M-CSF Receptor Signaling Research Articles

Macrophage colony-stimulating factor potentially induces recruitment and maturation of macrophages in recurrent pituitary neuroendocrine tumors.

Although pituitary neuroendocrine tumors (PitNETs) are usually benign, some are highly invasive and recurrent. Recurrent PitNETs are often treatment-resistant and there is currently no effective evidence-based treatment. Tumor-associated macrophages (TAMs) promote tumor growth in many cancers, but the effect of TAMs on PitNETs remains unclear. This study investigated the role of TAMs in the incidence of recurrent PitNETs. Immunohistochemical analysis revealed that the densities of CD163- and CD204-positive TAMs tended to increase in recurrent PitNETs. Compared with TAMs in primary lesions, those in recurrent lesions were enlarged. To clarify the cell-cell interactions between TAMs and PitNETs, in vitro experiments were performed using a mouse PitNET cell line AtT20 and the mouse macrophage cell line J774. Several cytokines related to macrophage chemotaxis and differentiation, such as M-CSF, were elevated significantly by stimulation with macrophage conditioned medium. When M-CSF immunohistochemistry analysis was performed using human PitNET samples, M-CSF expression increased significantly in recurrent lesions compared with primary lesions. Although no M-CSF receptor (M-CSFR) expression was observed in tumor cells of primary and recurrent PitNETs, flow cytometric analysis revealed that the mouse PitNET cell line expressed M-CSFR. Cellular proliferation in mouse PitNETs was inhibited by high concentrations of M-CSFR inhibitors, suggesting that cell-to-cell communication between PitNETs and macrophages induces M-CSF expression, which in turn enhances TAM chemotaxis and maturation in the tumor microenvironment. Blocking the M-CSFR signaling pathway might be a novel therapeutic adjuvant in treating recurrent PitNETs.

M-CSF supports medullary erythropoiesis and erythroid iron demand following burn injury through its activity on homeostatic iron recycling

M-CSF receptor signaling supports the development and survival of mononuclear phagocytes and is thought to play a role in post burn anemia by promoting myeloid lineage bias. We found M-CSF secretion was increased in burn patients and a murine model of post burn ACI, so we neutralized M-CSF in ACI mice to determine if erythropoiesis was improved. Instead, M-CSF blockade further impaired erythropoiesis and erythroid cells access to iron. M-CSF blockade enhanced inflammatory cytokine secretion, further increased systemic neutrophil counts, and led to tissue iron sequestration that was dependent, in part, on augmented IL-6 secretion which induced hepcidin. Deleterious effects of post burn M-CSF blockade were associated with arrest of an iron recycling gene expression signature in the liver and spleen that included Spi-C transcription factor and heme oxygenase-1, which promote heme metabolism and confer a non-inflammatory tone in macrophages. Hepatic induction of these factors in ACI mice was consistent with a recovery of ferroportin gene expression and reflected an M-CSF dependent expansion and differentiation of Spi-C+ monocytes into Kupffer cells. Together, this data indicates M-CSF secretion supports a homeostatic iron recycling program that plays a key role in the maintenance of erythroid cells access to iron following burn injury.

Diabetes induces myeloid bias in bone marrow progenitors associated with enhanced wound macrophage accumulation and impaired healing.

Diabetes induces dysregulation throughout the spectrum of myeloid lineage cells from progenitors to terminally differentiated cells. Another complication of diabetes is persistent inflammation, including prolonged accumulation of macrophages, which contributes to impaired wound healing. However, it remains unclear whether diabetes disrupts the response of bone marrow progenitors to peripheral injury and whether such dysregulation leads to sustained inflammation and impaired healing. Here, we demonstrated that diabetic mice (db/db, referred to here as DB) exhibit myeloid lineage bias during homeostasis and following injury. In addition, cells in the LSK (Lin- Sca-1+ cKit+ ) population of DB mice are preprogrammed towards myeloid commitment at the transcriptional level, and cultured myeloid progenitors from DB mice produce more monocytes ex vivo than their non-diabetic counterparts. We also show via bone marrow transfer between interleukin-1 receptor 1 KO (Il1r1-/- ) and DB mice that IL-1R1 signaling is likely not involved in myeloid skewing in DB mice. Furthermore, in vitro experiments indicated that macrophage colony-stimulating factor receptor signaling is not likely involved in enhanced myeloid transcription factor expression in LSK cells of DB mice. Our findings indicate that myeloid lineage commitment in bone marrow may contribute to increased macrophage numbers observed in diabetic skin wounds, and that strategies to regulate monopoiesis during homeostasis or post-wounding may improve diabetic wound healing. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Potential anti-lymphoma effect of M-CSFR inhibitor in adult T-cell leukemia/lymphoma.

