Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway

Oxidized LDL (oxLDL) is known to induce endothelial adhesion molecule and monocyte chemoattractant protein 1 expression and this is thought to be involved in monocyte recruitment into atherosclerotic lesions. oxLDL has also been found to induce macrophage proliferation. The purpose of the present study was to determine whether oxLDL might also have the ability to increase macrophage populations by inhibiting apoptosis. We found that oxLDL caused a dose-dependent inhibition of the apoptosis that occurs in cultured bone marrow-derived macrophages after macrophage colony-stimulating factor (M-CSF) withdrawal without inducing proliferation. Incubation of macrophages with either native LDL or acetylated LDL had no effect on apoptosis. The prosurvival effect of oxLDL was not inhibited by neutralizing antibodies to granulocyte-macrophage colony-stimulating factor, was maintained in mice homozygous for a mutation in the M-CSF gene, and was not due to other secreted cytokines or growth factors. oxLDL caused activation of the mitogen-activated protein kinases ERK1/2 (extracellular signal-regulated kinases 1 and 2) as well as protein kinase B (PKB), a target of phosphatidylinositol 3-kinase (PI 3-kinase). Furthermore, there was phosphorylation of two important prosurvival PKB targets, I-kappaBalpha(Ser-32) and Bad(Ser-136). The MEK inhibitors PD 98059 and U0126 blocked ERK1/2 activation but did not diminish survival. Conversely, the PI 3-kinase inhibitors LY 294002 and wortmannin blocked PKB activation, and the ability of oxidized LDL to promote macrophage survival.Taken together, these results indicate that oxLDL can directly activate a PI 3-kinase/PKB-dependent pathway that permits macrophage survival in the absence of growth factors.     

PMA activation of macrophages alters macrophage metabolism of aggregated LDL

Aggregation of LDL may contribute to its retention in atherosclerotic lesions. Previously, we showed that aggregated LDL induces and enters surface-connected compartments (SCCs) in human monocyte-derived macrophages by a process we have named patocytosis. Aggregated LDL was disaggregated and released from SCCs of macrophages when exposed to human lipoprotein-deficient serum. The serum factor that mediated aggregated LDL release and disaggregation was plasmin generated from plasminogen by macrophage urokinase plasminogen activator. We now show that activation of macrophages with PMA inhibits plasmin-mediated release of aggregated LDL from macrophages. With macrophage activation, plasminogen released about 60% less cholesterol and 63% less TCA-insoluble (125)I-aggregated LDL than when macrophages were not activated. Electron microscopy showed that PMA did not cause SCCs to close, which could have trapped aggregated LDL within the SCCs and limited protease access to aggregated LDL. Rather, PMA decreased macrophage generation of plasmin by 61%, and stimulated lysosomal degradation of aggregated LDL by more than 2-fold. Degradation was mediated by protein kinase C, shown by the finding that degradation was inhibited by the protein kinase C inhibitor G?6976. PMA-stimulated degradation of aggregated LDL was associated with a 3-fold increase in cholesterol esterification, consistent with hydrolysis and re-esterification of aggregated LDL-derived cholesteryl ester. In conclusion, macrophage activation with PMA causes more of the aggregated LDL that enters macrophage SCCs to be metabolized by lysosomes. This results in more cholesterol to be stored in macrophages and less aggregated LDL to be available for plasmin-mediated release from macrophage SCCs.     

