Saara Aittomäki

Identification of p100 as a coactivator for STAT6 that bridges STAT6 with RNA polymerase II

STAT6 is a central mediator of IL‐4‐induced gene responses. STAT6‐mediated transcription is depend ent on the C‐terminal transcription activation domain (TAD), but the mechanisms by which STAT6 activates transcription are poorly understood. Here, we have identified the staphylococcal nuclease (SN)‐like domain and tudor domain containing protein p100 as a STAT6 TAD interacting protein. p100 was originally characterized as a transcriptional coactivator for Epstein–Barr virus nuclear antigen 2. STAT6 interacted with p100 in vitro and in vivo. The interaction was mediated by the TAD domain of STAT6 and the SN‐like domain of p100. p100 did not affect the immediate activation events of STAT6, but enhanced STAT6‐mediated transcriptional activation and the IL‐4‐induced Igϵ gene transcription in human B‐cell line. Finally, p100 associated with the large subunit of RNA polymerase II and was mediating interaction between STAT6 and RNA polymerase II. These findings identify p100 as a novel coactivator for STAT6 and suggest that p100 functions as a bridging factor between STAT6 and the basal transcription machinery.

Local Hypomethylation in Atherosclerosis Found in Rabbit ec-sod Gene

Extracellular superoxide dismutase (EC-SOD) protects arteries against deleterious effects of superoxide anions and the development of atherosclerosis. In this study, we cloned and characterized rabbit ec-sod gene. We identified 6 rabbit C-elements and 5 CpG clusters in the cloned sequence. One of the CpG clusters is located on the coding sequence. Because CpG clusters are potential sites for methylation and may explain the occurrence of mutations, methylation status of each of the CpG dimers located in the coding sequence CpG cluster was characterized using direct genomic sequencing. Unexpectedly, a marked reduction in the amount of methylated CpG dinucleotides in ec-sod gene was detected in atherosclerotic aortas as compared with normal aortic intima-media. Although alterations in DNA methylation are well characterized in malignant tumors, the presence of methylation changes in atherosclerosis has not been studied even though both diseases are characterized by excess cellular proliferation and alterations in gene expression. Further analysis of the whole genomic methylation by high-pressure liquid chromatography in normal and atherosclerotic aortas revealed a tendency for a decreased 5-methylcytosine (5-mC) content in atherosclerotic aortas as compared with normal arteries. Hypomethylation in atherosclerotic aortas occurred at the same level as has been reported from malignant tumors. Although a causal relationship between the methylation level and expression of EC-SOD cannot be proven, our results show that ec-sod hypomethylation is associated with the development of atherosclerosis and suggest that it may affect structure and function of ec-sod and other genes possibly involved in the development of atherosclerotic lesions.

Cooperation Among Stat1, Glucocorticoid Receptor, and PU.1 in Transcriptional Activation of the High-Affinity Fcγ Receptor I in Monocytes

IFN-γ and glucocorticoids regulate inflammatory and immune responses through Stat1 and glucocorticoid receptor (GR) transcription factors, respectively. The biological responses to these polypeptides are determined by integration of various signaling pathways in a cell-type and promoter-dependent manner. In this study we have characterized the molecular basis for the functional cooperation between IFN-γ and dexamethasone (Dex) in the induction of the high-affinity Fcγ receptor I (FcγRI) in monocytes. Dex did not affect IFN-γ-induced Stat1 DNA binding activity or induce novel DNA-binding complexes to the FcγRI promoter. By using cell systems lacking functional GR or Stat1, we showed that GR stimulated Stat1-dependent transcription in a ligand-dependent manner, while Stat1 did not influence GR-dependent transcription. The cooperation required phosphorylation of Tyr701, DNA binding, and the trans-activation domain of Stat1, but did not involve Ser727 phosphorylation of Stat1 or physical interaction between GR and Stat1. The costimulatory effect of Dex was not dependent on a consensus glucocorticoid response element in the Stat1-responsive promoters, but required the DNA-binding and trans-activation functions of GR, and Dex-induced protein synthesis. GR activated the natural FcγRI promoter construct, and this response required both Stat1 and the Ets family transcription factor PU.1. Previously, physical association between GR and Stat5 has been shown to enhance Stat5-dependent and suppress GR-dependent transcription. The results shown here demonstrate a distinct, indirect mechanism of cross-modulation between cytokine and steroid receptor signaling that integrates Stat1 and GR pathways with cell type-specific PU.1 transcription factor in the regulation of FcγRI gene transcription.

