M-CSF Receptor Research Articles

Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival

Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec(-/-)Btk(-/-) BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec(-/-)Btk(-/-) macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor alpha (GM-CSFRalpha) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage-specific regulation of GM-CSFRalpha expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling-induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM-CSFRalpha expression.

Down-regulation of Notch-1 expression decreases PU.1-mediated myeloid differentiation signaling in acute myeloid leukemia

The Notch receptor-mediated signaling pathways control cell fate in many types of organisms including neurogenesis, myogenesis and hematopoiesis in mammalian species. During normal hematopoiesis, Notch-1 promotes myeloid differentiation through up-regulation of the transcriptional factor PU.1. We therefore speculated that down-regulation of Notch-1 expression might be involved in the leukemogenesis of acute myeloid leukemia (AML). Here we investigated Notch-1 expression and its association with PU.1-mediated differentiation signaling in AML. The transcriptional level of Notch-1 and PU.1 was evaluated in 6 AML cell lines and 54 AML patient samples using real-time PCR analysis, and Western blot analysis of Notch-1, PU.1 and one of its downstream targets, the M-CSF receptor (MCSFR), was performed to test for confirmation. A significant decrease in the transcription levels of Notch-1 was noted in AML cell lines and patient samples, and decreased Notch-1 protein expression in AML was confirmed by Western blotting. Down-regulation of Notch-1 expression was associated with a decrease in PU.1/MCSFR expression in AML. Co-immunoprecipitation experiments showed that partial disruption of the Notch-1/PU.1 complex was noted in AML cells. No detectable mutation of Notch-1 (ANK, PEST) and PU.1 (PEST, DBD) was noted by PCR-single-strand conformation polymorphism (SSCP) assay. These results suggest that down-regulation of Notch-1 expression decreases PU.1/MCSFR expression and disrupts the Notch-1/PU.1 complex, which may impede the PU.1-mediated myeloid signaling and contribute to the leukemogenesis of AML.

Increased TNF expression in CD43 ++ murine blood monocytes

Monocyte heterogeneity has been studied extensively in man but only recently tools have been developed to study blood monocyte populations in the mouse. We have used the MacGreen mouse model, which expresses the green fluorescent protein under the control of the promoter of the murine M-CSF receptor (CSF1 receptor, c-fms). Since both monocytes and granulocytes show GFP expression in this model the latter cells were excluded by staining with the Ly6G granulocyte marker. GFP + Ly6G − blood monocytes were found to account for an average of 246 ± 121 cells/μl in these mice. These monocytes can be subdivided into CD43 + GR-1 + cells and CD43 ++ GR-1 − cells, with the latter cells accounting for 140 ± 77 cells/μl, i.e. about 60% of all blood monocytes. After intraperitoneal injection of lipopolysaccharide (LPS) both blood monocyte subpopulations were depleted. The same was true after intranasal infection with Streptococcus pneumoniae but here the CD43 ++ subpopulation was preferentially reduced to 4 cells/μl. For the study of TNF expression cells were stimulated in vitro with LPS from Salmonella abortus equi in the presence of Brefeldin A followed by intracellular staining and multicolor flow cytometry. Over a dose range of 10–100 ng LPS/ml, TNF protein production was significantly higher in the CD43 ++ monocyte subset. At 1000 ng LPS/ml 90% of all CD43 ++ monocytes stained positive for TNF and in terms of fluorescence intensity TNF was 5-fold higher compared to the CD43 + monocytes. These data indicate that the murine CD43 ++ monocyte subset exhibits features of pro-inflammatory monocytes and is functionally homologeous to the human CD14 +CD16 + monocytes.

Multiple myeloma cells directly stimulate bone resorption in vitro by down-regulating mature osteoclast apoptosis

Multiple myeloma (MM) is characterized by devastating bone destruction mainly due to stimulation of osteoclastogenesis. However, whether MM cells can directly influence osteoclast apoptosis, a mechanism that would contribute to increase the number of active osteoclasts, has not been addressed yet. Herein, using authentic mature rabbit osteoclasts, we demonstrated that conditioned media (CM) prepared from U266 and RPMI8226 cells but not from LP-1 and OPM-2 cells, stimulated bone resorption and inhibited osteoclast apoptosis in a dose-dependent manner. The MM cells which exerted an anti-apoptotic effect secreted high amounts of M-CSF and addition of a neutralizing antibody against M-CSF reversed the CM effects. Imatinib mesylate, a tyrosine kinase inhibitor that can target the M-CSF receptor, also prevented the effect of CM. These findings suggest that M-CSF originating from MM cells may play a critical role in MM bone disease by decreasing osteoclast apoptosis.

