M-CSF Receptor Research Articles

M-CSF mediates TNF-induced inflammatory osteolysis

TNF-alpha is the dominant cytokine in inflammatory osteolysis. Using mice whose BM stromal cells and osteoclast precursors are chimeric for the presence of TNF receptors, we found that both cell types mediated the cytokine's osteoclastogenic properties. The greater contribution was made, however, by stromal cells that express the osteoclastogenic cytokine M-CSF. TNF-alpha stimulated M-CSF gene expression, in vivo, only in the presence of TNF-responsive stromal cells. M-CSF, in turn, induced the key osteoclastogenic cytokine receptor, receptor activator of NF-kappaB (RANK), in osteoclast precursors. In keeping with the proproliferative and survival properties of M-CSF, TNF-alpha enhanced osteoclast precursor number only in the presence of stromal cells bearing TNF receptors. To determine the clinical relevance of these observations, we induced inflammatory arthritis in wild-type mice and treated them with a mAb directed against the M-CSF receptor, c-Fms. Anti-c-Fms mAb selectively and completely arrested the profound pathological osteoclastogenesis attending this condition, the significance of which is reflected by similar blunting of the in vivo bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b). Confirming that inhibition of the M-CSF signaling pathway targets TNF-alpha, anti-c-Fms also completely arrested osteolysis in TNF-injected mice with nominal effect on macrophage number. M-CSF and its receptor, c-Fms, therefore present as candidate therapeutic targets in states of inflammatory bone erosion.

Selective Inhibition of Receptor Pathways for Macrophage-Specific Cytokines by HIV-1 Nef Protein.

HIV-1 Nef protein is a well-known major determinant of the pathogenicity of the virus and much attention has been given to Nef to explain its contribution to HIV-1 pathogenesis. One of the important features of Nef is the binding to a variety of host intracellular proteins. Hck, a member of Src family tyrosine kinases, is one of the binding partners of Nef. Meanwhile, Src kinases including Hck are key regulators for signaling pathways of several cytokine receptor systems. We therefore investigated whether Nef would affect cytokine receptor signaling pathways. Both GM-CSF and M-CSF are primary cytokines for monocytes/macrophages, and regulates their development and effector functions. We introduced a conditionally active Nef (Nef-estrogen receptor hormone binding-domain fusion protein) into the human leukemia cell line of which proliferation was dependent on those cytokines, and analyzed their ligand-dependent proliferation and receptor signaling events. Nef activation did not apparently affect the proliferation mediated by GM-CSF but markedly caused the inhibition of proliferation mediated by M-CSF, indicating that Nef selectively inhibited the M-CSF receptor pathways. In fact, Nef inhibited the tyrosine-phosphorylation/activation of M-CSF receptor and the molecular association with the activated receptor, which was the earliest signaling events induced by M-CSF. We found that Nef-activated Hck constitutively associated with M-CSF receptor complex. Since the formation of the molecular complex should occur under physiological conditions, i.e., upon M-CSF stimulation, the constitutive association occurred in Nef-active cells was an aberrant interaction. These results suggested that HIV-1 Nef interfered with M-CSF receptor signaling through Hck activation and thereby inhibited M-CSF bioactivity. The experiments in which Nef, Hck and M-CSF receptor were transiently expressed within 293 T cells in various combinations supported the notion (Suzu S et al., Blood 2005). The selective inhibition of Nef on cytokine signaling might result in the modification of macrophage functions such as cytokine/chemokine production, creating an immunologic environment favorable for HIV-1 virus production.

Gfi-1 Represses PU.1 Activity To Promote Granulocyte over Macrophage Differentiation.

