Regulation of cytokine and chemokine expression by histone lysine methyltransferase MLL1 in rheumatoid arthritis synovial fibroblasts.

Histone lysine methylation is thought to play a role in the pathogenesis of rheumatoid arthritis (RA). We previously reported aberrant expression of the gene encoding mixed-lineage leukemia 1 (MLL1), which catalyzes methylation of histone H3 lysine 4 (H3K4), in RA synovial fibroblasts (SFs). The aim of this study was to elucidate the involvement of MLL1 in the activated phenotype of RASFs. SFs were isolated from synovial tissues obtained from patients with RA or osteoarthritis (OA) during total knee joint replacement. MLL1 mRNA and protein levels were determined after stimulation with tumor necrosis factor α (TNFα). We also examined changes in trimethylation of H3K4 (H3K4me3) levels in the promoters of RA-associated genes (matrix-degrading enzymes, cytokines, and chemokines) and the mRNA levels upon small interfering RNA-mediated depletion of MLL1 in RASFs. We then determined the levels of H3K4me3 and mRNAs following treatment with the WD repeat domain 5 (WDR5)/MLL1 inhibitor MM-102. H3K4me3 levels in the gene promoters were also compared between RASFs and OASFs. After TNFα stimulation, MLL1 mRNA and protein levels were higher in RASFs than OASFs. Silencing of MLL1 significantly reduced H3K4me3 levels in the promoters of several cytokine (interleukin-6 [IL-6], IL-15) and chemokine (C-C motif chemokine ligand 2 [CCL2], CCL5, C-X-C motif chemokine ligand 9 [CXCL9], CXCL10, CXCL11, and C-X3-C motif chemokine ligand 1 [CX3CL1]) genes in RASFs. Correspondingly, the mRNA levels of these genes were significantly decreased. MM-102 significantly reduced the promoter H3K4me3 and mRNA levels of the CCL5, CXCL9, CXCL10, and CXCL11 genes in RASFs. In addition, H3K4me3 levels in the promoters of the IL-6, IL-15, CCL2, CCL5, CXCL9, CXCL10, CXCL11, and CX3CL1 genes were significantly higher in RASFs than OASFs. Our findings suggest that MLL1 regulates the expression of particular cytokines and chemokines in RASFs and is associated with the pathogenesis of RA. These results could lead to new therapies for RA.

Essentiality of Nfatc1 short isoform in osteoclast differentiation and its self-regulation.

During osteoclast differentiation, the expression of the transcription factor nuclear factor of activated T cell 1 (Nfatc1) increases in an autoproliferative manner. Nfatc1 isoforms are of three sizes, and only the short isoform increases during osteoclast differentiation. Genetic ablation of the whole Nfatc1 gene demonstrated that it is essential for osteoclastogenesis; however, the specific role of the Nfatc1 short form (Nfatc1/αA) remains unknown. In this study, we engineered Nfatc1 short form-specific knockout mice and found that these mice died in utero by day 13.5. We developed a novel osteoclast culture system in which hematopoietic stem cells were cultured, proliferated, and then differentiated into osteoclasts in vitro. Using this system, we show that the Nfatc1/αA isoform is essential for osteoclastogenesis and is responsible for the expression of various osteoclast markers, the Nfatc1 short form itself, and Nfatc1 regulators.

Synovial Tissue Heterogeneity in Japanese Patients With Rheumatoid Arthritis Elucidated Using a Cell-Type Deconvolution Approach.

