Possible Role of Activin in the Adiponectin Paradox-Induced Progress of Alzheimer's Disease.

Accumulating evidence suggests that the adiponectin (APN) paradox might be involved in promoting aging-associated chronic diseases such as Alzheimer's disease (AD). In human brain, APN regulation of the evolvability of amyloidogenic proteins (APs), including amyloid-ß (Aß) and tau, in developmental/reproductive stages, might be paradoxically manifest as APN stimulation of AD through antagonistic pleiotropy in aging. The unique mechanisms underlying APN activity remain unclear, a better understanding of which might provide clues for AD therapy. In this paper, we discuss the possible relevance of activin, a member of transforming growth factor ß (TGFß) superfamily of peptides, to antagonistic pleiotropy effects of APN. Notably, activin, a multiple regulator of cell proliferation and differentiation, as well as an endocrine modulator in reproduction and an organizer in early development, might promote aging-associated disorders, such as inflammation and cancer. Indeed, serum activin, but not serum TGFß increases during aging. Also, activin/TGFß signal through type II and type I receptors, both of which are transmembrane serine/threonine kinases, and the serine/threonine phosphorylation of APs, including Aß42 serine 8 and αS serine 129, may confer pathological significance in neurodegenerative diseases. Moreover, activin expression is induced by APN in monocytes and hepatocytes, suggesting that activin might be situated downstream of the APN paradox. Finally, a meta-analysis of genome-wide association studies demonstrated that two SNPs relevant to the activin/TGFß receptor signaling pathways conferred risk for major aging-associated disease. Collectively, activin might be involved in the APN paradox of AD and could be a significant therapeutic target.

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 ßE 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-ß 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.

Dual-therapy strategy for modification of adiponectin receptor signaling in aging-associated chronic diseases.

Given the paradigm of anti-insulin resistance in therapies for metabolic syndrome, there has been considerable interest in adiponectin (APN), an adipocyte-derived sensitizer of insulin receptor signaling. In contrast to hypoadiponectinemia in metabolic syndrome, evidence suggests that Alzheimer's disease (AD) and other diseases, including chronic heart failure (CHF) and chronic kidney disease (CKD), are characterized by hyperadiponectinemia as well as the APN/obesity paradoxes, indicating that a decrease in APN might also be beneficial for these diseases. Thus, distinct from metabolic syndrome, it is anticipated that APN receptor antagonists rather than agonists might be effective in therapy for some chronic diseases.

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.

Biosynthesis of ribosomal RNA in nucleoli regulates pluripotency and differentiation ability of pluripotent stem cells.

Pluripotent stem cells have been shown to have unique nuclear properties, for example, hyperdynamic chromatin and large, condensed nucleoli. However, the contribution of the latter unique nucleolar character to pluripotency has not been well understood. Here, we show that fibrillarin (FBL), a critical methyltransferase for ribosomal RNA (rRNA) processing in nucleoli, is one of the proteins highly expressed in pluripotent embryonic stem (ES) cells. Stable expression of FBL in ES cells prolonged the pluripotent state of mouse ES cells cultured in the absence of leukemia inhibitory factor (LIF). Analyses using deletion mutants and a point mutant revealed that the methyltransferase activity of FBL regulates stem cell pluripotency. Knockdown of this gene led to significant delays in rRNA processing, growth inhibition, and apoptosis in mouse ES cells. Interestingly, both partial knockdown of FBL and treatment with actinomycin D, an inhibitor of rRNA synthesis, induced the expression of differentiation markers in the presence of LIF and promoted stem cell differentiation into neuronal lineages. Moreover, we identified p53 signaling as the regulatory pathway for pluripotency and differentiation of ES cells. These results suggest that proper activity of rRNA production in nucleoli is a novel factor for the regulation of pluripotency and differentiation ability of ES cells.

TIF1beta regulates the pluripotency of embryonic stem cells in a phosphorylation-dependent manner.

