Holly Cherwinski

Raf-induced proliferation or cell cycle arrest is determined by the level of Raf activity with arrest mediated by p21Cip1.

The Raf family of protein kinases display differences in their abilities to promote the entry of quiescent NIH 3T3 cells into the S phase of the cell cycle. Although conditional activation of deltaA-Raf:ER promoted cell cycle progression, activation of deltaRaf-1:ER and deltaB-Raf:ER elicited a G1 arrest that was not overcome by exogenously added growth factors. Activation of all three deltaRaf:ER kinases led to elevated expression of cyclin D1 and cyclin E and reduced expression of p27Kip1. However, activation of deltaB-Raf:ER and deltaRaf-1:ER induced the expression of p21Cip1, whereas activation of deltaA-Raf:ER did not. A catalytically potentiated form of deltaA-Raf:ER, generated by point mutation, strongly induced p21Cip1 expression and elicited cell cycle arrest similarly to deltaB-Raf:ER and deltaRaf-1:ER. These data suggested that the strength and duration of signaling by Raf kinases might influence the biological outcome of activation of this pathway. By titration of deltaB-Raf:ER activity we demonstrated that low levels of Raf activity led to activation of cyclin D1-cdk4 and cyclin E-cdk2 complexes and to cell cycle progression whereas higher Raf activity elicited cell cycle arrest correlating with p21Cip1 induction and inhibition of cyclin-cdk activity. Using green fluorescent protein-tagged forms of deltaRaf-1:ER in primary mouse embryo fibroblasts (MEFs) we demonstrated that p21Cip1 was induced by Raf in a p53-independent manner, leading to cell cycle arrest. By contrast, activation of Raf in p21Cip1(-/-) MEFs led to a robust mitogenic response that was similar to that observed in response to platelet-derived growth factor. These data indicate that, depending on the level of kinase activity, Raf can elicit either cell cycle progression or cell cycle arrest in mouse fibroblasts. The ability of Raf to elicit cell cycle arrest is strongly associated with its ability to induce the expression of the cyclin-dependent kinase inhibitor p21Cip1 in a manner that bears analogy to alpha-factor arrest in Saccharomyces cerevisiae. These data are consistent with a role for Raf kinases in both proliferation and differentiation of mammalian cells.

Characterization of the CD200 Receptor Family in Mice and Humans and Their Interactions with CD200

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.

Molecular Mechanisms of CD200 Inhibition of Mast Cell Activation

CD200 and its receptor CD200R are both type I membrane glycoproteins that contain two Ig-like domains. Engagement of CD200R by CD200 inhibits activation of myeloid cells. Unlike the majority of immune inhibitory receptors, CD200R lacks an ITIM in the cytoplasmic domain. The molecular mechanism of CD200R inhibition of myeloid cell activation is unknown. In this study, we examined the CD200R signaling pathways that control degranulation of mouse bone marrow-derived mast cells. We found that upon ligand binding, CD200R is phosphorylated on tyrosine and subsequently binds to adapter proteins Dok1 and Dok2. Upon phosphorylation, Dok1 binds to SHIP and both Dok1 and Dok2 recruit RasGAP, which mediates the inhibition of the Ras/MAPK pathways. Activation of ERK, JNK, and p38 MAPK are all inhibited by CD200R engagement. The reduced activation of these MAPKs is responsible for the observed inhibition of mast cell degranulation and cytokine production. Similar signaling events were also observed upon CD200R engagement in mouse peritoneal cells. These data define a novel inhibitory pathway used by CD200R in modulating mast cell function and help to explain how engagement of this receptor in vivo regulates myeloid cell function.

Regulation of myeloid cell function through the CD200 receptor

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-γ- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.

Induction of beta3-integrin gene expression by sustained activation of the Ras-regulated Raf-MEK-extracellular signal-regulated kinase signaling pathway.

