulating subunit of the GM-CSF receptor has been described19. Such a mechanism may explain the apparent, but as yet unidentified, crossreactive GM-CSF/IL-3 receptor(s) 2°.
G proteins Inhibition of growth factor responses by pertussis toxin (PT) has been used to investigate the involvement of guanine-nucleotide-binding proteins (G proteins) in growth factor signal transduction. PT can inhibit the
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Table 1. Relationship of CSF-induced responses to their mitogenic action Response
Stimuli CSF
Receptor targets PI-3 kinase phosphorylation
Cell Type Nonmitogen
CSF-1 CSF-1 CSF-1
G proteins GTP binding/GTPase CSF-1 activity p21 ras phosphorylation GM-CSF p62 'GAP'-associated CSF-1 protein tyrosine phosphorylation Phospholipid hydrolysis DAG turnover CSF-1/GMCSF/IL-3 CSF-1 Protein kinases PKC (translocation) PKC substrate phosphorylation
RAF-1 kinase
Cyclic nucleotides I" cGMP $ cAMP
Ion fluxes Na+,H + exchange
Refs
Cycling
Poorly-cycling
A31 fibroblasts (c-fms) 3T3 (c-fins) BMM/BAC1.2F5
P388D1 a 13 3T3 (fms 809 F) 59 14 PBM
CFC 3T3/R2C1 fibroblasts (c-fins)
Zymosan
BMM
(not RPM
30,31
PBM
22
FDC-P1 NSF.60 NSF.60.8
32 22 33 33 33
B6SUtA1 BAC1.2F5 FDC-P1 DA3
34 16 42 42
PBM
PMN
GM-CSF CSF-1 GM-CSF
BMM PMN
CSF-1/GMCSHIL-3/G-CSF LPS/TNF-c~ CSF-1/GMCSF/IL-3 CSF-1 PAF GM-CSF GM-CSF IL-3
activity of some (but not all) G proteins by ADPribosylation of the active, % subunit of the heterotrimeric G protein complex21-23. CSF-1 increases GTP binding and elevates GTPase activity in human monocyte membrane preparations, and a variety of responses of these cells to CSF-1 are PT sensitive (Refs 21,22 and Fig. 1(c)). The actions of CSF-1, GM-CSF and 1L-3 on murine haemopoietic cells23,24 appear to be less sensitive to the effects of PT. It therefore appears that PT-sensitive G proteins have only a minor role, if any, in the CSF response in mouse cells, although PT-insensitive G proteins may still be involved. The rasGAP system, which may be functionally related to prototypic G proteins, has also been implicated in the action of both CSF-1 and GM-CSF (Refs 14,25,26 and Table 1).
1mrnunology Today
26 14
FDC-P1
IL-3 CSF-1 IL-3 IL-3/G-CSF IL-3/G-CSF (not GM-CSF) IL-3/GM-CSF CSF-1 IL-3 GM-CSF/IL-3
21
52 50 52
CFC
36
BMM
37,55, 6
PBM BMM AML 193 FDC-P1
21 30 56 35
Phospholipid hydrolysis The activation of a phosphatidylinositol 4,5-bisphosphate (HP2)-specific phospholipase C (PLC), which hydrolyses membrane inositol phospholipids to yield the so-called second messengers, inositol-l,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DAG), could play an important role in the action of various growth factors9. However, studies involving a variety of CSF-responsive cell types have so far failed to detect enhanced inositol phosphate turnover in response to CSFs22,27-29. The hydrolysis of phosphatidylcholine (PC) by a PC-specific PLC or phospholipase-D (PLD) is a major alternative pathway for DAG production9. Elevated DAG turnover, possibly via PLC-mediated PC hydrolysis, has been described in human monocytes in response
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Table 1. Relationship of CSF-induced responses to their mitogenic action Response
Na+,K+-ATPase
Stimuli Cycling
Poorly-cycling
CSF-1/GMCSF/IL-3 CSF-1
LPS/ConA/ TNF-c~
BMM
RPM
37, b
PBM
21
CSF-1/GMCSF/IL-3 CSF-1 CSF-1/GM-CSF c-myc mRNA CSF-1/GMCSF/IL-3 CSF-1/GM-CSF c-jun mRNA CSF-1 CSF-1 c-fgr mRNA CSF-1/GM-CSF hck mRNA CSF-1 Ornithine decarboxylase GM-CSF mRNA
u-PA mRNA G1 cyclin mRNA
Refs
Nonmitogen
Gene expression c-fos mRNA
Fbn.R (13)mRNA [3-actin mRNA JE mRNA KC mRNA
Cell Type
CSF
LPS/zymosan/ TNF-c~
Zymosan
LPS/IFN-y
28,57, b
3T3 (c-fms) BAC1.2F5 BMM
3T3 (fins 809 F) 59 Aut.l a 58 28,57
BAC1.2F5 3T3 (fins)
NSF.60.8
Aut.l a 58 3T3 (fms 809 F) 59 PBM 48 47 PBM 48 53
BAC1.2F5 BAC1.2F5
PBM PBM Aut.1 a Aut.1 a
BMM
CSF-1 CSF-1 CSF-1/GM-CSF CSF-I (not GM-CSF) CSF-1/GMConA CSF/IL-3 CSF-1
Other responses Ornithine decarboxylase GM-CSF/ G-CSF/IL-3 G1 cyclin expression CSF-1 Protein synthesis CSF-1 RNA synthesis CSF-1 Glucose uptake CSF-1/GMCSF/IL-3 IL-3 Pinocytosis CSF-1/GMCSF/IL-3 u-PA production CSF-1/GMCSF/IL-3
BMM
48 48 58 58
BMM
24
BAC1.2F5
62
NSF.60.8
53
LPS/ConA
BAC1.2F5 BMM BMM BMM
62 66 55 38
LPS/zymosan
FDC-P2 BMM
