CSF-1 Signaling Research Articles

The promotion of breast cancer metastasis caused by inhibition of CSF-1R/CSF-1 signaling is blocked by targeting the G-CSF receptor.

Treatment options are limited for patients with breast cancer presenting with metastatic disease. Targeting of tumor-associated macrophages through the inhibition of colony-stimulating factor-1 receptor (CSF-1R), a key macrophage signaling pathway, has been reported to reduce tumor growth and metastasis, and these treatments are now in clinical trials. Here, we report that, surprisingly, treatment with neutralizing anti-CSF-1R and anti-CSF-1 antibodies, or with two different small-molecule inhibitors of CSF-1R, could actually increase spontaneous metastasis without altering primary tumor growth in mice bearing two independently derived mammary tumors. The blockade of CSF-1R or CSF-1 led to increased levels of serum G-CSF, increased frequency of neutrophils in the primary tumor and in the metastasis-associated lung, as well as increased numbers of neutrophils and Ly6C(hi) monocytes in the peripheral blood. Neutralizing antibody against the G-CSF receptor, which regulates neutrophil development and function, reduced the enhanced metastasis and neutrophil numbers that resulted from CSF-1R blockade. These results indicate that the role of the CSF-1R/CSF-1 system in breast cancer is far more complex than originally proposed, and requires further investigation as a therapeutic target.

Development and characterisation of monoclonal antibodies reactive with porcine CSF1R (CD115)

Macrophage colony-stimulating factor (CSF1) controls the proliferation and differentiation of cells of the mononuclear phagocyte system. CSF1, alongside a second ligand, interleukin-34 (IL-34), acts by binding to a cell surface receptor (CSF1R). We previously cloned and expressed pig CSF1 and IL-34. Here we produced a pig CSF1R-Ig+pFUSE Fc fusion protein and used it as an immunogen to produce three monoclonal antibodies (ROS8G11, ROS3A5 and ROS3B10) targeted against porcine CSF1R. Specific binding of each monoclonal antibody was confirmed by ELISA, Western blot, flow cytometry and immunocytochemistry. The antibodies did not block CSF1 signalling. The surface expression of CSF1R in pig peripheral blood was restricted to CD14-positive monocytes and was also detected on lung macrophages. These antibodies provided an opportunity to investigate the increase of available CSF1R during pig BMDM differentiation. The new monoclonal antibodies provide useful reagents to support the study of monocyte and macrophage biology in the pig.

CSF1 over‐expression has pleiotropic effects on microglia in vivo (844.13)

Increased microglial numbers and activated microglial phenotypes are associated with many central nervous system (CNS) disorders including gliomas, neurodegeneration and brain injury. Thus, microglia are being tested as potential drug targets. Macrophage Colony Stimulating Factor (CSF1), an important cytokine for microglia and macrophages, is upregulated in these CNS disorders, but the effects of CSF1 on microglial phenotypes have not been well‐characterized in vivo. To study the effects of CSF1 over‐expression on microglia in the unperturbed CNS in vivo, we generated a transgenic mouse model in which CSF1 is inducibly over‐expressed in the GFAP compartment. We found that CSF1 over‐expression results in increased microglial proliferation, increased microglial numbers and an altered microglial phenotype compared to control mice. Microglia in CSF1 over‐expressing mice do not exhibit gene expression profiles typical of either M1 or M2 polarization, however they are defective in their response to the inflammatory stimulus LPS. Treatment with a small molecule inhibitor of the CSF1 receptor (CSF1R) and related kinases, decreased microglial numbers through apoptosis in both CSF1 over‐expressing and control mice. These results indicate that CSF1 signaling has multiple effects on microglia in vivo, and pharmacological inhibition of this signaling pathway may have efficacy in reversing the pathological levels of microglial accumulation in various CNS diseases by promoting microglial apoptosis. Moreover, our CSF1 over‐expressing genetic model should have utility for studying the impact of CSF1 over‐expression in mouse models of human disease.Grant Funding Source: Goldhirsh Foundation, the University of Wisconsin Graduate School and the NIH

Role of alveolar macrophages in chronic obstructive pulmonary disease.