The c-fms proto-oncogene is also known as macrophage colony stimulating factor receptor (M-CSFR) or colony-stimulating factor-1 receptor (CSF-1R), and is expressed on several types of malignant tumor cells and myeloid cells. In the present study, we found that overexpression of M-CSFR was present in adult T-cell leukemia/lymphoma (ATLL) cases. M-CSFR signaling was associated with lymphoma cell proliferation, and M-CSFR inhibition induced apoptosis in lymphoma cells. The ATLL cell line ATL-T expressed M-CSF/CSF-1 and interleukin (IL)-34, which are both M-CSFR ligands. M-CSF and IL-34 expression was seen in ATLL cases, and co-expression of these ligands was detected in 11 of 13 ATLL cases. M-CSFR inhibition suppressed programmed death-1 and -2 ligand in ATL-T cells and macrophages stimulated with conditioned medium from ATL-T cells. Thus, an M-CSFR inhibitor may be useful as additional therapy against ATLL due to direct and indirect mechanisms.

Beyond the M-CSF receptor - novel therapeutic targets in tumor-associated macrophages.

Tumor-associated macrophages (TAM) are by now established as important regulators of tumor progression by impacting on tumor immunity, angiogenesis, and metastasis. Hence, a multitude of approaches are currently pursued to intervene with TAM's protumor activities, the most advanced of which being a blockade of macrophage-colony stimulating factor (M-CSF)/M-CSF receptor (M-CSFR) signaling. M-CSFR signaling largely impacts on the differentiation of macrophages, including TAM, and hence strongly influences the numbers of these cells in tumors. However, a repolarization of TAM toward a more antitumor phenotype may be more elegant and may yield stronger effects on tumor growth. In this respect, several aspects of TAM behavior could be altered, such as their intratumoral localization, metabolism and regulatory pathways. Intervention strategies could include the use of small molecules but also new generations of biologicals which may complement the current success of immune checkpoint blockers. This review highlights current work on the search for new therapeutic targets in TAM.

Functional Relationship between Tumor-Associated Macrophages and Macrophage Colony-Stimulating Factor as Contributors to Cancer Progression.

The current review article describes the functional relationship between tumor-associated macrophages (TAM) as key cellular contributors to cancer malignancy on the one hand and macrophage-colony-stimulating factor (M-CSF or CSF-1) as an important molecular contributor on the other. We recapitulate the available data on expression of M-CSF and the M-CSF receptor (M-CSFR) in human tumor tissue as constituents of a stromal macrophage signature and on the limits of the predictive and prognostic value of plasma M-CSF levels. After providing an update on current insights into the nature of TAM heterogeneity at the level of M1/M2 phenotype and TAM subsets, we give an overview of experimental evidence, based on genetic, antibody-mediated, and pharmacological disruption of M-CSF/M-CSFR signaling, for the extent to which M-CSFR signaling can not only determine the TAM quantity, but can also contribute to shaping the phenotype and heterogeneity of TAM and other related tumor-infiltrating myeloid cells (TIM). Finally, we review the accumulating information on the – sometimes conflicting – effects blocking M-CSFR signaling may have on various aspects of cancer progression such as tumor growth, invasion, angiogenesis, metastasis, and resistance to therapy and we thereby discuss in how far these different effects actually reflect a contribution of TAM.

The role of Src family kinases in mediating M-CSF receptor signaling and monocytic cell survival

Previously, we reported that M-CSF induced monocyte survival through the activation of Akt, p38MAPK and Erk1/2 kinases. Here, we found that Src family kinases were upstream of these kinases and played a central role in regulating M-CSF-induced monocyte survival. We observed that M-CSF promoted c-Src activation in monocytes and MDMs in a time-dependent manner. Src inhibitors reduced M-CSF-mediated phosphorylation of the M-CSF receptor (M-CSFR), Akt, Erk1/2, and p38 MAPK. We also observed that Src directly phosphorylated the M-CSFR. Notably, the inhibitors blocked phosphorylation of specific tyrosine residues within the M-CSFR. We further demonstrated that the Src inhibitor, PP2, attenuated M-CSF-induced NF-κB activation and M-CSF-induced monocyte survival. These findings indicated that Src family kinases mediate monocyte survival through the regulation of receptor phosphorylation and modulation of downstream signaling events. Thus, we predict that targeting Src family kinases may have therapeutic implication in inflammatory diseases.

THOC5 couples M-CSF receptor signaling to transcription factor expression

THOC5 is a nuclear/cytoplasmic protein member of the spliceosome complex which potentiates C/EBP expression in adipocyte differentiation. As C/EBP family members are important regulators of myelopoiesis and THOC5 is highly expressed in neutrophil/macrophage progenitor cells we assessed the role of THOC5 in cytokine-stimulated monocytic development. M-CSF stimulated maturation of the NFS60 cell line was associated with enhanced THOC5 expression and phosphorylation. THOC5 was also shown to form a complex with C/EBPβ. Ectopic expression of THOC5 mimicked M-CSF mediated cell maturation and enhanced protein expression of the myeloid transcription factors C/EBPβ, C/EBPα, Pu-1 and also GAB2 (a PI-3 Kinase and macrophage development regulator). Increased THOC5 expression also mimicked M-CSF stimulated increases in the lipid second messenger PtdInsP 3. Inhibition of THOC5-induced increases in PtdInsP 3 levels abrogated the elevated levels of C/EBPβ. Thus THOC5 expression can potentiate receptor signalling to transcription factor expression and monocyte differentiation.

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.

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.

