Inhibition of the signaling pathways for macrophage proliferation by cyclic AMP. Lack of effect on early responses to colony stimulating factor-1.

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in quiescent murine bone marrow-derived macrophages (BMM). CSF-1 action has been shown to involve activation of the CSF-1 receptor kinase. The protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (PMA), is itself weakly mitogenic and synergises with CSF-1 for stimulation of BMM DNA synthesis suggesting a possible role for protein kinase C in the stimulation of BMM DNA synthesis. In this report we show that several agents which raise intracellular cAMP (8-bromoadenosine 3':5'-cyclic monophosphate, 3-isobutyl-1-methylxanthine, cholera toxin, and prostaglandin E2) reversibly inhibit DNA synthesis in BMM induced by CSF-1, granulocyte macrophage-colony stimulating factor, interleukin-3, and PMA. The suppressive action of cAMP elevation on the proliferative response to CSF-1 can be manifested even late in the G1 phase of the cell cycle. Several CSF-1-stimulated earlier responses, viz. protein synthesis, Na+/H+ exchange, Na+,K(+)-ATPase and c-myc-mRNA expression, were not inhibited thus showing a striking difference from some other cellular systems involving growth factor-mediated responses. c-fos-mRNA levels were raised and stabilized by the cAMP-elevating agents, and this modulation was not altered by CSF-1. Thus, the signaling pathways in the macrophages involving tyrosine kinase and protein kinase C activation are associated with increased proliferation while those involving elevation of cAMP (and presumably activation of cAMP-dependent protein kinases) appear to have an inhibitory effect.     

Inhibition of colony-stimulating factor-stimulated macrophage proliferation by tumor necrosis factor-alpha, IFN-gamma, and lipopolysaccharide is not due to a general loss of responsiveness to growth factor

The role of stimulatory factors, such as the CSF, in the regulation of hemopoiesis has been extensively documented. Less is known of the negative regulators of hemopoiesis. In this report, we show that the macrophage activating agents, TNF-alpha, IFN-gamma, and LPS, are all potent inhibitors of CSF-1-stimulated murine bone marrow-derived macrophage (BMM) DNA synthesis and increase in cell numbers. The inhibitory effects of TNF-alpha and IFN-gamma do not appear to be due to endotoxin contamination in the recombinant cytokine preparations. The inhibition of proliferation is reversible and is not due to a general loss of growth factor responsiveness, inasmuch as the three agents do not inhibit CSF-1-stimulated BMM survival, protein synthesis, or fluid phase pinocytosis. Because TNF-alpha and LPS are known to rapidly and potently down-modulate CSF-1 receptor levels in BMM, the results also suggest that low levels of receptor occupancy are sufficient for biological responses to CSF-1. The inhibitory effects of TNF-alpha, IFN-gamma, or LPS were also seen when granulocyte-macrophage-CSF or IL-3 was used to stimulate BMM DNA synthesis. The results suggest that TNF-alpha, IFN-gamma, and LPS appear to be inhibiting CSF-stimulated proliferation by acting at a post-receptor level, possibly by regulation of some critical event(s) in the mitogenic signaling pathway.     

Activation and proliferation signals in murine macrophages: relationships among c-fos and c-myc expression, phosphoinositide hydrolysis, superoxide formation, and DNA synthesis

Murine bone marrow-derived macrophages (BMM) undergo DNA synthesis in response to growth factors such as colony stimulating factor-1 (CSF-1) and granulocyte-macrophage CSF (GM-CSF). These macrophages can also be "activated," but without subsequent DNA synthesis, by a number of other agents, including lipopolysaccharide (LPS), concanavalin A, zymosan, formyl-methionyl-leucyl-phenylalanine (FMLP), and the Ca2+ ionophore, A23187. When BMM are treated with a range of stimuli, there is some, although not perfect, correlation between transient elevations in both c-myc mRNA and c-fos mRNA levels and increases in DNA synthesis. However, enhanced DNA synthesis and oncogene expression are readily dissociated from rises in inositol phosphates and, by implication, phospholipase C-mediated hydrolysis of phosphatidyl inositol 4,5-bisphosphate. Superoxide formation in BMM can also be dissociated from the other responses and does not necessarily depend on protein kinase C activation.     

