Follistatin and its role as an activin-binding protein

Follistatin (FS), a specific binding protein for activin, neutralizes the diverse actions of activin by forming an inactive complex with activin. FS is a monomer derived from two polypeptide core sequences of 288 (FS-288) and 315 (FS-315) amino acids originated from alternatively spliced mRNA. We purified six molecular forms of FS from porcine ovaries. Their structural differences were caused by truncation of the COOH-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-288, FS-315 and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites. All six molecular species have almost the same activin binding activity (Kd=540-680 pM). By contrast, the COOH-terminal truncated form, FS-288, showed much higher affinity for heparan sulfate proteoglycans of the cell surface than FS-303, whereas the intact form of FS, FS-315, had no affinity. Furthermore, FS-288 more effectively blocked the suppression of follicle-stimulating hormone (FSH) secretion from rat pituitary cells by activin. This implies that activin binds to the cell surface through FS-288 which adheres to the cell surface. To clarify the physiological role of cell-associated FS, we then investigated the binding of activin to cell-associated FS and the fate of cell surface-bound activin and FS using primary cultured rat pituitary and ovarian granulosa cells. When the cells were incubated with 125I-activin A in the presence of FS-288 or 315, the binding of activin A to the cell surface was promoted much more markedly by FS-288 than by FS-315. The amounts of radioactivity recovered in trichloroacetic acid-soluble fractions (degraded activin) from the incubation medium were greatly increased by the addition of FS-288. This increase was abolished by heparan sulfate, monensin (an endocytosis inhibitor), chloroquine (a lysosome function inhibitor) and several lysosomal enzyme inhibitors. These results suggest that cell-associated FS-288 accelerates the internalization of activin into the cells, leading to its degradation by lysosomal enzymes, and that cell surface-associated FS therefore plays a role in the clearance system of activin

ALK7 is a novel marker for adipocyte differentiation

Transforming growth factor-β (TGF-β) family members regulate a variety of cellular functions and play important roles in cell differentiation. Activin receptor-like kinase 7 (ALK7), a receptor for TGF-β family members, was initially cloned from rats as an orphan receptor and has been recently shown to be a type I receptor for nodal, activin B and activin AB. ALK7 is expressed not only in neurons, but also in insulin-producing islet β cells and white and brown adipose tissues however, the specific functions of ALK7 in these tissues are not known. In order to test whether ALK7 is involved in adipocyte differentiation, we analyzed its expression during adipocyte differentiation. ALK7 expression was detected in the late phase of adipocyte differentiation by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunofluorescence staining in 3T3-L1 cells. We also detected the expression of ALK7 by RT-PCR in stromal vascular fraction (SVF) cells. These results indicated that ALK7 is a novel marker specifically expressed during the late phase of adipocyte differentiation. Furthermore, our results suggest the possible involvement of nodal or activin B in adipocyte differentiation

Analysis of skin graft survival using green fluorescent protein transgenic mice

Skin grafting has become a basic and established operation technique ; however, it is not clear how skin grafts adapt to recipient beds and replace their functions. In this study, we analyzed the origin of cells in adapted transplants by using green fluorescent protein (GFP) transgenic mice, which emits green fluorescence in the whole body. The dorsal skins of GFP transgenic mice were transplanted to the back of wild-type mice. Similarly, wild-type skins were transplanted to the back of GFP transgenic mice. Since transplantation with full thickness back skin was not successful due to severe immunorejection, tail skins, which contain fewer epidermal Langerhans cells, were used for the experiments. Six months after transplantation, immunohistochemical analysis of the grafts revealed that tissues derived from ectodermal origin such as the epidermis, hair follicles, and sebaceous glands survived in transplanted grafts, but that other tissues such as the dermis, nerves and blood vessels are partly replaced by tissues from recipient beds. Our results further demonstrated that transplantation analyses with GFP transgenic mice could be a useful approach to study the origin of cells in transplants

Identification of novel proteins differentially expressed in pluripotent embryonic stem cells and differentiated cells

Mammalian pluripotent stem cells possess properties of self-renewal and pluripotency. These abilities are maintained by the strict regulation of pluripotent stem cell-specific transcription factor network and unique properties of chromatin in the stem cells. Although these major signaling pathways robustly control the characteristics of stem cells, other regulatory factors, such as metabolic pathways, are also known to modulate stem cell proliferation and differentiation. In this study, we fractionated protein samples from mouse embryonic stem (ES) cells cultured with or without the leukemia inhibitory factor (LIF). Protein expression was quantified by 2-dimensional differential gel electrophoresis (2D-DIGE). In total, 44 proteins were identified as being differentially expressed in the pluripotent stem cells and the differentiated cells. Surprisingly, half of the identified proteins were the proteins localized in mitochondria, which supply cellular energy and regulate cell cycle, development, and cell death. Some of these identified proteins are involved in the metabolic function and the regulation of pluripotency. Further analysis of the identified proteins could provide new information for the manipulation of pluripotency in ES cells