Direct Comparison of G-CSF Receptor and M-CSF Receptor Signaling Leading to AP-1 and STAT Induction and Identification of Novel Signaling Pathways.

External signals often influence developmental decisions. Whether hematopoietic cytokines contribute to lineage specification or serve to amplify decisions made by an intrinsic transcription factor program remains unresolved. Although much is known about signaling events emanating from the G-CSF Receptor (GR) and the M-CSF Receptor (MR), these signals have not been directly compared in a uniform cellular environment. Reasoning that differences seen could contribute to myeloid lineage choice in bipotent cells such as the granulocyte monocyte progenitor (GMP) or the CFU-GM, we have compared GR and MR signaling via endogenous receptors present in the U937 human leukemia cell line and using a subclone derived from the IL-3 dependent Ba/F3 stem cell line expressing both exogenous GR and MR, introduced by retroviral transduction. FACS analysis confirmed that the large majority of cells in our Ba/F3 line express both MR and GR at similarly high levels. Ba/F3 cells were IL-3 starved for 1hr (which caused no change in signaling for the pathways tested) prior to G-CSF, M-CSF stimulation, or mock addition. Both receptors reproducibly activated JNK and induced c-Jun phosphorylation, and both GR and MR signals activated ERK increasing total c-Fos levels, likely via c-Fos serine phosphorylation. Although only the cytoplasmic domain of MR has been demonstrated to interact with PLCγ, two different PLC-inhibitors (U-73122, a PLCγ-specific inhibitor, and edelfosine, a general PLC inhibitor) reduced ERK phosphorylation and c-Fos expression at 30 min downstream of both G-CSF and M-CSF stimulation. A connection between PLCγ and the ERK pathway had not been previously described within these signaling pathways. Two different ERK inhibitors (U0126 and PD9899) reduced c-Fos expression, confirming a link between ERK and c-Fos in hematopoietic cells. PLC and ERK inhibition not only reduced Fos induction, but also increased JNK activation and c-Jun phosphorylation at 30 min, identifying a novel connection between the ERK and JNK branches of the MAPK pathways. Conversely, JNK inhibition with SP600125 reduced c-Jun phosphorylation without altering ERK activation or c-Fos expression. The balance between c-Jun and c-Fos activation via the signaling pathways we have characterized may contribute to granulocyte versus monocyte lineage specification, as exogenous c-Jun directs monocyte development in cooperation with PU.1 and C/EBPα. For example, perhaps increased c-Fos diverts c-Jun to facilitate granulopoiesis. We did not detect differences in the effects of GR or MR on C/EBPα or PU.1 levels, DNA-binding, or C/EBPα serine 21 phosphorylation in U937 or Ba/F3 cells. On the other hand, GR but not MR signals potently induced STAT1, STAT3, and STAT5 tyrosine phosphorylation and DNA-binding at 30 min in Ba/F3 cells, of potential relevance to both proliferation and granulocytic differentiation induced by G-CSF. We now intend to further delineate G-CSF versus M-CSF activation of the JNK/c-Jun, PLCγ/ERK/c-Fos, and STAT pathways and their effects on granulocyte versus monocyte differentiation, using lineage-negative murine myeloid progenitors.

Cyclin-Dependent Kinase Phosphorylation of RUNX1/AML1 on Three Sites Increases Trans-Activation Potency and Stimulates Cell Proliferation.

