Glutamine deprivation induces metabolic adaptations associated with beta cell dysfunction and exacerbate lipotoxicity
Rodrigo Carlessi, Jordan Rowlands, Gaewyn Ellison, Heloisa Helena de Oliveira Alves, ... Cyril Mamotte
In Press, Corrected Proof, Available online 21 April 2019
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Abstract
Abstract
Studies have reported that plasma glutamine is reduced in type 2 diabetes (T2D) patients. Glutamine supplementation improves glycaemic control, however the mechanisms are unclear. Here, we evaluated in vitro the pancreatic beta cell bioenergetic and insulin secretory responses to various levels of glutamine availability, or treatment in the presence of an inhibitor of intracellular glutamine metabolism. The impact of glutamine deprivation to the pathological events induced by the saturated fatty acid palmitate was also investigated. Glutamine deprivation induced a reduction in mitochondrial respiration and increase in glucose uptake and utilization. This phenotype was accompanied by impairment in beta cell function, as demonstrated by diminished insulin production and secretion, and activation of the unfolded protein response pathway. Palmitate led to insulin secretory dysfunction, loss of viability and apoptosis. Importantly, glutamine deprivation significantly exacerbated these phenotypes, suggesting that low glutamine levels could participate in the process of beta cell dysfunction in T2D.

Alternative retinoid X receptor (RXR) ligands
Wojciech Krężel, Ralph Rühl, Angel R. de Lera
In Press, Corrected Proof, Available online 23 April 2019
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Abstract
Abstract
Retinoid X receptors (RXRs) control a wide variety of functions by virtue of their dimerization with other nuclear hormone receptors (NRs), contributing thereby to activities of different signaling pathways. We review known RXR ligands as transcriptional modulators of specific RXR-dimers and the associated biological processes. We also discuss the physiological relevance of such ligands, which remains frequently a matter of debate and which at present is best met by member(s) of a novel family of retinoids, postulated as Vitamin A5. Through comparison with other natural, but also with synthetic ligands, we discuss high diversity in the modes of ligand binding to RXRs resulting in agonistic or antagonistic profiles and selectivity towards specific subtypes of permissive heterodimers. Despite such diversity, direct ligand binding to the ligand binding pocket resulting in agonistic activity was preferentially preserved in the course of animal evolution pointing to its functional relevance, and potential for existence of other, species-specific endogenous RXR ligands sharing the same mode of function.

7TM proteins are not necessarily GPCRs
Ieva Vasiliauskaité-Brooks, Robert D. Healey, Sébastien Granier
In Press, Corrected Proof, Available online 24 April 2019
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Abstract
Abstract
In this review article, we summarize the current knowledge on a large and diverse superfamily of seven-pass transmembrane proteins functionally independent from the GPCR superfamily. We include the newest research findings about their physiological roles and their mechanism of action. In particular, we concentrate on the structural basis for the newly discovered amide hydrolase activity, with a focus on adiponectin receptors for which structures are available. Finally, we discuss the remaining challenges in understanding the activation and signaling of these intramembrane proteins and suggest how regulation of the amide hydrolase activity may help in development of new therapeutic agents.

Relationships between eating disorder psychopathology, sexual hormones and sexual behaviours
Giovanni Castellini, Lorenzo Lelli, Emanuele Cassioli, Valdo Ricca
In Press, Corrected Proof, Available online 24 April 2019
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Abstract
Abstract
A growing body of evidences demonstrated that sexuality is an important topic in the clinical research of eating disorders (EDs), due to its association with specific psychopathological features, and etiological factors. The present review took into consideration the complex relationship between sexual behaviours, hormonal alterations and EDs psychopathology. Studies pertaining sexual behaviours in EDs were divided into those focusing on sexual dysfunctions, and those related to risky sexual behaviours. The limited number of studies on sexual dysfunctions, reported a controversial association with weight status and hormonal alterations, and a clear relationship with severity of specific psychopathology (e.g. body image disturbance). Risky sexual behaviours have been associated with impulsivity and dissociation, as well as with abnormal stress response. Finally, both restriction and uncontrolled eating have been found to be responsible for several complex metabolic alterations, determining varied sexual and gynecologic problems, such as amenorrhea, hypogonadism, genital vascular problems, infertility, and miscarriage, although it is also possible that alterations in feeding and stress hormones contribute to altered eating behaviour.