Alveolar macrophages (AMs) represent a unique leukocyte population that responds to airborne irritants and microbes. This distinct microenvironment coordinates the maturation of long-lived AMs, which originate from fetal blood monocytes and self-renew through mechanisms dependent on GM-CSF and CSF-1 signaling. Peripheral blood monocytes can also replenish lung macrophages; however, this appears to occur in a stimuli specific manner. In addition to mounting an appropriate immune response during infection and injury, AMs actively coordinate the resolution of inflammation through efferocytosis of apoptotic cells. Any perturbation of this process can lead to deleterious responses. In chronic obstructive pulmonary disease (COPD), there is an accumulation of airway macrophages that do not conform to the classic M1/M2 dichotomy. There is also a skewed transcriptome profile that favors expression of wound-healing M2 markers, which is reflective of a deficiency to resolve inflammation. Endogenous mediators that can promote an imbalance in inhibitory M1 vs. healing M2 macrophages are discussed, as they are the plausible mechanisms underlying why AMs fail to effectively resolve inflammation and restore normal lung homeostasis in COPD.

Production and characterisation of a monoclonal antibody that recognises the chicken CSF1 receptor and confirms that expression is restricted to macrophage-lineage cells

Macrophages contribute to innate and acquired immunity as well as many aspects of homeostasis and development. Studies of macrophage biology and function in birds have been hampered by a lack of definitive cell surface markers. As in mammals, avian macrophages proliferate and differentiate in response to CSF1 and IL34, acting through the shared receptor, CSF1R. CSF1R mRNA expression in the chicken is restricted to macrophages and their progenitors. To expedite studies of avian macrophage biology, we produced an avian CSF1R-Fc chimeric protein and generated a monoclonal antibody (designated ROS-AV170) against the chicken CSF1R using the chimeric protein as immunogen. Specific binding of ROS-AV170 to CSF1R was confirmed by FACS, ELISA and immunohistochemistry on tissue sections. CSF1 down-regulated cell surface expression of the CSF1R detected with ROS-AV170, but the antibody did not block CSF1 signalling. Expression of CSF1R was detected on the surface of bone marrow progenitors only after culture in the absence of CSF1, and was induced during macrophage differentiation. Constitutive surface expression of CSF1R distinguished monocytes from other myeloid cells, including heterophils and thrombocytes. This antibody will therefore be of considerable utility for the study of chicken macrophage biology.

CSF-1 receptor-mediated differentiation of a new type of monocytic cell with B cell-stimulating activity: its selective dependence on IL-34.

With the use of a mouse FDC line, FL-Y, we have been analyzing roles for FDCs in controlling B cell fate in GCs. Beside these regulatory functions, we fortuitously found that FL-Y cells induced a new type of CD11b⁺ monocytic cells (F4/80⁺, Gr-1⁻, Ly6C⁻, I-A/E(-/lo), CD11c⁻, CD115⁺, CXCR4⁺, CCR2⁺, CX₃CR1⁻) when cultured with a Lin⁻c-kit⁺ population from mouse spleen cells. The developed CD11b⁺ cells shared a similar gene-expression profile to mononuclear phagocytes and were designated as FDMCs. Here, we describe characteristic immunological functions and the induction mechanism of FDMCs. Proliferation of anti-CD40 antibody-stimulated B cells was markedly accelerated in the presence of FDMCs. In addition, the FDMC-activated B cells efficiently acquired GC B cell-associated markers (Fas and GL-7). We observed an increase of FDMC-like cells in mice after immunization. On the other hand, FL-Y cells were found to produce CSF-1 as well as IL-34, both of which are known to induce development of macrophages and monocytes by binding to the common receptor, CSF-1R, expressed on the progenitors. However, we show that FL-Y-derived IL-34, but not CSF-1, was selectively responsible for FDMC generation using neutralizing antibodies and RNAi. We also confirmed that FDMC generation was strictly dependent on CSF-1R. To our knowledge, a CSF-1R-mediated differentiation process that is intrinsically specific for IL-34 has not been reported. Our results provide new insights into understanding the diversity of IL-34 and CSF-1 signaling pathways through CSF-1R.