OBJECTIVE: Recent advances in single-cell RNA sequencing technology have improved our understanding of the immunological landscape of rheumatoid arthritis (RA). We aimed to stratify the synovium from East Asian patients with RA by immune cell compositions and gain insight into the inflammatory drivers of each synovial phenotype. METHODS: Synovial tissues were obtained from East Asian patients in Japan with RA (n = 41) undergoing articular surgery. The cellular composition was quantified by a deconvolution approach using a public single-cell-based reference. Inflammatory pathway activity was calculated by gene set variation analysis, and chromatin accessibility was evaluated using assay of transposase accessible chromatin-sequencing. RESULTS: We stratified RA synovium into three distinct subtypes based on the hierarchical clustering of cellular composition data. One subtype was characterized by abundant HLA-DRAhigh synovial fibroblasts, autoimmune-associated B cells, GZMK+ GZMB+ CD8+ T cells, interleukin (IL)1-ß+ monocytes, and plasmablasts. In addition, tumor necrosis factor (TNF)-α, interferons (IFNs), and IL-6 signaling were highly activated in this subtype, and the expression of various chemokines was significantly enhanced. Moreover, we found an open chromatin region overlapping with RA risk locus rs9405192 near the IRF4 gene, suggesting the genetic background influences the development of this inflammatory synovial state. The other two subtypes were characterized by increased IFNs and IL-6 signaling, and expression of molecules associated with degeneration, respectively. CONCLUSION: This study adds insights into the synovial heterogeneity in East Asian patients and shows a promising link with predominant inflammatory signals. Evaluating the site of inflammation has the potential to lead to appropriate drug selection that matches the individual pathology.

Dual effects of interleukin-10 on natural killer cells and monocytes and the implications for adult-onset Still's disease.

OBJECTIVES: We compared the serum levels of multiple cytokines in patients with adult-onset Still's disease (AOSD) and healthy controls to assess the effects of humoral factors on natural killer (NK) cells and monocytes. METHODS: We quantified the serum levels of 10 cytokines in the patients using bead-based multiplex immunoassays, along with interleukin (IL-)18 using ELISA. We then sorted NK cells and monocytes from the peripheral blood mononuclear cells (PBMCs) of healthy volunteers, cultured them in the presence or absence of cytokines that were detected in some or all of the serum samples from the AOSD patients and their combinations in vitro, and analysed the culture supernatant. RESULTS: IL-6 and IL-18 were the main cytokines increased in the serum of AOSD patients. When NK cells were cultured with the cytokines and IL-10, the combination of IL-10 and IL-18 substantially induced interferon (IFN-)γ. IL-6 had little effect on NK cells, probably because they barely expressed the IL-6 receptor and glycoprotein 130 (gp130). IFN-γ induced monocytes to produce IL-1ß, IL-6 and tumour necrosis factor (TNF-)α whereas IL-10 inhibited the induction of these proinflammatory cytokines. CONCLUSIONS: IL-10 evidently has dual effects on NK cells (stimulation) and on monocytes (inhibition). Better understanding the roles of the cytokine network would shed light on the pathogenesis of AOSD.

Characterization and Function of Tumor Necrosis Factor and Interleukin-6-Induced Osteoclasts in Rheumatoid Arthritis.

OBJECTIVE: We have previously reported that stimulation of mouse bone marrow-derived macrophages with tumor necrosis factor (TNF) and interleukin-6 (IL-6) induces differentiation of osteoclast-like cells. We undertook this study to clarify the characterization and function of human TNF and IL-6-induced osteoclasts using peripheral blood collected from patients with rheumatoid arthritis (RA) and healthy donors. METHODS: Peripheral blood monocytes were cultured with a combination of TNF and IL-6, TNF alone, IL-6 alone, or with RANKL, and their bone resorption ability was evaluated. Expression levels of NFATc1, proinflammatory cytokines, and matrix metalloproteinase 3 were analyzed. The effects of NFAT inhibitor and JAK inhibitor were examined. Furthermore, the relationship between the number of TNF and IL-6-induced osteoclasts or RANKL-induced osteoclasts differentiated from peripheral blood mononuclear cells (PBMCs) in patients with RA and the modified total Sharp score (mTSS) or whole-body bone mineral density (BMD) was examined. RESULTS: Peripheral blood monocytes stimulated with a TNF and IL-6-induced osteoclasts were shown to demonstrate the ability to absorb bone matrix. Cell differentiation was not inhibited by the addition of osteoprotegerin. Stimulation with a combination of TNF and IL-6 promoted NFATc1 expression, whereas the NFAT and JAK inhibitors prevented TNF and IL-6-induced osteoclast formation. Expression levels of IL1ß, TNF, IL12p40, and MMP3 were significantly increased in TNF and IL-6-induced osteoclasts, but not in RANKL-induced osteoclasts. The number of TNF and IL-6-induced osteoclasts differentiated from PBMCs in patients with RA positively correlated with the mTSS, whereas RANKL-induced osteoclast numbers negatively correlated with the whole-body BMD of the same patients. CONCLUSION: Our results demonstrate that TNF and IL-6-induced osteoclasts may contribute to the pathology of inflammatory arthritis associated with joint destruction, such as RA.