RUNX1/AML1 regulates lineage-specific genes during hematopoiesis and also stimulates G1 cell cycle progression. CBFβ-SMMHC or AML1-ETO dominantly inhibit RUNX1 and slow G1 progression in hematopoietic cell lines or in murine or human marrow progenitors, cdk4, cyclin D2, or c-Myc overcome inhibition of proliferation by these CBF oncoproteins, exogenous RUNX1 stimulates G1 progression, and stimulation of G1 via deletion of p16INK4a or expression of E7 cooperates with CBFβ-SMMHC or TEL-AML1 to induce acute leukemia in mice. Induction of cdk4 or cyclin D3 transcription may underlie stimulation of G1 progression by RUNX1. Remarkably, the C. elegans ortholog of RUNX1, RNT-1, also stimulates G1 progression and couples stem cell proliferation with differentiation. Not only does RUNX1 regulate cell cycle progression, but in addition RUNX1 levels increase as hematopoietic cells progress from G1 to S and from S to G2/M. Within RUNX1, S48, S303, and S424 fit the cdk phosphorylation consensus, (S/T)PX(R/K). Phosphorylation of RUNX1 by cyclin dependent kinases on serine 303 was shown to mediate destabilization of RUNX1 in G2/M. We now find that S48 and S424 are also phosphorylated by cdk1 or cdk6. S48, S303, or S424 phosphopeptide antiserum that we developed specifically recognized kinased GST-RUNX1 and interacted with RUNX1 expressed in 293T cells or in the Ba/F3 hematopoietic cell line. S48 phosphorylation of RUNX1 paralleled total RUNX1 levels during cell cycle progression, S303 was more effectively phosphorylated in G2/M, and S424 in G1. Single, double, and triple mutation to alanine or to the partially phosphomimetic aspartic acid progressively diminished or increased trans-activation, such that the tripleA mutant activated a RUNX1 reporter 5-fold less potently than the tripleD mutant. Aspartic acid does not perfectly mimic serine phosphorylation, as illustrated by the much greater affinity of our antisera for wild-type RUNX1 versus RUNX1(tripleD), suggesting that the biologic effect of RUNX1 cdk phosphorylation is even more significant. The p300 co-activator retained interaction with the tripleA variant. The tripleD RUNX1 mutant rescued Ba/F3 cells from inhibition of proliferation by CBFβ-SMMHC more effectively than the tripleA mutant. Cdk phosphorylation of RUNX1 on three sites increases its ability to active transcription and to stimulate proliferation, potentially coupling entry of stem/progenitors into cycle with induction of genes required for hematopoietic lineage progression, such as those encoding myeloperoxidase, neutrophil elastase, the M-CSF receptor, and PU.1.

Stromal Cells Support a Myeloid Pathway of Human NK Cell Differentiation.

Natural killer (NK) cells belong to the lymphocyte lineage; however a myeloid origin has been debated in the past based on nascent experimental evidence. We studied the in vitro development of human NK cells from UCB-derived CD34+ cells following culture with cytokines (IL15, IL7, SCF, FLT3L, IL3) on a murine fetal stromal cell line EL08.1D2 (Blood , 2006; 108: 3824–3833). We investigated the differential requirement of CD34+ subsets for stromal cell support. Limiting dilution experiments showed that CD34+ cells negative for phenotypic markers of NK commitment (CD7, CD161, integrin B7, CD122, CD45RA) absolutely require stromal cells and/or addition of hydrocortisone (HC) to differentiate into functional NK cells. Without stromal cells or HC those progenitors give rise to myeloid lineage cells, but not NK cells. Thus, we hypothesized that stromal cells could instruct myeloid precursors to convert to the NK lineage. Indeed, CD56+ cells generated in stroma supported cultures frequently co-express CD33 and CD13. To determine whether myeloid cells developing from CD34+ cells after 2–3 wk cultures could give rise to NK cells, we FACS sorted the CD56−CD33+CD13high and CD56−CD14+ populations. Such CD33+CD13high and CD14+ cells express macrosialin (CD68) and acquire lyzozyme (by FACS), confirming their myeloid characteristics. Sorted cells cultured further in cytokines alone (IL15, IL7, SCF, FLT3L) did not give rise to NK cells. However, in the presence of cytokines, stromal cells and HC, NK cells were generated. To exclude the possibility of NK cell contamination, CD33+CD13high and CD14+ cells were isolated from cultures of CD34+ cells in conditions not supportive of NK cell development (GM-CSF, IL3, FLT3L, SCF, without stroma, IL15 or IL7). Such cells gave the same results as above (i.e., NK cells developed only with stroma and HC). In additional studies, a fraction (∼16%) of CFU-GM colonies isolated from methylocellulose cultures could generate NK cells only in the presence of stromal cells, HC and cytokines, but not cytokines alone. As more of a definitive marker of the monocytic lineage, we used the surface expression of M-CSF receptor (CD115) on hematopoietic precursors. CD56−CD117+CD115+ and CD56−CD117+CD115− fractions were FACS sorted from 2–3 wk cultures of CD34+ cells. While both populations could differentiate into NK cells, only the CD115+ monocytic precursors required stromal cells. Quantitatively the CD117+CD115− cells were the main source of NK cells in this culture system. Notably the NK cells derived from CD115+ precursors were remarkably different, showing significantly higher expression of Killer Immunoglobulin-like Receptors (KIR: CD158a, CD158b and CD158e) than their CD115− derived counterparts (52% vs 15% KIR+, n=3, p=0.002). With respect to the repertoire of HLA-specific inhibitory receptors, NK cells derived from monocytic precursors resemble the dominant fraction of peripheral blood NK cells, including potentially alloreactive NK cells (KIR+CD94/NKG2A−). Collectively we present evidence that NK cells can be derived from developmental intermediates of the monocytic lineage and this differentiation pathway is dependent upon interaction with stroma. Our data indicate that the developmental trajectory shapes the pattern of inhibitory receptor expression on mature NK cells. Such findings have bearing on our understanding of NK cell biology, post transplant NK cell reconstitution and could explain the paucity of recognized immature NK cell leukemias coinciding with the occurrence of AML variants with NK specific antigen expression.

