Cells of the macrophage lineage serve diverse homeostatic and immunologic functions by differentiating into functionally distinct end cells that proliferate and differentiate under the influence of M-CSF, GM-CSF and other locally produced regulatory molecules (10
). Studies of the osteopetrotic op/op
mouse indicate that the macrophage lineage can be considered to consist of M-CSF-dependent and M-CSF-independent populations (47
). Mice homozygous for the op
mutation lack M-CSF (46
) and exhibit striking defects in some but not all macrophage populations. Osteoclasts, peritoneal macrophages, and lymph node subcapsular macrophages, for example, are severely reduced or absent. Other populations, however, including phagocytes in the splenic red pulp, thymic cortex, Kupffer cells, alveolar macrophages, and microglia, are largely unaffected in the absence of M-CSF (47
). These observations indicate the presence of multiple pathways for macrophage differentiation that give rise to cells that have overlapping but nonidentical functions.
The CSFs responsible for M-CSF-independent macrophage populations appear to be highly redundant. Disruptions of the genes encoding GM-CSF or the common β subunit of the GM-CSF, IL-3, and IL-5 receptors have had relatively little effect on any of the macrophage lineages, with the exception of a functional defect in the ability of alveolar macrophages to clear surfactant components (8
). Thus, GM-CSF is not essential for the development of M-CSF-independent populations of macrophages. Nevertheless, the potent activities of GM-CSF in promoting the development of macrophage colonies in vitro and its expression by many cell types in vivo support the idea that it plays important physiologic roles in establishing M-CSF-independent macrophage populations.
Although M-CSF and GM-CSF promote different sets of specialized functions, many of the phenotypic characteristics of M-CSF- and GM-CSF-derived macrophages are similar. At the gene level, M-CSF and GM-CSF induce the expression of many macrophage- or myeloid-specific genes in parallel, including the SR-A gene. Because the receptors for M-CSF and GM-CSF are not related, these observations have raised the question of whether coordinate regulation of these genes reflects (i) activation of distinct sets of transcription factors that function independently or (ii) convergence of signal transduction pathways on a common set of transcription factors.
In this study, we have examined the mechanisms by which M-CSF and GM-CSF coordinately regulate the expression of the macrophage SR-A gene. SR regulatory elements consisting of a minimal 245-bp promoter and a 400-bp upstream enhancer were sufficient to confer macrophage-specific expression and M-CSF-dependent regulation in transgenic mice. Several lines of evidence indicate that transcriptional activation of the SR-A gene by M-CSF is mediated by a Ras-dependent signal transduction cascade that stimulates the expression and activities of AP-1 and Ets domain transcription factors. We have previously shown that c-Jun, JunB, and Ets2 bind to regulatory elements in the M-CSF-dependent enhancer and act synergistically to stimulate transcription (49
). In the present study, we have shown that mutation of these binding sites abolishes SR-A promoter responses to M-CSF in transgenic mice, while expression of a constitutive form of Ras is sufficient to activate the M-CSF-responsive enhancer.
These observations raised the possibility that GM-CSF acts to regulate the SR-A gene in a similar manner, as the GM-CSF receptor also couples to Ras-dependent signal transduction pathways and activates AP-1 and Ets transcription factors (1
). Consistent with this possibility, GM-CSF was indeed capable of activating the M-CSF-responsive enhancer in Ba/F3-GMR cells, and this effect was mediated by AP-1 and Ets transcription factors. However, the response of the M-CSF-dependent enhancer to GM-CSF was very transient in Ba/F3 cells, corresponding to the temporal profile of an immediate-early gene. Furthermore, when evaluated in transgenic mice, GM-CSF not only was unable to activate SR-A promoter activity through the M-CSF-responsive enhancer but inhibited activation by M-CSF.
Regulation of the SR-A gene by GM-CSF in transgenic mice was instead found to depend on a distinct GM-CSF-responsive enhancer. This enhancer could be activated by both Ras and GM-CSF in a cell line expressing functional GM-CSF receptors, with maximal activation observed 72 h following GM-CSF treatment. DNase I footprinting experiments of the GM-CSF-responsive promoter using nuclear extracts prepared from these cells indicated the presence of both constitutively expressed DNA binding proteins, as well as GM-CSF-inducible proteins that become maximally expressed 12 h following GM-CSF treatment. The identities of these proteins remain to be established, but they do not appear to correspond to known STAT factors. The constitutively expressed proteins are likely to be subject to posttranslational regulation by Ras-dependent signal transduction pathways, because the GM-CSF-responsive enhancer exhibited relatively little transcriptional activity in Ba/F3-GMR resting cells in which these proteins were found to be present but was very active following forced expression of constitutively active Ras. Similarly, truncation of the distal C terminus of the GM-CSF/IL-3/IL-5 receptor β subunit, which couples to Ras, significantly reduced GM-CSF-dependent transcription. While immediate-early effects of Ras on the GM-CSF-dependent enhancer are likely mediated by intracellular signaling pathways, delayed effects could potentially reflect the induction of other cytokines that indirectly activate the GM-CSF-dependent promoter in an autocrine manner.
In concert, our findings are most consistent with a model in which M-CSF and GM-CSF regulate the expression of the SR-A gene by fundamentally different mechanisms (Fig. ). While Ras appears to play an important role in regulating both enhancers, cross talk between GM-CSF-dependent signal transduction pathways and targets of the M-CSF receptor that are activated by Ras is insufficient to mediate activation of the M-CSF-responsive enhancer in primary bone marrow progenitor cells. The relatively restricted utilization of Ras signaling pathways by M-CSF and GM-CSF may reflect the differential assembly of receptor-associated signaling complexes that are relatively dedicated to specific downstream targets (43
). Differential utilization of Ras signal transduction pathways by M-CSF and GM-CSF is consistent with the distinct phenotypic properties of M-CSF- and GM-CSF-derived macrophages. It will be of considerable interest to identify the transcription factors responsible for activation of the SR-A gene by GM-CSF, as these factors are likely to be important in establishing the phenotypic properties of the M-CSF-independent population of macrophages.
FIG. 8 Mechanisms of transcriptional control of the SR-A gene by M-CSF and GM-CSF. Activation of the SR-A gene by M-CSF is mediated by a M-CSF-dependent enhancer that is recognized by AP-1 and cooperating Ets domain transcription factors. These transcription (more ...)