Mice lacking the zinc finger transcriptional repressor protein Gfi-1 are neutropenic. These mice generate abnormal immature myeloid cells exhibiting characteristics of both macrophages and granulocytes. Interestingly these immature Gfi-1−/− myeloid cells overexpress target genes of the PU.1 transcription factor: M-CSF receptor (M-CSFR) and PU.1 itself. Because of this we sought to determine if Gfi-1 modulates the transcriptional activity of PU.1. Here we demonstrate that Gfi-1 physically associates with PU.1 and represses PU.1- dependent transcription from the M-CSFR promoter. This interaction is functionally relevant as Gfi-1 blocks PU.1- dependent macrophage differentiation, but not granulocyte differentiation of a multipotential hematopoietic progenitor cell line. Additionally infection of primary bone marrow progenitors with a Gfi-1 retrovirus promotes granulocyte differentiation at the expense of macrophage differentiation. In contrast, a Gfi-1 mutant lacking repressor activity strongly promotes macrophage differentiation of hematopoietic progenitors. In transient transfections this Gfi-1 mutant not only blocks Gfi-1 repression of PU.1 transcriptional activity but C/EBP alpha mediated repression as well. From this data we hypothesize that Gfi-1 along with other transcriptional regulators target PU.1 for repression in the granulocyte-macrophage progenitor (GMP) in order to direct granulocyte differentiation. If true decreasing the concentration of PU.1 may decrease the need for Gfi-1 in directing granulocyte development. We are currently testing this in vivo by examining myeloid development in PU.1+/−Gfi-1−/− mice.

Phosphorylation of AML1 (RUNX1) Controls Transcription, Nuclear Localization, and Is Regulated during DNA Damage.

We demonstrate that AML1 (RUNX1) is phosphorylated on S276, S293, T300, S303 and S462 after phorbol ester treatment in K562 cells, and these phosphorylations control the transcriptional activity of this protein and regulate varied promoters including TCRb, GM-CSF, M-CSF receptor, and myeloperoxidase gene (JBC 279: 53116–53125, 2004). In addition, we find that phosphorylation of the AML1 protein controls the binding of AML1 to the nuclear matrix, and regulates its degradation (MCR 3: 391–401, 2005). We have now derived rabbit phosphopeptide antisera to S276, S303, and S462 in the AML1 protein. Using these antibodies, we find that in unsynchronized Jurkat T cells AML1 contain low levels of phosphorylation at all three sites. To examine whether this phosphoryaltion is cell cycle regulated, we blocked cells in G1/S with aphidicolin or in G2/M with nocodazole. In contrast to G1/S block that had no effect on phosphorylation, nocodazole treatment induces marked increases in phosphorylation of S276, S303, and S462. The nocodazole increase in phosphorylation was markedly inhibited by staurosporine, a PKC inhibitor, and to a lesser extent by the JNK inhibitor SP600125, and the p38 inhibitor, SB202190. Using elutriation to examine untreated Jurkat cells, we demonstrate increases in the level of AML1 protein as cells progress through the cell cycle to G2/M. However, the actual level of phosphorylated protein as judged by phospho303 and 462 antibodies decreases. Nocodazole treatment of Jurkat cells not only induces G2/M arrest, but also induces apoptosis. We are currently investigating the hypothesis that DNA damage causes phosphorylation of AML1 that regulates both its activity and location.

