Nicola Na

SOCS1 Is a Critical Inhibitor of Interferon γ Signaling and Prevents the Potentially Fatal Neonatal Actions of this Cytokine

Mice lacking suppressor of cytokine signaling-1 (SOCS1) develop a complex fatal neonatal disease. In this study, SOCS1-/- mice were shown to exhibit excessive responses typical of those induced by interferon gamma (IFNgamma), were hyperresponsive to viral infection, and yielded macrophages with an enhanced IFNgamma-dependent capacity to kill L. major parasites. The complex disease in SOCS1-/- mice was prevented by administration of anti-IFNgamma antibodies and did not occur in SOCS1-/- mice also lacking the IFNgamma gene. Although IFNgamma is essential for resistance to a variety of infections, the potential toxic action of IFNgamma, particularly in neonatal mice, appears to require regulation. Our data indicate that SOCS1 is a key modulator of IFNgamma action, allowing the protective effects of this cytokine to occur without the risk of associated pathological responses.

Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor.

Two cDNA clones encoding a receptor for human granulocyte‐macrophage colony‐stimulating factor (hGM‐CSF‐R) were isolated by expression screening of a library made from human placental mRNA. Pools of recombinant plasmid DNA were electroporated into COS cells which were then screened for their capacity to bind radioiodinated hGM‐CSF using a sensitive microscopic autoradiographic approach. The cloned GM‐CSF‐R precursor is a 400 amino acid polypeptide (Mr 45,000) with a single transmembrane domain, a glycosylated extracellular domain and a short (54 amino acids) intracytoplasmic tail. It does not contain a tyrosine kinase domain nor show homology with members of the immunoglobulin super gene family, but does show some significant sequence homologies with receptors for several other haemopoietic growth factors, including those for interleukin‐6, erythropoietin and interleukin‐2 (beta‐chain) and also to the prolactin receptor. When transfected into COS cells the cloned cDNA directed the expression of a GM‐CSF‐R showing a single class of affinity (KD = 2(‐8) nM) and specificity for human GM‐CSF but not interleukin‐3. Messenger RNA coding for this receptor was detected in a variety of haemopoietic cells known to display hGM‐CSF binding, and cross‐linking experiments revealed a similar size for the glycosylated receptors in transfected COS and haemopoietic cells.

Molecular cloning and expression of cDNA encoding a murine myeloid leukaemia inhibitory factor (LIF).

Leukaemia inhibitory factor (LIF) can induce macrophage differentiation in M1 murine myeloid leukaemic cells and suppress their proliferation in vitro. It does not stimulate the proliferation of normal progenitor cells and is apparently distinct from known colony‐stimulating factors. We have used oligo‐nucleotides complementary to partial amino acid sequence of LIF to isolate a LIF clone from a T lymphocyte cDNA library. When this cDNA was coupled to a yeast expression vector (YEpsec1) and introduced into yeast cells, a molecule with the biological properties characteristic of native LIF was secreted into the growth medium. The amino acid sequence of LIF established it to be a unique molecular entity, distinct from the other known haemopoietic growth factors. Since LIF is encoded by a unique gene, two biochemically separable forms of LIF probably represent post‐transcriptional or posttranslational variants of the same gene product. In contrast to several other haemopoietic regulators, the 0.8‐ to 1‐kb LIF mRNA was expressed constitutively in two murine T lymphocyte cell lines examined, and its abundance was not enhanced by stimulation with concanavalin A. Cloning, sequencing and expressing LIF has resolved several discrepancies in the literature concerning the identity of factors capable of inducing differentiation of murine myeloid leukaemic cells in vitro.

Cloning of a murine IL-11 receptor alpha-chain; requirement for gp130 for high affinity binding and signal transduction.

An adult mouse liver cDNA library was screened with oligonucleotides corresponding to the conserved WSXWS motif of the haemopoietin receptor family. Using this method, cDNA clones encoding a novel receptor were isolated. The new receptor, named NR1, was most similar in sequence and predicted structure to the alpha‐chain of the IL‐6 receptor and mRNA was expressed in the 3T3‐L1 pre‐adipocytic cell line and in a range of primary tissues. Expression of NR1 in the factor‐dependent haemopoietic cell line Ba/F3 resulted in the generation of low affinity receptors for IL‐11 (Kd approximately 10 nM). The capacity to bind IL‐11 with high affinity (Kd = 300‐800 pM) appeared to require coexpression of both NR1 and gp130, the common subunit of the IL‐6, leukaemia inhibitory factor (LIF), oncostatin M (OSM) and ciliary neurotrophic factor (CNTF) receptors. The expression of both NR1 and gp130 was also necessary for Ba/F3 cells to proliferate and M1 cells to undergo macrophage differentiation in response to IL‐11.

