Pamela H. Correll

Fv2 encodes a truncated form of the Stk receptor tyrosine kinase

The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.

Macrophage-Stimulating Protein, the Ligand for the Stem Cell-Derived Tyrosine Kinase/RON Receptor Tyrosine Kinase, Inhibits IL-12 Production by Primary Peritoneal Macrophages Stimulated with IFN-γ and Lipopolysaccharide

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-γ by NK and γδT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d’origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-γ and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-γ and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON−/− animals produce increased levels of IFN-γ. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-γ and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.

Activation of the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase by macrophage-stimulating protein results in the induction of arginase activity in murine peritoneal macrophages.

Regulation of macrophage activities in response to inflammatory stimuli must be finely tuned to promote an effective immune response while, at the same time, preventing damage to the host. Our lab and others have previously shown that macrophage-stimulating protein (MSP), through activation of its receptor RON, negatively regulates NO production in response to IFN-γ and LPS by inhibiting the expression of inducible NO synthase (iNOS). Furthermore, activated macrophages from mice harboring targeted mutations in RON produce increased levels of NO both in vitro and in vivo, rendering them more susceptible to LPS-induced endotoxic shock. In this study, we demonstrate that stimulation of murine peritoneal macrophages with MSP results in the RON-dependent up-regulation of arginase, an enzyme associated with alternative activation that competes with iNOS for the substrate l-arginine, the products of which are involved in cell proliferation and matrix synthesis. Expression of other genes associated with alternative activation, including scavenger receptor A and IL-1R antagonist, is also up-regulated in MSP-stimulated murine macrophages. Stimulation of cells with IFN-γ and LPS blocks the ability of MSP to induce arginase activity. However, pretreatment of cells with MSP results in the up-regulation of arginase and inhibits their ability to produce NO in response to IFN-γ and LPS, even in the presence of excess substrate, suggesting that the inhibition of NO by MSP occurs primarily through its ability to regulate iNOS expression.

Sf-Stk kinase activity and the Grb2 binding site are required for Epo-independent growth of primary erythroblasts infected with Friend virus.

During the initial stage of Friend virus-induced erythroleukemia in mice, interaction of the viral protein gp55 with the erythropoietin receptor, and other host factors, drives the expansion of erythroid precursor cells. Recently, we demonstrated that the Friend virus susceptibility locus, Fv2, which is required for the expansion of infected cells, encodes a naturally occurring, N-terminally truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Here we show that in vitro expression of Sf-Stk confers Friend virus sensitivity to erythroid progenitor cells from Fv2rr mice. Furthermore, our data reveal that Sf-Stk kinase activity and Y436, but not Y429, are required for Epo-independent colony formation following Friend virus infection. Introduction of a mutation that results in failure to bind Grb2 abrogates the ability of Sf-Stk to induce colonies in response to Friend virus, while the Grb2 binding site from EGFR restores this response. Consistent with the ability of Grb2 to recruit SOS and Gab1, the Ras/MAPK and PI3K pathways are activated by Sf-Stk, and both of these pathways are required for gp55-mediated erythroblast proliferation. These data clearly demonstrate a requirement for signaling through Sf-Stk in the Epo-independent expansion of Friend virus-infected cells, and suggest a pivotal role for Grb2 in this response.

