Tracy A. Willson

A family of cytokine-inducible inhibitors of signalling

Cytokines are secreted proteins that regulate important cellular responses such as proliferation and differentiation. Key events in cytokine signal transduction are well defined: cytokines induce receptor aggregation, leading to activation of members of the JAK family of cytoplasmic tyrosine kinases. In turn, members of theSTAT family of transcription factors are phosphorylated, dimerize and increase the transcription of genes with STAT recognition sites in their promoters. Less is known of how cytokine signal transduction is switched off. We have cloned a complementary DNA encoding a protein SOCS-1, containing an SH2-domain, by its ability to inhibit the macrophage differentiation of M1 cells in response to interleukin-6. Expression of SOCS-1 inhibited both interleukin-6-induced receptor phosphorylation and STAT activation. We have also cloned two relatives of SOCS-1, named SOCS-2 and SOCS-3, which together with the previously described CIS (ref. 5) form a new family of proteins. Transcription of all four SOCS genes is increased rapidly in response to interleukin-6, in vitro and in vivo, suggesting they may act in a classic negative feedback loop to regulate cytokine signal transduction.

SOCS3 negatively regulates IL-6 signaling in vivo.

Members of the suppressor of cytokine signaling (SOCS) family are potentially key physiological negative regulators of interleukin-6 (IL-6) signaling. To examine whether SOCS3 is involved in regulating this signaling, we have used conditional gene targeting to generate mice lacking Socs3 in the liver or in macrophages. We show that Socs3 deficiency results in prolonged activation of signal transducer and activator of transcription 1 (STAT1) and STAT3 after IL-6 stimulation but normal activation of STAT1 after stimulation with interferon-γ (IFN-γ). Conversely, IL-6-induced STAT activation is normal in Socs1-deficient cells, whereas STAT1 activation induced by IFN-γ is prolonged. Microarray analysis shows that the pattern of gene expression induced by IL-6 in Socs3-deficient livers mimics that induced by IFN-γ. Our data indicate that SOCS3 and SOCS1 have reciprocal functions in IL-6 and IFN-γ regulation and imply that SOCS3 has a role in preventing IFN-γ-like responses in cells stimulated by IL-6.

Mutational analyses of the SOCS proteins suggest a dual domain requirement but distinct mechanisms for inhibition of LIF and IL‐6 signal transduction

SOCS‐1 (suppressor of cytokine signaling‐1) is a representative of a family of negative regulators of cytokine signaling (SOCS‐1 to SOCS‐7 and CIS) characterized by a highly conserved C‐terminal SOCS box preceded by an SH2 domain. This study comprehensively examined the ability of several SOCS family members to negatively regulate the gp130 signaling pathway. SOCS‐1 and SOCS‐3 inhibited both interleukin‐6 (IL‐6)‐ and leukemia inhibitory factor (LIF)‐induced macrophage differentiation of murine monocytic leukemic M1 cells and LIF induction of a Stat3‐responsive reporter construct in 293T fibroblasts. Deletion of amino acids 51–78 in the N‐terminal region of SOCS‐1 prevented inhibition of LIF signaling. The SOCS‐1 and SOCS‐3 N‐terminal regions were functionally interchangeable, but this did not extend to other SOCS family members. Mutation of SH2 domains abrogated the ability of both SOCS‐1 and SOCS‐3 to inhibit LIF signal transduction. Unlike SOCS‐1, SOCS‐3 was unable to inhibit JAK kinase activity in vitro, suggesting that SOCS‐1 and SOCS‐3 act on the JAK–STAT pathway in different ways. Thus, although inhibition of signaling by SOCS‐1 and SOCS‐3 requires both the SH2 and N‐terminal domains, their mechanisms of action appear to be biochemically different.

Gigantism in mice lacking suppressor of cytokine signalling-2.

Suppressor of cytokine signalling-2 (SOCS-2) is a member of the suppressor of cytokine signalling family, a group of related proteins implicated in the negative regulation of cytokine action through inhibition of the Janus kinase (JAK) signal transducers and activators of transcription (STAT) signal-transduction pathway. Here we use mice unable to express SOCS-2 to examine its function in vivo. SOCS-2-/- mice grew significantly larger than their wild-type littermates. Increased body weight became evident after weaning and was associated with significantly increased long bone lengths and the proportionate enlargement of most organs. Characteristics of deregulated growth hormone and insulin-like growth factor-I (IGF-I) signalling, including decreased production of major urinary protein, increased local IGF-I production, and collagen accumulation in the dermis, were observed in SOCS-2-deficient mice, indicating that SOCS-2 may have an essential negative regulatory role in the growth hormone/IGF-I pathway.

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.

