Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

Characterization of the molecular mechanisms that govern anti-Müllerian hormone synthesis and activity.

The roles of anti-Müllerian hormone (AMH) continue to expand, from its discovery as a critical factor in sex determination, through its identification as a regulator of ovarian folliculogenesis, its use in fertility clinics as a measure of ovarian reserve, and its emerging role in hypothalamic-pituitary function. In light of these actions, AMH is considered an attractive therapeutic target to address diverse reproductive needs, including fertility preservation. Here, we set out to characterize the molecular mechanisms that govern AMH synthesis and activity. First, we enhanced the processing of the AMH precursor to >90% by introducing more efficient proprotein convertase cleavage sites (RKKR or ISSRKKRSVSS [SCUT]). Importantly, enhanced processing corresponded with a dramatic increase in secreted AMH activity. Next, based on species differences across the AMH type II receptor-binding interface, we generated a series of human AMH variants and assessed bioactivity. AMHSCUT potency (EC50 4 ng/mL) was increased 5- or 10-fold by incorporating Gln484 Met/Leu535 Thr (EC50 0.8 ng/mL) or Gln484 Met/Gly533 Ser (EC50 0.4 ng/mL) mutations, respectively. Furthermore, the Gln484 Met/Leu535 Thr double mutant displayed enhanced efficacy, relative to AMHSCUT . Finally, we identified residues within the wrist pre-helix of AMH (Trp494 , Gln496 , Ser497 , and Asp498 ) that likely mediate type I receptor binding. Mutagenesis of these residues generated gain- (Trp494 Phe or Gln496 Leu) or loss- (Ser497 Ala) of function AMH variants. Surprisingly, combining activating type I and type II receptor mutations only led to modest additive increases in AMH potency/efficacy. Our study is the first to characterize AMH residues involved in type I receptor binding and suggests a step-wise receptor-complex assembly mechanism, in which enhancement in the affinity of the ligand for either receptor can increase AMH activity beyond the natural level.

Effects of lactate, super-GDF9, and low oxygen tension during bi-phasic in vitro maturation on the bioenergetic profiles of mouse cumulus-oocyte complex†.

In vitro maturation (IVM) is an alternative assisted reproductive technology with reduced hormone-related side effects and treatment burden compared to conventional IVF. Capacitation (CAPA)-IVM is a bi-phasic IVM system with improved clinical outcomes compared to standard monophasic IVM. Yet, CAPA-IVM efficiency compared to conventional IVF is still suboptimal in terms of producing utilizable blastocysts. Previously, we have shown that CAPA-IVM leads to a precocious increase in cumulus cell (CC) glycolytic activity during cytoplasmic maturation. In the current study, considering the fundamental importance of CCs for oocyte maturation and cumulus-oocyte complex (COC) microenvironment, we further analyzed the bioenergetic profiles of maturing CAPA-IVM COCs. Through a multi-step approach, we (i) explored mitochondrial function of the in vivo and CAPA-IVM matured COCs through real-time metabolic analysis with Seahorse analyzer, and to improve COC metabolism (ii) supplemented the culture media with lactate and/or super-GDF9 (an engineered form of growth differentiation factor 9) and (iii) reduced culture oxygen tension. Our results indicated that the pre-IVM step is delicate and prone to culture-related disruptions. Lactate and/or super-GDF9 supplementations failed to eliminate pre-IVM-induced stress on COC glucose metabolism and mitochondrial respiration. However, when performing pre-IVM culture under 5% oxygen tension, CAPA-IVM COCs showed similar bioenergetic profiles compared to in vivo matured counterparts. This is the first study providing real-time metabolic analysis of the COCs from a bi-phasic IVM system. The currently used analytical approach provides the quantitative measures and the rational basis to further improve IVM culture requirements.

Targeting activins and inhibins to treat reproductive disorders and cancer cachexia.

Although originally characterised as proteins involved in the control of reproductive function, activins, and to a lesser degree inhibins, are also important regulators of homeostasis in extragonadal tissues. Accordingly, disrupted inhibin/activin expression can have detrimental effects not only on fertility and fecundity but also on the regulation of muscle, fat and bone mass. Indeed, only recently, two complementary mouse models of inhibin designed to lack bioactivity/responsiveness revealed that inhibin A/B deficiency during pregnancy restricts embryo and fetal survival. Conversely, hyper-elevated levels of activin A/B, as are frequently observed in patients with advanced cancers, can not only promote gonadal tumour growth but also cancer cachexia. As such, it is not surprising that inhibin/activin genetic variations or altered circulating levels have been linked to reproductive disorders and cancer. Whilst some of the detrimental health effects associated with disrupted inhibin/activin levels can be attributed to accompanied changes in circulating follicle-stimulating hormone (FSH) levels, there is now abundant evidence that activins, in particular, have fundamental FSH-independent tissue homeostatic roles. Increased understanding of inhibin/activin activity, garnered over several decades, has enabled the development of targeted therapies with applications for both reproductive and extra-gonadal tissues. Inhibin- or activin-targeted technologies have been shown not just to enhance fertility and fecundity but also to reduce disease severity in models of cancer cachexia. Excitingly, these technologies are likely to benefit human medicine and be highly valuable to animal breeding and veterinary programmes.

