Induction of DNA Methylation by Artificial piRNA Production in Male Germ Cells

SummaryGlobal DNA demethylation and subsequent de novo DNA methylation take place in mammalian male embryonic germ cells [1–3]. P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs), which are germline-specific small RNAs, have been postulated to be critically important for de novo DNA methylation of retrotransposon genes, and many proteins, including PIWI family proteins, play pivotal roles in this process [4–6]. In the embryonic mouse testis, two mouse PIWI proteins, mouse PIWI-like (MILI) and mouse PIWI2 (MIWI2), are involved in the biogenesis of piRNAs through the so-called ping-pong amplification cycle [7–10], and long single-stranded RNAs transcribed from the gene regions of piRNA clusters have been proposed to be the initial material [11–16]. However, it remains unclear whether transcription from the piRNA clusters is required for the biogenesis of piRNAs. To answer this question, we developed a novel artificial piRNA production system by simple expression of sense and antisense EGFP mRNAs in embryonic male germ cells in the piRNA biogenesis phase. EGFP expression was silenced by piRNA-dependent DNA methylation, indicating that concomitant expression of sense and antisense RNA transcripts is necessary and sufficient for piRNA production and subsequent piRNA-dependent gene silencing. In addition, we demonstrated that this artificial piRNA induction paradigm could be applied to an endogenous gene essential for spermatogenesis, DNMT3L [3, 17, 18]. This study not only provides novel insights into the molecular mechanisms of piRNA production, but also presents an innovative strategy for inducing epigenetic modification in germ cells

Recombinant human FGF-2 for the treatment of early-stage osteonecrosis of the femoral head: TRION, a single-arm, multicenter, Phase II trial

Aim: This study aimed to evaluate the 2-year outcomes from a clinical trial of recombinant human FGF-2 (rhFGF-2) for osteonecrosis of the femoral head (ONFH). Patients & methods: Sixty-four patients with nontraumatic, precollapse and large ONFHs were percutaneously administered with 800 μg rhFGF-2 contained in gelatin hydrogel. Setting the end point of radiological collapse, we analyzed the joint preservation period of the historical control. Changes in two validated clinical scores, bone regeneration and safety were evaluated. Results: Radiological joint preservation time was significantly higher in the rhFGF-2 group than in the control group. The ONFHs tended to improve to smaller ONFHs. The postoperative clinical scores significantly improved. Thirteen serious adverse events showed recovery. Conclusion: rhFGF-2 treatment increases joint preservation time with clinical efficacy, radiological bone regeneration and safety

Th22 Cells Promote Osteoclast Differentiation via Production of IL-22 in Rheumatoid Arthritis

T helper (Th) cells can differentiate into functionally distinct subsets and play a pivotal role in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). Th22 cells have been identified as a new subset secreting interleukin (IL)-22. Although elevated levels of IL-22 in the synovial fluids of RA patients were reported, its pathological roles remain unclear. Here, we demonstrated that IL-22 was characteristically produced from CD3+CD4+CC-chemokine receptor (CCR)4+CCR6+CCR10+ cells and their ability of the production of IL-22 markedly exceeded that of other Th subsets and the subset, thereby, designated Th22 cells. Th22 cells were efficiently induced by the stimulation with tumor necrosis factor-α, IL-6, and IL-1β. Th22 cells were markedly infiltrated in synovial tissue in patients with active RA, but not in patients with osteoarthritis (OA). CCL17, CCL20, and CCL28, which are chemokine ligands of CCR4, CCR6, and CCR10, respectively, were abundantly expressed in RA synovial tissue compared to OA. By in vitro Trans-well migration assay, Th22 cells efficiently migrated toward CCL28. Co-culture of Th22 cells, which were sorted from peripheral blood, with monocytes in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor (NF)-κB ligand induced osteoclasts formation more efficiently than that of either Th1 cells or Th17 cells. Furthermore, IL-22 markedly augmented osteoclast differentiation by promoting nuclear factor of activated T cells c1 expression in CD14+ monocytes. Contrarily, the addition of IFN-γ to the culture significantly decreased osteoclasts number, whereas IL-17 had marginal effects. IL-22 neutralizing antibody inhibited osteoclast formation in the co-culture of Th22 cells with CD14+ monocytes. Collectively, the results indicated that Th22 cells, which co-express chemokine receptors CCR4, CCR6, and CCR10, possess strong potency of tissue migration and accumulate into inflamed synovial tissues where the ligands such as CCL28 are highly expressed. Thus, Th22 cells have the capacity to promote osteoclast differentiation through production of IL-22 and thus play a pivotal role in bone destruction in patients with RA

Small RNA class transition from siRNA/piRNA to miRNA during pre-implantation mouse development

