Activin-binding Protein Research Articles

Functional interaction of Clock genes and bone morphogenetic proteins in the adrenal cortex.

The bone morphogenetic protein (BMP) system in the adrenal cortex plays modulatory roles in the control of adrenocortical steroidogenesis. BMP-6 enhances aldosterone production by modulating angiotensin (Ang) II-mitogen-activated protein kinase (MAPK) signaling, whereas activin regulates the adrenocorticotropin (ACTH)-cAMP cascade in adrenocortical cells. A peripheral clock system in the adrenal cortex was discovered and it has been shown to have functional roles in the adjustment of adrenocortical steroidogenesis by interacting with the BMP system. It was found that follistatin, a binding protein of activin, increased Clock mRNA levels, indicating an endogenous function of activin in the regulation of Clock mRNA expression. Elucidation of the interrelationships among the circadian clock system, the BMP system and adrenocortical steroidogenesis regulated by the hypothalamic-pituitary-adrenal (HPA) axis would lead to an understanding of the pathophysiology of adrenal disorders and metabolic disorders and the establishment of better medical treatment from the viewpoint of pharmacokinetics.

New aspects of activin biology in epididymal function and immunopathology.

The activins (A and B) and their binding protein, follistatin, play crucial roles in development, immunoregulation and inflammation throughout the body. In the male reproductive tract of the mouse, activin A and B production is largely confined to the initial segment and proximal caput of the epididymis and the efferent ducts, under normal conditions, with very low expression in the corpus, cauda and vas deferens. However, activin A protein is present throughout the epididymis and vas deferens and is largely associated with the epithelium and interstitial macrophages. Conversely, the activin-binding protein follistatin is produced in the distal epididymis, with very high expression in the vas deferens. Activin activity in the distal tract is inhibited by follistatin, and the activin-follistatin balance is important for regulating coiling of the duct during epididymal development. In further experiments, as described in this report, in situ hybridisation was used to localise activin A mRNA principally to cells in the periductal zone and interstitium in the efferent ducts and proximal caput. Activin B mRNA, on the other hand, was localised to periductal cells in the efferent ducts and proximal epididymis and, most notably, to epithelial cells in the initial segment. Activin A is implicated in the regulation of mononuclear phagocyte function and immune responses in the caput and stimulates the expression of the key immunoregulatory protein, indoleamine 2,3-dioxygenase in this region. Activin A production in the corpus and cauda increases dramatically during bacterial epididymitis in mice, promoting inflammation and fibrosis and causing damage to the epithelium and obstruction of the epididymal duct. Consequently, it appears that the activin-follistatin axis is crucial for maintaining normal epididymal structure and function, but disruption of this balance during inflammation has deleterious effects on male fertility. Follistatin has therapeutic potential in ameliorating the proinflammatory and profibrotic effects of activins.

Experimental Cryptorchidism Causes Chronic Inflammation and a Progressive Decline in Sertoli Cell and Leydig Cell Function in the Adult Rat Testis.

Cryptorchidism causes spermatogenic failure and reduced serum androgen levels, as well as testicular oedema and fibrosis, which are hallmarks of inflammation. However, the role of inflammation and the effects of cryptorchidism on Sertoli cell and Leydig cell function at the molecular level remain ill-defined. Bilateral cryptorchidism was surgically induced in adult rats for 7 and 14 weeks. Testis weights decreased to 40% of normal within 7 weeks, due to loss of all developing spermatogenic cells except spermatogonia, but did not decrease further at 14 weeks. Serum FSH and LH were increased at both time points, consistent with a loss of feedback by inhibin and testosterone. This damage was accompanied by progressive accumulation of interstitial fluid and peritubular fibrosis, and a progressive decline of several critical Sertoli cell genes (Sox9, Inha (inhbin α-subunit), Cldn11 (claudin 11), Gja1 (connexin 43), and Il1a (interleukin-1α)) and the Leydig cell steroidogenic enzymes, Cyp11a1, Hsd3b1, and Hs17b3. Activin B and the activin-binding protein, follistatin, also declined, but the intratesticular concentration of activin A, which is a regulator of inflammatory responses, was largely unaffected at either time point. Expression of genes involved in inflammation (Tnf, Il10, Il1b, Mcp1) and fibrosis (Acta2, Col1a1) were considerably elevated at both time points. These data indicate that induction of experimental cryptorchidism, which causes complete failure of spermatogenesis in the adult rat, also induces chronic testicular inflammation, manifesting in oedema and fibrosis, and a progressive decline of Sertoli and Leydig cell gene expression and function.