ConA
BMM
RPM
60 7,39, c RPM
24
The relationshipof CSF-inducedresponsesto their mitogenicactionwas examined.The responsesof the CSFs,when examinedin the one study, were compared with other nonmitogenicstimuli. The responses of cells that progress through the cell cyclein response to CSFs (cycling)were also comparedwith cellsthat showa poor proliferativeresponse (poorlycycling).DAG: 1,2-diacylglycerol;PI: phosphatidyl inositol; Fbn.R ([3): fibronectinreceptor [3subunit; GAP: GTPase-activatingprotein; ConA: concanavalinA; IFN-y: gamma-interferon; LPS: lipopolysaccharide;PAF: platelet-activatingfactor; TNF-(x:turnout necrosis factor ~; BMM: bone-marrow-derivedmacrophages; CFC: colony-formingprogenitor cells; c-fins: cells transfectedwith wild type CSF-1 receptor; fins 809 F: cells transfectedwith human CSF-I receptor containing phenylalanine substitution at position 809; PBM: human peripheral blood monocytes; PMN: human polymorphonuclearcells; RPM: murine residentperitoneal macrophages, aConstitutivelygrowingcell lines for which CSFsdo not act as mitogens, bG. Vairo, A.K. Royston and J.A. Hamilton, (unpublished).CK.R.Knight,G. Vairo and J.A. Hamilton, (unpublished). to CSF-1 (Ref. 22 and Table 1). CSF-1 has also been found to increase DAG turnover in bone-marrowderived macrophages but not in the poorly cycling murine resident peritoneal macrophage population 3°, suggesting that such enhanced DAG turnover may represent an important early signal for macrophage proliferation (Fig. l(d)). Enhanced DAG turnover also occurs in
Immunology Today
bone-marrow-derived macrophages in response to both GM-CSF and IL-3 (Ref. 31 and Table 1), Further evidence suggesting a role for PLC in CSF action comes from the findings that exogenous, nonspecific PLC is itself a weak mitogen for bone-marrow-derived macrophages and elicits a variety of responses common to CSF-1, GMCSF and IL-3 (Refs 24,28). The nonspecific PLC also
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elevates DAG turnover in these cells3° without a detectable increase in inositol phosphate levels28. Protein kinases Protein kinases play an important role in regulating the activities of many of the key enzymes and other proteins involved in growth factor responses. The serine/ threonine kinase, PKC, which is activated by DAG, has been implicated in the action of many growth factors 9. CSF-1, GM-CSF and IL-3 have been reported to induce a translocation and/or activation of PKC in a number of haemopoietic cell types (Refs 22,32-34 and see Table 1 and Fig. l(e)). Evidence for a role of PKC in CSF action also comes from the findings that phorbol esters and other direct PKC activators elicit a similar series of early responses to CSFs24,28,3°,35-39, and are themselves weakly mitogenic 33-37 and synergize with CSFs for stimulation of DNA synthesis 33,4°. The inhibition of PKC with pharmacological agents or by chronic phorbol ester pretreatment inhibits responses to different CSFs, including Na+,H + exchange, activation and proliferation =,35,41. The possible involvement of PKC in these CSF-1 responses is shown in Fig. l(f). Although there is increasing evidence that PKC activation may be involved in CSF action, at least in some cell types, PKC-independent mechanisms may also be involved. PKC-independent serine phosphorylation has been described in response to IL-3 or GM-CSF 34, and CSFs stimulate RAF-1 kinase activity in several factordependent cell lines (Refs t6, 42 and see Table 1). Whereas the known receptors for IL-3 and GM-CSF do not contain a consensus sequence for tyrosine kinase activity 3, these CSFs can induce the rapid tyrosine phosphorylation of a variety of cellular substrates in a number of cell types 34,43. Whether this activity is due to an uncharacterized receptor(s) for these CSFs, which possesses tyrosine kinase activity, or whether the nontyrosine kinase receptors activate a nonreceptor tyrosine kinase remains to be determined. Evidence for a regulatory role for tyrosine phosphorylation in the proliferative response to such CSFs comes from the findings that the protein tyrosine phosphatase inhibitor, vanadate, which enhances tyrosine phosphorylation in response to GMCSF or IL-3 in a factor-dependent cell line, also enhances the proliferative