Activin A/erythroid differentiation factor induces thromboxane A2 synthetic activity in murine erythroleukemia cells

Activin A, a protein homologous to transforming growth factor beta, was shown to induce hemoglobin synthesis in murine erythroleukemia (MEL) cells and was also termed erythroid differentiation factor (EDF) (Eto, Y., Tsuji, T., Takezawa, M., Takano, S., Yokogawa, Y., and Shibai, H. (1987) Biochem. Biophys. Res. Commun. 142, 1095-1103). We found that activin A/EDF also induced thromboxane (TX) A2 synthetic activity in these cells. Synthesis of TXA2 from arachidonic acid is catalyzed by cyclooxygenase and TX synthase. Activin A/EDF induced the latter TX synthase activity, whereas the cyclooxygenase activity was constitutively expressed. The induction of this enzyme activity was inhibited by cycloheximide, suggesting that activin A/EDF induced de novo protein synthesis of TX synthase. Furthermore, we studied the relationship between the induction of TXA2 synthetic activity and erythroid differentiation in MEL cells, since the former is not an erythroid phenotype. We found 1) that the two responses to activin A/EDF were distinctly affected by the initial cell density; 2) that the dose-response curves for activin A/EDF were similar (ED50 = approximately 100 pM), whereas the time course of induction of TXA2 synthetic activity was much faster; and 3) that other erythroid differentiation inducers of MEL cells, namely dimethyl sulfoxide and hexamethylene bisacetamide, had little or no effect on TXA2 synthesis. These results indicate that activin A/EDF induces TXA2 synthetic activity independently of erythroid differentiation.     

Identification of a specific receptor for erythroid differentiation factor on follicular granulosa cell

A cellular receptor for erythroid differentiation factor (EDF) was demonstrated by incubation of 125I-labeled EDF with rat follicular granulosa cell cultures. The specific binding of labeled EDF to the cells showed saturation; Scatchard analysis of binding data indicated a single class of receptors having Kd = 3.4 x 10(-10) M. A large excess of unlabeled EDF reduced labeled EDF binding almost completely, whereas similar doses of inhibin and transforming growth factor type beta, which are quite similar to EDF in protein structure and subunit organization, had no effect; EDF did not share receptors with those factors. EDF receptor (activin A receptor) expression was enhanced in granulosa cells cultured in the presence of follicle-stimulating hormone; follicle-stimulating hormone raised the number of EDF binding sites/cell from 13,000 to 96,000 without altering the binding affinity.     

Activities of Mesoderm-Inducing Factors Secreted by Mammalian Cells in Culture1

We have examined the activities of several mesoderm-inducing factors contained in the culture fluids of phorbol ester (4beta-phorbol 12-myristate 13-acetate;PMA)-stimulated human cell lines. Mesoderm induction was assayed by examining the differentiation of mesoderm tissues reacted with presumptive ectoderm of the Cynops blastula. The assay system also examined erythroid differentiation activity (EDF activity) in order to test the relationship between mesoderm induction and activin A (EDF). Of 22 human cell lines examined, six strains were positive for both mesoderm-inducing activity and EDF activity. Four strains showed only mesoderm inducing activity, and one showed only EDF activity. The remaining 11 strains showed neither activity. Therefore, most cell lines secreting mesoderm-inducing activity also possessed EDF activity. Furthermore, culture fluid of a strain (K-562) that exhibited both types of activities, was partially fractionated by DEAE-Toyopearl column chromatography and examined in the same way. The fractions that showed the highest amount of EDF activity were coincident with those displaying mesoderm-inducing activity. These results suggest that a number of PMA-stimulated mammalian cell lines have the ability to secrete mesoderm-inducing factors which are similar to activin A (EDF).     

The existence of activin A/erythroid differentiation factor and its inhibitor in human serum: comparison of normal and chronic renal failure sera.