Inhibition of AIF-1 expression by constitutive siRNA expression reduces macrophage migration, proliferation, and signal transduction initiated by atherogenic stimuli

Allograft inflammatory factor-1 (AIF-1) is a cytoplasmic, calcium-binding, inflammation-responsive scaffold protein. Several studies have reported increased AIF-1 expression in activated macrophages and have implicated AIF-1 as a marker of activated macrophages. However, the function of AIF-1 in macrophages and the mechanism whereby it participates in macrophage activation are unknown at this time. Immunohistochemical analysis colocalized AIF-1 expression with CD68-positive macrophages in atherosclerotic human coronary arteries. Subsequent experiments were designed to determine a role for AIF-1 in macrophage activation in response to atherogenic stimuli. Stimulation of human and murine macrophages with oxidized LDL significantly increased AIF-1 expression above basal levels. Stable transfection of AIF-1 small interfering RNA (siRNA) in macrophages reduced AIF-1 protein expression by 79% and reduced macrophage proliferation by 52% (P < 0.01). Inhibition of proliferation was not due to induction of apoptosis. Sequences that did not knock down AIF-1 expression had no effect on proliferation. AIF-1 siRNA expression reduced macrophage migration by 60% (P < 0.01). Both proliferation and migration of siRNA-expressing macrophages could be restored by adenoviral expression of AIF-1 (P < 0.001 and 0.005, respectively), suggesting a tight association between AIF-1 expression and macrophage activation. Phosphorylation of Akt, p44/42 MAPK, and p38 kinase were significantly reduced in siRNA macrophages challenged with oxidized LDL (P < 0.05). Phosphorylation of p38 kinase was significantly inhibited in siRNA macrophages stimulated with T lymphocyte conditioned medium (P < 0.05). These data indicate that AIF-1 mediates atherogenesis-initiated signaling and activation of macrophages. allograft inflammatory factor-1; cell activation; small interfering RNA     

Identification of a signal-transduction pathway shared by haematopoietic growth factors with diverse biological specificity.

The haematopoietic growth factors multi-colony-stimulating factor, granulocyte/macrophage colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2 specifically control the production and proliferation of distinct leucocyte series. Each growth factor acts on a unique surface receptor associated with an appropriate signal-transduction apparatus. In this report we identify a 68 kDa substrate which is phosphorylated after stimulation of different cell types with multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2. The 68 kDa substrate is also phosphorylated in each cell line stimulated with synthetic diacylglycerol, a direct activator of protein kinase C. Interestingly, granulocyte/macrophage colony-stimulating factor does not induce phosphorylation of the 68 kDa molecule. The 68 kDa molecule that is phosphorylated after stimulation with each ligand yielded similar peptide maps after chymotryptic digestion; furthermore, the substrate was always phosphorylated on threonine residues. Phosphorylation of the same residues in the 68 kDa substrate suggests that activation of protein kinase C is one common signal-transduction event associated with the action of multi-colony-stimulating factor, granulocyte colony-stimulating factor and interleukin 2.     

Effect of cytokines on thymic hematopoietic precursors

Summary We have previously shown that the interaction of thymocytes with thymic accessory cells (macrophages and/or interdigitating cells) is one of the factors required for thymocyte activation. Precursors of both thymic accessory cell and thymocytes are included in the CD4^- CD8^- Mac-1^- Ia^- subpopulation, and their respective maturation and/or activation may be modulated by granulocyte-macrophage colony-stimulating factor, interleukin 1 and interleukin 2. When CD4^- CD8^- thymic cells are activated with granulocyte-macrophage colony-stimulating factor plus interleukin 2, both macrophages and interdigitating-like cells are present, as shown by electron microscopy. When activated with interleukin 1 plus interleukin 2, the interdigitating-like cells is the only accessory cell present. In both culture conditions, large clusters are formed between interdigitating cells and lymphoid cells. These results have led us to propose two-step signals for thymocyte proliferation: first, the maturation of macrophages under granulocyte-macrophage colony-stimulating factor control and the production of interleukin 1, and secondly, the maturation of interdigitating cells under interleukin 1 control, their clustering with thymocytes which are then activated.Abbreviations CFU-S colony-forming units in the spleen - CSF colony-stimulating factor - DC dendritic cells - DN double negative cells (CD4^- CD8^-) - EC epithelial cells - GM-CFC granulocyte/macrophage colony-forming cells - GM-CSF granulocytemacrophage CSF - IDC interdigitating cell - IL-1 interleukin 1 - IL-2 interleukin 2 - MØ macrophage - P-TR phagocytic cell of the thymic reticulum     

