Catherine M. Owczarek

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.

A novel member of the STOMATIN/EPB72/mec-2 family, stomatin-like 2 (STOML2), is ubiquitously expressed and localizes to HSA chromosome 9p13.1

A cDNA encoding a novel second member of the Band7/stomatin-like/SPFH domain family in humans designated stomatin-like 2 (STOML2) has been isolated using the technique of cDNA Representational Difference Analysis. The STOML2 cDNA encoded a 356 amino acid residue polypeptide with a predicted molecular weight of 38.5 kDa. The predicted polypeptide sequence of STOML2 could be delineated into three major domains: an N-terminal α-helical region; a domain with significant similarity to a 172 amino acid region of the HSA stomatin polypeptide, composed of an alternating α-helical and β-sheet structure and a C-terminal domain that was mostly α-helical. The stomatin-like domain was observed in 51 other proteins with potentially diverse functions. Based on its homology to stomatin, STOML2 was predicted to be cytoplasmically located. However, unlike most of the other proteins containing stomatin-like domains, the predicted STOML2 polypeptide does not contain a transmembrane region although the presence of N-myristoylation sites suggest that it has the potential to be membrane-associated. Northern blot analysis of a panel of poly(A)+ mRNA from normal human adult tissues showed that a single 1.3-kb mRNA transcript encoding STOML2 was ubiquitously expressed, with relatively higher levels in skeletal muscle and heart compared to other tissues. Comparison of the STOML2 cDNA sequence with human genomic DNA indicated that the gene encoding STOML2 was 3,250 bp long and consisted of ten exons interrupted by nine introns. We have mapped STOML2 to HSA chromosome 9p13.1, a region that is rearranged in some cancers and thought to contain the gene responsible for acromesomelic dysplasia.

Multiple regions within the promoter of the murine Ifnar-2 gene confer basal and inducible expression.

The (murine) type I interferon (IFN) receptor, muIfnar-2, is expressed ubiquitously, and exists as both transmembrane and soluble forms. In the present study we show that the gene encoding muIfnar-2 spans approx. 33 kb on mouse chromosome 16, and consists of nine exons and eight introns. The three mRNA splice variants resulting in one transmembrane (muIfnar-2c) and two soluble (muIfnar-2a/2a) mRNA isoforms are generated by alternative RNA processing of the muIfnar-2 gene. Treatment of a range of murine cell lines with a combination of type I and II IFN showed that the muIfnar-2a and -2c mRNA isoforms were up-regulated independently of each other in L929 fibroblasts and hepa-1c1c7 hepatoma cells, but not in M1 myeloid leukaemia cells. Analysis of the 5 flanking region of muIfnar-2 using promoter-luciferase reporter constructs defined three regulatory regions: a region proximal to exon 1, conferring high basal expression, a distal region conferring inducible expression, and a negative regulatory region between the two. These data represent the first promoter analysis of a type I IFN receptor and, taken together with our previous data demonstrating high expression levels and dual biological functions for muIfnar-2a protein, suggests that the regulation of muIfnar-2 isoform expression may be an important way of modulating type I IFN responses.

A Type I Interferon Signaling Factor, ISF21, Encoded on Chromosome 21 Is Distinct from Receptor Components and Their Down-regulation and Is Necessary for Transcriptional Activation of Interferon-regulated Genes

The type I interferons (IFNs) are a family of cytokines, comprising at least 17 subtypes, which exert pleiotropic actions by interaction with a multi-component cell surface receptor and at least one well characterized signal transduction pathway involving JAK/STAT (Janus kinase/signaltransducer and activator oftranscription) proteins. In a previous report, we showed that a signaling factor, encoded by a gene located on the distal portion of chromosome 21, distinct from the IFNAR-1 receptor, was necessary for 2′-5′-oligoadenylate synthetase activity and antiviral responses, but not for high affinity ligand binding. In the present studies using hybrid Chinese hamster ovary cell lines containing portions of human chromosome 21, we show that the type I IFN signaling molecule, designated herein as ISF21, is distinct from the second receptor component, IFNAR-2, which is expressed in signaling and non-signaling cell lines. The location of the gene encoding ISF21 is narrowed to a region between the 10;21 and the r21 breakpoints, importantly eliminating the Mx gene located at 21q22.3 (the product of which is involved in IFN-induced antiviral responses) as a candidate for the signaling factor. To characterize the action of this factor in the type I IFN signaling pathway, we show that it acts independently of receptor down-regulation following ligand binding, both of which occur equally in the presence or absence of the factor. In addition, we demonstrate that ISF21 is necessary for transcriptional activation of 2′-5′-oligoadenylate synthetase, 6–16, and guanylate-binding protein gene promoter reporter constructs, which are mediated by several signaling pathways. ISF21 represents a novel factor as the localization to chromosome 21, and the data presented in this study exclude any of the known type I IFN signal-transducing molecules.