response to these CSFs44. In addition, introduction and expression of nonreceptor tyrosine kinase oncogenes, such as v-src or v-abl, into CSFdependent cell lines induces factor independence via a nonautocrine mechanism 4s,46. Increased mRNA levels for the src-related genes, c-fgr and hck, in response to CSF-1 or GM-CSF have been reported (Refs 47, 48 and see Table 1). The activation of other nonreceptor 'effector' kinases by CSF-1 is shown in Fig. l(g). Possible roles for such kinases in mediating the numerous responses to CSF-1 are represented in Fig. l(h). cAMP-dependent protein kinases (PKA), which mediate the second messenger function of cAMP, have also been implicated in modulation of growth factor action. The pleiotropic role of cAMP which is seen in many cellular systems is also evident in the haemopoietic system: increased levels enhance IL-3- and phorbol-esterinduced megakaryocyte colony formation, have a weaker effect on erythroid colony formation and inhibit granulo-
Immunology Today
cyte colony formation from murine bone marrow 49. Recent reports indicate that elevation of cAMP levels inhibits CSF-stimulated macrophage proliferation 5°,51. For bone-marrow-derived macrophages, CSF-1 reverses the inhibition of DNA synthesis caused by agents that activate adenylyl cyclase but not direct PKA activators. This suggests that CSF-1 inhibits cAMP accumulation, perhaps by inhibiting adenylyl cyclase and/or increasing cAMP phosphodiesterase activities s°. Interestingly, GMCSF inhibits adenylyl cyclase activity and enhances guanylyl cyclase activity and cGMP levels in human neutrophils (Ref. 52 and Table 1). The inhibition of bone-marrow-derived macrophage DNA synthesis by cAMP elevation occurs, even quite late, in the G1 phase of the cell cycle without affecting a variety of other CSF- 1 responses s°, suggesting that cAMP acts at some late G1 control point to inhibit S phase progression. Consistent with this, cAMP inhibition of GM-CSF-stimulated proliferation of a factor-dependent cell line is associated with an inhibition of the late induction in ornithine decarboxylase gene expression and enzyme activity 53. cAMP elevation has been found to inhibit CSF-t-, GM-CSF- and IL-3-stimulated urokinasetype plasminogen activator (u-PA) mRNA levels and enzyme production in bone-marrow-derived macrophages, although this response does not appear to be necessary for their proliferation 24. Ion fll~txes
Stimulation of the amiloride-sensitive Na+,H + exchanger is a ubiquitous early response to growth factors. Inhibition of this intracellular pH (pHi) regulator often abrogates the proliferative response, suggesting that antiport activation is an important early mitogenic signal (reviewed in Ref. 54). Certainly, in the absence of HCO 3- this antiporter can be the major pH i regulator, and as such is required to maintain a sufficiently alkaline pH i to allow DNA synthesis. However, in the presence of HCO3 , other HCO3--dependent pH i regulatory processes can also contribute to pH i homeostasis 54. Under these more physiological conditions the role of the Na+,H + exchange-mediated increase in pH i as a signal for growth factor action has been questioned 54. CSF-I, GM-CSF and IL-3 have all been found to activate Na+,H + exchange activity in a number of cell types (see Table 1). Inhibition of CSF-stimulated proliferation by amiloride analogues in the absence 29,35,36,ss or presence 55,56 of HCO3- indicates an important role for Na+,H + exchange in CSF action under these conditions. The inhibition of CSF-stimulated proliferation in the presence of HCO 3- suggests that the Na + influx, rather than any pH i increase, may be the more important consequence of Na+,H + exchange activation s5,56. The stimulation of Na+,H + exchange activity by CSF-1 is shown in Fig. 1(i). The Na + influx accompanying Na+,H + exchange activation has been shown in turn to activate the Na-,K+-ATPase (Na+-pump) (see Fig. l(j)), which is responsible for maintaining intracellular Na + ([Na+]i) levels21,37,55. This co-ordinated activation of both of these ion transporters has been referred to as a 'Na + cycle '37,55. The Na +, K+-ATPase, via its effects on intracellular Na +, K + and ATP levels, may also be important for growth factor action.