The differential time-course of extracellular-regulated kinase activity correlates with the macrophage response toward proliferation or activation

Bone marrow-derived macrophages proliferate in response to specific growth factors, including macrophage colony-stimulating factor (M-CSF). When stimulated with activating factors, such as lipopolysaccharide (LPS), macrophages stop proliferating and produce proinflammatory cytokines. Although triggering opposed responses, both M-CSF and LPS induce the activation of extracellular-regulated kinases (ERKs) 1 and 2. However, the time-course of ERK activation is different; maximal activation by M-CSF and LPS occurred after 5 and 15 min of stimulation, respectively. Granulocyte/macrophage colony-stimulating factor, interleukin 3, and TPA, all of which induced macrophage proliferation, also induced ERK activity, which was maximal at 5 min poststimulation. The use of PD98059, which specifically blocks ERK 1 and 2 activation, demonstrated that ERK activity was necessary for macrophage proliferation in response to these factors. The treatment with phosphatidylcholine-specific phospholipase C (PC-PLC) inhibited macrophage proliferation, induced the expression of cytokines, and triggered a pattern of ERK activation equivalent to that induced by LPS. Moreover, PD98059 inhibited the expression of cytokines induced by LPS or PC-PLC, thus suggesting that ERK activity is also required for macrophage activation by these two agents. Activation of the JNK pathway did not discriminate between proliferative and activating stimuli. In conclusion, our results allow to correlate the differences in the time-course of ERK activity with the macrophagic response toward proliferation or activation.     

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL

Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²⁵I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an ∼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.     

A modification of apolipoprotein B accounts for most of the induction of macrophage growth by oxidized low density lipoprotein

It has recently been shown that macrophage proliferation occurs during the progression of atherosclerotic lesions and that oxidized low density lipoprotein (LDL) stimulates macrophage growth. Possible mechanisms for this include the interaction of oxidized LDL with integral plasma membrane proteins coupled to signaling pathways, the release of growth factors and autocrine activation of growth factor receptors, or the potentiation of mitogenic signal transduction by a component of oxidized LDL after internalization. The present study was undertaken to further elucidate the mechanisms involved in the growth-stimulating effect of oxidized LDL in macrophages. Only extensively oxidized LDL caused significant growth stimulation, whereas mildly oxidized LDL, native LDL, and acetyl LDL were ineffective. LDL that had been methylated before oxidation (to block lysine derivatization by oxidation products and thereby prevent the formation of a scavenger receptor ligand) did not promote growth, even though extensive lipid peroxidation had occurred. The growth stimulation could not be attributed to lysophosphatidylcholine (lyso-PC) because incubation of oxidized LDL with fatty acid-free bovine serum albumin resulted in a 97% decrease in lyso-PC content but only a 20% decrease in mitogenic activity. Similarly, treatment of acetyl LDL with phospholipase A2 converted more than 90% of the initial content of phosphatidylcholine (PC) to lyso-PC, but the phospholipase A2-treated acetyl LDL was nearly 10-fold less potent than oxidized LDL at stimulating growth. Platelet-activating factor receptor antagonists partly inhibited growth stimulation by oxidized LDL, but platelet-activating factor itself did not induce growth. Digestion of oxidized LDL with phospholipase A2 resulted in the hydrolysis of PC and oxidized PC but did not attenuate growth induction. Native LDL, treated with autoxidized arachidonic acid under conditions that caused extensive modification of lysine residues by lipid peroxidation products but did not result in oxidation of LDL lipids, was equal to oxidized LDL in potency at stimulating macrophage growth. Albumin modified by arachidonic acid peroxidation products also stimulated growth, demonstrating that LDL lipids are not essential for this effect. These findings suggest that oxidatively modified apolipoprotein B is the main growth-stimulating component of oxidized LDL, but that oxidized phospholipids may play a secondary role.     

