Ecrg4 peptide is the ligand of multiple scavenger receptors.

Esophageal cancer-related gene 4 (Ecrg4) encodes a hormone-like peptide that is believed to be involved in a variety of physiological phenomena, including tumour suppression. Recent progress in the study of Ecrg4 has shown that Ecrg4 is a proinflammatory factor and induces the expression of several cytokines and chemokines in macrophages/microglia. However, the detailed molecular mechanisms of Ecrg4 signalling, especially the Ecrg4 receptors, remain poorly understood. Here, using retrovirus-mediated expression cloning, we identified lectin-like oxidised low-density lipoprotein receptor-1 (LOX-1) as a membrane protein that binds amino acid residues 71-132 of Ecrg4 (Ecrg4(71-132)). Moreover, in addition to LOX-1, several scavenger receptors, such as Scarf1, Cd36 and Stabilin-1, facilitated the efficient internalisation of Ecrg4(71-132) into cells. A broad competitive inhibitor of scavenger receptors, polyinosinic acid, reduced both the binding of Ecrg4(71-132) and the activation of NF-κB in microglia. This activation was dependent on MyD88, an adaptor protein that recruits signalling proteins to Toll-like receptors (TLRs), with the consequent induction of various immune responses. These data suggest that multiple scavenger receptors recognise Ecrg4(71-132) and transduce its signals, together with TLRs, in microglia.

The dynamics of mucosal-associated invariant T cells in multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelination, gliosis and axonal loss in the Central Nervous System. Although the etiology of the disease has remained enigmatic, recent studies have suggested a role of the innate-like T cells, called Mucosal Associated Invariant T cells (MAITs) in the pathophysiology. In the present study, we have analyzed the relative frequency of MAITs and the expression of the cell surface antigens in MAITs to seek a possible link to the disease. RESULTS: There was little difference in the frequency of total MAITs between healthy donors (HDs) and untreated MS patients, whereas the latter harbored more CD8(lo/neg) (DN) MAITs concomitant with a decrease in CD8(high) MAITs and in CD4 MAITs compared with those in HDs. While the expression of CCR5, CCR6, CD95, CD127, and CD150 has increased in untreated subjects compared with that in HDs, CD45RO has declined in untreated subjects in both DN MAITs and CD8(hi) MAITs. FTY720 therapy has increased the relative frequency of total MAITs in a time-dependent fashion up to 2 years. Intriguingly, FTY720 therapy for 3 years reversed the above phenotype, engendering more CD8(high) MAITs accompanied with decreased DN MAITs. FTY720 therapy affected the cytokine production from CD4 T cells and also enhanced the relative frequency of cells producing both TNF-α and IFN-γ from MAITs, CD8 T cells, and CD4 T cells compared with that in untreated subjects. CONCLUSIONS: FTY 720 therapy enhanced the relative frequency of MAITs in MS patients in a time-dependent manner. Although the expression of CD8 in MAITs has been affected early by FTY720, longer treatment has reversed the phenotypic change. These data demonstrated that FTY720 induced dynamic change in the relative frequency and in the phenotype of MAITs in MS.

Ecrg4 contributes to the anti-glioma immunosurveillance through type-I interferon signaling.

Esophageal cancer-related gene 4 (Ecrg4), a hormone-like peptide, is thought to be a tumor suppressor, however, little is known about the mechanism of how Ecrg4 suppresses tumorigenesis. Here, we show that the ecrg4 null glioma-initiating cell (GIC) line, which was generated from neural stem cells of ecrg4 knockout (KO) mice, effectively formed tumors in the brains of immunocompetent mice, whereas the transplanted ecrg4 wild type-GIC line GIC(+/+) was frequently eliminated. This was caused by host immune system including adaptive T cell responses, since depletion of CD4+, CD8+, or NK cells by specific antibodies in vivo recovered tumorigenicity of GIC(+/+). We demonstrate that Ecrg4 fragments, amino acid residues 71-132 and 133-148, which are produced by the proteolitic cleavage, induced the expression of pro-inflammatory cytokines in microglia in vitro. Moreover, blockades of type-I interferon (IFN) signaling in vivo, either depleting IFN-α/ß receptor 1 or using stat1 KO mice, abrogated the Ecrg4-dependent antitumor activity. Together, our findings indicate a major antitumor function of Ecrg4 in enhancing host immunity via type-I IFN signaling, and suggest its potential as a clinical candidate for cancer immunotherapy.

