Isolation and characterization of native activin B.

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of 125I-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.     

FLRG, a new ADAM12-associated protein, modulates osteoclast differentiation

BACKGROUND INFORMATION: FLRG (follistatin-related gene) is a secreted glycoprotein that is highly homologous with follistatin. These proteins are involved in the regulation of various biological effects mediated by their binding to TGF-beta (transforming growth factor-beta) superfamily members, activin A and bone morphogenetic proteins. To characterize further the function of FLRG, we used a yeast two-hybrid screen to look for other possible functional partners. RESULTS: We report a direct interaction between the cysteine-rich domain of FLRG and ADAM12 (a disintegrin and metalloprotease 12). ADAMs are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Several studies have reported that ADAM12 protein, as well as activin A, are important regulators of osteoclast differentiation. We observed that the expressions of ADAM12 and activin A are modulated during osteoclast formation, whereas the FLRG expression seemed to remain quite constant. We showed that the FLRG protein inhibits osteoclast differentiation from murine primary spleen cells and macrophage RAW264.7 cells cultured in the presence of RANK-L (receptor activator of nuclear factor kappaB ligand) and M-CSF (macrophage colony-stimulating factor). Addition of FLRG protein to precursors significantly reduces the number of osteoclasts, as well as the average number of nuclei in each osteoclast. CONCLUSIONS: Our study indicates that the FLRG protein may contribute to bone formation by inhibiting osteoclast differentiation.     

Regulation of myostatin in vivo by growth and differentiation factor-associated serum protein-1: a novel protein with protease inhibitor and follistatin domains

Myostatin, a member of the TGFbeta superfamily, is a potent and specific negative regulator of skeletal muscle mass. In serum, myostatin circulates as part of a latent complex containing myostatin propeptide and/or follistatin-related gene (FLRG). Here, we report the identification of an additional protein associated with endogenous myostatin in normal mouse and human serum, discovered by affinity purification and mass spectrometry. This protein, which we have named growth and differentiation factor-associated serum protein-1 (GASP-1), contains multiple domains associated with protease-inhibitory proteins, including a whey acidic protein domain, a Kazal domain, two Kunitz domains, and a netrin domain. GASP-1 also contains a domain homologous to the 10-cysteine repeat found in follistatin, a protein that binds and inhibits activin, another member of the TGFbeta superfamily. We have cloned mouse GASP-1 and shown that it inhibits the biological activity of mature myostatin, but not activin, in a luciferase reporter gene assay. Surprisingly, recombinant GASP-1 binds directly not only to mature myostatin, but also to the myostatin propeptide. Thus, GASP-1 represents a novel class of inhibitory TGFbeta binding proteins.     

Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.     

Regulation of prostate branching morphogenesis by activin A and follistatin

Ventral prostate development occurs by branching morphogenesis and is an androgen-dependent process modulated by growth factors. Many growth factors have been implicated in branching morphogenesis including activins (dimers of beta(A) and beta(B) subunits); activin A inhibited branching of lung and kidney in vitro. Our aim was to examine the role of activins on prostatic development in vitro and their localization in vivo. Organ culture of day 0 rat ventral prostates for 6 days with activin A (+/- testosterone) inhibited prostatic branching and growth without increasing apoptosis. The activin-binding protein follistatin increased branching in vitro in the absence (but not presence) of testosterone, suggesting endogenous activins may reduce prostatic branching morphogenesis. In vivo, inhibin alpha subunit was not expressed until puberty, therefore inhibins (dimers of alpha and beta subunits) are not involved in prostatic development. Activin beta(A) was immunolocalized to developing prostatic epithelium and mesenchymal aggregates at ductal tips. Activin beta(B) immunoreactivity was weak during development, but was upregulated in prostatic epithelium during puberty. Activin receptors were expressed throughout the prostatic epithelium. Follistatin mRNA and protein were expressed throughout the prostatic epithelium. The in vitro evidence that activin and follistatin have opposing effects on ductal branching suggests a role for activin as a negative regulator of prostatic ductal branching morphogenesis.     

Isolation and characterization of activin receptor from mouse embryonal carcinoma cells. Identification of its serine/threonine/tyrosine protein kinase activity.