New tumor markers: CA125 and beyond

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

New tumor markers: CA125 and beyond

A variety of biomarkers have been developed to monitor growth of ovarian cancer and to detect disease at an early interval. CA125 (MUC16) has provided a useful serum tumor marker for monitoring response to chemotherapy, detecting disease recurrence, distinguishing malignant from benign pelvic masses, and potentially improving clinical trial design. A rapid fall in CA125 during chemotherapy predicts a favorable prognosis and could be used to redistribute patients on multiarmed randomized clinical trials. Several studies now document that CA125 can serve as a surrogate marker for response in phase II trials. Serial measurement of CA125 might also provide a useful marker for monitoring stabilization of disease with cytostatic targeted therapeutic agents. The greatest potential for serum markers may be in detecting ovarian cancer at an early stage. A rising CA125 can be used to trigger transvaginal sonography (TVS) in a small fraction of patients. An algorithm has been developed that calculates risk of ovarian cancer based on serial CA125 values and refers patients at highest risk for TVS. Use of the algorithm is currently being evaluated in a trial with 200,000 women in the UK that will test critically the ability of a two-stage screening strategy to improve survival in ovarian cancer. Whatever the outcome, as 20% of ovarian cancers have little or no expression of CA125, additional serum markers will be required to detect all patients in an initial phase of screening. More than 30 serum markers have been evaluated alone and in combination with CA125 by different investigators. Some of the most promising include: HE4, mesothelin, M-CSF, osteopontin, kallikrein(s), and soluble EGF receptor. Two proteomic approaches have been used: one examines the pattern of peaks on mass spectroscopy and the other uses proteomic analysis to identify a limited number of critical markers that can be assayed by more conventional methods. Both approaches are promising and require further development. Several groups are placing markers on multiplex platforms to permit simultaneous assay of multiple markers with very small volumes of serum. Mathematical techniques are being developed to analyze combinations of marker levels to improve sensitivity and specificity. In the future, serum markers should improve the sensitivity of detecting recurrent disease as well as facilitate earlier detection of ovarian cancer.

FMS receptor for M-CSF (CSF-1) is sensitive to the kinase inhibitor imatinib and mutation of Asp-802 to Val confers resistance

The kinase inhibitor imatinib is used in the treatment of chronic myeloid leukaemia, where it targets the intracellular Bcr-Abl tyrosine kinase, and gastrointestinal stromal tumours, where it targets either the KIT or PDGF tyrosine kinase receptors. Here, we report that imatinib is also an effective inhibitor of the closely related FMS receptor for macrophage colony stimulating factor and that mutation of Asp 802 of FMS to Val confers imatinib resistance. Imatinib readily reverted the transformed phenotype of haemopoietic and fibroblast cell lines that express the oncogene v-fms and also inhibited the growth of the Bacl.2F5 macrophage cell line. The cellular IC50 value of imatinib for FMS was similar to those for Bcr-Abl and KIT. Consequently, imatinib may also prove effective for the treatment of diseases whose progression is dependent upon macrophage-colony stimulating factor, this includes certain aspects of cancer and inflammation.

Modulation of cell cycle and gene expression in pancreatic tumor cell lines by methionine deprivation (methionine stress): implications to the therapy of pancreatic adenocarcinoma

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Human stem cell factor-antibody [anti-SCF] enhances chemotherapy cytotoxicity in human CD34+ resistant myeloid leukaemia cells

Acute myeloid leukaemia (AML) is a heterogenous malignant disease with diverse biological features in which disease progression at the level of CD34+ cells has a major impact on the resistance to chemotherapy and relapse. The AML blast cells in these elderly patients are often characterised by several unfavourable covariates that predict the poor treatment outcome, including high stem cell marker CD34 expression, minimally or undifferentiated features, high P-glycoprotein expression, high bcl-2/bax ratio, unfavourable karyotype and more frequent internal tandem duplications (ITDs) and mutations of class III receptor-type tyrosine kinase for key haematopoietic cytokines: Flt-3 (receptor for Flt-ligand), c-kit (receptor for stem cell factor) and fms (receptor for M-CSF). Testing the new and more specific molecular-targeted therapeutic approaches in CD34+ AML cells can provide the basis for a more effective combined molecular/chemotherapy regimen and may consequently improve the treatment outcome in elderly AML patients. Therefore, the present study was performed to evaluate whether stem cell factor-antibody (anti-SCF) can enhance the efficacy of the two main chemotherapeutic drugs used in AML therapy: cytarabine and daunorubicin at low doses in human-resistant CD34+ AML cells, in an attempt to identify a novel effective regimen with tolerable side-effects for elderly AML patients. The effect of anti-SCF on each of the two chemotherapeutic drugs-induced apoptosis and necrosis was investigated in KG1a human-resistant CD34+ AML cells expressing P-glycoprotein to determine its enhancing activity. Anti-SCF has significantly enhanced the low dose cytarabine- and daunorubicin-induced apoptosis+necrosis in KG1a CD34+ AML cells from 12.0±1.7 to 40.9±5.9% and from 16.3±0.9 to 48.9±1.0%, respectively, p<0.01. It has also exerted its significant enhancement activity on the low dose cytarabine- and daunorubicin-induced apoptosis+necrosis in KG1a CD34+ AML cells in the presence of SCF, p<0.05. Anti-SCF has significantly enhanced the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells from 26.7±0.6 to 64.6±1.0% and from 59.8±3.1 to 80.1±7.9%, respectively, p<0.01. The addition of SCF has not altered the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells (Table 4). Anti-SCF has also significantly enhanced the low dose cytarabine- and daunorubicin-induced bcl-2 reduction in KG1a CD34+ AML cells in the presence of SCF, p<0.05. The unique potent enhancing activity of anti-SCF on low dose chemotherapy-induced apoptosis and necrosis in extremely resistant AML cells suggest a novel promising role for the treatment of elderly AML patients. Further studies are warranted to evaluate a similar enhancing effect for anti-SCF in blast cells from elderly AML patients in primary cultures before its introduction in a pilot clinical study. In conclusion, the combination of anti-SCF and the low dose cytarabine provides a promising solution for the dilemma of therapy in elderly AML patients.