CSF-1 signalling is involved in repair following AKI

CSF-1 signalling is involved in repair following AKI

CSF-1 signaling mediates recovery from acute kidney injury

Renal tubule epithelia represent the primary site of damage in acute kidney injury (AKI), a process initiated and propagated by the infiltration of macrophages. Here we investigated the role of resident renal macrophages and dendritic cells in recovery from AKI after ischemia/reperfusion (I/R) injury or a novel diphtheria toxin-induced (DT-induced) model of selective proximal tubule injury in mice. DT-induced AKI was characterized by marked renal proximal tubular cell apoptosis. In both models, macrophage/dendritic cell depletion during the recovery phase increased functional and histologic injury and delayed regeneration. After I/R-induced AKI, there was an early increase in renal macrophages derived from circulating inflammatory (M1) monocytes, followed by accumulation of renal macrophages/dendritic cells with a wound-healing (M2) phenotype. In contrast, DT-induced AKI only generated an increase in M2 cells. In both models, increases in M2 cells resulted largely from in situ proliferation in the kidney. Genetic or pharmacologic inhibition of macrophage colony-stimulating factor (CSF-1) signaling blocked macrophage/dendritic cell proliferation, decreased M2 polarization, and inhibited recovery. These findings demonstrated that CSF-1-mediated expansion and polarization of resident renal macrophages/dendritic cells is an important mechanism mediating renal tubule epithelial regeneration after AKI.

Donor and Recipient Cell Surface Colony Stimulating Factor-1 Promote Neointimal Formation in Transplant-Associated Arteriosclerosis

Transplant-associated arteriosclerosis manifests as progressive vascular neointimal expansion throughout the arterial system of allografted solid organs, and eventually compromises graft perfusion and function. Allografts placed in colony stimulating factor (CSF)-1-deficient osteopetrotic (Csf1(op)/Csf1(op)) mice develop very little neointima, a finding attributed to impaired recipient macrophage function. We examined how CSF-1 affects neointima-derived vascular smooth muscle cells, tested the significance of CSF-1 expressed in donor tissue, and evaluated the contribution of secreted versus cell surface CSF-1 isoforms in transplant-associated arteriosclerosis. CSF-1 activated specific signaling pathways to promote migration, survival, and proliferation of cultured vascular smooth muscle cells. Tumor necrosis factor-α addition increased CSF-1 and CSF-1 receptor expression, and tumor necrosis factor-α-driven proliferation was blocked by anti-CSF-1 antibody. In a mouse vascular allograft model, lack of recipient or donor CSF-1 impaired neointima formation; the latter suggests local CSF-1 function within the allograft. Moreover, reconstitution of donor or recipient cell surface CSF-1, without secreted CSF-1, restored neointimal formation. Vascular smooth muscle cells activation, including that mediated by tumor necrosis factor-α, can be driven in an autocrine/juxtacrine manner by CSF-1. These studies provide evidence for local function of CSF-1 in neointimal expansion, and identify CSF-1 signaling in vascular smooth muscle cells, particularly cell surface CSF-1 signaling, as a target for therapeutic strategies in transplant-associated arteriosclerosis.

Allosteric competitive inactivation of hematopoietic CSF-1 signaling by the viral decoy receptor BARF1

Hematopoietic human colony-stimulating factor 1 (hCSF-1) is essential for innate and adaptive immunity against viral and microbial infections and cancer. The human pathogen Epstein-Barr virus secretes the lytic-cycle protein BARF1 that neutralizes hCSF-1 to achieve immunomodulation. Here we show that BARF1 binds the dimer interface of hCSF-1 with picomolar affinity, away from the cognate receptor-binding site, to establish a long-lived complex featuring three hCSF-1 at the periphery of the BARF1 toroid. BARF1 locks dimeric hCSF-1 into an inactive conformation, rendering it unable to signal via its cognate receptor on human monocytes. This reveals a new functional role for hCSF-1 cooperativity in signaling. We propose a new viral strategy paradigm featuring an allosteric decoy receptor of the competitive type, which couples efficient sequestration and inactivation of the host growth factor to abrogate cooperative assembly of the cognate signaling complex.