The transcription factor Erg is essential for definitive hematopoiesis and the function of adult hematopoietic stem cells

Ets-related gene (ERG), which encodes a member of the Ets family of transcription factors, is a potent oncogene. Chromosomal rearrangements involving ERG are found in acute myeloid leukemia, acute lymphoblastic leukemia, Ewings sarcoma and more than half of all prostate cancers; however, the normal physiological function of Erg is unknown. We did a sensitized genetic screen of the mouse for regulators of hematopoietic stem cell function and report here a germline mutation of Erg. We show that Erg is required for definitive hematopoiesis, adult hematopoietic stem cell function and the maintenance of normal peripheral blood platelet numbers.

SOCS2 negatively regulates growth hormone action in vitro and in vivo

Mice deficient in SOCS2 display an excessive growth phenotype characterized by a 30-50% increase in mature body size. Here we show that the SOCS2-/- phenotype is dependent upon the presence of endogenous growth hormone (GH) and that treatment with exogenous GH induced excessive growth in mice lacking both endogenous GH and SOCS2. This was reflected in terms of overall body weight, body and bone lengths, and the weight of internal organs and tissues. A heightened response to GH was also measured by examining GH-responsive genes expressed in the liver after exogenous GH administration. To further understand the link between SOCS2 and the GH-signaling cascade, we investigated the nature of these interactions using structure/function and biochemical interaction studies. Analysis of the 3 structural motifs of the SOCS2 molecule revealed that each plays a crucial role in SOCS2 function, with the conserved SOCS-box motif being essential for all inhibitory function. SOCS2 was found to bind 2 phosphorylated tyrosines on the GH receptor, and mutational analysis of these amino acids showed that both were essential for SOCS2 function. Together, the data provide clear evidence that SOCS2 is a negative regulator of GH signaling.

ChIP-seq analysis reveals distinct H3K27me3 profiles that correlate with transcriptional activity

Transcriptional control is dependent on a vast network of epigenetic modifications. One epigenetic mark of particular interest is tri-methylation of lysine 27 on histone H3 (H3K27me3), which is catalysed and maintained by Polycomb Repressive Complex 2 (PRC2). Although this histone mark is studied widely, the precise relationship between its local pattern of enrichment and regulation of gene expression is currently unclear. We have used ChIP-seq to generate genome-wide maps of H3K27me3 enrichment, and have identified three enrichment profiles with distinct regulatory consequences. First, a broad domain of H3K27me3 enrichment across the body of genes corresponds to the canonical view of H3K27me3 as inhibitory to transcription. Second, a peak of enrichment around the transcription start site (TSS) is commonly associated with ‘bivalent’ genes, where H3K4me3 also marks the TSS. Finally and most surprisingly, we identified an enrichment profile with a peak in the promoter of genes that is associated with active transcription. Genes with each of these three profiles were found in different proportions in each of the cell types studied. The data analysis techniques developed here will be useful for the identification of common enrichment profiles for other histone modifications that have important consequences for transcriptional regulation.

Biological Evidence That SOCS-2 Can Act Either as an Enhancer or Suppressor of Growth Hormone Signaling

Suppressor of cytokine signaling (SOCS)-2 is a member of a family of intracellular proteins implicated in the negative regulation of cytokine signaling. The generation of SOCS-2-deficient mice, which grow to one and a half times the size of their wild-type littermates, suggests that SOCS-2 may attenuate growth hormone (GH) signaling. In vitro studies indicate that, while SOCS-2 can inhibit GH action at low concentrations, at higher concentrations it may potentiate signaling. To determine whether a similar enhancement of signaling is observed in vivo or alternatively whether increased SOCS-2 levels repress growth in vivo, we generated and analyzed transgenic mice that overexpress SOCS-2 from a human ubiquitin C promoter. These mice are not growth-deficient and are, in fact, significantly larger than wild-type mice. The overexpressed SOCS-2 was found to bind to endogenous GH receptors in a number of mouse organs, while phosphopeptide binding studies with recombinant SOCS-2 defined phosphorylated tyrosine 595 on the GH receptor as the site of interaction. Together, the data implicate SOCS-2 as having dual effects on GH signaling in vivo.

The SOCS box of suppressor of cytokine signaling-1 is important for inhibition of cytokine action in vivo

Suppressor of Cytokine Signaling-1 (SOCS-1) is an essential physiological inhibitor of IFN-γ signaling. Mice lacking this gene die in the early postnatal period from a disease characterized by hyperresponsiveness to endogenous IFN-γ. The SOCS box is a C-terminal domain shared with over 30 other proteins that links SOCS proteins to an E3 ubiquitin ligase activity and the proteasome, but whether it contributes to inhibition of cytokine signaling is currently disputed. We have deleted only the SOCS box of the SOCS-1 gene in mice and show that such mice have an increased responsiveness to IFN-γ and slowly develop a fatal inflammatory disease. These results demonstrate that deletion of the SOCS box leads to a partial loss of function of SOCS-1.