Morisada Hayakawa

Soluble ST2 Blocks Interleukin-33 Signaling in Allergic Airway Inflammation

The ST2 gene produces a soluble secreted form and a transmembrane form, referred to as soluble ST2 and ST2L, respectively. A recent study has reported that interleukin (IL)-33 is a specific ligand of ST2L and induces production of T helper type 2 (Th2) cytokines. Although soluble ST2 is highly produced in sera of asthmatic patients and plays a critical role for production of Th2 cytokines, the function of soluble ST2 in relation to IL-33 signaling remains unclear. Here we show antagonistic effects of soluble ST2 on IL-33 signaling using a murine thymoma EL-4 cells stably expressing ST2L and a murine model of asthma. Soluble ST2 directly bound to IL-33 and suppressed activation of NF-κB in EL-4 cells stably expressing ST2L, suggesting that the complex of soluble ST2 and IL-33 fails to bind to ST2L. In a murine model of asthma, pretreatment with soluble ST2 reduced production of IL-4, IL-5, and IL-13 from IL-33-stimulated splenocytes. These results indicate that soluble ST2 acts as a negative regulator of Th2 cytokine production by the IL-33 signaling. Our study provides a molecular mechanism wherein soluble ST2 modulates the biological activity of IL-33 in allergic airway inflammation.

Increased Levels of Interleukin 33 in Sera and Synovial Fluid from Patients with Active Rheumatoid Arthritis

Objective. To determine levels of interleukin 33 (IL-33) in serum and synovial fluid (SF) and their clinical associations in patients with rheumatoid arthritis (RA). To evaluate the ability of activated peripheral blood mononuclear cells (PBMC) and fibroblast-like synoviocytes (FLS) from RA patients to release IL-33. Methods. Sera were obtained from 59 patients with RA, 10 patients with infectious diseases, and 42 healthy volunteers. SF samples were obtained from 15 patients with RA and 13 with osteoarthritis. IL-33 levels were measured using a sandwich ELISA after removal of rheumatoid factor with protein A-Sepharose beads. FLS were stimulated with IL-1ß and tumor necrosis factor, and treated with or without chemical damage. PBMC were stimulated with anti-CD3/CD28 antibodies. The levels of IL-33 were measured in the culture supernatants and cell lysates by ELISA or immunoblotting. Results. Serum IL-33 levels were significantly higher in RA patients, especially in the high disease activity group compared to the moderate or low activity group. IL-33 levels in SF were elevated in all 15 RA patients measured. IL-33 levels were higher in SF samples than in sera in 7 RA patients measured simultaneously. The 30-kDa IL-33 precursor was detected in the culture supernatants of damaged FLS but was not detected in those of activated PBMC and non-damaged FLS. Conclusion. IL-33 levels were elevated in sera and SF samples from patients with RA, and correlated with disease activity. IL-33 was produced mainly in inflamed joints; IL-33/ST2L signaling might play an important role in joint inflammation of human RA.

TRAF6 is a critical signal transducer in IL-33 signaling pathway.

IL-33 has been shown to induce Th2 responses by signaling through the IL-1 receptor-related protein, ST2L. However, the signal transduction pathways activated by the ST2L have not been characterized. Here, we found that IL-33-induced monocyte chemoattractant protein (MCP)-1, MCP-3 and IL-6 expression was significantly inhibited in TNF receptor-associated Factor 6 (TRAF6)-deficient MEFs. IL-33 rapidly induced the formation of ST2L complex containing IL-1 receptor-associated kinase (IRAK), however, lack of TRAF6 abolished the recruitment of IRAK to ST2L. Consequently, p38, JNK and Nuclear factor-kappaB (NF-kappaB) activation induced by IL-33 was completely inhibited in TRAF6-deficient MEFs. On the other hand, IL-33-induced ERK activation was observed regardless of the presence of TRAF6. The introduction of TRAF6 restored the efficient activation of p38, JNK and NF-kappaB in TRAF6 deficient MEFs, resulting in the induction of MCP-1, MCP-3 and IL-6 expression. Moreover, IL-33 augmented autoubiquitination of TRAF6 and the reconstitution of TRAF6 mutant (C70A) that is defective in its ubiquitin ligase activity failed to restore IL-33-induced p38, JNK and NF-kappaB activation. Thus, these data demonstrate that TRAF6 plays a pivotal role in IL-33 signaling pathway through its ubiquitin ligase activity.