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The inhibition of CSF-l-stimulated bone-marrowderived macrophage DNA synthesis by an amiloride analogue occurs even if added several hours after the growth factor, suggesting that Na + cycle activity late in the G1 stage of their cell cycle is required for S phase progression ss. Consistent with this proposal are the reports that CSF-1 and GM-CSF result in a persistent stimulation of Na+,H + exchange activity in these cells29,s5. Although Na ÷ cycle activity appears necessary for CSF-l-stimulated bone-marrow-derived macrophage proliferation, this is not so for a variety of other early CSF-1 responses 24,3s,55, suggesting that Na ÷ cycle activation itself is unlikely to be a general mediator of CSF action. The lack of effect of CSFs on intracellular Ca 2+ levels is consistent with a lack of production of the CaZ+-mobilizing inositol phosphates in the same systems 27-29,36. Furthermore, for bone-marrow-derived macrophages at least, a lack of Ca 2+ involvement in CSF action is suggested by the findings that Ca p+ ionophores are not mitogenic 28 and do not elicit many of the responses induced by CSFs 24,28.
sequent DNA synthesis. As shown in Table 1, CSFinduced responses are not specific (indeed they can also be elicited by nonmitogens) and many of the CSFs elicit similar responses in both cycling and poorly cycling cells. Thus, the early events alone are not sufficient to induce a proliferative response and it remains to be determined whether or not many of the events play a direct role in the mitogenic action of the CSFs. Some of the parameters listed have even been dissociated from the proliferative response to CSFs; for instance, stimulation of macrophage proliferation can occur in the absence of u-PA production 24 or KC gene expression s8. On the other hand, inhibition of some parameters, including Na+,H + exchange 29,3s,36,ss,s6 and PKC 3s,41 activities, prevents CSF-stimulated proliferation. Induction of these early events, although not sufficient, is necessary for the proliferative response to CSFs. The observation that CSF-1 is required throughout the G1 phase of the bone-marrowderived macrophage cell cycle for S phase progression 61 is consistent with the notion that the early transient responses are necessary, but not sufficient on their own, to induce DNA synthesis. Clearly, events later in the G1 phase of the cell cycle play an important role in S phase Gene expression progression, as evidenced by the ability of inhibitors of Binding of growth factor to its receptor results in the CSF-stimulated proliferation, such as cAMP, to exert transmission of a 'signal' to the nucleus, presumably via their effect late in G1 (Refs 50, 55 and Fig. 2). The recent the action of pre-existing cytosolic effectors, whereupon report of CSF-l-induced expression of novel G1 cyclins a programmed process of gene expression is initiated. (putative S phase regulators) in the BAC1.2F5 macroCSFs induce the expression of a variety of genes (see phage cell line 62 is also relevant to this proposal. Table 1) that encode kinases (for example c-fgr, hck) 47,48 Under certain conditions the proliferative actions of and transcription factors (for example c-fos, c-jun and CSFs can be inhibited without affecting CSF-mediated c-myc) ~s,48,57-59, which can further amplify the growth survival 7,s°,51,ss or certain functional responses ~4. These factor signal; housekeeping proteins with enzymatic or results imply that distinct signalling pathways may mestructural activities, including metabolic enzymes (for diate the pleiotropic actions of the CSFs. example ornithine decarboxylase) s3, matrix and cytoskeletal proteins (for example fibronectin receptor, Comparison of the actions of the CSFs [3-actin) 48, which are required to prepare for the proDo the different CSFs which show mitogenic actions liferative or functional response of the cell; and secreted for the one cell type use a common signalling pathway? proteins, which are involved in intercellular communi- For bone-marrow-derived macrophages, which repcation (for example the products of the KC and JE resent a homogeneous normal haemopoietic cell type, genes) ss, or functional activation (for example u-PA) 24. there is potent synergism between CSF-1 and GM-CSF or The stimulation of gene expression by CSF-1 is represen- IL-3 for DNA synthesis 4°. The simplest explanation for ted in Fig. l(k). the synergism is that there is a co-operative interaction via common post-receptor pathways initiated by the Glucose transport different CSFs (see Fig. 2). The common pattern of early The stimulation of cell metabolism accompanying events in response to the different CSFs also suggests that growth factor action places an increased