Treatment of psoriatic arthritis complicated by systemic lupus erythematosus with the IL-17 blocker secukinumab and an analysis of the serum cytokine profile.

Psoriasis is a chronic disease of the skin that often affects the joints (psoriatic arthritis, PsA). Biologic agents such as TNF-α, IL-23 and IL-17 blockers have been proven to be quite effective against psoriasis and PsA, indicating the importance of those cytokines in the pathogenesis of the diseases. The importance of the IL-23/IL-17 axis has also been reported in systemic lupus erythematosus (SLE), but the safety and effectiveness of IL-17 blockers in SLE remain largely unknown. We encountered a patient with PsA and SLE. We treated him with an IL-17 blocker, secukinumab, and quantified the serum levels of various cytokines before and after the initiation of secukinumab therapy. As expected, the treatment was effective against the symptoms of PsA. No serious adverse events were observed in terms of SLE. Interestingly, serum IL-6 was substantially decreased after the initiation of therapy, whereas serum IL-17 was under the detection limit. These data indicate that IL-17 is produced locally, upstream of IL-6 production.

Plasma pentraxin 3 is associated with progression of radiographic joint damage, but not carotid atherosclerosis, in female rheumatoid arthritis patients: 3-year prospective study.

Background: Pentraxin 3 (PTX3) has an important role in inflammation, immunity, and atherosclerosis. Rheumatoid arthritis (RA) is a chronic inflammatory disease featuring both joint damage and atherosclerosis. We investigated whether the plasma PTX3 level was associated with progression of joint destruction and subclinical atherosclerosis in RA patients.Methods: Plasma PTX3 levels were measured in 72 women with RA and 80 female control subjects. In RA patients, we also evaluated clinical characteristics, medications, and at one and three years, joint damage and atherosclerosis. Then we investigated whether PTX3 was associated with progression of joint destruction or an increase of carotid intima-media thickness (IMT).Results: Plasma PTX3 levels were significantly higher in the RA patients than in healthy controls (4.05 ± 2.91 ng/mL vs. 1.61 ± 1.05 ng/mL, p < .001). By multivariate linear regression analysis, the plasma pentraxin 3 level was independently associated with radiographic progression of joint damage for 3 years in the RA patients after adjustment for age, disease duration, body mass index, rheumatoid factor, MMP-3, Disease Activity Score 28-ESR, postmenopausal status, current use of corticosteroids and biologic use. On the other hands, pentraxin 3 was not associated with an increase of carotid intima-media thickness in RA patients.Conclusion: Female RA patients had elevated plasma PTX3 levels compared with control female subjects. PTX3 was independently associated with radiographic progression of joint damage in the RA patients, but not with carotid atherosclerosis.

Altered gene expression profiles of histone lysine methyltransferases and demethylases in rheumatoid arthritis synovial fibroblasts.

OBJECTIVES: Aberrant histone lysine methylation (HKM) has been reported in rheumatoid arthritis (RA) synovial fibroblasts (SFs). As histone lysine methyltransferases (HKMTs) and demethylases (HKDMs) regulate HKM, these enzymes are believed to be dysregulated in RASFs. The aim of this study is to clarify whether gene expressions of HKMTs and HKDMs are altered in RASFs. METHODS: SFs were isolated from synovial tissues obtained from RA or osteoarthritis (OA) patients during total knee joint replacement. The mRNA levels of 34 HKMTs and 22 HKDMs were examined after stimulation with tumour necrosis factor α (TNF-α) in RASFs and OASFs. RESULTS: The gene expression of the 12 HKMTs, including MLL1, MLL3, SUV39H1, SUV39H2, PRDM2, EZH2, SETD2, NSD2, NSD3, SMYD4, DOT1, and PR-set7, that catalyse the methylation of H3K4, H3K9, H3K27, H3K36, H3K79, or H4K20 was higher after TNFα stimulation in RASFs vs. OASFs. The gene expression of the 4 HKDMs, including FBXL10, NO66, JMJD2D, and FBXL11, that catalyse the methylation of H3K4, H3K9, or H3K36 was higher after TNFα stimulation in RASFs vs. OASFs. CONCLUSIONS: The study findings suggest that the HKM-modifying enzymes are involved in the alteration of HKM, which results in changes in the gene expression of RASFs.