Involvement of clock gene expression, bone morphogenetic protein and activin in adrenocortical steroidogenesis by human H295R cells.

Functional interactions between the levels of clock gene expression and adrenal steroidogenesis were studied in human adrenocortical H295R cells. Fluctuations of Bmal1, Clock, Per2 and Cry1 mRNA levels were found in H295R cells treated with forskolin (FSK) in a serum-free condition. The changes of clock gene expression levels were diverged, with Clock mRNA level being significantly higher than Cry1 and Per2 mRNA levels after 12-h stimulation with FSK. After FSK induction, mRNA levels of StAR and CYP11B2 were highest at 12 hours and CYP17 mRNA level reached a peak at 6 hours, but HSD3B1 mRNA level was transiently decreased at 3 hours. The expression levels of Clock mRNA showed a significant positive correlation with StAR among the interrelationships between mRNA levels of key steroidogenic factors and clock genes. Knockdown of Clock gene by siRNA led to a significant reduction of FSK-induced expression of StAR and CYP17 after 12-h treatment with FSK. BMP-6 and activin, which modulate adrenal steroidogenesis, had inhibitory effects on Clock mRNA expression, whereas treatment with follistatin, a binding protein of activin, increased Clock mRNA levels in the presence of FSK, suggesting an endogenous function of activin in regulation of Clock mRNA expression. Collectively, the results indicated that changes of Clock mRNA expression, being upregulated by FSK and suppressed by BMP-6 and activin, were tightly linked to StAR expression by human adrenocortical cells.

Acute heat-treatment disrupts inhibin-related protein production and gene expression in the adult rat testis

Heat reversibly disrupts spermatogenesis, but the effects on Sertoli cell (SC) function and inhibin/activin-related proteins are less well-defined. Adult rat testis weights decreased by 40% within 2 weeks after heat-treatment (43 °C, 15 min), due to loss of pachytene spermatocytes and round spermatids. Coincident effects were reduced SC nuclear volume at one week and >50% reduction in expression of several critical SC genes (Inha, Cld11, Gja1, Tjp1, Cldn3) by 2 weeks. Leydig cell steroidogenic enzymes, Cyp11a1, Hsd3b1, were also reduced. Activin gene expression was unaffected at this time, but expression of the activin-binding protein, follistatin (Fst), increased >2-fold. At 4–8 weeks, coincident with the recovery of spermatocytes and early spermatids, but progressive loss of elongated spermatids, most SC genes had recovered; however, testicular activin A was reduced and activin B increased. At 8 weeks, serum inhibin was decreased and, consequently, serum FSH increased. Crucially, germ cell damage was not associated with a significant inflammatory response. At 14 weeks, most testicular parameters had returned to normal, but testis weights remained slightly reduced. These data indicate that, following acute heat-treatment, expression of several key Sertoli and Leydig cell genes declined in parallel with the initial loss of meiotic germ cells, whereas activins were responsive to the subsequent loss of mature spermatids, leading to an increase in testicular activin B production relative to activin A.

Activins, follistatin and immunoregulation in the epididymis.

