Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1.
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The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation. (+info)
Increase of hematopoietic responses by triple or single helical conformer of an antitumor (1-->3)-beta-D-glucan preparation, Sonifilan, in cyclophosphamide-induced leukopenic mice.
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It has been suggested that the immunopharmacological activity of soluble (1-->3)-beta-D-glucan depends on its conformation in mice. In this study, we examined the relationship between the conformation of Sonifilan (SPG) and hematopietic responses in cyclophosphamide (Cy)-induced leukopenic mice. SPG, a high molecular weight (1-->3)-beta-D-glucan, has a triple helical conformation in water, and it was changed by treatment with aqueous sodium hydroxide to the single helical conformer (SPG-OH). The effects of SPG or SPG-OH on hematopoietic responses in cyclophosphamide induced leukopenic mice were investigated by monitoring i) gene expression of cytokines by RT-PCR, ii) protein synthesis of interleukin 6 (IL-6) by ELISA and iii) colony formation of bone marrow cells (BMC). The mice administered Cy and SPG or SPG-OH expressed and produced higher levels of IL-6 mRNA and protein than the mice administered only Cy. Gene expression of NK1.1 was also induced by Cy/SPG (or SPG-OH) treatment. Induced gene expression of stem cell factor (SCF) and macrophage-colony stimulating factor (M-CSF) by SPG/SPG-OH were also found in in vitro culture of BMC from Cy treated mice. These results strongly suggested that conformation of the glucans, single and triple helix, are independent of the hematopietic response. (+info)
Enhanced production of tumor necrosis factor-alpha and interleukin-6 due to prolonged response to lipopolysaccharide in human macrophages infected in vitro with human immunodeficiency virus type 1.
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Elevated levels of circulating tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 have been detected in human immunodeficiency virus (HIV) type 1 infection. The overproduction of these cytokines could contribute to AIDS pathogenesis. Thus, the expression of TNF-alpha and IL-6 in human macrophages infected with HIV-1 was investigated. HIV-1 infection, per se, did not induce any TNF-alpha or IL-6 production or cytokine-specific mRNA expression. In contrast, HIV-1 primed macrophages to a prolonged TNF-alpha and IL-6 response to lipopolysaccharide (LPS) stimulation with respect to uninfected cells. Time-course analysis and flow cytometry demonstrated that cytokine production stopped at 6 h in uninfected macrophages but continued up to 24 h in HIV-1-infected cells. RNA studies suggested that HIV-1 interfered with late steps of cytokine synthesis. No modulation of membrane CD14 was found to account for the enhanced response to LPS. Finally, the effect of HIV-1 on cytokine response could not be abolished by the antiviral compound U75875. (+info)
The Ets2 transcription factor inhibits apoptosis induced by colony-stimulating factor 1 deprivation of macrophages through a Bcl-xL-dependent mechanism.
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Bcl-xL, a member of the Bcl-2 family, inhibits apoptosis, and its expression is regulated at the transcriptional level, yet nothing is known about the transcription factors specifically activating this promoter. The bcl-x promoter contains potential Ets binding sites, and we show that the transcription factor, Ets2, first identified by its sequence identity to v-ets of the E26 retrovirus, can transactivate the bcl-x promoter. Transient expression of Ets2 results in the upregulation of Bcl-xL but not of Bcl-xS, an alternatively spliced gene product which induces apoptosis. Ets2 is ubiquitously expressed at low levels in a variety of cell types and tissues but is specifically induced to abundant levels during macrophage differentiation. Since Bcl-xL is also upregulated during macrophage differentiation, we asked whether the bcl-x could be a direct downstream target gene of Ets2 in macrophages. BAC1.2F5 macrophages, which are dependent on macrophage colony-stimulating factor 1 (CSF-1) for their growth and survival, were used in these studies. We show that CSF-1 stimulation of BAC1.2F5 macrophages results in the upregulation of expression of ets2 and bcl-xL with similar kinetics of induction. In the absence of CSF-1, these macrophages undergo cell death by apoptosis, whereas constitutive expression of Ets2 rescues these cells from cell death, and bcl-xL is upregulated. These results strongly suggest a novel role of Ets2 in affecting apoptosis through its regulation of Bcl-xL transcription. (+info)
Lipopolysaccharide can block the potential of monocytes to differentiate into dendritic cells.
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We examined whether priming monocytes (MO) with lipopolysaccharide (LPS) influenced their further differentiation into either macrophages (Mphi) or dendritic cells (DC). LPS-primed MO differentiated into Mphi when cultured further with Mphi colony-stimulating factor (M-CSF) but, if cultured then with granulocyte/Mphi (GM)-CSF and IL-4 (interleukin-4), only about 30% of the cells differentiated into CD1a+ CD14- DC and half became CD1a- CD14+ Mphi. Cytokines present during LPS priming could affect subsequent MO differentiation. Relative to priming with LPS alone, adding M-CSF to LPS did not modify differentiation of MO to Mphi in further culture with M-CSF, nor did it change the way of differentiation of MO into DC was altered if culture was later switched to GM-CSF/IL-4. Using GM-CSF/IL-4 plus LPS upon priming did not modify differentiation of MO to Mphi in further culture with M-CSF, as compared to priming with GM-CSF/IL-4 alone, but it counteracted the effect of LPS on the differentiation of MO to DC in further culture with GM-CSF/IL-4: about 75% of cells then became DC. Alternatively, despite activation by LPS, mature M-CSF-induced Mphi preserved the potential to differentiate into DC on subsequent culture with GM-CSF/IL-4. Thus, LPS, a bacterial product known to sustain maturation of MO/Mphi as well as of DC, may block the differentiation of MO into DC, except if signal triggering DC differentiation is delivered concomitantly, and modulate in this manner the induction of adaptive immune responses to infection. (+info)
Endotoxin-mediated S-nitrosylation of p50 alters NF-kappa B-dependent gene transcription in ANA-1 murine macrophages.