Src tyrosine kinase signaling regulates microglial protection of neurons from excitotoxicity

Microglial activation is thought to be important in Alzheimer's disease (AD). However, signal transduction pathways mediating microglia activation are incompletely understood. Src tyrosine kinase is important in the regulation of a variety of cellular functions, including inflammation. We previously found that overexpressing the M-CSF receptor (M-CSFR; encoded by c-fms) on microglia leads to rescue of neurons from NMDA-induced excitotoxicity. We sought to determine if Src signaling mediates M-CSFR-induced neuroprotection. We examined the effects of M-CSFR overexpression on Src kinase levels and activity. We also tested whether LPS, a potent inflammatory stimulus, activated Src. To determine the specificity of M-CSFR activation of Src, we quantified the activity of the Src-family kinase Lyn. A mutagenesis approach was used to block M-CSFR-induced Src activation, and the effects on microglial neuroprotection were examined. We also tested whether a Src dominant negative (DN) construct would block M-CSFR-induced neuroprotection. Western analysis showed that microglial M-CSFR activation resulted in a greater than 2 fold increase in Src expression, whereas an enzymatic assay showed a greater than 3.5 fold increase in Src kinase activity. Microglia treatment with LPS did affect Src activity. M-CSFR activation had no effect on Lyn activity. In the M-CSFR, tyrosine position c-fmsY559 binds Src and induces activation by phosphorylation. Position c-fmsY807, the global c-fms activation site, indirectly affects Src activation. Using co-cultures consisting of microglia overexpressing the M-CSFR and rat organotypic hippocampal cultures subjected to NMDA-induced excitotoxicity, overexpression of the Y559F or Y807F c-fms mutant constructs in microglia resulted in a loss of neurprotection. Similarly, microglial expression of a Src DN form resulted in loss of M-CSFR-induced neuroprotection, and also inhibited microglial proliferation. Increasing the number of microglia expressing the Src DN form in co-culture only partially restored neuroprotection. Adding exogenous M-CSF also partially restored neuroprotection by Src DN-treated microglia in co-cultures. Our results show that the Src signal transduction pathway is important in microglial neuroprotection. Identification of downstream Src kinase targets in microglia could lead to new pharmacological treatments for AD (Funding: Alzheimer's Association and NIH MH57833).