Macrophage-colony stimulating factor is required for the production of neutrophil-promoting activity by mouse embryo fibroblasts deficient in G-CSF and GM-CSF

G-CSF and GM-CSF play important roles in regulating neutrophil production, survival, differentiation, and function. However, we have shown previously that G-CSF/GM-CSF double-deficient [knockout (KO)] mice still develop a profound neutrophilia in bone marrow and blood after infection with Candida albicans. This finding suggests the existence of other systems, which can regulate emergency neutrophil production. We have now developed an "in vitro" technique to detect and characterize a neutrophil-promoting activity (NPA) in the media conditioned by mouse embryonic fibroblasts (MEFs) derived from G-CSF(-/-)/GM-CSF(-/-) mice. NPA is produced in vitro by the MEFs after stimulation with LPS or heat-inactivated C. albicans. Although M-CSF added directly to bone marrow cultures does not sustain granulocyte production, our studies indicate that production of NPA requires activation of the M-CSF receptor (c-fms). First, G-CSF(-/-)/GM-CSF(-/-) MEFs produce high levels of NPA after stimulation with LPS or C. albicans, and G-CSF/GM-CSF/M-CSF triple-KO MEFs do not. Second, the production of NPA by the G-CSF(-/-)/GM-CSF(-/-) MEFs is reduced significantly upon incubation with neutralizing antibodies to M-CSF or c-fms. Third, NPA production by G-CSF(-/-)/GM-CSF(-/-)/M-CSF(-/-) fibroblasts is enhanced by supplementing culture medium with M-CSF. Thus, stimulation of c-fms by M-CSF is a prerequisite for the production of NPA.

MicroRNAs 17-5p–20a–106a control monocytopoiesis through AML1 targeting and M-CSF receptor upregulation

We investigated the role of microRNAs (miRNA) 17-5p, 20a and 106a in monocytic differentiation and maturation. In unilineage monocytic culture generated by haematopoietic progenitor cells these miRNAs are downregulated, whereas the transcription factor acute myeloid leukaemia-1 (AML1; also known as Runt-related transcription factor 1, Runx1) is upregulated at protein but not mRNA level. As miRNAs 17-5p, 20a and 106a bind the AML1 mRNA 3'UTR, their decline may unblock AML1 translation. Accordingly, transfection with miRNA 17-5p-20a-106a suppresses AML1 protein expression, leading to M-CSF receptor (M-CSFR) downregulation, enhanced blast proliferation and inhibition of monocytic differentiation and maturation. Treatment with anti-miRNA 17-5p, 20a and 106a causes opposite effects. Knockdown of AML1 or M-CSFR by short interfering RNA (siRNA) mimics the action of the miRNA 17-5p-20a-106a, confirming that these miRNAs target AML1, which promotes M-CSFR transcription. In addition, AML1 binds the miRNA 17-5p-92 and 106a-92 cluster promoters and transcriptionally inhibits the expression of miRNA 17-5p-20a-106a. These studies indicate that monocytopoiesis is controlled by a circuitry involving sequentially miRNA 17-5p-20a-106a, AML1 and M-CSFR, whereby miRNA 17-5p-20a-106a function as a master gene complex interlinked with AML1 in a mutual negative feedback loop.

