Engineering the Ovarian Hormones Inhibin A and Inhibin B to Enhance Synthesis and Activity.

Ovarian-derived inhibin A and inhibin B (heterodimers of common α- and differing ß-subunits) are secreted throughout the menstrual cycle in a discordant pattern, with smaller follicles producing inhibin B, whereas the dominant follicle and corpus luteum produce inhibin A. The classical function for endocrine inhibins is to block signalling by activins (homodimers of ß-subunits) in gonadotrope cells of the anterior pituitary and, thereby, inhibit the synthesis of FSH. Whether inhibin A and inhibin B have additional physiological functions is unknown, primarily because producing sufficient quantities of purified inhibins, in the absence of contaminating activins, for preclinical studies has proven extremely difficult. Here, we describe novel methodology to enhance inhibin A and inhibin B activity and to produce these ligands free of contaminating activins. Using computational modeling and targeted mutagenesis, we identified a point mutation in the activin ß A-subunit, A347H, which completely disrupted activin dimerization and activity. Importantly, this ß A-subunit mutation had minimal effect on inhibin A bioactivity. Mutation of the corresponding residue in the inhibin ß B-subunit, G329E, similarly disrupted activin B synthesis/activity without affecting inhibin B production. Subsequently, we enhanced inhibin A potency by modifying the binding site for its co-receptor, betaglycan. Introducing a point mutation into the α-subunit (S344I) increased inhibin A potency ~12-fold. This study has identified a means to eliminate activin A/B interference during inhibin A/B production, and has facilitated the generation of potent inhibin A and inhibin B agonists for physiological exploration.

Activin-binding protein from rat ovary is follistatin.

Activin, a member of the transforming growth factor beta protein family, was originally isolated from gonadal fluids and stimulates the release of pituitary follicle-stimulating hormone (FSH). Activin has numerous functions in both normal and neoplastic cells. Various cells synthesize activin and have a specific binding site for this peptide. However, the molecular basis for its actions is unknown. A binding protein for activin was purified from rat ovary and was identical to follistatin, a specific inhibitor of FSH release. It is likely that the binding protein participates in the diverse regulatory actions of activin.

Isolation, structure, and synthesis of a human seminal plasma peptide with inhibin-like activity.

A basic peptide isolated from pooled human seminal plasma exhibited inhibin-like activity by suppressing pituitary follicle-stimulating hormone secretion in vitro and in vivo. The peptide has been characterized and sequenced, and a 31-amino-acid synthetic replicate showed full biological activity in vitro.

Inhibins, activins, and follistatins: gonadal proteins modulating the secretion of follicle-stimulating hormone.

The endocrine system displays highly complex interactions among its components. Excesses or deficiencies of hormone production in one gland may alter the production of hormones by others. Several physiological functions are affected by a balance among hormones acting either together or in sequence. For example, FSH secretion has been demonstrated to be affected by hypothalamic influences upon the anterior pituitary through a specific releasing factor, the decapeptide LRF. This decapeptide stimulates the release of both LH and FSH by the pituitary, and these gonadotropins cause the production of steroids by the testes and the ovaries. Gonadal steroids in the blood act directly upon the anterior pituitary to regulate the output of gonadotropins as originally proposed by Moore and Price in 1932 (3), or act indirectly upon the hypothalamus to adjust the output of pituitary hormones in accordance with the needs of the reproductive system. However, such a simple negative feedback of steroids on the hypothalamic-hypophysial axis cannot account for the differential secretion of FSH observed during the estrus cycle. Therefore, the concept that a gonadal protein, inhibin, specifically regulates FSH secretion was proposed. This concept has now been validated by the isolation and characterization of two forms of inhibin that exert their effects on the pituitary to suppress FSH secretion both in vitro and probably in vivo. Furthermore, the production of inhibin is stimulated by FSH, thus establishing a reciprocal relationship between the release of FSH and inhibin. Since hormones in the body are controlled through interlocking complexes of factors, a variety of secondary factors, in one way or another, may also exert influence on the regulation of FSH secretion. As an example, TGF beta, a protein growth factor found in all tissues, promotes the basal secretion of FSH by the pituitary and enhances FSH-mediated estrogen production by the granulosa cells. It is therefore not surprising that two forms of a novel protein, activin and activin A, isolated from the same FF from which inhibins were isolated, show bioactivities similar to those of TGF beta. These activins are formed as dimers of the two beta-subunits of inhibin, probably as a result of the rearrangement of the gene products. This novel observation that different arrangements of gene products can result in opposite biological activities may thus reflect a wholly different level of control of FSH secretion. If such a phenomenon occurs in other biosystems, it would represent an important form of homeostatic mechanism for controlling biologically active substances.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibin A and B in vitro bioactivities are modified by their degree of glycosylation and their affinities to betaglycan.