Effect of neonatal exposure to endosulfan on myometrial adaptation during early pregnancy and labor in rats
Ramiro Alarcón, Jorgelina Varayoud, Enrique H. Luque, María M. Milesi
In Press, Corrected Proof, Available online 25 April 2019
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Abstract
Abstract
Proper myometrial adaptation during gestation is crucial for embryo implantation, pregnancy maintenance and parturition. Previously, we reported that neonatal exposure to endosulfan alters uterine development and induces implantation failures. The present work investigates the effects of endosulfan exposure on myometrial differentiation at the pre-implantation period, and myometrial activation during labor. Newborn female rats were s.c. injected with corn oil (vehicle) or 600 μg/kg/day of endosulfan (Endo600) on postnatal days (PND) 1, 3, 5 and 7. On PND90, the rats were mated to evaluate: i) the myometrial differentiation on gestational day 5 (GD5, pre-implantation period), by assessment myometrial histomorphology, smooth muscle cells (SMCs) proliferation, and expression of proteins involved in myometrial adaptation for embryo implantation (steroid receptors, Wnt7a and Hoxa10); ii) the timing of parturition and myometrial activation during labor by determining the uterine expression of contraction-associated genes (oxytocin receptor, OTXR; prostaglandin F2α receptor, PTGFR and connexin-43, Cx-43). Endosulfan decreased the thickness of both myometrial layers, with a concomitant decrease in the collagen remodeling. Blood vessels relative area in the interstitial connective tissue between muscle layers was also decreased. Endo600 group showed lower myometrial proliferation in association with a downregulation of Wnt7a and Hoxa10. Although in all females labor occurred on GD23, the exposure to endosulfan altered the timing of parturition, by inducing advancement in the initiation of labor. This alteration was associated with an increased uterine expression of OTXR, PTGFR and Cx-43. In conclusion, neonatal exposure to endosulfan produced long-term effects affecting myometrial adaptation during early pregnancy and labor. These alterations could be associated with the aberrant effects of endosulfan on the implantation process and the timing of parturition.

Fish as models for understanding the vertebrate endocrine regulation of feeding and weight
Helene Volkoff
In Press, Corrected Proof, Available online 2 May 2019
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Abstract
Abstract
The frequencies of eating disorders and obesity have increased worldwide in recent years. Their pathophysiologies are still unclear, but recent evidence suggests that they might be related to changes in endocrine and neural factors that regulate feeding and energy homeostasis. In order to develop efficient therapeutic drugs, a more thorough knowledge of the neuronal circuits and mechanisms involved is needed. Although to date, rodents have mostly been used models in the area of neuroscience and neuroendocrinology, an increasing number of studies use non-mammalian vertebrates, in particular fish, as model systems. Fish present several advantages over mammalian models and they share genetic and physiological homology to mammals with close similarities in the mechanisms involved in the neural and endocrine regulation of appetite. This review briefly describes the regulation of feeding in two model species, goldfish and zebrafish, how this regulation compares to that in mammals, and how these fish could be used for studies on endocrine regulation of eating and weight and its dysregulations.

Role of regulatory T cells in Schistosoma-mediated protection against type 1 diabetes
Chun-lian Tang, Yan-ru Gao, Li-xia Wang, Ya-wen Zhu, ... Ying Xiong
In Press, Corrected Proof, Available online 9 May 2019
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Abstract
Abstract
The prevalence of T1D in developed societies is partly based on the hygiene hypothesis, that is, the loss of exposure to infectious agents accompanies the loss of immune stimuli shaping the immune system during development. Indeed, the components of parasites, such as Schistosoma, have been reported to ameliorate or prevent the development of T1D, which might be associated with immune cell activity especially that of regulatory T cells (Tregs). Schistosoma infection can lead to the expansion of Treg. Herein, we provide a comprehensive overview of the involvement of Tregs in the response against Schistosoma infection and the mechanism of Schistosoma-associated host protection against T1D.