Inhibition of S-phase progression in macrophages is linked to G1/S-phase suppression of DNA synthesis genes.

Some of the important controlling events regulating eukaryotic S-phase progression are considered to occur late in the G1 stage of the cell cycle. We show here that stimulation of DNA synthesis in bone marrow-derived macrophages (BMM) by macrophage CSF-1 is preceded by G1 expression of three genes which encode proteins associated with the DNA synthesis machinery--the M1 and M2 subunits of ribonucleotide reductase and proliferating cell nuclear Ag (PCNA). Increased expression for these genes correlated well with the mitogenic response and sustained expression required de novo RNA and protein synthesis and also the presence of CSF-1 for at least most of G1. Inhibitors of BMM proliferation (LPS, TNF-alpha, IFN-gamma, and cAMP elevating agents) suppressed CSF-1-induced expression of M1, M2, and PCNA mRNA measured at 22 h. This suppression occurred even when added up to 12 h after the CSF-1, a period coinciding with the G1/S-phase boundary. The delayed kinetics of this effect parallels the ability of these agents to maximally inhibit CSF-1-induced BMM DNA synthesis when added at similar times. Decreased expression of M1, M2, and PCNA was not merely a consequence of DNA synthesis inhibition because the S-phase inhibitor, hydroxyurea, did not suppress CSF-1-induced gene expression. These results suggest that inhibition of DNA synthesis by antiproliferative agents involves inhibition of expression of several genes associated with the DNA synthesis machinery.     

Roles of the mitogen-activated protein kinase family in macrophage responses to colony stimulating factor-1 addition and withdrawal.

Colony stimulating factor-1 (CSF-1) (or macrophage CSF) is involved in the survival, proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Because the mitogen-activated protein kinase family members extracellular signal-regulated kinases (ERKs), p38, and c-Jun N-terminal kinase are widely implicated in such cellular functions, we measured their activity in growing and growth-arrested cultures of bone marrow-derived macrophages (BMM), as well as their stimulation by saturating concentrations of CSF-1. ERK activity was approximately 2-fold higher in cycling BMM compared with growth-arrested BMM; in addition, CSF-1-stimulated BMM DNA synthesis was partially inhibited by PD98059, a specific inhibitor of MEK activation, suggesting a role for a mitogen-activated protein-ERK kinase (MEK)/ERK pathway in the control of DNA synthesis but surprisingly not in the control of cyclin D1 mRNA or c-myc mRNA expression. The suppression of BMM apoptosis by CSF-1, i.e. enhanced survival, was not reversed by PD98059, suggesting that a MEK/ERK pathway is not involved in this process. Using a quantitative kinase assay, it was found that CSF-1 gave a slight increase in BMM p38 activity, supporting prior data that CSF-1 is a relatively weak stimulator of inflammatory cytokine production in monocytes/macrophages. Relatively high concentrations of the p38 inhibitor, SKB202190, suppressed CSF-1-stimulated BMM DNA synthesis. No evidence could be obtained for the involvement of p38 activity in BMM apoptosis following CSF-1 withdrawal. We were not able to show that CSF-1 enhanced BMM JNK-1 activity to a significant extent; again, no role could be found for JNK-1 activity in the BMM apoptosis occurring after CSF-1 removal.     

Colony stimulating factor-1 stimulates diacylglycerol generation in murine bone marrow-derived macrophages, but not in resident peritoneal macrophages

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in murine bone marrow-derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol-4,5-bisphosphate hydrolysis is not associated with CSF-1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF-1 transiently elevated both [3H]myristoyl- and [3H]arachidonyl-diacylglycerol (DAG) in BMM in a dose-dependent fashion. CSF-1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF-1 in macrophages. Other mitogenic agents, 12-0-tetradecanoyl phorbol 13-acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl- and [3H]arachidonyl-DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF-alpha increased only [3H]arachidonyl-DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.     