SCL Expression at Critical Points in Human Hematopoietic Lineage Commitment

The stem cell leukemia (SCL or tal-1) gene was initially identified as a translocation partner in a leukemia that possessed both lymphoid and myeloid differentiation potential. Mice that lacked SCL expression showed a complete block in hematopoiesis; thus, SCL was associated with hematopoietic stem cell (HSC) function. More recent studies show a role for SCL in murine erythroid differentiation. However, the expression pattern and the role of SCL during early stages of human hematopoietic differentiation are less clear. In this study we chart the pattern of human SCL expression from HSCs, through developmentally sequential populations of lymphoid and myeloid progenitors to mature cells of the hematopoietic lineages. Using recently defined surface immunophenotypes, we fluorescence-activated cell-sorted (FACS) highly purified populations of primary human hematopoietic progenitors for reverse transcription-polymerase chain reaction (RT-PCR) analysis of SCL expression. Our data show that SCL mRNA is easily detectable in all hematopoietic populations with erythroid potential, including HSCs, multipotential progenitors, common myeloid progenitors, megakaryocyte/erythrocyte progenitors, and nucleated erythroid lineage cells. SCL mRNA expression was present but rapidly downregulated in the common lymphoid progenitor and granulocyte/monocyte progenitor populations that lack erythroid potential. SCL expression was undetectable in immature cells of nonerythroid lineages, including pro-B cells, early thymic progenitors, and myeloid precursors expressing the M-CSF receptor. SCL expression was also absent from all mature cells of the nonerythroid lineages. Although low levels of SCL were detected in lymphoid- and myeloid-restricted progenitors, our studies show that abundant SCL expression is normally tightly linked with erythroid differentiation potential.

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.

Macrophage colony-stimulating factor receptor induces tyrosine phosphorylation of SKAP55R adaptor and its association with actin

The production, survival, and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor. M-CSF receptor activates multiple cytoplasmic pathways in which adaptor and scaffolding proteins play a central role. In this study, we showed that SKAP55-related (SKAP55R) adaptor protein is expressed in myeloid cells and macrophages and is rapidly and transiently tyrosine-phosphorylated in response to M-CSF. M-CSF induced SKAP55R association with other tyrosine-phosphorylated proteins and with actin. When overexpressed in myeloid cells, SKAP55R decreased M-CSF-dependent proliferation without affecting differentiation. Altogether, these results demonstrate that SKAP55R adaptor is implicated in the M-CSF signaling pathway and suggest its role as a negative regulator of growth. Moreover, specific association between SKAP55R and actin support the idea that SKAP55R is implicated in the regulation of actin dynamics under the control of M-CSF.

Exogenous C/EBPα Increases the Monocytic Maturation of Normal Murine Myeloid Progenitors.