Autocrine CSF-1 and CSF-1 Receptor Coexpression Promotes Renal Cell Carcinoma Growth

Renal cell carcinoma is increasing in incidence but the molecular mechanisms regulating its growth remain elusive. Coexpression of the monocytic growth factor colony-stimulating factor (CSF)-1 and its receptor CSF-1R on renal tubular epithelial cells (TEC) will promote proliferation and antiapoptosis during regeneration of renal tubules. Here, we show that a CSF-1-dependent autocrine pathway is also responsible for the growth of renal cell carcinoma (RCC). CSF-1 and CSF-1R were coexpressed in RCCs and TECs proximally adjacent to RCCs. CSF-1 engagement of CSF-1R promoted RCC survival and proliferation and reduced apoptosis, in support of the likelihood that CSF-1R effector signals mediate RCC growth. In vivo CSF-1R blockade using a CSF-1R tyrosine kinase inhibitor decreased RCC proliferation and macrophage infiltration in a manner associated with a dramatic reduction in tumor mass. Further mechanistic investigations linked CSF-1 and epidermal growth factor signaling in RCCs. Taken together, our results suggest that budding RCC stimulates the proximal adjacent microenvironment in the kidney to release mediators of CSF-1, CSF-1R, and epidermal growth factor expression in RCCs. Furthermore, our findings imply that targeting CSF-1/CSF-1R signaling may be therapeutically effective in RCCs.

Abstract 1131: Differential induction of c-fms promoters by TGFβ in cervical cancer and macrophage cell lines

Abstract Cervical cancer is the second most common gynecological malignancy in the world. An expected 12,200 total new diagnosed cases were estimated by the American Cancer Society for 2010 (Cancer Facts and Figures, 2010). Human papillomavirus infection, a major cause for cervical cancer, results in a persistent and pathological inflammatory response, contributing to establishment of disease and malignant progression. Our previous studies have shown that inflammatory factors, like Cox2, tumor associated macrophages, and cytokines such as macrophage colony-stimulating factor (CSF-1) and transforming growth factor beta (TGFβ) are elevated in cervical carcinomas. The receptors for CSF-1 (encoded by the c-fms oncogene) and TGFβ were also elevated in cervical carcinomas compared to normal cervical tissue. Both CSF-1 and c-fms have been implicated in aggressive gynecological malignancies, including cervical cancer. CSF-1/c-fms signaling is involved in proliferation, differentiation and recruitment of macrophages as well as cancer cell growth. We have previously demonstrated that TGFβ induced the expression of c-fms mRNA in cervical cancer cell lines and macrophage cell line THP-1. Using flow cytometry in this study, we have confirmed induction of c-fms protein at the cell surface due to TGFβ treatment, suggesting that TGFβ may enhance CSF-1 signaling by inducing cell surface expression of its receptor. Our goal was to examine the mechanisms underlying the regulation of c-fms in macrophages, relevant leukocytes associated with inflammation and pro-malignant growth within the tumor microenvironment. Two promoters for the c-fms gene exist, promoter one driving expression in trophoblasts (trophoblast specific promoter) and promoter two driving expression in macrophages (macrophage specific promoter). Consistent with our previous findings, TGFβ induced c-fms expression via MAPK p38 mainly at the trophoblast specific promoter in cervical cancer cells, as shown by the use of the p38 inhibitor SB203580. Our data also suggest that Casein kinase 1 mediates c-fms expression via this promoter in cervical cancer cells. In the macrophage cell line THP-1, p38 mediated the expression of c-fms via both promoters, as demonstrated by using exon specific RT-PCR primer sets. The c-fms mRNA induced at the macrophage specific promoter is the major c-fms transcript in THP-1 cells and is likely to mainly contribute to c-fms expression in these cells. Understanding the regulation of c-fms in macrophages and cervical cancer cells may pave the way to targeting c-fms expression to interrupt aberrant CSF-1 signaling in cervical cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1131. doi:10.1158/1538-7445.AM2011-1131

