Yoshimi Aizaki

The (pro)renin receptor is cleaved by ADAM19 in the Golgi leading to its secretion into extracellular space

The (pro)renin receptor ((P)RR), which is a recently discovered molecule of the renin–angiotensin system, plays an important role in the development of cardiovascular diseases. However, the molecular properties and the subcellular distribution of (P)RR remain controversial. In this study, (P)RR-Venus in Chinese hamster ovary (CHO) cells ((P)RR-Venus-CHO) or endogenous (P)RR in human vascular smooth muscle cells (VSMC) were constitutively cleaved without any stimulation, and secretion of the amino-terminal fragment (NTF-(P)RR) into the media was determined using western blot analysis. Immunofluorescent analysis showed robust expression of (P)RR in the endoplasmic reticulum (ER) or the Golgi but not in the plasma membrane. Moreover, we identified ADAM19, which is expressed in the Golgi, as one of cleaving proteases of (P)RR. Transfected ADAM19 evoked the shedding of (P)RR, whereas transfected dominant negative ADAM19 suppressed it. Although (P)RR contains a furin cleavage site, neither the furin-deficient LoVo cells nor furin inhibitor-treated VSMC lost NTF-(P)RR in the media. The secreted NTF-(P)RR induced the renin activity of prorenin in the extracellular space. We describe that (P)RR is mainly localized in the subcellular organelles, such as the ER and Golgi, and (P)RR is cleaved by ADAM19 in the Golgi resulting in two fragments, NTF-(P)RR and CTF-(P)RR. These results may suggest that (P)RR is predominantly secreted into the extracellular space.

Aberrant histone acetylation contributes to elevated interleukin-6 production in rheumatoid arthritis synovial fibroblasts.

Accumulating evidence indicates that epigenetic aberrations have a role in the pathogenesis of rheumatoid arthritis (RA). However, reports on histone modifications are as yet quite limited in RA. Interleukin (IL)-6 is an inflammatory cytokine which is known to be involved in the pathogenesis of RA. Here we report the role of histone modifications in elevated IL-6 production in RA synovial fibroblasts (SFs). The level of histone H3 acetylation (H3ac) in the IL-6 promoter was significantly higher in RASFs than osteoarthritis (OA) SFs. This suggests that chromatin structure is in an open or loose state in the IL-6 promoter in RASFs. Furthermore, curcumin, a histone acetyltransferase (HAT) inhibitor, significantly reduced the level of H3ac in the IL-6 promoter, as well as IL-6 mRNA expression and IL-6 protein secretion by RASFs. Taken together, it is suggested that hyperacetylation of histone H3 in the IL-6 promoter induces the increase in IL-6 production by RASFs and thereby participates in the pathogenesis of RA.

Short-Chain Fatty Acids Induce Intestinal Transient Receptor Potential Vanilloid Type 6 Expression in Rats and Caco-2 Cells

Fructooligosaccharides (FOS) are indigestible oligosaccharides that increase calcium absorption by the colorectum in rats, but the underlying mechanisms remain unclear. We therefore investigated the effects of FOS on expressions of genes involved with calcium absorption in rat colorectal mucosa cells. After feeding a diet containing FOS (100 g/kg diet) to rats for 2 d, we investigated gene transcripts of transient receptor potential vanilloid type 6 (TRPV6), calbindin-D9k, and plasma membrane calcium-ATPase 1b (PMCA1b). The FOS diet increased expression of TRPV6 and calbindin-D9k but did not affect PMCA1b expression. Because FOS could not directly affect gene expression, SCFA formed as fermentation products of FOS were considered as likely intermediates. SCFA (2.0 mmol/L) were thus added to Caco-2 human colonic epithelial cells, resulting in significantly increased mRNA expression of TRPV6. To ascertain the effects of SCFA on mRNA expression, a genomic clone of TRPV6 was isolated. Using luciferase reporter assay, a segment between -71 nucleotides and the translation start site was found to contain a positive responsive element to SCFA. These results suggest that FOS increase calcium absorption by increasing mRNA expression of TRPV6 in rat colorectum, and cell culture analysis indicated that SCFA, as fermentation products of FOS, are involved in the increased mRNA expression of TRPV6. We found for the first time, to our knowledge, that regulation of TRPV6 gene expression by SCFA may be a molecular mechanism involved in the promotion of calcium absorption by FOS in rats.

Histone Methylation and STAT‐3 Differentially Regulate Interleukin‐6–Induced Matrix Metalloproteinase Gene Activation in Rheumatoid Arthritis Synovial Fibroblasts

Synovial fibroblasts (SFs) produce matrix‐degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.