Within hematopoiesis C/EBPα is expressed specifically in both the granulocytic and monocytic myeloid lineages. Mice lacking C/EBPα have reduced neutrophil and monocyte production, due in part to diminished expression of the G-CSF, M-CSF, and GM-CSF receptor mRNAs. C/EBPα-ER directs the granulocytic differentiation of 32Dcl3 cells, and induction of late markers of granulopoiesis by C/EBPα-ER in 32Dcl3 cells was dependent upon its ability to slow G1 to S progression. We have now assessed the effect of C/EBPα-ER on the myeloid maturation of murine marrow progenitors. Regulation of the transgene using estradiol avoids biasing cell populations during transduction. Mononuclear cells isolated from 5-FU-treated mice were transduced with pBabePuro-C/EBPαER and selected for 2 days with puromycin. Surviving cells were isolated using a density gradient, subjected to lineage-depletion, which removed 98% of Mac1+ and Gr1+ cells, and placed in liquid or methylcellulose culture with IL3/IL6/SCF +/− estradiol. By 48 hrs, 80% of the cells had acquired myeloid surface markers. Mac1+/Gr1+ cells were granulocytes and Mac1+/Gr1− cells were monocytes, as confirmed by Wright’s-Giemsa staining of cytospins. Estradiol increased the proportion of monocytes 1.7-fold and reduced the proportion of granulocytes 2.5-fold in 5 independent assessments. Real-time PCR analysis indicated a 5-fold increase in CD14 and CD68/macrosialin RNA, a 2-fold increase in M-CSF receptor and PU.1 RNA, and a 4-fold decrease in NE RNA by 48 hrs. In contrast, estradiol had little effect on the development of monocytes or granulocytes from vector, pBabePuro-transduced cells. Consistent with these findings, in an initial experiment we observed increased granulocytes when progenitors from floxxed C/EBPα mice (kindly provided by P. Johnson) were transduced with pBabeNeo-CRE, compare with pBabeNeo, subjected to lineage-depletion, and cultured in IL3/IL6/SCF. Loss of C/EBPα was demonstrated by PCR and Western blotting. During the first 48 hrs after transfer of transduced wild-type cells to estradiol, C/EBPα-ER slowed the proliferation of myeloid progenitors 2.6-fold, increased their G1/S ratio 2.8-fold, and reduced myeloid CFUs 5.1-fold. CFU-M, CFU-G, and CFU-GM were affected equally. Transduction with C/EBPαL12V-ER, harboring a defective leucine zipper, or with C/EBPαBR3-ER, mutant in 4 basic region residues, slowed proliferation in liquid culture 2-fold, reduced myeloid CFUs 1.5-fold, and had little effect on the G1/S ratio, perhaps indicating a role for DNA-binding in the regulation of the cell cycle by C/EBPα in normal myeloid progenitors (CFUs) and precursors. The L12V variant did not affect lineage output, whereas C/EBPαBR3 reduced monocytic maturation 1.4-fold and increased granulocyte formation 1.4-fold, in 4 experiments. C/EBPαp30, expressed in a subset of AML cases and lacking an N-terminal transactivation domain, increased the G1/S ratio and reduced myeloid CFUs 1.6-fold and had no effect on myeloid maturation. In summary, exogenous C/EBPα increases the monocytic and reduces the granulocytic maturation of transduced myeloid progenitors cultured in cytokines whose receptors are not regulated by C/EBPα. Cell cycle inhibition may play a role in this lineage bias, as the the L12V and p30 variants had both a reduced ability to slow G1 progression and to induce monocytes. Induction of PU.1 may also favor monocytic maturation. Finally, results with BR3 compared with L12V, neither of which bind DNA, suggest that alteration of a protein interaction with the non-DNA contact surface of the C/EBPα basic region favors granulocyte formation.

Flt3 Receptor Expression Rescues Dendritic Cell Developmental Potential in Megakaryocyte/Erythrocyte Progenitor Cells.

Flt3/Flt3-ligand has a non-redundant role in dendritic cell (DC) and interferon producing cell (IPC) development from hematopoietic progenitor cells. Flt3-ligand−/− mice display a severe reduction of DCs and IPCs, and both, Flt3-ligand injection and conditional over- expression of Flt3-ligand in mice leads to an increased pool size of both cellular subsets. Furthermore, DC and IPC developmental precursor activity segregates with Flt3+ progenitor populations in mouse bone marrow. We have reported that common myeloid progenitors (CMPs) and their progeny granulocyte/macrophage progenitors (GMPs) as well as common lymphoid progenitors (CLPs) and pro-T cells can give rise to DCs in vitro and in vivo. DC developmental potential was lost, however, once definitive megakaryocyte/erythrocyte or B cell commitment occurs, both steps that are accompanied by loss flt3 receptor expression. To test if DC/IPC developmental potential can be regained, we evaluated the effect of ecotropic expression of human Flt3 in lin−c-kit+Flt3−Thy1.1−IL-7Ra− cells (flt3− progenitors) and in megakaryocyte/erythrocyte progenitors (MEPs).hFlt3 was transduced into purified flt3− progenitors, MEPs, and GMPs using a retrovirus vector carrying both hFlt3 and GFP. The transduction efficiencies were 20~25%. hFlt3-transduced flt3− progenitors gave rise to functional DCs and IPCs, but not control-GFP-transduced flt3− progenitors in vitro. Similarly, hFlt3-transduced MEPs differentiated into functional DCs in Flt3L, SCF, GM-CSF, IL-4, and TNF-a supplemented cultures but not into TER119+ erythroid cells. DC developmental potential of hFlt3-transduced MEPs was comparable to GFP-transduced GMPs. Over expression of hFlt3 in GMPs resulted in augmentation of its DC developmental potential. Interestingly, both hFlt3-tarnsduced MEPs and GMPs preferentially differentiated into IPCs in Flt3L supplemented medium. RT-PCR analysis revealed that enforced Flt3 signaling in MEPs lead to up-regulation of GM- and/or DC-development affiliated genes such as G-CSF receptor, M-CSF receptor, and GM-CSF receptor, PU.1, C/EBPa and STAT3. Furthermore, hFlt3-transduced MEPs gave rise to myeloid colonies (CFU-GM/G/M) but not CFU-Mix colonies in GM-CSF, IL-3, SCF, Flt3L, Tpo, and Epo supplemented methylcellulose medium. These results show that artificial Flt3 signaling in otherwise Flt3− progenitors is sufficient to reestablish DC/IPC differentiation and GM-differentiation programs.