Inhibin A and B, important regulators of normal function in tissues of the reproductive axis, are glycosylated at either Asn(268) or Asn(268) and Asn(302) in the alpha-subunit to produce 31- and 34-kDa isoforms, respectively. In this study, glycosylated isoforms of recombinant human inhibin A and B were purified from conditioned medium using immunoaffinity chromatography and reversed-phase HPLC. The masses of the purified inhibin preparations were determined by several inhibin immunoassays, and their in vitro bioactivities were based on suppression of FSH release by rat pituitary cells in culture. Based on a ratio of in vitro bioactivity to immunoactivity (B:I ratio), the monoglycosylated 31-kDa inhibin A was 5-fold more potent than the diglycosylated 34-kDa inhibin A (B:I ratio, 1.22 +/- 0.15 vs. 0.24 +/- 0.05; P < 0.001, respectively). The 31-kDa inhibin B was significantly (P < 0.001) more potent (1.75 +/- 0.29) than the 34-kDa form (1.08 +/- 0.20). Because inhibin biological activity is dependent upon interactions with the coreceptor betaglycan, the effect of inhibin glycosylation on betaglycan binding was assessed. Analogous to the pattern of in vitro bioactivity, 31-kDa inhibin A was 12-fold more active (IC(50), 0.68 nM) than the 34-kDa isoform (IC(50), 8.2 nM) at displacing [(125)I]inhibin A from COS7 cells expressing betaglycan. However, the 1.6-fold difference in bioactivity of the inhibin B isoforms was not matched by differences in their affinities for betaglycan. It is concluded that glycosylation of Asn(302) of the alpha-subunit of inhibin A and B results in a decrease in bioactivity, and the effect on inhibin A, at least, is explained by its reduced affinity to betaglycan.

Human prostatic inhibin suppresses tumor growth and inhibits clonogenic cell survival of a model prostatic adenocarcinoma, the Dunning R3327G rat tumor.

Prostatic inhibin (PI) is a M(r) 10,700 protein found in human seminal plasma and is secreted by the prostate. Recognition of alteration of PI levels in prostatic diseases prompted us to investigate its effect on an animal prostatic adenocarcinoma model, the Dunning R3327G rat tumor. PI not only inhibited in vitro growth of tumor cells but also suppressed tumor growth in vivo. A dose-dependent inhibition of both the clonogenic cell growth and rate of proliferation (DNA synthesis) was observed in tumor cell cultures incubated with purified PI. These inhibitory activities were similar in both androgen-dependent and androgen-independent Dunning tumor cell lines. A functional decapeptide of PI was also found to inhibit Dunning tumor cell colonies in a dose-dependent manner. Daily injection of purified PI into tumor-bearing rats suppressed the tumor growth. A 58% reduction in tumor weight and a 2-fold reduction in tumor growth rate were observed over a 15-day treatment period. Continued treatment with PI significantly suppressed the tumor growth rate by nearly 3-fold. These findings clearly demonstrate a potential application of PI for treating human prostatic adenocarcinoma.

The N-myc oncogene in human neuroblastoma cells: down-regulation of an angiogenesis inhibitor identified as activin A.