The interface between the epididymis and the immune system is implicated in many male reproductive pathologies. The resident immune cell populations and immune-environment within the epididymis are significantly different from the testis, which is an immune-privileged site. Moreover, the immune cell subsets and immunological responses between different regions of the epididymis vary considerably. The cauda epididymis is more susceptible to autoimmune responses than the caput in rodent models of active immunization or suppressed immune tolerance, and in men with congenital or physical damage to the reproductive tract. Activins are members of the transforming growth factor-β family of cytokines that are crucial for testis and epididymal development; however, they also have complex immunoregulatory properties and may play an essential role in the regulation of immunity in the reproductive tract. Our recent research and relevant publications by other researchers identified following a PubMed search are reviewed. The caput epididymis displays elevated endogenous expression of activins A and B and the immunoregulatory gene, indoleamine-2,3-dioxygenase, co-existing with an extensive population of intra-epithelial and interstitial macrophages and dendritic cells, which appear to be involved in regulating tolerance against sperm antigens. The caput is also relatively resistant to inflammatory damage caused by autoimmunity or bacterial infection, but the cauda, which exhibits low activin expression and high levels of the activin-binding protein, follistatin, is highly susceptible to inflammatory damage. Paradoxically, inflammation in the cauda induces increased activin production, and inhibition of activin activity reduces inflammatory responses. Studies using mouse models with altered levels of activins and follistatin indicate a relationship between the activins and genes involved in inflammation and immunoregulation. The existing data indicate that activins play a complex role in controlling inflammation and immunity in the epididymis and vas deferens.

Exogenous GDF11 attenuates non-canonical TGF-β signaling to protect the heart from acute myocardial ischemia-reperfusion injury.

Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor beta 1 (TGF-β1) superfamily that reverses age-related cardiac hypertrophy, improves muscle regeneration and angiogenesis, and maintains progenitor cells in injured tissue. Recently, targeted myocardial delivery of the GDF11 gene in aged mice was found to reduce heart failure and enhance the proliferation of cardiac progenitor cells after myocardial ischemia-reperfusion (I-R). No investigations have as yet explored the cardioprotective effect of exogenous recombinant GDF11 in acute I-R injury, despite the convenience of its clinical application. We sought to determine whether exogenous recombinant GDF11 protects against acute myocardial I-R injury and investigate the underlying mechanism in Sprague-Dawley rats. We found that GDF11 reduced arrhythmia severity and successfully attenuated myocardial infarction; GDF11 also increased cardiac function after I-R, enhanced HO-1 expression and decreased oxidative damage. GDF11 activated the canonical TGF-β signaling pathway and inactivated the non-canonical pathways, ERK and JNK signaling pathways. Moreover, administration of GDF11 prior to reperfusion protected the heart from reperfusion damage. Notably, pretreatment with the activin-binding protein, follistatin (FST), inhibited the cardioprotective effects of GDF11 by blocking its activation of Smad2/3 signaling and its inactivation of detrimental TGF-β signaling. Our data suggest that exogenous GDF11 has cardioprotective effects and may have morphologic and functional recovery in the early stage of myocardial I-R injury. GDF11 may be an innovative therapeutic approach for reducing myocardial I-R injury.

Physiological roles of activins in the human ovary

Abstract Initially discovered in the pituitary as stimulators of follicle stimulating hormone, activins are homo- or heterodimers of inhibin subunits, which belong to the transforming growth factor-β superfamily. Subsequent studies have demonstrated that these growth factors play multifaceted roles in regulating various functions in multiple organs, including the ovary. The spatial and temporal expression of inhibin subunits (α, βA, βB, and βC), their cognate receptors, and activin-binding proteins (inhibins and follistatins) in the principal cells of growing follicles in human ovaries indicates that these activin isoforms are involved in ovarian biology. Information collected from animal studies and clinical samples suggests that these locally produced growth factors are crucial modulators of various ovarian functions, including primordial germ cell development, follicular growth and development, ovarian steroidogenesis, extracellular matrix remodeling, oocyte maturation, ovulation, and luteal function. Along with gonadotropins, intrafollicular activins exert synergistic and complementary effects on growing follicles to help them develop a mature, competent oocyte that is prepared for fertilization. Abnormal activin expression, an imbalanced activin/follistatin ratio, and the dysregulation of the activin signaling pathway have been observed in several ovarian pathologies, such as reproductive aging, polycystic ovary syndrome, and ovarian cancers. Recent advancements in our understanding of the molecular interactions and mechanisms that underlie activins and the development of related ovarian abnormalities have provided insights into disease pathogenesis and increased opportunities to achieve efficient and safe therapies.