Transcription networks composed of various transcriptional factors specifically expressed in undifferentiated embryonic stem (ES) cells have been implicated in the regulation of pluripotency in ES cells. However, the molecular mechanisms responsible for self-renewal, maintenance of pluripotency, and lineage specification during differentiation of ES cells are still unclear. The results of this study demonstrate that a phosphorylation-dependent chromatin relaxation factor, transcriptional intermediary factor-1beta (TIF1beta), is a unique regulator of the pluripotency of ES cells and regulates Oct3/4-dependent transcription in a phosphorylation-dependent manner. TIF1beta is specifically phosphorylated in pluripotent mouse ES cells at the C-terminal serine 824, which has been previously shown to induce chromatin relaxation. Phosphorylated TIF1beta is partially colocalized at the activated chromatin markers, and forms a complex with the pluripotency-specific transcription factor Oct3/4 and subunits of the switching defective/sucrose nonfermenting, ATP-dependent chromatin remodeling complex, Smarcd1 [corrected], Brg-1, and BAF155, all of which are components of an ES-specific chromatin remodeling complex, esBAF. Phosphorylated TIF1beta specifically induces ES cell-specific genes and enables prolonged maintenance of an undifferentiated state in mouse ES cells. Moreover, TIF1beta regulates the reprogramming process of somatic cells in a phosphorylation-dependent manner. Our results suggest that TIF1beta provides a phosphorylation-dependent, bidirectional platform for specific transcriptional factors and chromatin remodeling enzymes that regulate the cell differentiation process and the pluripotency of stem cells.

Activin plays a key role in the maintenance of long-term memory and late-LTP.

A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin betaA, a member of the TGF-beta superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology. To clarify the role of activin in the synaptic plasticity of the adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal long-term potentiation (LTP). We found that follistatin, a specific inhibitor of activin, blocked the maintenance of late LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP (E-LTP). We generated forebrain-specific activin- or follistatin-transgenic mice in which transgene expression is under the control of the Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear-conditioning test, we found that follistatin blocked the formation of long-term memory (LTM) without affecting short-term memory (STM). Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory.

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells.

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self-renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2-D PAGE-based analysis using fluorescently labeled proteins and shotgun-based analysis with isotope-labeled peptides identified 338 proteins, including transmembrane, membrane-binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.

Follistatin suppresses the production of experimental multiple-organ metastasis by small cell lung cancer cells in natural killer cell-depleted SCID mice.

PURPOSE: Follistatin (FST), an inhibitor of activin, regulates a variety of biological functions, including cell proliferation, differentiation, and apoptosis. However, the role of FST in cancer metastasis is still unknown. Previous research established a multiple-organ metastasis model of human small cell lung cancer in natural killer cell-depleted SCID mice. In this model, i.v. inoculated tumor cells produced metastatic colonies in multiple organs including the lung, liver, and bone. The purpose of this study is to determine the role of FST in multiple-organ metastasis using this model. EXPERIMENTAL DESIGN: A human FST gene was transfected into the small cell lung cancer cell lines SBC-3 and SBC-5 and established transfectants secreting biologically active FST. The metastatic potential of the transfectants was evaluated using the metastasis model. RESULTS: FST-gene transfection did not affect the cell proliferation, motility, invasion, or adhesion to endothelial cells in vitro. I.v. inoculated SBC-3 or SBC-5 cells produced metastatic colonies into multiple organs, including the lung, liver, and bone in the natural killer cell-depleted SCID mice. FST transfectants produced significantly fewer metastatic colonies in these organs when compared with their parental cells or vector control clones. Immunohistochemical analyses of the liver metastases revealed that the number of proliferating tumor cells and the tumor-associated microvessel density were significantly less in the lesions produced by FST transfectants. CONCLUSIONS: These results suggest that FST plays a critical role in the production of multiple-organ metastasis, predominantly by inhibiting the angiogenesis. This is the first report to show the role of FST in metastases.

Transgenic expression of a myostatin inhibitor derived from follistatin increases skeletal muscle mass and ameliorates dystrophic pathology in mdx mice.

Myostatin is a potent negative regulator of skeletal muscle growth. Therefore, myostatin inhibition offers a novel therapeutic strategy for muscular dystrophy by restoring skeletal muscle mass and suppressing the progression of muscle degeneration. The known myostatin inhibitors include myostatin propeptide, follistatin, follistatin-related proteins, and myostatin antibodies. Although follistatin shows potent myostatin-inhibiting activities, it also acts as an efficient inhibitor of activins. Because activins are involved in multiple functions in various organs, their blockade by follistatin would affect multiple tissues other than skeletal muscles. In the present study, we report the characterization of a myostatin inhibitor derived from follistatin, which does not affect activin signaling. The dissociation constants (K(d)) of follistatin to activin and myostatin are 1.72 nM and 12.3 nM, respectively. By contrast, the dissociation constants (K(d)) of a follistatin-derived myostatin inhibitor, designated FS I-I, to activin and myostatin are 64.3 microM and 46.8 nM, respectively. Transgenic mice expressing FS I-I, under the control of a skeletal muscle-specific promoter showed increased skeletal muscle mass and strength. Hyperplasia and hypertrophy were both observed. We crossed FS I-I transgenic mice with mdx mice, a model for Duchenne muscular dystrophy. Notably, the skeletal muscles in the mdx/FS I-I mice showed enlargement and reduced cell infiltration. Muscle strength is also recovered in the mdx/FS I-I mice. These results indicate that myostatin blockade by FS I-I has a therapeutic potential for muscular dystrophy.

Activin increases the number of synaptic contacts and the length of dendritic spine necks by modulating spinal actin dynamics.

Long-lasting modifications in synaptic transmission depend on de novo gene expression in neurons. The expression of activin, a member of the transforming growth factor beta (TGF-beta) superfamily, is upregulated during hippocampal long-term potentiation (LTP). Here, we show that activin increased the average number of presynaptic contacts on dendritic spines by increasing the population of spines that were contacted by multiple presynaptic terminals in cultured neurons. Activin also induced spine lengthening, primarily by elongating the neck, resulting in longer mushroom-shaped spines. The number of spines and spine head size were not significantly affected by activin treatment. The effects of activin on spinal filamentous actin (F-actin) morphology were independent of protein and RNA synthesis. Inhibition of cytoskeletal actin dynamics or of the mitogen-activated protein (MAP) kinase pathway blocked not only the activin-induced increase in the number of terminals contacting a spine but also the activin-induced lengthening of spines. These results strongly suggest that activin increases the number of synaptic contacts by modulating actin dynamics in spines, a process that might contribute to the establishment of late-phase LTP.

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.

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-beta (TGF-beta) 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.

Effects of preactivation by portal vein ligation on liver regeneration following massive hepatectomy in rats.

BACKGROUND/AIMS: We performed a long-term assessment of liver regeneration with or without PVL after massive hepatectomy in similar sized remnant livers to evaluate effects of regenerating livers preactivated by PVL following massive hepatectomy. METHODOLOGY: Rats were randomly divided into two groups, PVL-88%Hx and sham 88%Hx. As the initial operation, PVL or sham operation was performed by ligation of the portal vein of the left and median lobes or a similar manipulation without ligation, respectively. Four days after PVL, the volume of the posterior caudate lobe (5%) increased approximately two-fold (12%) and was the same size as the whole caudate lobe (12%) in the sham animals. Subsequently, 88%Hx was performed in the two groups. RESULTS: Survival rates were not significantly different between the two groups. Relative liver weight in PVL-88%Hx group was significantly higher up to 24hr, but after 48hr no significant difference was evident between the two groups. PCNA LI in sham-89%Hx group was significantly higher than that in PVL-88%Hx group after 48hr. The mRNA expression levels of activin A and ActRIIA were significantly higher in PVL-88%Hx group than in sham-88% group at 72 hr. CONCLUSIONS: The regenerating liver preactivated by PVL is restricted late-phase liver regeneration after massive hepatectomy.

