Regulation of CXCR4 Expression by Taurine in Macrophage-Like Cells.

Taurine (2-aminoethanesulfonic acid) is a free ß-amino acid found at high concentrations in many mammalian tissues. Taurine plays a role in several essential biological processes, including anti-oxidation, anti-inflammation, and osmoregulation. However, its regulatory mechanisms, especially at the genetic and molecular levels, have not been elucidated. Here, we targeted immune-related genes and investigated the effects of taurine on immune-related gene expression in macrophage-like cells. J774.1 cell line was used, and the effect of taurine on mRNA expression of immune-related genes such as cytokines, their receptors, and toll-like receptors was examined. Among these, taurine significantly increased the mRNA levels of C-X-C chemokine receptor 2 (CXCR2), chemokine receptor. Furthermore, we found that the taurine-induced increase in CXCR4 mRNA levels was higher than that in CXCR2 mRNA levels. Taurine increased both mRNA and protein expression levels of CXCR4. Additionally, we examined the effects of taurine analogs, including hypotaurine, ß-alanine, and γ-aminobutyric acid (GABA). While GABA increased the mRNA expression of CXCR4, hypotaurine slightly increased this expression, and ß-alanine had no effect, although these taurine analogs are the substrates of taurine transporter. These findings demonstrate that taurine specifically affects CXCR4 mRNA expression in macrophage-like cells.

Leukotriene B4 receptor 1 exacerbates inflammation following myocardial infarction.

Leukotriene B4 receptor 1 (BLT1), a high-affinity G-protein-coupled receptor for leukotriene B4 (LTB4 ), is expressed on various inflammatory cells and plays critical roles in several inflammatory diseases. In myocardial infarction (MI), various inflammatory cells are known to be recruited to the infarcted area, but the function of BLT1 in MI is poorly understood. Here, we investigated the role of BLT1 in MI and the therapeutic effect of a BLT1 antagonist, ONO-4057, on MI. Mice with infarcted hearts showed increased BLT1 expression and LTB4 levels. BLT1-knockout mice with infarcted hearts exhibited attenuated leukocyte infiltration, proinflammatory cytokine production, and cell death, which led to reduced mortality and improved cardiac function after MI. Bone-marrow transplantation studies showed that BLT1 expressed on bone marrow-derived cells was responsible for the exacerbation of inflammation in infarcted hearts. Furthermore, ONO-4057 administration attenuated the inflammatory responses in hearts surgically treated for MI, which resulted in reduced mortality and improved cardiac function after MI. Our study demonstrated that BLT1 contributes to excessive inflammation after MI and could represent a new therapeutic target for MI.

Effect of Taurine on Cell Function via TXNIP Induction in Caco-2 Cells.

Taurine (2-aminoethanesulfonic acid), a sulfur-containing ß-amino acid, is a free amino acid present in high concentrations in mammalian tissues. Taurine has pivotal roles in anti-oxidation, membrane stabilization, osmoregulation, anti-inflammation, and other process. In a DNA microarray analysis, we previously found that taurine markedly increases the mRNA expression of thioredoxin interacting protein (TXNIP) in Caco-2 cells. In this study, we investigated the effect of these taurine-induced changes in TXNIP on the function of Caco-2 cells. We found that taurine decreases glucose uptake in a dose-dependent manner. The taurine-induced decrease in glucose uptake was completely abolished by transfection with siRNA against TXNIP, suggesting that TXNIP is involved in the taurine-induced down-regulation of glucose uptake. We also revealed that taurine induces AMPK activation and further increases the intracellular ATP content in Caco-2 cells. These results suggest that taurine could regulate the function of Caco-2 cells via TXNIP induction, leading to extend our understanding of the functions of taurine.

GRK6 phosphorylates IκBα at Ser(32)/Ser(36) and enhances TNF-α-induced inflammation.

