In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF-β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor-derived factors induces cachexia, we used recombinant serotype 6 adeno-associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose-dependent manner, to a maximum of -12.4% (-4.2±1.1 g). These reductions in body mass in rAAV6:activin-injected mice correlated inversely with elevated serum activin A levels (7- to 24-fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy-related ubiquitin ligases, decreased Akt/mTOR-mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes.

When it was initially discovered in 1923, inhibin was characterized as a hypophysiotropic hormone that acts on pituitary cells to regulate pituitary hormone secretion. Ninety years later, what we know about inhibin stretches far beyond its well-established capacity to inhibit activin signaling and suppress pituitary FSH production. Inhibin is one of the major reproductive hormones involved in the regulation of folliculogenesis and steroidogenesis. Although the physiological role of inhibin as an activin antagonist in other organ systems is not as well defined as it is in the pituitary-gonadal axis, inhibin also modulates biological processes in other organs through paracrine, autocrine, and/or endocrine mechanisms. Inhibin and components of its signaling pathway are expressed in many organs. Diagnostically, inhibin is used for prenatal screening of Down syndrome as part of the quadruple test and as a biochemical marker in the assessment of ovarian reserve. In this review, we provide a comprehensive summary of our current understanding of the biological role of inhibin, its relationship with activin, its signaling mechanisms, and its potential value as a diagnostic marker for reproductive function and pregnancy-associated conditions.

Transforming growth factor-␤1 (TGF-␤1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-␤1 propeptide (latency-associated peptide (LAP)), and latent TGF-␤-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-␤1 complex. Transforming growth factor-␤1 (TGF-␤1) 2 is the prototypical member of a large family of structurally related proteins, with well documented roles in cellular proliferation, differentiation, apoptosis, adhesion, and extracellular matrix deposition (1, 2). Like other family members, TGF-␤1 is synthesized as a large precursor molecule consisting of an N-terminal prodomain (latency-associated peptide (LAP)) and a C-terminal mature domain. Within this precursor, LAP acts as a multifunctional peptide capable of regulating the crucial roles TGF-␤1 plays throughout development and in the maintenance of tissue homeostasis in adult life (3-5).Initially, hydrophobic residues near the N terminus of LAP bind to mature TGF-␤1 (6), and this interaction is necessary to maintain the molecule in a conformation competent for dimerization. Two precursors are then covalently linked at sites within both the mature growth factor and LAP to form the small latent complex (SLC) (7-10). The SLC can be cleaved by proprotein convertases (11, 12), but LAP remains non-covalently associated with the mature TGF-␤1 dimer (13,14). During the secretory process, LAP also interacts covalently with latent TGF-␤-binding proteins (LTBPs) to form the large latent complex (LLC), and it is in this form that TGF-␤1 is secreted from the cell (7, 15). In the absence of LTBPs, the reactive cysteine (Cys 33 ) within LAP forms an incorrectly paired disulfide bond with a free cysteine in mature TGF-␤1, ensuring the SLC is secreted in an inactive form (10).Extracellularly, LAP confers latency to TGF-␤1 by shielding the type II receptor-binding epitope on the outer convex surface of the mature dimer (16, 17). Lacking additional secreted antagonists, this is the major point of regulation of TGF-␤1 biological activity. Interestingly, of the 33 TGF-␤ ligands, only TGF-␤1, -␤2, -␤3, myostatin, and GDF-11 (growth and differentiation factor-11) bind their propeptides with high enough affinity to confer latency (16, 18 -21). For other family members (e.g. activins and bone morphogenetic proteins), affinity for receptors is greater than affinity for propeptides, and they are secreted in an "active" form (6,22).LTBPs associate with LAP via signature 8-Cys domains, which are unique to these proteins and the structurally related molecules, fibrillin-1 and fibrillin-2 (23-25). Once secreted, LTBPs target latent TGF-␤1 to fibrillin microfibrils within the extracellular matrix. TGF-␤1 signaling is dependant upon liberation of the mature ligand from the LLC, a process mediated by activators, such as thrombospondin-1 and integrins, that bind to specific residues in LAP ( 54 LSKL and 244 RGD, respectively) (4, 26 -28). By altering the co...