Combination therapy of human pancreatic cancer implanted in nude mice by oral fluoropyrimidine anticancer agent (S-1) with interferon-alpha.

PURPOSE: We evaluated the antitumor and antiangiogenic activities of human natural interferon-alpha (IFN-alpha) alone or in combination with S-1 against human pancreatic cancer cells. METHODS: Three days after the subcutaneous (s.c.) implantation of tumor cells, mice (n = 12) were received s.c. injection with IFN-alpha alone (10,000 U six times a week), oral administration with S-1 alone (8 mg/kg six times a week), or both with IFN-alpha and S-1 (8, 10, 12 mg/kg six times a week). RESULTS: Administration of IFN-alpha in combination with S-1 significantly decreased progressive growth and angiogenesis of human pancreatic cancer cells. The combination therapy produced more significant inhibition in expression of the representative proangiogenic molecules, vascular endothelial growth factor and basic fibroblast growth factor than individual treatment either IFN-alpha or S-1 alone did. These treatments also decreased the staining of proliferating cell nuclear antigen, induced apoptosis and decreased microvessel density. In order to better understand the precise molecular mechanisms by which IFN-alpha and S-1 exert its effects, we have utilized cDNA microarray including 124 known genes to determine the gene expression profile altered by IFN-alpha and S-1 treatment. We found a total of seven genes which showed a twofold change after IFN-alpha and S-1 treatment in addition to VEGF, bFGF, CD31, MMP-2, MMP-7 and MMP-9. Among these genes, we found down-regulation of six genes and up-regulation of one gene, which are related to angiogenesis, tumor cell invasion and metastasis. CONCLUSIONS: These data suggest that administration of IFN-alpha in combination with S-1 may provide a novel and effective approach to the treatment of human pancreatic cancer.

ALK7 is a novel marker for adipocyte differentiation.

Transforming growth factor-beta (TGF-beta) 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-beta 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 beta 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.

Identification of tocopherol-associated protein as an activin/TGF-beta-inducible gene in mast cells.

Previous studies have demonstrated that treatment with activin A and TGF-beta(1), members of the TGF-beta family, stimulated maturation of mouse bone marrow-derived cultured mast cells (BMMC), which was characterized by morphology and gene expression of mouse mast cell proteases (mmcps). In order to gain a better understanding of activin A- and TGF-beta(1)-induced maturation in mast cells, we investigated the genes that were up-regulated in response to treatment with these two members of the TGF-beta family. The cDNA microarray analyses indicated that in BMMC, five genes were induced by treatment with 4 nM activin A for 2 h. Tocopherol-associated protein (Tap) was one of the induced genes, and the Tap induction in response to activin A treatment was confirmed by real-time RT-PCR analyses. Treatment with TGF-beta(1) at 200 pM but not BMP-2 at 4 nM also increased Tap gene transcript in BMMC. Activin A-induced Tap expression was detected in BMMC but not in RAW264 macrophage-like cells, B16 melanoma cells or P19 embryonic carcinoma cells. Treatment with >1 muM SB431542, an inhibitor of activin and TGF-beta type I receptors ALK4/5, reduced responsiveness of Tap expression to TGF-beta(1), whereas <0.5 microM SB431542 effectively reduced TGF-beta(1)-induced expression of mmcp-1 and mmcp-7. These results suggest that inhibitory effects of SB431542 are different between TGF-beta-induced genes. Reporter assays indicated that Tap expression enhances transcription mediated by the activin/TGF-beta pathway. Thus, the present results suggest that Tap induction in response to activin/TGF-beta occurs predominantly in mast cells and serves as a positive regulator in activin/TGF-beta signaling.

Requirement of Smad3 for mast cell growth.