Rabbit extracellular superoxide dismutase: expression and effect on LDL oxidation.

Extracellular superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme with an abundant mRNA expression in kidney and arterial wall. In order to study expression and antioxidative function of EC-SOD, we cloned the rabbit ec-sod cDNA and produced the recombinant protein in cell culture. In vitro studies did not show a direct relationship between the amounts of synthesized mRNA and secreted protein activity, suggesting post-transcriptional regulation. The antiatherogenic role of EC-SOD was studied by determining the effect of EC-SOD on the oxidation (ox) of low density lipoprotein (LDL), and subsequent degradation of oxLDL in RAW 264 macrophages in vitro. It was found that recombinant EC-SOD reduced both the degradation of LDL in RAW 264 macrophages by 28-36% and its electrophoretic mobility caused by endothelial cell-mediated oxidation. It is therefore suggested that EC-SOD can act as a protective enzyme against the development of atherosclerosis.

Therapeutic Targeting of the JAK/STAT Pathway

Antibodies that block cytokine function provide a powerful therapeutic tool especially for the treatment of autoimmune diseases. Cytokines are a group of small hydrophilic glycoproteins that bind their receptors on the cell surface and subsequently activate intracellular signalling cascades, such as the JAK/STAT pathway. A bulk of evidence has demonstrated that genetic mutations in signalling molecules can cause immunodeficiencies and malignant cell growth. As a result, several drug companies have begun to develop therapeutics that inhibit the function of JAK tyrosine kinases. Currently, two JAK inhibitors, tofacitinib and ruxolitinib, are used in the clinic for treating rheumatoid arthritis and myeloproliferative diseases, respectively. Inhibiting JAK function has been shown to efficiently prevent the uncontrolled growth of cancerous cells and to harness overly active immune cells. In the future, other small molecule compounds are likely to come into clinical use, and intense work is ongoing to develop inhibitors that specifically target the constitutively active mutant JAKs. This MiniReview will summarize the basic features of the JAK/STAT pathway, its role in human disease and the therapeutic potential of JAK/STAT inhibitors.

Monocyte anergy is present in patients with severe acute pancreatitis and is significantly alleviated by granulocyte-macrophage colony-stimulating factor and interferon-gamma in vitro.