Activin A/EDF, initially found as a differentiation inducer of murine Friend erythroleukemia, also has a stimulatory effect on erythropoiesis in vitro and in vivo. Here we proved activin A/EDF activity in human serum. The activin A/EDF level in 18 normal human serum samples was measured by a specific bioassay and was found to be 8.3 +/- 4.6 ng/ml, indicating that there exists sufficient activity to affect erythropoiesis in normal serum. In contrast, activin A/EDF activity was reduced in the chronic renal failure patients and 23/26 serum samples examined showed levels below 1.2 ng/ml. Further analysis using HPLC revealed that chronic renal failure serum actually contained as much activin A/EDF as normal serum, and that the difference between normal and patient serum existed in the content of a specific inhibitor of activin A/EDF. This observation suggests the possibility that the inhibitor is participating in the regulation of activin A/EDF activity in vivo in chronic renal failure patients and also the possibility of activin A/EDF could be utilized in the therapy of the anemia of such patients.     

A new bacterial cellulose substrate for mammalian cell culture

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.     

Identification of the two types of specific receptor for activin/EDF expressed on Friend leukemia and embryonal carcinoma cells

Activin and inhibin are polypeptide factors which control the release of follicle stimulating hormone(FSH) from pituitary cells. The recent finding that erythroid differentiation factor(EDF) is identical to activin showed the multifunctional feature of this protein. To identify the specific receptor for activin, the binding of 125I-labeled activinA was investigated for a number of culture cell lines. Friends leukemia cell, which can be differentiated by activin, and embryonal carcinoma(EC) cells(PCC3, P19 and F9), were found to express 3500-20,000 per cell of activin receptors. Scatchard plot analysis of the binding data shows that the receptors on those cells could be divided into two groups with different Kd values. The Kd values of high and low affinity receptors are 0.15-0.4 nM and 1.5-3.0 nM respectively. The proportion of the number of the high and low affinity receptors was varied in each cell. Inhibin was able to compete for activin binding to both types of receptors, although the binding affinity was about 50-200 fold lower than that of activinA. Transforming growth factor-beta had no binding ability to the activin receptors.     

Phorbol ester-treated human acute myeloid leukemia cells secrete G-CSF, GM-CSF and erythroid differentiation factor into serum-free media in primary culture.

Upon treatment with the phorbol ester, tetradecanoylphorbol 13-acetate (PMA), peripheral mononuclear blood cells from patients with acute myeloid leukemia secrete into serum-free cell-conditioned media (PMA-CCM) at least three distinct nondialysable 'hematopoietic' factors: granulocyte-colony-stimulating factor (G-CSF), granulocyte/macrophage-colony-stimulating factor (GM-CSF) and erythroid differentiation factor (EDF, activin A). G-CSF was identified by its stimulation of [3H]thymidine incorporation into a G-CSF-responsive cell line, NSF-60, and the inhibition of its stimulation by a G-CSF-specific monoclonal antibody (MAB). GM-CSF was identified by its stimulation of [3H]thymidine incorporation into a GM-CSF-responsive line, TALL-101, and the inhibition of its stimulation by a GM-CSF-specific MAB. EDF was identified by its ability to stimulate erythroid differentiation in mouse erythroleukemia cell lines, its identical retention times to those of authentic EDF on three successive reverse-phase HPLC columns and characterization of its penultimate N-terminal residue as leucine which is the same as that of authentic EDF. Both authentic EDF and the erythroid-stimulating activity in PMA-CCM were found to act synergistically with a suboptimal inducing concentration of a well-studied inducing agent, dimethyl sulfoxide, in inducing erythroid differentiation. In addition, a fourth activity was observed in PMA-CCM: normal human fetal bone marrow cell-proliferation stimulating activity (FBMC-PSA). FBMC-PSA was identified by its ability to stimulate the growth of granulocytes and macrophages in FBMC suspension cultures, which neither recombinant G-CSF or GM-CSF were found to do.     