Regulation of Osteoclast Apoptosis and Motility by Small GTPase Binding Protein Rac1

The role of Rac1 in osteoclast survival and bone-resorbing activity was examined using adenovirus vector expression systems. Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling primarily through PI3K/Akt pathways. Rac1 also plays a significant role in bone resorptive activity, probably by regulating the motility of osteoclasts. Rac1 is a member of Rho family small G-proteins, and recent studies have revealed that it mediates anti-apoptotic signals in some types of cells. Rac1 is reported to be required for the cytoskeletal organization and bone-resorbing activity of osteoclasts, but their roles in osteoclast survival and function are not fully elucidated. We constructed the adenovirus vector carrying cDNA of either the dominant negative Rac1 (Rac1(DN)) or constitutively active Rac1 (Rac1(CA)) gene, and osteoclast-like cells (OCLs) generated in mouse co-culture system were infected with these viruses. To examine the role of Rac1 in osteoclast survival and function, we performed pit formation assays, survival assays, and Western blotting, including an activated-Rac1 pull-down assay using adenovirus-infected OCLs. To further clarify the mechanism of Rac1 regulation in osteoclast survival, some specific inhibitors and adenovirus vectors of signal transduction molecules were used. To quantify membrane movement before and after macrophage colony-stimulating factor (M-CSF) treatment, OCLs expressing either enhanced green fluorescent protein (EGFP) or Rac1(DN) were recorded with a time-lapse video microscope. Adenovirus vector-mediated dominant negative Rac1 (Rac1(DN)) expression significantly reduced pit formation, and promoted their apoptosis. M-CSF rapidly activated Rac1, and the prosurvival effect of M-CSF for OCLs was abrogated by Rac1(DN) overexpression. Constitutively active Rac1 enhanced OCL survival, which was completely suppressed by phosphatidylinositol 3'-kinase (PI3K) inhibitors, whereas a Mek inhibitor had only partial effect. Rac1(DN) also partially blocked the activation of Akt induced by the overexpressing catalytic subunit of PI3K. Using time-lapse video microscopy, we found that Rac1(DN) expression reduced membrane ruffling and the spreading of OCLs in response to M-CSF. Small guanosine triphosphatase (GTPase) Rac1 is critically involved in M-CSF receptor signaling and mediates survival signaling of osteoclasts primarily by modulating PI3K/Akt pathways. Rac1 also plays a significant role in the bone resorptive activity of cells, probably by regulating the motility of osteoclasts.

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.

HIV-1 Nef interferes with M-CSF receptor signaling through Hck activation and inhibits M-CSF bioactivities

AbstractHIV-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 disease progression. Here, we show for the first time that Nef interferes with the macrophage colony-stimulating factor (M-CSF)/M-CSF receptor signal 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 the M-CSF receptor complex. The formation of the molecular complex should occur under physiologic conditions, that is, on M-CSF stimulation. Because of 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 signaling through Hck activation and thereby inhibits M-CSF functions in monocytes/macrophages. (Blood. 2005;105:3230-3237)

Signal Transduction in Macrophages: Negative Regulation for Macrophage Colony-Stimulating Factor Receptor Signaling

The receptor for macrophage colony-stimulating factor (M-CSF) is expressed in monocytes/macrophages and their progenitor cells and stimulates both the growth and development of the blood-cell lineage. Although the specific components positively regulating M-CSF receptor signaling have been relatively well defined, it is now clear that important mechanisms to control the signaling cascades also exist. This review discusses the most recent results concerning the negative regulatory molecules for M-CSF receptor signaling. In particular, we focus on negative molecules for both proliferation of monocytes/macrophages and differentiation into mature cells.

Early Events in M-CSF Receptor Signaling

The M-CSF receptor (M-CSFR) is expressed in monocytes-macrophages and their progenitors, and drives growth and development of this blood cell lineage. The M-CSFR is a member of a small family of growth factor receptors exhibiting related structures but distinct tissue-specific functions. This review discusses the early molecular events in the M-CSF signaling mechanisms, positive signals, negative signals, the possible organization of individual signaling pathways, and the problem of achieving specificity in the signal transduction mechanism.

The Low-Molecular-Weight Phosphotyrosine Protein Phosphatase, when Overexpressed, Reduces the Mitogenic Response to Macrophage Colony-Stimulating Factor and Tyrosine Phosphorylation of Its Receptor

The interference of low-molecular-weight phosphotyrosine protein phosphatase with the macrophage response to macrophage colony-stimulating factor was investigated. This paper shows that this phosphatase, already known to be involved in platelet-derived growth factor receptor signaling, is physiologically expressed in murine macrophages and dephosphorylatesin vitromacrophage colony-stimulating factor receptor molecules immunoprecipitated from macrophage colony-stimulating factor-stimulated macrophages. We obtained the first demonstration that a phosphotyrosine-specific protein phosphatase dephosphorylates the macrophage colony-stimulating factor receptorin vivoand reduces the mitogenic response to macrophage colony-stimulating factor. The data indicate that low-molecular-weight phosphotyrosine protein phosphatase is a negative regulator of macrophage colony-stimulating factor receptor signaling.