Association of serum follistatin levels with histological types and progression of tumor in human lung cancer

BackgroundFollistatin (FST), an activin-binding protein, inhibits activin action by interfering with activin binding to its receptor. The prognostic value of FST has been studied in various cancers. However, these studies rarely focus on lung cancer. In our study, we investigated the relationship between serum FST levels and lung cancer with histologic types, TNM staging, and recurrence.MethodsA total of 150 serum samples were collected, including 91 from patients with SCLC or NSCLC, 22 from patients with benign lung diseases, and 37 from healthy subjects. Enzyme-linked immunosorbent assay was used to determine serum FST levels in healthy subjects, patients with benign lung diseases and patients with lung cancers.ResultsSerum FST levels in patients with LADC, SCC, LASC, LCLC, and SCLC were much higher than those in healthy subjects and in patients with lung benign disease. A ROC curve was constructed for differentiating the lung cancer from the healthy subjects and benign lung diseases. The results indicated that the area under the ROC curve (AUC) was 0.971 and 0.728 respectively. According to TNM staging, serum FST level increased significantly in patients with stage III and IV of LADC. Moreover, serum FST expression were increased in LADC patients with different TNM category. Furthermore, we found that a higher expression of serum FST was correlated with recurrence in LADC patients.ConclusionsThe serum FST levels gradually increased with the rise of TNM staging and category in lung cancer patients. These data suggest that serum FST levels not only can be used in auxiliary diagnosis for lung cancer but also might be associated with the disease progression and metastasis of lung cancers.

Induction of experimental autoimmune orchitis in mice: responses to elevated circulating levels of the activin-binding protein, follistatin

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood-testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Activin/follistatin system in grass carp pituitary cells: - Regulation by local release of growth hormone and luteinizing hormone and its functional role in growth hormone synthesis and secretion.

Gonadotrophin regulation by activin/follistatin system is well-documented, but the corresponding effect on growth hormone (GH) has not been fully characterized and with little information available in lower vertebrates, especially in fish models. In grass carp, local interactions of GH and luteinizing hormone (LH) can induce GH release and gene expression at pituitary level via autocrine/paracrine mechanisms. To shed light on the role of activin/follistatin system in GH regulation by local actions of GH and LH, grass carp activin βA and βB were cloned, shown to be single-copy genes expressed in the pituitary, and confirmed to encode activin proteins capable of transactivating promoter with activin-responsive elements. In grass carp pituitary cells, activin A and B were effective in reducing GH secretion and GH cell content with concurrent drop in GH mRNA level whereas the opposite was true for follistatin, the activin-binding protein known to neutralize the effects of endogenous activin. Treatment with activin A and B not only could suppress basal but also inhibit GH mRNA expression induced by GH and human chorionic gonadotropin (hCG), a functional analogue of LH in fish model. Apparently, down-regulation of GH mRNA by activin was mediated by reducing GH transcript stability with concurrent inhibition on GH promoter activity via the SMAD pathway. In reciprocal experiments, GH treatment was found to up-regulate activin βA, activin βB and follistatin mRNA levels in carp pituitary cells but the opposite was noted by removing endogenous GH with GH antiserum. Interestingly, parallel treatment with hCG could also inhibit basal as well as GH-induced activin βA, activin βB and follistatin gene expression. These results, as a whole, indicate that the pituitary activin/follistatin system can serve as a regulatory target for local interactions of GH and LH and contribute to GH regulation by autocrine/paracrine mechanisms in the carp pituitary.