Recent studies showed that small interfering RNAs (siRNAs) and Piwi-interacting RNA (piRNA) in mammalian germ cells play important roles in retrotransposon silencing and gametogenesis. However, subsequent contribution of those small RNAs to early mammalian development remains poorly understood. We investigated the expression profiles of small RNAs in mouse metaphase II oocytes, 8–16-cell stage embryos, blastocysts and the pluripotent inner cell mass (ICM) using high-throughput pyrosequencing. Here, we show that during pre-implantation development a major small RNA class changes from retrotransposon-derived small RNAs containing siRNAs and piRNAs to zygotically synthesized microRNAs (miRNAs). Some siRNAs and piRNAs are transiently upregulated and directed against specific retrotransposon classes. We also identified miRNAs expression profiles characteristic of the ICM and trophectoderm (TE) cells. Taken together, our current study reveals a major reprogramming of functional small RNAs during early mouse development from oocyte to blastocyst

Authentic role of ATP signaling in micturition reflex

Adenosine triphosphate (ATP) is a signaling molecule that regulates cellular processes. Based on previous studies of bladder function over the past decade, bladder ATP signaling was thought to have an essential role in the normal micturition reflex. In this study, we performed detailed analyses of bladder function in purinergic receptor-deficient mice using the automated voided stain on paper method and video-urodynamics. Unexpectedly, a lack of P2X2 or P2X3 receptors did not affect bladder function under normal physiological conditions, indicating that bladder ATP signaling is not essential for normal micturition reflex. In contrast, we found that lipopolysaccharide (LPS) induced markedly high levels of ATP release from the urothelium. In addition, LPS-induced rapid bladder hyperactivity was attenuated in P2X2 -/- and P2X3 -/- mice. Contrary to the previous interpretation, our present findings indicate that bladder ATP signaling has a fundamental role in the micturition reflex, especially in bladder dysfunction, under pathological conditions. Therefore, the bladder ATP signaling pathway might be a highly promising therapeutic target for functional bladder disorders. This study newly defines an authentic role for bladder ATP signaling in the micturition reflex

Safety confirmation of induced pluripotent stem cell-derived cardiomyocyte patch transplantation for ischemic cardiomyopathy: first three case reports

IntroductionWith the expected increase in patients with heart failure and ischemic 15 cardiomyopathy, the development of myocardial regenerative medicine using cell transplantation as a novel treatment method is progressing. This first-in-human clinical trial aimed to confirm the safety of cardiomyocyte patch transplantation derived from allogeneic induced pluripotent stem (iPS) cells based on the results of several preclinical studies.Study designThe inclusion criteria were left ventricular ejection fraction of 35% or less; heart failure symptoms of New York Heart Association class III or higher despite existing therapies such as revascularization; and a 1-year observation period that included a 3-month immunosuppressive drug administration period after transplantation of iPS cell-derived cardiomyocyte patches to evaluate adverse events, cardiac function, myocardial blood flow, heart failure symptoms, and immune response.ResultsIn the first three cases of this trial, no transplanted cell-related adverse events were observed during the 1-year observation period, and improvement in heart failure symptoms was observed. In addition, improvements in left ventricular contractility and myocardial blood flow were observed in two of the three patients. Regarding immune response, an increase in transplant cell-specific antibody titer was observed in all three patients after immunosuppressive drug administration. In one patient with poor improvement in cardiac function and myocardial blood flow, an increase in antibody titer against HLA-DQ was observed even before cell transplantation.ConclusionsOur case findings demonstrate that the transplantation of iPS cell-derived cardiomyocyte patches for ischemic cardiomyopathy can be safely performed; however, further investigation of the therapeutic effect and its relationship with an immune response is needed by accumulating the number of patients through continued clinical trials

A novel underuse model shows that inactivity but not ovariectomy determines the deteriorated material properties and geometry of cortical bone in the tibia of adult rats

Our goal in this study was to determine to what extent the physiologic consequences of ovariectomy (OVX) in bones are exacerbated by a lack of daily activity such as walking. We forced 14-week-old female rats to be inactive for 15 weeks with a unique experimental system that prevents standing and walking while allowing other movements. Tibiae, femora, and 4th lumbar vertebrae were analyzed by peripheral quantitative computed tomography (pQCT), microfocused X-ray computed tomography (micro-CT), histology, histomorphometry, Raman spectroscopy, and the three-point bending test. Contrary to our expectation, the exacerbation was very much limited to the cancellous bone parameters. Parameters of femur and tibia cortical bone were affected by the forced inactivity but not by OVX: (1) cross-sectional moment of inertia was significantly smaller in Sham-Inactive rat bones than that of their walking counterparts; (2) the number of sclerostin-positive osteocytes per unit cross-sectional area was larger in Sham-Inactive rat bones than in Sham-Walking rat bones; and (3) material properties such as ultimate stress of inactive rat tibia was lower than that of their walking counterparts. Of note, the additive effect of inactivity and OVX was seen only in a few parameters, such as the cancellous bone mineral density of the lumbar vertebrae and the structural parameters of cancellous bone in the lumbar vertebrae/tibiae. It is concluded that the lack of daily activity is detrimental to the strength and quality of cortical bone in the femur and tibia of rats, while lack of estrogen is not. Our inactive rat model, with the older rats, will aid the study of postmenopausal osteoporosis, the etiology of which may be both hormonal and mechanical

Contribution of Intragenic DNA Methylation in Mouse Gametic DNA Methylomes to Establish Oocyte-Specific Heritable Marks

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells