Yogeshwar Makanji

Elevated expression of activins promotes muscle wasting and cachexia

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.—Chen, J. L., Walton, K. L., Winbanks, C. E., Murphy, K. T., Thomson, R. E., Makanji, Y., Qian, H., Lynch, G. S., Harrison, C. A., Gregorevic, P. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 28, 28–1711 (1723). www.fasebj.org

Two Distinct Regions of Latency-associated Peptide Coordinate Stability of the Latent Transforming Growth Factor-β1 Complex

Transforming growth factor-β1 (TGF-β1) is secreted as part of an inactive complex consisting of the mature dimer, the TGF-β1 propeptide (latency-associated peptide (LAP)), and latent TGF-β-binding proteins. Using in vitro mutagenesis, we identified the regions of LAP that govern the cooperative assembly and stability of the latent TGF-β1 complex. Initially, hydrophobic LAP residues (Ile53, Leu54, Leu57, and Leu59), which form a contiguous epitope on one surface of an amphipathic α-helix, interact with mature TGF-β1 to form the small latent complex. TGF-β1 binding is predicted to alter LAP conformation, exposing ionic residues (Arg45, Arg50, Lys56, and Arg58) on the other side of the α-helix, which form the binding site for latent TGF-β-binding proteins. The stability of the resultant large latent complex is dependent upon covalent dimerization of LAP, which is facilitated by key residues (Phe198, Asp199, Val200, Leu208, Phe217, and Leu219) at the dimer interface. Significantly, genetic mutations in LAP (e.g. R218H) that cause the rare bone disorder Camurati-Engelmann disease disrupted dimerization and reduced the stability of the latent TGF-β1 complex.

Expression of nodal signalling components in cycling human endometrium and in endometrial cancer

BackgroundThe 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.MethodsIn this study, the expression of Nodal, Cripto (co-receptor) and Lefty A (antagonist) was examined by RT-PCR and immunohistochemistry across the menstrual cycle and in endometrial carcinomas.ResultsNodal and Cripto were found to be expressed at high levels in both stromal and epithelial cells during the proliferative phase of the menstrual cycle. Although immunoreactivity for both proteins in surface and glandular epithelium was maintained at relatively steady-state levels across the cycle, their expression was significantly decreased within the stromal compartment by the mid-secretory phase. Lefty expression, as has previously been reported, was primarily restricted to glandular epithelium and surrounding stroma during the late secretory and menstrual phases. In line with recent studies that have shown that Nodal pathway activity is upregulated in many human cancers, we found that Nodal and Cripto immunoreactivity increased dramatically in the transition from histologic Grade 1 to histologic Grades 2 and 3 endometrial carcinomas. Strikingly, Lefty expression was low or absent in all cancer tissues.ConclusionThe expression of Nodal in normal and malignant endometrial cells that lack Lefty strongly supports an important role for this embryonic morphogen in the tissue remodelling events that occur across the menstrual cycle and in tumourogenesis.

A Common Biosynthetic Pathway Governs the Dimerization and Secretion of Inhibin and Related Transforming Growth Factor β (TGFβ) Ligands

The assembly and secretion of transforming growth factor β 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 α-subunit was characterized using in vitro mutagenesis. Three hydrophobic residues near the N terminus of the prodomain (Leu30, Phe37, Leu41) 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 (Phe271, Ile280, Pro283, Leu338, and Val340) on the outer convex surface of the mature α-subunit. Homology modeling indicated that these mature residues are located at the interface between two β-sheets of the α-subunit and that their side chains form a hydrophobic packing core. Mutation of these residues likely disturbs the conformation of this region, thereby disrupting non-covalent interactions with the prodomain. A similar hydrophobic interface was identified spanning the pro- and mature domains of the inhibin βA-subunit. Mutation of key residues, including Ile62, Leu66, Phe329, and Pro341, across this interface was disruptive for the production of both inhibin A and activin A. In addition, mutation of Ile62 and Leu66 in the βA-propeptide reduced its ability to bind, or inhibit the activity of, activin A. Conservation of the identified hydrophobic motifs in the pro- and mature domains of other transforming growth factor β superfamily ligands suggests that we have identified a common biosynthetic pathway governing dimer assembly.

Inhibin at 90: From Discovery to Clinical Application, a Historical Review

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.

Suppression of inhibin A biological activity by alterations in the binding site for betaglycan.