HIV-1 Nef Interferes with M-CSF Receptor Signalling through Hck Activation and Inhibits M-CSF Bioactivities.

HIV-1 Nef protein is a major determinant of the pathogenicity of the virus. It has been shown that Nef activates Hck, a member of Src family kinase, in monocytes/macrophages and that the interaction is critical for AIDS-like disease progression in a mouse model. However, it was unclear how the molecular interaction in monocytes/macrophages leads to the disease progression. Here, we show for the first time that Nef interferes with the M-CSF/M-CSF receptor pathway. In this study, we introduced a conditionally active Nef into myeloid leukemia TF-1-fms cells and analyzed their responsiveness to M-CSF. We found that Nef-activated Hck constitutively associated with M-CSF receptor complex. The formation of the molecular complex should occur under physiological conditions, i.e., upon M-CSF stimulation. Due to the aberrant molecular association, the tyrosine-phosphorylation/activation of the receptor in response to M-CSF was markedly diminished in Nef-active cells. Consequently, Nef activation caused the inhibition of M-CSF-mediated proliferation of TF-1-fms cells and macrophage differentiation of the cells induced by M-CSF and 12-O-tetradecanoylphorbol 13-acetate. These results indicate that HIV-1 Nef interferes with M-CSF receptor signalling through Hck activation and thereby inhibits M-CSF functions in monocytes/macrophages.

E47 Binds to PU.1 Inhibiting Its Ability To Bind DNA and Activate Gene Expression.

The transciption factor PU.1 is required for the development of macrophages, granulocytes, and B lymphocytes. Additionally its protein concentration in multipotential hematopoietic progenitor cells regulates cell fate decisions with high levels of PU.1 directing myeloid cell fate acquisition and low levels directing B cell fate acquisition. Potentially high levels of PU.1 are required for myeloid development in order to overcome repressive effects of B cell lineage specific factors. The essential B cell factor BSAP (Pax-5) was shown to associate with PU.1 and repress its transactivation activity. Here we investigate whether two other essential B cell factors, E2A and EBF could affect PU.1 activity. We observed that the E2A component, E47, but not EBF could associate with PU.1. Additionally E47 could repress PU.1 dependent transactivation of the M-CSFR promoter and that this repression is mediated by inhibiting PU.1 DNA binding. Exogenous E47 expression in an IL-3 dependent multipotential cell line could block PU.1 induced differentiation and expression of endogenous M-CSF receptor protein. Our data suggests that high PU.1 concentration is required for myeloid development in order to overcome repressive actions of other lineage specific transcriptional regulators such as E47.

Identification of the M‐CSF Receptor in Endometriosis by Immunohistochemistry and RT‐PCR

