Yuzuru Eto

Purification and characterization of Erythroid Differentiation Factor(EDF) isolated from human leukemia cell line THP-1

We isolated a protein, from a cell line of human origin, which exhibits extensive differentiation inducing activity toward Friend leukemia cells. The protein, called Erythroid Differentiation Factor (EDF), was found in a 4 day culture of THP-1 cells performed in the presence of 4 beta-phorbol 12-myristate 13-acetate(PMA). EDF is a homodimer of a molecular weight of 25,000, with an NH2-terminal sequence identical to that of the beta A-chain of porcine Inhibin. It was suggested that a single protein species is responsible for the activities of both EDF and FRP, a FSH releasing protein isolated from porcine ovarian follicular fluid.

Activin A: an autocrine inhibitor of initiation of DNA synthesis in rat hepatocytes.

The present study was conducted to examine the effect of activin A on growth of rat hepatocytes. EGF induced a 10-fold increase in DNA synthesis as assessed by [3H]thymidine incorporation in cultured hepatocytes. When activin A was added together with EGF, DNA synthesis induced by EGF was markedly inhibited. Inhibition was detected at a concentration of 10(-10) M, and 5 x 10(-9) M activin A almost completely blocked EGF-mediated DNA synthesis. Similarly, activin A completely blocked DNA synthesis induced by hepatocyte growth factor/scatter factor. Activin A was capable of inhibiting EGF-mediated DNA synthesis, even when added 36 h after the addition of EGF. With the same time interval, TGF-beta also blocked EGF-induced DNA synthesis. Although both activin A and TGF-beta inhibited growth of hepatocytes in a similar manner, either activin A or TGF-beta did not compete with each other in their binding when assessed by competitive binding using an iodinated ligand. When hepatocytes were incubated with EGF, release of bioactivity of activin A into culture medium was detected after 48 h or later. Activity of activin A was released from parenchymal cells but not from nonparenchymal cells. mRNA for beta A subunit of activin was detected only slightly in unstimulated hepatocytes, but markedly increased at 48 h after the addition of EGF. To determine whether endogenously produced activin A affects DNA synthesis, we examined the effect of follistatin, an activin-binding protein that blocks the action of activin A. An addition of follistatin significantly enhanced EGF-induced DNA synthesis. Finally, in partial hepatectomized rat, expression of mRNA for beta A subunit in liver was markedly increased 24 h after the partial hepatectomy. These results indicate that activin A inhibits initiation of DNA synthesis in hepatocytes by acting on its own receptor and that activin A acts as an autocrine inhibitor of DNA synthesis in rat hepatocytes.

Involvement of the calcium-sensing receptor in human taste perception.

By human sensory analyses, we found that various extracellular calcium-sensing receptor (CaSR) agonists enhance sweet, salty, and umami tastes, although they have no taste themselves. These characteristics are known as “kokumi taste” and often appear in traditional Japanese cuisine. Although GSH is a typical kokumi taste substance (taste enhancer), its mode of action is poorly understood. Here, we demonstrate how the kokumi taste is enhanced by the CaSR, a close relative of the class C G-protein-coupled receptors T1R1, T1R2, and T1R3 (sweet and umami receptors). We identified a large number of CaSR agonist γ-glutamyl peptides, including GSH (γ-Glu-Cys-Gly) and γ-Glu-Val-Gly, and showed that these peptides elicit the kokumi taste. Further analyses revealed that some known CaSR agonists such as Ca2+, protamine, polylysine, l-histidine, and cinacalcet (a calcium-mimetic drug) also elicit the kokumi taste and that the CaSR-specific antagonist, NPS-2143, significantly suppresses the kokumi taste. This is the first report indicating a distinct function of the CaSR in human taste perception.

Induction of follicle stimulating hormone receptor by erythroid differentiation factor on rat granulosa cell.

Erythroid differentiation factor (EDF), inhibin beta A-homodimer, induced expression of follicle stimulating hormone receptors on rat granulosa cells prepared from diethylstilbestrol primed immature female rats. After 3 day incubation with EDF, the number of FSH receptors on the granulosa cells was increased to about 3.5 times of the control value in a dose dependent manner with an ED50 value of 61 ng/ml. On the other hand, EDF related peptides, i.e., bovine 32K Da inhibin A and TGF beta, had no effect on the FSH receptor induction. The present observation suggests that EDF may play a role in the initiation of the cytodifferentiation of ovarian granulosa cells.

Branched-chain amino acids promote albumin synthesis in rat primary hepatocytes through the mTOR signal transduction system.

The administration of branched-chain amino acids (BCAAs) to cirrhosis patients increases serum albumin levels and improves the blood Fischers ratio. Although it has been reported that albumin synthesis in rat primary hepatocytes is diminished under lower Fishers ratio conditions compared to normal Fischers ratio conditions, the mode of action at the molecular level for these effects is still uncertain. It has been reported recently that the triggering signal for protein synthesis is transmitted through mTOR (mammalian target of rapamycin). We have had an interest in the mTOR signal transduction system. In the present study, we analyzed the mode of action of BCAA-induced albumin synthesis using rat primary hepatocytes. The BCAA mixture dose-dependently promoted the production of albumin, with leucine being the major effector half of which was inhibited by the mTOR inhibitor rapamycin. We also showed that only leucine induces P70 S6 kinase activation and 4E-BP1 phosphorylation which are mTORs downstream translational effectors. These activations were completely inhibited by rapamycin. Our results suggest that BCAAs, especially leucine, promote the production of albumin in rat primary hepatocytes through an mTOR signal transduction system.