Activin A level is associated with physical function in critically ill patients.

BACKGROUND: Activin A is a potent negative regulator of muscle mass elevated in critical illness. It is unclear whether muscle strength and physical function in critically ill humans are associated with elevated activin A levels. OBJECTIVES: The objective of this study was to investigate the relationship between serum activin A levels, muscle strength, and physical function at discharge from the intensive care unit (ICU) and hospital. METHODS: Thirty-six participants were recruited from two tertiary ICUs in Melbourne, Australia. Participants were included if they were mechanically ventilated for >48 h and expected to have a total ICU stay of >5 days. The primary outcome measure was the Six-Minute Walk Test distance at hospital discharge. Secondary outcome measures included handgrip strength, Medical Research Council Sum Score, Physical Function ICU Test Scored, Six-Minute Walk Test, and Timed Up and Go Test assessed throughout the hospital admission. Total serum activin A levels were measured daily in the ICU. RESULTS: High peak activin A was associated with worse Six-Minute Walk Test distance at hospital discharge (linear regression coefficient, 95% confidence interval, p-value: -91.3, -154.2 to -28.4, p = 0.007, respectively). Peak activin A concentration was not associated with the secondary outcome measures. CONCLUSIONS: Higher peak activin A may be associated with the functional decline of critically ill patients. Further research is indicated to examine its potential as a therapeutic target and a prospective predictor for muscle wasting in critical illness. STUDY REGISTRATION: ACTRN12615000047594.

Inhibin Inactivation in Female Mice Leads to Elevated FSH Levels, Ovarian Overstimulation, and Pregnancy Loss.

Inhibins are members of the transforming growth factor-ß family, composed of a common α-subunit disulfide-linked to 1 of 2 ß-subunits (ßA in inhibin A or ßB in inhibin B). Gonadal-derived inhibin A and B act in an endocrine manner to suppress the synthesis of follicle-stimulating hormone (FSH) by pituitary gonadotrope cells. Roles for inhibins beyond the pituitary, however, have proven difficult to delineate because deletion of the inhibin α-subunit gene (Inha) results in unconstrained expression of activin A and activin B (homodimers of inhibin ß-subunits), which contribute to gonadal tumorigenesis and lethal cachectic wasting. Here, we generated mice with a single point mutation (Arg233Ala) in Inha that prevents proteolytic processing and the formation of bioactive inhibin. In vitro, this mutation blocked inhibin maturation and bioactivity, without perturbing activin production. Serum FSH levels were elevated 2- to 3-fold in InhaR233A/R233A mice due to the loss of negative feedback from inhibins, but no pathological increase in circulating activins was observed. While inactivation of inhibin A and B had no discernible effect on male reproduction, female InhaR233A/R233A mice had increased FSH-dependent follicle development and enhanced natural ovulation rates. Nevertheless, inhibin inactivation resulted in significant embryo-fetal resorptions and severe subfertility and was associated with disrupted maternal ovarian function. Intriguingly, heterozygous Inha+/R233A females had significantly enhanced fecundity, relative to wild-type littermates. These studies have revealed novel effects of inhibins in the establishment and maintenance of pregnancy and demonstrated that partial inactivation of inhibin A/B is an attractive approach for enhancing female fertility.

Effect of cumulin and super-GDF9 in standard and biphasic mouse IVM.