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Nitric oxide (NO) regulates cellular function, in part, by S-nitrosylating active site thiol groups of proteins. Ex vivo S-nitrosylation of NF-kappa B p50 significantly decreases its capacity for DNA binding. To determine the cellular relevance of this observation, we utilized the ANA-l murine macrophage model of endotoxin (LPS)-mediated NO synthesis. In selected instances, the NO synthase inhibitor, L-arginine methyl ester (L-NAME; 100 microM), or the NO donor, S-nitroso-N-acetylcysteine (SNAC; 100 microM), was added. In contrast to that of LPS cells, nuclear extracts from LPS + L-NAME cells demonstrated increased NF-kappa B DNA binding on gel shift analysis. Addition of SNAC to LPS + L-NAME cells restored binding to a level equivalent to that of LPS cells. Spectrophotometric analysis of NF-kappa B p50 immunoprecipitates demonstrated S-NO bonds exclusively in LPS cells; these p50 protein isolates retained the same DNA binding characteristics as that of the nuclear extracts. Transfection assays utilizing NF-kappa B-dependent promoter-reporter constructs demonstrated increased activity in LPS + L-NAME cells compared with LPS cells; nuclear run-on assays confirmed increased transcription of the corresponding genes. These results suggest that LPS-mediated NO synthesis is associated with S-nitrosylation of NF-kappa B p50 and inhibition of NF-kappa B-dependent DNA binding, promoter activity, and gene transcription. We conclude that NO can regulate gene transcription by S-nitrosylation of NF-kappa B. (+info)
Mutation of tyrosine 960 within the insulin receptor juxtamembrane domain impairs glucose transport but does not inhibit ligand-mediated phosphorylation of insulin receptor substrate-2 in 3T3-L1 adipocytes.
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CSF-1 is equipotent to insulin in its ability to stimulate 2-[3H]deoxyglucose uptake in 3T3-L1 adipocytes expressing the colony stimulating factor-1 receptor/insulin receptor chimera (CSF1R/IR). However, CSF-1-stimulated glucose uptake and glycogen synthesis is reduced by 50% in comparison to insulin in 3T3-L1 cells expressing a CSF1R/IR mutated at Tyr960 (CSF1R/IRA960). CSF-1-treated adipocytes expressing the CSF1R/IRA960 were impaired in their ability to phosphorylate insulin receptor substrate 1 (IRS-1) but not in their ability to phosphorylate IRS-2. Immunoprecipitation of IRS proteins followed by Western blotting revealed that the intact CSF1R/IR co-precipitates with IRS-2 from CSF-1-treated cells. In contrast, the CSF1R/IRA960 co-precipitates poorly with IRS-2. These observations suggest that Tyr960 is important for interaction of the insulin receptor cytoplasmic domain with IRS-2, but it is not essential to the ability of the insulin receptor tyrosine kinase to use IRS-2 as a substrate. These observations also suggest that in 3T3-L1 adipocytes, tyrosine phosphorylation of IRS-2 by the insulin receptor tyrosine kinase is not sufficient for maximal stimulation of receptor-regulated glucose transport or glycogen synthesis. (+info)
Protein phosphatase 2A is expressed in response to colony-stimulating factor 1 in macrophages and is required for cell cycle progression independently of extracellular signal-regulated protein kinase activity.
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Colony-stimulating factor 1 (CSF-1) is required for the development of monocytes/macrophages from progenitor cells and for the survival and activation of mature macrophages. The receptor for CSF-1 is the product of the c-fms proto-oncogene, which, on binding ligand, can stimulate a mitogenic response in the appropriate cells. To investigate which genes are regulated in response to CSF-1-stimulation in murine bone-marrow-derived macrophages (BMM), we employed mRNA differential display reverse transcriptase-mediated PCR to identify cDNA species induced by CSF-1. Both Northern and Western blot analyses confirmed the increased expression of one of the cDNA species identified as coding for the catalytic subunit of protein phosphatase 2A (PP2A), an observation not previously reported during the response to a growth factor. To determine the significance of the increased expression of PP2A in response to CSF-1, the PP2A inhibitor okadaic acid (OA) was added to CSF-1-treated BMM and found to inhibit DNA synthesis in a dose-dependent manner. Further analysis with flow cytometry in the presence of OA led to the novel conclusion that PP2A activity is critical for CSF-1-driven BMM cell cycle progression in both early G1 and S phases. Surprisingly, in the light of previous studies with other cells, the PP2A-dependent proliferation could be dissociated from activation by extracellular signal-regulated protein kinase (ERK) in macrophages because OA did not affect either the basal or CSF-1-induced ERK activity in BMM. Two-dimensional SDS/PAGE analysis of lysates of 32P-labelled BMM, which had been treated with CSF-1 in the presence or absence of OA, identified candidate substrates for PP2A. (+info)