Na+/H+ exchange involvement in colony-stimulating factor-1-stimulated macrophage proliferation. Evidence for a requirement during late G1 of the cell cycle but not for early growth factor responses

Na+/H+ exchange activation by growth factors is proposed to be an important early signal for mitogenesis; however, little is known of its duration and requirement during later stages of the cell cycle. Macrophage-specific colony factor (CSF-1) rapidly activates murine bone marrow-derived macrophage Na+/H+ exchange, resulting in stimulation of Na+,K(+)-ATPase activity. The response to CSF-1 is maintained for at least 24 h. Inhibition of Na+/H+ exchange with 5-N,N-dimethylamiloride prevents CSF-1-stimulated DNA synthesis and cell growth. This is unlikely to be due to cytoplasmic acidosis, but more likely reflects a requirement for Na+/H+ exchange-mediated Na+ influx. DMA addition even up to 8 h after the growth factors suppresses S-phase progression. Na+/H+ exchange appears not to be involved in the induction of other early growth factor responses (c-fos and c-myc mRNA induction and general RNA and protein synthesis). We propose that growth factor-stimulated Na+/H+ exchange late in G1 of the cell cycle is required for S-phase progression but not for certain early growth factor responses.     

Colony-stimulating factor 1-induced Na+ influx into human monocytes involves activation of a pertussis toxin-sensitive GTP-binding protein

Colony-stimulating factor 1 (CSF-1) regulates the survival, growth, and differentiation of monocytes through binding to a single class of high affinity receptors. The present studies demonstrate that the interaction of CSF-1 with monocyte membranes is associated with a 2.4-fold increase in specific binding of the GTP analogue, GTP gamma S. Scatchard analysis of the GTP gamma S binding data indicated that CSF-1 stimulates GTP binding by increasing the affinity, rather than the number, of available sites. This stimulation of GTP binding by CSF-1 was also associated with an increase in GTPase activity. Furthermore, the CSF-1-induced stimulation of GTPase activity was sensitive to pertussis toxin. We also demonstrate that CSF-1 stimulates Na+ influx into monocytes by an amiloride-sensitive mechanism, presumably the Na+/H+ antiport. This CSF-1-stimulated influx of Na+ was further associated with an increase in Na+,K+-ATPase activity. Moreover, this stimulation of Na+ influx and Na+,K+-ATPase activity by CSF-1 was sensitive to pertussis toxin. Finally, we demonstrate that CSF-1-induced proliferation is also a pertussis toxin-sensitive event. The present findings thus suggest: 1) that the CSF-1 receptor is linked to a pertussis toxin-sensitive G protein; and 2) that a pertussis toxin-sensitive G protein is involved in the induction of Na+ influx by CSF-1.     

Suppression of growth factor-induced CYL1 cyclin gene expression by antiproliferative agents.

A recently identified novel mammalian cyclin (CYL1), induced by growth factors and apparently functional during the G1 phase of the cell cycle, is of potential significance, given that cell division is primarily controlled in G1. We have measured CYL1 gene expression in murine bone marrow-derived macrophages (BMM), a normal cell type dependent upon colony-stimulating factors (CSFs) for survival and proliferation. The induction of CYL1 mRNA levels correlated strongly with stimulation of DNA synthesis, since elevated CYL1 mRNA levels occurred in response to the mitogenic stimuli, CSF-1, and granulocyte/macrophage CSF, but not to nonmitogenic macrophage-activating agents. BMM are subject to cell cycle arrest by numerous agents, including tumor necrosis factor alpha, interferon gamma, bacterial lipopolysaccharide, and agents that increase cAMP. These antiproliferative agents suppressed CSF-1-stimulated CYL1 gene expression, even when added late in G1. This pattern of CYL1 gene expression was remarkably consistent with the ability of these agents to inhibit progression into S phase. The mechanisms of negative growth regulation are largely unknown, and given the likely importance of G1 cyclins in the control of cell division, we propose that antiproliferative agents may exert their effects by suppressing G1 cyclin gene expression.     