Activin E Controls Energy Homeostasis in Both Brown and White Adipose Tissues as a Hepatokine

Brown adipocyte activation or beige adipocyte emergence in white adipose tissue (WAT) increases energy expenditure, leading to a reduction in body fat mass and improved glucose metabolism. We found that activin E functions as a hepatokine that enhances thermogenesis in response to cold exposure through beige adipocyte emergence in inguinal WAT (ingWAT). Hepatic activin E overexpression activated thermogenesis through Ucp1 upregulation in ingWAT and other adipose tissues including interscapular brown adipose tissue and mesenteric WAT. Hepatic activin E-transgenic mice exhibited improved insulin sensitivity. Inhibin βE gene silencing inhibited cold-induced Ucp1 induction in ingWAT. Furthermore, in vitro experiments suggested that activin E directly stimulated expression of Ucp1 and Fgf21, which was mediated by transforming growth factor-β or activin type I receptors. We uncovered a function of activin E to stimulate energy expenditure through brown and beige adipocyte activation, suggesting a possible preventive or therapeutic target for obesity

Characterization of follistatin-related gene as a negative regulatory factor for activin family members during mouse heart development

Follistatin-related gene (FLRG) encodes a secretory glycoprotein that has characteristic cysteine-rich follistatin domains. FLRG protein binds to and neutralizes several transforming growth factor-β (TGF-β) superfamily members, including myostatin (MSTN), which is a potent negative regulator of skeletal muscle mass. We have previously reported that FLRG was abundantly expressed in fetal and adult mouse heart. In this study, we analyzed the expression of FLRG mRNA during mouse heart development. FLRG mRNA was continuously expressed in the embryonic heart, whereas it was very low in skeletal muscles. By contrast, MSTN mRNA was highly expressed in embryonic skeletal muscles, whereas the expression of MSTN mRNA was rather low in the heart. In situ hybridization and immunohistochemical analysis revealed that FLRG expressed in smooth muscle of the aorta and pulmonary artery, valve leaflets of mitral and tricuspid valves, and cardiac muscles in the ventricle of mouse embryonic heart. However, MSTN was expressed in very limited areas, such as valve leaflets of pulmonary and aortic valves, the top of the ventricular and atrial septa. Interestingly, the expression of MSTN was complementary to that of FLRG, especially in the valvular apparatus. Biochemical analyses with surface plasmon resonance biosensor and reporter assays demonstrated that FLRG hardly dissociates from MSTN and activin once it bound to them, and efficiently inhibits these activities. Our results suggest that FLRG could function as a negative regulator of activin family members including MSTN during heart development

Study on the Youth Project to Support from Remote Place in the Early Stage of the Disaster : Case Study of Information Support Using SNS at the Kumamoto Earthquake in 2016

In our research, we investigated information support using SNS at the time of disaster. There is an advantage that anyone can easily get and spread information. Therefore, it is effective to provide information using SNS. In conclusion, from the distant place young people of nonprofessional that they can support by using SNS. Introducing on the case of "YA4K" of facebook group who actually carried out support activities.研究ノー

The rasGAP-binding protein, Dok-1, mediates activin signaling via serine/threonine kinase receptors

Activins, members of the transforming growth factor-β family, are pleiotropic growth and differentiation factors. Activin A induces B-cell apoptosis. To identify the genes responsible for activin-induced apoptosis, we performed retrovirus-mediated gene trap screening in a mouse B-cell line. We identified the rasGAP-binding protein Dok-1 (p62) as an essential molecule that links activin receptors with Smad proteins. In B cells overexpressing Dok-1, activin A-induced apoptotic responses were augmented. The expression of bcl-X(L) was down-regulated by inhibition of the ras/Erk pathway. Activin stimulation triggered association of Dok-1 with Smad3, as well as association of Smad3 with Smad4. Dok-1 also associated with both the type I and type II activin receptors. Dok-1 has been characterized previously as a tyrosine-phosphorylated protein acting downstream of the protein tyrosine kinase pathway: intriguingly, activin signaling did not induce tyrosine phosphorylation of Dok-1. These findings indicate that Dok-1 acts as an adaptor protein that links the activin receptors with the Smads, suggesting a novel function for Dok-1 in activin signaling leading to B-cell apoptosis