G protein-coupled receptor kinases (GRKs) comprise a family of seven serine/threonine kinases that phosphorylate agonist-activated G protein-coupled receptors (GPCRs). It has recently been reported that GRKs regulate GPCR-independent signaling through the phosphorylation of intracellular proteins. To date, several intracellular substrates for GRK2 and GRK5 have been reported. However, those for GRK6 are poorly understood. Here we identified IκBα, a negative regulator of NF-κB signaling, as a substrate for GRK6. GRK6 directly phosphorylated IκBα at Ser(32)/Ser(36), and the kinase activity of GRK6 was required for the promotion of NF-κB signaling after TNF-α stimulation. Knockout of GRK6 in peritoneal macrophages remarkably attenuated the transcription of inflammatory genes after TNF-α stimulation. In addition, we developed a bioluminescence resonance energy transfer (BRET) probe to monitor GRK6 activity. Using this probe, we revealed that the conformational change of GRK6 was induced by TNF-α. In summary, our study demonstrates that TNF-α induces GRK6 activation, and GRK6 promotes inflammatory responses through the phosphorylation of IκBα.

Induction of cardiac fibrosis by ß-blocker in G protein-independent and G protein-coupled receptor kinase 5/ß-arrestin2-dependent Signaling pathways.

G-protein coupled receptors (GPCRs) have long been known as receptors that activate G protein-dependent cellular signaling pathways. In addition to the G protein-dependent pathways, recent reports have revealed that several ligands called "biased ligands" elicit G protein-independent and ß-arrestin-dependent signaling through GPCRs (biased agonism). Several ß-blockers are known as biased ligands. All ß-blockers inhibit the binding of agonists to the ß-adrenergic receptors. In addition to ß-blocking action, some ß-blockers are reported to induce cellular responses through G protein-independent and ß-arrestin-dependent signaling pathways. However, the physiological significance induced by the ß-arrestin-dependent pathway remains much to be clarified in vivo. Here, we demonstrate that metoprolol, a ß(1)-adrenergic receptor-selective blocker, could induce cardiac fibrosis through a G protein-independent and ß-arrestin2-dependent pathway. Metoprolol, a ß-blocker, increased the expression of fibrotic genes responsible for cardiac fibrosis in cardiomyocytes. Furthermore, metoprolol induced the interaction between ß(1)-adrenergic receptor and ß-arrestin2, but not ß-arrestin1. The interaction between ß(1)-adrenergic receptor and ß-arrestin2 by metoprolol was impaired in the G protein-coupled receptor kinase 5 (GRK5)-knockdown cells. Metoprolol-induced cardiac fibrosis led to cardiac dysfunction. However, the metoprolol-induced fibrosis and cardiac dysfunction were not evoked in ß-arrestin2- or GRK5-knock-out mice. Thus, metoprolol is a biased ligand that selectively activates a G protein-independent and GRK5/ß-arrestin2-dependent pathway, and induces cardiac fibrosis. This study demonstrates the physiological importance of biased agonism, and suggests that G protein-independent and ß-arrestin-dependent signaling is a reason for the diversity of the effectiveness of ß-blockers.

Self-organization, quality control, and preclinical studies of human iPSC-derived retinal sheets for tissue-transplantation therapy.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Safety and stable survival of stem-cell-derived retinal organoid for 2 years in patients with retinitis pigmentosa.

Transplantation of induced pluripotent stem cell (iPSC)-derived retinal organoids into retinal disease animal models has yielded promising results, and several clinical trials on iPSC-derived retinal pigment epithelial cell transplantation have confirmed its safety. In this study, we performed allogeneic iPSC-derived retinal organoid sheet transplantation in two subjects with advanced retinitis pigmentosa (jRCTa050200027). The primary endpoint was the survival and safety of the transplanted retinal organoid sheets in the first year post-transplantation. The secondary endpoints were the safety of the transplantation procedure and visual function evaluation. The grafts survived in a stable condition for 2 years, and the retinal thickness increased at the transplant site without serious adverse events in both subjects. Changes in visual function were less progressive than those of the untreated eye during the follow-up. Allogeneic iPSC-derived retinal organoid sheet transplantation is a potential therapeutic approach, and the treatment's safety and efficacy for visual function should be investigated further.

Generation and purification of ACTH-secreting hPSC-derived pituitary cells for effective transplantation.