PURPOSE: In vitro maturation (IVM) is a technology that generates mature oocytes following culture of immature cumulus-oocyte complexes (COC) in vitro. IVM is characterized by minimal patient stimulation, making it attractive for certain patient groups. Recently, a biphasic IVM system, capacitation (CAPA)-IVM, has shown improved clinical outcomes relative to standard IVM; however, it remains less efficient than IVF. This study assessed whether supplementation of CAPA-IVM culture media with the novel TGFß superfamily proteins cumulin and super-GDF9 improves subsequent mouse embryo development. METHODS: Immature mouse COCs were cultured by standard IVM or biphasic IVM ± cumulin or super-GDF9. RESULTS: Both cumulin and super-GDF9 in standard IVM significantly improved day-6 blastocyst rate (53.9% control, 73.6% cumulin, 70.4% super-GDF9; p = 0.006; n = 382-406 oocytes). Cumulin or super-GDF9 in CAPA-IVM did not alter embryo yield or blastocyst cell allocation in an unstimulated model. Moreover, cumulin did not alter these outcomes in a mild PMSG stimulation model. Cumulin in CAPA-IVM significantly increased cumulus cell expression of cumulus expansion genes (Ptgs2, Ptx3, Adamts1, Gfat2) and decreased Lhr expression relative to control. However, cumulin-induced mRNA expression of cumulus cell (Ptgs2, Ptx3) and oocyte genes (Gdf9, Bmp15, Oct4, Stella) in CAPA-IVM remained significantly lower than that of in vivo matured cells. CONCLUSION: Cumulin did not provide an additional beneficial effect in biphasic IVM in terms of blastocyst yield and cell allocation; however in standard IVM, cumulin and super-GDF9 significantly improve oocyte developmental competence.

TGFBR3L is an inhibin B co-receptor that regulates female fertility.

Follicle-stimulating hormone (FSH), a key regulator of ovarian function, is often used in infertility treatment. Gonadal inhibins suppress FSH synthesis by pituitary gonadotrope cells. The TGFß type III receptor, betaglycan, is required for inhibin A suppression of FSH. The inhibin B co-receptor was previously unknown. Here, we report that the gonadotrope-restricted transmembrane protein, TGFBR3L, is the elusive inhibin B co-receptor. TGFBR3L binds inhibin B but not other TGFß family ligands. TGFBR3L knockdown or overexpression abrogates or confers inhibin B activity in cells. Female Tgfbr3l knockout mice exhibit increased FSH levels, ovarian follicle development, and litter sizes. In contrast, female mice lacking both TGFBR3L and betaglycan are infertile. TGFBR3L's function and cell-specific expression make it an attractive new target for the regulation of FSH and fertility.

Structure of AMH bound to AMHR2 provides insight into a unique signaling pair in the TGF-ß family.

Anti-Müllerian hormone (AMH), or Müllerian-inhibiting substance, is a protein hormone that promotes Müllerian duct regression during male fetal sexual differentiation and regulation of folliculogenesis in women. AMH is a member of the transforming growth factor beta (TGF-ß) family, which has evolved to signal through its own dedicated type II receptor, AMH receptor type II (AMHR2). Structures of other TGF-ß family members have revealed how ligands infer specificity for their cognate receptors; however, it is unknown how AMH binds AMHR2 at the molecular level. Therefore, in this study, we solved the X-ray crystal structure of AMH bound to the extracellular domain of AMHR2 to a resolution of 2.6Å. The structure reveals that while AMH binds AMHR2 in a similar location to Activin and BMP ligand binding to their type II receptors, differences in both AMH and AMHR2 account for a highly specific interaction. Furthermore, using an AMH responsive cell-based luciferase assay, we show that a conformation in finger 1 of AMHR2 and a salt bridge formed by K534 on AMH and D81/E84 of AMHR2 are key to the AMH/AMHR2 interaction. Overall, our study highlights how AMH engages AMHR2 using a modified paradigm of receptor binding facilitated by modifications to the three-finger toxin fold of AMHR2. Furthermore, understanding these elements contributing to the specificity of binding will help in the design of agonists or antagonists or the selection of antibody therapies.

TMEPAI/PMEPA1 Is a Positive Regulator of Skeletal Muscle Mass.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-ß (TGF-ß1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models of disease.

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.

A variant of human growth differentiation factor-9 that improves oocyte developmental competence.

Growth differentiation factor-9 (GDF9) and bone morphogenetic protein-15 (BMP15) are co-expressed exclusively in oocytes throughout most of folliculogenesis and play central roles in controlling ovarian physiology. Although both growth factors exist as homodimers, recent evidence indicates that GDF9 and BMP15 can also heterodimerize to form the potent growth factor cumulin. Within the cumulin complex, BMP15 "activates" latent GDF9, enabling potent signaling in granulosa cells via type I receptors (i.e. activin receptor-like kinase-4/5 (ALK4/5)) and SMAD2/3 transcription factors. In the cumulin heterodimer, two distinct type I receptor interfaces are formed compared with homodimeric GDF9 and BMP15. Previous studies have highlighted the potential of cumulin to improve treatment of female infertility, but, as a noncovalent heterodimer, cumulin is difficult to produce and purify without contaminating GDF9 and BMP15 homodimers. In this study we addressed this challenge by focusing on the cumulin interface formed by the helix of the GDF9 chain and the fingers of the BMP15 chain. We demonstrate that unique BMP15 finger residues at this site (Arg301, Gly304, His307, and Met369) enable potent activation of the SMAD2/3 pathway. Incorporating these BMP15 residues into latent GDF9 generated a highly potent growth factor, called hereafter Super-GDF9. Super-GDF9 was >1000-fold more potent than WT human GDF9 and 4-fold more potent than cumulin in SMAD2/3-responsive transcriptional assays in granulosa cells. Our demonstration that Super-GDF9 can effectively promote mouse cumulus cell expansion and improve oocyte quality in vitro represents a potential solution to the current challenges of producing and purifying intact cumulin.