Gene therapy for follistatin decreases systemic inflammation and pain sensitivity following knee injury in high-fat diet induced obese mice

Purpose: Both obesity and joint trauma are risk factors for osteoarthritis (OA) and OA-related pain. In addition, muscle may have a role in knee OA development as changes in muscle strength could affect kinematics of joint loading. Furthermore, because of its key function in glucose storage and utilization, muscle strength and physiology are particularly vulnerable to obesity-associated inflammation. However, the mechanical, inflammatory, and metabolic links among obesity, muscle, and OA remain unclear. Follistatin (FST), an activin-binding protein, has been used as an effective treatment for muscle degenerative diseases by increasing muscle growth due to its ability to inhibit myostatin, a negative regulator of muscle mass. Moreover, FST has been reported to reduce the infiltration of inflammatory cells in the synovial membrane of the knee joint. Therefore, we hypothesized that overexpression of FST using a gene therapy approach will mitigate obesity-associated inflammation and pain sensitization in a high-fat diet (HFD) induced obesity model of mouse OA. Methods: Six-week old male C57BL/6 mice were fed either a chow control-diet or HFD (60% fat by kcal, n = 16/dietary group). At 7-weeks of age, either an AAV9-mediated FST 314 gene vector or GFP-labeled control vector (n = 10 for each dietary groups) was delivered via tail vein at a final dose of 6 × 1011 vg/ mouse. At 16-weeks of age, mice in each group (n = 10) underwent surgery to destabilize the medial meniscus (DMM) on the left knee. The remaining mice served as non-surgical controls. At time of surgery, Dual Energy X-ray Absorptiometry was conducted to measure body composition and bone mineral density (BMD). Six-weeks post-surgery (22-weeks of age), blood was collected and analyzed using a 31-plex Luminex® assay. Peripheral thermal sensitivity was determined using a hot/cold plate, while mechanical pain algesia at the knee was evaluated by a pressure-pain test. A two-way ANOVA between groups or a paired t-test within each group was performed to determine statistical significance, as appropriate (α = 0.05). Results: Prior to surgery, HFD resulted in increased body weight for both GFP- and FST-treated animals compared to chow (p < 0.05). However, overexpression of FST significantly decreased percentage of body fat in both chow and HFD treated mice as compared to their own corresponding GFP-controls. There was a main effect of both diet (p = 0.028) and FST-treatment (p = 0.007) on bone mineral density, such that HFD decreased BMD (HFD-GFP: 0.46 ± 0.23g/cm2 vs. chow-GFP: 0.51 ± 0.15g/cm2) while FST increased BMD (HFD-FST: 0.52g/cm2 ± 0.17, chow-FST: 0.56g/cm2 ± 0.17). Six weeks post-surgery, serum inflammatory profiles for HFD-GFP animals demonstrated increased systemic inflammation compared to chow diet, which was attenuated in the HFD-FST animals across fourteen markers (Table 1). HFD increased hot-plate paw-withdrawal latency time in GFP-treated animals compared to all other groups (p < 0.05), and the phenotype was rescued by FST-treatment in the HFD animals. Similarly, reduced cold plate responsiveness was also observed in HFD animals, which was rescued by FST-treatment. No differences in thermal peripheral pain were found with surgery. Additionally, FST treatment protected HFD animals from mechanical algesia at the knee post-DMM surgery (Fig. 1). Conclusions: Overexpression of FST decreased body fat, pain sensitivity, and obesity-associated systemic inflammation, while increasing whole body BMD in mice with HFD-induced obesity. These findings further support the important role of muscle in obesity and inflammation, which are well-known risk factors associated with OA onset and progression. We are currently evaluating the relationship among these factors in these mice, and whether FST treatment will mitigate OA development due to obesity and joint injury.Tabled 1Serum Markers Decreased with FST Treatment in HFD Animals compared to HFD animals with GFP.MarkerHFD-GFP Mean ± SEM (ng/mL)HFD-FST Mean ± SEM (ng/mL)p-valueCXCL1/KC0.25 ± 0.030.16 ± 0.020.050CXCL10/IP-100.09 ± 0.010.06 ± 0.040.005, #IL-1α1.03 ± 0.170.53 ± 0.080.022*IL-1β0.06 ± 0.020.03 ± 0.010.028*Leptin137.93 ± 17.4317.69 ± 4.640.001*Insulin25.22 ± 4.884.30 ± 0.650.001** indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals. Open table in a new tab * indicates p < 0.05; # indicates p < 0.05 between DMM animals and control surgery animals.