Estradiol stimulates adipogenesis and Slc2a4/GLUT4 expression via ESR1-mediated activation of CEBPA
Luciana A. Fatima, Raquel S. Campello, João N. Barreto-Andrade, Marisa Passarelli, ... Ubiratan F. Machado
In Press, Corrected Proof, Available online 14 May 2019
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Abstract
Graphical abstract
Abstract
The ability of adipose tissue to expand is dependent on adipocyte differentiation and adipose tissue glucose disposal. The CCAAT/enhancer-binding protein alpha (CEBPA) enhances the expression of the Slc2a4 gene and GLUT4 protein, which are markers of adipocyte differentiation/glucose disposal. We hypothesized estradiol (E2) facilitates adipocyte differentiation/glucose disposal by an estrogen receptor 1 (ESR1)-dependent and CEBPA-mediated mechanism. Our results suggest that E2 (10 nM) has a positive effect on 3T3-L1 adipocyte differentiation (days 2–8), lipid accumulation, Slc2a4 and Cebpa mRNA expression, total GLUT4 and nuclear CEBPA contents, and CEBP/Slc2a4-binding activity. Esr1 silencing (∼50%) in mature adipocytes abrogates the 24-h E2 effects on nuclear CEBPA content, Slc2a4/GLUT4 expression and GLUT4 translocation to the cell membrane. Thus, E2 stimulates adipocyte differentiation and Slc2a4/GLUT4 expression in an ESR1/CEBPA-mediated pathway. Our data provide mechanistic insight demonstrating E2 participates in adipose-tissue differentiation and glucose transporter expression which ultimately can improve adipose tissue expandability and glycemic control.

Associations between neuropsychological performance and appetite-regulating hormones in anorexia nervosa and healthy controls: Ghrelin's putative role as a mediator of decision-making
Georgios Paslakis, Zaida Agüera, Roser Granero, Isabel Sánchez, ... Fernando Fernández-Aranda
In Press, Corrected Proof, Available online 20 May 2019
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Abstract
Abstract
Anorexia nervosa (AN) is a severe eating disorder accompanied by alterations in endocrinological circuits and deficits in neuropsychological performance. In this study, a series of appetite-regulating hormones (ghrelin, leptin, cholecystokinin, PYY, adiponectin, and visfatin) were measured under fasting conditions in female patients with AN and female healthy controls. All of the participants also underwent a battery of neuropsychological assessment [namely the Iowa Gambling Task (IGT), the Wisconsin Card Sorting Test (WCST), and the Stroop Color and Word Test (SCWT)]. As the main finding, we found that higher ghrelin levels predict better performance in the IGT. Ghrelin may be a putative mediator of decision-making, a finding that has not been described so far. The role of ghrelin in decision-making can only be described as speculative, as there are hardly any additional evidence-based data published up to date. Further studies are warranted.

Identification and functional characterization of microRNAs in rat Leydig cells during development from the progenitor to the adult stage
Hongxia Chen, Xiaoping Guo, Xue Xiao, Leping Ye, ... Zhijian Su
In Press, Accepted Manuscript, Available online 23 May 2019
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Abstract
Abstract
The aim of the present study was to identify microRNAs (miRNAs) that regulate the proliferation and differentiation of Leydig cells (LCs) of rat. Three small RNA libraries derived from progenitor LCs (PLCs), immature LCs (ILCs) and adult LCs (ALCs) were analyzed by microarrays. In total, 68 differentially expressed miRNAs (DEMs) were identified. Based on the trend of DEM expression from PLCs to ALCs, primary LCs were transfected with miRNA mimics or inhibitors. Five miRNAs (miR-30a-5p, miR-3585-5p, miR-212-3p, miR-369-5p and miR-434-3p) promoted PLC proliferation, and 3 miRNAs (miR-17-5p, miR-532-3p and miR-329-3p) activated caspase-3, which triggered LC apoptosis. For steroidogenesis, 18 miRNAs could elevate or inhibit androsterone release at the PLC stage. Eleven and 9 miRNAs inhibited the production of 5α-androstane-3α,17β-diol in ILCs and testosterone in ALCs, respectively. miR-17-5p, miR-29a-3p and miR-299a-5p decreased androgen production by LCs at all developmental stages. Furthermore, the miR-299a-5p-mediated decrease in androgen production by the LC lineage was primarily achieved by downregulating the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and 3β-hydroxysteroid dehydrogenase 1 (HSD3B1). These findings provide insights into the regulatory roles of miRNAs during the postnatal development of LCs and suggest potential strategies for the treatment of steroid-related disorders.