Activin A-Induced Cachectic Wasting Is Attenuated by Systemic Delivery of Its Cognate Propeptide in Male Mice.

In cancer, elevated activin levels promote cachectic wasting of muscle, irrespective of tumor progression. In excess, activins A and B use the myostatin signaling pathway in muscle, triggering a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to atrophy. Recently, we demonstrated that local delivery of engineered activin and myostatin propeptides (natural inhibitors of these growth factors) could induce profound muscle hypertrophy in healthy mice. Additionally, the expression of these propeptides effectively attenuated localized muscle wasting in models of dystrophy and cancer cachexia. In this study, we examined whether a systemically administered recombinant propeptide could reverse activin A-induced cachectic wasting in mice. Chinese hamster ovary cells stably expressing activin A were transplanted into the quadriceps of nude mice and caused an 85-fold increase in circulating activin A levels within 12 days. Elevated activin A induced a rapid reduction in body mass (-16%) and lean mass (-10%). In agreement with previous findings, we demonstrated that adeno-associated virus-mediated delivery of activin propeptide to the tibialis anterior muscle blocked activin-induced wasting. In addition, despite massively elevated levels of activin A in this model, systemic delivery of the propeptide significantly reduced activin-induced changes in lean and body mass. Specifically, recombinant propeptide reversed activin-induced wasting of skeletal muscle, heart, liver, and kidneys. This is the first study to demonstrate that systemic administration of recombinant propeptide therapy effectively attenuates tumor-derived activin A insult in multiple tissues.

Serum Concentrations of Oocyte-Secreted Factors BMP15 and GDF9 During IVF and in Women With Reproductive Pathologies.

Oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) are critical for folliculogenesis and fertility. This study developed ELISAs for the measurement of BMP15 and GDF9 in serum and investigated their usefulness as biomarkers of female reproductive function. Serum samples were obtained from women undergoing infertility treatments (n = 154) and from perimenopausal and postmenopausal women (n = 28). Serum concentrations of BMP15 and GDF9 were analyzed in women relative to age, anti-Müllerian hormone, number of oocytes retrieved, and polycystic ovary syndrome (PCOS) after superovulation for in vitro fertilization. BMP15 and GDF9 immunoassays were validated for specificity, sensitivity (24 and 26 pg/mL, respectively), and reproducibility. BMP15 and GDF9 were detectable in 61% and 29% of women, respectively. BMP15 and GDF9 varied 64-fold and 15-fold, respectively, between women, but they did not change within subjects following ovarian stimulation with gonadotropins. Serum GDF9 concentration, but not BMP15 concentration, was associated with oocyte number retrieved in patients without PCOS (P = 0.018). GDF9 and BMP15 associations with oocyte number differed significantly (P < 0.05) with PCOS status. GDF9 concentrations were lower in poor responders (women with fewer than four oocytes retrieved or with cancelled cycles; P = 0.020). Serum BMP15, but not GDF9, was lower in women >55 years of age, compared with women of reproductive age (P < 0.01). This study develops and validates immunoassays to quantitate BMP15 and GDF9 in human serum and to correlate concentrations with female reproductive potential. Although assay sensitivities require improvement, this study demonstrates the diagnostic potential of oocyte-secreted BMP15 and GDF9 as serum biomarkers in reproductive medicine.

Cumulin and FSH Cooperate to Regulate Inhibin B and Activin B Production by Human Granulosa-Lutein Cells In Vitro.