Soluble IFN Receptor Potentiates In Vivo Type I IFN Signaling and Exacerbates TLR4-Mediated Septic Shock

Circulating levels of a soluble type I IFNR are elevated in diseases, such as chronic inflammation, infections, and cancer, but whether it functions as an antagonist, agonist, or transporter is unknown. In this study, we elucidate the in vivo importance of the soluble type I IFNAR, soluble (s)IFNAR2a, which is generated by alternative splicing of the Ifnar2 gene. A transgenic mouse model was established to mimic the 10–15-fold elevated expression of sIFNAR2a observed in some human diseases. We generated transgenic mouse lines, designated SolOX, in which the transgene mRNA and protein-expression patterns mirrored the expression patterns of the endogenous gene. SolOX were demonstrated to be more susceptible to LPS-mediated septic shock, a disease model in which type I IFN plays a crucial role. This effect was independent of “classical” proinflammatory cytokines, such as TNF-α and IL-6, whose levels were unchanged. Because the increased levels of sIFNAR2a did not affect the kinetics of the increased interferonemia, this soluble receptor does not potentiate its ligand signaling by improving IFN pharmacokinetics. Mechanistically, increased levels of sIFNAR2a are likely to facilitate IFN signaling, as demonstrated in spleen cells overexpressing sIFNAR2a, which displayed quicker, higher, and more sustained activation of STAT1 and STAT3. Thus, the soluble IFNR is an important agonist of endogenous IFN actions in pathophysiological processes and also is likely to modulate the therapeutic efficacy of clinically administered IFNs.

Characterization and transcriptional analysis of the mouse Chromosome 16 cytokine receptor gene cluster.

The class II cytokine receptor (CIICR) genes Il10r2 and Ifnar1 are localized on mouse Chr 16 in a cluster that also contains the CIICR genes Ifnar2 and Ifngr2. The structure of the Il10r2 gene was deduced and consisted of 7 exons and 6 introns arrayed in an organization similar to its human ortholog. We also present a revised Il10r2 cDNA sequence with a total of 100 bp of additional nucleotide sequence in the 5′ and 3′ untranslated regions, and report the first extensive profiles of Il10r2 and Ifnar1 mRNA developmental stage and adult tissue expression. Promoter-luciferase reporter constructs were used to define the major region (−108 to +67) that conferred basal expression of the Il10r2 gene. Long-range comparative genomic sequence analysis between the mouse and the orthologous human CIICR genomic loci revealed several conserved non-coding regions. The most proximal conserved non-coding sequence was a 204-bp element located 1.6 kb upstream of the transcriptional start site of Ifnar2 that had repressor-like activity in transient transfection assays with an SV40 promoter-luciferase reporter construct. The identification of multiple conserved non-coding sequences will provide the basis for further investigations to elucidate CIICR gene regulation.

Identification and characterization of polymorphisms at the HSA alpha1-acid glycoprotein (ORM*) gene locus in Caucasians

Abstract Human α 1 -acid glycoprotein (AGP) or orosomucoid (ORM) is a major acute phase protein that is thought to play acrucialroleinmaintaininghomeostasis.HumanAGPistheproductofaclusterofatleasttwoadjacentgeneslocatedon HSA chromosome 9. Using a range of restriction endonucleases we have investigated DNA variation at the locusencoding the AGP genes in a panel of healthy Caucasians. Polymorphisms were identified using Bam HI, Eco RI,BglII,Pvu II,Hin dIII, TaqI and Msp I. Non-random associations were found between the Bam HI, Eco RI, BglII RFLPs.The RFLPs detected with Pvu II, TaqI and Msp I were all located in exon 6 of both AGP genes. The duplication of anAGP gene was observed in 11% of the indiviuals studied and was in linkage disequilibrium with the TaqI RFLP. Theidentificationandcharacterizationofthesepolymorphismswillproveusefulforotherpopulationandforensicstudies. Key words :Humanα 1 -acidglycoprotein,RFLP,linkagedisequilibrium.Received:March5,2002;accepted:March25,2002. Introduction