Mesodermal induction in early amphibian embryos by activin A (erythroid differentiation factor)

Summary Recently the mesoderm-inducing effects of the transforming growth factor (TGF-) family of proteins have been widely examined. In an attemt to elucidate the functions of these proteins, porcine inhibin A and activin A (erythroid differentiation factor; EDF) were examined. Treatment of explants with activin A led to differentiation of mesodermal derivatives such as mesenchyme, notochord, blood cells and muscle, but inhibin A had a much lesser effect. The mesodermal differentiation induced by activin A was also comfirmed by analyses using a polyclonal antibody against muscle myosin. By indirect immunofluorescence analysis, the differentiation of muscle blocks was observed in the activin-A-treated explants, whereas no differentiation was observed in inhibin-A-treated and control explants. These findings confirm that this protein of the TGF- family has mesoderm-inducing ability.     

Gene expression of activin, activin receptors, and follistatin in intestinal epithelial cells

Gene expression of activin, activin receptors, and follistatin was investigated in vivo and in vitro using semiquantitative RT-PCR in intestinal epithelial cells. Rat jejunum and the intestinal epithelial cell line IEC-6 expressed mRNA encoding the betaA-subunit of activin, alpha-subunit of inhibin, activin receptors IB and IIA, and follistatin. An epithelial cell isolation study focused along the crypt-villus axis in rat jejunum showed that betaA mRNA levels were eight- to tenfold higher in villus cells than in crypt cells. Immunohistochemistry revealed the expression of activin A in upper villus cells. The human intestinal cell line Caco-2 was used as a differentiation model of enterocytes. Four- to fivefold induction of betaA mRNA was observed in postconfluent Caco-2 cells grown on filter but not in those cells grown on plastic. In contrast, follistatin mRNA was seen to be reduced after reaching confluence. Exogenous activin A dose-dependently suppressed the proliferation and stimulated the expression of apolipoprotein A-IV gene, a differentiation marker, in IEC-6 cells. These results suggest that the activin system is involved in the regulation of such cellular functions as proliferation and differentiation in intestinal epithelial cells.     

Modulation by cAMP of 1alpha,25-dihydroxyvitamin D3 sensitivity of murine erythroleukemia cells.

As we previously reported, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC(50): 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h. In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.     

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.     

Activin/EDF as an inhibitor of neural differentiation

Activin/EDG, a stimulator of the secretion of follicle stimulating hormone (FSH) from pituitary gland and an inducer of erythroid differentiation for Friend leukemia cells, has since been implicated in a variety of biological roles. Here, we show some novel effects of activin on murine embryonal carcinoma cells (EC cells). First, activin acts as a growth factor on undifferentiated P19 cells, a well characterized EC cell line for the study of mammalian development. Second, activin inhibits the retinoic acid (RA) induced differentiation of P19 cells to neurons and glial cells. The inhibitory effect of activin on neural differentiation, which has yet to be proved in other physiological peptides, is confirmed also on the differentiation of various neuroblastoma cell lines. Our results suggest a possible role of activin as a negative regulator of neural differentiation in mammalian development.     

Possible involvement of protein kinases and Smad2 signaling pathways on osteoclast differentiation enhanced by activin A.

Bone tissues reportedly contain considerable amounts of activin A and follistatin, an activin A-binding protein. In the present study, we found that follistatin strongly inhibited osteoclast formation in cocultures of mouse bone marrow cells and primary osteoblasts induced by 1alpha,25 dihydroxyvitamin D(3), prostaglandin E(2), and interleukin-1alpha. Antibody aganist activin A also inhibited the osteoclast formation. Furthermore, activin A synergistically stimulated osteoclast differentiation mediated by receptor activator NF-kappaB ligand (RANKL). RT-PCR analysis revealed that osteoblasts produced not only activin A but also follistatin. Western blot analysis of a panel of phosphorylated proteins revealed that activin A stimulated the phosphorylation of p44/42 mitogen activated protein (MAP) kinase (ERK1/2) and p38 MAP kinase in macrophage colony-stimulating factor-dependent bone marrow macrophages (M-BMMPhis). In addition, phosphorylation of Smad2 was observed in M-BMMPhis stimulated with activin A. These findings indicate that the phosphorylation of p44/42 MAP kinase, p38 MAP kinase, and Smad2 is involved in activin A-enhanced osteoclast differentiation induced by RANKL. Taken together, these results suggest that both activin A and follistatin produced by osteoblasts may play an important role in osteoclast differentiation through MAP kinases and Smad2 signaling pathways.     