The oocyte-secreted factors bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) interact functionally, and it is hypothesized that this interaction may be mediated by formation of a GDF9:BMP15 heterodimer termed cumulin. GDF9 and BMP15 regulate folliculogenesis and ovulation rate and have been shown to regulate inhibin and activin, local regulators of folliculogenesis. The objective of this study was to determine whether cumulin regulates granulosa cell inhibin and activin production and whether this requires cooperation with FSH. Human granulosa-lutein (hGL) cells collected from patients undergoing in vitro fertilization were cultured with or without FSH with various forms of recombinant cumulin (native and cysteine mutants, with or without the prodomains), and cysteine mutant GDF9 or BMP15. Messenger RNA expression of the subunits of inhibins/activins (INHA, INHBA, INHBB) and secretion of inhibin A, inhibin B, and activin B were measured. Mature forms and proforms of cumulin stimulated comparable INHBB mRNA expression and secretion of inhibin B and activin B, whereas GDF9 or BMP15 exhibited no effect. Cumulin, but not GDF9 or BMP15, interacted synergistically with FSH to increase INHBB mRNA and inhibin B expression. FSH markedly stimulated INHA, which encodes the α subunit of inhibin A/B, and suppressed activin B. Cumulin with or without FSH did not significantly alter inhibin A. Together these data demonstrate that cumulin, but not GDF9 or BMP15, exerts paracrine control of FSH-induced regulation of inhibin B and activin B. The prodomains of cumulin may have a minimal role in its actions on granulosa cells.

Inhibition of activin signaling in lung adenocarcinoma increases the therapeutic index of platinum chemotherapy.

Resistance to platinum chemotherapy is a long-standing problem in the management of lung adenocarcinoma. Using a whole-genome synthetic lethal RNA interference screen, we identified activin signaling as a critical mediator of innate platinum resistance. The transforming growth factor-ß (TGFß) superfamily ligands activin A and growth differentiation factor 11 (GDF11) mediated resistance via their cognate receptors through TGFß-activated kinase 1 (TAK1), rather than through the SMAD family of transcription factors. Inhibition of activin receptor signaling or blockade of activin A and GDF11 by the endogenous protein follistatin overcame this resistance. Consistent with the role of activin signaling in acute renal injury, both therapeutic interventions attenuated acute cisplatin-induced nephrotoxicity, its major dose-limiting side effect. This cancer-specific enhancement of platinum-induced cell death has the potential to dramatically improve the safety and efficacy of chemotherapy in lung cancer patients.

Molecular characterization of latent GDF8 reveals mechanisms of activation.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-ß. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-ß and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

Targeting TGF-ß Mediated SMAD Signaling for the Prevention of Fibrosis.

Fibrosis occurs when there is an imbalance in extracellular matrix (ECM) deposition and degradation. Excessive ECM deposition results in scarring and thickening of the affected tissue, and interferes with tissue and organ homeostasis - mimicking an exaggerated "wound healing" response. Many transforming growth factor-ß (TGF-ß) ligands are potent drivers of ECM deposition, and additionally, have a natural affinity for the ECM, creating a concentrated pool of pro-fibrotic factors at the site of injury. Consequently, TGF-ß ligands are upregulated in many human fibrotic conditions and, as such, are attractive targets for fibrosis therapy. Here, we will discuss the contribution of TGF-ß proteins in the pathogenesis of fibrosis, and promising anti-fibrotic approaches that target TGF-ß ligands.

Specific targeting of TGF-ß family ligands demonstrates distinct roles in the regulation of muscle mass in health and disease.

The transforming growth factor-ß (TGF-ß) network of ligands and intracellular signaling proteins is a subject of intense interest within the field of skeletal muscle biology. To define the relative contribution of endogenous TGF-ß proteins to the negative regulation of muscle mass via their activation of the Smad2/3 signaling axis, we used local injection of adeno-associated viral vectors (AAVs) encoding ligand-specific antagonists into the tibialis anterior (TA) muscles of C57BL/6 mice. Eight weeks after AAV injection, inhibition of activin A and activin B signaling produced moderate (∼20%), but significant, increases in TA mass, indicating that endogenous activins repress muscle growth. Inhibiting myostatin induced a more profound increase in muscle mass (∼45%), demonstrating a more prominent role for this ligand as a negative regulator of adult muscle mass. Remarkably, codelivery of activin and myostatin inhibitors induced a synergistic response, resulting in muscle mass increasing by as much as 150%. Transcription and protein analysis indicated that this substantial hypertrophy was associated with both the complete inhibition of the Smad2/3 pathway and activation of the parallel bone morphogenetic protein (BMP)/Smad1/5 axis (recently identified as a positive regulator of muscle mass). Analyses indicated that hypertrophy was primarily driven by an increase in protein synthesis, but a reduction in ubiquitin-dependent protein degradation pathways was also observed. In models of muscular dystrophy and cancer cachexia, combined inhibition of activins and myostatin increased mass or prevented muscle wasting, respectively, highlighting the potential therapeutic advantages of specifically targeting multiple Smad2/3-activating ligands in skeletal muscle.