Abstract 1349: Ablation of E2f3 in macrophages reduces the incidence of lung metastasis

Abstract The tumor stroma is predominantly composed of cells of the immune system, one of them are the tumor-associated macrophages (TAMs). Tumor macrophages are recruited to the tumor in response to cytokines secreted by the tumor epithelial cells. TAMs secrete proteases into the extracellular matrix (ECM) that contributes to tumor cell intravasation. Rb-E2F axis represents one of the important pathways involved during cell cycle and is deregulated in number of cancers. E2Fs have distinct roles in the control of cell proliferation, apoptosis and differentiation pathways. We have previously shown that E2F are important downstream targets in CSF-1 signaling cascade that are involved in macrophage survival and proliferation. Present study is aimed at exploring the link between conditional ablation of E2f3 in TAM and its effect on lung metastasis using MMTV-PyMT mouse model of breast cancer. Ablation of E2f3 in the tumor macrophages resulted in a reduction of lung metastasis without affecting tumor onset or macrophage numbers. Microarray analysis of E2f3 deleted TAMs showed an up-regulation in expression of genes involved during metastasis. Our results show that E2f3 regulates the physiological processes in TAMs that inhibit lung metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1349.

Therapeutic targeting of the c-fms oncogene diminishes the growth of breast cancer cells and ductal hyperplasia in the mammary gland of the CSF-1 transgenic mouse model.

Abstract Abstract #3120 The macrophage colony-stimulating factor (CSF-1) is a cytokine produced by macrophages as well as epithelial cancer cells. By signaling via its receptor c-fms, CSF-1 induces growth/survival pathways in breast cancer cells. Defects in mammary gland development were observed in mice deficient in CSF-1/c-fms signaling, implicating this pathway in normal mammary development as well. In breast cancer, elevated levels of both CSF-1 and c- fms have been detected, with 80% of invasive breast carcinomas abnormally expressing c-fms. To study the role of CSF-1 signaling in breast cancer, we have previously generated mammary specific transgenic mouse models that over-express these factors (termed MMTV-CSF-1 and MMTV-c-fms). Both these transgenic mouse strains developed ductal hyperplasia and dysplasia as well as tumor formation. Our previous evidence also showed increase in cellular proliferation factors such as Cyclin D1 and PCNA in these mice. To examine which signaling pathways are altered in these mice, we have compared in this study the expression profile of target genes of signaling pathways in MMTV-CSF-1 mice and wild type mice using the Signal Transduction Pathway Finder RT-PCR array (Superarrray). Wnt signaling pathway was induced in the mammary gland of the MMTV-CSF-1 mouse strain compared to wild type, suggesting that this pathway may mediate CSF-1 signaling. In a proof-of-principle experiment, an MMTV-CSF-1 mouse was injected subcutanuously with neutralizing anti-c-fms antibody (Santacruz Biotech, 2-4A5) at a dose of 5 μg daily for 7 days. The treated mouse exhibited a decrease in the elevated ductal branching and hyperplasia that are characteristics of untreated MMTV-CSF-1 mice. This treatment also resulted in reversing many of expression alterations in MMTV-CSF-1 mice. For example, expression of the CSF-1 responsive gene Cyclin D1 was reduced by over 3-fold due to antibody treatment. Fas, the receptor for the pro-apoptotic factor Fas ligand, decreased by 5-folds in the MMTV-CSF-1 strain relative to wild type but increased 14-folds due to c-fms antibody treatment. The expression of the proapoptotic factor Bax increased about 4-fold, whereas the angiogenic factor Angiopoietin 1 was reduced by 60% due to anti-cfms antibody treatment. The treatment also reversed alterations in the expression of the wnt transcription factors Lef-1/TCF7. Because small molecule tyrosine kinase inhibitors that target c-fms, Gleevecâ (Imatinib, obtained from Novartis) and GW2580 (Calbiochem), are emerging, we examined their effects on the growth of SKBR3 breast cancer cells that express both CSF-1 and c-fms. Our data showed both these drugs inhibited the growth of SKBR3 cells, providing the rationale to test their effects on tumor formation and underlying biochemical effects in our transgenic preclinical models. The current studies warrant further investigation of treating breast cancer using antibody and small molecule drugs targeting CSF-1/c-fms signaling. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3120.