The activin receptor protein was isolated from the mouse embryonal carcinoma (EC) cell line P19 by three cycles of affinity chromatography on an activin A-immobilized column. The purified receptor had a specific and high affinity for activins A, AB, and B (Kd = 345 pM), but not for transforming growth factor beta. The purified activin receptor was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ligand blotting analysis as a single protein of 70 kDa. The amino acid sequence of the first 18 NH2-terminal residues revealed that the receptor is a member of the activin receptor family. The purified receptor phosphorylated itself and exogenous substrate proteins on serine, threonine, and tyrosine residues, indicating that the activin receptor is a transmembrane serine/threonine/tyrosine protein kinase. These results suggest that signal transduction of activin employs a novel pathway via a new class of cellular receptor in EC P19 cells.     

A novel role for fibronectin type I domain in the regulation of human hematopoietic cell adhesiveness through binding to follistatin domains of FLRG and follistatin

FLRG and follistatin belong to the family of follistatin proteins involved in the regulation of various biological effects, such as hematopoiesis, mediated by their binding to activin and BMP, both members of the TGFbeta family. To further characterize the function of FLRG, we searched for other possible functional partners using a yeast two-hybrid screen. We identified human fibronectin as a new partner for both FLRG and follistatin. We also demonstrated that their physical interaction is mediated by type I motifs of fibronectin and follistatin domains. We then analyzed the biological consequences of these protein interactions on the regulation of hematopoiesis. For the first time, we associated a biological effect with the regulation of human hematopoietic cell adhesiveness of both the type I motifs of fibronectin and the follistatin domains of FLRG and follistatin. Indeed, we observed a significant and specific dose-dependent increase of cell adhesion to fibronectin in the presence of FLRG or follistatin, using either a human hematopoietic cell line or primary cells. In particular, we observed a significantly increased adhesion of immature hematopoietic precursors (CFC, LTC-IC). Altogether these results highlight a new mechanism by which FLRG and follistatin regulate human hematopoiesis.     

Activin incorporation into vitellogenic oocytes of Xenopus laevis.

Activin uptake into Xenopus oocytes was studied by several complementary methods. Immunocytochemistry of adult ovary localized activin and follistatin in the cytoplasm of vitellogenic oocytes and surrounding follicle cells. Surface plasmon resonance analysis of protein interaction kinetics indicated that while follistatin or a complex of activin-follistatin bound to yolk vitellogenin, activin alone did not. Radioactive tracer analysis measured specific incorporation of activin by viable oocytes in vitro. Together, the results suggest that vitellogenic oocytes can import activins from follicle cells and that follistatin may act as a chaperone for binding activin to vitellogenin in yolk platelets.     

Synthesis, purification and bioactivity of recombinant human activin A expressed in the yeast Pichia pastoris

The transforming growth factor-beta (TGF-β) superfamily member, activin A, plays a central role in the regulation of multiple physiological processes including cell differentiation, mitogenesis, embryogenesis, apoptosis and inflammation. In normal cells, activin A signalling is regulated to maintain cellular and tissue health and suppress tumour growth. Disruption of activin A signalling has been implicated in tumour formation and progression. Hence, the availability of activin A is an important target for the development of diagnostics and drugs for therapeutic intervention. To this end, we have expressed human activin A in Pichia pastoris, permitting its secretion into culture medium and purification as the mature homodimer. A construct was engineered encoding the monomeric precursor protein with a N-terminal FLAG affinity tag (DYKDDDDK) and a cleavage site (EKR) for Kex2p protease. Procedures for the two-step purification of human activin A by ion-exchange and anti-FLAG antibody affinity chromatography, and for the removal of the FLAG affinity tag from purified recombinant human activin A by enteropeptidase, are described. The molecular weights of the FLAG-tagged and de-tagged human activin A were confirmed by MALDI-TOF mass spectroscopy. The biological activity of these recombinant activins was assessed for their effects on modulating the secretion of Endothelin-1 (ET-1) by human umbilical vein endothelial cells (HUVECs). The recombinant human activin A containing the intact FLAG tag resulted in a reduced ET-1 secretion from HUVECs, whereas upon removal of this affinity purification tag the purified recombinant human activin A restored ET-1 secretion to levels comparable to the positive control. These results document an approach of considerable potential for the simple, large-scale expression and purification of this important human growth factor for use in diagnostic and therapeutic purposes.     

Specific erythroid differentiation of mouse erythroleukemia cells by activins and its enhancement by retinoic acids.