Distinct roles of the DRY motif in rat melanin-concentrating hormone receptor 1 in signaling control

Rhodopsin family (class A) G protein-coupled receptors possess common key residues or motifs that appear to be important for receptor function. To clarify the roles of the highly conserved amino acid triplet Asp(3.49)-Arg(3.50)-Tyr(3.51) (DRY motif), we examined how single-substitution mutations of the amino acids in the motif influenced specific features of rat melanin-concentrating hormone receptor 1 (MCH1R) activity. Substitution of either Asp140(3.49) or Tyr142(3.51) to Ala resulted in nonfunctional receptors, despite the retention of apparent potencies for agonist binding. These loss-of-function phenotypes may be caused by the lack of stimulation for GDP-GTP exchange observed in GTPgammaS-binding assays. On the other hand, substitution of Arg141(3.50) to Ala caused a 4-fold reduction in the agonist binding affinity and, concomitantly, a rightward shift of the dose-dependency curve for calcium mobilization and inhibition of cyclic AMP production. Although many experimental studies have suggested that the DRY motif is involved in maintaining the receptor in its ground state, none of the DRY motif substitutions to Ala in MCH1R led to constitutive activation, in terms of the basal signaling level for ERK1/2 activation or GTPgammaS binding. These data suggest that the major contribution of the DRY motif in MCH1R is to govern receptor conformation and G protein coupling/recognition.

Regulation of melanin-concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) belongs to the class A G protein-coupled receptors (GPCRs). The MCH-MCH1R system plays a central role in energy metabolism, and thus the regulation of signaling pathways activated by this receptor is of particular interest. Regulator of G protein signaling (RGS) proteins work by increasing the GTPase activity of G protein alpha subunits and attenuate cellular responses coupled with G proteins. Recent evidence has shown that RGS proteins are not simple G protein regulators but equally inhibit the signaling from various GPCRs. Here, we demonstrate that RGS8, which is highly expressed in the brain, functions as a negative modulator of MCH1R signaling. By using biochemical approaches, RGS8 was found to selectively and directly bind to the third intracellular (i3) loop of MCH1R in vitro. When expressed in HEK293T cells, RGS8 and MCH1R colocalized to the plasma membrane and RGS8 potently inhibited the calcium mobilization induced by MCH. The N-terminal 9 amino acids of RGS8 were required for the optimal capacity to downregulate the receptor signaling. Furthermore, Arg(253) and Arg(256) at the distal end of the i3 loop were found to comprise a structurally important site for the functional interaction with RGS8, since coexpression of RGS8 with R253Q/R256Q mutant receptors resulted in a loss of induction of MCH-stimulated calcium mobilization. This functional association suggests that RGS8 may represent a new therapeutic target for the development of novel pharmaceutical agents.

Histone methylation and STAT3 differentially regulate IL‐6‐induced MMP gene activation in rheumatoid arthritis synovial fibroblasts

Synovial fibroblasts (SFs) produce matrix‐degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.

OP0322 A novel hierarchical relationship between interleukin-17a and interferon-alpha is indicated by analysis of multiple cytokines in the serum of adult-onset still's disease and behÇet's disease

Background Adult-onset Stills disease (AOSD) and Behçets disease (BD) are both systemic inflammatory diseases, the causes of which are largely unknown. They have been recently classified as autoinflammatory diseases, a group of diseases in which innate rather than acquired immunity plays important roles in their pathogenesis. As AOSD and BD are clinically distinct diseases, their cytokine networks should also be different. Objectives In this study, we attempted to quantify the levels of multiple cytokines in the serum of patients by utilizing a beads-array technique and ELISA, and then compared the serum cytokine profiles of the two diseases by factor analysis. We then sought to clarify the hierarchical relationship between interleukin (IL-) 17A and interferon (IFN-) α using peripheral blood mononuclear cells (PBMCs). Methods We quantified the serum levels of 10 cytokines (IFN-α, IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-17A and tumor necrosis factor α) in 16 AOSD patients and 28 BD patients using multiplex bead array assays and IL-18 along with ELISA. The data were then subjected to factor analysis. We next stimulated PBMCs from three healthy volunteers in vitro with class A CpG oligodeoxynucleotides (ODNs) in the presence or absence of IL-17A for 15 hours. We performed flowcytometric analysis to examine the expression of intracellular IFN-α in plasmacytoid dendritic cells (pDCs). Results Two factors were extracted from the factor analysis using the data on 8 cytokines that were detectable in the serum of the patients. IL-17A and IFN-α, the levels of which showed a strong positive correlation in the serum of BD patients (Fig. A), were the main components of Factor 1, while Factor 2 consisted of IL-6, IL-10 and IL-18 (Fig. B). Patients were also plotted on a plane determined by Factors 1 and 2 according to each patients factor scores. Those who were high in Factor 1 but low in Factor 2 were likely to be BD patients and vice versa, many of those who were high in Factor 2 but low in Factor 1 were AOSD patients. In terms of flowcytometric analysis, IL-17A alone did not induce IFN-α expression in pDCs, but it did substantially increase IFN-α-positive pDCs induced by CpG ODNs. Conclusions The cytokines examined were clearly separated into distinct groups by the factor analysis. Similarly, the AOSD and BD patients could be separated, although roughly. High levels of serum IL-6, -10 and -18 suggest AOSD while high levels of IFN-α and IL-17A indicate BD. To establish patterns of correlation among cytokines, it is important to focus on the cytokine concentrations in each patient, rather than the average cytokine concentrations in each of the diseases. In terms of the hierarchical relationship between IFN-α and IL-17A, a previous report suggested that IFN-α blocks IL-17A production in PBMCs from BD patients. Thus, we examined the effect of IL-17A on IFN-α production. It should be noted that the real stimulus for IFN-α release in BD is unknown. In addition, cells other than pDCs may be involved in the production of IFN-α. Nevertheless, understanding the hierarchical relationship among cytokines should prove to be helpful in clarifying the pathogenesis of various inflammatory diseases. Disclosure of Interest None declared