Effects of the cFMS Kinase Inhibitor 5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in Normal and Arthritic Rats

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005). In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.

Protein tyrosine phosphatase 1B negatively regulates macrophage development through CSF-1 signaling

Protein tyrosine phosphatase 1B (PTP-1B) is a ubiquitously expressed cytosolic phosphatase with the ability to dephosphorylate JAK2 and TYK2, and thereby down-regulate cytokine receptor signaling. Furthermore, PTP-1B levels are up-regulated in certain chronic myelogenous leukemia patients, which points to a potential role for PTP-1B in myeloid development. The results presented here show that the absence of PTP-1B affects murine myelopoiesis by modifying the ratio of monocytes to granulocytes in vivo. This bias toward monocytic development is at least in part due to a decreased threshold of response to CSF-1, because the PTP-1B -/- bone marrow presents no abnormalities at the granulocyte-monocyte progenitor level but produces significantly more monocytic colonies in the presence of CSF-1. This phenomenon is not due to an increase in receptor levels but rather to enhanced phosphorylation of the activation loop tyrosine. PTP-1B -/- cells display increased inflammatory activity in vitro and in vivo through the constitutive up-regulation of activation markers as well as increased sensitivity to endotoxin. Collectively, our data indicate that PTP-1B is an important modulator of myeloid differentiation and macrophage activation in vivo and provide a demonstration of a physiological role for PTP-1B in immune regulation.

The Colony-Stimulating Factor 1 Receptor Is Expressed on Dendritic Cells during Differentiation and Regulates Their Expansion

The lineage of dendritic cells (DC), and in particular their relationship to monocytes and macrophages, remains obscure. Furthermore, the requirement for the macrophage growth factor CSF-1 during DC homeostasis is unclear. Using a transgenic mouse in which the promoter for the CSF-1R (c-fms) directs the expression of enhanced GFP in cells of the myeloid lineage, we determined that although the c-fms promoter is inactive in DC precursors, it is up-regulated in all DC subsets during differentiation. Furthermore, plasmacytoid DC and all CD11c(high) DC subsets are reduced by 50-70% in CSF-1-deficient osteopetrotic mice, confirming that CSF-1 signaling is required for the optimal differentiation of DC in vivo. These data provide additional evidence that the majority of tissue DC is of myeloid origin during steady state and supports a close relationship between DC and macrophage biology in vivo.

Chromosomal location of murine Disabled-2 gene and structural comparison with its human ortholog

Disabled-2 (Dab2) is one of the two mammalian orthologs of the Drosophila Disabled. The three spliced forms, p96, p93, and p67 of murine Dab2 cDNAs were first isolated as phosphoproteins functioning in the macrophage CSF-1 signal transduction pathway. Subsequently, the involvement of Dab2 in ovarian cancer development has been investigated: Dab2 expression is lost or greatly diminished in breast and ovarian cancers, and gene deletions have been found. Regulation of Disabled-2 expression is also found to be important in development and physiological functions. Structural information of the murine Dab2 gene is essential for studies of transcription regulation and gene function in mouse models. In this study, the mouse Dab2 gene coding sequence was identified and sequenced from three lambda phage clones containing the gene. Two BAC clones of mouse genomic DNA were also used to identify the sequences of the non-coding first exon and promoter. The first exon is separated from the second exon by a large (15 kb) intron. The mouse gene is about 40 kb in size and consists of 15 exons, producing a 3.6 kb message. The translation initiation site resides in the middle of the second exon. The mouse Dab2 gene structure is very similar to that of its human ortholog in exon/intron sizes and promoter sequences. The chromosomal localization of mouse Dab2 was mapped by FISH to chromosome 15A2, a site of syntax with the human 5p12 where human Dab2 gene resides. The information on the mouse Dab2 gene structure and promoter will be invaluable in studies of the involvement of Dab2 gene in cancer, expression, physiological function, and development in mouse models.