Mature interleukin-33 is produced by calpain-mediated cleavage in vivo

Interleukin (IL)-33 is a novel member of the IL-1 family. IL-33 is primarily synthesized as a 30-kDa precursor (pro-IL-33). Pro-IL-33 is cleaved by caspase-1 into an 18-kDa mature form (mature IL-33) in vitro. Recombinant mature IL-33 has been known to induce T-helper type-2 (Th2)-associated cytokines and inflammatory cytokines via its receptor, ST2L. However, processing of pro-IL-33 in vivo has not been clarified yet. Here, we report that calpain mediates pro-IL-33 processing in vivo. Pro-IL-33 was expressed by stimulating human epithelial cells with phorbol 12-myristate 13-acetate. Calcium ionophore induced pro-IL-33 cleavage and mature IL-33 production. This cleavage was inhibited by treatment with a calcium chelator and calpain inhibitors. Moreover, short interfering RNA-mediated knockdown of calpains suppressed pro-IL-33 cleavage. These results indicate that calpains play a critical role in pro-IL-33 processing in vivo.

ST2 gene expression is proliferation-dependent and its ligand, IL-33, induces inflammatory reaction in endothelial cells

ST2 gene products that are members of IL-1 receptor family are expressed in various cells such as growth-stimulated fibroblasts and Th2 helper T-cells, and recently, IL-33, which belongs to IL-1 family, was identified as the ligand for ST2L, the receptor type product of the ST2 gene. Subsequently, IL-33 and ST2L have been reported to be involved in Th2 immunity and inflammation, however, their functions on non-immunological cells are still obscure. Among non-immunological adhesive cells, vascular endothelial cells were reported to express both ST2 gene products and IL-33, therefore, we investigated the expression manner of the ST2 gene in vascular endothelial cells and the effect of IL-33 on endothelial cells. ST2 gene was expressed in each of the vascular endothelial cell types tested, and the expression was growth-dependent and down-regulated when the cells were differentiated to form vascular structures on the extracellular membrane matrix. IL-33 scarcely affected the growth and tube formation of the endothelial cells, but induced IL-6 and IL-8 secretion from endothelial cells with the rapid activation of extracellular signal-regulated kinase (ERK) 1/2, so IL-33 is supposed to involve in inflammatory reaction of vascular endothelial cells through its receptor, ST2L.

SNIP1 Is a Candidate Modifier of the Transcriptional Activity of c-Myc on E Box-Dependent Target Genes

Using a yeast two-hybrid screen, we found that SNIP1 (Smad nuclear-interacting protein 1) associates with c-Myc, a key regulator of cell proliferation and transformation. We demonstrate that SNIP1 functions as an important regulator of c-Myc activity, binding the N terminus of c-Myc through its own C terminus, and that SNIP1 enhances the transcriptional activity of c-Myc both by stabilizing it against proteosomal degradation and by bridging the c-Myc/p300 complex. These effects of SNIP1 on c-Myc likely contribute to synergistic effects of SNIP1, c-Myc, and H-Ras in inducing formation of foci in an in vitro transformation assay and also in supporting anchorage-independent growth. The significant association of SNIP1 and c-Myc staining in a non-small cell lung cancer tissue array is further evidence that their activities might be linked and suggests that SNIP1 might be an important modulator of c-Myc activity in carcinogenesis.

Differential Regulation of Exonic Regulatory Elements for Muscle-specific Alternative Splicing during Myogenesis and Cardiogenesis

Muscle-specific isoform of the mitochondrial ATP synthase γ subunit (F1γ) was generated by alternative splicing, and exon 9 of the gene was found to be lacking particularly in skeletal muscle and heart tissue. Recently, we reported that alternative splicing of exon 9 was induced by low serum or acidic media in mouse myoblasts, and that this splicing required de novo protein synthesis of a negative regulatory factor (Ichida, M., Endo, H., Ikeda, U., Matsuda, C., Ueno, E., Shimada, K., and Kagawa, Y. (1998) J. Biol. Chem. 273, 8492–8501; Hayakawa, M., Endo, H., Hamamoto, T., and Kagawa, Y. (1998)Biochem. Biophys. Res. Commun. 251, 603–608). In the present report, we identified a cis-acting element on the muscle-specific alternatively spliced exon of F1γ gene by an in vivo splicing system using cultured cells and transgenic mice. We constructed a F1γ wild-type minigene, containing the full-length gene from exon 8 to exon 10, and two mutants; one mutant involved a pyrimidine-rich substitution on exon 9, whereas the other was a purine-rich substitution, abbreviated as F1γ Pu-del and F1γ Pu-rich mutants, respectively. Based on an in vivo splicing assay using low serum- or acid-stimulated splicing induction system in mouse myoblasts, Pu-del mutation inhibited exon inclusion, indicating that a Pu-del mutation would disrupt an exonic splicing enhancer. On the other hand, the Pu-rich mutation blocked muscle-specific exon exclusion following both inductions. Next, we produced transgenic mice bearing both mutant minigenes and analyzed their splicing patterns in tissues. Based on an analysis of F1γ Pu-del minigene transgenic mice, the purine nucleotide of this element was shown to be necessary for exon inclusion in non-muscle tissue. In contrast, analysis of F1γ Pu-rich minigene mice revealed that the F1γ Pu-rich mutant exon had been excluded from heart and skeletal muscle of these transgenic mice, despite the fact mutation of the exon inhibited muscle-specific exon exclusion in myotubes of early embryonic stage. These results suggested that the splicing regulatory mechanism underlying F1γ pre-mRNA differed between myotubes and myofibers during myogenesis and cardiogenesis.