Follistatin treatment suppresses SERCA1b levels independently of other players of calcium homeostasis in C2C12 myotubes.

Follistatin (FS) is a high affinity activin-binding protein, neutralizing the effects of the Transforming Growth Factor-beta (TGF-β) superfamily members, as myostatin (MSTN). Since MSTN emerged as a negative regulator, FS has been considered as a stimulator of skeletal muscle growth and differentiation. Here, we studied the effect of FS administration on the Ca2+-homeostasis of differentiating C2C12 skeletal muscle cells. FS-treatment increased the fusion index, the size of terminally differentiated myotubes, and transiently elevated the expression of the calcium-dependent protein phosphatase, calcineurin, at the beginning of differentiation. Functional experiments did not detect any alterations in the Ca2+ transients following the stimulation by KCl or caffeine in myotubes. On the other hand, decreased Ca2+-uptake capability was determined by calculating the maximal pump rate (332±17 vs. 279±11µM/s, in control and FS-treated myotubes, respectively; p<0.05). In the same way, the expression and ATPase activity of the neonatal sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA1b) were decreased (0.59±0.01 vs. 0.19±0.01mM ATP/min, in control and FS-treated myotubes, respectively; p<0.05). However, the expression level of other proteins involved in Ca2+-homeostasis and differentiation (calsequestrin, STIM1, MyoD) were not affected. Our results suggest that the FS controlled myotube growth is paralleled with the tight regulation of cytosolic calcium concentration, and the decline of SERCA1b appears to be one of the key components in this process.

Activin and follistatin interactions in the male reproductive tract: activin expression and morphological abnormalities in mice lacking follistatin 288.

Activin A is an important regulator of testicular and epididymal development and function, as well as inflammation and immunity. In the adult murine reproductive tract, activin A mRNA (Inhba) expression levels are highest in the caput epididymis and decrease progressively towards the distal vas deferens. The activin-binding protein, follistatin (FST), shows the opposite expression pattern, with exceptionally high levels of the Fst288 mRNA variant in the vas deferens. This unique pattern of expression suggests that activin A and follistatin, in particular FST288, play region-specific roles in regulating the epididymis and vas deferens. The cellular distribution of activin and follistatin and structural organization of the male reproductive tract was examined in wild-type and transgenic (TghFST315) mice lacking FST288. Compared to wild-type littermates, TghFST315 mice showed a 50% reduction in serum follistatin and a significant elevation of both activin A and B. Testicular, epididymal and seminal vesicle weights were reduced, but intra-testicular testosterone was normal. A decrease in the epididymal duct diameter in the corpus and thickening of the peritubular smooth muscle in the cauda, together with increased coiling of the proximal vas deferens, were observed in TghFST315 mice. No immune cell infiltrates were detected. Immunohistochemistry indicated that epithelial cells are the main source of activins and follistatin in the epididymis and vas deferens. Activin A, but not activin B, was also localized to sperm heads in the lumen of the epididymis and vas deferens. Expression of Inhba and another immunoregulatory gene, indoleamine-2,3-dioxygenase (Ido-1), was increased approximately twofold in the TghFST315 caput epididymis, but several other genes associated with immunoregulation, inflammation or fibrosis were unaffected. Our novel data indicate that disruption of follistatin expression has significant effects on the testis and epididymis, and suggest an association between activin A and indoleamine-2,3-dioxygenase in the caput epididymis, with implications for the epididymal immunoenvironment.

Uropathogenic Escherichia coli causes fibrotic remodelling of the epididymis.