Pituitary organoids are promising graft sources for transplantation in treatment of hypopituitarism. Building on development of self-organizing culture to generate pituitary-hypothalamic organoids (PHOs) using human pluripotent stem cells (hPSCs), we established techniques to generate PHOs using feeder-free hPSCs and to purify pituitary cells. The PHOs were uniformly and reliably generated through preconditioning of undifferentiated hPSCs and modulation of Wnt and TGF-ß signaling after differentiation. Cell sorting using EpCAM, a pituitary cell-surface marker, successfully purified pituitary cells, reducing off-target cell numbers. EpCAM-expressing purified pituitary cells reaggregated to form three-dimensional pituitary spheres (3D-pituitaries). These exhibited high adrenocorticotropic hormone (ACTH) secretory capacity and responded to both positive and negative regulators. When transplanted into hypopituitary mice, the 3D-pituitaries engrafted, improved ACTH levels, and responded to in vivo stimuli. This method of generating purified pituitary tissue opens new avenues of research for pituitary regenerative medicine.

Signaling Pathway of Taurine-Induced Upregulation of TXNIP.

Taurine, a sulfur-containing ß-amino acid, is present at high concentrations in mammalian tissues and plays an important role in several essential biological processes. However, the genetic mechanisms involved in these physiological processes associated with taurine remain unclear. In this study, we investigated the regulatory mechanism underlying the taurine-induced transcriptional enhancement of the thioredoxin-interacting protein (TXNIP). The results showed that taurine significantly increased the luciferase activity of the human TXNIP promoter. Further, deletion analysis of the TXNIP promoter showed that taurine induced luciferase activity only in the TXNIP promoter region (+200 to +218). Furthermore, by employing a bioinformatic analysis using the TRANSFAC database, we focused on Tst-1 and Ets-1 as candidates involved in taurine-induced transcription and found that the mutation in the Ets-1 sequence did not enhance transcriptional activity by taurine. Additionally, chromatin immunoprecipitation assays indicated that the binding of Ets-1 to the TXNIP promoter region was enhanced by taurine. Taurine also increased the levels of phosphorylated Ets-1, indicating activation of Ets-1 pathway by taurine. Moreover, an ERK cascade inhibitor significantly suppressed the taurine-induced increase in TXNIP mRNA levels and transcriptional enhancement of TXNIP. These results suggest that taurine enhances TXNIP expression by activating transcription factor Ets-1 via the ERK cascade.

Publisher Correction: Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Preconditioning the Initial State of Feeder-free Human Pluripotent Stem Cells Promotes Self-formation of Three-dimensional Retinal Tissue.

A three-dimensional retinal tissue (3D-retina) is a promising graft source for retinal transplantation therapy. We previously demonstrated that embryonic stem cells (ESCs) can generate 3D-retina in vitro using a self-organizing stem cell culture technique known as SFEBq. Here we show an optimized culture method for 3D-retina generation from feeder-free human pluripotent stem cells (hPSCs). Although feeder-free hPSC-maintenance culture was suitable for cell therapy, feeder-free hPSC-derived aggregates tended to collapse during 3D-xdifferentiation culture. We found that the initial hPSC state was a key factor and that preconditioning of the hPSC state by modulating TGF-beta and Shh signaling improved self-formation of 3D-neuroepithelium. Using the preconditioning method, several feeder-free hPSC lines robustly differentiated into 3D-retina. In addition, changing preconditioning stimuli in undifferentiated hPSCs altered the proportions of neural retina and retinal pigment epithelium, important quality factors for 3D-retina. We demonstrated that the feeder-free hiPSC-derived 3D-retina differentiated into rod and cone photoreceptors in vitro and in vivo. Thus, preconditioning is a useful culture methodology for cell therapy to direct the initial hPSC state toward self-organizing 3D-neuroepithelium.

Phagocytosis Assay of Necroptotic Cells by Cardiac Myofibroblasts.

In myocardial infarction (MI), a plenty of cardiomyocytes undergo necrosis and necroptosis due to the lack of oxygen and nutrients. The dead cardiomyocytes are promptly engulfed by phagocytes. When the dead cells are not engulfed, the noxious contents of the cells are released outside, and thus, induce inflammation, and obstruct the function of organs. Therefore, phagocytosis is crucial for maintaining homeostasis of organs. Herein, we describe a protocol of an in vitro phagocytosis assay of necroptotic cells.

An Assay to Determine Phagocytosis of Apoptotic Cells by Cardiac Macrophages and Cardiac Myofibroblasts.

In myocardial infarction (MI), a number of cardiomyocytes undergo apoptosis. These apoptotic cardiomyocytes are promptly engulfed by phagocytes. If the dead cells are not engulfed, their noxious contents are released outside, resulting in induction of inflammation. Therefore, the removal of these dead cells is necessary. However, the contribution of each phagocyte type to the removal of apoptotic cells in infarcted hearts remains unresolved. Here, we describe an in vitro protocol for a phagocytosis assay to compare the engulfment ability of cardiac macrophages and cardiac myofibroblasts.

Cardiac myofibroblast engulfment of dead cells facilitates recovery after myocardial infarction.

Myocardial infarction (MI) results in the generation of dead cells in the infarcted area. These cells are swiftly removed by phagocytes to minimize inflammation and limit expansion of the damaged area. However, the types of cells and molecules responsible for the engulfment of dead cells in the infarcted area remain largely unknown. In this study, we demonstrated that cardiac myofibroblasts, which execute tissue fibrosis by producing extracellular matrix proteins, efficiently engulf dead cells. Furthermore, we identified a population of cardiac myofibroblasts that appears in the heart after MI in humans and mice. We found that these cardiac myofibroblasts secrete milk fat globule-epidermal growth factor 8 (MFG-E8), which promotes apoptotic engulfment, and determined that serum response factor is important for MFG-E8 production in myofibroblasts. Following MFG-E8-mediated engulfment of apoptotic cells, myofibroblasts acquired antiinflammatory properties. MFG-E8 deficiency in mice led to the accumulation of unengulfed dead cells after MI, resulting in exacerbated inflammatory responses and a substantial decrease in survival. Moreover, MFG-E8 administration into infarcted hearts restored cardiac function and morphology. MFG-E8-producing myofibroblasts mainly originated from resident cardiac fibroblasts and cells that underwent endothelial-mesenchymal transition in the heart. Together, our results reveal previously unrecognized roles of myofibroblasts in regulating apoptotic engulfment and a fundamental importance of these cells in recovery from MI.

Identification of a Potent and Selective GPR4 Antagonist as a Drug Lead for the Treatment of Myocardial Infarction.

GPR4, a pH-sensing G protein-coupled receptor, is highly expressed in endothelial cells and may be activated in myocardial infarction due the decreased tissue pH. We are interested in GPR4 antagonists as potential effective pharmacologic tools and/or drug leads for the treatment of myocardial infarction. We investigated the structure-activity relationship of a known GPR4 antagonist 1 as a lead compound to identify 3b as the first potent and selective GPR4 antagonist, whose effectiveness was demonstrated in a mouse myocardial infarction model.

Multiple functions of G protein-coupled receptor kinases.

Desensitization is a physiological feedback mechanism that blocks detrimental effects of persistent stimulation. G protein-coupled receptor kinase 2 (GRK2) was originally identified as the kinase that mediates G protein-coupled receptor (GPCR) desensitization. Subsequent studies revealed that GRK is a family composed of seven isoforms (GRK1-GRK7). Each GRK shows a differential expression pattern. GRK1, GRK4, and GRK7 are expressed in limited tissues. In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed throughout the body. The roles of GRKs in GPCR desensitization are well established. When GPCRs are activated by their agonists, GRKs phosphorylate serine/threonine residues in the intracellular loops and the carboxyl-termini of GPCRs. Phosphorylation promotes translocation of ß-arrestins to the receptors and inhibits further G protein activation by interrupting receptor-G protein coupling. The binding of ß-arrestins to the receptors also helps to promote receptor internalization by clathrin-coated pits. Thus, the GRK-catalyzed phosphorylation and subsequent binding of ß-arrestin to GPCRs are believed to be the common mechanism of GPCR desensitization and internalization. Recent studies have revealed that GRKs are also involved in the ß-arrestin-mediated signaling pathway. The GRK-mediated phosphorylation of the receptors plays opposite roles in conventional G protein- and ß-arrestin-mediated signaling. The GRK-catalyzed phosphorylation of the receptors results in decreased G protein-mediated signaling, but it is necessary for ß-arrestin-mediated signaling. Agonists that selectively activate GRK/ß-arrestin-dependent signaling without affecting G protein signaling are known as ß-arrestin-biased agonists. Biased agonists are expected to have potential therapeutic benefits for various diseases due to their selective activation of favorable physiological responses or avoidance of the side effects of drugs. Furthermore, GRKs are recognized as signaling mediators that are independent of either G protein- or ß-arrestin-mediated pathways. GRKs can phosphorylate non-GPCR substrates, and this is found to be involved in various physiological responses, such as cell motility, development, and inflammation. In addition to these effects, our group revealed that GRK6 expressed in macrophages mediates the removal of apoptotic cells (engulfment) in a kinase activity-dependent manner. These studies revealed that GRKs block excess stimulus and also induce cellular responses. Here, we summarized the involvement of GRKs in ß-arrestin-mediated and G protein-independent signaling pathways.

ß-arrestin2 in infiltrated macrophages inhibits excessive inflammation after myocardial infarction.

Beta-arrestins (ß-arrestin1 and ß-arrestin2) are known as cytosolic proteins that mediate desensitization and internalization of activated G protein-coupled receptors. In addition to these functions, ß-arrestins have been found to work as adaptor proteins for intracellular signaling pathways. ß-arrestin1 and ß-arrestin2 are expressed in the heart and are reported to participate in normal cardiac function. However, the physiological and pathological roles of ß-arrestin1/2 in myocardial infarction (MI) have not been examined. Here, we demonstrate that ß-arrestin2 negatively regulates inflammatory responses of macrophages recruited to the infarct area. ß-arrestin2 knockout (KO) mice have higher mortality than wild-type (WT) mice after MI. In infarcted hearts, ß-arrestin2 was strongly expressed in infiltrated macrophages. The production of inflammatory cytokines was enhanced in ß-arrestin2 KO mice. In addition, p65 phosphorylation in the macrophages from the infarcted hearts of ß-arrestin2 KO mice was increased in comparison to that of WT mice. These results suggest that the infiltrated macrophages of ß-arrestin2 KO mice induce excessive inflammation at the infarct area. Furthermore, the inflammation in WT mice transplanted with bone marrow cells of ß-arrestin2 KO mice is enhanced by MI, which is similar to that in ß-arrestin2 KO mice. In contrast, the inflammation after MI in ß-arrestin2 KO mice transplanted with bone marrow cells of WT mice is comparable to that in WT mice transplanted with bone marrow cells of WT mice. In summary, our present study demonstrates that ß-arrestin2 of infiltrated macrophages negatively regulates inflammation in infarcted hearts, thereby enhancing inflammation when the ß-arrestin2 gene is knocked out. ß-arrestin2 plays a protective role in MI-induced inflammation.

GRK6 deficiency in mice causes autoimmune disease due to impaired apoptotic cell clearance.

Efficient engulfment of apoptotic cells is critical for maintaining tissue homoeostasis. When phagocytes recognize 'eat me' signals presented on the surface of apoptotic cells, this subsequently induces cytoskeletal rearrangement of phagocytes for the engulfment through Rac1 activation. However, the intracellular signalling cascades that result in Rac1 activation remain largely unknown. Here we show that G-protein-coupled receptor kinase 6 (GRK6) is involved in apoptotic cell clearance. GRK6 cooperates with GIT1 to activate Rac1, which promotes apoptotic engulfment independently from the two known DOCK180/ELMO/Rac1 and GULP1/Rac1 engulfment pathways. As a consequence, GRK6-deficient mice develop an autoimmune disease. GRK6-deficient mice also have increased iron stores in splenic red pulp in which F4/80(+) macrophages are responsible for senescent red blood cell clearance. Our results reveal previously unrecognized roles for GRK6 in regulating apoptotic engulfment and its fundamental importance in immune and iron homoeostasis.