Robyn Starr

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.

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.

Growth Hormone Preferentially Induces the Rapid, Transient Expression of SOCS-3, a Novel Inhibitor of Cytokine Receptor Signaling

Four members (SOCS-1, SOCS-2, SOCS-3, and CIS) of a family of cytokine-inducible, negative regulators of cytokine receptor signaling have recently been identified. To address whether any of these genes are induced in response to growth hormone (GH), serum-starved 3T3-F442A fibroblasts were incubated with GH for various time points, and the expression of the SOCS gene family was analyzed by Northern blotting. GH stimulated the rapid, transient induction ofSOCS-3 mRNA, peaking 30 min after the initiation of GH exposure and declining to basal levels by 2 h. Expression of the other SOCS genes (SOCS-1, SOCS-2,CIS) was also up-regulated by GH, although to a lesser extent than SOCS-3 and with differing kinetics.SOCS-3 expression was also strongly induced in 3T3-F442A cells treated with leukemia-inhibitory factor (LIF), with weaker induction of SOCS-1 and CIS being observed. The preferential induction of SOCS-3 mRNA was also observed in hepatic RNA isolated from the livers of mice that had received a single supraphysiological dose of GH intraperitoneally. Co-transfection studies revealed that constitutive expression of SOCS-1 and SOCS-3, but not SOCS-2 or CIS, blocked GH-induced transactivation of the GH-responsive serine protease inhibitor 2.1 gene promoter.

Negative regulation of the JAK/STAT pathway

Cytokines induce a variety of biological responses by binding to specific cell surface receptors and activating cytoplasmic signal transduction pathways, such as the JAK/STAT pathway. Although these responses are generally transient, few molecules have been characterised that switch the signal off. Several different steps of the signal transduction pathway appear to be targeted by negative regulators, including the receptor/ligand complex, JAK kinases, and STAT transcription factors. Negative regulation is achieved by dephosphorylation of signalling intermediates by protein tyrosine phosphatases such as SHP‐1, and by proteolytic degradation. Recent studies have identified two new families of negative regulatory molecules, SOCS and PIAS, which function in novel ways to suppress signal transduction pathways. The duration and intensity of a cells response to cytokine therefore appear to be determined by the net effect of several regulatory mechanisms. BioEssays 1999;21:47–52.

Suppressor of cytokine signaling 1 regulates the immune response to infection by a unique inhibition of type I interferon activity

Suppressor of cytokine signaling 1 (SOCS1) is a critical regulator of cytokine signaling and immune responses. SOCS1-deficient mice develop severe inflammatory disease, but are very resistant to viral infections. Using neutralizing antibody to type I interferon (IFN-α and IFN-β) and mice deficient in interferon-γ or type I interferon receptor components (IFNAR1 or IFNAR2), we demonstrate here that SOCS1 deficiency amplified type I interferon antiviral and proinflammatory actions independently of interferon-γ. The mechanism of the suppression of type I interferon responses by SOCS1 was distinct from that of other cytokines. SOCS1 associated with and regulated IFNAR1- but not IFNAR2-specific signals, abrogating tyrosine phosphorylation of transcription factor STAT1 and reducing the duration of antiviral gene expression. Thus, SOCS1 is an important in vivo inhibitor of type I interferon signaling and contributes to balancing its beneficial antiviral versus detrimental proinflammatory effects on innate immunity.

Suppressor of Cytokine Signaling-1 Is a Critical Regulator of Interleukin-7-Dependent CD8+ T Cell Differentiation

To determine the tissue-specific functions of SOCS-1, mice were generated in which the SOCS-1 gene could be deleted in individual tissues. A reporter gene of SOCS-1 promoter activity was also inserted. Using the reporter, high SOCS-1 expression was found at the CD4(+)CD8(+) stage in thymocyte development. To investigate the function of this expression, the SOCS-1 gene was specifically deleted throughout the thymocyte/T/NKT cell compartment. Unlike SOCS-1(-/-) mice, these mice did not develop lethal multiorgan inflammation but developed multiple lymphoid abnormalities, including enhanced differentiation of thymocytes toward CD8(+) T cells and very high percentages of peripheral CD8(+) T cells with a memory phenotype (CD44(hi)CD25(lo)CD69(lo)). These phenotypes were found to correlate with hypersensitivity to the gamma-common family of cytokines.

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.

SOCS-6 Binds to Insulin Receptor Substrate 4, and Mice Lacking the SOCS-6 Gene Exhibit Mild Growth Retardation

ABSTRACT SOCS-6 is a member of the suppressor of cytokine signaling (SOCS) family of proteins (SOCS-1 to SOCS-7 and CIS) which each contain a central SH2 domain and a carboxyl-terminal SOCS box. SOCS-1, SOCS-2, SOCS-3, and CIS act to negatively regulate cytokine-induced signaling pathways; however, the actions of SOCS-4, SOCS-5, SOCS-6, and SOCS-7 remain less clear. Here we have used both biochemical and genetic approaches to examine the action of SOCS-6. We found that SOCS-6 and SOCS-7 are expressed ubiquitously in murine tissues. Like other SOCS family members, SOCS-6 binds to elongins B and C through its SOCS box, suggesting that it might act as an E3 ubiquitin ligase that targets proteins bound to its SH2 domain for ubiquitination and proteasomal degradation. We investigated the binding specificity of the SOCS-6 and SOCS-7 SH2 domains and found that they preferentially bound to phosphopeptides containing a valine in the phosphotyrosine (pY) +1 position and a hydrophobic residue in the pY +2 and pY +3 positions. In addition, these SH2 domains interacted with a protein complex consisting of insulin receptor substrate 4 (IRS-4), IRS-2, and the p85 regulatory subunit of phosphatidylinositol 3-kinase. To investigate the physiological role of SOCS-6, we generated mice lacking the SOCS-6 gene. SOCS-6−/− mice were born in a normal Mendelian ratio, were fertile, developed normally, and did not exhibit defects in hematopoiesis or glucose homeostasis. However, both male and female SOCS-6−/− mice weighed approximately 10% less than wild-type littermates.

Suppressors of cytokine signaling (SOCS): negative regulators of signal transduction.

SOCS‐1 was originally identified as an inhibitor of interleukin‐6 signal transduction and is a member of a family of proteins (SOCS‐1 to SOCS‐7 and CIS) that contain an SH2 domain and a conserved carboxyl‐terminal SOCS box motif. Mutation studies have established that critical contributions from both the amino‐terminal and SH2 domains are essential for SOCS‐1 and SOCS‐3 to inhibit cytokine signaling. Inhibition of cytokine‐dependent activation of STAT3 occurred in cells expressing either SOCS‐1 or SOCS‐3, but unlike SOCS‐1, SOCS‐3 did not directly interact with or inhibit the activity of JAK kinases. Although the conserved SOCS box motif appeared to be dispensable for SOCS‐1 and SOCS‐3 action when over‐expressed, this domain interacts with elongin proteins and may be important in regulating protein turnover. In gene knockout studies, SOCS‐1−/− mice were born but failed to thrive and died within 3 weeks of age with fatty degeneration of the liver and hemopoietic infiltration of several organs. The thymus in SOCS‐1−/− mice was small, the animals were lymphopenic, and deficiencies in B lymphocytes were evident within hemopoietic organs. We propose that the absence of SOCS‐1 in these mice prevents lymphocytes and liver cells from appropriately controlling signals from cytokines with cytotoxic side effects. J. Leukoc. Biol. 66: 588–592; 1999.