GM-CSF mediates alveolar macrophage proliferation and type II cell hypertrophy in SP-D gene-targeted mice

Mice deficient in surfactant protein (SP) D develop increased surfactant pool sizes and dramatic changes in alveolar macrophages and type II cells. To test the hypothesis that granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates alveolar macrophage proliferation and activation and the type II cell hypertrophy seen in SP-D null mice, we bred SP-D and GM-CSF gene-targeted mice to obtain littermate double null, single null, and wild-type mice. Bronchoalveolar lavage levels of phospholipid, protein, SP-D, SP-A, and GM-CSF were measured from 1 to 4 mo. There was an approximately additive accumulation of phospholipid, total protein, and SP-A at each time point. Microscopy showed normal macrophage number and morphology in GM-CSF null mice, numerous giant foamy macrophages and hypertrophic type II cells in SP-D null mice, and large but not foamy macrophages and mostly normal type II cells in double null mice. These results suggest that the mechanisms underlying the alveolar surfactant accumulation in the SP-D-deficient and GM-CSF-deficient mice are different and that GM-CSF mediates some of the macrophage and type II cell changes seen in SP-D null mice.     

Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin-3 and granulocyte-macrophage colony-stimulating factor mediate rapid phosphorylation and activation of cytosolic c-raf.

Interleukin-3 (IL-3) and granulocyte-macrophage colony-stimulating factor induce the rapid phosphorylation of the c-raf protein in the growth factor-dependent FDC-P1 and DA-3 murine myeloid cell lines. Furthermore, immunoprecipitates of c-raf isolated from growth factor-stimulated cells demonstrate a marked increase in intrinsic protein kinase activity as measured in vitro. IL-3 and granulocyte-macrophage colony-stimulating factor induce phosphorylation of c-raf at both serine and tyrosine residues. Antiphosphotyrosine immunoprecipitates from IL-3-stimulated cells demonstrate the rapid and coordinate phosphorylation of both c-raf and a protein co-migrating with the 140-kDa putative IL-3 receptor component. Collectively, the findings of rapid and coordinate ligand-induced phosphorylation of a potential IL-3 growth factor receptor component and cytoplasmic c-raf with concomitant c-raf activation provide a cogent sequential molecular model for linking external growth stimuli to intracellular signal transduction events.     

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression.

Adenosine is produced during inflammation and modulates different functional activities in macrophages. In murine bone marrow-derived macrophages, adenosine inhibits M-CSF-dependent proliferation with an IC50 of 45 microM. Only specific agonists that can activate A2B adenosine receptors such as 5'-N-ethylcarboxamidoadenosine, but not those active on A1 (N6-(R)-phenylisopropyladenosine), A2A ([p-(2-carbonylethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine), or A3 (N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide) receptors, induce the generation of cAMP and modulate macrophage proliferation. This suggests that adenosine regulates macrophage proliferation by interacting with the A2B receptor and subsequently inducing the production of cAMP. In fact, both 8-Br-cAMP (IC50 85 microM) and forskolin (IC50 7 microM) inhibit macrophage proliferation. Moreover, the inhibition of adenylyl cyclase and protein kinase A blocks the inhibitory effect of adenosine and its analogues on macrophage proliferation. Adenosine causes an arrest of macrophages at the G1 phase of the cell cycle without altering the activation of the extracellular-regulated protein kinase pathway. The treatment of macrophages with adenosine induces the expression of p27kip-1, a G1 cyclin-dependent kinase inhibitor, in a protein kinase A-dependent way. Moreover, the involvement of p27kip-1 in the adenosine inhibition of macrophage proliferation was confirmed using macrophages from mice with a disrupted p27kip-1 gene. These results demonstrate that adenosine inhibits macrophage proliferation through a mechanism that involves binding to A2B adenosine receptor, the generation of cAMP, and the induction of p27kip-1 expression.     

