Skeletal muscle functions as an endocrine organ. Exosomes, small vesicles containing mRNAs, miRNAs, and proteins, are secreted from muscle cells and facilitate cell-to-cell communication. Our recent work found greater exosome release from oxidative compared to glycolytic muscle. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a key driver of mitochondrial biogenesis, a characteristic of oxidative muscle. It was hypothesized that PGC1α regulates exosome biogenesis and secretion in skeletal muscle. The purpose of this study is to determine if PGC-1α regulates skeletal muscle exosome biogenesis and secretion. On day 4 of differentiation, human primary myotubes from vastus lateralis biopsies from lean donors (BMI < 25.0 kg/m2) were exposed to adenovirus encoding human PGC-1α or GFP control. On day 6 of differentiation, culture media was replaced with exosome-free media. On day 8, cells were collected for mRNA and protein analysis, and culture media was collected for exosome isolation. Overexpression of PGC-1α increases regulators of exosome biogenesis in the endosomal sorting complexes required for transport (ESCRT) pathway: Alix (CON: 1.0 ± 0.2 vs. PGC-1α: 7.6 ± 3.8), TSG-101 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.1), CD63 (CON: 1.0 ± 0.17 vs. PGC-1α: 3.7 ± 0.4), Clathrin (CON: 1.0 ± 0.2 vs. PGC-1α: 11.6 ± 2.5), and the secretion pathway: Rab27b (CON: 1.0 ± 0.3 vs. PGC-1α: 3.2 ± 0.3), STAM (CON: 1.0 ± 0.3 vs. PGC-1α: 7.3 ± 0.6), and VTA1 (CON: 1.0 ± 0.1 vs. PGC-1α: 7.3 ± 2.4). Exosome count and total extracellular vesicle count were not significantly different from control. Overexpression of PGC-1α increases gene 9 expression of regulators of exosome biogenesis and secretion in human primary myotubes. In the future, in vitro studies assessing exosomal content from PGC-1 OE cells as well as in vivo effects of PGC-1 OE on exosome production and release should be investigated to further understand the role PGC-1 plays in exosome secretion.

Erythroid terminal differentiation couples sequential cell divisions with progressive reductions in cell size. The erythropoietin receptor (EpoR) is essential for erythroblast survival, but its other functions are not well characterized. I used Epor−/− mouse erythroblasts endowed with survival signaling to identify novel non-redundant EpoR functions. I found that, paradoxically, EpoR signaling increases red cell size while also increasing the number and speed of erythroblast cell cycles. Specifically, I found that high levels of EpoR signaling increase the size and shorten the cycle of early erythroblasts, which are amongst the fastest cycling somatic cells. I confirmed the effect of erythropoietin (Epo) on red cell size in human volunteers, whose mean corpuscular volume (MCV) increases following Epo administration. Our work shows that EpoR signaling alters the expected inverse relationship between cell cycle length and cell size. Further, diagnostic interpretations of increased MCV should now include high Epo levels and hypoxic stress. The ability of EpoR signaling to increase cell size in rapidly cycling early erythroblasts suggests that these cells have exceptionally efficient EpoR-driven mechanisms for growth. I found evidence for this in ongoing work, where Epor−/− and Stat5−/− single-cell transcriptomes show dysregulated expression of ribosomal proteins and rRNA transcription and processing genes. Global rates of ribosomal rRNA transcription and protein synthesis increase in an EpoR dependent manner during a narrow developmental window in early ETD, coincident with the time of cell cycle shortening. Our work therefore suggests EpoR-driven regulation of ribosome biogenesis and translation orchestrating rapid cycling and cell growth during early ETD.

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosomal biogenesis, and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of some of the most fundamental processes in mammalian cells.

No description available.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24516号 / 医博第4958号 / 新制||医||1064(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 貴浩, 教授 岩田 想, 教授 武藤 学 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo- iv PabABCE1 increases >30 fold from ~30 µM to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge () to being dependent on  with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1’s ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with v ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.

No description available.

No description available.