M-CSF Regulates the Cytoskeleton via Recruitment of a Multimeric Signaling Complex to c-Fms Tyr-559/697/721

M-CSF is known to induce cytoskeletal reorganization in macrophages and osteoclasts by activation of phosphatidylinositol 3-kinase (PI3K) and c-Src, but the detailed mechanisms remain unclear. We find, unexpectedly, that tyrosine (Tyr) to phenylalanine (Phe) mutation of Tyr-721, the PI3K binding site in the M-CSF receptor c-Fms, fails to suppress cytoskeletal remodeling or actin ring formation. In contrast, mutation of c-Fms Tyr-559 to Phe blocks M-CSF-induced cytoskeletal reorganization by inhibiting formation of a Src Family Kinase SFK.c-Cbl.PI3K complex and the downstream activation of Vav3 and Rac, two key mediators of actin remodeling. Using an add-back approach in which specific Tyr residues are reinserted into c-Fms inactivated by the absence of all seven functionally important Tyr residues, we find that Tyr-559 is necessary but not sufficient to transduce M-CSF-dependent cytoskeletal reorganization. Furthermore, this same add-back approach identifies important roles for Tyr-697 and Tyr-721 in collaborating with Tyr-559 to recruit a multimeric signaling complex that can transduce signals from c-Fms to the actin cytoskeleton.

Understanding the mechanisms by which Reactive Oxygen Species (ROS) activate mononuclear phagocytes in inflammatory diseases of the lung.

Previously, we found that ROS activated the Akt survival pathway and MAP kinases in monocytes. Thus, we hypothesized that ROS mediates signals through the activation of the macrophage colony-stimulating factor receptor (M-CSFR). In NIH 3T3 cells stably expressing the M-CSFR, we found that H2O2 induced M-CSFR phosphorylation and this phosphorylation event occurred in the absence of receptor dimerization suggesting that other kinases were involved. We examined the effect of either the general tyrosine kinase inhibitor genistein or the Src kinase inhibitor PP2. In the presence of the inhibitors, we found that H2O2-induced phosphorylation of the M-CSFR, Akt, and Erk was greatly reduced in NIH3T3/M-CSFR cells and monocytes. Upon H2O2 stimulation of SYF fibroblast cell line that lacks Src kinases, phosphorylation of Akt and Erk was reduced compared to cells expressing c-Src. A decrease in tyrosine phosphorylated proteins was observed in platelet derived growth factor (PDGF)-treated SYF cells whereas expression of c-Src restored activation. These observations suggest that ROS activate Src kinases to induce both PDGF and M-CSF receptor activation and downstream signaling events. In monocytes, this occurs without receptor dimerization. We predict that these events result in prolonged activation of these cells and the development of inflammatory diseases within the lung.

HIV-1 Nef Inhibits M-CSF Signals by Down-Regulating Its Receptor Fms through Hck.

Nef, an accessory protein of HIV−1, has been shown to be critical for both high viral loads and progression to AIDS. We recently demonstrated that Nef inhibited the bioactivities of M-CSF, a monocytes/macrophages-specific cytokine (Blood 105: 3230–3237, 2005). The inhibitory effect of Nef is a likely explanation for dysregulated functions in HIV−1−infected macrophages. Here, we examined the specificity and molecular mechanism of the newly-characterized function of Nef, to clarify its relevance to the pathogenetic activity of Nef. By analyzing cytokine-dependent cell lines expressing the conditionally active Nef, we found that Nef had a profound inhibitory effect on M-CSF signal, but not on IL-4- and GM-CSF signals. We also found that Nef down-regulated the surface expression of M-CSF receptor (M-CSFR) but not of GM-CSF receptors. The M-CSFR down-regulation by Nef was reproducible in 293 cells when co-transfected with Hck, a member of Src kinases. Interestingly, the down-regulation of cell surface M-CSFR in the presence of Nef and Hck correlated with the concomitant increase of the immature form of M-CSFR. These results suggested that Nef perturbed M-CSFR maturation through Hck and thereby selectively inhibited M-CSF signal.

A Novel Cytokine, FPT025, Regulates Myeloid Growth and Differentiation Via the M-CSF Receptor.

To identify novel protein therapeutics we have utilized an integrated screening system in which a collection of full-length human cDNA clones, encoding virtually all secreted proteins and receptors, was assembled. Soluble secreted proteins were expressed in a high-throughput format using human cells as the expression host and tested in high-throughput cell-based assays. Through this approach, a novel cytokine, FPT025, was identified in a human monocyte proliferation assay. FPT025 has no apparent sequence homology to known cytokines or any other genes and is expressed in human spleen, skin, brain, and other tissues. The purified recombinant FPT025 protein stimulated human primary monocyte proliferation and/or survival. FPT025 protein specifically bound to human primary monocytes and activated ERK1/2 phosphorylation in primary monocytes as well as in a human monocytic cell line, THP-1. FPT025 promoted formation of the myeloid lineage colonies, CFU-M, in a human bone marrow colony formation assay and enhanced proliferation of cells with myeloid cell surface markers from human monocytes. To identify the receptor of FPT025, a collection of extracellular domains (ECD) of transmembrane proteins was screened for their ability to block FPT025 activation of monocyte proliferation. In this screen, we found that FPT025 binds to the M-CSF receptor (M-CSFR) with high affinity. Furthermore, we showed that the binding of FPT025 to M-CSFR was specific and could be competed by M-CSF. The soluble ECD of M-CSFR inhibited both the binding of FPT025 to M-CSFR and the activity of FPT025 in monocyte proliferation. Therefore, FPT025 functions as a novel ligand of the M-CSF receptor and participates in the regulation of myeloid lineage differentiation, proliferation, and survival.

