M-CSF Signals through the MAPK/ERK Pathway via Sp1 to Induce VEGF Production and Induces Angiogenesis In Vivo

BACKGROUND: M-CSF recruits mononuclear phagocytes which regulate processes such as angiogenesis and metastases in tumors. VEGF is a potent activator of angiogenesis as it promotes endothelial cell proliferation and new blood vessel formation. Previously, we reported that in vitro M-CSF induces the expression of biologically-active VEGF from human monocytes. METHODOLOGY AND RESULTS: In this study, we demonstrate the molecular mechanism of M-CSF-induced VEGF production. Using a construct containing the VEGF promoter linked to a luciferase reporter, we found that a mutation reducing HIF binding to the VEGF promoter had no significant effect on luciferase production induced by M-CSF stimulation. Further analysis revealed that M-CSF induced VEGF through the MAPK/ERK signaling pathway via the transcription factor, Sp1. Thus, inhibition of either ERK or Sp1 suppressed M-CSF-induced VEGF at the mRNA and protein level. M-CSF also induced the nuclear localization of Sp1, which was blocked by ERK inhibition. Finally, mutating the Sp1 binding sites within the VEGF promoter or inhibiting ERK decreased VEGF promoter activity in M-CSF-treated human monocytes. To evaluate the biological significance of M-CSF induced VEGF production, we used an in vivo angiogenesis model to illustrate the ability of M-CSF to recruit mononuclear phagocytes, increase VEGF levels, and enhance angiogenesis. Importantly, the addition of a neutralizing VEGF antibody abolished M-CSF-induced blood vessel formation. CONCLUSION: These data delineate an ERK- and Sp1-dependent mechanism of M-CSF induced VEGF production and demonstrate for the first time the ability of M-CSF to induce angiogenesis via VEGF in vivo

Src-family kinases play an essential role in differentiation signaling downstream of macrophage colony-stimulating factor receptors mediating persistent phosphorylation of phospholipase C-gamma2 and MAP kinases ERK1 and ERK2.

Macrophage colony-stimulating factor (M-CSF) has been found to be involved in multiple developmental processes, especially production of cells belonging to the mononuclear phagocyte system. The decision of myeloid progenitor cells to commit to differentiation depends on activation levels of the mitogen-activated protein kinases (MAPK), ERK1 and ERK2. Using the murine myeloid progenitor cell line FD-Fms, we show here that persistent activity of Src-family kinases (SFK) is necessary for FD-Fms cell differentiation to macrophages in response to M-CSF. Chemical inhibition of SFK blocked FD-Fms cell differentiation while it caused strong inhibition of the late phosphorylation of phospholipase C (PLC)-gamma2 and MAPK. The PLC inhibitor U73122, previously shown to block M-CSF-induced differentiation, strongly decreased long-term MAPK phosphorylation. Interestingly, inhibiting SFK with SU6656 or the MAPK kinases MEK with U0126 significantly impaired development of mononuclear phagocytes in cultures of mouse bone marrow cells stimulated with M-CSF. Collectively, results support a model in which SFK are required for sustained PLC activity and MAPK activation above threshold required for commitment of myeloid progenitors to macrophage differentiation.Leukemia advance online publication, 1 November 2007; doi:10.1038/sj.leu.2404986

Ptdlns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox)