FOXE1 inhibits cell proliferation, migration and invasion of papillary thyroid cancer by regulating PDGFA
Zheng Ding, Ronghu Ke, Yong Zhang, Youben Fan, Jianxia Fan
In Press, Accepted Manuscript, Available online 23 May 2019
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Abstract
Abstract
Purpose
Forkhead box E1 (FOXE1) plays an important role in the development, proliferation and differentiation of thyroid cells. However, the biological functions of FOXE1 in papillary thyroid cancer (PTC) remain unclear.

Materials and methods
In this study, the level of FOXE1 expression was examined in human PTC tissues and cells. Then, the high expression of FOXE1 was specifically silenced by RNA interference in vitro. Subsequently, FOXE1-shRNA was transfected into PTC cells (TPC-1 and K1). The effects on cell proliferation, migration and invasion were evaluated. In addition, FOXE1 targets were screened by cDNA microarray assays. The correlation between the expression of target gene platelet-derived growth factor A (PDGFA) and clinicopathological features of PTC patients was analysed.

Results
FOXE1 is highly expressed in PTC tissues and PTC cell lines. The silencing of FOXE1 significantly promotes PTC cell proliferation, migration and invasion in vitro. The cDNA microarray analyses show that PDGFA is a critical downstream target gene of FOXE1 in PTC cells. It was also observed that PDGFA is negatively regulated by FOXE1 in PTC. The clinical data indicate that the low expression level of PDGFA is correlated with the small size of PTC.

Conclusion
Collectively, the results indicate for the first time that high expression of FOXE1 may function as a tumour suppressor in the early stage of PTC and restrain the proliferation, migration and invasion of PTC by negatively regulating PDGFA expression. Thus, FOXE1 could serve as a prognostic biomarker for PTC.

Correlation between renin-angiotensin system (RAS) related genes, type 2 diabetes, and cancer: Insights from metanalysis of transcriptomics data
Luciana Xavier Pereira, Laryssa Cristina Alves da Silva, Alexya de Oliveira Feitosa, Ricardo Jansen Santos Ferreira, ... Carlos Alberto de Carvalho Fraga
In Press, Accepted Manuscript, Available online 28 May 2019
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Abstract
Abstract
Although studies have provided significant evidence about the role of RAS in mediating cancer risk in type 2 diabetes mellitus (DM), conclusions about the central molecular mechanisms underlying this disease remain to be reached, because this type of information requires an integrative multi-omics approach. In the current study, meta-analysis was performed on type 2 diabetes and breast, bladder, liver, pancreas, colon and rectum cancer-associated transcriptome data, and reporter biomolecules were identified at RNA, protein, and metabolite levels using the integration of gene expression profiles with genome-scale biomolecular networks in diabetes samples. This approach revealed that RAS biomarkers could be associated with cancer initiation and progression, which include metabolites (particularly, aminoacyl-tRNA biosynthesis and ABC transporters) as novel biomarker candidates and potential therapeutic targets. We detected downregulation and upregulation of differentially expressed genes (DEGs) in blood, pancreatic islets, liver and skeletal muscle from normal and diabetic patients. DEGs were combined with 211 renin-angiotensin-system related genes. Upregulated genes were enriched using Pathway analysis of cancer in pancreatic islets, blood and skeletal muscle samples. It seems that the changes in mRNA are contributing to the phenotypic changes in carcinogenesis, or that they are as a result of the phenotypic changes associated with the malignant transformation. Our analyses showed that Ctsg and Ednrb are downregulated in cancer samples. However, by immunohistochemistry experiments we observed that EDNRB protein showed increased expression in tumor samples. It is true that alterations in mRNA expression do not always reflect alterations in protein expression, since post-translational changes can occur in proteins. In this study, we report valuable data for further experimental and clinical analysis, because the proposed biomolecules have significant potential as systems biomarkers for screening or for therapeutic purposes in type 2 diabetes and cancer-associated pathways.