Angiogenesis, the formation of new blood vessels, is seen during embryonic development and tumor progression, but the mechanisms have remained unclear. Recent data indicate that developmental and tumor angiogenesis can be induced by cellular oncogenes, leading to the enhanced activity of molecules stimulating angiogenesis. However, activated oncogenes might also facilitate angiogenesis by down-regulating endogenous inhibitors of angiogenesis. We report here that enhanced expression of the N-myc oncogene in human neuroblastoma cells down-regulates an inhibitor of endothelial cell proliferation, identified by amino acid sequencing as being identical with activin A, a developmentally regulated protein. Down-regulation appears to involve interaction of the N-Myc protein with the activin A promoter. In addition, activin A inhibits both endothelial cell proliferation in vitro and angiogenesis in vivo, and it induces hemorrhage in vivo. We suggest that the N-myc-induced down-regulation of activin A could contribute to developmental and tumor angiogenesis.

Purification and characterization of recombinant human activin B.

Recombinant human activin B has been isolated to more than 95% purity from a mammalian kidney cell line. Activin B is a covalently-linked homodimer with an apparent molecular mass of 25.9 kDa (unreduced) and 15.2 kDa (reduced) as determined by SDS-polyacrylamide-gel electrophoresis. On gel filtration in 6 M guanidine hydrochloride, activin B chromatographs with an apparent molecular mass of 11 kDa, whether reduced or not. The amino-terminal sequence of the purified protein is consistent with the expected sequence derived from the beta subunit of inhibin B. The amino acid composition of the purified molecule agrees with the expected theoretical composition of the beta subunit of inhibin B. Activin B has an apparent pI of 4.6 as determined by isoelectric focusing in 6 M urea and 4.7 as determined by chromatofocusing in 6 M urea. The extinction coefficient is 1.8.

Developmental biology of amphibians after Hans Spemann in Germany.

After the Hans Spemann and Hilde Mangold discovery of the importance of the dorsal blastopore lip for axis formation in the early embryo (Nobelprize for Spemann, 1935), the scientific community tried in a goldrush-like manner to find the inducing factors responsible for the programming of early embyronic determination and differentiation. The slow progress towards a solution of this problem caused a fading of interest on behalf of most laboratories. This article describes the activities of a few laboratories in Finland, Japan and Germany, which continued their studies despite tremendous experimental difficulties. Finally only Heinz Tiedemann's group in Berlin was the first which could isolate a mesoderm/endoderm inducing factor in highly purified form, the so-called vegetalizing factor, now known as activin. Furthermore this article describes the identification of neuralizing factors like Chordin, Cerberus and Dickkopf in the zone of the Spemann-Mangold organizer. The finding that BMP-4 acts as an antagonist to these factors located on the dorsal side led to a new understanding of the mechanisms of action of inducing (neuralizing) factors and early embryonic pattern formation. Moreover, the observations that closely related genes and their products were also found in Drosophila, Zebrafish, Mice and Human were the basis for new concepts of evolutionary mechanisms (dorsal/ventral and anterior/posterior polarity or conserved processes in eye-development of all 7 animal phyla).

The isolation of activin from ovine amniotic fluid.

During a study of the levels of inhibin and follistatin in ovine amniotic fluid, we noted that although detectable levels of immunoactive inhibin and follistatin were found throughout gestation, the addition of amniotic fluid to a rat anterior pituitary cell culture resulted in a stimulation, rather than the expected suppression, of FSH concentrations. These data suggested the possibility that activin was present in amniotic fluid. We, therefore, set out to isolate the molecules responsible for this activin-like activity and determine their structure. Amniotic fluid, collected from pregnant sheep between 120-140 days gestation, was used as starting material in the purification and diluted in parallel to a human activin-A standard in the activin RIA employed to monitor the purification. A total pool of 7.4 liters amniotic fluid was processed by dye affinity chromatography, hydrophobic interactive chromatography, gel filtration, and a series of reverse phase HPLC steps. Polyacrylamide gel electrophoresis of fractions from the final HPLC step, which showed both activin immunoactivity and bioactivity, revealed a band with a mol wt of 25.3 kilodaltons (kDa), which reduced to 15.8 kDa, and a minor band of 45 kDa, which reduced to 25 kDa. NH2-terminal amino acid sequences of several active fractions from the same region were identical to the known sequence of ovine activin-A. The identification of immunoactive activin, follistatin, and inhibin in amniotic fluid raises the question of the sites of production of these proteins and their interactions and role in fetal physiology.