Expansion of functional human mucosal-associated invariant T cells via reprogramming to pluripotency and redifferentiation.

Mucosal-associated invariant T (MAIT) cells play an important physiological role in host pathogen defense and may also be involved in inflammatory disorders and multiple sclerosis. The rarity and inefficient expansion of these cells have hampered detailed analysis and application. Here, we report an induced pluripotent stem cell (iPSC)-based reprogramming approach for the expansion of functional MAIT cells. We found that human MAIT cells can be reprogrammed into iPSCs using a Sendai virus harboring standard reprogramming factors. Under T cell-permissive conditions, these iPSCs efficiently redifferentiate into MAIT-like lymphocytes expressing the T cell receptor Vα7.2, CD161, and interleukin-18 receptor chain α. Upon incubation with bacteria-fed monocytes, the derived MAIT cells show enhanced production of a broad range of cytokines. Following adoptive transfer into immunocompromised mice, these cells migrate to the bone marrow, liver, spleen, and intestine and protect against Mycobacterium abscessus. Our findings pave the way for further functional analysis of MAIT cells and determination of their therapeutic potential.

G-CSF promotes the proliferation of developing cardiomyocytes in vivo and in derivation from ESCs and iPSCs.

During a screen for humoral factors that promote cardiomyocyte differentiation from embryonic stem cells (ESCs), we found marked elevation of granulocyte colony-stimulating factor receptor (G-CSFR) mRNA in developing cardiomyocytes. We confirmed that both G-CSFR and G-CSF were specifically expressed in embryonic mouse heart at the midgestational stage, and expression levels were maintained throughout embryogenesis. Intrauterine G-CSF administration induced embryonic cardiomyocyte proliferation and caused hyperplasia. In contrast, approximately 50% of csf3r(-/-) mice died during late embryogenesis because of the thinning of atrioventricular walls. ESC-derived developing cardiomyocytes also strongly expressed G-CSFR. When extrinsic G-CSF was administered to the ESC- and human iPSC-derived cardiomyocytes, it markedly augmented their proliferation. Moreover, G-CSF-neutralizing antibody inhibited their proliferation. These findings indicated that G-CSF is critically involved in cardiomyocyte proliferation during development, and may be used to boost the yield of cardiomyocytes from ESCs for their potential application to regenerative medicine.

In vitro pharmacologic testing using human induced pluripotent stem cell-derived cardiomyocytes.

The lethal ventricular arrhythmia Torsade de pointes (TdP) is the most common reason for the withdrawal or restricted use of many cardiovascular and non-cardiovascular drugs. The lack of an in vitro model to detect pro-arrhythmic effects on human heart cells hinders the development of new drugs. We hypothesized that recently established human induced pluripotent stem (hiPS) cells could be used in an in vitro drug screening model. In this study, hiPS cells were driven to differentiate into functional cardiomyocytes, which expressed cardiac markers including Nkx2.5, GATA4, and atrial natriuretic peptide. The hiPS-derived cardiomyocytes (hiPS-CMs) were analyzed using a multi electrode assay. The application of ion channel inhibitors resulted in dose-dependent changes to the field potential waveform, and these changes were identical to those induced in the native cardiomyocytes. This study shows that hiPS-CMs represent a promising in vitro model for cardiac electrophysiologic studies and drug screening.

Cardiomyocyte proliferation and protection against post-myocardial infarction heart failure by cyclin D1 and Skp2 ubiquitin ligase.