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

OBJECTIVE: 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. METHODS: MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6. RESULTS: The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs. CONCLUSION: Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

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 increase the level of calbindin-D9k messenger RNA in Caco-2 cells.

Fructooligosaccharides (FOS) are indigestible oligosaccharides that increase the expression of calbindin-D9k and consequently increase calcium absorption by the colon in rats. The molecular mechanism of the increased expression of calbindin-D9k resulting from FOS ingestion has not been elucidated. Short-chain fatty acids (SCFAs), namely, fermentation products of FOS by intestinal bacteria have been hypothesized as direct effectors of calbindin-D9k gene expression. To test this hypothesis, SCFAs were added to Caco-2 human intestinal epithelial cells, and changes in the levels of transcription of genes for calbindin-D9k, and transcription factors (vitamin D receptor: VDR, caudal homeobox-2: Cdx-2, hepatocyte nuclear factor 1-α: HNF1-α) were determined by quantitative reverse transcription polymerase chain reaction. Addition of sodium propionate or sodium butyrate to cell cultures increased levels of calbindin-D9k mRNA to 731% (p<0.05) and 321% (p<0.05), respectively. However, addition of these SCFAs did not affect the levels of mRNA VDR, Cdx-2, or HNF1-α. In conclusion, addition of SCFAs to cultured Caco-2 cells results in elevation of calbindin-D9k mRNA, consistent with the expected role of SCFAs as mediators of the increase of calcium absorption in rats that were fed with FOS.

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.

New perspectives on secretion of (pro)renin receptor into extracellular space.

(Pro)renin receptor is a new molecule of the renin-angiotensin system. The (pro)renin receptor binds both renin and prorenin leading to protease activity. Furthermore, the binding of renin/prorenin to (pro)renin receptor activates intracellular signaling. Although these studies show the classical function of the (pro)renin receptor on the plasma membrane as a receptor, subcellular distribution and extracellular secretion of (pro)renin receptor remained controversial until recently when Cousin et al. reported possible existence of the soluble form of (pro)renin receptor. Chinese hamster ovary (CHO) cells transfected with human (pro)renin tagged with Venus showed bands at 74 kDa and 35 kDa without any stimulation in Western blot analysis. Moreover, these cells secreted a 29 kDa form, which was the amino-terminal fragment of the (pro)renin receptor. In immunofluorescent staining, (pro)renin receptor tagged with Venus was mainly stained on the endoplasmic reticulum and in vesicle-like structures, but not on the plasma membrane. These data suggest that the (pro)renin receptor may be cleaved in the intracellular compartments of cells and secreted into the extracellular space.

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.

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.

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.

Berberine alters the processing of Alzheimer's amyloid precursor protein to decrease Abeta secretion.

Berberine is an isoquinoline alkaloid isolated from Coptidis rhizoma, a major herb widely used in Chinese herbal medicine. Berberine's biological activity includes antidiarrheal, antimicrobial, and anti-inflammatory effects. Recent findings show that berberine prevents neuronal damage due to ischemia or oxidative stress and that it might act as a novel cholesterol-lowering compound. The accumulation of amyloid-beta peptide (Abeta) derived from amyloid precursor protein (APP) is a triggering event leading to the pathological cascade of Alzheimer's disease (AD); therefore the inhibition of Abeta production should be a rational therapeutic strategy in the prevention and treatment of AD. Here, we report that berberine reduces Abeta levels by modulating APP processing in human neuroglioma H4 cells stably expressing Swedish-type of APP at the range of berberine concentration without cellular toxicity. Our results indicate that berberine would be a promising candidate for the treatment of AD.