Expression and function of members of the cytokine receptor superfamily on breast cancer cells.

Receptors for the cytokines leukemia inhibitory factor (LIF), interleukin-6 (IL-6), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and interleukin-11 (IL-11) are members of the structurally conserved hemopoietin receptor superfamily. In addition, they all share the transmembrane signalling protein gp130. In this paper the expression and function of this family of receptors in breast cancer cells was examined. RT – PCR analyses demonstrated that gp130 was expressed in 12/12 breast cell lines and the specific receptor α-chains for IL-6, LIF, IL-11 and CNTF were expressed in the majority of these cell lines. This was in contrast to other hemopoitin receptors. Examination of 50 clinical samples of malignant breast tissue by RT – PCR showed a similar pattern of expression of gp130 associated receptors. Treatment of breast cancer cell lines with OSM resulted in changes in cellular morphology. Cellular proliferation was inhibited following exposure to OSM (3/4 cell lines), IL-11 (2/4 cell lines), and by IL-6 and LIF (1/4 cell lines). Cell surface binding of LIF and OSM was also documented. The expression of these receptors in 12/12 cell lines and greater than 95% of clinical samples suggests that these molecules may be important in regulating the growth of breast cells.

Aberrant hematopoiesis in mice with inactivation of the gene encoding SOCS-1

Mice with homozygous inactivation of the gene encoding the suppressor of cytokine signaling-1 (SOCS-1) protein die within 21 days of birth with low body weight, fatty degeneration and necrosis of the liver, infiltration of the lung, pancreas, heart and skin by macrophages and granulocytes and a profound depletion of T- and B-lymphocytes. In the present study, SOCS-1 −/− mice were found to have a moderate neutrophilia, and reduced platelet and hematocrit levels. Replacement of the SOCS-1 gene by a lac-Z reporter gene allowed documentation by FACS sorting that at least a proportion of granulocyte–macrophage progenitor cells transcribe SOCS-1. Most hematopoietic progenitor cell frequencies were normal in −/− marrow as were the size and cellular content of colonies formed by −/− progenitor cells in response to various stimulating factors. However, there was an increased frequency of macrophage progenitor cells in −/− mice and, abnormally, one quarter of all progenitor cells were located in the liver. Progenitor cells from −/− mice were hyper-responsive to stimulation by GM-CSF but not by M-CSF or Multi-CSF (IL-3). Progenitor cells from −/− mice were also hypersensitive to inhibition by interferon-gamma (IFN-γ), the degree of inhibition varying markedly with the stimulating factor used. The suppressive effects of IFN-γ therefore appear to involve interactions with particular growth factor-initiated signals in −/− cells – interactions that are strongly modulated by the action of the SOCS-1 protein.

DISTINCT ROLES FOR LEUKEMIA INHIBITORY FACTOR RECEPTOR ALPHA -CHAIN AND GP130 IN CELL TYPE-SPECIFIC SIGNAL TRANSDUCTION

Leukemia inhibitory factor (LIF) induces a variety of disparate biological responses in different cell types. These responses are thought to be mediated through the functional LIF receptor (LIFR), consisting of a heterodimeric complex of LIFR α-chain (LIFRα) and gp130. The present study investigated the relative capacity of the cytoplasmic domains of each receptor subunit to signal particular responses in several cell types. To monitor the signaling potential of LIFRα and gp130 individually, we constructed chimeric receptors by linking the extracellular domain of granulocyte colony-stimulating factor receptor (GCSFR) to the transmembrane and cytoplasmic regions of either LIFRα or gp130. Both chimeric receptors and the full-length GCSFR in expressed in M1 myeloid leukemic cells to measure differentiation induction, in embryonic stem cells to measure differentiation inhibition, and in Ba/F3 cells to measure cell proliferation. Our results demonstrated that whereas GCSFR-gp130 receptor homodimer mediated a GCSF-induced signal in all three cell types, the GCSFR-LIFRα receptor homodimer was only functional in embryonic stem cells. These findings suggest that the signaling potential of gp130 and LIFRα cytoplasmic domains may differ depending upon the tissue and cellular response initiated.