Defective endoplasmic reticulum export causes proinsulin misfolding in pancreatic β cells
Ruimin Zhu, Xin Li, Jialu Xu, Cesar Barrabi, ... Ming Liu
In Press, Accepted Manuscript, Available online 31 May 2019
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Abstract
Abstract
Endoplasmic reticulum (ER) homeostasis is essential for cell function. Increasing evidence indicates that, efficient protein ER export is important for ER homeostasis. However, the consequence of impaired ER export remains largely unknown. Herein, we found that defective ER protein transport caused by either Sar1 mutants or brefeldin A impaired proinsulin oxidative folding in the ER of β-cells. Misfolded proinsulin formed aberrant disulfide-linked dimers and high molecular weight proinsulin complexes, and induced ER stress. Limiting proinsulin load to the ER alleviated ER stress, indicating that misfolded proinsulin is a direct cause of ER stress. This study revealed significance of efficient ER export in maintaining ER protein homeostasis and native folding of proinsulin. Given the fact that proinsulin misfolding plays an important role in diabetes, this study suggests that enhancing ER export may be a potential therapeutic target to prevent/delay β-cell failure caused by proinsulin misfolding and ER stress.

Studying GPCR conformational dynamics by single molecule fluorescence
Robert B. Quast, Emmanuel Margeat
In Press, Accepted Manuscript, Available online 1 June 2019
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Abstract
Graphical abstract
Abstract
Over the last decades, G protein coupled receptors (GPCRs) have experienced a tremendous amount of attention, which has led to a boost of structural and pharmacological insights on this large membrane protein superfamily involved in various essential physiological functions. Recently, evidence has emerged that, rather than being activated by ligands in an on/off manner switching from an inactive to an active state, GPCRs exhibit high structural flexibility in the absence and even in the presence of ligands. So far the physiological as well as pharmacological impact of this structural flexibility remains largely unexplored albeit its potential role in precisely fine-tuning receptor function and regulating the specificity of signal transduction into the cell. By complementing other biophysical approaches, single molecule fluorescence (SMF) offers the advantage of monitoring structural dynamics in biomolecules in real-time, with minimal structural invasiveness and in the context of complex biological environments. In this review a general introduction to GPCR structural dynamics is given followed by a presentation of SMF methods used to explore them. Particular attention is paid to single molecule Förster resonance energy transfer (smFRET), a key method to measure actual distance changes between two probes, and highlight conformational changes occurring at timescales relevant for protein conformational movements. The available literature reporting on GPCR structural dynamics by SMF is discussed with a focus on the newly gained biological insights on receptor activation and signaling, in particular for the β2 adrenergic and the metabotropic glutamate receptors.

The development of the serotonergic and dopaminergic systems during chicken mid-late embryogenesis
Xiaohong Huang, Shihuan Kuang, Todd J. Applegate, Tsang-long Lin, Heng-wei Cheng
In Press, Accepted Manuscript, Available online 2 June 2019
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Abstract
Graphical abstract
Abstract
Serotonin (5-HT) acts as a morphogen influencing embryonic brain development, and as a neurotransmitter regulating multiple biological functions with lifelong effects on animal physical, physiological and mental health, especially during the rapid growth phase prior to birth when embryos face many challenges to reach structural and functional completion. In this study, the development of the serotoninergic (5-HTergic) system and its modulatory effect on the dopaminergic (DAergic) system and related neural circuits were investigated during the mid-late embryogenesis, embryonic day (E)12-E20, in the chicken's brain. During 5-HTergic neuronal maturation, a growth-related anatomical and functional remodeling was highlighted: the 5-HT neurons continuously grew during E12-E20 except for a remarkable regression during E14-E16. Correspondingly, there was a time-dependent change in the 5-HT synthetic capacity. Specifically, 5-HT concentrations in the raphe nuclei increased from E12 to E14, reaching a first plateau during E14-E16, then continuously increased up to E19, and reaching a second plateau between E19-E20. The second plateau of the 5-HT concentration was in correspondence with the establishment of the 5-HTergic autoregulatory loop during E19-E20 and the development of the DAergic system. The DA concentrations remained unchanged from E12 to E16, then started to increase at E16, reaching a maximum at E19, and diminished before hatching. The unique developing time sequence between the 5-HTergic and DAergic systems suggests that the 5-HTergic system may play a critical role in forming the 5-HT – DA neural circuit during chicken embryogenesis. These results provide new insights for understanding the functional organization of the 5-HTergic system during embryonic development and raise the possibility that prenatally modulating the 5-HTergic system may lead to long-lasting brain structural and functional alterations.