The regulation of macrophage protein turnover by a colony stimulating factor (CSF-1)

CSF-1 is a hemopoietic growth factor that specifically regulates the survival, proliferation, and differentiation of mononuclear phagocytic cells. A homogeneous population of mononuclear phagocytes, bone marrow derived macrophages (BMM), were used to study the regulation of protein turnover by CSF-1. Removal of CSF-1 (approximately 0.4 nM) from exponentially growing BMM cultured in 15% fetal calf serum containing medium decreases the rate of DNA synthesis by more than 100-fold. Addition of CSF-1 to these cells causes them to resume DNA synthesis within 12 h. More immediate effects of CSF-1 were observed on BMM protein metabolism. BMM cultured for 24 h in the absence of CSF-1 reduce their protein synthetic rate by 50-60%. The protein synthetic rate commences to decrease at 2-3 h after CSF-1 removal. Readdition of CSF-1 to BMM previously incubated in its absence causes a return to the protein synthetic rate of exponentially growing cells within 2 h. In the presence of CSF-1, BMM synthesize protein at a rate of approximately 8.7%/h and degrade it at a rate of approximately 0.9%/h. Removal of CSF-1 results in a decrease in the protein synthetic rate to approximately 3.4%/h and an increase in the rate of protein degradation to approximately 3.4%/h. The rate of protein synthesis by BMM increases linearly with CSF-1 concentration over the range of concentrations stimulating both survival and proliferation, while the rate of protein degradation decreases exponentially over the range of concentrations stimulating survival without proliferation. Therefore, it appears that the stimulation of the rate of protein synthesis and inhibition of the rate of protein degradation are two distinct effects of CSF-1, both part of the pleiotropic response to this growth factor. The inhibition of the rate of protein degradation by CSF-1 may be most significant for its survival inducing effect.     

cAMP inhibits CSF-1-stimulated tyrosine phosphorylation but augments CSF-1R-mediated macrophage differentiation and ERK activation

Macrophage colony stimulating factor (M-CSF) or CSF-1 controls the development of the macrophage lineage through its receptor tyrosine kinase, c-Fms. cAMP has been shown to influence proliferation and differentiation in many cell types, including macrophages. In addition, modulation of cellular ERK activity often occurs when cAMP levels are raised. We have shown previously that agents that increase cellular cAMP inhibited CSF-1-dependent proliferation in murine bone marrow-derived macrophages (BMM) which was associated with an enhanced extracellular signal-regulated kinase (ERK) activity. We report here that increasing cAMP levels, by addition of either 8-bromo cAMP (8BrcAMP) or prostaglandin E(1) (PGE1), can induce macrophage differentiation in M1 myeloid cells engineered to express the CSF-1 receptor (M1/WT cells) and can potentiate CSF-1-induced differentiation in the same cells. The enhanced CSF-1-dependent differentiation induced by raising cAMP levels correlated with enhanced ERK activity. Thus, elevated cAMP can promote either CSF-1-induced differentiation or inhibit CSF-1-induced proliferation depending on the cellular context. The mitogen-activated protein kinase/extracellular signal-related protein kinase kinase (MEK) inhibitor, PD98059, inhibited both the cAMP- and the CSF-1R-dependent macrophage differentiation of M1/WT cells suggesting that ERK activity might be important for differentiation in the M1/WT cells. Surprisingly, addition of 8BrcAMP or PGE1 to either CSF-1-treated M1/WT or BMM cells suppressed the CSF-1R-dependent tyrosine phosphorylation of cellular substrates, including that of the CSF-1R itself. It appears that there are at least two CSF-1-dependent pathway(s), one MEK/ERK dependent pathway and another controlling the bulk of the tyrosine phosphorylation, and that cAMP can modulate signalling through both of these pathways.     

Activation and proliferation signals in murine macrophages: stimulation of glucose uptake by hemopoietic growth factors and other agents