Inhibin/activin beta-subunit monomer: isolation and characterization.

Using an activin RIA that showed limited cross-reaction with inhibin, activin immunoactivity was monitored throughout the isolation of activin from bovine follicular fluid and side-fractions during the isolation of human recombinant inhibin. Two peaks of activin immunoactivity were identified in both materials and isolated to homogeneity by dye affinity chromatography, hydrophobic interaction and gel permeation chromatography, and reverse phase HPLC. The purified proteins in all four peaks had terminal amino acid sequences identical to those of the inhibin/activin beta-subunit. The molecular masses determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing (and reducing) conditions were 25 and 15 and 15 and 15 kilodaltons (kDa) for each pair of proteins from both sources. Based on these criteria, the bovine and human recombinant 25-kDa proteins correspond to the inhibin/activin beta A-subunit dimer (activin-A), while the 15-kDa proteins correspond to the inhibin/activin beta A-subunit monomer. The activity of the monomer was 17% of the activity of the dimer in the activin RIA. Based on this level of cross-reaction and the proportion of monomer to dimer immunoactivity found after reverse phase HPLC of bovine follicular fluid, it is estimated that the levels of monomer in bovine follicular fluid are 25-60% those of the dimer. The biological activities of the human recombinant activin monomer and dimer were investigated in two different cell culture systems. In a rat pituitary cell system the activity of the activin monomer was 19% of the activity of the dimer in stimulating FSH release, while in rat thymocyte cultures the activity of the monomer was 45% the activity of the dimer in suppressing lectin-stimulated [3H]thymidine uptake. It is concluded that the beta A-subunit monomer is found in bovine follicular fluid at a level 25-60% that of the beta A-subunit dimer (activin-A). The monomer displays in vitro responses similar to those of the dimer, although the monomer is less active (18-45%) than the dimer. It is unclear if dimerization of the monomer is a necessary prerequisite for biological activity.

Purification and characterization of high molecular weight forms of inhibin from bovine follicular fluid.

High molecular mass forms [95 kilodaltons (kDa)] of bovine inhibin-A as well as the known forms of intermediate (55 kDa) and low (32 kDa) mass were purified from bovine follicular fluid by ion exchange chromatography on DEAE-Sepharose, immunoaffinity chromatography using a monoclonal antibody directed against bovine 32-kDa inhibin-A, gel permeation HPLC on TSK-gel, and reverse phase HPLC. The 95-kDa inhibin-A had similar suppressive activity on FSH secretion from cultured rat anterior pituitary cells as the 55- and 32-kDa inhibins. There is, however, a possibility that the inhibin activity detected with larger forms may be due to that of the 32-kDa form that results from proteolytic processing during incubation with rat pituitary cells. Both sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis using monoclonal antibodies specific for 32-kDa inhibin alpha- or beta A-subunits revealed that the 95-kDa inhibin preparation contained two forms of inhibin (105 and 95 kDa), which were composed of either a 50- or a 40-kDa alpha-subunit linked by a disulfide bond(s) to a 55-kDa beta A-subunit. Amino-terminal sequence analysis showed that the 50-kDa alpha-subunit and the 55-kDa beta A-subunit were generated by removal of a signal peptide from each corresponding primary translation product [the first NH2-terminal 17 residues of the inhibin alpha-subunit (residues 1-360) and the first 20 residues of the inhibin beta A-subunit (residues 1-425)] and suggested that the 40-kDa alpha-subunit was formed by proteolytic processing of the 50-kDa alpha-subunit. On the basis of our findings, we propose that in bovine follicular fluid, the larger 105-kDa form of inhibin is processed successively to form the lowest molecular mass form, 32 kDa inhibin, through the smaller 95- and 55-kDa forms.

Identification of activins and follistatin proteins in human follicular fluid and placenta.