Effect of macrophages on the translocation of protein kinase C in concanavalin A-stimulated lymphocytes

The concanavalin A (Con A)-induced proliferation of lymph node lymphocytes is dependent on the presence of macrophages. When lymphocytes are depleted of macrophages, Con A is no longer mitogenic. Either 12-0-tetradecanoylphorbol-13-acetate (TPA), interleukin 1 (IL1), or macrophages in combination with Con A can restore proliferation. To establish where the proliferation process is blocked in the absence of macrophages, an early step in the signalling pathway, the activation of protein kinase C, was examined. It was found that although Con A caused translocation of protein kinase C from the cytosol to the membrane of lymph node cells, when the lymph node cells were depleted of macrophages and exposed to Con A, this translocation of protein kinase C did not occur. Instead, protein kinase C activity decreased in the membrane fraction and increased in the cytosol. On the other hand, TPA caused translocation of protein kinase C (PKC) from the cytosol to the membrane regardless of the presence of macrophages. However, the macrophage product, IL1, alone or in combination with Con A did not cause translocation of protein kinase C. In a reconstitution experiment, in which lymph node cells were depleted of macrophages and then macrophages were added back, the addition of Con A again lead to translocation of protein kinase C from the cytosol to the membrane. This combination also restored cell proliferation. Therefore, the Con A induced PKC translocation in T lymphocytes is macrophage mediated. TPA overcomes the macrophage requirement by directly activating PKC, while IL1 appears to act at a different step in proliferation.     

Constitutive receptor-independent low density lipoprotein uptake and cholesterol accumulation by macrophages differentiated from human monocytes with macrophage-colony-stimulating factor (M-CSF)

Recently, we have shown that macrophage uptake of low density lipoprotein (LDL) and cholesterol accumulation can occur by nonreceptor mediated fluid-phase macropinocytosis when macrophages are differentiated from human monocytes in human serum and the macrophages are activated by stimulation of protein kinase C (Kruth, H. S., Jones, N. L., Huang, W., Zhao, B., Ishii, I., Chang, J., Combs, C. A., Malide, D., and Zhang, W. Y. (2005) J. Biol. Chem. 280, 2352-2360). Differentiation of human monocytes in human serum produces a distinct macrophage phenotype. In this study, we examined the effect on LDL uptake of an alternative macrophage differentiation phenotype. Differentiation of macrophages from human monocytes in fetal bovine serum with macrophage-colony-stimulating factor (M-CSF) produced a macrophage phenotype demonstrating constitutive fluid-phase uptake of native LDL leading to macrophage cholesterol accumulation. Fluid-phase endocytosis of LDL by M-CSF human macrophages showed non-saturable uptake of LDL that did not down-regulate over 48 h. LDL uptake was mediated by continuous actin-dependent macropinocytosis of LDL by these M-CSF-differentiated macrophages. M-CSF is a cytokine present within atherosclerotic lesions. Thus, macropinocytosis of LDL by macrophages differentiated from monocytes under the influence of M-CSF is a plausible mechanism to account for macrophage foam cell formation in atherosclerotic lesions. This mechanism of macrophage foam cell formation does not depend on LDL modification or macrophage receptors.     

Monocyte colony-stimulating factor enhances uptake and degradation of acetylated low density lipoproteins and cholesterol esterification in human monocyte-derived macrophages