Activin A has been shown to induce hemoglobin production in various hematopoietic cells. Such activities of three structurally distinct activins (activin A, activin AB, and activin B) were compared using F5-5 mouse erythroleukemia cells. Activin A and AB had similarly potent inducing activities whereas that of activin B was much lower. The erythroid inducing activity of activins was suppressed by follistatin, an activin-binding protein but not by inhibin A and inhibin B. Retinoic acids (both all-trans and 13-cis) had weak erythroid differentiation activity. In addition, clear synergistic erythroid induction occurred when retinoic acid and activin A were mixed together. These results indicate that retinoic acid may modulate activin-induced erythropoiesis in vivo.     

人修饰型降钙素和鼠酰胺化酶基因在昆虫细胞中的偶联表达

人降钙素 (hCT)是 32氨基酸的多肽激素 ,C-端为α脯氨酰胺结构 ,具有调节体内钙、磷代谢等许多重要生理功能。用重组昆虫杆状病毒表达系统 ,偶联表达合成的人修饰型降钙素 (hmCT)基因与GST融合基因和大鼠酰胺化酶 (PAM)基因 ,再用抗hmCT或抗PAM抗体 ,既检测到由昆虫细胞表达的GSThmCT产物也检测到PAM产物。经GSH 琼脂糖凝胶亲和层析 ,分离纯化GSThmCT融合蛋白。这种蛋白修饰酶与底物在真核细胞偶联表达也将适用于其他生物活性肽的体外表达     

Cloning,sequencing, and expressional analysis of the chick homologue of follistatin

Follistatin, a secreted glycoprotein, has been shown to act as a potent neural inducer during early amphibian development. The function of this protein during embryogenesis in higher vertebrates is unclear, and to further our understanding of its role we have cloned, sequenced, and performed an in-depth expressional analysis of the chick homologue of follistatin. In addition we also describe the expression pattern of activin βA and activin β B, proteins that have previously been shown to be able to interact with follistatin. In this study we show that the expression of follistatin and the activins do not always overlap. Follistatin was first detected in Hensen's node and subsequently in the region described by Spratt [1952] as the neuralising area. In older embryos it was also expressed in a highly dynamic manner in the hind-brain as well as in the somites. We also present evidence that follistatin may have a later role in the resegmentation of the somites. We were unable to detect the expression of activin βA during early embryogenesis, whereas activin βB was first expressed in the extending primitive streak and subsequently in the neural folds. The results from this study are consistent with a role for follistatin in neural induction but suggest it has additional functions unrelated to its inhibitory actions on activins. © 1995 Wiley-Liss, Inc.     

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate. When granulosa cells were treated with glycosaminoglycan-degrading enzymes before or after addition of follistatin to the cultures, heparinase and heparitinase treatments resulted in significant suppression of the binding, whereas treatment with chondroitinase ABC had no effect. A competition study of the binding using heparin derivatives demonstrated that follistatin seemed to recognize O-sulfate groups in the heparin molecule. Heparitinase-treated granulosa cells exhibited almost full responsiveness to activin, indicating that the enzyme treatment had no effect on activin and receptor interaction. These results suggest that follistatin/activin-binding protein binds to heparan sulfate side chains of proteoglycans on the granulosa cell surface to regulate the various actions of activin.     

Control of digit formation by activin signalling

Major advances in the genetics of vertebrate limb development have been obtained in recent years. However, the nature of the signals which trigger differentiation of the mesoderm to form the limb skeleton remains elusive. Previously, we have obtained evidence for a role of TGFbeta2 in digit formation. Here, we show that activins A and B and/or AB are also signals involved in digit skeletogenesis. activin betaA gene expression correlates with the initiation of digit chondrogenesis while activin betaB is expressed coincidently with the formation of the last phalanx of each digit. Exogenous administration of activins A, B or AB into the interdigital regions induces the formation of extra digits. follistatin, a natural antagonist of activins, is expressed, under the control of activin, peripherally to the digit chondrogenic aggregates marking the prospective tendinous blastemas. Exogenous application of follistatin blocks physiological and activin-induced digit formation. Evidence for a close interaction between activins and other signalling molecules, such as BMPs and FGFs, operating at the distal tip of the limb at these stages is also provided. Chondrogenesis by activins is mediated by BMPs through the regulation of the BMP receptor bmpR-1b and in turn activin expression is upregulated by BMP signalling. In addition, AER hyperactivity secondary to Wnt3A misexpression or local administration of FGFs, inhibits activin expression. In correlation with the restricted expression of activins in the course of digit formation, neither activin nor follistatin treatment affects the development of the skeletal components of the stylopod or zeugopod indicating that the formation of the limb skeleton is regulated by segment-specific chondrogenic signals.