Inter-species chimeras of leukaemia inhibitory factor define a major human receptor-binding determinant.

Human leukaemia inhibitory factor (hLIF) binds to both human and mouse LIF receptors (LIF‐R), while mouse LIF (mLIF) binds only to mouse LIF‐R. Moreover, hLIF binds with higher affinity to the mLIF‐R than does mLIF. In order to define the regions of the hLIF molecule responsible for species‐specific interaction with the hLIF‐R and for the unusual high‐affinity binding to the mLIF‐R, a series of 15 mouse/human LIF hybrids has been generated. Perhaps surprisingly, both of these properties mapped to the same region of the hLIF molecule. The predominant contribution was from residues in the loop linking the third and fourth helices, with lesser contributions from residues in the third helix and the loop connecting the second and third helices in the predicted three‐dimensional structure. Since all chimeras retained full biological activity and receptor‐binding activity on mouse cells, and there was little variation in the specific biological activity of the purified proteins, it can be concluded that the overall secondary and tertiary structures of each chimera were intact. This observation also implied that the primary binding sites on mLIF and hLIF for the mLIF‐R were unaltered by inter‐species domain swapping. Consequently, the site on the hLIF molecule that confers species‐specific binding to the hLIF‐R and higher affinity binding to the mLIF‐R, must constitute an additional interaction site to that used by both mLIF and hLIF to bind to the mLIF‐R. These studies define a maximum of 15 amino acid differences between hLIF and mLIF that are responsible for the different properties of these proteins.

Genetic Deletion of Murine SPRY Domain-Containing SOCS Box Protein 2 (SSB-2) Results in Very Mild Thrombocytopenia

ABSTRACT The SSB family is comprised of four highly homologous proteins containing a C-terminal SOCS box motif and a central SPRY domain. No function has yet been ascribed to any member of this family in mammalian species despite a clear role for other SOCS proteins in negative regulation of cytokine signaling. To investigate its physiological role, the murine Ssb-2 gene was deleted by homologous recombination. SSB-2-deficient mice were shown to have a reduced rate of platelet production, resulting in very mild thrombocytopenia (25% decrease in circulating platelets). Tissue histology and other hematological parameters were normal, as was the majority of serum biochemistry, with the exception that blood urea nitrogen (BUN) levels were decreased in mice lacking SSB-2. Quantitative analysis of SSB mRNA levels indicated that SSB-1, -2, and -3 were ubiquitously expressed; however, SSB-4 was only expressed at very low levels. SSB-2 expression was observed in the kidney and in megakaryocytes, a finding consistent with the phenotype of mice lacking this gene. Deletion of SSB-2 thus perturbs the steady-state level of two tightly controlled homeostatic parameters and identifies a critical role for SSB-2 in regulating platelet production and BUN levels.

Negative regulation of gp130 signalling mediated through tyrosine-757 is not dependent on the recruitment of SHP2.

Cytokines of the interleukin-6 family utilize the shared cytokine receptor gp130 in the formation of active signalling complexes. Tyrosine-757 (Y757) on this receptor is critical for negative regulation of gp130-mediated signalling. Two signalling regulators, suppressor of cytokine signalling 3 (SOCS3) and Src homology 2 domain-containing tyrosine phosphatase-2 (SHP2), are recruited to Y757 following receptor activation; however, the relative contribution made by each of these in down-regulating gp130 signalling is not known. In the present study, we show the design of a mutant gp130 receptor that can recruit SHP2, but not SOCS3. This receptor maintains the critical Y757 residue, but contains mutations in other surrounding residues which are also important for interactions with the Src homology 2 domains of SOCS3 and SHP2. Cells transfected with a chimaeric receptor containing the SHP2-selective gp130 intracellular domain showed an enhanced response to cytokine stimulation, which was similar to that shown by a chimaeric gp130 receptor mutant carrying a Y757F point mutation that failed to recruit either SOCS3 or SHP2. These results demonstrate that the recruitment of SHP2 alone is not sufficient for Y757-dependent negative regulation of gp130 signalling and that this activity must therefore be dependent on SOCS3.