Inhibins A and B negatively regulate the production and secretion of follicle-stimulating hormone from the anterior pituitary, control ovarian follicle development and steroidogenesis, and act as tumor suppressors in the gonads. Inhibins regulate these reproductive events by forming high affinity complexes with betaglycan and activin or bone morphogenetic protein type II receptors. In this study, the binding site of inhibin A for betaglycan was characterized using inhibin A mutant proteins. An epitope for high affinity betaglycan binding was detected spanning the outer convex surface of the inhibin α-subunit. Homology modeling indicates that key α-subunit residues (Tyr50, Val108, Thr111, Ser112, Phe118, Lys119, and Tyr120) form a contiguous epitope in this region of the molecule. Disruption of betaglycan binding by the simultaneous substitution of Thr111, Ser112, and Tyr120 to alanine yielded an inhibin A variant that was unable to suppress activin-induced follicle-stimulating hormone release by rat pituitary cells in culture. Together these results indicate that a high affinity interaction between betaglycan and residues Val108–Tyr120 of the inhibin α-subunit mediate inhibin A biological activity.

Inhibin B is a more potent suppressor of rat follicle-stimulating hormone release than inhibin a in vitro and in vivo.

Mature 31- and 34-kDa inhibin A and B negatively regulate the release of FSH from the anterior pituitary; however, a direct comparison of these hormones in vivo has not been undertaken. The bioactivities of highly purified preparations of recombinant human 31-kDa inhibin A and B were determined in rat pituitary cells in vitro, and in ovariectomized adult rats in vivo based on suppression of plasma FSH. The 31-kDa inhibin B was 4.2-fold more bioactive than inhibin A in vitro and 1.45 (1.01-2.79)-fold more bioactive in vivo than 31-kDa inhibin A. However, the corresponding relative binding affinities of 31-kDa inhibin B for betaglycan, betaglycan+activin type II receptor (ActRII)-A, and betaglycan+ActRIIB were lower (IC(50) 2200, 400, and 750 pm, respectively) compared with 31-kDa inhibin A (IC(50) 190, 80, and 290 pm, respectively). A 2.7- and 2.5-fold reduction in in vitro bioactivity was observed between the 31- and 34-kDa inhibin A and 31- and 34-kDa inhibin B, respectively, and these decreases in bioactivities were matched by a parallel reduction in binding to betaglycan and betaglycan+ActRIIA/B. It is concluded that the increased in vitro and in vivo bioactivities of 31-kDa inhibin B cannot be explained by a higher affinity to betaglycan or activin type II receptors; thus, additional factors mediate inhibin Bs action. In addition, similar reductions in in vitro bioactivity and betaglycan+ActRIIA/B binding between 31- and 34-kDa inhibins A and B are attributed to hindrance by the additional carbohydrate group at Asn(302) in the formation of a functional inhibin+betaglycan+ActRIIA/B complex.

Feedback Regulation by Inhibins A and B of the Pituitary Secretion of Follicle-Stimulating Hormone

Inhibins A and B are gonadal factors that negatively regulate FSH synthesis by the anterior pituitary. Across the menstrual cycle, women show a strong inverse correlation between circulating FSH and inhibin B, estradiol, and anti-Mullerian hormone (AMH), but not with inhibin A. Estradiol is believed to provide a tonic inhibitory effect while the inhibitory role of AMH is unknown. In human males, inhibin B is the primary testicular factor regulating FSH with limited effects by gonadal steroids. In vitro and in vivo studies in rats indicate that inhibin B is more biologically active than inhibin A but showed a lower affinity for the activin type II receptors and the co-receptor, betaglycan, suggesting an alternative mechanism. While this review reinforces the important role inhibin plays in regulating FSH, the observed differences in mode of action of inhibins A and B and their interplay with other gonadal factors are still poorly understood.

Tumour necrosis factor-α stimulates human neutrophils to release preformed activin A

Activin A, a member of the transforming growth factor‐β superfamily, is a critical early mediator of acute inflammation. Activin A release coincides with the release of tumour necrosis factor‐α (TNF‐α) 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‐α, but were unable to respond to LPS directly. Although TNF‐α 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‐α stimulation did not change between 1 and 12 h. Specific inhibition of the p38 MAP kinase signalling pathway blocked TNF‐α‐induced activin release, and the secretion of activin A was not due to TNF‐α‐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‐α may contribute to the early peak in circulating activin A levels during acute inflammation.

Activin-βc reduces reproductive tumour progression and abolishes cancer-associated cachexia in inhibin-deficient mice

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α‐subunit and by an activin‐binding protein, follistatin. The activin‐βC subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin‐βC forms heterodimers with activin‐βA and activin‐C antagonizes activin‐A in vitro. Thus, it is proposed that overexpression, rather than loss of activin‐βC, regulates activin‐A bioactivity. In order to prove biological efficacy, inhibin α‐subunit knockout mice (α‐KO) were crossed with mice overexpressing activin‐βC (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin‐A, and cancer‐associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α‐KO/ActC++ mice, where excessive activin‐A is the underlying cause. Accordingly, a reduction in activin‐A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α‐KO/ActC++ mice. Overexpression of activin‐βC antagonized the activin signalling cascade; thus, the tumourigenic effects of activin‐A were abrogated. This study provides proof of the biological relevance of activin‐βC. Being a regulator of activin‐A, it is able to abolish cachexia and modulate reproductive tumour development in α‐KO mice.