Cathepsin E: a novel target for regulation by class II transactivator

The aspartic proteinase cathepsin E (CatE) has been implicated in Ag processing. In this study we report that CatE expression is negatively regulated by the MHC class II transactivator (CIITA). CIITA-deficient murine and human B cells expressed greater CatE than wild-type B cells, whereas overexpression of CIITA in a human gastric carcinoma cell line, AGS, resulted in decreased CatE mRNA and protein. AGS cells expressing CIITA also exhibited decreased processing of OVA Ag. Inhibition of CatE expression is specific to the type III CIITA isoform and maps to the acidic and proline/serine/threonine-rich (PST) protein domains of CIITA. We found that CatE expression is inducible by PU.1 and p300, and that this induction can be reversed by CIITA. These findings demonstrate a novel phenomenon: regulation of CatE Ag processing by CIITA in an isoform-dependent manner.     

A positive regulatory role for suppressor of cytokine signaling 1 in IFN-gamma-induced MHC class II expression in fibroblasts

Suppressor of cytokine signaling 1 (SOCS1) is rapidly induced following stimulation by several cytokines. SOCS1 negatively regulates cytokine receptor signal transduction by inhibiting Janus family tyrosine kinases. Lack of such feedback regulation underlies the premature death of SOCS1(-/-) mice due to unbridled IFN-gamma signaling. We used mouse embryo fibroblasts derived from SOCS1(-/-) mice to investigate the role of SOCS1 in IFN-gamma signaling pathways. SOCS1(-/-) fibroblasts were exquisitely sensitive to the IFN-gamma-mediated growth arrest and showed sustained STAT1 phosphorylation. However, SOCS1(-/-) fibroblasts were inefficient in MHC class II surface expression following IFN-gamma stimulation, despite a marked induction of the MHC class II transactivator and MHC class II gene expression. Retroviral transduction of wild-type SOCS1 relieved the growth-inhibitory effects of IFN-gamma in SOCS1(-/-) fibroblasts by inhibiting STAT1 activation. SOCS1R105K, carrying a mutation within the phosphotyrosine-binding pocket of the Src homology 2 domain, did not inhibit STAT1 phosphorylation, yet considerably inhibited IFN-gamma-mediated growth arrest. Strikingly, expression of SOCS1R105K restored the IFN-gamma-induced MHC class II expression in SOCS1(-/-) cells, indicating that expression of SOCS1 facilitates MHC class II expression in fibroblasts. Our results show that SOCS1, in addition to its negative regulatory role of inhibiting Janus kinases, has an unanticipated positive regulatory function in retarding the degradation of IFN-gamma-induced MHC class II proteins in fibroblasts.     

Mitogen-activated protein kinase ERK1/2 regulates the class II transactivator

The expression of major histocompatibility class II genes is necessary for proper antigen presentation and induction of an immune response. This expression is initiated by the class II transactivator, CIITA. The establishment of the active form of CIITA is controlled by a series of post-translational events, including GTP binding, ubiquitination, and dimerization. However, the role of phosphorylation is less clearly defined as are the consequences of phosphorylation on CIITA activity and the identity of the kinases involved. In this study we show that the extracellular signal-regulated kinases 1 and 2 (ERK1/2) interact directly with CIITA, targeting serine residues in the amino terminus of the protein, including serine 288. Inhibition of this phosphorylation by dominant-negative forms of ERK or by treatment of cells with the ERK inhibitor PD98059 resulted in the increase in CIITA-mediated gene expression from a class II promoter, enhanced the nuclear concentration of CIITA, and impaired its ability to bind to the nuclear export factor, CRM1. In contrast, inhibition of ERK1/2 activity had little effect on serine-to-alanine mutant forms of CIITA. These data suggest a model whereby ERK1/2-mediated phosphorylation of CIITA down-regulates CIITA activity by priming it for nuclear export, thus providing a means for cells to tightly regulate the extent of antigen presentation.     