Despite antibiotic treatment, up to 40% of patients have impaired fertility after epididymitis due to serovars of Escherichia coli, a frequent pathogen. The reasons for infertility are unclear, but it may result from epididymal duct obstruction. To determine whether E. coli infection of the epididymis causes obstruction due to fibrosis, and to identify the key mediators, tissues from patients with epididymitis were assessed. Additionally, epididymitis was induced with uropathogenic E. coli (UPEC) or commensal serovars in wild-type and MyD88(-/-) mice, which are relatively unresponsive to bacterial pathogens. Epididymal organ cultures were treated with activin A and bacteria and their histology and levels of cytokines and fibrosis markers were analysed. Patients with epididymitis showed severe fibrosis of the epididymal duct. In mice, UPEC infection also caused fibrosis and ductal obstruction in the cauda epididymis. Levels of mRNA for fibrotic markers (α-smooth muscle actin, fibronectin) and cytokines (activin A, TNFα, IL-1α, IL-1β, IL-6) and total collagen levels were significantly elevated. This fibrotic response was blunted by the loss of MyD88. Activin A induced fibrosis in cultured epididymis, which was inhibited by the activin-binding protein follistatin. In summary, bacterial epididymitis causes fibrosis and obstruction. The milder tissue damage in Myd88(-/-) UPEC epididymitis highlights the importance of the host response to infection in causing epididymal damage. Elevated levels of activin A in vivo and fibrotic remodelling elicited by activin A in vitro indicate that this cytokine is a potential target for supplementary treatment to antibiotic therapy. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Role of activin A in the pathogenesis of endothelial cell dysfunction in preeclampsia.

Circulating markers for endothelial activation such as endothelin-1 (ET-1), ICAM-1 and VCAM-1 are elevated in women with preeclampsia. Using human umbilical vein endothelial cells (HUVECs) as an in vitro model of the maternal vasculature, we show that activin A and preeclamptic serum upregulate ET-1, ICAM-1, and VCAM-1 in HUVECs. Further, we show that follistatin, a specific binding protein for activin, mitigates the upregulation of ET-1, ICAM-1 and VCAM-1 in HUVECs exposed to either activin A or preeclamptic serum. These data are consistent with activin A contributing to the pathophysiology of preeclampsia and suggest that therapies targeting activin signalling are worth exploring.

Activin A and follistatin during the oestrous cycle and early pregnancy in ewes.

The activin pathway has been postulated to be involved in regulation of multiple reproductive processes important for survival of the conceptus. These processes include luteinisation of the follicular cells and thus function of the corpus luteum, early embryo development and uterine function including implantation of the conceptus. Therefore, the aim of the current study was to determine whether the concentrations of activin A and follistatin (FST), an activin-binding protein, differed between ewes with a lifetime history of enhanced or reduced embryonic survival (ES). The mRNAs encoding FST and activin A (inhibin beta A subunit; INHBA) were present in the uterus and abundant in the uterine luminal or glandular epithelia by day 18 of gestation. A peak of activin A was observed in the systemic circulation around the time of oestrus, and activin A concentrations were elevated in animals with reduced ES during the oestrous cycle and early gestation. Concentrations of activin A in uterine fluid were approximately twofold greater on day 16 of gestation in ewes with reduced ES compared to those with enhanced ES. No consistent differences in FST were observed between these groups. Treatment of luteinising ovine granulosa cells with activin A in vitro suppressed progesterone secretion providing evidence of a potential pathway whereby increased concentrations of activin A may decrease ES.

A novel role for follistatin in hypersensitivity following cystitis.

Previous studies have shown that the activin-binding protein follistatin reduces inflammation in several mouse models of colitis. To determine whether follistatin also has a beneficial effect following bladder inflammation, we induced cystitis in mice using cyclophosphamide (CYP) and examined the relationship between bladder hypersensitivity and bladder follistatin expression. Adult female C57BL/6 mice were treated with CYP (100 mg/kg) or vehicle (saline) three times over 5 days. Bladder hypersensitivity was assessed by recording the visceromotor response (VMR) to urinary bladder distension and in vitro single-fiber bladder afferent recording. Follistatin gene expression was measured using qRT-PCR. Immunohistochemistry was employed for further characterization. Bladder hypersensitivity was established by day 6 and persisted to day 14 in CYP-treated mice. On day 14, hypersensitivity was accompanied by increases in follistatin gene expression in the bladder. Follistatin-like immunoreactivity colocalized with laminin, and the percentage of structures in the lamina propria that were follistatin-positive was increased in CYP-treated mice. Exogenous follistatin increased VMR and afferent responses to bladder distension in CYP- but not vehicle-treated mice. Chronic bladder pain following CYP treatment is associated with increased follistatin expression in the bladder. These results suggest a novel, pro-nociceptive role for follistatin in cystitis, in contrast with its proposed therapeutic role in colitis. This protein has exciting potential as a biomarker and therapeutic target for bladder hypersensitivity. Neurourol. Urodynam. 36:286-292, 2017. © 2015 Wiley Periodicals, Inc.