The measurement of activin/EDF in mouse serum: evidence for extragonadal production.

Many studies have shown that activin/EDF mediates local physiological events at various sites. In this study, the authors confirmed the presence of activin in mouse serum by high performance liquid chromatography (HPLC) monitored by a specific bioassay. The retention time of the active fraction in HPLC was identical to that of authentic activin A, and the activity was neutralized by follistatin. That the serum activin levels in ovariectomized and aged mice were decreased suggests that the serum activin was generated partly by ovary (35%), but also by extragonadal organs. Activin and inhibin are structurally closely related, and both are involved in many physiological processes including control of follicle stimulating hormone secretion by the pituitary. The regulation of serum activin, however, appeared to differ from that of inhibin.     

Follistatin inhibits activin-induced differentiation of rat follicular granulosa cells in vitro.

The effect of follistatin on activin-induced granulosa cell differentiation was investigated in freshly harvested granulosa cells from diethylstilbestrol (DES)-treated rats. Activin induced a remarkable change in granulosa cellular morphology from elongated fibroblast-like to round cells, which follistatin prevented. Follistatin itself had no influence on the cellular morphology. We studied the action of follistatin on activin-induced differentiation of granulosa cells cultured in a chemically defined medium. Addition of activin (30 ng/ml) to the culture increased the FSH binding site approximately 2-fold compared with the control (spontaneous expression) level, whereas follistatin reduced the activin-induced expression level to the control level in a concentration-dependent manner. Activin (30 ng/ml) markedly augmented FSH-induced hCG binding and progesterone production by approximately 20-fold, and these effects were suppressed by follistatin in a concentration-dependent manner. Similarly, addition of follistatin to the culture induced a concentration-dependent decrease of activin-enhanced inhibin-producing activity, but had no effect on FSH-induced inhibin production. These results suggest that follistatin/activin-binding protein binds to activin stoichiometrically to suppress the activin-induced differentiation of rat granulosa cell in vitro, but follistatin itself has no direct effect on activin-independent reactions.     

The vegetalizing factor from chicken embryos: its EDF (activin A)-like activity

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.     

Activin A induces neuronal differentiation and survival via ALK4 in a SMAD-independent manner in a subpopulation of human neuroblastomas

Activin A is a multifunctional homo-dimeric protein that belongs to the transforming growth factor (TGF)-β superfamily. In neurons, activin has neuroprotective effects both in vitro and in vivo, but it inhibits neuronal differentiation in some cell lines. Here we report that activin A can promote neuronal differentiation in particular cases. We examined activin A-induced neuronal differentiation and survival in a selected subpopulation of a human neuroblastoma cell line, SK-N-SH, grown in low-serum (differentiation-inducing) conditions. Activin A caused dramatic neurite outgrowth, and increased the expression of neuronal markers and the transactivation of dopamine β-hydroxylase. We demonstrated that the activin A signal is transduced through the activin A type 1 receptor, ALK4, and transactivates several TGF-β target genes in a SMAD-independent manner. That is, activin A did not induce the phosphorylation of SMAD2/3, the interaction of SMAD2/3 with SMAD4, the binding of SMAD2/3 to the promoter of TGF-β target genes, or the accumulation of SMAD2/3 in the nucleus. These results suggest that, in particular cases, activin A can induce neuronal differentiation and support neuronal survival in vitro. These findings may reflect previously unknown functions of activin A in neuronal cells in vivo.