Characterization of ST2 transgenic mice with resistance to IL-33

IL‐33, a member of the IL‐1 family, activates MAPK and NF‐κB through its receptor ST2L and IL‐1RAcP. ST2, a member of the IL‐1R superfamily, is a secreted form of ST2 gene products, which has been shown to act as a decoy receptor for IL‐33 and to inhibit the IL‐33/ST2L/IL‐1RAcP signaling pathway. In this work, we generated ST2 transgenic mice. In control mice, intraperitoneal administration of IL‐33 caused an increased number of eosinophils in blood and in peritoneal cavity, an increased number of peritoneal MΦ, splenomegaly, accumulation of periodic acid‐Schiff‐positive material in the lung, and high concentrations of serum IL‐5 and IL‐13. However, these alterations were hardly detectable in ST2 Tg mice. In peritoneal MΦ from IL‐33‐stimulated mice, mRNA expression of M2 MΦ marker genes were increased compared with thioglycollate‐elicited peritoneal MΦ. The IL‐33‐stimulation also increased the secretion of IL‐6 from MΦ. However, when the IL‐33 was preincubated with ST2 prior to its addition to the MΦ cultures, the secretion of IL‐6 was attenuated. These data suggest that, though IL‐33 induced the Th2‐type immune responses and infiltration of M2 type MΦ into the peritoneal cavity, ST2 can downregulate these reactions both in vivo and in vitro.

A novel splice variant of mouse interleukin-1-receptor-associated kinase-1 (IRAK-1) activates nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)

Interleukin-1 (IL-1)-receptor-associated kinase (IRAK) is an indispensable signalling molecule for host-defence responses initiated by a variety of ligands that bind to members of the Toll/IL-1 receptor family. Here we report a novel splice variant of mouse IRAK-1, IRAK-1-S, which is generated by utilizing a new splicing acceptor site within exon 12. IRAK-1-S cDNA is shorter than the originally reported IRAK-1 (IRAK-1-W) cDNA by 271 nucleotides, and the subsequent frameshift causes a premature termination of translation after 23 amino acids, which are unique to the IRAK-1-S protein. To elucidate the physiological function of IRAK-1-S, we overexpressed it in 293T cells and studied the effects on the IL-1 signalling cascade. As it lacks the C-terminal region of IRAK-1-W that has been reported to contain the TRAF6 (tumour necrosis factor receptor-associated factor 6) binding domain, IRAK-1-S was unable to bind TRAF6 protein, which is a proposed downstream signalling molecule. However, IRAK-1-S overexpressed in 293T cells induced constitutive activation of nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK) independent of stimulation by IL-1, as did IRAK-1-W. To clarify the mechanism of NF-kappaB activation by IRAK-1-S in the absence of binding to TRAF6, we demonstrated that IRAK-1-S binds to IRAK-1-W through its death domain; the findings suggested that overexpressed IRAK-1-S may bind endogenous IRAK-1-W and activate TRAF6 through IRAK-1-W. These results also indicate that this novel variant may play roles in the activation of NF-kappaB and JNK by IL-1 and other ligands whose signal transduction is dependent on IRAK-1 under physiological conditions.

Genes of Human ATP Synthase: Their Roles in Physiology and Aging

The reaction of ATP synthase (F0F1) is the final step in oxidative phosphorylation (OXPHOS). Although OXPHOS has been studied extensively in bacteria, no tissue-specific functions nor bioenergetic disease, such as mitochondrial encephalomyopathy and aging occur in these organisms. Recent developments of the Human Genome Project will become an important factor in the study of mammalian bioenergetics. To elucidate the physiological roles of human F0F1, genes encoding the subunits of F0F1 were sequenced, and their expression in human cells was analyzed. The following results were obtained: A. The roles of the residues in F0F1 are not only to transform the energy of the electrochemical potential (ΔμH+) across the membrane, but also to respond rapidly to the changes in the energy demand by regulating the intramolecular rotation of F0F1 with the ΔμH+ and the inhibitors of the ATPase. B. The roles of the control regions of the F0F1 genes, are to coordinate both mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) depending on the energy demand of the cells, especially in muscle. C. The cause of the age-dependent decline of ATP synthesis has been attributed to the accumulation of mutations in mtDNA. However, the involvement of nDNA in the decline is also important because of telomere shortening in somatic cells, and age-dependent mtDNA expression analyzed with ρ° cells (cells without mtDNA).