The aim of this paper is to provide further evidence that the dystopic proliferation of endometriotic epithelia is caused by the stimulation of peritoneal macrophages. It is essential to show that endometriotic epithelial cells express the macrophage colony-stimulating factor receptor (M-CSFR) which binds the M-CSF produced by the peritoneal macrophages. For the detection of M-CSFR, samples of ectopic endometrium (n = 79) and eutopic endometrium (n = 18) were compared. The specimens were gained at operative laparoscopy in the proliferative phase of the cycle. Cryostat sections were used for immunohistochemical detection. For in vitro reverse transcriptase polymerase chain reaction (RT-PCR) tests, the tissue was immediately shock frozen on paraffin sections. For the in situ RT-PCR technique the specimens were placed in a para-formaldehyde solution, embedded in paraffin and later processed. The Gene Amp 1000 in situ PCR system (Perkin Elmer) was used as the thermal cycler. M-CSF and the M-CSF receptor are present in eutopic and ectopic endometrium. Qualitatively, with both PCR techniques we found the M-CSF receptor to be present in all samples examined. Using the histochemical detection technique, the M-CSF receptor was found in nearly 70% of endometriosis patients compared with a statistically significant lower percentage in normal endometrium. The in situ RT-PCR technique and immunohistochemistry elaborated the need to trace the cellular sources of the M-CSF receptor. The identification of the M-CSF receptor in endometriotic tissue and in endometrium is apt to open a new experimental field in endometriosis research.

Intracellular glutathione status regulates mouse bone marrow monocyte-derived macrophage differentiation and phagocytic activity

Although a redox shift can regulate the development of cells, including proliferation, differentiation, and survival, the role of the glutathione (GSH) redox status in macrophage differentiation remains unclear. In order to elucidate the role of a redox shift, macrophage-like cells were differentiated from the bone marrow-derived monocytes that were treated with a macrophage colony stimulating factor (M-CSF or CSF-1) for 3 days. The macrophagic cells were characterized by a time-dependent increase in three major symptoms: the number of phagocytic cells, the number of adherent cells, and the mRNA expression of c-fms, a M-CSF receptor that is one of the macrophage-specific markers and mediates development signals. Upon M-CSF-driven macrophage differentiation, the GSH/GSSG ratio was significantly lower on day 1 than that observed on day 0 but was constant on days 1–3. To assess the effect of the GSH-depleted and -repleted status on the differentiation and phagocytosis of the macrophages, GSH depletion by BSO, a specific inhibitor of the de novo GSH synthesis, inhibited the formation of the adherent macrophagic cells by the down-regulation of c-fms, but did not affect the phagocytic activity of the macrophages. To the contrary, GSH repletion by the addition of NAC, which is a GSH precursor, or reduced GSH in media had no effect on macrophage differentiation, and led to a decrease in the phagocytic activity. Furthermore, we observed that there is checkpoint that is capable of releasing from the inhibition of the formation of the adherent macrophagic cells according to GSH depletion by BSO. Summarizing, these results indicate that the intracellular GSH status plays an important role in the differentiation and phagocytosis of macrophages.

Integrin engagement regulates monocyte differentiation through the forkhead transcription factor Foxp1.

The precise signals responsible for differentiation of blood-borne monocytes into tissue macrophages are incompletely defined. "Outside-in" signaling by integrins has been implicated in modulation of gene expression that affects cellular differentiation. Herein, using differential display PCR, we have cloned an 85-kDa forkhead transcription factor (termed Mac-1-regulated forkhead [MFH] and found subsequently to be identical to Foxp1) that is downregulated in beta(2)-integrin Mac-1-clustered compared with Mac-1-nonclustered monocytic THP-1 cells. MFH/Foxp1 is expressed in untreated HL60 cells, and its expression was markedly reduced during phorbol ester-induced monocyte differentiation, but not retinoic acid-induced granulocyte differentiation. Overexpression of MFH/Foxp1 markedly attenuated phorbol ester-induced expression of c-fms, which encodes the M-CSF receptor and is obligatory for macrophage differentiation. This was accompanied by decreased CD11b expression, cell adhesiveness, and phagocytosis. Using electromobility shift and reporter assays, we have established that MFH/Foxp1 binds to previously uncharacterized sites within the c-fms promoter and functions as a transcriptional repressor. Deficiency of Mac-1 is associated with altered regulation of MFH/Foxp1 and monocyte maturation in vivo. Taken together, these observations suggest that Mac-1 engagement orchestrates monocyte-differentiation signals by regulating the expression of the forkhead transcription repressor MFH/Foxp1. This represents a new pathway for integrin-dependent modulation of gene expression and control of cellular differentiation.