Follistatin, an activin-binding protein, is able to neutralize the various activities of activin by forming an inactive complex with it. The widespread tissue localization of follistatin is very similar to that of activin, which suggests that it plays a local modulatory role in the various paracrine/autocrine actions of activin. We detected significant activin-binding activities in human follicular fluid and placental homogenates, although they were much lower than those in porcine and bovine follicular fluids, which raised the possibility that follistatin is present in human follicular fluid and placenta. Therefore, we attempted to identify the protein molecules responsible for this activin-binding activity in human follicular fluid and placental homogenates. Human follicular fluid, collected from in vitro fertilization patients, was processed by affinity chromatography successive steps on sulfated gel matrices and reverse phase high performance liquid chromatography (HPLC). The final HPLC yielded abundant follistatin and almost equimolar amounts of activin-A, -AB, and -B. The follistatin protein showed characteristic multiple bands when analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ability of each band to bind activin specifically was determined by activin binding assay and ligand blotting analysis. Several pieces of evidence, including the immunoblotting analysis and functional assay results, demonstrated the presence of three activin isoforms, A, AB, and B, in the follicular fluid. In contrast, human placental homogenates were found to contain follistatin and activin-A proteins only. Activin-AB and -B were not detected in any HPLC fraction, indicating that activin-A is the major form of activin in the human placenta. The present data indicate that the three activin isoforms and multiple forms of follistatin exist in human follicular fluid, and the activin-A isoform and follistatin exist in human placenta. They suggest that the physiological functions of the activin isoforms during embryonic development differ and that follistatin plays a functional role in the local control system(s) that regulates human reproduction.

Quantification of inhibin pro-alpha C-containing forms in human serum by a new ultrasensitive two-site enzyme-linked immunosorbent assay.

Precursor forms of the alpha-subunit of inhibin are abundant in human follicular fluid and possibly plasma, although their function is uncertain. We now describe the development of a new enzyme-linked immunosorbent assay to measure inhibin forms containing both the pro and alpha C regions of the alpha-subunit. The assay has a detection limit for purified human pro-alpha C of 0.5 pg/mL and less than 0.02% cross-reaction with recombinant forms of inhibin, activin, and follistatin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of follicular fluid extracts demonstrated that the assay is likely to detect pro-containing precursor forms of both the free alpha-subunit and intact dimeric inhibin. The serum concentration was measured in normal men (446 +/- 28 pg/mL), postmenopausal women (45.8 +/- 3.8 pg/mL), and women treated with FSH before in vitro fertilization (1827 pg/mL). Pooled human follicular fluid contained 488 ng/mL. The mean serum concentration in the female menstrual cycle rose from 150.6 +/- 26.1 pg/mL in the early follicular phase to 692.2 +/- 113 pg/mL in the midluteal phase. This assay offers a useful tool for investigation of the role of inhibin-related proteins in human reproduction. There may be particular clinical value under circumstances in which other assays for inhibin forms have insufficient sensitivity.

Extragonadal alpha-inhibin precursor proteins circulate in human male serum.

The role of inhibin as a negative feedback regulator of pituitary FSH secretion in men remains controversial inasmuch as serum inhibin and FSH levels are often not correlated, in part because of the known alpha-inhibin subunit cross-reactivity of the most widely used inhibin antiserum (Monash #1989). The objective of this study was to identify the nature of these alpha-inhibin proteins in male serum using antisera specific for the alpha-inhibin precursor. Three polyclonal antisera were raised against synthetic peptide fragments from the proregion (amino acids 21-35 = precursor alpha inhibin (PIN) 1 and 42-56 = PIN 2) and alpha-N segment (113-127 = PIN 3) of the human alpha-inhibin precursor protein. These antisera were then used in individual RIAs with the homologous peptide as both standard and radioligand. Because pure human alpha-inhibin subunit proteins are not available, recombinant alpha-inhibin medium and porcine follicular fluid were used as reference preparations in the PIN assays. All assays were specific for alpha-inhibin proteins, i.e. 1) they showed no significant cross-reactivity with other alpha-inhibin peptide fragments, dimeric 32-kDa inhibin, recombinant activin, FSH, human albumin, or the inhibin binding proteins alpha-2 macroglobulin and follistatin, and 2) they recognized native protein in alpha-inhibin precursor-containing porcine follicular fluid and in medium from a recombinant alpha-inhibin-only secreting cell line. Furthermore, immunoreactivity of all serum proteins seen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting with the PIN antisera was abolished or significantly reduced (40-100%) by preincubation of each PIN antiserum with the homologous peptide. Serial dilutions of serum from normal, GnRH-deficient, and castrate males exhibited equivalent displacement curves in each precursor (PIN) assay, and there were no significant group differences in PIN 2 immunoreactivity between normal (n = 14), GnRH-deficient (n = 8), and castrate men (n = 3). Western blotting of serum samples from a normal and a GnRH-deficient male revealed immunoreactive proteins of approximately 57 and 29 kDa under reducing conditions with all three PIN antisera. An additional 40-kDa protein was observed with the pro-alpha-inhibin antisera, PIN 1 and 2, and a protein of more than 97 kDa was seen with the PIN 2 antiserum.(ABSTRACT TRUNCATED AT 400 WORDS)