CSF-1 signal transduction.

Colony-stimulating factor-1 (CSF-1) or macrophage-CSF (M-CSF) is a growth factor involved in the proliferation, differentiation, and activation of cells of the monocyte/macrophage lineage. Its receptor is the homodimeric, tyrosine kinase product of the c-fms proto-oncogene, which contains a so-called kinase insert domain. This review focuses mainly on recent studies of signal transduction events that are initiated on interaction of CSF-1 and its receptor. A summary is given of the tyrosine autophosphorylation sites on c-Fms identified to date, including their interaction with various substrates and their possible significance for signal transduction and cellular function. In addition, the signal transduction pathways that have been identified to lie downstream of activated c-Fms are reviewed. Although it is apparent that there have been many recent significant developments in our understanding of CSF-1 signaling, a number of examples are mentioned of significant discrepancies in the literature, some possible reasons for which can sometimes be offered. It is also apparent that any particular biochemical response or signal transduction pathway, even though widespread in other ligand receptor/cellular systems, including those with similar receptor structures to c-Fms, may not be relevant to CSF-1 signaling. The relevance of any potentially important molecular signaling pathway activated by CSF-1 in cells in vitro will ultimately have to be related to the functions of monocytes/macrophages in vivo.

Phosphatidylcholine signaling in response to CSF-1.

The formation of cell membrane following CSF-1 stimulation of a macrophage cell line is coordinated with cell cycle progression. The majority of membrane phospholipid accumulates during the S phase and results from cell-cycle dependent oscillations in the rates of phosphatidylcholine biosynthesis and degradation. Both synthesis and degradation are enhanced during the G1 phase, resulting in a high rate of phosphatidylcholine turnover. Degradation of phosphatidylcholine after CSF-1 stimulation is mediated by a phospholipase C, and the release of diacylglycerol during G1 phase is biphasic. The degradation essentially stops during the S phase, thus allowing biosynthesis to supply the necessary membrane for cell division and doubling. The degradation of phosphatidylcholine during G1 signals the downstream activation of c-fos and junB transcription and can be mimicked by incubation of the macrophage cells with exogenous bacterial phospholipase C. In contrast, the expression of c-myc transcripts normally associated with CSF-1 stimulation is severely compromised in phospholipase C-treated cells, indicating that the diacylglycerol signals a pathway distinct from the pathway that governs c-myc activation. Constitutive expression of c-myc complements phospholipase C activity and permits the growth of cells in the presence of exogenous bacterial enzyme and the absence of CSF-1. Protein kinase C is not required to mediate the diacylglycerol signal that supports cell growth. GTP exchange on Ras is not enhanced, and MAP kinase activity is not stimulated in response to phosphatidylcholine degradation by exogenous phospholipase C. The 85 kDa cytoplasmic phospholipase A2 is activated, however, as well as a novel protein we have called p96. Rapid serine phosphorylation of p96 follows stimulation of cells with either CSF-1 or exogenous phospholipase C. Analysis of the murine cDNA encoding p96 reveals an amino-terminal domain with significant similarity to the amino-terminal domain of the Drosophila-disabled gene product and a carboxy-terminal domain containing proline-rich sequences characteristic of SH3 binding regions. The sequence of p96 suggests an interactive role for this unique protein in the CSF-1 signal transduction cascade.