The involvement of the TGF-beta family in cell growth of bone marrow-derived mast cells (BMMC) cultured with medium containing pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM) was examined. Doubling time of BMMC from Smad3-null mice was longer than that from wild-type (WT) mice, and the differences tended to be larger with time of culture. Consistent with the results, uptake and reduction of [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; MTS] was lower in Smad3-deficient BMMC. Cell cycle analyses revealed no apparent differences between WT BMMC and Smad3-deficient BMMC, suggesting that longer doubling time in Smad3-deficient BMMC resulted from increased cell death. TGF-beta and activin A were supplied by PWM-SCM rather than by self-production by BMMC. Blocking the TGF-beta pathway by anti-TGF-beta neutralizing antibody or an inhibitor for the type I receptors for ligands including TGF-beta and activin, SB431542, inhibited MTS uptake and reduction in WT BMMC, whereas anti-activin A antibody and SB431542 tended to inhibit them in Smad3-deficient BMMC. The present results suggest that TGF-beta-induced and Smad3-mediated signaling is essential for maximal cell growth in mast cells, and that the activin pathway may be required for it when mast cell context is modulated by Smad3 depletion.

Purification of recombinant activin A using the second follistatin domain of follistatin-related gene (FLRG).

Activins are multifunctional growth factors belonging to the transforming growth factor-beta superfamily. Isolation of activins from natural sources requires many steps and only produces limited quantities. Even though recombinant preparations have been used in recent studies, purification of recombinant activins still requires multiple steps. To purify recombinant activin A, we have developed a simple method using the second follistatin domain of an activin-binding protein follistatin-related gene (FLRG). An affinity column was prepared with a partial FLRG fusion protein. The partial FLRG protein contained the second follistatin domain and the C-terminus acidic domain, and was tagged with six histidine residues at its N-terminus. The fusion protein was expressed in Escherichia coli and purified with nickel affinity column. Thereafter, the purified fusion protein was coupled to NHS-activated column. Recombinant activin A was produced in Chinese hamster ovary (CHO) cells, which were stably transfected with rat inhibin/activin betaA-subunit cDNA. After 48-h suspension culture of the cells in a serum free medium, the culture media was recovered and passed through the FLRG-coupled column. After washing with phosphate-buffered saline, bound protein was eluted out with an acidic buffer. Any significant contaminations were not detected when the purified protein was analyzed by SDS-PAGE. Apparent sizes of the protein were 14 and 28 kDa under the reduced and non-reduced conditions, respectively. Western blot analysis confirmed that the purified protein was activin A. The purified recombinant activin stimulated p3TP-lux reporter activity in CHO cells and follicle-stimulating hormone secretion from rat pituitary cells.

Involvement of p38 MAP kinase and Smad3 in TGF-beta-mediated mast cell functions.

Transforming growth factor-beta (TGF-beta) modulates functions of bone marrow-derived cultured mast cells (BMMCs); cell maturation (up-regulation of mouse mast cell proteases (mmcps)), growth arrest and migration. We investigated the roles of p38 MAP kinase and Smad3 in TGF-beta-mediated cell responses in BMMCs. Treating BMMCs with TGF-beta induced the phosphorylation of p38 within 2 h and persisted for 24 h. The involvement of p38 in TGF-beta-induced cell responses depended upon mast cell functions; it was necessary for up-regulation of mmcp-1 and migration, but not for up-regulation of mmcp-7 and inhibition of metabolic activity. New protein synthesis was required for the up-regulation of mmcp-1 but not mmcp-7 in response to TGF-beta treatment, and stabilization of mRNA was partially responsible for the increase in gene transcript of mmcp-1. The decrease in metabolic activity in response to TGF-beta treatment was smaller in Smad3-deficient BMMCs compared to wild-type BMMCs. Maximal migration was detected at a TGF-beta concentration of 40 fM in wild-type BMMCs, whereas TGF-beta-induced migration was absent in Smad3-deficient BMMCs. Thus, the roles of p38 and Smad3 are different among TGF-beta-mediated cell responses in BMMCs.