Purified colony-stimulating factor (CSF-1) (or macrophage colony stimulating factor [M-CSF]) stimulated the glucose uptake of murine bone marrow-derived macrophages (BMM) and resident peritoneal macrophages (RPM) as measured by 3H-2-deoxyglucose (2-DOG) uptake. Similar concentrations of CSF-1 stimulated the 2-DOG uptake and DNA synthesis in BMM. Other purified hemopoietic growth factors, granulocyte-macrophage CSF (GM-CSF) and interleukin-3 (IL-3) (or multi-CSF), and the tumor promoter, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), even though differing in their mitogenic capabilities on BMM, were also stimulators of 2-DOG uptake in BMM and RPM. The nonmitogenic agents, lipopolysaccharide (LPS) and concanavalin A (Con A), were also active. The inhibition by cytochalasin B and by high concentrations of D-glucose suggest that the basal and stimulated 2-DOG uptake occurred via a carrier-facilitated D-glucose transport system. The responses of the two macrophage populations to the hemopoietic growth factors and to the other agents were quite similar, suggesting that events that are important for the induction of DNA synthesis are not tightly coupled to the earlier rise in glucose uptake. For the BMM, the ability of a particular agent to stimulate glucose uptake did not parallel its ability to promote cell survival. However, stimulation of glucose uptake could still be a necessary but insufficient early macrophage response for cell survival and subsequent DNA synthesis.     

Long-term regulation of Na+, K+-ATPase pump in human lymphocytes: the role of JAK/STAT- and MAPK-signaling pathways

In interleukin-2 (IL-2)-induced human blood lymphocytes, the Na+/K+ pump function (assessed by ouabain-sensitive Rb+ influx), the abundance of Na+, K+-ATPase alpha1-subunit (determined by Western blotting) and the alpha1- and beta1-subunits mRNA of Na+, K+-ATPase (RT-PCR), as well as the phosphorylation of STAT5 and STAT3 family proteins and ERK1/2 kinase have been examined. A 3.5-4.0-fold increase in the expression of alpha1- and beta1-subunits mRNA of Na+, K+-ATPase was found at 24 h of IL-2 stimulation. The inhibitors of JAK3 kinase (B-42, WHI-P431) was shown to decrease both the phosphorylation of STATs and the rise in the oubain-sensitive rubidium influx as well as the increased abundance of Na+, K+-ATPase alpha1-subunit. The inhibition of the protein kinases ERK1/2 by PD98059 (20 microM) suppressed the alpha1-subunit accumulation. All the kinase inhibitors tested did not alter the intracellular content ofmonovalent cations in resting and IL-2-stimulated lymphocytes. It is concluded that MAPK and JAK/STAT signaling pathways mediate the IL-2-dependent regulation of the Na+, K+-ATPase expression during the lymphocyte transition from resting stage to proliferation.     

Synergistic effect of tumor necrosis factor-alpha and interferon-gamma on collagen synthesis of human skin fibroblasts in vitro

The effect of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on collagen metabolism by human diploid fibroblasts in confluent monolayer culture was examined. Recombinant TNF alpha reduced collagen mRNA levels 2-fold and stimulated collagenase mRNA levels 5-fold, while recombinant IFN gamma affected only collagen mRNA levels. The combination of TNF alpha (10 ng/ml) and IFN gamma (100 ng/ml) resulted in a much stronger (about 30-fold) reduction of collagen mRNA levels indicating that the two cytokines act synergistically. In contrast no such synergism was observed with respect to collagenase mRNA levels. The effect of TNF alpha and IFN gamma on collagen metabolism reported here indicates a complex interaction of different cytokines in the control of tissue remodeling that occurs during inflammation, repair, or atrophy.     

Antisense inhibition of c-myc expression reveals common and distinct mechanisms of growth inhibition by TGF beta and TNF alpha

Downregulation of the c-myc gene in HL-60 cells is associated with growth inhibition and induction of differentiation. Previous studies have reported that the growth inhibitors TGF beta and TNF alpha downregulate c-myc mRNA levels, suggesting the possibility that these agents may exert some of their phenotypic effects via c-myc downregulation. Our study demonstrates that although both growth inhibitors produce a similar decrease in c-myc protein synthesis, TNF alpha produces a greater growth inhibition and differentiation induction in HL-60 cells. Combined addition of anti-myc oligomer with either growth inhibitor produces no additive effect. In fact, 4 microM anti-myc oligomer produces the same growth and differentiation effects as does 10 ng/ml TGF beta 1. We conclude that downregulation of c-myc expression represents a common mechanism of growth inhibition by TGF beta and TNF alpha, but that TNF alpha possesses an additional effect that is independent of c-myc expression.