Receptor tyrosine kinases and the regulation of macrophage activation

It is becoming increasingly clear that macrophages are a diverse and dynamic population of cells that can be activated down a number of distinct developmental pathways (recently reviewed in refs. [1, 2]). These cells have the capacity to perform a wide range of critical functions including the recognition, phagocytosis, and clearance of invading pathogens through the expression of pattern recognition receptors (PRRs) and the up-regulation of cytotoxic molecules; immune modulation through the production of cytokines and chemokines, antigen presentation, and the regulation of T cell activation and differentiation; and the resolution of inflammation and the promotion of healing through induction of matrix synthesis, fibroblast proliferation, angiogenesis, and the clearance of cellular debris. To perform these disparate tasks, macrophages must be capable of inducing the expression of specific subsets of genes in a coordinated manner in response to environmental stimuli. The basal activity of tissue resident macrophages as well as their ability to respond to environmental stimuli vary considerably. This response must be tightly controlled to stimulate immunity to infection and at the same time, protecting host tissues from inflammatory damage and promoting normal tissue homeostasis. Here, we propose that receptor tyrosine kinases (RTKs) play a role in fine-tuning this system. Expression of at least three distinct families of RTKs has been reported in the monocyte/macrophage lineage. The receptor for macrophage colony-stimulating factor 1 (M-CSF-1R; c-fms) is widely expressed in the monocyte/macrophage lineage [3] and is required for the development of a number of tissue-resident macrophage populations [4]. This receptor is a member of the platelet-derived growth factor receptor (PDGFR) superfamily of RTKs, containing five immunoglobulin (Ig)-like domains in the extracellular portion of the receptor and a split kinase domain (Fig. 1A). In contrast, the Axl/Tyro3/Mer family of receptors [5, 6] contains two Ig-like domains and two fibronectin type III repeats in the ectodomain and a contiguous kinase domain with two tandem tyrosines in the activation loop. The murine STK/human RON receptor [7, 8], a member of the MET family of RTKs, is closely related to the Tyro3 family and shares a similar kinase structure. The ectodomain of STK, however, is composed of a disulfide-linked extracellular chain and transmembrane chain, the structure of which is poorly characterized. Recent studies using the MET receptor suggest that the c-terminal half of the extracellular domain contains four atypical Ig domains, and the N-terminal ligandbinding region adopts a -propeller fold similar to that displayed by the V integrin [9]. Although M-CSF has been implicated in the proliferation but not the activity of terminally differentiated macrophages [10], the Tyro3 family of receptors and STK appear to play a role in regulating the activation of these cells in response to environmental stimuli [11, 12]. Consistent with the activation of nearly all RTKs, the ligands for the Tyro3 family of RTKs (Gas6 and protein S [13–15]) and STK [macrophage-stimulating protein (MSP); ref. 16] bind to the extracellular domain of their respective receptors, resulting in the up-regulation of kinase activity. However, RTKs also appear to participate in the recognition of a wide range of exogenous and endogenous ligands. Listeria monocytogenes has been shown to gain entry into hepatocytes through the interaction of InlB with the Met receptor [17]. In addition, Tyro3 receptors play a direct role in the recognition of apoptotic cells through the interaction of Gas6 with phosphatidyl serine on the surface of apoptotic cells, and macrophages from Mer knockout (KO) mice are defective in the clearance of apoptotic thymocytes [18]. Alternatively, these RTKs can also regulate the expression or activity of classic PRRs. For example, signaling through the STK receptor regulates the activation of the M 2 integrin, complement receptor 3 (CR3), which recognizes a variety of exogenous and endogenous ligands, including C3bi, intercellular adhesion molecule-1 (ICAM-1), LPS, and zymosan. CR3 activation by the MSP/STK signaling pathway results in the phagocytosis of C3bi-coated erythrocytes and enhanced macrophage binding to ICAM-1 via a phosphatidylinositol-3 kinase (PI-3K)/protein kinase C-dependent mechanism [19]. In addition, MSP stimulation of primary peritoneal macrophages induces the expression of scavenger receptor A [20], which recognizes the exogenous ligands lipid A and lipoteichoic acid as well as oxidized low-density lipoprotein and apoptotic cells, resulting in enhanced uptake of acetylated LDL by these cells. Although the Tyro3 family of receptors is reported to be widely expressed by monocytes and their derivatives, expression of the STK receptor by monocyte/macrophage populations is highly regulated. STK/RON is not expressed on circulating monocytes, bone marrow-derived macrophages, splenic red

Activation of CR3-mediated phagocytosis by MSP requires the RON receptor, tyrosine kinase activity, phosphatidylinositol 3-kinase, and protein kinase C ζ

Macrophage‐stimulating protein (MSP) promotes the phagocytosis of C3bi‐coated erythrocytes by resident peritoneal macrophages, although the mechanism by which this occurs is largely unknown. We show that MSP‐induced complement‐mediated phagocytosis requires the RON receptor tyrosine kinase and the αMβ2 integrin, as evidenced by the inability of RON−/− and αM−/− peritoneal macrophages to augment phagocytosis of complement‐coated sheep erythrocytes in response to MSP. MSP stimulation of macrophages results in tyrosine phosphorylation and AKT activation, and inhibitor studies demonstrate a phagocytic requirement for tyrosine kinase and phosphatidylinositol 3‐kinase (PI‐3K) activity as well as activity of the atypical protein kinase C (PKC) isoform ζ, which localizes to MSP‐induced phagosomes containing complement‐coated beads. Additionally, MSP augments the ability of peritoneal macrophages to bind to intercellular adhesion molecule‐1 (ICAM‐1) via the αMβ2 integrin. MSP‐induced ICAM‐1 adhesion is also dependent on tyrosine kinase activity, PI‐3K, and PKC ζ, indicating that these signaling requirements are upstream of complement receptor 3 activation.