ABSTRACT Alterations in the expression of integrin receptors for extracellular matrix (ECM) proteins are strongly associated with the acquisition of invasive and/or metastatic properties by human cancer cells. Despite this, comparatively little is known of the biochemical mechanisms that regulate the expression of integrin genes in cells. Here we demonstrate that the Ras-activated Raf–MEK–extracellular signal-regulated kinase (ERK) signaling pathway can specifically control the expression of individual integrin subunits in a variety of human and mouse cell lines. Pharmacological inhibition of MEK1 in a number of human melanoma and pancreatic carcinoma cell lines led to reduced cell surface expression of α6- and β3-integrin. Consistent with this, conditional activation of the Raf-MEK-ERK pathway in NIH 3T3 cells led to a 5 to 20-fold induction of cell surface α6- and β3-integrin expression. Induced β3-integrin was expressed on the cell surface as a heterodimer with αv-integrin; however, the overall level of αv-integrin expression was not altered by Ras or Raf. Raf-induced β3-integrin was observed in primary and established mouse fibroblast lines and in mouse and human endothelial cells. Consistent with previous reports of the ability of the Raf-MEK-ERK signaling pathway to induce β3-integrin gene transcription in human K-562 erythroleukemia cells, Raf activation in NIH 3T3 cells led to elevated β3-integrin mRNA. However, unlike immediate-early Raf targets such as heparin binding epidermal growth factor and Mdm2, β3-integrin mRNA was induced by Raf in a manner that was cycloheximide sensitive. Surprisingly, activation of the Raf-MEK-ERK signaling pathway by growth factors and mitogens had little or no effect on β3-integrin expression, suggesting that the expression of this gene requires sustained activation of this signaling pathway. In addition, despite the robust induction of cell surface αvβ3-integrin expression by Raf in NIH 3T3 cells, such cells display decreased spreading and adhesion, with a loss of focal adhesions and actin stress fibers. These data suggest that oncogene-induced alterations in integrin gene expression may participate in the changes in cell adhesion and migration that accompany the process of oncogenic transformation.

Differential abilities of activated Raf oncoproteins to abrogate cytokine dependency, prevent apoptosis and induce autocrine growth factor synthesis in human hematopoietic cells

Raf is a key serine-threonine protein kinase which participates in the transmission of growth, anti-apoptotic and differentiation messages. These signals can be initiated after receptor ligation and are transmitted to members of the MAP kinase cascade that subsequently activate transcription factors controlling gene expression. Raf is a member of a multigene family which includes: Raf-1, A-Raf and B-Raf. The roles that individual Raf kinases play in the regulation of normal and malignant hematopoietic cell growth are not clear. The following studies show that all three Raf kinases are functionally present in certain human hematopoietic cells, and their aberrant expression can result in abrogation of cytokine dependency. Cytokine-dependent TF-1 cells were infected with retroviruses encoding amino-terminal deleted (Δ) A-Raf, B-Raf and Raf-1 proteins. These Raf proteins were conditionally inducible as they were fused to the hormone-binding domain of the estrogen receptor (ER). A hierarchy in the abilities of Raf-containing retroviruses to abrogate cytokine dependency was observed as ΔA-Raf:ER was 20- to 200-fold more efficient than either ΔRaf-1:ER or ΔB-Raf:ER, respectively. This result was unexpected as A-Raf is an intrinsically weaker kinase than either Raf-1 or B-Raf. The activated Raf proteins induced downstream MEK and MAP (ERK1 and ERK2) kinase activities in the cells which proliferated in response to Raf activation. Furthermore, a functional MEK signaling pathway was necessary as treatment of the cells with a MEK1-inhibitor suppressed Raf-mediated proliferation. To determine whether the regulatory phosphorylation residues contained in the modified Raf oncoproteins were necessary for transformation, they were altered by site-directed mutagenesis. Substitution of the regulatory phosphorylation tyrosine residues with phenylalanine in either A-Raf or Raf-1 reduced the capacity of these oncoproteins to abrogate cytokine dependency. In contrast, changing the critical aspartic acid residues of B-Raf to either tyrosine or phenylalanine increased the frequency of estradiol-responsive cells. Thus, the amino acids present in the regulatory residues modulated the capability of Raf proteins to abrogate the cytokine dependency of TF-1 cells. Differences in the levels of Raf and downstream kinase activities were observed between cytokine-dependent and estradiol-responsive ΔRaf:ER-infected cells as estradiol-responsive cells usually expressed more Raf and MEK activity than GM-CSF-dependent, ΔRaf:ER-infected cells. Abrogation of cytokine dependency by the activated ΔRaf:ER proteins was associated with autocrine growth factor synthesis which was sufficient to promote the growth of uninfected TF-1 cells. In summary, these observations indicate that the aberrant expression of certain activated ΔRaf:ER oncoproteins can alter the cytokine dependency of human hematopoietic TF-1 cells. These cells will be useful in evaluating the roles of the individual Raf oncoproteins in signal transduction, cell cycle progression, autocrine transformation, regulation of apoptosis and differentiation. Moreover, these Raf-infected cells may be important in evaluating the efficacy of novel anticancer drugs designed to inhibit Raf and downstream signal transduction molecules.