The vegetalizing factor. A member of the evolutionarily highly conserved activin family.

The mesoderm and endoderm inducing vegetalizing factor was partially sequenced after BrCN cleavage. A sequence which is highly conserved in activin A near the C-terminal end was identified. This shows that the factor belongs to the activin family. The activins are not confined to embryos and gonads, but widely distributed in other tissues like calf kidney and calf liver. Functional aspects are discussed.

Peptides of postulated inhibin activity. Lack of in vitro inhibin activity of a 94-residue peptide isolated from human seminal plasma, and of a synthetic replicate of its C-terminal 28-residue segment.

A 94-residue polypeptide isolated from human seminal plasma and its chemically synthesized C-terminal 28-residue segment were studied in an in vitro inhibin bioassay utilizing rat pituitary cell cultures. Both peptides have previously been claimed to have inhibin activities, and the effects on the secretion and cellular content of gonadotrophins (FSH and LH) were now assessed in the in vitro assay. No inhibition was found. After 72 h of culture, both the cellular content and the spontaneous as well as the LHRH-stimulated release of bioactive or immunoactive FSH and LH remained unaffected. Similarly, no effects were found on the storage and/or release of prolactin, growth hormone, or thyrotropin. We conclude that both the native 94-residue peptide and the synthetic replicate of its C-terminal 28-residue segment, do not influence the pituitary FSH secretion when assessed in this in vitro system.

Complete amino acid sequence of human seminal plasma beta-inhibin. Prediction of post Gln-Arg cleavage as a maturation site.

The complete sequence of a 94 amino acid human seminal plasma polypeptide exhibiting inhibin-like activity is presented. This molecule, called beta-inhibin, selectively and specifically suppresses the release of pituitary FSH in vivo as well as in vitro. It does not affect the secretion of LH. Such a novel acidic protein contains a very basic C-terminal segment which is easily cleaved by mild tryptic digestion. It is predicted that the FSH inhibiting activity may reside within this region of the molecule. This would imply a post Gln-Arg cleavage to release the basic C-terminal active moiety.

Induction of apoptosis in B lineage cells by activin A derived from macrophages.

A factor produced by P388D1 cell line murine macrophages showed a profound suppressive effect on the in vitro proliferation of B lineage cells. It was purified to homogeneity from conditioned media of P388D1 cells stimulated with phorbol 12-myristate 13-acetate for 48 h by a three-step procedure. The purified factor gave a single band of protein with a molecular mass of 16 kD on SDS-polyacrylamide gel electrophoresis. We show here that exposure of B lineage cells to this factor results in the induction of a cytotoxic effect and a significant increase in the proportion of fragmented DNA. DNA fragmentation was detected in B lineage cells after 3 h culture with the factor in the quantitative colorimetric determination. The mechanism of cell death was characterized by a ladder-like electrophoretic pattern of degraded chromosomal DNA, indicating that the factor induces apoptosis. The NH2-terminal amino acid sequence of this factor was identical with that of activin A over the 26 amino acid residues identified. We sought to determine whether apoptosis could be modulated by two kinds of inhibitor of protein kinases, H7 and HA1004, in concentrations that are below their toxicity limits. Apoptosis induced by the factor was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals by protein kinases may regulate apoptotic B cell death by the factor activin A, derived from macrophages.