demand for activation of common effector mechanisms, such as tyroenergy on the cell. Increases in glucose uptake and sine kinases, PKC and RAF-1 kinase, are likely. Simiglycolytic flux are, therefore, common responses to many larities in the negative modulation of CSF-I-, GM-CSFgrowth factors. CSF-1, GM-CSF and IL-3 have all been and IL-3-stimulated bone-marrow-derived macrophage found to increase glucose uptake in bone-marrow- DNA synthesis by cAMP 5° and other proliferation derived macrophages and murine resident peritoneal inhibitors, such as TNF-% gamma-interferon and macrophages 38. In contrast to the factor-dependent lipopolysaccharide 7, also suggests a common level of FDC-P2 cell line 6°, increased glucose uptake alone was control. The synergism of all three CSFs with phorbol not sufficient to maintain bone-marrow-derived macro- esters 4° suggests a convergence at, or prior to, PKC phage survival, suggesting that the relationship between activation, with the negative control by cAMP occurring enhanced glucose uptake and cell survival in normal later (Ref. 50 and Fig. 2). haemopoietic cell types may differ from those of There are some differences in the responses among haemopoietic cell lines. CSFs even within the same cell type. For instance, GMCSF, unlike CSF-1, does not elevate KC mRNA levels in Relationship of CSF responses to DNA synthesis the BAC1.2F5 macrophage cell line58; GM-CSF primes It is not clear whether there is a causal relationship bone-marrow-derived macrophages for subsequent trigbetween the early response triggered by CSFs and sub- gering of the respiratory burst whereas CSF-1 actually Immunology Today
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IL-3
CSF-1 Phorbol esters
GM-CSF
F•2
1
Adenylyl~ cyclase IL-3R ~1~
CSF-1R (c
Plasma membrane
-fms)
cAMP
/ RESPONSE I
v
G1 phase I Sphase
GO
Fig. 2. interactions between signalling pathways for CSF-I, GM-CSF and IL-3 are shown. Each CSF is proposed to initiate multiple pathways. Activation of putative common effectors results in a convergence or overlap in early responses, some of which may be involved (+) in the subsequent proliferative response (see Table 1). Such a scheme may account for the synergism between CSF-1 and GM-CSF or IL-3 for D NA synthesis 4°. It may be that synergism could also involve other mechanisms, such as autocrine growth factor production, increased CSF receptor affinity and~or numbers, or inhibition of CSF internalization or destruction (but see Refs 5 and 65 for evidence against these proposals in the murine system). Inhibition of CSFinduced S phase progression (-) by agents that elevate cAMP levels occurs later.
inhibits such priming of these cells63; and GM-CSF stimulates major histocompatibility complex (MHC) class II expression in these cells whereas CSF-1 inhibits it 64. The differences in responses between CSFs may reflect unique pathways for each CSF (Fig. 2). This may explain why GM-CSF and IL-3 show a weaker mitogenic effect for bone-marrow-derived macrophages than CSF-1 while inducing other responses to similar levels: perhaps GM-CSF and IL-3 lack the ability to fully activate some critical event(s) (possibly later in the cell cycle?) for the proliferative response. Conclusion The three major concepts presented in this review are: (1) CSFs initiate a series of rapid responses, some of which are necessary, but alone are not sufficient, to induce a proliferative response; (2) later events in the G1 stage of the cell cycle have an important role in determining S phase progression and need further analysis; (3) different CSFs, which can act upon the one cell type, Immunology Today
initiate multiple signalling pathways, one or more of which converge or overlap, resulting in stimulation of many common responses. The synergism between CSFs for stimulation of DNA synthesis may reflect cooperative interactions of these post-receptor signalling pathways. The characterization of receptors for most of the CSFs will undoubtedly be followed by further identification of CSF receptor targets and/or effectors. The use of techniques allowing specific inhibition ('knockout') of these and other cellular proteins by strategies such as genetic manipulation, antisense oligonucleotides or the use of more specific pharmacological inhibitors will establish the importance of individual components of the signalling cascades for the various effects mediated by different CSFs.
Our work described in this review was supported by the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria. We would like to
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thank Sharon Wong for typing the manuscript and Nurin Veis and Wayne Phillips for helpful discussions.
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