FRI0042 Altered Profiles of Histone Lysine Methylation Affect Mmp Gene Transcription in Rheumatoid Arthritis Synovial Fibroblasts

Background Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes progressive joint destruction. A line of evidence suggests that synovial fibroblasts (SFs) play an important role in the pathogenesis of RA. RASFs produce matrix metalloproteinases (MMPs) that degrade articular cartilage. Although interleukin-6 (IL-6) is an inflammatory cytokine and is proved to be involved in the pathogenesis of RA, it is unknown whether IL-6 affects MMPs gene transcription in RASFs. Furthermore, recent advances have revealed that epigenetic mechanisms, including histone modifications, are considered to be important regulators in gene transcription. We hypothesized that epigenetic dysregulation might induce RASF activation. Objectives The aim of this study is to examine whether IL-6 regulates MMPs expression in RASFs and whether histone modifications are associated with the MMPs gene activation in RASFs. Methods We compared MMPs gene expression by quantitative RT-PCR and histone methylation in the MMP promoters by chromatin immunoprecipitation (ChIP) assay after stimulation with IL-6 and/or soluble IL-6 receptor α (sIL-6Rα) in RASFs and control, osteoarthritis (OA) SFs. Chromatin structures in the MMP promoters were evaluated with micrococcal nuclease (MNase) assay in RASFs and OASFs. We investigated the change in the MMPs gene expression after silencing of WDR5 that is required for generating H3K4me3, an active histone marker. IL-6 signal induces Signal Transducer and Activator of Transcription 3 (STAT3) activation. To elucidate the mechanisms of IL-6-induced MMPs gene activation in RASFs, we investigated cell surface expression of the IL-6 receptor (gp130 and membrane-bound IL-6Rα) by flow cytometry, phospho-STAT3 (p-STAT3) expression by immunoblotting, and binding of STAT3 to the MMPs 1, 3, 9, and 13 promoters after IL-6 stimulation by ChIP assay in RASFs and OASFs. Results MMPs 1, 3, 9 and 13 genes were actively transcribed in RASFs. The histone methylation profiles (H3K4me3 and H3K27me3) and the result of MNase assay indicated that chromatin structures were open in the MMPs 1, 3, 9 and 13 promoters in RASFs. The depletion of WDR5 reduced the levels of H3K4me3 as well as the MMPs 1, 3, 9 and 13 gene expression. Interestingly, IL-6 and sIL-6Rα significantly increased the expression of MMPs 1, 3 and 13, but not MMP-9, in RASFs. Although the expression levels of gp130 as well as membrane-bound IL-6Rα were comparable and STAT3 was similarly phosphorylated after IL-6 stimulation in RASFs and OASFs, STAT3 bound to the MMPs 1, 3 and 13 promoters, but not the MMP-9 promoter, after stimulation with IL-6 and sIL-6Rα only in RASFs. It was suggested that binding of STAT3 to the promoters resulted in MMPs 1, 3 and 13 gene activation after IL-6 stimulation in RASFs. Conclusions Our data indicate that altered profiles of histone methylation and binding of STAT3 to the promoters regulate constitutive and IL-6-induced MMPs gene activation in RASFs and possibly arthritogenic properties of RASFs. References Araki Y et al. Arthritis Rheum. Epub. 2015 Dec 29. Disclosure of Interest None declared

Histone methylation and STAT3 differentially regulate IL-6-induced MMP gene activation in rheumatoid arthritis synovial fibroblasts: Histone methylation and STAT3 regulate MMP gene activation in RASFs

Synovial fibroblasts (SFs) produce matrix‐degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.