Local administration of activin promotes fracture healing in the rat fibula fracture model

Osteogenic activities of activin, a member of the transforming growth factor (TGF)-beta superfamily, have been demonstrated in both in vitro and in vivo studies. The present study investigates the effects of topical application of activin on fracture healing using rat fibula fracture models. Activin (0.4-10 microg/day) was injected locally to the fracture once a day for 2 weeks. Activin promoted callus formation in a dose-dependent manner and both callus volume and callus weight were significantly increased with doses of 2-10 microg/day activin. Also, 3 weeks of activin treatment increased the mechanical strength of the healing bone in addition to the callus mass. Histological study 2 weeks after the fracture revealed that activin promoted endochondral bone formation. Immunohistochemical examination of the fractured tibia revealed that activin was localized to osteoblasts and chondrocytes in the region ossified both endochondrally and intramembranously. These findings suggest that activin expressed during fracture healing promotes the healing process through an autocrine/paracrine mode of action.

Erythroid differentiation factor can modulate follicular granulosa cell functions.

The action of human erythroid differentiation factor (EDF) on the functions of rat granulosa cells cultured in a chemically defined medium was investigated. In the presence of FSH that induced LH receptor expression and progesterone synthesis during culture of the cells, EDF augmented both responses in a dose- and time-dependent manner. Unlike FSH, EDF itself did not have such an inducing effect at all. Furthermore, in the absence of FSH, EDF was found to strongly enhance the ability of granulosa cells to produce inhibin. Thus, EDF may play an important role in the regulation of granulosa cell function and differentiation during follicle development.

Calcium‐sensing receptor mediates phenylalanine‐induced cholecystokinin secretion in enteroendocrine STC‐1 cells

Intraluminal l‐phenylalanine (Phe) stimulates cholecystokinin (CCK) secretion in vivo and in vitro. However, the cellular mechanism by which CCK‐producing enteroendocrine cells sense Phe is unknown. The calcium‐sensing receptor (CaR) can sense amino acids, and is expressed in the gastrointestinal tract. In the present study, we examined whether CaR functions as a receptor for Phe in CCK‐producing enteroendocrine cells. CCK secretion and intracellular Ca2+ concentration in response to Phe were measured in the murine CCK‐producing enteroendocrine cell line STC‐1 at various extracellular Ca2+ concentrations or after treatment with a CaR antagonist. At more than 20 mm, Phe induced dose‐dependent CCK secretion and intracellular Ca2+ mobilization in STC‐1 cells. In the presence of 3.0 mm extracellular Ca2+, 10 and 20 mm Phe induced significantly higher CCK secretion than under normal conditions (1.2 mm extracellular Ca2+). Intracellular Ca2+ mobilization, induced by 10 or 20 mm Phe, was also enhanced by increasing extracellular Ca2+ concentrations. In addition, intracellular Ca2+ mobilization induced by addition of extracellular Ca2+ was augmented by the presence of Phe. These results closely match the known CaR properties. Treatment with a specific CaR antagonist (NPS2143) completely inhibited Phe‐induced CCK secretion and the latter phase of intracellular Ca2+ mobilization. CaR mRNA expression was demonstrated by RT‐PCR in STC‐1 cells, as well as in other mouse tissues including the kidney, thyroid, stomach and intestine. In conclusion, CaR functions as a receptor for Phe, stimulating CCK secretion in enteroendocrine STC‐1 cells.

A new bacterial cellulose substrate for mammalian cell culture. A new bacterial cellulose substrate

A new substrate for mammalian cell culture was developed using a cellulose membrane produced byAcetobacter aceti. Modification of the ionic charge of the membrane and adsorption of collagen to it promoted cellular adhesion to the membrane surface. The growth of eight kinds of cells on the membrane, was comparable to that achieved in plastic Petri dishes. The membrane was tested for use in the production of recombinant Erythroid Differentiation Factor (EDF)/activin A using genetically engineered Chinese hamster ovary cells. Both the viability of the cells and production of EDF/activin A were maintained for about 1 month, while cultures on plastic dishes lasted only 12 days. It was considered that the mechanism of improved cell viability was related to the ultrastructure of the cellulose membrane.

Activin increases bone mass and mechanical strength of lumbar vertebrae in aged ovariectomized rats

Activin is a member of the transforming growth factor-beta superfamily and is thought to be involved in the regulation of bone formation due to its presence in bone tissue and its osteogenic activity both in vitro and in vivo. We recently found that systemic administration of activin increased both tibial bone mass and mechanical strength in young growing rats. The present study investigated the effects of activin in aged ovariectomized (ovx) rats. Twelve-month-old Fischer rats were ovariectomized and maintained for 10 months. Recombinant human activin A (activin) or human parathyroid hormone 1-34 (PTH) was administered intramuscularly three times a week for 12 weeks. Activin (1 and 5 microg/kg) markedly increased lumbar vertebral bone mineral content and bone mineral density. Activin also increased the mechanical strength of the vertebral body, which was highly correlated to the bone mineral density of the vertebral body. The maximal response in bone mass and strength was observed at 1 microg/kg of activin, which was approximately equal to that induced by PTH at 40 microg/kg. Peripheral quantitative computed tomography revealed that activin enlarged the cross-sectional size of the vertebrae without changing the foramen area, indicating its effects on cortical shells. Histomorphometric analysis of cancellous bone of vertebral body in similar experiment showed that activin (3 microg/kg) increased bone volume and the mineralizing surface, although its effects were less than PTH. The present results indicate that low doses of activin are effective against vertebral bone loss in aged ovx rats.