AIMS: Cyclins and other cell-cycle regulators have been used in several studies to regenerate cardiomyocytes in ischaemic heart failure. However, proliferation of cardiomyocytes induced by nuclear-targeted cyclin D1 (D1NLS) stops after one or two rounds of cell cycles due in part to accumulation of p27Kip1, an inhibitor of cyclin-dependent kinase (CDK). Thus, expression of S-phase kinase-associated protein 2 (Skp2), a negative regulator of p27Kip1, significantly enhances the effect of D1NLS and CDK4 on cardiomyocyte proliferation in vitro. Here, we examined whether Skp2 can also improve cardiomyocyte regeneration and post-ischaemic cardiac performance in vivo. METHODS AND RESULTS: Wistar rats underwent ischaemia/reperfusion injury by ligation of the coronary artery followed by injection of adenovirus vectors for D1NLS and CDK4 with or without Skp2. Enhanced proliferation of cardiomyocytes in the presence of Skp2 was demonstrated by increased expression of Ki67, a marker of proliferating cells (1.95% vs. 4.00%), and mitotic phosphorylated histone H3 (0.24% vs. 0.58%). Compared with rats that received only D1NLS and CDK4, expression of Skp2 improved left ventricular function as measured by the maximum and minimum rates of change in left ventricular pressure, the left ventricle end-diastolic pressure, left ventricle end-diastolic volume index, and the lung/body weight ratio. CONCLUSION: Expression of Skp2 enhanced the effect of D1NLS and CDK4 on the proliferation of cardiomyocytes and further contributed to improved post-ischaemic cardiac function. Skp2 might be a versatile tool to improve the effect of cyclins on post-ischaemic regeneration of cardiomyocytes in vivo.

Embryonic undifferentiated cells show scattering activity on a surface coated with immobilized E-cadherin.

Rearrangement of cell-cell adhesion is a critical event in embryonic development and tissue formation. We investigated the regulatory function of E-cadherin, a key adhesion protein, in the developmental process by using E-cadherin/IgG Fc fusion protein as an adhesion matrix in cell culture. F9 embryonal carcinoma cells usually form colonies when cultured on gelatin or fibronectin matrices. However, F9 cells cultured on the E-cadherin/IgG Fc fusion protein matrix formed a scattered distribution, with a different cytoskeletal organization and E-cadherin-rich protrusions that were regulated by Rac1 activity. The same scattering activity was observed in P19 embryonal carcinoma cells. In contrast, three types of differentiated cells, NMuMG mammary gland cells, MDCK kidney epithelial cells, and mouse primary isolated hepatocytes, did not show the scattering activity observed in F9 and P19 cells. These results suggest that migratory behavior on an E-cadherin-immobilized surface is only observed in embryonic cells, and that the regulatory mechanisms underlying E-cadherin-mediated cell adhesion vary with the state of differentiation.

E-cadherin-coated plates maintain pluripotent ES cells without colony formation.

Embryonic stem (ES) cells cultured on gelatin-coated plates or feeder layers form tight aggregated colonies by the E-cadherin-mediated cell-cell adhesions. Here we show that murine ES cells do not make cell-cell contacts or form colonies when cultured on the plate coated with a fusion protein of E-cadherin and IgG Fc domain. The cells in culture retain all ES cell features including pluripotency to differentiate into cells of all three germ layers and germ-line transmission after extended culture. Furthermore, they show a higher proliferative ability, lower dependency on LIF, and higher transfection efficiency than colony-forming conditions. Our results suggest that aggregated colony formation might inhibit diffusion of soluble factors and increase cell-cell communication, which may result in a heterogeneous environment within and between surrounding cells of the colony. This method should enable more efficient and scalable culture of ES cells, an important step towards the clinical application of these cells.

Transient inhibition of BMP signaling by Noggin induces cardiomyocyte differentiation of mouse embryonic stem cells.

Embryonic stem (ES) cells are a promising source of cardiomyocytes, but clinical application of ES cells has been hindered by the lack of reliable selective differentiation methods. Differentiation into any lineage is partly dependent on the regulatory mechanisms of normal early development. Although several signals, including bone morphogenetic protein (BMP), Wnt and FGF, are involved in heart development, scarce evidence is available about the exact signals that mediate cardiomyocyte differentiation. While investigating the involvement of BMP signaling in early heart formation in the mouse, we found that the BMP antagonist Noggin is transiently but strongly expressed in the heart-forming region during gastrulation and acts at the level of induction of mesendoderm to establish conditions conducive to cardiogenesis. We applied this finding to develop an effective protocol for obtaining cardiomyocytes from mouse ES cells by inhibition of BMP signaling.

Hepatocyte growth factor is essential for migration of myogenic cells and promotes their proliferation during the early periods of tongue morphogenesis in mouse embryos.

Temporal and spatial occurrence of hepatocyte growth factor (HGF) and its cognate receptor c-Met in the mouse mandibular development was investigated by immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction. HGF was first recognized in the mesenchymal cells of the first branchial arch at the 10th day of gestation (E10), before tongue formation, whereas HGF receptor (c-Met) -positive myogenic cells first appeared at E11 in the center of mandibles. By E12, HGF turned to be colocalized with c-Met in the differentiating tongue myoblasts. Between E14 and E16, HGF disappeared, whereas c-Met remained, in the tongue myoblasts. The levels of HGF mRNA in the developing tongue decreased in accordance with the increase of desmin mRNA levels from E11 to E17. These in vivo results strongly suggest that the HGF/c-Met system takes part in the earlier stages of tongue development. To elucidate this hypothesis, the antisense oligodeoxyribonucleotide (A-ODN) for mouse HGF mRNA was added to the organ culture system of mandible with serumless, defined medium. Mandibular arches from E10 mouse embryos were cultured at 37 degrees C for 10 days in the absence or presence of A-ODN, control (sense) oligonucleotide (C-ODN), or A-ODN plus recombinant HGF. In the control mandibular explants cultured without HGF or ODN, the anterior two-third of the tongue derived from the first branchial arch was formed. It contained abundant desmin-positive myoblasts and was equivalent to the tongue of E14-E15. In contrast, in the presence of A-ODN in the medium, neither the swelling nor myogenic cells were found in the tongue-forming region of explants, and myogenic cells accumulated behind the tongue-forming region. Such dysplasia of tongue was never induced in the presence of C-ODN or A-ODN plus recombinant HGF in the medium. The effect of A-ODN appeared to be developmental stage-specific, because tongue dysplasia occurred when A-ODN was present during the earlier 4 days but not during the later 4 days of the culture. Furthermore, recombinant HGF added to the culture without ODNs during the earlier 4 days caused elevation in the number of mitotic myoblasts. These results suggest that HGF regulates both the migration and proliferation of myogenic cells during the earlier stages of tongue development.

Impaired spermatogenic ability of testicular germ cells in mice deficient in the LIM-kinase 2 gene.

LIM-Kinase (LIMK), including LIMK1 and LIMK2, is the only known catalytic protein among LIM-family molecules. It is well known that LIMK phosphorylates and inactivates cofilin, an actin-depolymerizing factor regulating actin reorganization, while in vivo functions have remained to be elucidated. In the present study, we generated Limk2 gene-deficient mice in which three LIMK2 isoforms were disrupted in a Cre-mediated fashion. Impaired cofilin phosphorylation was clearly observed in Limk2-/- fibroblasts stimulated with bradykinin or lysophosphatidic acid, thereby suggesting that Cdc42 or Rho-dependent LIMK activation did not occur. However, Limk2-/- mice did not exhibit embryonic lethality or any phenotypic abnormalities in postnatal growth and development, except for spermatogenesis in the testis. The testes of Limk2-/- mice were smaller in size and partial degeneration of spermatogenic cells in the seminiferous tubules was apparent in association with increased apoptosis. In addition, the viability of Limk2-/- spermatogenic cells, when cultured under stressed conditions, was diminished. Furthermore, the potential for germ cells to differentiate in a regenerative state was severely impaired in Limk2-/- testis. Experimental hyperthermia induced impairment of ADF/cofilin phosphorylation and the formation of intranuclear cofilin inclusions in mutant germ cells. Based on these findings, we propose that LIMK2, especially the testis-specific isoform tLIMK2, plays an important role in proper progression of spermatogenesis by regulation of cofilin activity and/or localization in germ cells.