Gene trapping of two novel genes, Hzf and Hhl, expressed in hematopoietic cells

Using an expression gene trapping strategy, we have identified and characterized two novel hematopoietic genes, Hzf and Hhl. Embryonic stem (ES) cells containing a gene trap vector insertion were cultured on OP9 stromal cells to induce hematopoietic differentiation and screened for lacZ reporter gene expression. Two ES clones displaying lacZ expression within hematopoietic cells in vitro were used to generate mice containing the gene trap integrations. Paralleling this in vitro expression pattern, both Hzf and Hhl were expressed in a tissue-specific manner during hematopoietic development in vivo. Hzf encodes a novel protein containing three C(2)H(2)-type zinc fingers predominantly expressed in megakaryocytes and CFU-GEMM. Hhl encodes a novel protein containing a putative phosphotyrosine binding (PTB) domain expressed in megakaryocytes, CFU-GEMM and BFU-E. These results demonstrate the utility of expression trapping to identify novel hematopoietic genes. Future studies of Hzf and Hhl should provide valuable information on the role these genes play during megakaryocytopoiesis.

A novel Stat3 binding motif in Gab2 mediates transformation of primary hematopoietic cells by the Stk/Ron receptor tyrosine kinase in response to Friend virus infection.

ABSTRACT Friend erythroleukemia virus has long served as a paradigm for the study of the multistage progression of leukemia. Friend virus infects erythroid progenitor cells, followed by an initial polyclonal expansion of infected cells, which is driven by the activation of a naturally occurring truncated form of the Stk receptor tyrosine kinase (Sf-Stk). Subsequently, the accumulation of additional mutations in p53 and the activation of PU.1 result in full leukemic transformation. The early stages of transformation induced by Friend virus are characterized in vitro by the Epo-independent growth of infected erythroblasts. We have shown previously that this transforming event requires the kinase activity and Grb2 binding site of Sf-Stk and the recruitment of a Grb2/Gab2 complex to Sf-Stk. Here, we demonstrate that Stat3 is required for the Epo-independent growth of Friend virus-infected cells and that the activation of Stat3 by Sf-Stk is mediated by a novel Stat3 binding site in Gab2. These results underscore a central role for Stat3 in hematopoietic transformation and describe a previously unidentified role for Gab2 in the recruitment and activation of Stat3 in response to transforming signals generated by tyrosine kinases.

GRB2-mediated recruitment of GAB2, but not GAB1, to SF-STK supports the expansion of Friend virus-infected erythroid progenitor cells.

Friend virus induces the development of erythroleukemia in mice through the interaction of a viral glycoprotein, gp55, with a truncated form of the Stk receptor tyrosine kinase, short form-Stk (Sf-Stk), and the EpoR. We have shown previously that the ability of Sf-Stk to participate in the transformation of Friend virus-infected cells requires the kinase activity and Grb2-binding site of Sf-Stk. Here we show that Grb2 heterozygous mice exhibit decreased susceptibility to Friend erythroleukemia and that expansion of erythroid progenitors in response to infection requires the C-terminal SH3 domain of Grb2. A fusion protein in which the Grb2-binding site in Sf-Stk is replaced by Gab2, supports the growth of progenitors from mice lacking Sf-Stk, whereas a Sf-Stk/Gab1 fusion protein does not. Gab2 is expressed in spleens from Friend virus-infected mice, co-immunoprecipitates with Sf-Stk and is tyrosine phosphorylated in the presence of Sf-Stk. Mice with a targeted deletion in Gab2 are less susceptible to Friend erythroleukemia and the expansion of erythroid progenitor cells in response to infection can be rescued by expression of Gab2, but not Gab1. Taken together, these data indicate that a Sf-Stk/Grb2/Gab2 complex mediates the growth of primary erythroid progenitor cells in response to Friend virus.

RON Receptor Tyrosine Kinase, a Negative Regulator of Inflammation, Inhibits HIV-1 Transcription in Monocytes/Macrophages and Is Decreased in Brain Tissue from Patients with AIDS

Activation of macrophages and microglia cells after HIV-1 infection and their production of inflammatory mediators contribute to HIV-associated CNS diseases. The mechanisms that initiate and maintain inflammation after HIV-1 infection in the brain have not been well studied. Furthermore, it is not understood why in HIV-associated CNS disease, macrophages and microglia are biased toward inflammation rather than production of mediators that control inflammation. We have focused on the receptor tyrosine kinase RON, a critical negative regulator of macrophage function and inflammation, to determine whether this receptor regulates HIV-1 expression. Overexpressing RON in monocytes/macrophages demonstrates that RON inhibits HIV-1 proviral transcription in part by decreasing the binding activity of NF-κB to the HIV-1 long terminal repeat. Because macrophages and microglia cells are a critical reservoir for HIV-1 in the CNS, we examined brain tissues for RON expression and detected RON in astrocytes, cortical neurons, and monocytoid cells. RON was detected in all control patients who were HIV seronegative (n = 7), whereas six of nine brain samples obtained from AIDS patients exhibited reduced RON protein. These data suggest that RON initiates signaling pathways that negatively regulate HIV-1 transcription in monocytes/macrophages and that HIV-1 suppresses RON function by decreasing protein levels in the brain to assure efficient replication. Furthermore, HIV-1 infection would compromise the ability of RON to protect against inflammation and consequent CNS damage.