The production of reactive oxygen species (ROS) by neutrophils has a vital role in defence against a range of infectious agents, and is driven by the assembly of a multi-protein complex containing a minimal core of five proteins: the two membrane-bound subunits of cytochrome b(558) (gp91(phox) and p22(phox)) and three soluble factors (GTP-Rac, p47(phox) and p67(phox) (refs 1, 2). This minimal complex can reconstitute ROS formation in vitro in the presence of non-physiological amphiphiles such as sos, p40(phox) has subsequently been discovered as a binding partner for p67(phox) (ref. 3), but its role in ROS formation is unclear, Phosphoinositide-3-OH kinases (PI(3)Ks) have been implicated in the intracellular signalling pathways coordinating ROS formation(4,5) but through an unknown mechanism. We show that the addition of p40(phox) to the minimal core complex allows a lipid product of PI(3)Ks, phosphatidylinositol 3-phosphate (PtdIns(3)P), to stimulate specifically the formation of ROS, This effect was mediated by binding of PtdIns(S)P to the PX domain(6) of p40(phox). These results offer new insights into the roles for PI(3)Ks and p40(phox) in ROS formation and define a cellular ligand for the orphan PX domain

The p110δ isoform of PI 3-kinase negatively controls RhoA and PTEN

Inactivation of PI 3-kinase (PI3K) signalling is critical for tumour suppression by PTEN. This is thought to be a unidirectional relationship in which PTEN degrades the lipids produced by PI3K, thus controlling cell proliferation, survival and migration. We now show that this relationship is in fact bidirectional, whereby PI3K reciprocally controls PTEN. We report that the p110δ PI3K negatively regulates PTEN, through a pathway involving inhibition of RhoA. Inactivation of p110δ in macrophages led to reduced Akt and Rac1 activation, but paradoxically to increased RhoA and PTEN activity. Partial inactivation of p190RhoGAP and a reduced binding of cytoplasmic RhoA to the cyclin-dependent kinase inhibitor p27 both contributed to the increased RhoA-GTP levels upon p110δ inactivation. Pharmacological inhibition of ROCK, a downstream effector kinase of RhoA, restored all signalling and functional defects of p110δ inactivation, including Akt phosphorylation, chemotaxis and proliferation. This work identifies the RhoA/ROCK pathway as a major target of p110δ-mediated PI3K signalling, and establishes for the first time that PI3K controls itself, via a feedback loop involving PTEN

PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions

Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110gamma in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110gamma(+/-) and LDLR(-/-)p110gamma(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110gamma(+/-) and p110gamma(-/-) mice. Lack of p110gamma in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110gamma(-/-) mice were smaller than in LDLR(-/-)p110gamma(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110gamma(-/-) mouse lesions. This proliferation defect was also observed in p110gamma(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110gamma(-/-) mice. Moreover, p110gamma deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110gamma. Nonetheless, p110gamma deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation

Ets-2 Repressor Factor Silences Extrasynaptic Utrophin by N-Box–mediated Repression in Skeletal Muscle

Utrophin is the autosomal homologue of dystrophin, the protein product of the Duchenne's muscular dystrophy (DMD) locus. Utrophin expression is temporally and spatially regulated being developmentally down-regulated perinatally and enriched at neuromuscular junctions (NMJs) in adult muscle. Synaptic localization of utrophin occurs in part by heregulin-mediated extracellular signal-regulated kinase (ERK)-phosphorylation, leading to binding of GABPα/β to the N-box/EBS and activation of the major utrophin promoter-A expressed in myofibers. However, molecular mechanisms contributing to concurrent extrasynaptic silencing that must occur to achieve NMJ localization are unknown. We demonstrate that the Ets-2 repressor factor (ERF) represses extrasynaptic utrophin-A in muscle. Gel shift and chromatin immunoprecipitation studies demonstrated physical association of ERF with the utrophin-A promoter N-box/EBS site. ERF overexpression repressed utrophin-A promoter activity; conversely, small interfering RNA-mediated ERF knockdown enhanced promoter activity as well as endogenous utrophin mRNA levels in cultured muscle cells in vitro. Laser-capture microscopy of tibialis anterior NMJ and extrasynaptic transcriptomes and gene transfer studies provide spatial and direct evidence, respectively, for ERF-mediated utrophin repression in vivo. Together, these studies suggest “repressing repressors” as a potential strategy for achieving utrophin up-regulation in DMD, and they provide a model for utrophin-A regulation in muscle