Objectives: Severe acute pancreatitis (AP) is frequently associated with immune suppression, which increases the risk of infections, organ failure, and death. Our aims were to measure monocyte function (ie, HLA-DR expression and tumor necrosis factor-α [TNF-α] production as markers of immune suppression) in patients with severe AP and to determine whether treatment of blood samples with granulocyte-macrophage colony-stimulating factor (GM-CSF) and/or interferon-γ (IFN-γ) corrected the functional defects of monocytes in vitro. Methods: The study consisted of 28 patients with severe AP who were treated at intensive care unit and in whom the proportion of HLA-DR-positive monocytes in the circulation was less than 70%, and 28 matched control subjects who were selected from healthy laboratory personnel. HLA-DR density was determined by whole blood flow cytometry. Monocyte TNF-α production in response to bacterial lipopolysaccharides (LPSs) was studied in a whole blood assay. Aliquots of blood were supplemented with IFN-γ (all 28 patients), GM-CSF (the last 24 patients), or both (the last 12 patients). Results: The median proportion of HLA-DR-positive monocytes was 45% in patients (range, 18%-73%) and was 98% in controls (range, 86%-100%; P < 0.001). TNF-α levels in response to LPSs were lower in patients (545 pg/mL; range, 84-1990 pg/mL) than in controls (1415 pg/mL; range, 660-5490 pg/mL; P < 0.001). The proportion of HLA-DR-positive cells correlated positively with TNF-α levels (r = 0.56; P < 0.01). Both GM-CSF and IFN-γ increased HLA-DR expression of monocytes in patients (98%; range, 74%-100% for GM-CSF; 99%; range, 86%-100% for IFN-γ; both P < 0.001). The combination restored monocyte HLA-DR expression (99%; range, 96%-100%; P = 0.002). Compared with basal levels, GM-CSF increased TNF-α production of monocytes both in blood samples from patients (median, 1320 pg/mL; range, 35-8015 pg/mL) and controls (median, 3450 pg/mL; range, 1040-9835 pg/mL; both P < 0.001). IFN-γ increased TNF-α production by monocytes in patients (683 pg/mL; range, 186-2705 pg/mL; P < 0.05) but not in controls (1658 pg/mL; range, 765-4755 pg/mL; P = 0.31). With the combination of GM-CSF and IFN-γ, the TNF-α levels of monocytes in patients (3185 pg/mL; range, 545-8280 pg/mL) and in controls (2800 pg/mL; range, 1080-6860 pg/mL) were comparable. Conclusions: The proportion of HLA-DR-positive monocytes correlates with TNF-α production, and they both reflect the degree of immune suppression. The low proportion of HLA-DR-positive monocytes in AP can be reversed in vitro by GM-CSF and/or IFN-γ. The GM-CSF and IFN-γ treatments also increase LPS-induced TNF-α production. By the combination of GM-CSF and IFN-γ, but not by either agent alone, LPS-induced TNF-α production of monocytes was equally high in patients and in controls.

PU.1 is required for transcriptional activation of the Stat6 response element in the Igε promoter

Signal transducer and activator of transcription 6 (Stat6) has a crucial role in regulation of IL‐4‐induced gene responses. Stat6‐binding sites are present in the promoters of both ubiquitously and cell‐type‐specifically expressed genes. The promoter regions of IL‐4‐inducible genes contain cis‐acting elements for several transcription factors that act in concert with Stat6 and are also likely to modulate lineage‐specific gene expression. We have observed that the Stat6 response element from the B‐cell‐specific Igϵ promoter is readily activated upon IL‐4 stimulation in B cells but not in non‐hematopoietic cells. A minimal low‐affinity PU.1‐core‐binding sequence (5′‐AGAA‐3′) was identified within the Stat6 DNA‐binding site in the Igϵ promoter. Ectopic expression of the myeloid‐ and B‐cell‐specific transcription factor PU.1 restored the IL‐4‐inducibility of the Igϵ‐Stat6 response element in HepG2 cells, and the induction required an intact PU.1‐binding sequence. Both the transactivation and the DNA‐binding domains of PU.1 were required for induction of Stat6‐mediated transcription. The co‐operation between PU.1 and Stat6 in transactivation of the Igϵ gene represents a molecular mechanism for the fine‐tuning of cell‐type‐restricted expression of IL‐4‐induced gene responses.