High level class II trans-activator induction does not occur with transient activation of the IFN-gamma signaling pathway

Gene activation in early development is highly dependent on precise concentrations of trans-acting factors for the activation of different genes at differing points in the embryo. Thus, not only is the presence or absence of a particular trans-activator or repressor relevant in determining gene activation, but also the concentration of the regulatory protein must be above or below a certain threshold for proper gene regulation. Signaling pathways in somatic cells are thought to represent cascades of on/off switches, mediated most commonly by phosphorylation. Here we demonstrate a quantitative mechanism for regulating the level of a component of the IFN-gamma signaling pathway that in effect represents the differential sensitivities of STAT1, IFN-regulatory factor-1, and class II trans-activator (CIITA) to IFN-gamma. Unlike developmental gene regulation, in which specificity of gene activation is a function of regulatory protein concentrations, specificity of gene activation in the IFN-gamma signaling pathway is regulated by the duration of the activation of the primary IFN-gamma-regulatory protein, STAT1. This result most likely explains previously reported data indicating that a minimum amount of IFN-gamma is required for MHC class II gene activation despite the fact that the level of the IFN-gamma-inducible factor directly required for MHC class II induction, CIITA, directly correlates with the level of MHC class II expression. The induction of a high level of CIITA is dependent on sustained IFN-gamma signaling. The possible implications of this result for tumorigenesis are discussed.     

Suppressor of cytokine signaling 1 inhibits cytokine induction of CD40 expression in macrophages

CD40 is a type I membrane-bound molecule belonging to the TNFR superfamily that is expressed on various immune cells including macrophages and microglia. The aberrant expression of CD40 is involved in the initiation and maintenance of various human diseases including multiple sclerosis, arthritis, atherosclerosis, and Alzheimer's disease. Inhibition of CD40 signaling has been shown to provide a significant beneficial effect in a number of animal models of human diseases including the aforementioned examples. We have previously shown that IFN-gamma induces CD40 expression in macrophages and microglia. IFN-gamma leads to STAT-1alpha activation directly and up-regulation of NF-kappaB activity due to the secretion and subsequent autocrine signaling of TNF-alpha. However, TNF-alpha alone is not capable of inducing CD40 expression in these cells. Suppressor of cytokine signaling 1 protein (SOCS-1) is a cytokine-inducible Src homology 2-containing protein that regulates cytokine receptor signaling by inhibiting STAT-1alpha activation via a specific interaction with activated Janus kinase 2. Given the important role of CD40 in inflammatory events in the CNS as well as other organ systems, it is imperative to understand the molecular mechanisms contributing to both CD40 induction and repression. We show that ectopic expression of SOCS-1 abrogates IFN-gamma-induced CD40 protein expression, mRNA levels, and promoter activity. Additionally, IFN-gamma-induced TNF-alpha secretion, as well as STAT-1alpha and NF-kappaB activation, are inhibited in the presence of SOCS-1. We conclude that SOCS-1 inhibits cytokine-induced CD40 expression by blocking IFN-gamma-mediated STAT-1alpha activation, which also then results in suppression of IFN-gamma-induced TNF-alpha secretion and subsequent NF-kappaB activation.     

Re-examination of the role of suppressor of cytokine signaling 1 (SOCS1) in the regulation of toll-like receptor signaling

Suppressor of cytokine signaling 1 (SOCS1) is an obligate negative regulator of cytokine signaling and most importantly in vivo, signaling via the interferon-gamma (IFN-gamma) receptor. SOCS1, via its Src homology 2 domain, binds to phosphotyrosine residues in its targets, reducing the amplitude of signaling from cytokine receptors. SOCS1 is also implicated in blocking Toll-like receptor (TLR) signaling in macrophages activated by TLR agonists such as lipopolysaccharide (LPS), thus regulating multiple steps in the activation of innate immune responses. To rigorously test this, we isolated macrophages from Socs1-/- mice on multiple genetic backgrounds. We found no evidence that SOCS1 blocked TLR-activated pathways, endotoxin tolerance, or nitric oxide production. However, Socs1-/-;IFN-gamma-/- mice were extremely susceptible to LPS challenge, confirming previous findings. Because LPS induces IFN-beta production from macrophages, we tested whether SOCS1 regulates IFN-alpha/beta receptor signaling. We find that SOCS1 is required to inhibit IFN-alpha/beta receptor signaling in vitro. Furthermore, the absence of a single allele encoding TYK2, a JAK (Janus kinase) family member essential IFN-alpha/beta receptor signaling, rescued Socs1-/- mice from early lethality, even in the presence of IFN-gamma. We conclude that previous reports linking SOCS1 to TLR signaling are most likely due to effects on IFN-alpha/beta receptor signaling.