The assembly and secretion of transforming growth factor ␤ superfamily ligands is dependent upon non-covalent interactions between their pro-and mature domains. Despite the importance of this interaction, little is known regarding the underlying regulatory mechanisms. In this study, the binding interface between the pro-and mature domains of the inhibin ␣-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu 30 , Phe 37 , Leu 41 ) were identified that, when mutated to alanine, disrupted heterodimer assembly and secretion. It is postulated that these residues mediate dimerization by interacting non-covalently with hydrophobic residues (Phe 271 , Ile 280 , Pro 283 , Leu 338 , and Val 340 ) on the outer convex surface of the mature ␣-subunit. Homology modeling indicated that these mature residues are located at the interface between two ␤-sheets of the ␣-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting noncovalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro-and mature domains of the inhibin ␤ A -subunit. Mutation of key residues, including Ile 62 , Leu 66 , Phe 329 , and Pro 341 , across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile 62 and Leu 66 in the ␤ A -propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the proand mature domains of other transforming growth factor ␤ superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.Inhibin A and B, members of the transforming growth factor ␤ (TGF␤) 3 superfamily, negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary (1, 2), control ovarian follicle development and steroidogenesis (3), and act as tumor suppressors in the gonads (4). Outside the hypothalamic pituitary gonadal axis, inhibins contribute to the endocrine regulation of bone metabolism (5) and play critical roles in adrenal gland growth and function (6, 7). It is recognized that inhibins regulate these processes by inhibiting the stimulatory actions of the structurally related proteins, activins (8). Inhibins are heterodimers of an 18-kDa ␣-subunit disulfide linked to one of two 13-kDa ␤-subunits (␤ A and ␤ B ), resulting in inhibin A or inhibin B, respectively. Activins are composed of two ␤-subunits: ␤ A -␤ A (activin A), ␤ A -␤ B (activin AB), and ␤ B -␤ B (activin B). Inhibin antagonism of activin-related ligands is dependent upon interactions with betaglycan, a cell surface proteoglycan that also acts as a TGF␤2 co-receptor (9). Betaglycan binds inhibin A directly and promotes the formation of a stable high affinity complex involving activin type II receptors (10). Sequestration of type II receptors in this way prevents their ...

No abstract

Background: The human endometrium is unique in its capacity to remodel constantly throughout adult reproductive life. Although the processes of tissue damage and breakdown in the endometrium have been well studied, little is known of how endometrial regeneration is achieved after menstruation. Nodal, a member of the transforming growth factor-beta superfamily, regulates the processes of pattern formation and differentiation that occur during early embryo development.

Activin A, a member of the transforming growth factor-b superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor-a (TNF-a) in models of lipopolysaccharide (LPS)-induced inflammation. The source of circulating activin A during acute inflammation has not been identified and the potential contribution of leukocyte subsets was examined in the following study. Human leukocytes from healthy volunteers were fractionated using Ficoll gradients and cultured under serum-free conditions. Freshly isolated human neutrophils contained 20-fold more activin A than blood mononuclear cells as measured by enzyme-linked immunosorbent assay (ELISA), and both dimeric and monomeric forms of activin A were detected in these cells by western blotting. Activin A was predominantly immunolocalized in the neutrophil cytoplasm. Purified neutrophils secreted activin A in culture when stimulated by TNF-a, but were unable to respond to LPS directly. Although TNF-a stimulated activin A release from neutrophils within 1 h, activin subunit mRNA expression did not increase until 12 h of culture, and the amount of activin A released following TNF-a stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF-a-induced activin release, and the secretion of activin A was not due to TNF-a-induced neutrophil apoptosis. These data provide the first evidence that neutrophils are a significant source of mature, stored activin A. Stimulation of the release of neutrophil activin A by TNF-a may contribute to the early peak in circulating activin A levels during acute inflammation. Keywords: cytokine; immunoassay; inflammation; leukocytes; lipopolysaccharide Activin A, a member of the transforming growth factor-b superfamily of proteins, is a disulphide-linked homodimer of the inhibin bA subunits. 1 Recently, activin A has been established as a critical cytokine released early in endotoxaemia and other inflammatory syndromes. 2 Further, inhibiting activin A with follistatin, a high affinity activinbinding protein, results in decreased mortality in a mouse model of endotoxic shock. 3 Following a lipopolysaccharide (LPS) challenge, activin A is released into the circulation in a biphasic pattern. 3,4 The first peak occurs 1 h following LPS injection and coincides with the peak release of tumour necrosis factor (TNF)-a. This is suggestive of release of stored activin A rather than secretion from de novo synthesis, as induction of new activin A synthesis appears to require several hours to manifest following inflammatory stimuli in most cell types. [5][6][7][8][9] The source of this rapid increase in circulating activin A during acute inflammation has not yet been established.One potential source of activin A during acute inflammation is the circulating leukocyte subsets. Monocytes, B lymphocytes and eosinophils are known to be capable of synthesizing activin A in vitro, 10-12 but none of these cell types have been shown to co...