76: Regulation of activins and the activin-binding protein, follistatin, by Toll-like receptor ligands in the adult mouse

The activins, A and B, are members of the transforming growth factor- β cytokine family, and are regulators of inflammation, immunity and fibrosis. Production of activin A and the activin-binding protein, follistatin, increases during inflammation, although aspects of this regulation require clarification and activin B has not been studied in inflammation, previously. Adult male mice were injected with Toll-like receptor (TLR) ligands, acting though MyD88-dependent (TLR2, 4, 7 and 9) and MyD88-independent (TLR3, 4) signalling pathways. Activation of TLR4 by lipopolysaccharide (LPS) was also examined in MyD88-deficient mice, and in mice pre-treated with cycloheximide and actinomycin D. Activin A, B and follistatin were measured in serum by specific immunoasssays, and mRNA expression was measured in the liver by qRT-PCR. Activin A secretion, but not mRNA expression, was stimulated via activation of TLR2, 3, 4 and 7, reaching a peak in the serum around 2–3 h. By contrast, all ligands stimulated activin B at both protein and mRNA level. Compared with activin A, activin B in serum increased more slowly (3–5 h), but reached much higher maximum concentrations. Follistatin was stimulated at the mRNA level by TLR2, 3, 4 and 7. In the absence of MyD88, induction of activin A and follistatin by LPS was prevented, but activin B production was unaffected. While production of activin A was inhibited by cycloheximide, but not by actinomycin D, activin B was inhibited to a similar degree by both compounds. These data indicate that viral and bacterial ligands, acting through both MyD88-dependent and MyD88-independent signaling pathways, stimulate production of activin A, B and follistatin. These data further indicate that activin B, like follistatin, but unlike activin A, is primarily regulated at the transcriptional level during inflammation. These studies suggest that delayed production of activin B may complement the more rapid release of activin A during inflammatory responses.

Regulation and actions of activin A and follistatin in myocardial ischaemia–reperfusion injury

Activin A, a member of the transforming growth factor-β superfamily, is stimulated early in inflammation via the Toll-like receptor (TLR) 4 signalling pathway, which is also activated in myocardial ischaemia–reperfusion. Neutralising activin A by treatment with the activin-binding protein, follistatin, reduces inflammation and mortality in several disease models. This study assesses the regulation of activin A and follistatin in a murine myocardial ischaemia–reperfusion model and determines whether exogenous follistatin treatment is protective against injury. Myocardial activin A and follistatin protein levels were elevated following 30min of ischaemia and 2h of reperfusion in wild-type mice. Activin A, but not follistatin, gene expression was also up-regulated. Serum activin A did not change significantly, but serum follistatin decreased. These responses to ischaemia–reperfusion were absent in TLR4−/− mice. Pre-treatment with follistatin significantly reduced ischaemia–reperfusion induced myocardial infarction. In mouse neonatal cardiomyocyte cultures, activin A exacerbated, while follistatin reduced, cellular injury after 3h of hypoxia and 2h of re-oxygenation. Neither activin A nor follistatin affected hypoxia-reoxygenation induced reactive oxygen species production by these cells. However, activin A reduced cardiomyocyte mitochondrial membrane potential, and follistatin treatment ameliorated the effect of hypoxia-reoxygenation on cardiomyocyte mitochondrial membrane potential. Taken together, these data indicate that myocardial ischaemia–reperfusion, through activation of TLR4 signalling, stimulates local production of activin A, which damages cardiomyocytes independently of increased reactive oxygen species. Blocking activin action by exogenous follistatin reduces this damage.