Myeloid cell expressed proprotein convertase FURIN attenuates inflammation

The proprotein convertase enzyme FURIN processes immature pro-proteins into functional end- products. FURIN is upregulated in activated immune cells and it regulates T-cell dependent peripheral tolerance and the Th1/Th2 balance. FURIN also promotes the infectivity of pathogens by activating bacterial toxins and by processing viral proteins. Here, we evaluated the role of FURIN in LysM+ myeloid cells in vivo. Mice with a conditional deletion of FURIN in their myeloid cells (LysMCre-fur(fl/fl)) were healthy and showed unchanged proportions of neutrophils and macrophages. Instead, LysMCre-fur(fl/fl) mice had elevated serum IL-1β levels and reduced numbers of splenocytes. An LPS injection resulted in accelerated mortality, elevated serum pro-inflammatory cytokines and upregulated numbers of pro-inflammatory macrophages. A genome-wide gene expression analysis revealed the overexpression of several pro-inflammatory genes in resting FURIN-deficient macrophages. Moreover, FURIN inhibited Nos2 and promoted the expression of Arg1, which implies that FURIN regulates the M1/M2-type macrophage balance. FURIN was required for the normal production of the bioactive TGF-β1 cytokine, but it inhibited the maturation of the inflammation-provoking TACE and Caspase-1 enzymes. In conclusion, FURIN has an anti-inflammatory function in LysM+ myeloid cells in vivo.

IL-7Rα Expression Regulates Murine Dendritic Cell Sensitivity to Thymic Stromal Lymphopoietin

Thymic stromal lymphopoietin (TSLP) and IL-7 are related cytokines that mediate growth and differentiation events in the immune system. They signal through IL-7Rα–containing receptors. Target cells of TSLP in Th2 responses include CD4 T cells and dendritic cells (DCs). Although it has been reported that expression of TSLP receptor (TSLPR) on CD4 T cells is required for OVA-induced lung inflammation, DCs have also been shown to be target cells of TSLP. In this study, we show that murine ex vivo splenic DCs are unresponsive to TSLP, as they fail to phosphorylate STAT5, but in vitro overnight culture, especially in presence of IL-4, renders DCs responsive to both TSLP and IL-7. This induced responsiveness is accompanied by dramatic upregulation of IL-7Rα on DCs with little change in expression of TSLPR or of γc. In splenic DCs, the induction of IL-7Rα occurs mainly in CD8− DCs. In vivo, we found that IL-4 has a differential regulatory role on expression of IL-7Rα depending on the cell type; IL-4 decreases IL-7Rα expression on CD4 T cells whereas it upregulates the expression on DCs. Our results indicate that the induction of IL-7Rα expression on DCs is critical for TSLP responsiveness and that IL-4 can upregulate IL-7Rα on DCs.

Proprotein convertase FURIN regulates T cell receptor-induced transactivation

Antigen emergence rapidly stimulates T cells, which leads to changes in cytokine production, cell proliferation, and differentiation. Some of the key molecules involved in these events, such as TGF‐β1 and NOTCH1, are synthesized initially as inactive precursors and are proteolytically activated during T cell activation. PCSKs regulate proprotein maturation by catalyzing the proteolytic cleavage of their substrates. The prototype PCSK FURIN is induced upon TCR activation, and its expression in T cells is critical for the maintenance of peripheral immune tolerance. In this study, we tested the hypothesis that FURIN regulates T cell activation. Our data demonstrate that IL‐2 is increased initially in FURIN‐deficient mouse CD4+ T cells, but the TCR‐induced IL‐2 mRNA expression is not sustained in the absence of FURIN. Accordingly, the inhibition of FURIN in human Jurkat T cell lines also results in a decrease in IL‐2 production, whereas the overexpression of WT FURIN is associated with elevated IL‐2 levels. In Jurkat cells, FURIN is dispensable for immediate TCR signaling steps, such as ERK, ZAP70, or LAT phosphorylation. However, with the use of gene reporter assays, we demonstrate that FURIN regulates the AP‐1, NFAT, and NF‐κB transcription factors. Finally, by performing a transcription factor‐binding site enrichment analysis on FURIN‐dependent transcriptomes, we identify the FURIN‐regulated transcription factors in mouse CD4+ T cell subsets. Collectively, our work confirms the hypothesis that the TCR‐regulated protease FURIN plays an important role in T cell activation and that it can specifically modulate TCR‐activated transactivation.