We have investigated effects of monocyte colony-stimulating factor (M-CSF) on the uptake of acetylated low density lipoproteins (acetyl-LDL) and the activity of cholesterol esterification in human monocyte-derived macrophage. The cells were cultured with M-CSF for 10 days and then incubated with acetyl-LDL for 24 h. M-CSF (128 ng/ml) enhanced the uptake and degradation of 10 micrograms/ml of 125I-acetyl LDL 7.5-fold (n = 6) and the effect of M-CSF was dose-dependent at the concentrations of 0.5-32 ng/ml. The binding experiments at 4 degrees C demonstrated that the number of acetyl-LDL receptor was increased by the addition of M-CSF. Supporting this, ligand blotting analysis revealed a significant increase in a receptor protein for acetyl-LDL (240 kDa). Binding of LDL was also enhanced by M-CSF but less significantly than that of acetyl-LDL. Cellular cholesterol esterification in the presence of 10 micrograms/ml acetyl-LDL was enhanced 24.1-fold (n = 13) by 128 ng/ml M-CSF. It was evident that M-CSF enhanced cholesterol esterification to a greater extent than the cellular uptake of acetyl-LDL (24.1- versus 7.5-fold). Cholesterol esterification was also enhanced by the addition of granulocyte-macrophage colony-stimulating factor and interleukin 1. We conclude that M-CSF enhances the uptake of both acetyl-LDL and LDL by increasing their receptor number, and further enhances the process of cholesterol esterification, resulting in a remarkable increase in cholesterol esterification in macrophages. These findings strongly suggest the significant involvement of cytokines such as M-CSF in cholesterol metabolism of macrophages.     

Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) ameliorate atherosclerotic diseases. Macrophages play an important role in the development and subsequent stability of atherosclerotic plaques. We reported previously that oxidized low density lipoprotein (Ox-LDL) induced macrophage proliferation through the secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) and the consequent activation of p38 MAPK. The present study was designed to elucidate the mechanism of the inhibitory effect of statins on macrophage proliferation. Mouse peritoneal macrophages were used in our study. Cerivastatin and simvastatin each inhibited Ox-LDL-induced [(3)H]thymidine incorporation into macrophages. Statins did not inhibit Ox-LDL-induced GM-CSF production, but inhibited GM-CSF-induced p38 MAPK activation. Farnesyl transferase inhibitor and geranylgeranyl transferase inhibitor inhibited GM-CSF-induced macrophage proliferation, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the effect of statins. GM-CSF-induced p38 MAPK phosphorylation was also inhibited by farnesyl transferase inhibitor or geranylgeranyl transferase inhibitor, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the suppression of GM-CSF-induced p38 MAPK phosphorylation by statins. Furthermore, we found that statin significantly inhibited the membrane translocation of the small G protein family members Ras and Rho. GM-CSF-induced p38 MAPK activation and macrophage proliferation was partially inhibited by overexpression of dominant negative Ras and completely by that of RhoA. In conclusion, statins inhibited GM-CSF-induced Ras- or RhoA-p38 MAPK signal cascades, thereby suppressing Ox-LDL-induced macrophage proliferation. The significant inhibition of macrophage proliferation by statins may also explain, at least in part, their anti-atherogenic action.     

c-Fms tyrosine 559 is a major mediator of M-CSF-induced proliferation of primary macrophages

The molecular mechanisms by which binding of monocyte/macrophage colony-stimulating factor to its receptor c-Fms promotes replication in primary macrophages are incompletely understood, as all previous studies involved overexpression of receptor mutants in transformed cells not endogenously expressing the receptor. To address this issue we retrovirally expressed, in bone marrow-derived macrophages, a chimeric receptor containing a range of tyrosine to phenylalanine mutations in the c-Fms cytoplasmic tail. We measured incorporation of bromodeoxyuridine as a marker of proliferation and phosphorylation of ERKs, Akt, and the receptor itself. Our data indicate that tyrosine 559 is the major mediator of receptor activation and cell death, intracellular signaling, and cell proliferation and that the tyrosine residues at positions 697 and 807 play lesser roles in these events. Importantly, we find that activation of the ERK and Akt pathways is necessary but not sufficient for induction of macrophage proliferation. Using specific small molecule inhibitors we find that a combination of the Src family kinase, phosphatidylinositol 3-kinase/Akt, phospholipase C, and ERK pathways mediates macrophage proliferation in response to M-CSF.