MicroRNAs 17-5p/20a/106a Function as a Master Gene Complex Controlling Monocytopoiesis through AML1 Targeting.

MicroRNAs (miRs) are a novel regulatory class of non-coding, single-stranded RNAs of ~ 22 nucleotides, identified in plants and animals. MiRs repress protein expression at post-transcriptional level, mostly through base pairing to the 3′ untranslated region (UTR) of the target mRNA, thus leading to its degradation and/or reduced translation. MiRs have been shown to control basic biological functions, such as cell proliferation and differentiation. However, little is known on their role in hematopoiesis. Furthermore, in diverse types of cancer miRs may function as “oncomirs”.We investigated the role of miR-17-5p, -20a and -106a, located in the miR-17-5p-92 and miR-106a-92 clusters respectively, in monocytic-macrophage (Mo) differentiation-maturation. In unilineage Mo culture generated by cord blood (CB) hematopoietic progenitor cells (HPC), these miRs are gradually and markedly downmodulated, whereas AML1 is upmodulated at protein but not at mRNA level. The acute myeloid leukaemia-1 protein (AML1, also known as Runt-related transcription factor 1, Runx1) is the DNA-binding subunit of the hematopoietic transcription factor CBF, which plays a pivotal role in Mo differentiation-maturation. Luciferase assay shows that miR-17/20/106 bind the AML1 mRNA 3′UTR, implying that during monocytopoiesis their decline unblocks AML1 translation. Mo culture transfection with miR-17/20/106 stimulates blast cell proliferation and inhibits differentiation-maturation: this is coupled with AML1 translation blockade, leading in turn to inhibition of M-CSF receptor (M-CSFR) expression. Equivalent results were observed in Mo clonogenic culture of HPCs. Knockdown of endogenous miR-17/20/106 by anti-miR oligonucleotide transfection causes opposite effects. Altoghether, our studies indicate that miR-17/20/106 downmodulation during monocytopoiesis “unblocks” the translation of AML1, which in turn transactivates the M-CSFR, thereby allowing Mo differentiation and maturation. We hence propose that these miRs function as a master gene complex underlying the monocytopoiesis gene program, at least in part through the regulation of two pivotal proteins, AML1 and M-CSFR.This study also sheds light on other relevant aspects:the regulatory action of miR-17/20/106 action on AML1 mRNA is restricted to the Mo lineage, implying a role for lineage-specific factor(s) to modulate the interaction of these miRs with their mRNA target;the subtle complexity of mechanisms controlling the transcription/processing of the miR-17-5p-92 cluster, implied by the differential expression patterns of the miRs comprised in the 17-5p-92 polycistron in Mo culture;the leukemogenesis mediated by AML1 fusion proteins.

PU.1 and C/EBPalpha Expression in Acute Myeloid Leukemia.

The myeloid transcription factors C/EBPalpha and PU.1 play a pivotal role in normal hematopoiesis and alterations of their function are involved in the pathogenesis of Acute Myeloid Leukemia (AML). So far, different mechanisms have been shown to affect their function and are important in some AML subsets. However most AML patients do not apparently show any alteration of these transcription factors. Here, we investigated C/EBPalpha and PU.1 mRNA levels by real time RT-PCR in 109 AML patients and correlated these data to morphology, FLT3 mutations and cytogenetics. C/EBPalpha and PU.1 levels were expressed as percentage of 18S. Twelve normal bone marrow mononuclear cells, four CD34+ cells isolated from normal bone marrow samples and 8 peripheral blood granulocytes and monocytes, were used as controls.Heterogeneous PU.1 expression was observed in AML patients (median 0.657, range 0.004 – 24.148), while PU.1 levels were more homogeneous in normal bone marrows (median 1.5, range 0.328 – 4.737). In particular, 55 AML patients (50.5%) had PU.1 levels similar to controls, while 37 patients (33.9%) and 17 patients (15.6%) expressed PU.1 levels at levels lower and higher, than the control range, respectively. In the same way, also C/EBPalpha mRNA expression was variable (median 0.047, range 0.0002 – 1.858 in AML and median 0.064, range 0.008 – 0.138 in normal bone marrows). Fourty-five AML patients (41.%) displayed C/EBPalpha levels similar to the normal range, while 26 patients (23.8%) had lower and 37 (33.9%) higher C/EBPalpha expression.Looking at different AML subsets, we found low C/EBPalpha mRNA in patients carrying recurrent chromosomal abnormalities, such as t(8;21) and inv16, as previously reported. On the other hand, patients carrying 11q23 rearrangements showed higher PU.1 levels than normal controls. No association was found between C/EBPalpha and PU.1 levels and therapy-related AML, AML with normal karyotype, AML with multilineage dysplasia, and AML not otherwise characterized (including previous F.A.B. categories).Although experimental models showed that FLT3 internal tandem duplications (ITD) downregulate both transcription factors, we did not find any association between the presence of FLT3 ITD and D835 mutations and C/EBPalpha and PU.1 levels, both in the whole patient group and in patients with normal karyotype.We then analyzed expression of two PU.1 and C/EBPalpha target genes, the M-CSF and G-CSF receptors, in patients expressing high and low levels of these transcription factors. A direct correlation was found between C/EBPalpha and G-CSFR levels (Spearman r = 0.5; p=0.02, 95% C.I. 0.07 – 0.78), while there was a tendency to correlation between PU.1 and M-CSFR, that did not reach the statistical significance.Since mutations and post-trascriptional events may affect C/EBPalpha and PU.1 function, we analyzed protein expression of 18 patients by Western Blotting. PU.1 protein was expressed by all patients. The functional p42 C/EBPalpha isoform was absent in 2 patients that expressed only the 30 kDa isoform, and was undetectable in 5 of 18 patients.In conclusion, down regulation of PU1 mRNA was found in one third of AML patients, consistently with the oncosuppressive role recently described. On the other side, C/EBPalpha is down-regulated in specific AML subsets, with recurrent cytogenetic abnormalities, while mutations and post-translational events could affect C/EBPalpha expression in other patients.

MicroRNA Function in Human Hematopoiesis: Identification of Lineage- and Stage-Specific Expression Profiles, Pivotal Targets and Regulatory Circuitries.

MicroRNAs (miRs) play a key role in the regulation of gene expression in normal and abnormal cell growth/differentiation, via suppression of translation or degradation of target mRNAs mainly by base pairing to their 3′ UTR sequences. However, little is known on miR expression/function in normal hematopoiesis. We have assayed the level of miRs in serum-free unilineage culture of cord blood (CB) CD34+ hematopoietic progenitor cells (HPC) through the erythroid (E), megakaryocytic (Mk), granulocytic (G) and monocytic (Mo) series at discrete stages of differentiation up to terminal maturation. The assay included control CB T cells. The analysis was performed by microarray and confirmed by Northern blot. Altogether, 49 miRs are expressed at significant levels in hematopoiesis. Cluster analysis indicates lineage-specific expression patterns. Furthermore, miR levels are mostly downmodulated through HPC differentiation/maturation. A group of 12 single or clustered miRs is sharply modulated (> 10-fold) in hematopoiesis according to specific patterns, thus suggesting their key functional role. This group includes:miR-223, downmodulated in the E series, but upmodulated in the other lineages;miR-221/222, miR-146 and miR-155 declining more sharply in E than other lineages;miR-17/20/106 and miR-25, miR-32, declining prevailingly in the G and Mo series;a miR group showing little downmodulation in the Mk or Mo lineages (miR-123, -130a or miR-181a, b, respectively);miR-99b, -100 downmodulated in all lineages;miR-210 upmodulated in all lineages.Bioinformatic analysis suggests that these miRs have important targets, which have been in part verified by the luciferase target assay (see below). Relevant examples include miR-221/222 and -130a targeting kit receptor, miR-146 targeting SDFR/CXCR4, miR 181a, b, 99b and 100, 210 targeting diverse HOX genes (respectively, HOXB5, A1 and A3). Novel miR-based regulatory circuitries have been identified:(a) miR-221/222 modulate erythropoietic and erythroleukemia cell growth via kit receptor targeting (N. Felli et al, PNAS, 2005);(b) miR-17-5p/20a/106 function as a master gene complex controlling monocytopoiesis through AML1 targeting, leading to M-CSF receptor transactivation (L. Fontana et al, this Meeting);miR-155 and -221/222 control megakaryopoiesis at progenitor and precursor level through Ets-1 multitargeting (G. Marziali et al, this Meeting).

Pivotal Advance: Vasoactive intestinal peptide inhibits up-regulation of human monocyte TLR2 and TLR4 by LPS and differentiation of monocytes to macrophages

Vasoactive intestinal peptide (VIP) is an immunoregulatory peptide, which inhibits LPS-induced cytokine secretion in myeloid cells and has beneficial effects in animal models of inflammatory diseases. We show for the first time that VIP decreases LPS-induced up-regulation of TLR2 and TLR4 by human monocytic THP1 cells and peripheral blood monocytes (PBM). VIP inhibited up-regulation of TLR4 expression in THP1 cells in response to LPS from Escherichia coli or the periodontal pathogen Porphyromonas gingivalis within 6 h poststimulation but had less of an effect on TLR2. After 24 h, P. gingivalis LPS-stimulated monocytic THP1 cells to differentiate into macrophages, which predominantly expressed TLR2, and E. coli LPS-stimulated THP1 differentiation to predominantly TLR4-expressing macrophages. VIP decreased monocyte differentiation to macrophages induced by LPS from either species and also reduced overall TLR2 and TLR4 expression in these cells. VIP had a similar effect on human PBM. The transcription factor PU.1 regulates TLR expression and has a central role in myeloid cell differentiation. VIP inhibited the nuclear translocation of PU.1 in LPS-stimulated THP-1 monocytes. VIP also inhibited the expression of the M-CSF receptor, which is regulated by PU.1. In summary, VIP inhibited LPS-induced differentiation of monocytes with a concomitant reduction in TLR2 and TLR4 expression. Although there was differential induction of TLR expression by LPS from P. gingivalis and E. coli, VIP inhibited the action of both of these LPS types on monocytes. The mechanism of action of VIP on monocyte differentiation may be via inhibition of the transcription factor PU.1.

Ability of breast cancer cell lines to stimulate bone resorbing activity of mature osteoclasts correlates with an anti-apoptotic effect mediated by macrophage colony stimulating factor

We compared the effect of conditioned medium (CM) from several human breast carcinoma cell lines on osteoclast bone resorbing activity and osteoclast apoptosis. Our findings indicate that ability of cancer cell line to increase the in vitro bone resorbing activity is linked to their potential to inhibit osteoclast apoptosis. Cancer cells producing the higher level of M-CSF have the higher osteolytic activity, suggesting that M-CSF originating from cancer cells may contribute, at least in part, to the osteoclast activity at the metastatic site by enhancing their survival. Given that M-CSF plays an important role in the anti-apoptotic effect, we speculated that blocking M-CSF pathway would prevent the CM effects. Small interfering RNA (siRNA) targeting M-CSF and imatinib, a protein tyrosine kinase inhibitor targeting M-CSF receptor, almost completely reversed the CM effect on both osteoclast apoptosis and bone resorption. Blockade of M-CSF pathway could be thus of clinical value in the treatment of breast cancer related bone destruction.

Toll-like Receptors on Hematopoietic Progenitor Cells Stimulate Innate Immune System Replenishment

Toll-like receptors (TLRs) are best known for their ability to recognize microbial or viral components and initiate innate immune responses. We showed here that TLRs and their coreceptors were expressed by multipotential hematopoietic stem cells, whose cell cycle entry was triggered by TLR ligation. TLR expression also extended to some of the early hematopoietic progenitors, although not the progenitor cells dedicated to megakaryocyte and erythroid differentiation. TLR signaling via the Myd88 adaptor protein drove differentiation of myeloid progenitors, bypassing some normal growth and differentiation requirements, and also drove lymphoid progenitors to become dendritic cells. CD14 contributed to the efficiency of lipopolysaccharide (LPS) recognition by stem and progenitor cells, and LPS interacted directly with the TLR4/MD-2 complex on these cells in bone marrow. Thus, the preferential pathogen-mediated stimulation of myeloid differentiation pathways may provide a means for rapid replenishment of the innate immune system during infection.

Th-W46:5 Oxidized LDL induces monocyte-to-macrophage differentiation through activation of the M-CSF receptor

Th-W46:5 Oxidized LDL induces monocyte-to-macrophage differentiation through activation of the M-CSF receptor