The CD200 Receptor Is a Novel and Potent Regulator of Murine and Human Mast Cell Function

CD200R is a member of the Ig supergene family that is primarily expressed on myeloid cells. Recent in vivo studies have suggested that CD200R is an inhibitory receptor capable of regulating the activation threshold of inflammatory immune responses. Here we provide definitive evidence that CD200R is expressed on mouse and human mast cells and that engagement of CD200R by agonist Abs or ligand results in a potent inhibition of mast cell degranulation and cytokine secretion responses. CD200R-mediated inhibition of FcεRI activation was observed both in vitro and in vivo and did not require the coligation of CD200R to FcεRI. Unlike the majority of myeloid inhibitory receptors, CD200R does not contain a phosphatase recruiting inhibitory motif (ITIM); therefore, we conclude that CD200R represents a novel and potent inhibitory receptor that can be targeted in vivo to regulate mast cell-dependent pathologies.

Differential abilities of the Raf family of protein kinases to abrogate cytokine dependency and prevent apoptosis in murine hematopoietic cells by a MEK1-dependent mechanism

In this study, the abilities of constitutive and conditional forms of the three Raf kinases to abrogate the cytokine dependency of FDC-P1 cells were examined. The constitutively active forms (Δ) of all three Raf kinases were fused to the hormone-binding domain of the estrogen receptor (ER), rendering their activities conditionally dependent upon exogenous β-estradiol. The vast majority of ΔRaf:ER-infected FDC-P1 cells remained cytokine-dependent; however, cells were obtained at low frequency in which expression of ΔRaf:ER abrogated cytokine dependency. Isoform specific differences between the Raf kinases were observed as cytokine-independent cells were obtained more frequently from ΔA-Raf:ER than either ΔRaf-1:ER or ΔB-Raf:ER infected cells. To determine whether the regulatory phosphorylation sites in the Raf proteins were necessary for abrogation of cytokine dependency, they were changed by site-directed mutagenesis. Substitution with phenylalanine eliminated the transforming ability of the ΔB-Raf:ER and ΔRaf-1:ER kinases. However, a similar substitution in A-Raf did not extinguish its transforming activity. The activated Raf proteins induced essential downstream MEK1 activity as treatment with the MEK1 inhibitor, PD98059, suppressed Raf-mediated growth. Activated MAP kinases (ERK1 and ERK2) were detected in ΔRaf:ER-transformed cells, and their presence was dependent upon a functional MEK1 protein. The cytokine-independent phenotype required the continued activity of the ΔRaf:ER proteins as removal of β-estradiol caused the cells to stop growing and undergo apoptosis. The Raf-responsive cells were found to express autocrine growth factors, which promoted their growth. Constitutive activation of the Raf-1 oncogene resulted in malignant transformation as cytokine-independent FDC-P1 cells infected with a retrovirus encoding an activated Raf-1 protein formed tumors upon injection of immunocompromised mice. In summary, Raf kinases can abrogate cytokine dependency, prevent apoptosis and induce the tumorigenicity of a certain subpopulation of FDC-P1 cells by a MEK1-dependent mechanism.