Editorial for Special Issue on “Alternative Nuclear Receptor Ligands”

Terry W. Moore, Jonna Frasor

In Press, Accepted Manuscript, Available online 4 June 2019

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Altered miR-186 and miR-135a contribute to granulosa cell dysfunction by targeting ESR2: A possible role in polycystic ovary syndrome
Yuxia Song, Guo Yu, Yungai Xiang, Yan Li, ... Li Tan
In Press, Accepted Manuscript, Available online 5 June 2019
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Abstract
Abstract
MicroRNAs (miRNAs) are a group of negative regulators of gene expression that function at the posttranscriptional level. Dysregulation of miRNAs is involved in many pathophysiological processes, including polycystic ovary syndrome (PCOS). In this study, we first detected the expression levels of 6 candidate miRNA in granulosa cells (GCs) from 63 women with PCOS and 20 healthy controls. We found that miR-186 and miR-135a were overexpressed in GCs from PCOS patients. Subsequently, the direct targets of miR-186 and miR-135a were predicted using bioinformatics analysis and verified by luciferase assays and immunoblotting. The present study determined that miR-186 and miR-135a repressed ESR2 expression in GCs, which further inhibited CDKN1A expression, promoted GC proliferation and repressed GC apoptosis. Meanwhile, the levels of miR-186 and miR-135a in GCs were found to positively correlate with serum estradiol levels in patients with PCOS. Furthermore, estradiol treatment directly increased miR-186 and miR-135a levels in KGN and primary GCs, which provides new insight into understanding the pathophysiology of PCOS.

Adiponectin is required for pioglitazone-induced improvements in hepatic steatosis in mice fed a high-fat diet
Mariana de Mendonça, Bruna de Araújo Cardoso dos Santos, Érica de Sousa, Alice Cristina Rodrigues
In Press, Accepted Manuscript, Available online 6 June 2019
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Abstract
Graphical abstract
Abstract
Pioglitazone has been used for the treatment of nonalcoholic fatty liver disease (NAFLD) related to diabetes. The role of adiponectin in pioglitazone-induced improvements in NAFLD was studied by using wild-type (adipoWT) and adiponectin knockout (adipoKO) mice. High-fat diet fed mice were insulin resistant, glucose intolerant and had increased hepatic lipid accumulation as evidenced by increased NAFLD activity score. Despite pioglitazone has improved insulin resistance in both genotypes, hepatic steatosis was only improved in adipoWT obese mice. Amelioration of NAFLD in adipoWT mice promoted by pioglitazone was associated with up-regulation of Pparg, Fgf21 and down-regulation of Pepck liver expression. On the other hand, resistance to pioglitazone treatment in adipoKO mice was associated with increased expression of miR-192 and Hsl, which was not followed by increased fatty acid oxidation. In conclusion, our data provides evidence that increased adiponectin production by pioglitazone is necessary for its beneficial action on NAFLD.

Introducing article numbering to Molecular and Cellular Endocrinology

In Press, Corrected Proof, Available online 6 June 2019

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ALK4-SMAD2/3-SMAD4 signaling mediates the activin A-induced suppression of PTX3 in human granulosa-lutein cells
Chang Liu, Hsun-Ming Chang, Yuyin Yi, Ying Fang, ... Xiaokui Yang
In Press, Accepted Manuscript, Available online 8 June 2019
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Abstract
Abstract
As one of the members of the transforming growth factor-β (TGF-β) superfamily, activin A plays an important role in regulating follicular development and oocyte maturation. Pentraxin 3 (PTX3) is the key component that promotes the process of cumulus expansion during mammalian ovulation. At present, the regulation of PTX3 expression in human granulosa cells remains largely unknown. This study aimed to examine the effects of activin A on the expression of PTX3 in human granulosa-lutein (hGL) cells and to investigate the underlying molecular mechanisms. Using an established immortalized hGL cell line (SVOG) and primary hGL cells as study models, we demonstrated that activin A significantly increased the phosphorylation of SMAD2 and SMAD3, which suppressed the expression of PTX3 at both the mRNA and protein levels. Additionally, these effects induced by activin A were completely reversed by pretreatment with the TGF-β type I receptor inhibitor SB431542 and knockdown of ALK4. Furthermore, knockdown of SMAD2, SMAD3, or SMAD4 completely reversed the activin A-induced suppressive effects on PTX3 expression. Notably, the ChIP analyses demonstrated that phosphorylated SMADs could bind to human PTX3 promoter. Collectively, our results showed that the ALK4-SMAD2/3-SMAD4 signaling pathway most likely mediates the suppressive effect of activin A on PTX3 expression in hGL cells.