2B4-mediated activation of human natural killer cells.

2B4 is a member of the CD2 subset of the immunoglobulin superfamily of cell surface receptors. Other members of this family include CD2, CD48, CD58, CD84, signaling lymphocytic activation molecule and Ly-9. Some of these molecules are activating structures expressed by natural killer cells and T cells. We have recently cloned and characterised the human homologue of 2B4 and found that the cytoplasmic domain of 2B4 can interact with SAP, a signaling adaptor protein that is mutated in the immunodeficiency X-linked lymphoproliferative disease (XLP). Additionally, the natural ligand of 2B4 has been identified as CD48. These findings have facilitated the investigation of the functional role of this receptor-ligand pair, and associated signal transduction pathways, on immune cells. In this study, it was found that the interaction between 2B4 on effector cells and CD48 on target cells induced NK-cell activation, as evidenced by increased cytotoxicity and secretion of IFN-gamma. The responses induced by ligation of 2B4 could be reduced by the co-ligation of inhibitory receptors expressed by NK cells, demonstrating that activation signals delivered via 2B4 can be regulated by the action of certain inhibitory receptors. Because the signalling pathway of 2B4 involves SAP, it is possible that 2B4-mediated NK-cell activation may be compromised in patients with XLP due to mutations in SAP. This may contribute to the phenotype and progression of this disease.

Mutations of critical amino acids affect the biological and biochemical properties of oncogenic A-Raf and Raf-1

The catalytic domains of the Raf family of protein kinases (ΔRaf) differ in their ability to activate MEK in vitro and in vivo and in their ability to oncogenically transform mammalian cells. The kinase domain of B-Raf is more active than the equivalent portion of Raf-1 which in turn is more active than A-Raf. In Raf-1 the phosphorylation or mutation to aspartic acid of two key tyrosine residues upstream of the ATP binding site has been demonstrated to significantly potentiate catalytic activity. In A-Raf the analogous amino acids are also tyrosine whereas in B-Raf they are aspartic acid. To determine if these differences in amino acid sequence influence the relative catalytic activity of the Raf kinase domains we constructed forms of ΔA-Raf, DB-Raf and ΔRaf-1 that encode either aspartic acid [DD], phenylalanine [FF] or tyrosine [YY] at these positions. These proteins were expressed both in mammalian cells as fusions with the hormone binding domain of the estrogen receptor and as epitope-tagged proteins in Sf9 insect cells to test their oncogenic and catalytic potentials. When expressed in Rat1 or 3T3 cells in the presence of hormone all of the ΔRaf-1:ER and ΔA-Raf:ER proteins were transforming with the exception of the [FF] form of ΔA-Raf. In general the [DD] forms of the ΔRaf-1:ER and ΔA-Raf:ER proteins were the most potently oncogenic which correlated with their ability to elicit activation of the MAP kinase pathway. Consistent with the transformation data, the catalytic activity of the [DD] forms of ΔA-Raf:ER and ΔRaf-1:ER was about ten times greater than the cognate [FF] and [YY] forms of the proteins. By contrast all of the ΔB-Raf:ER proteins were highly transforming and ΔB-Raf catalytic activity was largely unaffected by mutation of the aforementioned aspartic acids to either tyrosine or phenylalanine. Similar results were obtained with epitope-tagged forms of ΔA-Raf, ΔB-Raf and ΔRaf-1 expressed in Sf9 cells. These data provide support for the model that key tyrosine residues in the protein kinase domains of A-Raf and Raf-1 are important in the regulation of catalytic activity. In addition they demonstrate that the higher intrinsic activity of B-Raf cannot be explained simply by the presence of aspartic acids at the analogous positions.